key: cord- - x bch r authors: curran, janet a.; breneol, sydney; vine, jocelyn title: improving transitions in care for children with complex and medically fragile needs: a mixed methods study date: - - journal: bmc pediatr doi: . /s - - - sha: doc_id: cord_uid: x bch r background: children with medical complexity are a small yet resource intensive population in the canadian health care system. the process for discharging these children from hospital to home is not yet optimal. the overall goal of this project was to develop recommendations to be included in a provincial strategy to support transitions in care for children with complex and medically fragile needs. methods: a wide assortment of stakeholders participated in this mixed method, multiphase project. during phase , data was gathered from a range of sources to document families’ experiences transitioning from an inpatient hospital stay back to their home communities. in phase , pediatricians, nurses, and health administrators participated in key stakeholder interviews to identify barriers and facilitators to a successful transition in care for children and families with complex care needs. a multi-sector consensus meeting was held during phase to discuss study findings and refine key recommendations for inclusion in a provincial strategy. results: six case studies were developed involving children and families discharged home with a variety of complex care needs. children ranged in age from days to years old. nine telephone interviews were conducted in phase with pediatricians, nurses, and administrators from across the province. a variety of inter-institutional communication challenges were described as a major barrier to the transition process. a consistent message across all interviews was the need for improved coordination to facilitate transitions in care. the consensus meeting to review study findings included physicians, nurses, paramedics, senior administrators, and policy analysts from different health and government sectors and resulted in six recommendations for inclusion in a provincial strategy. conclusions: this project identified policy and practice gaps that currently exist related to transitions in care for children with complex and medically fragile needs and their families. our collaborative patient-centred approach to understanding how children and families currently navigate transitions in care provided a foundation for developing recommendations for a provincial wide strategy. children with complex and medically fragile needs are a small yet resource intensive population within the canadian pediatric health care system [ , ] . while these children represent less than % of the pediatric population, it is estimated they account for one third of pediatric health care spending [ ] . yet, discussion about the needs of children with medical complexity are often overshadowed in national health reform discussions by care of chronically ill adults [ ] . further, estimating the burden of illness on families, communities, and the health care system can be challenging due to the wide variation of health and social needs within this heterogeneous population. brenner et al. [ ] conceptualized "children's complex care needs [as] the multidimensional health and social care needs in the presence of a recognized medical condition or where there is no unifying diagnosis" [ ] (p. ). due to the complex nature of their care needs, these children often experience frequent hospitalizations [ , , ] , accounting for approximately % of pediatric admissions and % of hospital days [ ] . in comparison to children without complex chronic conditions, children with medical complexity experience almost times more inpatient visits and times greater inpatient costs [ ] . despite this high inpatient use, it is widely recognized that much of the care these children require could be provided in their home and home communities [ ] [ ] [ ] . with as many as % of children with complex chronic conditions being discharged from hospitalized settings [ ] , a well-coordinated and comprehensive transition from hospital to home is essential for improved patient and family outcomes and efficient use of health care resources. however, successful transitions in care for this population are characterized by a number of challenges. as many as physicians from distinct medical specialities and numerous other care team members across the health, educational, and community settings may be involved in the care of these children and families. this sizable care team extending across multiple services and sectors creates the potential for gaps in care coordination and communication [ ] . in addition to these factors, their care needs may include dependence on medical technology at discharge (i.e. ventilator, feeding tubes, etc.), requiring adequate support in their home community which can place this vulnerable population at an even greater risk for adverse outcomes or hospital re-admissions, particularly in geographically dispersed communities [ , , , ] . there has been increasing discussion in the literature over the past years surrounding pediatric complex care, including new models of service delivery to support children with complex care needs and their families in the community [ , ] . the american academy of pediatrics has advocated for the medical home model, grounded in family-centered primary care services, as a comprehensive, community based model of service for all children with complex care needs and their families [ ] . however, current health care structures in north american are not designed to effectively support these principles of care [ ] . with the multiple stakeholders involved in the care for this population, providing integrated and coordinated care for children with medical complexity in canada can prove to be challenging. a recent scoping review of the literature revealed a paucity of programs, interventions, or frameworks designed to support the transition from hospital to home for children with complex needs and their families [ ] . further, findings revealed a lack of patient and family-oriented outcome measures, indicating the need for researchers and policy makers to involve families in the research process and tailor forthcoming programs and policies to the individualized needs of this population [ ] . the need for clear guidelines and processes for the provision of high-quality care for children with complex and medically fragile care needs during care transitions is critical. while there are isolated tools and agreements in principle to collaboratively develop short-and longterm care plans for these children and their family, there are currently no generalizable transition processes that systemically guide transitions for this vulnerable population in nova scotia, canada. this project aimed to develop recommendations to improve the transition from hospital to home for children with complex and medically fragile needs in nova scotia. to achieve this aim, the following research objectives were addressed: ( ) describe the experience of patients with complex care needs and their families during the transition from a tertiary care facility to their home community; ( ) identify perceived barriers and enablers related to the existing transition process, and ( ) identify key components for inclusion in a strategy to enhance the transitioning of children and families with complex and medically fragile needs to their home community. using an integrated mixed methods approach [ ] , this project brought together a team of researchers, clinicians, parents/caregivers, and senior-level administrators across the province of nova scotia. data was collected from multiple sources across three phases to achieve a greater breadth and depth of understanding into the existing transition process (fig. ) [ ] . this study took place at a pediatric tertiary care facility located in nova scotia, canada, responsible for the care of children, youth, and women across the canadian maritime provinces. the canadian maritimes is composed of three provinces, with a combined population of almost , children and youth [ ] . this health centre has approximately baby and children beds, with estimated , acute inpatient admissions, , emergency department visits, and , outpatient visits annually [ , ] . ethical approval was obtained from the iwk research ethics board (project # ). data collection occurred between and . to address our first objective, a case study design was employed to develop cases representing the experiences of a range of children with complex and medically fragile needs transitioning from hospital to home. we employed maximum variation sampling to ensure a representation of different age groups, levels of complexity, family structures, geography and socioeconomic status levels when possible [ ] . eligibility for this study included parents or guardians of children aged - years old with one or more chronic conditions that were expected to require specialized care greater than year. this eligibility criteria was created to align with current literature describing children with complex care needs [ , , ] and based on expert opinion from our multidisciplinary research and clinical team. terminally ill patients were excluded from this study. clinical leads from each unit in the tertiary care facility invited potential participants according to the eligibility criteria by providing a letter of information describing the study and contact information for the research assistant. the research assistant reviewed the letter of informed consent with potential participants and they were given the opportunity to ask questions before participation. each case was informed by multiple sources of data: family, tertiary care provider, and community health care provider interviews and a structured chart audit. families were asked to identify one primary caregiver to complete the interviews and all data collection measures. all providers involved in the care of these children were invited to participate in our study. data collection occurred at four points in time: ( ) two weeks prior to discharge (t ); ( ) one day prior to scheduled discharge (t ); ( ) one week following discharge (t ); and four weeks following discharge (t ). families were asked to complete the sf- tool at t and t to estimate disease burden [ ] . the state trait anxiety inventory (stai) was used to assess parental anxiety and measured at t and t [ ] and the brief cope was used to assess parents coping strategies at t and t [ ] . our approach to data collection attempted to balance understanding a range of patient, family and health system factors relevant to the transition from hospital to home while being mindful of not contributing to caregiver burden during the transition home. the measures were chosen to capture the experiences and potential changes in stress and coping at different time points during the transition from hospital to home. the child's primary caregiver also participated in individual interviews at three time points: t , t , and t . a semi-structured interview tool, developed by the research team, was used to explore caregiver's knowledge, attitudes, and beliefs about the discharge process at each time point. tertiary care and community health care providers who were directly involved in providing care to the patient and family were also interviewed on a single occasion after the child's discharge from hospital. a semi-structured interview guide was also used to explore provider's knowledge, attitudes, and beliefs about the discharge process. all interviews were audio-recorded and transcribed verbatim. interview transcripts were sorted by case and managed in nvivo qualitative data analysis software [ ] . transcripts were coded by two independent reviewers. a content analysis approach was used to identify important barriers and enablers of the discharge process in each case [ ] . reviewers met to compare findings and coding discrepancies were resolved through discussion and consensus. to address our second objective, semi-structured telephone interviews were conducted with key stakeholders across the province to identify barriers and facilitators related to the transition from hospital to home for children with complex and medically fragile needs. an initial list of potential community stakeholder participants was identified through consultation with members of the executive leadership team of the tertiary care centre. subsequently, we used a snowball strategy inviting study participants to identify additional potential provincial stakeholders. a letter of invitation was sent to potential participants via email. data was collected through individual telephone interviews and verbal consent was obtained from all participants. a semi-structured interview guide, based on the tdf [ , ] , was used to explore participant's experiences, attitudes and beliefs about supporting children and families through transitions in care. this interview guide was initially drafted by the principle investigator (jc) and further refined by our team of researchers and clinicians. the interviewer (sb) used prompts when appropriate to encourage participants to elaborate on their experiences and opinions. interviews were recorded and transcribed verbatim by an experienced transcriptionist. one reviewer (sb) performed deductive content analysis to sort barriers and facilitators arising from each transcript according to the domains of the tdf [ , ] . coding began immediately after the first interview and a second reviewer (jc) validated the coding in % of the transcripts by independently coding the first and every other transcript. participant recruitment was deemed complete and data saturation was achieved when no new barriers and facilitators were identified in the transcripts [ ] . subsequently, one reviewer (sb) conducted a thematic analysis to identify important themes related to the barriers and enablers [ ] . frequent checks and collaboration with research team members occurred to discuss themes as they emerged. to address our final research objective, we hosted a half-day multi-disciplinary stakeholder meeting to review findings and identify and refine key recommendations for inclusion in a policy statement. our guiding framework for the meeting was underpinned by an adapted consensus-oriented decision-making process; steps included: ( ) framing the topic (through our data); ( ) open discussion (of data and analysis); ( ) identifying underlying concerns; ( ) collaborative strategy building; ( ) choosing a direction; ( ) synthesizing a final strategy/proposal; and ( ) identification of next steps and roles [ ] . six cases representing the transition experience for a range of children with complex and medically fragile needs were created. these cases included children with an age range of days to years and were followed by as many as seven different speciality services. case characteristics are summarized in table . table provides an overview of the brief cope inventory scores for all cases. a summary of the thematic cross case analysis can be found in table . the following section provides a brief overview of the key features of developed case studies. geographical distances from the tertiary care centre have been categorized dichotomously to: ( ) less than an hour drive and ( ) more than an hour drive. a total of health care providers were interviewed across the cases and represented a range of professions which included pediatricians, physiotherapists, social workers, pharmacists, neonatologists, transition coordinators, nurse practitioners, occupational therapists, family care coordinators, dieticians, oncologists, and plastic surgeons. this case examined the hospital to home transition of a month-old infant with a congenital atrial septal defect and hip dysplasia who was discharged home post-operatively for ongoing monitoring and rehabilitation. the family lived more than an hour drive from the tertiary care centre and had experienced multiple admissions. key features of this case included: ( ) family concerns regarding trust with certain members of the health care team and their level of knowledge regarding their child's condition and ( ) extensive collaboration between different care teams which was facilitated by a nurse practitioner. summary measures of physical (pcs) and mental (mcs) health and anxiety are unremarkable (table ) . when compared with other cases in this study, this family leveraged more dysfunctional versus problem focused strategies across the transition from hospital to home (table ) . this case involved a -day-old infant born at weeks gestation with fragility and a genetic anomaly. the family lived within h drive from the pediatric tertiary care centre which meant that certain elements of care could remain the responsibility of the tertiary care centre's perinatal team. key features of this case included: ( ) a strong sense of support surrounding the caregivers and child from family members and the tertiary and primary care team, which helped ease and support the transition home; and ( ) concerns expressed by a member of the tertiary care team regarding inadequate resources in the local community to address nutritional needs of the infant, which led the perinatal team to closely follow feeding and weight gain while at home. changes were noted in pcs scores from t to t ( . vs . ) ( table ). the family also leveraged a high degree of problem focused strategies during the transition home with a noted increase of positive framing techniques between t and t ( table ) . this case examined the experience of a -year-old child with complicated osteomyelitis requiring multiple debridement surgeries prior to discharge to their home community located less than an hour drive from the tertiary care centre. key features of this case included: ( ) a strong sense of trust in the tertiary care team prior to discharge; ( ) a decreased level of confidence in the care team post-discharge due to contradictory information received by various providers; ( ) communication gaps reported by the family between the home care nurse and the health centre regarding treatment needs; ( ) a high level of satisfaction with home nursing services and educational information received regarding their child's treatment; and ( ) an active involvement and cooperation of the child's school facilitating the accommodation of their medical needs. estimates of family anxiety and burden were stable (table ) . brief cope scores remained relatively consistent between t and t with a reliance on problem and emotional-focused coping strategies (table ) . this case explored the hospital to home transition of a month-old infant born at -weeks gestation with bronchopulmonary dysplasia, gastroesophageal reflux disease and required nasojejunal (nj) feeding. the more than one-hour drive from the home community to the tertiary care centre presented a challenge, as the there was another other child in home. key features of this case included: ( ) a high level of trust with the care team at the tertiary care centre; ( ) difficulty with feeling involved in all care decisions due to the division of time between the health centre and home; ( ) concerns from both the care team and the family about the lack of resources to manage the infant's nj tube at home; ( ) cost associated with formula and prescription; ( ) the process of accessing respite care; and ( ) limited information provided in discharge summary to adequately inform the primary care physician of the infant's condition, requiring the caregivers to provide more detailed information about the care plan to their provider. stai and sf- scores were unremarkable (table ) . when compared with other cases, this family reported limited use of coping strategies at both points in time with problemfocused strategies being most prevalent (table ) . this case examined the hospital to home transition of a -year-old infant with gastric atresia with additional complex care needs who had been hospitalized since birth. the family's home community was located more than h drive from the pediatric health centre. key features of this case included: ( ) a high level of trust in the tertiary care team; ( ) other parents on the inpatient unit act as key supports to the family; ( ) satisfaction with the care from the local community pediatrician, but dissatisfaction with care received from the community hospital following misplacement of the child's feeding tube; ( ) an advanced practice nurse guiding the discharge process and using a locally developed tool to guide discharge planning that was distributed to the family and home community to promote continuity of care; ( ) a high level of engagement from the family helping to facilitate a smooth transition as reported by the health care team; and ( ) a lack of pediatric expertise in community physiotherapy. compared with other cases this family reported the highest pcs across t and t and reported higher anxiety scores across t and t . (table ) this family also reported the highest rate of dysfunctional strategies at t when compared with other cases (table ) . further, there was a reported decrease in the use of instrumental support once home in the community (table ) . this case examined the transition of a -year-old child diagnosed with leukemia and ulcerative wounds who was discharged to a home community over an hour away following a four-month inpatient stay. key features of this case included: ( ) a high level of trust with the tertiary care centre and the development of strong relationships with the nurses and other families on the unit; ( ) concerns about inconsistencies regarding access to supports and resources for parents during inpatient stay; ( ) high level of satisfaction with home care services, but a desire for more continuity in nursing care; ( ) a 'family care coordinator' acted as key contact at the pediatric health centre and kept the family informed regarding the care plan; and ( ) the family feeling prepared to care for their child's medical needs at home but not prepared for the socio-emotional challenges and stress of caring for their child within the home. this case reported the highest anxiety at t and t with an increase in mcs from t to t ( . to . ) ( table ). the reported brief cope scores suggest a preference for problem and emotional focused strategies following their discharge home (table ) . telephone interviews were conducted with nine health care providers from five health regions in nova scotia who support children with medical complexity and their families. interview participants included four pediatric registered nurses, four community health care physicians, and one administrator. data saturation was determined after the th interview as no new themes emerged [ ] . data was organized using the tdf domains and highlighted potential barriers and enablers to the hospital to home transition for children with medical complexity (see table ) [ ] . the majority of participants were not aware of formal guidelines or policies to help support or inform the transition from hospital to home for children with medical complexity and their families. guidelines that were discussed included the medical home model from the american academy of pediatrics [ ] , toronto sick kids complex care program [ ] , and condition-specific health centre policies. a number of health care providers indicated a lack of awareness of the range of resources accessible in the community and the tertiary care facility for both families and health care providers to support this transition in care. further, given that some smaller communities may go many years in-between assuming care for a child with complex care needs, a lack of experience of how the transition process should occur was reported. participants also mentioned specific psychomotor skills that may be required by the community health care providers to care for the complex medical needs of these children. participants spoke of several systems and resources that could be used to support smoother, more effective, and efficient transitions from hospital to home for children with medical complexity and their families. this included: ( ) prompt and consistent communication to the appropriate health care providers/services; ( ) care coordinators located in the community and the tertiary care centre to oversee the coordination of care and support families; ( ) teleconferences between all members of the care team; ( ) written care plans provided to both families and providers in advance of discharge from the tertiary care facility; ( ) access to additional speciality health care services within the community; and ( ) online repository or system to store and organize existing beliefs about capabilities health care provider confidence "but the overwhelming theme there is that we have a system that isn't tuned to the needs of children and care for children outside of the specialized centre. so you know… and people will say to you very directly, "i don't have pediatric experience, and i'm not comfortable." parental confidence "i've seen it with our newborns that come back that have been born premature and have been in nicu, and now they're doing well, they're there just to grow. you know, they don't need the monitoring, they don't need that intensive level of care. and we find parents have a very difficult time understanding that the baby is now theirs to take care of. you know, they're not ready that now they're on the normal care track that they would have been on had baby been born at term." beliefs about consequences family burdens "because you're worried enough about your child, you don't need to worry that, you know, is the community ready, are the staff prepared? that, you know, should we land in the emergency room in days' time, are they going to know who my child is? are they going to know what he or she needs? are the staff at the nearest pediatric unit prepared to take care of them or know what to do if something should arise?" hospital readmissions "i think the consequences is sometimes it leads to increased admissions into the acute care unit." breaks in the continuum of care "because sometimes they might end up presenting back to our emergency department, for example, before they've seen any of us….they might end up coming in on a friday night and seeing one of my other pediatric colleagues that didn't get any of the documentation i received. preventable adverse outcomes "well, i think we may miss some opportunities to avoid or to monitor for complications. and so in that way, you know, even if nothing happens, we haven't been there being as diligent perhaps as we could be for quality assurance and ensuring that the best health care is being provided." health care providers highlighted the need for both expertise and confidence in caring for the complex pediatric population to ensure optimal care is provided. given the rurality of many of these communities, community health care providers may have limited encounters with pediatric patients resulting in a decreased confidence in caring for their unique needs. practitioners also spoke of the varying parental confidence levels during the shift in care responsibility. parents must be adequately prepared to transition from a secure hospital environment to their homes where health care providers will not be directly overseeing their child at all times. study participants discussed various consequences that may arise without proper structures and processes in place to support the hospital to home transition for children with medical complexity and their families. these included: ( ) family burdens (i.e. travelling unnecessarily to and from the tertiary care centre); ( ) hospital readmission; ( ) interruptions in continuum of care (i.e. providers not having access to the appropriate information); ( ) preventable adverse events; and ( ) poor reintegration into community (i.e. returning back to school). all study participants believed the transition from hospital to home for this vulnerable population was of great importance. when asked to rate the importance of improving this transition in care on a scale from to , with being not important, and being very important, participants reported an average of . / (range - ). participants highlighted the importance of improving this transition in care to ensure the delivery of patient-centred care. a common theme across all interviews was the perceived gap in efficient communication between the tertiary care centre and the receiving community providers. several community practitioners noted that being consulted early on was a key facilitator to the transition process. health care providers also discussed the inability to access certain discharge summaries and patient charts and reported not receiving care plan information in a timely manner. the variability in resources located in the home communities was discussed as both a barrier and enabler to the transition process. some communities reported having resources such as advance practice nurses, travel funds, and home care services that help support these children and families in the home. however, lack of speciality resources was highlighted as a barrier to providing adequate care for children with complex needs in some rural communities. participants also discussed various resources located at the pediatric health centre that were helpful in supporting smooth transitions in care. these included care coordinators, perinatal follow-up teams, social workers, clinical nurse specialists, specialty physicians and nurse practitioner. participants in the community frequently mentioned the importance of a having access to a key worker or coordinator assigned to each child and family. two members of the research team (jc, sb) facilitated a consensus meeting with key stakeholders from across nova scotia. consensus meeting participants included community and tertiary care health care providers, paramedics, senior administrators and policy analysts from the health and education sectors who were not involved in phase data collection. the consensus meeting lasted hours. key findings from phase and were presented along with proposed recommendations for inclusion in a provincial policy statement. recommendations were developed using a behaviour change lens to act as a starting point in addressing the challenges arising from phase and findings. key priorities in the development of the recommendations included building on existing programs and infrastructures and working with a range of stakeholder groups to co-develop new resources. we also recognized the need to make these resources accessible to end-users across the province, regardless of geographical location. each recommendation was discussed and revisions were made through a process of consensus as described previously in the methods section. two points of discussions resulted in modifications to the recommendations. first, consensus meeting participants agreed there was a need for a shared conceptual definition for children with complex and medically fragile needs to orient the present discussion, as well as future clinical and research initiatives examining this population. a number of published definitional frameworks were discussed and participants chose cohen et al.'s definitional framework as the most suitable given the recent work using this framework in a canadian context [ , ] . second, one of the recommendations included the need for a care coordinator within each provincial health zone to act as a liaison and resource for families and health care providers. following a lengthy discussion, consensus meeting participants recommended that the coordinator role should be specified as an advanced practice nurse. see table for a complete list of final recommendations. this project aimed to develop recommendations to improve the hospital to home transition for children with complex and medically fragile needs and their families in one canadian province. this multi-phased research initiative incorporated the perspectives and experiences from families, administrators, and health care professionals to co-develop key recommendations for policy and practice change. in addition to these recommendations, examination of study findings revealed a variety of gaps and areas for future research to optimally support these families during transitions from hospital to home. understanding the experiences and perspectives of families of children with medical complexity is essential for designing policies and systems to improve transitions in care for this vulnerable populations [ ] . cases included in this study represented a range of experiences and complexity. across all cases, there were a number of similarities between family experiences transitioning from hospital to home. prior to discharge, families reported feeling prepared to manage their child's medical care, and eager to return home once their child was medically stable. nurses were identified as a key member of a child's care team within the inpatient setting. they acted as both an educational resource, care coordinator, and emotional support, facilitating a sense of empowerment and security within families. this finding is consistent with the literature describing the role of nurses in supporting seamless discharge from hospital to home [ , ] . similar to other studies, parents in this case sample also identified other parents as an important source of support [ ] . divergence appeared across cases in regard to the availability of resources and support after returning home from hospital. while a number of parents reported having access to appropriate resources in their community, some families reported feeling inadequately supported to assume the various medical, social, and emotional care needs of their child in addition to their daily responsibilities (e.g. work, family, personal needs). parent readiness for caregiving at discharge can shape the families ongoing experience [ ] . ideally, the shift in responsibility for care from hospital to home should be negotiated with parents prior to discharge [ ] . the health care system is beginning to address the shift in care of these children from hospitalized to community-based settings, but work still remains to be completed to optimally support these children and families to flourish in the home [ ] . there have been a number of emerging models of care to support advanced health care in the community. these proposed models range greatly in regards to their characteristics, including being both hospital-and primary caremanaged, further highlighting the lack of agreement surrounding the optimal model of care for these children [ , ] . it is additionally important to recognize that caring for the health of the family as a whole is critical, as the child's health is often linked with that of their caregiver [ , ] . in designing an optimal health care system for individuals with complex care needs, the commonwealth fund released recommendations for policymakers [ ] . of these table recommendations for inclusion in provincial policy statement adopt the definitional framework for children with medical complexity (cohen et al., ) to identify children with intensive care needs in the province of nova scotia that are not easily met under existing policies and services. work with existing provincial programs and services (i.e. continuing care) to develop policies and tools that are unique to a pediatric population. develop a role for a pediatric advanced practice nurse in each health zone in nova scotia to act as a liaison/resource between the tertiary care facility and children discharged with medical complexities, their families and their health care providers to coordinate care and lead capacity building and education initiatives with local health care providers, children, and families. develop a comprehensive discharge plan for every child with complex care needs. the plan must be co-developed and approved by a discharge planner/advanced practice nurse, parent or caregiver-home, or community discharge coordinator prior to discharge from the pediatric tertiary care facility and will consider the medical, psychosocial and developmental requirements for patients to successfully transition back to their home community. develop a complex care information repository (ccir) for health care providers, administrators, patients and families to store and organize key resources (contact information for key personnel, clinical practice guidelines, community/hospital resources, etc). develop an educational outreach strategy to address the knowledge, skills and competency needs of health care providers across nova scotia who care for children with medical complexity. recommendations, one clearly outlines the need for greater support for caregivers, suggesting that "support might include respite care to provide relief for caregivers and assistance to help them look after their own health" ( [ ] , p. ). the crucial need to enact this recommendation is further confirmed by families of children with complex care needs reporting higher levels of depression [ , ] , stress [ ] , poorer general health [ ] and feelings of isolation [ ] . one scoping review conducted by edelstein, schippke, sheffe, and kingsnorth ( ) identified a range of interventions aimed to mitigate the stress experienced by caregivers of children with medical complexity living in the community [ ] . these programs included care coordinator models, respite care, telemedicine, financial benefits and peer support programs [ ] . with the promising results emerging from this review, future high-quality research evaluating the effectiveness of these interventions on caregivers is warranted to improve the support provided to families of children with complex care needs [ ] . attending to the complex multidimensional health and social care needs of this population of children requires a multidisciplinary approach [ ] . timely and effective communication between all members of the care team was identified as an essential component to a successful transition home. health care providers often reported turning to specific nurses when unsure about details of a certain care plan, with most all providers expressing the desire for a key coordinator assigned to each child with complex care needs and their family to facilitate effective communication. the role of this care coordinator, or otherwise referred to as a 'key worker', in the care transition process has been gaining increasing attention over the past number of years [ , , , ] . most recently, the canadian association of pediatric health centres has recommended the development of a key worker role to facilitate transition planning and management for all children with medical complexity and their families [ ] . further, the need of a care coordinator role is also mentioned in the commonwealth fund's recommendations for optimal health care for individuals with complex care needs to reduce the risk of fragmented health care delivery and facilitate timely communication amongst the care team [ ] . to support this transition, key workers act as a single point of entry, enable team collaboration and streamline essential communication amongst families and health care providers [ , ] . this key worker is a proposed strategy to mitigate the care coordination and communication challenges associated with what is often a large health team across multiple services and sectors managing the care of these children and their families. key workers not only streamline team communication, but also play a pivotal role in empowering and engaging families in the child's care [ ] . as the development of this role continues, it is critical that patient-oriented research is conducted to evaluate its effectiveness, make appropriate changes to the roles based on specific contexts and to ensure optimal care delivery for this vulnerable population. an economic evaluation would also be important to estimate the relative costs and consequences of implementing this new role. health care providers reported a paucity of knowledge of specific formal guidelines or policies to guide transitions from hospital to home for children with complex care needs. however, informal processes within each discipline were followed and many teams had developed their own discharge planning tools to ensure all appropriate actions and processes were taken to ensure smooth transitions in care. while there are a number of recommendations and guidelines aimed at the supporting the care management of this population [ , , , ] , there remains a limited number of empirically evaluated programs and interventions informing the transition from hospital to home [ ] . although the transition process should be customized to the individualized needs of children and families, overarching guidelines and structures should to be in place to ensure timely transition planning and continuity in care. however, to ensure the development of relevant, comprehensive, and sustainable policy recommendations, engaging a variety of key stakeholder is crucial [ ] . leveraging research techniques to bring family experiences and perspectives to the forefront of decision making is essential to ensuring family-oriented practice and policy changes are pursued. further, adopting an integrated knowledge translation approach by partnering with knowledge users throughout the research process can help ensure the knowledge being co-created will be relevant to their needs, and therefore more likely to be adopted into practice and policy [ , ] . the recommendations stemming from this study were developed by integrating the experiential knowledge from families with the perspectives of stakeholders across the tertiary care, community care, and government sectors. results from this research serves as a critical foundation for future intervention and program design to support the entire care team, including health care providers, administrators, and families, during the often complex, dynamic, and unique transition from the hospitalized setting back into a child's home and home community. there are several limitations to note in this study. although a range of children, families, and health care providers participated in this study and we leveraged a range of data collection sources and strategies, these findings may not be generalizable to the transition experiences of all children and families with complex care needs. we developed a total of cases to explore a range of experiences transitioning from hospital to home, however we recognize that this is a heterogeneous population of children and our cases may not represent the experiences of all children and families with complex care needs. further, a comprehensive understanding of the impact of the hospital to home transitions may not have been fully captured within the month follow-up time in the case studies [ ] . only individuals able to speak english were eligible to participate. children and families speaking other languages may have different experiences transitioning from hospital to home that were not captured by this study. children with medical complexity represent a small but resource intensive portion of the pediatric population in the canadian health care system. transitions from tertiary care to home can pose many challenges for children, families and health care providers in the recipient communities. our project identified policy and practice gaps that currently exist in one canadian province related to transitions from a tertiary care facility to home communities for children with medically fragile needs and families. our collaborative patient-centred approach to understanding how children, families, and health care providers currently navigate transitions in care provided a foundation for developing recommendations for a provincial wide strategy. children with medical complexity: an emerging population for clinical and research initiatives patterns and costs of health care use of children with medical complexity building systems that work for children with complex health care needs: editor's note children's complex care needs: a systematic concept analysis of multidisciplinary language children with medical complexity in canada children with complex chronic conditions in inpatient hospital settings in the united states variation in child health care utilization by medical complexity disabilities the cocw. home care of children and youth with complex health care needs and technology dependencies caring for children with specialized health care needs in the community: the challenges for primary care strategies to reduce hospitalizations of children with medical complexity through complex care: expert perspectives pediatric readmission prevalence and variability across hospitals medical complexity and pediatric emergency department and inpatient utilization the impacts of distance to hospital on families with a child with a chronic condition models of care delivery for children with medical complexity status complexicus? the emergence of pediatric complex care the landscape of medical care for children with medical complexity. overl park: child hosp assoc strategies to support transitions from hospital to home for children with medical complexity: a scoping review designing and conducting mixed methods research census families with children by age of children and children by age groups iwk health centre -about us report of the auditor general to the nova scotia house of assesmble. halifax: nova scotia auditor general pediatric deaths attributable to complex chronic conditions: a population-based study of washington state sf- v health survey manual for the stait-trait anxiety inventory you want to measure coping but your protocol's too long: consider the brief cope how to plan and perform a qualitative study using content analysis validation of the theoretical domains framework for use in behaviour change and implementation research the behaviour change wheel (behavior change wheel) -a guide to designing interventions are we there yet? data saturation in qualitative research using thematic analysis in psychology the basics of consensus decision making complex care program. sick kids families of children with medical complexity: a view from the front lines complex care packages: supporting seamless discharge for child and family advanced practice profiles and work activities of nurse navigators: an earlystage evaluation a qualitative study of parent to parent support for parents of children with special needs negotiating lay and professional roles in the care of children with complex health care needs experts' perspectives toward a population health approach for children with medical complexity designing a high-performing health care system for patients with complex needs: ten recommendations for policymakers. the commonwealth fund; health and social care a longitudinal study of families with technologydependent children changes over time in the health of caregivers of children with health problems: growth-curve findings from a -year canadian population-based study enabling young 'ventilator-dependent' people to express their views and experiences of their care in hospital getting out of the house: the challenges mothers face when their children have long-term care needs children with medical complexity: a scoping review of interventions to support caregiver stress. child care health dev the role of care coordinator for children with complex care needs: a systematic review canadian association of pediatric health centres. children and youth with medical complexity (cymc) caphc community of practice (cop). national guidelines collaborative care coordination: a priority for health reform families' priorities regarding hospital-to-home transitions for children with medical complexity importance and feasibility of transitional care for children with medical complexity: results of a multistakeholder delphi process strategy for patient-oriented research. ottawa: canadian institutes of health resarch guide to knowledge translation planning at cihr: integrated and end-of-grant approaches -cihr. canada: government of canada sharing and expanding academic and practitioner knowledge in health care a refined taxonomy of behaviour change techniques to help people change their physical activity and healthy eating behaviours: the calo-re taxonomy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all the children, families, and health care providers that committed their time to participate of this project. we also wish to acknowledge margaret champion and julia belliveau for their contributions with data collection. authors' contributions jc and jv conceived of the study, obtained funding to support the project. jc supervised the data analysis. sb participated in data collection, data analysis, and drafted the initial manuscript. both jc and jv made significant revisions to the manuscript. all authors read and approved the final manuscript. this research was funded through a translating research into care (tric) grant from the iwk health centre. funding to publish our study in an open access journal was provided through an open publication bursary from the maritime strategy for patient oriented research (spor) support unit. the datasets generated and/or analyzed during the current study are not publicly available due issues with privacy and confidentiality but are available from the corresponding author on reasonable request. ethical approval for this research was obtained at the iwk health centre by the iwk research ethics board (project #: ). written informed consent was obtained from parents or guardians for participation. consent from each individual participant was obtained following procedures approved by the iwk health centre's research ethics board. written consent appendix table theoretical domains framework definitions knowledge "an awareness of the existence of something" (p. ) skills "an ability or proficiency acquire through practice" (p. ) "the ability to retain information, focus selectively on aspects of the environment and choose between two or more alternatives" (p. )behavioural regulation "anything aimed at managing or changing objectively observed or measured actions" (p. ) "a coherent set of behaviours and displayed personal qualities of an individual in a social or work setting" (p. )beliefs about capabilities "acceptance of the truth, reality, or validity about an ability, talent, or facility that a person can put to constructive use" (p. )optimism "the confidence that things will happen for the best or that desired goals will be attained" (p. )beliefs about consequences "acceptance of the truth, reality, or validity about outcomes of a behaviour or a resolve to act in a certain way" (p. )intentions "a conscious decision to perform a behaviour or a resolve to act in a certain way" (p. ) goals "mental representations of outcomes or end states that an individual wants to achieve" (p. )reinforcement "increasing the probability of a response by arranging a dependent relationship, or contingency, between the response and a given stimulus" (p. )emotion "a complex reaction pattern, involving experiential, behavioural, and physiological elements, by which the individual attempts to deal with a personally significant matter or event" (p. ) "any circumstance of a person's situation or environment that discourages or encourages the development of skills and abilities, independent, social competence, and adaptive behaviour" (p. )social influences "those interpersonal processes that can cause individual to change their thoughts, feelings, or behaviours" (p. ) [ , ] was obtained from a parent or guardian on behalf of all participants under the age of . the authors declare that they have no competing interest.received: august accepted: may key: cord- -zgdvo n authors: biglan, anthony; elfner, karen; garbacz, s. andrew; komro, kelli; prinz, ronald j.; weist, mark d.; wilson, dawn k.; zarling, amie title: a strategic plan for strengthening america’s families: a brief from the coalition of behavioral science organizations date: - - journal: clin child fam psychol rev doi: . /s - - - sha: doc_id: cord_uid: zgdvo n despite significant progress in research on the treatment and prevention of psychological, behavioral, and health problems, the translation of this knowledge into population-wide benefit remains limited. this paper reviews the state of america’s children and families, highlighting the influence of stressful contextual and social conditions on child and family well-being and the concentration of disadvantage in numerous neighborhoods and communities throughout the nation. it then briefly reviews the progress that has been made in pinpointing policies that can reduce stressful contextual conditions such as poverty, discrimination, and the marketing of unhealthful foods and substances. it also describes numerous family and school interventions that have proven benefit in preventing psychological and behavioral problems as diverse as tobacco, alcohol, and other drug use; depression; antisocial behavior; academic failure; obesity prevention; and early childbearing. we argue that progress in translating existing knowledge into widespread benefit will require a nationwide effort to intervene comprehensively in neighborhoods and communities of concentrated disadvantage. we present a strategic plan for how such an effort could be organized. the first step in this organizing would be the creation of a broad and diverse coalition of organizations concerned with advancing public health and well-being. such a coalition could increase public support both for the policies needed to focus on these disadvantaged areas and the research needed to incrementally improve our ability to help these areas. this paper presents a strategic plan for reducing the prevalence of concentrated disadvantage in the usa. concentrated disadvantage consists of a tangle (sampson ) of inter-related environmental and behavioral problems such as poverty, discrimination, unemployment, substance use, single parenting, and academic failure that result in intergenerational poverty and the development of multiple psychological, behavioral, and health problems often resulting in premature death (national academy of sciences b). over the past fifty years, the prevalence of such disadvantage has increased dramatically in the usa (kristof and wudunn ; putnam ) . reversing this trend and thereby reducing the prevalence of these problems will require an unprecedented organization of our national resources over an indefinite period of time. we hope that this paper will contribute to such a nationwide effort. the paper begins with a brief summary of what we know about the contextual conditions that undermine family wellbeing and the resulting patterns of family life that contribute to life-long intergenerational psychological, behavioral, and health problems. it then summarizes the extensive evidence regarding programs and policies that have proven benefit in preventing or ameliorating one or more aspects of disadvantage, as well as the research that is needed to translate existing knowledge into widespread success in reducing concentrated disadvantage and its sequelae. we then present a strategy for building a nationwide effort to strengthen family well-being with special attention to neighborhoods and communities of concentrated disadvantage. the well-being of america's families can be gauged by the quality of the contextual conditions that affect families and by the quality of family life itself. even a brief summary of the evidence indicates that many families live in stressful social contexts and have high levels of family dysfunction. among the harmful contextual conditions that have been verified by previous large-scale studies are family poverty, homelessness, contacts with the criminal justice system, discrimination, and the marketing of health-compromising behaviors in youth (e.g., unhealthful food and substances). according to the u.s. census bureau (fontenot et al. ) , , , american families were living in poverty in . according to the organization for economic co-operation and development (oecd family database ), as of , the usa had the third-highest rate of poverty among developed countries ( . %). the federally defined poverty level for a family of four is $ , (families usa ). as of , % of children lived in homes with income below the poverty level, and . % were in homes below % of the poverty level (child trends databank ). a major reason for these rates of poverty is the high rate of single parenting. more than % of poor children live in single-mother families, and . % live in single-father families. a meta-analysis by stringhini et al. ( ) of studies involving . million people showed that socioeconomic status was associated with higher rates of mortality even when the researchers controlled for well-established risk factors for disease. children raised in poverty had significantly greater risk of most psychological, behavioral, and health problems (jarjoura et al. ; matthews and gallo ; pampel et al. ) and shorter life expectancy (galobardes et al. (galobardes et al. , miller et al. ) . distinct from the effects of poverty, economic inequality is a risk factor for multiple psychological, behavioral, and health problems. pickett and wilkinson, as well as other investigators (pickett and pearl ; pickett and wilkinson ; kaplan ) , have shown that premature death, obesity, infant mortality, depression, social anxiety, property crimes, and homicide are greater in economically unequal countries. many of these disparities in health are found among all but the very highest-earning segments of a population. the deleterious effects of inequality appear to be due to the fact that people living in economically unequal communities are more likely to have stressful interactions with people who are above or below them in the economic hierarchy (pickett and wilkinson ; wilkinson and pickett ) . according to a national system for estimating homelessness, there were about , homeless families with children on any given night in (national alliance to end homelessness ). homeless children have more developmental delays, poorer academic development, and more physical illnesses and conditions, such as stunted growth, anemia, asthma, lead poisoning, and infectious diseases (hart-shegos ) . the criminal justice system can have a devastating impact on families. the usa has the highest rate of incarceration in the world (wagner and walsh ) , and there is a much higher rate of incarceration of black people, a result of disparities at every point in the criminal justice process, from arrest through sentencing (alexander ) . a survey of american adults indicated that % of black people indicated that a member of their family had been incarcerated; the rate was % for whites and % for hispanics (enns et al. ) . families with a parent in prison are likely to experience poverty, homelessness, and residential instability (peterson et al. ) . parental imprisonment often contributes to children's behavior problems, depression, poor academic performance, and antisocial behavior (parke and clarke-stewart ; peterson et al. ) . children may also experience trauma through involvement with the criminal justice system at other points (e.g., if they witness the arrest of a parent or before or during visits with a parent in prison or jail; eddy and poehlmann ). discrimination is widespread in the usa (national public radio, robert wood johnson foundation, & harvard t.h. chan school of public health ) . in addition to welldocumented effects on the health of individuals (pascoe and smart richman ) , discrimination affects family wellbeing due to discriminatory hiring and pay and residential segregation. however, the most damaging aspect of discrimination may simply be the frequent exposure to experiences that are threatening or aversive and that contribute to inflammatory disease (american psychological association ). the marketing of tobacco, alcohol, and unhealthful food and beverages harms families. although the makers of these products often argue that their marketing is not to blame for underage use of tobacco and alcohol or childhood obesity, empirical research shows that marketers target children, their messages reach children, and these messages influence youths' purchases and consumption (biglan a; nestle ; pechmann et al. ) . moreover, the ready availability of illicit drugs in neighborhoods of concentrated disadvantage contributes to drug use disorders among both parents and children (ford et al. ; storr et al. ). each of the just-described conditions contributes to the development of psychological, behavioral, and health problems that undermine children's life chances and contribute to premature death. however, the risk of such outcomes is increased when multiple disadvantaging conditions are present. evidence for this comes from studies showing that the greater number of adverse experiences that children have, the more likely that their behavioral and physical health will be affected (anda et al. (anda et al. , felitti et al. ) . this underscores the necessity of targeting neighborhoods and communities of concentrated disadvantage if the nation is going to significantly reduce the number of children who are destined for multiple problems, impeded potential and premature death. a recent monograph by acevedo-garcia et al. documents the extent to which american communities have neighborhoods with extensive disadvantage (acevedo-garcia et al. ). the just-described contextual conditions contribute to the undermining of effective parenting. stressors such as poverty, economic inequality, and discrimination undermine parents' time, ability, and motivation to provide warm, responsive support of their children's development. poverty, discrimination, unemployment, and homelessness also make depression more likely. maternal depression is a risk factor for socio-emotional and cognitive delays in children (bernard-bonnin, society, health, & committee ) . there is also evidence that neighborhoods that are low in socioeconomic status have higher rates of some types of substance use (karriker-jaffe ). and, according to the substance abuse and mental health services administration (samhsa), about . % of children ( . million) live in homes where at least one parent has a substance use disorder (lipari and van horn ) . parental substance use is associated with a higher likelihood of child abuse (institute of medicine & national research council ) and later substance use by the child (anda et al. ) . finally, as the proportion of marriageable men declines due to unemployment and poverty, family stability is undermined, and the rate of single parenting increases. sawhill ( ) reported that, across ethnic groups, the rate of single parenting has increased in recent years among those without a college education. schools in high poverty neighborhoods and rural communities, generally have fewer resources than schools in more affluent areas, even though it is in high poverty areas that more resources are needed (duncombe ) . high poverty schools need more special education, more incentives for skilled teachers, and more effective supports for social and emotional learning. the absence of these resources is one of the reasons that students perform less well in high poverty communities (garcía and weiss ) . evidence across a wide variety of sub-areas of behavioral science converge in a consilience about how stressful social conditions result in a "fast life" strategy where threatening environments make such a strategy more likely to result in successful reproduction (wilson ) , but frequently culminate in premature death. figure depicts the impact of a stressful social context on parents and schools and the subsequent impact of all of these conditions on child and adolescent development. the conditions increase the likelihood that parents will have multiple problems and that, as a result, families will have frequent coercive interactions (dishion and snyder ) . it is well established that coercive interactions in families contribute to children's failure to develop self-regulation and to the development of aggressive and uncooperative behavior that puts them on a trajectory to fail in school and be rejected by peers dishion and snyder ; patterson et al. ). this rejection, in turn, leads to their joining with similarly rejected peers to form deviant peer groups. these groups become a training ground for the constellation of psychological and behavioral problems that include delinquency, substance use, risky sexual behavior, depression, and school dropout (debaryshe et al. ; patterson et al. patterson et al. , . this analysis is supported by research from a diversity of sub-disciplines (biglan et al. in press) . these include a wealth of evidence showing that multiple, interconnected problem behaviors result from a common set of adverse environmental conditions (biglan et al. ) . it is also supported by evolutionary analyses of the expression of fast life strategies that appear to have survival value in a dangerous environment (ellis and bjorklund ; wilson ) . it is shown by the evidence of epigenetic changes that make these strategies more likely (bergen et al. ). the analysis is also consistent with social psychological research contrasting the development of materialistic vs. prosocial values and the impact of threat on materialism and the association of materialism with diminished wellbeing (kasser ) . finally, there is clinical psychological research showing the benefits to human well-being of psychological flexibility, which is a pragmatic way of living that involves living intentionally according to one's values, a life strategy that benefits the individual and those around that individual (hayes ) . in addition to family influences, the quality of schools affects this developmental trajectory. as noted above, schools in high poverty neighborhoods and communities have higher rates of punitive practices and fewer resources for preventing the development of problem behavior (american psychological association, n.d.). there is one other point to be made about this developmental trajectory because it is so important for redirecting the expenditure of resources in our healthcare system. the u.s. per capita cost of healthcare is at least twice that of most other developed nations even though we lag behind these nations in health and longevity (biglan ) . a major yet frequently overlooked reason for these disparities is that ill health and premature death are made more likely by stressful social interactions in childhood (miller et al. ) . redirecting some of our healthcare expenditures to the prevention of stressful social relations in childhood is likely to make a bigger contribution to american's health than any advance in the treatment of chronic disease (biglan a) . when coupled with evidence from prevention research, these findings suggest a unifying framework about the kind of environments that are needed to nurture successful development (biglan a; biglan et al. ) . figure depicts the way in which nurturing conditions contribute to the development of prosocial behavior. by prosocial behavior, we refer to constellation of behaviors that contribute to not only the individual well-being, but the well-being of those around the person. these include caring, compassion, and an orientation toward helping others (kasser ) . they also include the development of a wide range of academic and social skills, as well as skill in art, music, and science. the evidence indicates that the development of prosocial behavior is promoted by environments that (a) minimize socially toxic coercive interactions, (b) richly reinforce all types of prosocial behavior, (c) limit both opportunities for problem behavior (such as unsupervised involvement with highrisk peers) and influences toward problem behavior such as the marketing of tobacco and alcohol, and (d) promote psychological flexibility, which is a values focused, mindful, and pragmatic way of living. thus, in the next section, we summarize the research suggesting that this problematic fast life trajectory can be prevented through the promotion of these nurturing conditions. the figure also indicates that we need to be concerned not only with the proximal family and school influences on development but on the neighborhood and community conditions that affect families and schools and often directly affect the child. as just suggested, focusing only on the proximal family and school influences on development is insufficient. we need to be concerned with multiple systems that can improve family well-being. in this section, we review evidence on interventions designed to affect the contextual conditions that we described above. in addressing family poverty, we rely on a recent nam report, a roadmap for reducing child poverty (nasem a), which thoroughly reviewed the evidence on the impact of poverty on children and the policies that have been shown to reduce it. the report identified ten policies or programs shown to reduce family poverty. contextual factors that have been instrumental in understanding family wellbeing involve many systems. we focus on policies below that could have a fundamental impact on these contextual factors. four policies both increase income and promote employment. the report deemed promotion of employment to be as valuable as increasing income because employment raises the probability that a family will continue to prosper over time. this view is consistent with our priorities in using a multi-level and systemic approach to improving family well-being. this policy supplements low paid workers' income by providing a tax credit on income they have earned. for example, people receive a refund of money previously withheld from their taxes. the credit is refundable in the sense that a worker may receive more money for their work than they would have had to pay in taxes. research on the eitc shows it reduces poverty, increases workforce participation (nichols and rothstein ), and increases the long-term health of family members. markowitz et al. ( ) found an association of higher tax credits with higher birth weights and longer gestation periods. unfortunately, states do not offer a state eitc and an additional six states have a nonrefundable eitc (tax policy center, nd.). a nonrefundable eitc can only offset state income taxes, so the benefit is limited for low-income families with little taxable income. also, many families who are eligible for federal and state eitcs do not apply for them, thus indicating a need to increase education and access to these opportunities. these subsidies impact family income, and at the same time, they increase workforce participation because they enable family members to work who might otherwise be providing childcare. the nam report estimated that the average annual cost for childcare for a young child (age through ) is $ , . the current federal minimum wage is $ . an hour, although states and the district of columbia have higher minimum wages. the nam report concluded that raising the wage could reduce the availability of jobs in some places if employers reduce their workforce in response, but also cited evidence that a higher minimum wage helps lift families out of poverty. an analysis by the robert wood johnson foundation ( ) concluded that minimum wage increases are associated with improved health. specifically, adult women who became pregnant and experienced an increase in the minimum wage had a reduced likelihood of smoking and fewer low birth weight babies. increases in state-level minimum wages are associated with decreased infant mortality, suicide rates, and heart disease death rates (kaufman et al. ; komro et al. ; van dyke, komro, shah et al. ) . at least two workforce development programs have been shown to increase employment and earnings for poor unemployed or underemployed workers. workadvance (schaberg ) was reviewed in the nam report. this program works with potential employers to identify the skills they need and then trains workers on those skills. four workforce development organizations ran randomized trials evaluating the program, and though not all replicated effects, the program was shown to increase employment, earnings, and job satisfaction. in addition, the arnold foundation, which funds and monitors results of randomized controlled trials evaluating government-funded interventions, reported that a randomized trial of quest, a job training program for low-income individuals, showed that it increased recipients' annual income % more than the control group over a nine-year follow-up period (straight talk on evidence ). like workadvance, quest works with employers to identify skill sets that companies need and provides training in those skills. based on this evidence, it appears that workforce-training programs focused on training for job skills that are in demand should be one component of improving family income. additional randomized controlled trials of such programs should take place and should include ongoing monitoring of their impact. the national academy of medicine report also examined the impact of a set of policies that would directly increase family income. one is housing vouchers. the federal government provides vouchers to families with very low incomes to subsidize rent, thus reducing expenditures and often enabling moves to better neighborhoods. a randomized trial of the impact of housing vouchers showed that they contributed to lower levels of obesity and depression (ludwig et al. ) and increased later earnings of those who moved to more affluent neighborhoods before the age of (chetty et al. ). the nam report (nasem b) indicated that increasing housing vouchers could result in moving % of children who currently live in poverty out of poverty. the national academy committee on poverty reduction also concluded that an increase in the number of families receiving assistance would contribute to reducing the proportion of children living in poverty by . %. the committee also estimated that this supplement to the income of poor families could reduce the number of children living in poverty by . %. note that while the nam report focused primarily on how these policies would increase family income and labor force participation, most have also improved health and wellbeing. for example, the report notes that the eitc improves children's educational and health outcomes. there is evidence that when people work together, each contributing to the group's success, prejudice diminishes. the most extensive research on this strategy involves cooperative learning procedures in schools, wherein small groups of students learn to work toward a common goal. this strategy has been shown to reduce prejudice and bullying (van ryzin and roseth a). paluck and green ( ) note that very little of this research has been done in real-world settings. given the extent of discrimination in the usa, its impact on public health, and the size of the workforce, it is imperative that foundations and the nih fund research to identify more effective ways to reduce prejudice in schools, work settings, and neighborhoods and communities. jurisdictions across the usa are beginning to recognize and address the harms that criminal justice involvement can cause in families. for example, some efforts are underway to reduce sentence length, eliminate incarceration, and use alternative sentences (e.g., enhanced community supervision) for certain nonviolent crimes. yet the country can do much more. the usa has not only a far higher rate of incarceration than other developed countries, it also has a much higher recidivism rate (fazel and wolf ) . comprehensive reform of the criminal justice system will require that its leaders make their primary goal a reduction in the incidence of crime. the pursuit of this goal will require a bigger investment in prevention, which, in turn, will require the system to become more involved in and supportive of the other sectors of society, including schools, family services, and healthcare. it will also require the adoption of evidence-based practices that can reduce recidivism. among the reforms that are likely to remedy this problem are the following. punitive practices in schools, such as zero-tolerance policies, often escalate suspensions and expulsions. this results in children's separation from school, extra-curricular activities, and peers and increases the likelihood of further offending as well as dropping out of school (american psychological association-zero tolerance task force ; insley ; martinez ; mitchell ) . moreover, school and justice policies that bring together at-risk youth amplify the development of antisocial behavior (dishion et al. ). these practices need to be replaced with evidence-based therapeutic interventions (lipsey ; lipsey et al. ) . involvement of parents in the criminal justice system can be traumatizing to their children at the same time that it reduces family income and the availability of a parent (peterson et al. ) . promising evidence points to enhanced family relationships through interventions such as parenting skills training, well-managed visitations, and improved communications (dallaire et al. ) . however, the evidence is not sufficient to justify widespread implementation. further research on how to reduce the harm to families affected by incarceration should be a high priority. in addition, immigration policy is currently doing substantial harm to children and families. the current highly punitive policies need to be examined in terms of the harm that they do. policy reform must start with the goal of enhancing rather than undermining family well-being-a reform which is unlikely under the current administration. as noted above, the marketing of tobacco, alcohol, and unhealthful food harms children's health and undermines parents' efforts to protect it. some progress is being made in regulating cigarette marketing because the evidence is so clear that such marketing contributes to underage smoking (biglan b ) and, ultimately, to the deaths of about a third of smokers (centers for disease control and prevention ) . similarly, the marketing of alcohol has been shown to increase underage drinking (sargent and babor ) . food marketing needs similar research. to the extent that research helps to determine whether marketing is a causal influence on alcohol or unhealthful food consumption, governments should regulate such marketing in the same way it regulates tobacco marketing. it is also clear that taxing tobacco and alcohol significantly reduces the proportion of young people who smoke or drink (komro et al. ) . and the evidence is beginning to emerge that taxation on sugar-sweetened beverages, can reduce the consumption of these products (redondo et al. ) . the harm that consumption of tobacco, alcohol, and unhealthful food does to children is substantial. this problem could be reduced if federal and state governments implement inflation-adjustable taxes on alcohol and unhealthful food and that those taxes are commensurate with taxes on tobacco products. in this section, we present a brief overview of the family and school interventions that have been shown to prevent problem development in youth. the evidence documents numerous family and school interventions that make these environments more nurturing. while it is true that addressing the above-described contextual conditions is likely to improve family well-being significantly, it is unlikely that reducing problems like poverty and discrimination will ensure that every family and school adopt the most effective ways of nurturing children's development . interventions are needed at both levels. research over the past thirty years has identified a broad array of family interventions that are available across the developmental spectrum. these interventions can reduce coercive interactions, promote parental skills in reinforcing prosocial behavior, set limits on risky behavior, and promote resilience in the face of challenges that stressful environments can pose. the strategies focus on two main areas: ( ) family promotion of children's social, emotional, and behavioral well-being and ( ) family promotion of children's healthy lifestyle behaviors to lower risk of obesity and other chronic disease conditions. this section briefly summarizes progress in each area and discusses what strategies can help achieve broad positive parenting and family support in society. over five decades, clinical and prevention professionals have developed a broad array of parenting and family support programs to foster social, emotional, and behavioral well-being of children. hundreds of studies document the benefits of such programs for families and children from infancy through adolescence. parenting and family support programming, if broadly implemented, has the potential to impact child mental health, child abuse, academic failure, bullying, antisocial or violent behavior, substance misuse, obesity, sexually transmitted disease in adolescence, teen parenthood. these programs increase positive and nurturing parent-child interactions, kindness, cooperation in peer interactions, and safe family environments many of these programs have common elements (prinz ) , including the following: • adopting a positive frame: no matter the circumstances, effective programs convey an optimistic and positive approach to each family. • action focus: parents actually do things instead of simply talking with a staff member. • problem-solving orientation: emphasis on solving current problems, not on casting blame. • specific, concrete, and practical parenting strategies: programs offer menus of strategies to address parenting challenges. • collaborative goal setting: parents and staff members collaborate to set programmatic goals for each family. • consultative rather than prescriptive attitude: programs favor providing useful consultation to parents without giving them orders. such programs reflect a well-honed set of parenting practices that are adaptable to children's ages and that offer parents many ways to foster nurturance, such as. • giving rules and instructions to children before situations begin. • paying frequent attention to positive behaviors. • providing supportive prompts, coaching, and correction. • setting clear limits and boundaries. • ignoring harmless but annoying behaviors so children don't get an "audience" for these. • recognizing and encouraging behaviors that are the opposite of any identified problem behaviors. • rearranging situations so that positive behaviors result in enjoyable activities. • expressing affection and caring frequently. • developing effective ways of solving daily and recurring problems. • envisioning the future and setting specific and attainable goals. families can positively influence youth health behaviors by providing a warm and supportive setting for learning, growth, and development (kitzman-ulrich et al. ; wilson et al. ) . it is increasingly apparent that positive parent-child communication is fundamental in promoting healthy behaviors in children. specifically, a supportive family climate has a positive association with adolescent health behaviors and healthy weight status (berge et al. ; halliday et al. ; st. george et al. ; wilson and sweeney ) . research shows that higher levels of family functioning (defined as communication, joint problem solving, closeness, and appropriate behavioral control) are related to healthier body mass index, nutrition, and physical activity among adolescents. haines and colleagues (haines et al. ) found similar results. they discovered relationships between higher family functioning, lower obesity risk, and less sedentary behavior. other evidence indicates that improving family climate through positive interventions reduces deleterious effects of unsupportive parenting on youth weight-related outcomes, with benefits that can be detected nearly years later (brody et al. ; wilson et al. ) . family programs that improve children's self-regulation also promote healthy lifestyles from early childhood through adolescence. for example, self-monitoring, which involves prompting families to keep a record of a specified behavior (e.g., in an electronic or written diary), is one of the most effective strategies for eliciting changes in diet and physical activity (michie et al. ). another common strategy is prompting specific goal setting. this includes details such as the frequency, intensity, or duration of behavior, as well as specifying when, where, and how the child plans to achieve the goal (gollwitzer ) . action planning, including encouraging families to identify potential barriers to performing a behavior and planning supportive ways to overcome them, is another often-used technique for promoting healthy lifestyle changes (ayotte et al. ; bandura ) . these behavior-change strategies have been increasing in family contexts to promote health-related behaviors among lowincome and ethnic minority populations. another approach for promoting healthy behaviors among youth focuses on supporting their autonomy, rather than telling them what they should do. motivational interviewing (mi) is designed to increase motivation for behavior change by listening to the child about their concerns and joining them around their personal desires to change their health or weight (miller and rose ) . rather than providing advice or urging specific actions, clinicians and parents use this strategy to elicit the motivation to change from individuals themselves using strategies through reflective listening and shared decision-making. the counselor or parent can then provide the child (or adolescent) with specific behavioral skills, including identifying goals, creating an action plan, anticipating potential barriers, and monitoring children's health behaviors (resnicow and mcmaster ) . ample evidence shows mi is an effective strategy when coupled with behavioral skills training for reducing weight problems and obesity (for a review, see armstrong et al. ) and for helping parents facilitate weight loss in their overweight children (resnicow et al. ; spear et al. ). in the mi framework, shared decision-making and autonomy support are critical skills for parents as youth transition from childhood to adolescence (ryan et al. ) . shared decision-making involves parents and children making decisions together in a way that facilitates sharing in ownership over plans. this encourages motivation for and enjoyment of healthy behaviors (ryan et al. ) . studies have demonstrated positive associations between parental autonomy support and adolescent intrinsic motivation on improving healthy lifestyle-related behaviors and adhering to weight-loss treatment (mâsse et al. ) . parents can also use this strategy to model problem solving and troubleshooting of barriers. research has shown that these behavioral and motivational approaches are effective for improving health behaviors from early childhood through adolescence (kahan et al. ; st. george et al. ; wilson and sweeney ; wilson et al. ) . however, in the case of these health behavior interventions, a gap between research and practice exists that limits the implementation of evidence-based interventions outside of research programs. this gap exists because we have not yet developed effective strategies for getting nonresearch settings to adopt these strategies. given the deleterious effects of parental substance use on children, interventions to address parental substance use are needed. the evidence for the value for children of treating parental substance use is currently limited. calhoun et al. ( ) reviewed four randomized trials of the impact of treating parental substance use on children. each of these studies provided some evidence that parental treatment could benefit children, either by reducing the likelihood of the children's substance use or by improving children's psychological or behavioral functioning. research on evidence-based methods for reducing family violence indicates promising effects of behavioral interventions. although much more research is required for strong recommendations about how to prevent child maltreatment, studies have shown positive effects of parenting programs that reduce risk factors such as parental depression and stress, parents' inappropriate attitudes toward child-rearing, abusive parenting behaviors, insufficient parenting skills, and minimal knowledge about child development (chen and chan ) . recent research on domestic violence interventions also indicates promising new efforts using cognitive-behavioral interventions. an intervention based on acceptance and commitment therapy is now being used in the state of iowa for all individuals convicted of domestic violence. the intervention joins an abusing partner around their own prosocial goals and helps them learn to step back from anger-inducing thoughts, and feelings in a self-compassionate way. in this context, they become better able to control anger and develop more nurturing ways of interacting with their family members. research on the program shows preliminary evidence that it is effective in reducing repeat domestic assault, in both community corrections and incarcerated populations (berta and zarling ; zarling et al. ) . evidence-based family interventions are essential but not sufficient for a successful impact on the well-being of children and their families. broad population reach is critical as well. reaching one family at a time (e.g., home visitation programs) has utility but does not preclude the use of collective and cost-efficient strategies to provide parenting and family support for large numbers of families. critical to population reach are multiple access points for programming, an array of delivery modalities including media, the involvement of personnel from several service sectors, de-stigmatized programming capitalizing on a range of intervention intensities, and recognition of how to take into account major social determinants of health (kaplan ; prinz ). substantial progress has also been made on school interventions that promote prosocial behavior and prevent the development of multiple problems. these programs have in common a focus on helping schools to replace punitive discipline practices with practices that promote prosocial behavior. here we briefly describe four programs that have proven benefit. pbis is a schoolwide system for promoting prosocial behavior. a team of staff members establishes and monitors pbis implementation. they establish a small number of clear rules for appropriate behavior. common rules are be responsible, be respectful, and be safe. students are then taught examples of behavior consistent with these rules in all of the venues of the school (classrooms, hallways, playground, buses). a system for increasing praise and rewards for appropriate behavior is put in place throughout the school. the pbis system monitors disruptive or inappropriate behavior throughout the school and uses the data to pinpoint settings or individuals with high rates of inappropriate behavior. this enables the pbis team to put in place more intensive interventions for particular students or settings in the school. bradshaw et al. ( ) reported a group randomized trial of the impact of positive behavior intervention and support and elementary schools. they found significant reductions in behavior and concentration problems and improved social-emotional functioning, and prosocial behavior. children in pbis schools were % less likely to be referred to the office for inappropriate behavior. pbs is now in more than , schools throughout the nation. this is a program that teaches students to engage in positive actions throughout the school. washburn et al. (washburn et al. ) reported on three randomized trials evaluating positive action in elementary schools. in all three studies, students who were randomized to the positive action intervention showed smaller declines in positive actions than those in controls. the program appears to have arrested the typical decline in positive actions that is seen as students enter early adolescence. the randomized trial conducted in chicago elementary schools showed that the students receiving positive action had significantly more positive affect and life satisfaction, and significantly lower levels of depression and anxiety (lewis a (lewis , b . cooperative learning was originally developed as a method of increasing student learning, especially among students who were performing poorly. this strategy involves having small teams of students work together in ways that require each student to make a contribution to the group's product. numerous studies have shown the benefit of cooperative learning in increasing academic success (roseth et al. ) . it has also shown a clear benefit in reducing intergroup prejudice (pettigrew and tropp ) . recently, a randomized trial of cooperative learning in middle schools has shown that the program significantly reduced bullying and victimization and that students in the cooperative learning condition reported less stress, fewer emotional problems, and less use of alcohol and tobacco (van ryzin and roseth b). like cooperative learning, the good behavior game involves small teams of students working together cooperatively. teams that meet the criteria for appropriate behavior receive rewards such as extra time for recess. extensive research over a period of years has shown that the good behavior game significantly reduces disruptive behavior (embry ) . a randomized trial of the benefit of the good behavior game among students in early elementary school in baltimore showed that students who played the game were significantly less likely to smoke or be arrested by middle school and that by young adulthood they were significantly more likely to graduate high school and attend college and significantly less likely to have problems with substance abuse, antisocial behavior, or suicidality (kellam et al. ). the paxis institute developed a version of the good behavior game that combines it with a set of simple behavior influence techniques called kernels. that version of the game has generally replicated the benefits of the program that were shown in previous studies (johannsson, biglan, & embry, under revision) . at this writing, more than , teachers have been trained in the implementation of this version of the game. there is also evidence of the benefit of increasing collaboration between families and schools. jeynes ( ) conducted a meta-analysis of the impact of a variety of parental involvement programs on academic success and found they increased the performance of students of all ages. similarly, sheridan, smith, moorman kim, beretvas, and park ( ) conducted a meta-analysis of studies on the impact of various types of family-school collaboration on students' social-emotional functioning and found significantly better social-behavioral competence and mental health among students of all ages; effects were strongest for african american children. the components of collaboration that made a difference included two-way communication between parents and teachers and trust between parents and teachers. also important was school provision of enrichment activities for the home, parents volunteering in the classroom, and specific help to parents on how to model and reinforce desirable behavior. despite the benefits of family-school collaboration, the collaborative practices sheridan et al. ( ) identified remain largely aspirational in typical school environments (garbacz et al. ) . perpetuating the gap between research and practice is a lack of trust families and educators have for each other. mistrust has intensified over time through certain practices, such as the disproportionate use of discipline (townsend ) . to promote family well-being, evidence-based family-school systems and practices (sheridan et al. ) should be aligned and integrated within existing school programs and initiatives (garbacz ) . such alignment and integration will provide the necessary context, through two-way communication, culturally sensitive practices, and family-centered services for parents and educators to build the skills and relationships necessary to strengthen families and support children. to advance this line of work, research is needed that examines the impact of strategies for promoting collaboration on (a) the level of parent-teacher collaboration; (b) the impact on teachers' attitudes and beliefs about parents and collaboration with them, and teacher burnout/morale; and (c) factors that explain the impact of a strategy on an outcome. in addition, an examination of the implementation process should consider alignment and integration in school and community systems; parent, youth, and teacher characteristics and experiences; the school infrastructure; and the external environment (feldstein and glasgow ). this brief review of family and school interventions documents the availability of numerous programs that can contribute to the well-being of families and the prosocial development of children. at the same time, additional research is needed if we are going to translate this body of knowledge into a widespread benefit. continued investment in strengthening these interventions is likely to improve their effectiveness. although there is evidence of the benefit of family and school interventions for poor and minority populations, further research on reaching and affecting this population is needed (van ryzin et al. ) . moreover, research on combining interventions at multiple levels and on how to get these interventions widely and effectively implemented will enhance our ability to make them available to neighborhoods and communities of concentrated disadvantage. the evidence presented thus far establishes three things. first, a large proportion of children and families are living in circumstances that undermine their well-being. indeed, over the past fifty years, conditions for families have deteriorated in the u.s. (kristof and wudunn ; putnam ) , and we have fallen behind other economically developed nations (unicef ) . second, those at greatest risk for future psychological, behavioral, and health problems are living in neighborhoods or communities of concentrated disadvantage. our impact on child and family well-being will be limited if we do not focus policies to alleviate structural barriers to well-being and how to further provide resources and assist these neighborhoods and communities. third, there is ample evidence that changes in public policy and the wider implementation of family, school, and community interventions can prevent or ameliorate many of the factors that undermine development. in principle, we have the knowledge needed to significantly improve wellbeing in american communities. we are, however, a long way from effectively addressing family well-being. despite the availability of policies and programs that could increase the number of families that are thriving, there appears to be no systematic plan for steadily improving well-being. the national academy's report fostering healthy mental, emotional, and behavioral development in children and youth (nasem b) called for a decade-long effort focused on promoting healthy development. the report further called attention to the fact that seven other academy reports had advocated for greater attention to the problems of poverty and concentrated disadvantage because these conditions constitute the greatest obstacle to successful development. in keeping with this focus, the remainder of this paper presents a strategic plan for how we might create an unprecedented effort to reduce concentrated disadvantage and thereby increase the proportion of families that support the successful development of children and adolescents. there are various sectors and policymaking bodies that are trying to address the challenges to family well-being. however, there is not a comprehensive and sufficiently cogent plan to fully impact family well-being in the u.s. for this reason, this strategic plan is addressed primarily to the key policymaking bodies, leaders in business, ngos, and foundations, child and family advocates, other key opinion leaders, and consumer groups. the proposed goals for this effort would be as follows. . increase the proportion of american families that meet an empirically defensible public health standard of wellbeing. . identify, monitor, and reduce the number of neighborhoods and communities of concentrated disadvantage. . promote the adoption and implementation of policies and programs that advantage families. . increase the amount of research relevant to reducing family disadvantage. this would include research on a. establishing a national standard for family wellbeing in the form of an index of family well-being. b. experimentally evaluating comprehensive strategies for reducing disadvantage in neighborhoods of concentrated disadvantage. there are two facets of our plan for furthering these goals. the first concerns organizing an unprecedented coalition of all sectors of society to make increasing the well-being of american families a national priority along the lines of what the nasem report on the promotion of mental, emotional, and behavioral health called for. the second is the creation of research agenda that would advance family well-being, especially in neighborhoods and communities of concentrated disadvantage. every sector of society has a role to play in reversing the deterioration of family well-being that has occurred over the past years (putnam ) . in each sector we need to identify practices that undermine family well-being and those that enhance well-being. the foundational value for this effort is a commitment to ensuring the well-being of every person. it is only when a growing number of people do so that we will have the support for the norms and public policies that select beneficial practices and diminish harmful ones (biglan a) . we envision a process in which each sector is prompted to examine whether its practices are contributing to the reduction of concentrated disadvantage. in particular, it would look for ways in which the sector undermines well-being and the degree to which it makes use of evidence-based policies and programs that enhance well-being. higher education is critical to a nationwide effort to reduce concentrated disadvantage. over the past forty years, a consilience has emerged in the human sciences regarding the conditions that people need to thrive and those that contribute to the development of psychological and behavioral problems and premature death (biglan (biglan , a biglan and embry ; biglan et al. ; biglan et al. ; catalano et al. ; dishion and snyder ; hawkins et al. ; institute of medicine ; kasse kasse , miller et al. ; nasem a nasem , b national research council & institute of medicine ; wilson et al. ) . the report of the national academy of medicine summarized the evidence this way: "the scientific foundation has been created for the nation to begin to create a society in which young people arrive at adulthood with the skills, interests, assets, and health habits needed to live healthy, happy, and productive lives in caring relationships with others." the research and training functions of our universities need to further develop strategies for translating what we know into effective interventions for disadvantaged families. according to the society for prevention research, there are only seven ph.d. programs in prevention science in the usa; there are seven masters level programs (society for prevention research ). increasing their numbers is a first step in advancing effective prevention. many other areas of the human sciences have a role to play in strengthening supports for family well-being. every university should be encouraged to examine how well it is preparing the scientists and practitioners that are needed to address the needs of families. higher education can also affect the trajectory of children living in disadvantaged communities. they can work with those communities to increase the number of children who attend college. they can reduce the costs of higher education for disadvantaged children. our healthcare system needs to shift from a primary emphasis on treating diseases once they develop to preventing them in the first place. here is just one example. there is an epidemic of childhood obesity in the usa and with it, a growing tendency for children to develop what used to be called adult-onset diabetes (u.s. department of health and human services ). a common practice among pediatricians is to screen children for obesity and counsel them and their parents (perrin et al. ). however, there is no evidence that intervention with an already obese child affects their trajectory (homer ). on the other hand, there is extensive evidence about the impact of food industry marketing on obesity and of the benefit of increased taxation on unhealthful food (biglan b) . restricting marketing and increasing the price of these foods are the practices that are needed. the same is true for the prevention of smoking and alcohol use (biglan c; pechmann et al. ) . strengthening the ability of the public health segment of the healthcare system to practice population health promotion will have a significant impact in reducing the incidence of a wide variety of disease that we currently wait to treat. the role of the public health sector in addressing the coronavirus epidemic may increase support for public health. in any case, this is the sector of the healthcare system that can lead the reforms that are needed. recent developments in business include a movement to evaluate business practices in terms of their contribution not just to investors, but to employees, customers, suppliers, and the society as whole. the business roundtable recently embraced these values and the b corp and conscious capitalism (business roundtable, n.d.; b corporation, n.d.; mackey and sisodia ) movements have been promoting them for some time. concrete steps that businesses can take include: (a) identifying and eliminating practices that harm, such as the marketing of harmful products (biglan b; c; d; e; f; g) ; (b) assessing the well-being of employees, customers, and the communities that they operate in; and (c) adopting practices that steadily increase family income and supports for child-rearing such as high quality day care and paid parental leave. a company that truly embraces the goal of ensuring that its employees are thriving, would strive to ensure that their wages are above the poverty level. the allies in reform of business norms and practices are the leaders of the conscious capitalism and b corp movements. as discussed above, the criminal justice system can reduce harm to families by reducing the use of imprisonment and investing more heavily in rehabilitation and the prevention of juvenile crime. there are numerous organizations working to reform this system (kerman, n.d.) . they are natural allies in any effort to reduce family disadvantage. we reviewed the array of family interventions that are available to help families reduce conflict and promote prosocial skills. every community has such services. they are increasingly guided by empirical evidence about effective practice. we need to encourage the transition from reactive intervention to instances of child abuse and neglect to a system of family check-ups and provision of support that prevents problems from developing. we also described some of the school-based programs that are available to schools. here too the challenge is to increase the proportion of schools that have the resources to make use of these programs. both the reform and the effectiveness of each of these sectors will be enhanced if organizations working in any given sector form a coalition with other sectors (biglan a) there are three reasons. first, the explicit embrace of the goal of enhancing population well-being by any give sector will build support for this goal in other sectors. for example, when people in criminal justice speak of reducing the incidence of juvenile crime and increasing the prevalence of prosocial youth it encourages those in other sectors to think in terms of the well-being of the entire population they serve rather than only those who seek or are remanded to intervention. second, progress in any sector makes the job easier for any other sector. for example, to the extent that criminal justice and family services improve their support of families, schools will have fewer students who lack self-regulation, engage in disruptive behavior, and fail academically. conversely, to the extent that schools are nurturing prosocial behavior, they will prevent crime. the third reason a coalition is needed is so that, speaking with one voice, the organizations in each of these sectors can look beyond a narrow focus on the problems each addresses and work to address contextual conditions that contribute to most psychological and behavioral problems as well as poor health. our systems for dealing with human problems have naturally evolved out of the need to address an emergent problem-a crime, an injury, and illness. as such they are reactive. however, the evidence presented above documents the impact of poverty, discrimination, homelessness, and concentrated disadvantage have on the entire range of problems. each sector has a stake in reducing these problems. speaking with one voice about the need to address these problems would contribute to greater support for the policies needed to address them. coalition building will need to proceed at the local, state, and national levels. increasingly community interventions are making use of the collective impact model to bring the sectors of the community together around a shared agenda (kania and karmer ) . at the same time, success at the local level will be enhanced to the extent that sector leaders at the state and national level are working together to achieve state and federal support for such efforts. in describing each sector, we mentioned groups within that sector that are working on reform. at the same time, there are numerous foundations and advocacy organizations that are working on the issues we address in this paper. two behavioral science organizations that are specifically working to advance effective action are the coalition of behavioral science organizations (cbso) which supported the writing of this paper and the national prevention science coalition, which is a member of the cbso and which has more than sixty national organizations affiliated with it. a formal coalition among all of these organizations is an important next step for advancing the goals we have proposed. the process of creating such a coalition would begin by inviting an initial group of organizations to formally create the coalition. the major impetus for changes in public policy and practice are the reports of the national academy of science, engineering, and medicine, surgeon generals' reports, monographs of the national institutes of health, and reports of the centers for disease control and prevention. with the publication of the recent nasem reports on poverty reduction and the promotion of mental, emotional, and behavioral health, the coalition of behavioral science organizations and the national prevention science coalition are working to publicize and build on the recommendations of these reports. behavioral scientists are beginning to do more to educate the public about how we can evolve a society that enhances everyone's well-being. npsc is regularly doing congressional briefings and publishing oped pieces in major national outlets. however, it is no longer possible to rely solely on mainstream media. since the advent of social media, it has become possible for numerous actors to reach a large audience with views, misinformation, and hateful rhetoric that would never have been widely disseminated previously (marantz ) . this fact has only recently come to the attention of people working mainstream media (haidt and rose-stockwell ) . it is imperative that those of us who are trying to influence movement in the direction of supporting everyone's well-being, make more and better use of social media to promote understanding of what is needed to improve human well-being. we believe that the two most important priorities for research are the development of a national system for monitoring both family well-being and neighborhood and community disadvantage and the development and testing of comprehensive interventions to reduce disadvantage and enhance family well-being. if the nation is going to be guided to steadily increase family well-being and reduce community disadvantage, then it needs valid and widely accepted indices of each type of well-being. ultimately, these indices should be reported annually by the federal government in the same way the indices of community economic well-being are monitored and reported. with respect to child and family well-being, the urban institute recently reported well-being and basic needs survey that is an example of the type of survey system that is needed. at the same time, systems are being created to track conditions in neighborhoods and communities. acevedo-garcia et al. ( ) have a system that reports on the conditions in communities that are likely to enhance or hinder family well-being. ultimately every community should be able to track well-being in their neighborhoods. the covid- pandemic has exposed virtually the entire nation to the practice of tracking well-being down to the level of the community. because of the danger of a high rate of covid- infections, people watch with great interest whether case rates were increasing or decreasing. states track what was working in other states and adopt practices that seem to be working. these same procedures are relevant to achieving changes in other aspects of well-being. granted the outcomes that would be focused on with a system that was monitoring family and community well-being will not attract as much attention as one that is tracking deaths due to a virus. however, that is in part because the public has not been educated about the fact that family and community well-being are critical for the prevention of premature death and virtually all of the psychological and behavioral problems that costs our nation so much. community-level monitoring of child and family wellbeing and the contextual conditions that affect families will provide the criteria by which a community can assess its progress and can maintain or modify its practices in light of the data. this process can shape the practices of each sector. for example, reductions in juvenile crime should result from adoption of evidence-based school, family, and juvenile justice practices. although it will not be possible to precisely attribute reductions in juvenile crime to one of these systems, each system can monitor how many youth and families it is reaching with evidence-based practices and can assess the impact of their services. evidence of the value of both economic development efforts and programs targeting families and schools suggests it is time to test comprehensive interventions that simultaneously address all risk factors for problems for which we have some effective solutions. interventions that only address family and school environments will leave many families in poverty. interventions that target only economic well-being will not ameliorate patterns of conflict in families, nor school cultures that undermine development and well-being . a variety of intervention strategies and evaluation methods have been used, but most have no connection to other work on this problem. there is research on workforce development, economic development, and family and school interventions that could contribute to reducing intergenerational poverty. there are community interventions focused on preventing adolescent problem behavior (hawkins et al. ) , efforts to strengthen supports for child and adolescent development, and collective impact approaches to bringing all sectors of communities together to address challenges. however, a truly comprehensive framework for addressing the tangle of problems will likely require all of these strategies. the principles of community organizing (hawkins et al. ; minkler ) and collective impact (cabaj and weaver ) are foundational for a comprehensive intervention. each of the sectors of the community needs to engage in a participatory process in which the community identifies the actions they want to take to improve wellbeing. as we indicated above, the efforts of each sector to enhance well-being will benefit from success of other sectors. to the extent that each sector supports other sectors efforts, synergistic benefits are likely. a second line of research that is essential for any comprehensive intervention consists of efforts to enhance economic well-being in the neighborhood and communities. the brookings institution (shambaugh and nunn ) reviewed research on place-based strategies to improve economic well-being in communities or neighborhoods of concentrated disadvantage. these included job training, tax benefits for investment in neighborhoods, subsidized employment, involving research universities in economic development, and extending the reach of existing health and nutrition programs. because of the limited number of experimental evaluations of these strategies, evidence for the benefit of programs such as enterprise and opportunity zones is, at best, weak. a third component of such interventions would be the implementation of family and school interventions that have proven benefit in enhancing family and school supports for successful development. table indicates activities that promote prosociality at every stage of development, from preconception through young adulthood. to our knowledge, no community has systematically implemented a comprehensive lifespan intervention and carefully assessed its impact. the closest we have found is the harlem children's zone, whose results are encouraging (whitehurst and croft ) . there are also likely to be synergistic effects of efforts at different phases of development. perhaps a child did not develop cognitive problems because her parents did everything they could to ensure her well-being in utero. as a result, a kindergarten class may have one less disruptive and uncooperative child. one more child will be able to read at grade level by third grade. if that child encounters adversity later in childhood (e.g., if a parent dies), she will benefit from good programs such as the one sandler and colleagues (sandler et al. ) developed to aid in coping with a death in the family. most evaluations of family and school programs with demonstrated benefits have been in contexts without other programs in place. and although the benefits of many interventions are significant, the size of their impact is generally small (jones et al. ) , and there is no evidence that their effectiveness has been increasing over time . one reason may be that these interventions are implemented in isolation both from many of the contextual conditions that affect behavior and from ameliorative interventions at other stages of development. it is likely that ensuring supports for families and schools at every stage of development can produce a larger impact on population well-being than any of these interventions in isolation. in addition to the many programs and policies with the potential to enhance well-being in communities, it may be beneficial to promote compassion and kindness. people who have faced numerous adverse experiences are likely to be hyper-vigilant and to read others as threatening; this is an adaptive orientation in such an environment . a community-wide effort to promote patient, caring, and kind interactions may contribute to reducing aversive interactions that not only maintain suspicion and distrust but lead to high levels of stress-related physiological processes that contribute to premature death (miller et al. ) . for example, imagine a family moving into a community that has increasingly organized around nurturing values, and that has explicit goals and procedures for promoting prosocial behavior. imagine this family has a long history of stressful contact with schools, the criminal justice system, healthcare providers, and perhaps neighbors. such a history is common in communities of concentrated disadvantage. the family's first experience in the community is likely to be enrolling their children in school, which may make them feel nervous and defensive. but suppose the school has a program to invite new families to discover how it supports children's prosocial behavior. suppose that staff convey that they like and admire the children. the family relaxes a bit, which is likely to promote positive interactions at home. now imagine that in every encounter in the community over the next few weeks, they meet people who show an interest in them and are welcoming and helpful. each positive encounter can contribute to family members becoming more comfortable while also conveying prosocial norms and reinforcing behavior. the complexities involved in evaluating comprehensive community interventions have hampered progress in helping neighborhoods of concentrated disadvantage. here we discuss what could strengthen this work. randomized trials are expensive and difficult to conduct in whole communities, especially if the intervention to be tested is multi-faceted. and, given the complexity of the multiple problems that need to be addressed, interventions need to be multi-faceted. randomized trials are useful for evaluating impact across cases, and they have demonstrated the value of numerous family and school interventions. however, they are not the best way to tease out functional relationships between environments and behavior. the family and school interventions that have been validated by randomized trials grew out of single-case experimental designs that revealed functional relationships between parents' and children's behavior (kazdin ) . similarly, before we can test complex, multi-faceted interventions through randomized trials in multiple communities, we need to show they are working reliably in individual communities. moreover, randomized trials require the assumption that all communities are alike in their needs and with regard to what will work. this makes it difficult to tailor an intervention to the needs of the community and still maintain consistency of the intervention across communities. greater use of interrupted time series designs would enhance research on communities (biglan et al. a, b) . these designs involve obtaining repeated measures of an individual behavior or group action and assessing the impact of a well-defined intervention or independent variable on that time series. such multiple baseline designs could help to evaluate the impact of comprehensive interventions on the wide range of aspects of child and family well-being, including economic well-being, quality of family interactions, and psychological and behavioral functioning of children and parents. by monitoring well-being in three communities or neighborhoods and implementing a comprehensive intervention in one of the communities, while continuing monitoring, but not intervening in the other two communities, we can assess whether the intervention affects measures of well-being. if an effect is detected in the first community over, for example, two years, it can be implemented in a second community. if its effects are replicated in the second community, it can be implemented in a third. such a design provides a valid experimental test of the impact of the intervention (biglan et al. a, b) . these methods are also useful for discovering the impact of individual interventions. for example, a community might implement the pax good behavior game (jiang et al. ) in a series of three elementary schools and track its impact on disruptive behavior and students' behavioral functioning. the community might initially evaluate introduction of an evidence-based family intervention in a multiple baseline across four families. it could similarly stage and evaluate a $ , per child allowance in terms of its impact on family relations. these assessments of individual programs would immediately show evidence of their efficacy, which could promote wider adoption. if they did not show benefits, the community could abandon the interventions or modify them until they worked. the beauty of these designs is that providing immediate experimental assessment of the impact of an intervention can enable its improvement. rather than requiring that each community get the same thing, these designs enable continuous quality improvement, so that successive communities receive an intervention modified by the improvements made in earlier implementations. although we believe that interrupted time series designs are the most efficient and effective strategy for developing effective interventions in neighborhoods of concentrated disadvantage, we also believe that it is impossible to say precisely what experimental strategies will be most productive. it would be a mistake to dictate that all interventions or all experimental procedures should have the same features. one experimental method that should also be tried is the most design (guastaferro and collins ). these factorial designs are valuable for efficiently identifying which components of an intervention and which combination of components are most effective. for example, an important question in neighborhood interventions is the relative and combined impact of economic development interventions and interventions focused on changing behavior in families and schools. a factorial design would efficiently address this question. it would involve randomly assigning neighborhoods to one of four conditions: (a) a combination of economic development and school and family interventions; (b) economic development interventions alone; (c) school and family strategies alone; or (d) no intervention. this design would allow assessment of the impact of each component, as well as the impact of the comprehensive intervention. we hope that this paper has made the case for a new initiative to address the problem of concentrated disadvantage in america. the extent of the problem is clear. its impact on the health and well-being of people living in these places is well-established. its harm to the rest of the nation is measured in the costs of poor health, drug use disorders, overdose deaths, and lost productivity. the policies and programs that can ameliorate these conditions are clear. this conclusion is being written on the day in which the u.s. became the country with the greatest number of covid- cases. the nation and the world are getting a lesson in how to think about public health. the entire world has been mobilized to do everything it can to prevent people from becoming infected. in the process millions of people have learned to think about the entire population. the incidence and prevalence of problems in entire populations has become as important as the individual case. this gives us an opportunity to bring attention to many other problems in society that would be best dealt with through public health strategies. every significant psychological, behavioral, and health problem can be thought of in terms of its incidence and prevalence. moreover, these problems are intertwined. this means that where we have high levels of one problem we are likely to high levels of others. and, specifically, these problems are concentrated in neighborhoods and communities that are high in poverty, substance use disorders, and social conflict. thus, efforts to greatly reduce the number of families that are affected by disadvantage will have limited success if we do not focus on reducing the prevalence of highly disadvantaged places. making a difference in these places will require policies and programs that reduce harmful contextual conditions such as poverty, homelessness, and discrimination, as well as programs that directly assist families and schools in adopting the most nurturing practices. the evidence that we can affect these problems is substantial. however, the challenge is to develop and evaluate comprehensive efforts to address multiple risk factors at every stage of development. putting in place such efforts will require a coalition of all sectors of society. if we can learn to speak with one voice about the harm that is being done by allowing children to be raised in concentrated disadvantage and the fact that we have policies and programs that could greatly reduce disadvantage, we may be able to achieve unprecedented improvements in the well-being of children and families in the usa. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any data from any studies with human participants that were performed by any of the authors. the geography of child opportunity: why 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approach to education the spirit level: why greater equality makes societies stronger behavioral medicine and integrated care: efficient delivery of the most effective treatments promoting social nurturance and positive social environments to reduce obesity in high-risk youth this view of life evolving the future: toward a science of intentional change. behavioral and brain science evaluation of acceptance and commitment therapy for domestic violence offenders. psychology of violence key: cord- -nmnssoeb authors: wall, jonathan t.; kaiser, bonnie n.; friis-healy, elsa a.; ayuku, david; puffer, eve s. title: what about lay counselors’ experiences of task-shifting mental health interventions? example from a family-based intervention in kenya date: - - journal: int j ment health syst doi: . /s - - - sha: doc_id: cord_uid: nmnssoeb background: a key focus of health systems strengthening in low- and middle-income countries is increasing reach and access through task-shifting. as such models become more common, it is critical to understand the experiences of lay providers because they are on the forefront for delivering care services. a greater understanding would improve lay provider support and help them provide high-quality care. this is especially the case for those providing mental health services, as providing psychological care may pose unique stressors. we sought to understand experiences of lay counselors, focusing on identity, motivation, self-efficacy, stress, and burnout. the goal was to understand how taking on a new provider role influences their lives beyond simply assuming a new task, which would in turn help identify actionable steps to improve interventions with task-shifting components. methods: semi-structured interviews (n = ) and focus group discussions (n = ) were conducted with three lay counselor groups with varying levels of experience delivering a community-based family therapy intervention in eldoret, kenya. thematic analysis was conducted, including intercoder reliability checks. a stress map was created to visualize stress profiles using free-listing and pile-sorting data collected during interviews and focus group discussions. results: counselors described high intrinsic motivation to become counselors and high self-efficacy after training. they reported positive experiences in the counselor role, with new skills improving their counseling and personal lives. as challenges arose, including client engagement difficulties and balancing many responsibilities, stress and burnout increased, dampening motivation and self-efficacy. in response, counselors described coping strategies, including seeking peer and supervisor support, that restored their motivation to persevere. at case completion, they again experienced high self-efficacy and a desire to continue. conclusions: findings informed suggestions for ways to incorporate support for lay providers into task-shifting interventions at initiation, during training, and throughout implementation. these include acknowledging and preparing counselors for challenges during training, increasing explicit attention to counselor stress in supervision, fostering peer support among lay providers, and ensuring a fair balance between workload and compensation. improving and building an evidence base around practices for supporting lay providers will improve the effectiveness and sustainability of lay provider-delivered interventions. background a major issue facing health systems in low-and middle-income countries (lmics) is improving healthcare accessibility resulting from health care professional shortages, especially for mental healthcare [ , ] . much work advocates for health systems strengthening through task-shifting, or task-sharing, by training non-specialists or non-professionals to provide services [ ] [ ] [ ] . most often, such programs employ community health workers (chws), more broadly referred to as lay providers, who deliver services ranging from hiv-tuberculosis care and management [ ] to mental healthcare, the topic of the current study [ , ] . task-shifting has become the de facto model of much mental healthcare delivery in lowresource settings globally due to the one-million-person shortage of mental health specialists [ , ] . despite the many benefits of task-shifting for increasing healthcare accessibility, a growing body of research points to challenges lay providers face. a key challenge is socioeconomic inequities between lay providers and employed health professionals. this reality is largely driven by interventionists pushing an ethic of volunteerism for lay provider programs [ , ] . some policymakers and community program leaders claim that lay providers are "priceless" and might lose intrinsic motivation to fulfill their responsibilities if paid [ , ] . additionally, many programs emphasize their costeffectiveness in increasing healthcare accessibility, which is premised on not paying lay providers [ , ] . this is a topic of ongoing debate in the field and has important implications for intervention delivery [ , ] . beyond economic impacts, the four most common areas of concern regarding lay provider experiences are motivation, self-efficacy, stress, and burnout ( table ) , any of which can contribute to poor retention among lay providers [ ] . these are concerning from an individual well-being perspective and because they might reduce quality and effectiveness of interventions. these outcomes are even more concerning among lay counselors, a specific type of lay provider focused on mental healthcare, which often requires more time commitment and raises the risk for unique stressors, including compassion fatigue. one proposed root cause of these problems is that lay providers are sometimes treated as a "means to an end, " rather than as individuals who may need support to optimize their services [ ] [ ] [ ] . therefore, some researchers propose fostering a humanistic view of lay providers, encouraging global health actors not to approach lay providers as technocratic solutions, but as people with unique skills, desires, and perspectives [ ] . although past studies have pointed toward workload and socioeconomic inequities as driving problems of demotivation and burnout among lay providers, another possible cause is the stress of taking on the new role itself [ ] . role identity theory provides a useful framing for exploring shifts in identity that lay providers may experience because of taking on a new role, and how these shifts relate to stress or resilience. the theory posits that all people have multiple, hierarchically arranged identities and roles that motivate behaviors [ ] . the roles are thought to be intimately related to each other, often influencing how other roles are performed and shaping the personal meaning of individual identities [ ] . as individuals learn new skills and interact socially, they constantly acquire new roles that add to their "role set, " like adding tools to a toolkit. role shifts are then defined as change or reshaping of a role set because of new relationship interactions, social positions, or duties. for lay providers, such shifts in social roles occur after they receive training and take on new health worker roles. the current study applies this theory with the goal of generating potential avenues for better lay provider support. we used a humanistic approach incorporating role identity theory to examine experiences of lay counselors trained in a family therapy intervention in eldoret, kenya. we aimed to describe how lay counselors experience role shifts and associated outcomes, such as keywords: mental health, lay counselors, task shifting, africa, burnout, motivation, self-efficacy, stress, family therapy, children intrinsic and extrinsic forces, beliefs, and ideals that incline an individual to pursue and maintain their position as a lay counselor [ ] an individual's perceptions and assessments of their capability to be competent and effective in their counseling role [ ] the physical, psychological, emotional, and social consequences that affect a lay counselor due to trying to fulfill the duties and responsibilities expected of them by their clients, supervisors, and communities combination of feelings of workrelated exhaustion, cynicism, and inefficacy due to the daily routine and duties required for a counselor, which can manifest in physical and behavioral changes [ , ] wall et al. int j ment health syst ( ) : motivation and burnout. where relevant, we also aimed to describe any variability in outcomes by comparing counselors with varying degrees of experience in their counseling roles (newly trained, months of experience, and years of experience). our overall goal was to use participant responses to inform strategies to improve counselor experiences, which should improve intervention delivery and quality. because the intervention was designed with the goal of minimizing the added burden for lay providers (described below), it offers a helpful lens and case study for understanding the specific contribution of role shifts in relation to other stressors associated with lay counselor experiences. semi-structured interviews and focus group discussions (fgds) were conducted to explore lay counselor experiences providing a family therapy intervention in eldoret, kenya. lay counselors were recruited from two pilot studies of the intervention in and . data collection occurred july-august . all study procedures were approved by the ethical review boards at duke university and moi university in kenya. written informed consent was obtained for all activities. the study was located in peri-urban communities surrounding the town of eldoret, kenya. eldoret is located in the rift valley province and is the fifth largest kenyan city [ ] . some residents have access to mental health services through moi teaching and referral hospital, which provides limited inpatient and outpatient care. available treatment focuses primarily on adults with serious mental illness and, to some extent, common mental disorders. very little child-or family-specific training or treatment is available, and community-based approaches are uncommon. the research team was composed of two doctoral level clinical psychologists who are co-principal investigators (one us-based, one kenyan-based), graduate students from the us, kenyan masters-level psychologists, and kenyan research assistants. team members collaborated throughout the planning, data collection, and data analysis stages of the project. tuko pamoja (tp, "we are together" in kiswahili) is a family therapy intervention designed to promote family functioning and child or adolescent mental health for families with difficulties in relationships. eligibility for tp is intentionally broad, with content designed to target problems related to family conflict, communication, and organization that occur alongside both internalizing and externalizing child symptoms. related, tp is components-based, tailored to fit the needs of each family, and focuses on generating solutions to influence the family system. it is not time-limited, with families allowed to move at their own pace. a full description of tp is provided in puffer et al. [ ] . for implementation in the pilot studies, lay counselors were recruited through existing social structures, such as religious and community organizations, and chosen because they already naturally engaged in informal helping roles and had shown sustained interest in such activities. none of the counselors in the study had any previous training related to counseling or formal counseling experience; rather, people sought them out for advice, and they reported spending significant time providing advice and listening to people experiencing individual or family problems. at times, this was performed in the context of another helping role, such as pastor, youth group leader, or village community leader (often referred to as "policy maker"). training in the family intervention was intended to augment their current, informal practices with evidence-based strategies. counselors completed approximately h of training. after being trained, counselors were expected to commit the same time to counseling as they dedicated to their advice-giving activities before they were trained, therefore not increasing their workload. this was important, as they were not paid for the counseling. the implementation approach was designed to be sustainable without external resources by integrating into existing volunteer activities and keeping counselors' time commitment the same. however, the lay counselors were paid for their participation in researchrelated activities, such as surveys and interviews. after training, counselors led the process of recruiting families to counsel, mirroring the natural ways they had already been connecting with families for informal counseling. in most cases, families had expressed a need to the counselor previously, but the counselor had not yet addressed the need in depth. the goal was to have counselors recruit from the types of families they were already helping so that the types and severity of problems would be representative of those they would have been expected to address in their day-to-day lives. given this, problems ranged in complexity but were appropriate for the intervention and the counselors, especially with access to support and supervision. counselors received supervision from local kenyan supervisors after each session (in-person or via phone) that included feedback, planning for next steps, and brief refresher training and practicing skills as needed. local supervisors received weekly consultation from clinical psychologists in kenya and the united states [ ] . the tp counseling sessions were held in the homes of the counseled families. in the first pilot trial of tp, counselors held an average of sessions with a mean length of min, totaling an average treatment exposure of contact hours. pilot results are promising for both family-and individual-level outcomes [ ] . lay counselors were included in this study if they completed tp training as part of a pilot study. we categorized them into three groups based on level of supervised tp counseling experience: "moderate" (n = ), "minimal" (n = ), and "training only" (n = ). the moderate experience group included individuals who started tp counseling in and had counseled - families (pilot study ), whereas the minimal group started tp counseling in and had been assigned a single family (pilot study ). the training only group were individuals from both pilot studies who had undergone training but had not yet provided tp for case-specific reasons, including changes in availability or difficulties engaging families. of the eligible counselors, were available, and all participated in this study. of the remaining five, three had moved away, and two had outdated contact information. demographically, the three counselor experience groups were similar (see additional file : appendix s for a detailed demographics table). the overall mean age was , with individual group means ranging between and . of all the counselors, half were women, were currently married, and were widowed. all had some formal education: five had primary-level education (two did not complete primary); six had some secondary education; three completed secondary education; and five had some post-secondary education, with one having a university degree. every counselor except one had a position, either formal or informal, that provided them income. this included four business owners, two casual workers, three policy makers, five farmers, three pastors, a school cook, and a village elder. all except one identified as christian, who identified as muslim. a kenyan research assistant completed all data collection activities following a -day training in study aims, data collection methods, and research ethics. semistructured interviews (n = ) were conducted in kiswahili using a guide developed and piloted collaboratively by the kenyan and us-based researchers. questions focused on lay counselors' approaches to counseling before tp training, changes in identity since becoming a tp counselor, and how tp counseling affected experiences of stress, burnout, motivation, and self-efficacy. for the training only group, questions related to formal counseling experiences were not included. the full interview guide is available in additional file : appendix s . during interviews, participants completed a free-listing activity where they listed current sources of stress, not necessarily associated with the counselor role. the participant then ranked the items from most to least stressful, with the option to rank multiple items at the same level. interviews were audio recorded and transcribed from kiswahili directly into english by a kenyan research assistant fluent in both languages. as each transcript was completed, we conducted preliminary data analysis using close reading of transcripts, memoing, and preliminary code development. this process helped to make iterative adjustments to the interview guide, such as selecting a simpler kiswahili translation or providing a clarifying metaphor. participants were compensated for their time with ksh (~ usd). after all participants from each counselor experience group completed their individual interviews, fgds were conducted for the purpose of member checking-the process of presenting preliminary results to ask participants to provide feedback about whether the interpretations of their responses are accurate. it is also a chance to expand upon their responses or delve deeper into interpretations [ ] . during each session, participants were presented with preliminary results from interviews with tp counselors from their group and asked to clarify, verify the information, and to expand on specific ideas that individuals mentioned. fgd participants also completed a pile sorting activity, a method used to understand how participants categorize items within a domain [ ] . as a group, participants sorted index cards listing the stressors from free lists, placing them into piles with as few or as many stressors as deemed appropriate and labeling the stressor categories. the goal was to consolidate the numerous stressors into higher-level groups to assist with later stress mapping, described below. fgds were conducted primarily in kiswahili, with occasional english. they were audio recorded and transcribed directly into english by a kenyan research assistant. participants were compensated for their time with refreshments and ksh (~ usd). thematic content analysis was used to analyze the data [ ] . all interview and fgd transcripts were first reviewed by the first author through close readings, where emergent themes were extracted and then reviewed with another researcher. themes were organized into a codebook containing parent and child codes, code definitions, examples, and inclusion/exclusion criteria [ ] . most codes reflected research topics on the guide (e.g., identity, stress, burnout, motivation, and self-efficacy), whereas others emerged from the data (e.g., role mixing, burnout resistance). to identify problematic codes and issues with code application, we used an intercoder reliability exercise. transcripts were independently coded one-by-one by two researchers. after each transcript, the researchers discussed discrepancies between coded segments and revised the codebook. this process was repeated until sufficient intercoder reliability was established (kappa coefficient of . and overall agreement percentage of %). all transcripts were then coded by the first author. code summaries were developed to facilitate understanding and interpretation of themes. we determined the data to be thematically saturated, as the final four individual interview transcripts yielded no new themes. this was further supported by the fact that in fgds, no new themes arose; participants only offered examples of previously-identified themes. code summaries were then compared across counselor experience groups to check for patterned differences. primarily, themes did not differ across groups; we indicate in results where they do differ. finally, a concept diagram was devised from data interpretations to provide a visual description of study domain interactions. a stress map was created for each experience group using the ranked lists of stressors from interviews and grouped categories from fgds. the goal of this visualization analysis was to better understand the stressors that were most influential in counselors' lives. methods from participatory risk mapping [ ] were adapted to produce stress maps that visually display relative prevalence and severity of stressors. such quantitative analyses of qualitative data are common with methods such as ranking and pile sorting, with results visually interpreted as reflecting relative rather than absolute values [ ] . the prevalence index was the proportion of a group that listed a stressor category at least once. the prevalence index is therefore ordered between and , with higher values representing stressors named by a higher proportion of group members. the severity index standardized each stressor rank within a participant's list, where each stressor (i) and its rank (r) with total (n) risks identified by each participant (j) was calculated as . if an individual listed multiple items within a category (e.g., daily provisions and school fees within the poverty category), their severity scores were averaged. the severity index is therefore ordered between and , with higher values representing stressors ranked as being more stressful. these individual severity values were then averaged across the group to produce a group severity index value for each stressor category. values were plotted in four quadrants that represent above/ below mean severity and prevalence to help visually identify key stressor clusters within and between groups. to understand counselors' personal expectations in their new formal counseling roles and their perceived counselor identity, participants were asked what they thought it means to be a tp counselor and to discuss key personal qualities of a tp counselor. their developing sense of "professional identity" was important to understand because their new formal role and training were not officially announced to the community but only to the individual families they worked with. across all groups, participants described a tp counselor as someone who is a good listener, empathetic, ready, flexible, has good communication skills, "takes their time, " and is a responsible leader. they noted specifically that a tp counselor differs from community elders in that tp counselors are not judgmental in their advice-giving; instead, they help families identify their own solutions. finally, being a tp counselor was described as being a role model in the community because "nobody will love your work if your actions are not straight" (male, , moderate group). participants described that before tp training, they offered advice and counsel anywhere they went: after church, on the streets, while running errands, or wherever they met someone in need. their advice focused on encouraging or teaching from bible scriptures, drawing from personal experiences, passing along wisdom from elders, praying with others, and giving direct advice on how to solve a specific problem. recipients of their "advising, " as they often called it, were either people who sought their advice directly or those referred to them by someone else. consistent with role identity theory, when counselors began to integrate their new formal counselor role, they experienced some positive role shifts as well as tensions between roles. some described that their new counseling skills generalized in ways that enhanced their performance in related professional or volunteer positions. for instance, "tp has affected me to some extent because i cannot use law enforcement like before; i no longer take people to the police so easily, and i try to solve the disputes" (male, , moderate group). in some cases, counselors described this resulting in increased respect and appreciation from others. on a personal level, counselors described improvements in their roles within their families as they applied the tp skills to their own lives (described below). the majority of tp counselors reported few problems with balancing roles because of the required responsibilities of the counselor role itself, suggesting the counseling role fit with their ongoing routines without significant interruptions. however, there were some cases where taking on the role of tp counselor sometimes conflicted with responsibilities associated with their other roles, especially when logistical challenges, like scheduling problems, required additional time. counselors described "sacrificing time" to engage in counseling, leaving less time to fulfill their many other responsibilities. one pastor described that "one cannot really have time to prepare a sermon, visit members of the congregation, and have time for your own family" (male, , minimal group). though not mentioned frequently, one female counselor described making sure that counseling did not interfere with income-generating activities, saying, "when i am helping others, i should not forget about my own family… since i am the sole provider for my children, if i don't work, what will they eat?" (female, , moderate group). though the tp intervention was designed so that counselors were not spending more total time on counseling, the shift to meeting with the same families on a regular basis restructured time demands in ways that necessitated shifts in how they balanced multiple roles. when asked why they became counselors, participants typically discussed intrinsic motivations first. as expected, every experience group mentioned a general desire to help others: "i was never happy at heart to see people languishing in their sorrows. i could never abandon or neglect them. i had to do something about it" (male, , training only). participants from each experience group also mentioned a deep-rooted calling from god and a passion for counseling and serving people after observing widespread community problems. the belief that they were making a difference and fulfilling god's work was highly motivating. when families engaged in counseling, attended sessions, and showed positive changes or outcomes, the counselors were motivated to continue working. many expressed that receiving training improved motivation to counsel families because they had increased knowledge and new skills to handle a broader set of cases. this in turn increased their sense of self-efficacy and helped sustain them even through challenges or negative feedback. participants also noted increased motivation after seeing the impact of applying some of the skills to their own lives, as described further below. additionally, counselors reported receiving encouragement, respect, and empowerment from supervisors and fellow counselors, as well as from family and community members. these were especially helpful in maintaining motivation when client families were struggling, not progressing, or having problems scheduling sessions. one counselor noted, "i really loved the fact that i wasn't left alone after the training… i got a lot of encouragement to continue" (male, , minimal group). to explore self-efficacy, counselors were asked about their feelings of preparedness and perceived ability to meet expectations. the resounding narrative was that counselors felt empowered in their abilities to fulfill their counselor role after completing tp training. with this improved self-confidence and empowerment from training came a change in how counselors viewed their counselor role. when discussing their counseling approach before, many described their role as "shallow, " "reckless, " and having "no consistency. " now, counselors felt they were more "professional" and were able to meet the expectations of their client families: "before, we were practicing in the dark, unlike now, we are working in the light. tp has given us knowledge, equipped us, and widened our minds" (female, , moderate group). despite overall improvements in self-efficacy, it was tested and often fluctuated based on client family trust, openness, and progress through therapy. though they clearly experienced expanded counseling capacity, the counselors consistently requested that tp continue to provide more trainings. they expressed a persistent desire to improve their counseling abilities to handle an even wider breadth of problems. the stress map (fig. ) helped identify top stress areas for each counselor experience group. as described in the methods, a top stress area is one that has both high prevalence (proportion of group listing the stressor) and severity (rated more stressful overall). for all counselor experience groups, a top stress category was poverty, describing both personal (e.g., acquiring daily provisions, paying school fees) and community (e.g., lack of employment, people not taking children to school) sources. another top category across multiple groups was family, describing both personal (e.g., marriage disputes) and community sources (e.g., homeless individuals, substance abuse). a third category was counseling, describing challenges with being a tp counselor both logistically and emotionally. the specific prevalence and severity of these stressors were different between experience groups. the top stress areas for moderate experience counselors were poverty, family, and poor parenting. for minimal experience counselors, they were counseling, education, poverty, and role balance. for training only counselors, they were family, informal counseling (not tp), and poverty. although participants mentioned a broad range of stressors in free-listing, they focused on a smaller set during individual interviews, as questions aimed to understand counseling-related stressors in depth. participants across groups expressed similar stressors related to therapy scheduling, family attendance and engagement, and perceptions of the family's progress. stress first came from becoming accustomed to their new formal counseling role and logistics, such as providing reports, recording sessions, and making follow-ups. then, family attendance problems were stressful, as family members were sometimes busy with other priorities or avoided counseling when it became difficult. counselors described arriving to empty, padlocked houses or wasting time waiting for family members to show up. at times, they questioned if they had done something wrong to cause a disinterest or avoidance of sessions. during therapy sessions, counselors noted stress if they perceived lack of family engagement, which was essential for conducting the counseling. one counselor noted difficulty "getting them to open up; it takes a lot of time sometimes when none of them believed that their problems will remain confidential" (female, , moderate group). one possibility raised by a counselor was research-specific activities (e.g., recordings and documents) might have made it harder for counselors to build rapport and trust. it was also difficult to maintain engagement and progress when families were undergoing acute hardships, which made it hard for them to focus on longer-term goals during the sessions. examples included losing a job, not having enough money to buy food, or experiencing a medical emergency. participants also noted stress when trying to navigate counseling across gender and age differences, such as a woman counselor advising a man, a younger counselor counseling elders, or a counselor attempting to facilitate inter-generational communication in the family. the interviewer used a "spring metaphor, " developed with the kenyan team during interview guide revisions, to help describe experiences of burnout. counselors were asked to think about any times they felt so compressed or stretched by their tp counseling work, they felt they could not continue working. twelve of the counselors endorsed feeling this way at times, including counselors from all experience groups. when discussing burnout, counselors often described instances where stress piled on continually without relief. this was most commonly discussed in connection to the scheduling and attendance stressors described previously. during times of burnout, counselors experienced pronounced negative thoughts or emotions related to these challenges that interfered with their drive to continue: "you find they are not there; then when you go there again, they tell you they are not ready for you; then it happens again…you feel like you are tired. that is what can make you lose hope" (male, , moderate group). additionally, some counselors experienced burnout when feeling like they could not meet the expectations of their supervisors, which led to hopelessness and the desire to avoid supervision-a required activity for counselors. participants were asked to reflect on how such situations made them feel in their head, heart, and body. all counselor groups expressed physical experiences of fatigue, pain, or feelings of sickness during such instances of overwhelming stress: "[it] makes you feel troubled. you lack peace. you feel even more tired as though you have been digging a shamba [field]" (male, , moderate group). counselors also expressed thoughts related to self-doubt and questioning of their capabilities as a counselor. at the core of the burnout experience was a struggle caused by the sincere desire to help-and belief that they had the skills to help-juxtaposed against families not engaging or making progress. in these situations, counselors noted "questioning myself " as they judged their counseling capabilities. emotionally, participants described an overall lack of peace, loss of hope, and feeling "burdened in my heart. " participants described similar coping mechanisms for dealing with both stress and burnout. for many counselors within all experience groups, stress relief came in the form of religious practices. these included praying, reading the bible because it "makes the burden lighter, " and gospel music because "the songs have encouraging words" or "help me remove my mind from the issues" (male, , moderate group). in addition, counselors invested in personal relationships, including talking to their family, spouse, or friends; playing with children; and "sharing ideas" with others. stress was also relieved by accessing support provided through tp, including information, supervisors, and peers. they described reviewing the therapy manual to reassure themselves they knew what they were doing, calling their supervisor, and meeting with other counselors. some also mentioned that returning to their other roles outside of counseling was helpful as a coping strategy because such actions helped counselors distract and distance themselves from the situation. one counselor said, "they [other tasks] help me to remove my mind from the issues that the family [is] going through and not dwell on it" (male, , moderate group). some counselors also mentioned reframing stressors as a way to cope, alongside reminding themselves that they are likely temporary: "the whole world encounters diverse challenges and stresses; for me, i have decided to take it as a normal thing" (female, , moderate group). in instances of burnout, counselors tended to focus on techniques that renewed their motivation and distracted them. they mentioned reviewing training documents to reassure themselves they were implementing the counseling correctly, as well as their case notes, which often showed some progress had been made. these strategies often restored feelings of self-efficacy. beyond these strategies counselors could perform, burnout also began to resolve if a family finally attended a session or showed progress; this often gave a sense of renewal and relief: "now i am feeling okay. you know when you help a family and they get healed, you feel even much better" for the eight counselors who reported not experiencing burnout, we asked them why they thought they avoided the experience of overwhelming stress. the first protective factor was a strong sense of self-efficacy because of having gone through training, having their capacity widened, and being "built up" in counseling skills. this was connected to intrinsic confidence in one's counseling capabilities. some described a feeling of imperviousness to burnout, with one saying he "never felt that they [a family] could overwhelm me" (male, , moderate group): "no, i didn't feel like that [burnout] . we underwent thorough training where my knowledge and capacity was widened. i have learned how to handle a lot of issues" (male, , moderate group). a second protective factor was supervision, received in person or via phone. these counselors felt they could receive help when needed, and supervision made them feel part of a team and appreciated and recognized for their hard work: "we have all the resources we need to use during the process like the mobile phone. we are also given constant advice on challenges we face. the team is also doing good in checking on us through phone calls" (male, , minimal group). a third protective factor was having a strict schedule for activities so that they could best manage their time. this helped the counselors better balance their multiple responsibilities and roles and perhaps even compartmentalize stress in a positive way: "i always try to ensure that i have a schedule of work so that i don't overstretch myself… there are days i have set for counseling, and there are days that i have set for my own work. " (female, , moderate group). a final protective factor was maintaining motivation based on the potential for overall improvement in a counselor's community, even when dealing with stressors related to a specific family. they took pride in being part of making their community more welcoming and harmonious. all counselor groups described applying tp counseling concepts to their own lives. some saw this as a necessary step for becoming role models and gaining credibility, and the changes they made contributed to positive role shifts on a personal level. counselors most commonly reported changing the ways they interacted with their own families, including their behavior toward their spouses and how they disciplined their children. they described using the problem-solving processes and communication skills taught in tp, which they noticed helped them to control their reactions to negative emotions. several counselors reported this led to positive relationship changes, such as increased love and togetherness in their home because of less quarreling and more open discussions, overall interactions, and time together. one counselor described reductions in marital conflict: another counselor described applying his new counseling skills directly to his spouse and children: in addition to family improvements, a few counselors also noted becoming more approachable, getting along easier, and having stronger bonds within their church and community, and subsequently receiving increased respect, encouragement, and appreciation-again contributing to positive role shifts. we identified a common cycle the counselors reported experiencing over time (fig. ) . following tp training, counselors experience an increase in self-efficacy because of their improved counseling abilities. the tp skills and manual made them feel that they could be more systematic when helping families and that they had an expanded scope of situations with which they could help. this high self-efficacy was accompanied by an increase in motivation to counsel because of a sense of readiness and preparation. after being assigned a family, counselors experienced the first challenges of formal counseling despite their preparations and increased self-efficacy: personal challenges from balancing roles in trying to make time for counseling and therapy-related challenges of engagement and slow (or no) progress in early sessions. for some, these caused stress to increase while motivation and self-efficacy decreased. with persistent challenges, the stress increased and persisted until a counselor could begin to experience burnout. this was driven by even further decreases in motivation and self-efficacy as a counselor questioned the commitment of the family they were counseling and their own capabilities. the struggles related to balancing roles as counselors with other responsibilities also continued, contributing to this negative cycle. when counselors had access to and engaged support and coping strategies, or the counseled family made progress, the negative effects of the challenges could be reduced. this included reduction in stress and burnout and an upswing in motivation and self-efficacy. notably, sometimes this cycle repeated multiple times during the course of counseling for just one family. finally, in most cases, families graduated from counseling, having succeeded in reaching some or all of their counseling goals. at this point, both those who had experienced more support and coping, as well as those who had experienced more prolonged stress, often reported feeling a sense of accomplishment reflected in reduced feelings of stress and burnout and restoration of motivation and self-efficacy. the purpose of this study was to conduct a humanistic examination of lay counselors' experiences of role shifts, in the context of a family therapy intervention in kenya. the lay counselors were already informally advising others in their communities, which provided a unique opportunity to examine changes due to a shift from an informal to formal counseling role. taking on a new formal counseling role produced changes in the lay counselors' lives due to their counseling duties and, more importantly, how their new counseling skills altered their approach to their other roles. this included changes in motivation, stress management techniques, and how they perceived their role in their community and families. results highlight both the positive and negative experiences of lay counselors, and the ways in which these interact. similar to other lay provider populations, counselors expressed clear intrinsic reasons for becoming tp counselors [ ] , and they maintained those foundational motivations throughout their experience. this is not surprising, given that such strong motivational forces have been found in a number of studies [ , ] . tp counselors saw their new role as an outlet to fulfill their intrinsic desires to help the community and achieve fulfillment. most connected this intrinsic motivation explicitly to their religious beliefs and values related to helping others through their abilities as counselors that were now even stronger than before. this idea of using one's talents to benefit others is one that is applicable across many religions and types of spirituality, and religiosity has been associated with more volunteering behaviors [ ] . further, those who perceive their work to be a calling from a higher power may be more involved and invested in their work [ ] . this body of research is small, but available findings resonate with our results and point to the importance of acknowledging the role of faith and the potential benefits of partnering with faith-based organizations in task-shifting efforts. tp counselors also applied the skills to their own lives, which they saw improve their relationships and ability to fulfill other roles, including professional and volunteer roles. this is perhaps related to the "helper therapy principle" described by riesman [ , ] , whereby lay providers who share similar characteristics or problems of their clients receive "helper benefits. " these benefits might promote retention, motivation, and effectiveness of a range of lay provider-delivered interventions [ ] [ ] [ ] . the new tp counselor role was manageable for most, perhaps because they were already engaged in informal counseling, showing a pre-existing interest in spending time on these activities. unlike mlotshaw et al. 's population of lay providers, who described multiple changes in identities and roles, tp counselors seemed to experience less of a role shift both in terms of identity change and adjusting to logistical requirements and maintenance of motivation [ ] . this suggests the model of working with these "natural counselors" might have benefits over models in which lay providers are given multiple new responsibilities related to multiple health needs in which they might lack a specific interest [ ] . in tp, the new roles also led to increased respect and status in the community for many counselors, which is also documented among lay providers in other studies, including those delivering home-based care in south africa [ ] . although tp counselors seemed to experience less of a strained role shift due to their previous informal experience and the positive outcomes they described, the challenges with which they needed to cope are important and informative. first, although entering the role of a formal counselor was positive in many ways and seemingly less disruptive than with other delivery models, the new counseling role did, at times, compete with other roles. some had to shift their schedules to guard against allowing the counseling role to interfere with their other responsibilities. second, counselors were now going beyond providing informal advising to providing more consistent care for families experiencing multiple difficulties in relationships that were often accompanied by unstable living situations, and overall lack of organization in the home that made it difficult for them to keep agreed upon appointments. although it is not surprising that this is a difficult population to engage in therapy, coping with attendance challenges can be defeating for counselors. third, even when families engage, family therapy is, by its nature, a difficult process; conflict is often necessary, clients often experience resistance, and progress can be slow. these clinical challenges lead to stress as counselors cope with the uncertainty about whether families will improve. although some of these stressors are likely unique, other studies have found that chws addressing a variety of health needs also have experienced stress related to caring for people with multiple complex needs that make delivery more difficult and lead to emotional distress, as they are unable to help with all of the presenting needs [ , ] . challenges experienced by counselors were associated clearly with experiences of stress and burnout that were particularly pronounced in the middle of the counseling process with a family, sandwiched between positive experiences. the initial motivation and high self-efficacy experienced at the beginning diminished as challenges persisted over time, leading over half of counselors to experience stress and some to experience burnout. similar to other studies, counselors experienced physical manifestations of stress; they questioned their abilities; and they began to lose hope that the families would ever make progress, feeling that their efforts may be futile [ , ] . their stories emphasize the importance of not only these case-by-case scenarios that caused changes in stress or burnout but the mounting interactions between them. when hitting these low points, counselors identified many effective coping strategies that led to a restoration of a positive perspective. some utilized the behavioral coping skills taught in the therapy for emotion regulation; they reached out to their social networks for emotional support; they met with their counselor peers to provide mutual support; and they used supervision to gain both emotional support alongside problem-solving. more literature on these naturally-occurring coping efforts of lay counselors across contexts and interventions would help in developing interventions that build on existing strengths. integrating counselor supports more intentionally and explicitly within interventions could buffer lay counselors' stress and enhance the benefits of taking on these types of roles. we recommend that lay provider trainings and supervision have more formalized components that discuss potential challenges they might face, allowing them to understand the role more fully and to consider together how to prepare. trainings should then include explicit activities to help counselors prepare for and buffer future stressors, such as forecasting and troubleshooting expected challenges ahead of time, normalizing expected negative emotions, and teaching and practicing coping strategies or problem-solving skills to prevent burnout-including the same skills that are included as part of the intervention itself. counselors in this study certainly applied skills from the intervention to their own lives, and this process could be formalized and facilitated by trainers and supervisors. in ongoing supervision, this process of support in coping and problem-solving should continue, providing structured, frequent opportunities for lay counselors to share their own emotional responses and difficulties that arise throughout the process. this process of implementation support is recognized as an important component of supervision in the training of mental health professionals in high-resource settings [ ] . because supervision efforts are already difficult to scale [ , ] , this can be a brief check-in to provide ongoing support with a mechanism for flagging concerns. additionally, connecting with peers can provide opportunities for mutual support during role transitions, stress, and burnout in a flexible and low-cost way. interventionists should foster a collaborative atmosphere among their workforce and coordinate peer-support strategies. this can be through formal mechanisms, such as in-person meetings and/or through telecommunication for virtual meetings or casual communication (e.g., whatsapp groups). in one tp pilot study, counselors organically developed peer support groups, citing these as important spaces for coping [ ] . in other programs, peer support is likely happening within the context of peer supervision, though their support of one another has not been closely studied. an example of a method to mobilize this organic peer support process was adopted in a study in myanmar through providing workshops for trauma management medics to learn techniques to promote personal and peer mental wellbeing [ ] . by providing lay providers with both the tools and collaborative spaces to work through their issues, such as with peer support groups or targeted workshops, they can become more proactive in dealing with problems that may arise in their work. future task-shifting interventions should carefully consider the demands placed on lay providers related to time, effort, and stress and identify ways to balance those demands with appropriate compensation or incentives. the who has recognized this need in their chw guidelines [ ] as important for promoting high motivation and retention, improving counselor experience, and recognizing that the needs and rights of providers are important in all task-shifting efforts. in our implementation model, the strategy is to lower demand and burden, with a maximum caseload of two families, and to integrate responsibilities into roles counselors had prior to tp training (e.g., informal counselors within their communities). this was not a perfect solution, as counselors met challenges such as requiring additional transport and frustration with participant attendance; at times, counseling demands interrupted their work-related activities, exceeding the anticipated burden. they benefitted from small additional incentives to continue, similar to other settings where training opportunities, certificates, and id cards serve as small, additional benefits [ , ] . even when intended demand on lay providers is relatively low, it is essential to carefully consider demand and value in developing a compensation plan, ideally in collaboration with lay provider input. monetary compensation should always be provided in cases where high workload prohibits the lay provider from meeting family financial demands through other employment avenues [ , ] . if financial resources are not available, it is essential to reduce demand and burden. one specific limitation related to analysis is that we coded the transcripts in english after translation from kiswahili, though the inclusion of member checking with fgds allowed opportunities to get feedback to clarify any questions about translations or interpretations. further, although it is a strength of this study that almost all of the eligible counselors participated, findings are limited by the fact that their experience was related to one specific intervention and geographical location. future work will be valuable in understanding the similarities and differences across contexts, types of mental health interventions, and more diverse groups of counselors. of particular interest is how socioeconomic status of counselors may influence levels of stress and burnout during non-compensated counseling activities-even with implementation strategies designed to minimize burden. in addition, while difference across counselors of different experience groups did not emerge across most domains in this study, future work should examine how counselors' experiences change over time. developing and evaluating lay counselor support strategies is also a clear future direction, as this is the essential step for improving outcomes and minimizing negative consequences for lay providers playing such an integral role in filling the global mental health treatment gap. as global health initiatives continue to involve lay providers, it is important to understand their experiences so they can be adequately supported. counselors in this study reported both positive and challenging aspects of their new roles, with most experiencing initial high motivation and self-efficacy that waned amidst challenges in providing treatment, creating stress and periods of burnout. they also reported effective coping strategies that restored motivation and self-efficacy on which future intervention approaches can build. task-shifting initiatives might improve lay provider experiences and ultimate success of the interventions themselves by providing more formal, intentional support structures. the mental health workforce gap in low-and middle-income countries: a needs-based approach south africa's rural mental health human resource crisis: a situation analysis and call for innovative task-shifting world health organization pu. task shifting: rational redistribution of tasks among health workforce teams: global recommendations and guidelines. geneva: world health organization task-sharing approaches to improve mental health care in rural and other low-resource settings: a systematic review the lancet commission on global mental health and sustainable 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in mental health interventions in low resource countries: an apprenticeship model for training local providers trauma and mental health of medics in eastern myanmar's conflict zones: a cross-sectional and mixed methods investigation evaluation of accredited social health activists (asha) springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we conducted this project in collaboration with ampath and moi teaching and referral hospital. we would like to thank joyce jeptum, mark nyalumbe, mercy korir, lydia jelagat, and purity nyangweso for their help with data collection and transcription processes. we also thank wilter rono for her local support in eldoret and savannah johnson for her help with intercoder reliability. supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file : appendix s . tp counselor demographics. appendix s . individual interview guide. lmic: low and middle income countries; chw(s): community health worker(s); fgds: focus group discussions; tp: tuko pamoja.authors' contributions jtw: design and analysis of the study and data and the writing of the first draft leading to the final draft. bnk: provided study design assistance and was a major contributor in writing the manuscript. eaf-h: supported design of methodology and study implementation at the field site; provided manuscript editing. da: advised on study conceptualization, design, and implementation at the field site. esp: advised on study design and analysis, and contributed significantly to manuscript writing. all authors read and approved the final manuscript. the author(s) disclosed receipt of the following financial support for the research, authorship, and or publication of this article: the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request ethics approval and consent to participate ethical approval for this study was obtained from duke university's irb ethics committee and the local ampath ethics committee at moi university, eldoret, kenya. informed, written consent was obtained from all participants prior to data collection. consent for publication of any individual person's data was obtained prior to data collection. all data were deidentified before analysis and incorporation in the final manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord- - ctgc n authors: barrett, alan j.; rawlings, neil d. title: families and clans of cysteine peptidases date: journal: perspect drug discov des doi: . /bf sha: doc_id: cord_uid: ctgc n the known cysteine peptidases have been classified into sequence families. we argue that these have arisen from at least five separate evolutionary origins, each of which is represented by a set of one or more modern-day families, termed a clan. clan ca is the largest, containing the papain family, c , and others with the cys/his catalytic dyad. clan cb (his/cys dyad) contains enzymes from rna viruses that are distantly related to chymotrypsin. the peptidases of clan cc are also from rna viruses, but have papain-like cys/his catalytic sites. clans cd and ce contain only one family each, those of interleukin- β-converting enzyme and adenovirus l proteinase, respectively. a few families cannot yet be assigned to clans. in view of the number of separate origins of enzymes of this type, one should be cautious in generalising about the catalytic mechanisms and other properties of cysteine peptidases as a whole. in contrast, it may be safer to generalise for enzymes within a single family or clan. peptidases in which the thiol group of a cysteine residue serves as the nucleophile in catalysis are defined as cysteine peptidases. in all the cysteine peptidases discovered so far, the activity depends upon a catalytic dyad, the second member of which is a histidine residue acting as a general base. the majority of cysteine peptidases are endopeptidases, but some act additionally or exclusively as exopeptidases. the archetypal cysteine peptidase is papain, from the latex of the unripe fruit of carica papaya. subsequently, cysteine peptidases have been found in nearly every kind of organism, from rna and dna viruses to eubacteria, protozoa, fungi, plants and animals. as yet, no archaebacterial cysteine peptidase has been sequenced, but there is evidence that it exists [ , ] . the first cysteine peptidase for which the amino acid sequence was determined was papain [ ] . for some time, it seemed that all cysteine peptidases might be relatives of papain, but as the data have accumulated, it has become clear that the true situation is very different. with about amino acid sequences of cysteine peptidases now available, the papain family is indeed by far the largest, but there are also many other families, each with its distinctive characteristics. the purpose of the present article is to describe these families, and to consider how they may be arranged in larger groups that reflect several separate evolutionary origins of cysteine peptidases. we shall also summarise the biologi-*to whom correspondence should be addressed. perspectives in drug discovery and design, vol. , pp. - - /$ . + . © escom science publishers b.v. cal functions of cysteine peptidases in the various families that make many of them potential therapeutic targets. we have compared all the available amino acid sequences of cysteine peptidases by methods described elsewhere [ ] . this has led us to identify different families of cysteine peptidases, which we number 'ci', 'c ', and so on (table ) . however, we consider it unlikely that there were unique evolutionary origins of cysteine peptidases. it is much more probable that some families have common ancestors, but that these are so ancient that no significant similarities in amino acid sequence survive. we term a set of families distantly related in this way a 'clan' (named 'ca', 'cb', and so on). since the members of a clan cannot be identified by the statistical analysis of amino acid sequences, other methods are needed. the best method of detecting very distant relationships between proteins, or excluding them, is the comparison of tertiary structures, when these are available. to date, three-dimensional structures have been determined for members of four families of cysteine peptidases. the available structures are those of papain and several other members of family c [ ] , hepatitis a virus endopeptidase (family c ) [ ] , interleukin-ll -converting enzyme (family c ) [ , ] and the human adenovirus- proteinase (c ) [ ] (fig. ). the structures seen in these four families are so different that it is virtually inconceivable that they had a common ancestor. we therefore take these families to represent four separate evolutionary origins of cysteine peptidases, or four separate clans. as we shall see, however, one of the clans of cysteine peptidases shares ancestors with a group of serine peptidases. relationships between families may also be revealed or excluded by examination of the amino acid sequences in the most highly conserved regions of the polypeptide chains, those forming the catalytic site. the order of occurrence of the residues of the catalytic dyad in the linear sequence can be crucial, since one can be certain that a peptidase in which the active site residues occur in the order cys, his cannot adopt the same fold as a peptidase in which the order is his, cys. both cys/his and his/cys arrangements of catalytic dyad residues occur amongst cysteine peptidases, and these help us to assign the families to clans. the amino acids surrounding the catalytic residues tend to be much more highly conserved than those elsewhere in the sequences and, accordingb; the sequences of these can give further clues to the grouping of families in clans. clan ca contains the families that had a common origin with papain. these have the cys/his type of catalytic dyad, and show other similarities in the structure of the catalytic site. in the papain family, gln , located in the active site wall, is important in defining the 'oxyanion hole', and asn has a role in orientating the imidazolium ring of the catalytic his ls [ ] . because of their important functions, these amino acids are absolutely conserved in the family, and also, the residue following the catalytic cys is invariably one with a bulky, hydrophobic side chain. of the other families of peptidases known to have the cys/his catalytic dyad, only those of calpain (family c ) and streptopain (c ) have the glutamine, asparagine (aspartic acid in streptopain) and bulky hydrophobic residues in the expected positions. the sequences forming the environments of the catalytic residues in these families also show other similarities to papain, and we therefore group families c , c and c t in clan ca. with few exceptions, the endopeptidases of clan ca show a strong preference for hydrolysis of bonds in which the p residue is a hydrophobic one. these enzymes are also more rapidly inactivated by iodoacetate than by iodoacetamide [ ] . this is contrary to the intrinsic reactivities of these compounds with free thiol groups, and is dictated by a charge distribution in the enzyme active site that seems to be peculiar to clan ca. this pattern of reactivity is not seen with clostripain in family c [ ] or legumain in c [ , ] . the papain family has more known members than any other family of cysteine peptidases. generally; these are synthesised as preproproteins that acquire activity only after the removal of the n-terminal peptides. exceptions are the cytoplasmic peptidases, bleomycin hydrolase, which is named for its detoxification of an anticancer drug, and bacterial aminopeptidase c. bleomycin hydrolase occurs, in yeast at least, as a hexamer with dnabinding activity [ ] . more typical members of the family are the proteinases of the food vacuoles of protozoa, and the lysosomal proteinases, cathepsins b, h, l, k, s and others in animals. as is fully described elsewhere in the present volume, the papain-like proteinases of parasites often play important roles in the destruction of host proteins [ ] , in the nutrition of the parasite [ ] , and in the neutralisation of the host immune response [ , ] . in the lysosome, enzymes of this type contribute to the turnover of cellular proteins, and also act in phagocytic cells to digest the proteins of bacteria. this activity contributes antigenic peptides to the major histocompatibility class ii system, leading to the production of antibodies [ ] . the lysosomal enzymes can also be secreted into the extracellular matrix, where they contribute to tissue remodelling in cartilage and bone [ , ] , and possibly also in tumour invasion [ ] . several studies have implicated endosomal/lysosomal endopeptidases of family" c in the pathology of alzheimer's disease [ , ] , and in another form of amyloidosis [ ] . endopeptidases of family c occur in just one group of viruses, the baculoviruses. one of these is responsible for the degradation of actin during the liquefaction of the tissues of the insect host [ ] . the endopeptidases of the calpain family, c , are cytoplasmic, calcium-dependent enzymes. their natural substrates include membrane proteins such as spectrin [ ] , and a number of cytoskeletal proteins [ , ] . calpain activity is strongly implicated in several forms of neurodegeneration [ ] . calpain is required for the normal muscle development [ ], but there is a muscle-specific calpain that is believed to be implicated in a form of muscular dystrophy [ ] . streptopain (family c ) is secreted as a proenzyme by the bacterial pathogen streptococcus pyogenes. it is a virulence factor, also known as pyrogenic exotoxin b. the enzyme contributes to the tissue injury of adult respiratory distress syndrome [ ] . the amino acid sequences of the cysteine peptidases of picornaviruses (picornains a and c) (family c ) hint at a relationship with chymotrypsin [ ] , and when the threedimensional structure of picornain c from the human hepatitis a virus was determined, it showed unmistakable similarities to that of chymotrypsin and other members of clan sa [ , ] . evidently, catalytic serine and cysteine residues have been interchanged in evolution, although it is not clear which came first. picornain c retains the equivalent of his of chymotrypsin, but has no homologue of asp ° , the third member of the catalytic triad [ ] . because of this distant relationship to chymotrypsin, the picornains and their relatives are commonly (if somewhat confusingly) termed 'chymotrypsin-like cysteine proteases'. there are many other eysteine peptidases with his/cys dyads in the rna viruses, and all of these are assigned to families in clan cb. the evolutionary relationships amongst the rna viruses are obscure, but there are good reasons for thinking that they have common ancestry. one line of evidence for this is the fact that the viral polyproteins contain some homologous proteins, which tend to be arranged in the same order. these include a helicase and an rna-directed rna polymerase, and, in the polyprotein of the polio virus, picornain c lies between the helicase and the polymerase. the helicase-peptidase-polymerase sequence of genes is also seen with the endopeptidases of tobacco etch virus (c ), feline calicivirus (c ), southampton virus (c ) and parsnip yellow fleck virus (c ). the endopeptidases of all five families contain the his/cys dyad, but they are too dissimilar to be placed in one family. the simplest explanation for this situation is that the viral polyproteins are homologous, but that the peptidase domain has a high rate of mutation relative to the helicase and polymerase. on these grounds, families c , ca, c , c and c are assigned to clan cb. peptidases from family c also have a his/cys dyad and are tentatively included in clan cb. all the peptidases in clan cb are from rna viruses, and they are responsible for proteotytic processing of the pol polyproteins (the polyproteins containing the rnadirected rna polymerase) in which they occur, commonly hydrolysing glutaminyl bonds. the picornains are not confined to action on the viral polyproteins, but also hydrolyse some host proteins. thus, picornain a also cleaves the p subunit of the eukaryotic initiation factor g into two fragments, one directing helicase activity [ ] . picornain c seems to be responsible for the degradation of microtubule-associated protein that is correlated with the collapse of the microtubules in the infected cell [ ] . through their actions on the polyprotein, and probably in other ways, the endopeptidases of clan cb perform vital functions in the replication of the viruses, many of which cause important diseases in animals and plants. this makes the cysteine peptidases of clan cb a target for the design of inhibitors that might have biomedical applications. there are additionally many polyprotein processing endopeptidases from rna viruses that have the cys/his catalytic dyad, and also show the bulky hydrophobic amino acid following the catalytic cysteine that is characteristic of the papain clan. accordingly, these cysteine peptidases are often termed 'papain-like' by the virologists. however, none of them possesses residues equivalent to gln w and asn of papain, which implies a different catalytic mechanism and quite possibly a different origin and tertiary structure. these viral peptidases are therefore not included in clan ca. there are no less than families of the 'papain-like' cys/his viral peptidases, and they are tentatively assigned to a separate clan, cc. there are structural similarities between the polyproteins from turnip yellow mosaic virus (c t), blueberry scorch virus (c ), apple chlorotic leaf spot virus (c ) and apple stem grooving virus (c ) implying a common ancestry, making it likely that their peptidases are homologous. in processing the viral polyproteins, the endopeptidases of clan cc most commonly hydrolyse glycyl or alanyl bonds. they do not show the specificity for a hydrophobic p residue that is seen in the great majority of the endopeptidases of clan ca. like the viral enzymes of clan cb, they probably also hydrolyse some host proteins. the l-peptidase of foot-and-mouth disease virus is known to cleave the p subunit of the eukaryotic initiation factor g at -gly +arg -, a requirement for successful viral replication [ ] . this clan is so tar represented by only a single family, family c of interleukin-l[ converting enzyme. these are cytosolic endopeptidases known only from animals. the three-dimensional structure of interleukin-l[ -converting enzyme shows a distinctive protein fold, and thus a separate origin for this group of cysteine peptidases [ , ] . remarkably, the several endopeptidases now known from family c all show strict specificity for the cleavage of aspartyl bonds. the interleukin-l[~-converting enzyme itself is synthesised as a proenzyme that is activated by the cleavage of aspartyl bonds, and then cleaves more aspartyl bonds to generate the active form of interleukin [~ [ ] . other members of the family are intimately involved in the process of apoptosis (programmed cell death) [ ] . again, clan ce contains just the single family (c ) of the adenovirus endopeptidase l p , which possesses a protein fold that is unique [ ] , and thus demands a further clan. the adenovirus endopeptidase has two activators: an -residue oligopeptide derived from the c-terminus of the structural protein pvi of the virus, and adenoviral dna [ , ] . the covalently bound activating oligopeptide is visible in the reported structure and, surprisingly, is not close to the active site [ ] . unlike the majority of the viruses that have been mentioned above, the adenoviruses are dna viruses. they therefore have no requirement for a polyprotein processing endopeptidase, and the virally encoded enzyme serves other functions essential for infection of the host cell [ ] . families not yet assigned to clans as described above, we have assigned the majority of the known families of cysteine peptidases to clans, but there remain a few that we cannot yet classify. family c l l contains clostripain, an endopeptidase secreted by the anaerobic, grampositive eubacterium ctostridium histoiyticum, which shows strict specificity for arginyl bonds. the enzyme is synthesised as a precursor that is activated by the removal of an nterminal propeptide and also the separation of two chains, with the loss of a nonapeptide, so that the mature enzyme has a light chain (containing the catalytic cysteine) and a heavy chain [ ] . unlike papain, clostripain is calcium-dependent, is more rapidly inactivated by iodoacetamide than by iodoacetate, and is not irreversibly inhibited by e- [ ] . families c and c contain ubiquitin c-terminal hydrolase and isopeptidase t, respectively. all members of these families seem to catalyse similar reactions, hydrolysing bonds involving the c-terminal carboxyl group of ubiquitin [ ] . ubiquitin molecules are conjugated to intracellular proteins and peptides as signals for rapid degradation by nonlysosomal endopeptidases such as the s proteasome, or to serve a chaperone function in the assembly of oligomeric proteins and ribosomes [ ] . ubiquitin is attached via the carboxyl group of its c-terminal glycine residue either to the n-terminus of another polypeptide or to the e-amino group of a lysine, and in the latter case forms an isopeptide bond. subsequently, the ubiquitin c-terminal hydrolases remove the ubiquitin moieties for re-use. there are many ubiquitin c-terminal hydrolases, perhaps as a reflection of a multiplicity of functions associated with subtle differences in specificity and function. ubiquitin chains are removed from other proteins, both following chaperonin-mediated folding, and concomitantly with proteolysis by the s proteasome. also, polyubiquitin chains are disassembled, and the activated form of ubiquitin, ubiquitin thiol ester, may be hydrolysed [ ] . family c contains peptidases with mr in the range - kda, whereas those in family c fall in the range .- kda. site-directed mutagenesis has implicated cys in catalysis by the peptidase that is the product of yeast gene doa [ ] , and the presumed catalytic histidine seems certain to be c-terminally located, so that these are cys/his peptidases. we have previously pointed out that there are slight similarities in the amino acid sequence around candidate cysteine and histidine residues in the two families, which, taken together with the similar activities, tend to suggest that families c and c belong together in a clan [ ] . family c contains peptidases including legumain, all of which show strict specificity for the hydrolysis of asparaginyl bonds. examples are known from plants and animals. in plants, the endopeptidase is vacuolar, and is implicated in both post-translational processing of seed proteins during maturation [ ] , and degradation of the same proteins during germination [ ] . the best-known animal member of the family is from the blood fluke schistosoma mansoni, in which the enzyme is implicated in host haemoglobin digestion, probably by activation of the schistosoma procathepsin b [ ] . this implies location in the lysosome. peptidases of the tegumain family differ from papain in their weak inhibition by cystatins and e- [ ] , and their more rapid inactivation by iodoacetamide than by iodoacetate [ , ] . family c is so far a small one, containing only the eubacterial, cytoplasmic pyroglutamylpeptidase i, an omega peptidase that hydrolyses the n-terminal pyroglutamyl group from a polypeptide. a mammalian enzyme with similar specificity exists, but has not yet been sequenced. family c contains primarily secreted endopeptidases from the anaerobic, gramnegative eubacterium porphyromonas gingivalis. the most fully characterised member of the family is the arginine-specific gingipain r, but there is also a gingipain k that cleaves lysyl bonds. p gingivalis is a causative agent of gingivitis, and the gingipains have been implicated in several aspects of the disease process [ ] . gingipains r and k are secreted as inactive precursors with n-terminal propeptides. the mature enzymes are multidomain proteins with the endopeptidases at the n-termini and the haemagglutinin domains at the c-termini [ ] . until recently, the cysteine peptidases of the papain family have been given so much more attention than the others that it was easy to assume that all cysteine peptidases were essentially papain-like, but it is now abundantly clear that this is not the case. the threedimensional structures of enzymes from three other families have been determined, and each has proved to have a unique protein fold, clearly unrelated and novel. it is therefore clear that there are a number of distinct evolutionary lines of cysteine peptidases, there is unequivocal evidence of the biomedical importance of the cysteine peptidases. this is seen in the pathophysiology of man and other animals, in the parasites and bacterial pathogens of man, in the physiology and pathology of plants, and in the replication of countless viruses. this demands that the peptidases from each of the clans be characterised with the thoroughness that has so far been accorded only to clan ca, and the work is now well underway. proc. natl. acad. sci. usa key: cord- -t yulofo authors: feltman, dalia m.; moore, gregory p.; beck, andrew f.; siffermann, emily; bellieni, carlo; lantos, john title: seeking normalcy as the curve flattens: ethical considerations for pediatricians managing collateral damage of covid- date: - - journal: j pediatr doi: . /j.jpeds. . . sha: doc_id: cord_uid: t yulofo nan fearful. uncertain. overwhelmed. committed. hopeful. pediatricians around the world have cycled through these emotions when considering the health and safety of patients and families during this coronavirus disease (covid- ) pandemic. we worry about the direct medical effects of covid- . we also worry about the ancillary effects of the lockdowns, stayat-home orders, closed clinics, schools, and daycare centers, and newly unemployed parents on the well-being of children. we will focuse on those concerns. we believe that responding to these ancillary effects of covid- will be a bigger challenge for pediatricians than the clinical care of children with covid- . these challenges will be with us for the foreseeable future. the covid- pandemic presents quickly-evolving ethical challenges for pediatricians. some of the issues are not specific to covid- . they include moral distress, reprioritizing clinical goals, responding to unique vulnerabilities of children, addressing uncertainty in shared decision-making, and considering how and when to proceed with pediatric clinical research. covid- puts a new spin on all of them. it also offers opportunities to rethink solutions. how we respond will matter. we must use the challenges of this pandemic as opportunities to improve the health of our children now and even after covid- recedes. this pandemic highlights long-standing deficiencies and inefficiencies in our current system of child health care. as advocates for children, pediatricians are in a unique position to redesign the system in a way that prioritizes children and families, addresses the social determinants of health, reduces inequalities and health disparities, and ensures ethical research on treatment and prevention of covid- . moral distress and utilitarian prioritization in the throes of crisis the covid- pandemic has created experiences of moral distress in our lives at home, in the clinical setting, and as citizens in our communities. moral distress is the psychological discomfort experienced when we feel that we must act in ways that violate our own deep moral commitments. as clinicians and as members of communities in this pandemic, our actions have initially and rightfully centered on crisis-mode utilitarian goals of saving the most lives. however, actions to conserve the medical resources that prevent iatrogenic spread of infection and protect health professionals and other patients have shifted priorities away from the best practices that are part of our usual care for children. we are committed to holistic and familycentered care. but the conditions of the pandemic constrain us; we cannot do what we know is best. therein lies our distress. we all prioritize goals of care within resource constraints. however, pandemic-era care is different in two ways. first, prioritization in the midst of a pandemic considers foremost the overall needs of groups of people instead of our usual prioritization of the individual person in front of us. second, the limited resources we must protect include us as healthcare workers. we are uncomfortable delaying a reintubation to don personal protective equipment (ppe) even though we know we must protect ourselves so that we can continue to treat our patients. we similarly worry about the patients we do not see because of decreased staffing levels in our clinics, or because families opt to reasonably defer medical care because of stay-at-home orders or fear of getting infected in a clinic. in our communities, we are conflicted in our inclinations to "reopen" or to remain "sheltered." we know lives have been saved by nonpharmaceutical interventions like transmission prevention measures. that said, we also recognize that many people have suffered because of school, business, and government shut-downs. there are clearly trade-offs. if everyone stayed home all the time, no one would become infected with covid- . but our children would not be educated, our stores would not be stocked, and our economy would grind to a halt. we seek the optimum balance between the risks of infection and the risks of perfect protection. our hearts ache for all children, but especially for those who have situational vulnerabilities that put them at greatest risk of adverse effects both from illness and from a shutdown society. situational vulnerabilities are conditions such as poverty or family instability that can worsen how individuals experience problems such as the current pandemic. these vulnerabilities deserve special ethical consideration because they often exist due to societal injustices. whether children are our patients or are outside of our immediate communities, we feel a sense of responsibility and with that, a deep uncertainty and powerlessness. we may not always know the best course of action, but we must help find that balance. we will gradually move out of a crisis mode; we can begin to shape a new normal right now. as we begin to imagine that new normal, we consider three domains that require attention to help children to stay well: medical, mental health, and educational needs. during the covid- crisis, many hospitals and clinics have limited procedures, numbers of staff, and patients, cancelling elective surgeries and nonessential pediatric visits. although this was necessary to reduce the risk of inadvertently spreading the virus and to conserve ppe, these practices have had unintended negative consequences. children have experienced delays in clinical care. immunization rates have fallen. children are at risk for health problems that are not directly related to covid- but that are a result of our necessary response to the pandemic. pediatricians and pediatric subspecialists are now required to triage patient procedures and visits, raising the question of what, exactly, is "elective." some surgical procedures that can be delayed without an immediate risk to the life or health of the patient may still have adverse consequences from waiting, as in the case of pyeloplasty and adolescent herniorrhaphy. , lengthy delays already exist for families trying to gain access to pediatric subspecialty appointments, such as for children with cleft palate. such delays increase anxiety and stress for families and could result in a worsening of the patient's clinical condition. temporary cessation of home health services for children with chronic medical conditions may increase the occurrence of acute-on-chronic complications. infants experiencing postponement of necessary early intensive developmental supports (e.g., speech and language therapy, occupational therapy, physical therapy) may lose a benefit proven to support their development, possibly lengthening time to advancement and/or stunting their ability to reach their true potential. to overcome covid- -mediated effects on medical care, primary care pediatricians, and pediatric subspecialists can respond with several measures. outreach, in order to check-in on patients and families regarding both their physical and mental health, will re-establish connections, nurture trust, and allow personalized counseling about needed services. virtual in-home physical, occupational, or speech therapies can keep children on track. primary care pediatricians, pediatric subspecialists, and pediatric surgeons must collaborate and carefully monitor all patients whose treatment was postponed to ensure that they are not getting worse. , communicating with patients on processes to make clinic visits safe may reassure anxious parents. all pediatricians should notify families about the options to connect with their care providers whether in person or via telephone, email, or telemedicine. , mobile clinics to reach children in underserved areas could decrease lags in immunizations. sometimes, home visits with adequate ppe might be necessary. finally, in looking further ahead, pediatricians can collaborate with allied healthcare professionals to facilitate "catch up" on the inevitable wait lists for those providers who have been limited to providing emergency services. alongside medical needs, pediatricians understand the relevance of social factors to their patients' health. , medical providers must acknowledge and address the needs of children whose situational vulnerabilities put them at greatest risks of harm during this pandemic. such a consideration is critical to prevent further widening, and to ideally narrow, existing health and socioeconomic disparities. the pandemic has already drastically altered fundamental disparities in the social determinants of health, defined by the world health organization as "conditions in which people are born, grow, work, live, and age, and the wider set of forces and systems shaping the conditions of daily life." there are ways that pediatricians can respond to mitigate the harmful effects of these changes, but we will need to be creative and proactive. we can leverage existing partnerships or forge new ones to best meet the needs of our patients and their families. for example, medical-legal partnerships (mlps) are local links between medical professionals and legal advocates. they can protect low-income families' legal rights that may be at risk in the current pandemic. they can help guard against eviction and utility shut-offs; help families navigate public benefit sign-ups, and ensure access to critical educational resources. other clinical-community partnerships (eg, with food banks) may have a similar, bold effect. , pediatricians may refer families to key social service agencies within their community that are responding in earnest to covid- 's harmful effects (e.g., united way , a free resource that can link families to local resources). we can also avail ourselves to help community groups that may benefit from a clinician's assistance. mental health needs within families. children in families with mental health needs will be vulnerable to further disruptions to their psychosocial stability due to covid- control measures. shuttered community resources upon which people rely will increase the risk for the most vulnerable children. a family member with mental illness or substance misuse may therefore experience barriers to their own usual coping supports. this could further endanger children in the home. these can all contribute to a dangerous escalating cycle if a family's coping skills were already precarious. stress also increases the risk of child maltreatment. rates of physical abuse increase during periods of economic crises and after natural disasters. maladaptive disciplinary behaviors, which put families at risk for escalating violence in the home, increase during recessions. as child sexual abuse is often perpetrated by someone known and trusted by the child, stay-at-home orders may increase perpetrators' access to children. abuse may go unreported, however. we were concerned to learn that, during the early weeks of the recent stay-at-home orders in illinois, there were sharply decreased call volumes to child maltreatment hotlines. we suspect that this is the result of children isolated from teachers, counselors, and health providers like pediatricians who typically generate most of the reports. not all abuse is going unnoticed, however. a tip line for reporting of online sexual exploitation of children noted a -fold increase in calls. social distancing will also affect visitation and placement for youth entering foster care. for those children who require alternative safe placements or children who need to be removed from a home due to maltreatment, foster parents and extended family members may be more hesitant to increase covid- exposures by accepting new children and child protection caseworkers into their homes. for many children, mortality and illness are unfamiliar concepts and they will be especially vulnerable to the psychological stresses caused by the disturbing news and images generated by stories about covid- . children may also see their parents worrying for the first time about their families' medical and economic health. some children may find themselves in difficult situations from being forcedly separated from one parent (e.g., children of separated couples or with a parent in jail). pediatricians can provide education to parents on using honest, age-appropriate language to discuss difficult topics such as disturbing news stories, illness and death, and unemployment. parents and older children can also learn from their pediatricians about moderating screen time and video gaming to minimize maladaptive effects on neurocognitive development, obesity, body malposition, and eye problems. , pediatricians need to review their records, identify children with mental health concerns or a history of maltreatment, and reach out to the families of these children. self-care to mitigate anxiety and stress will be protective and can build resilience. pediatricians can proactively provide education on mental health and effective discipline strategies. parents can also be directed to sites of professional organizations such as the american academy of pediatrics' healthychildren.org. pediatricians can connect patients to mental health professionals who develop and disseminate mental health interventions to support families with pandemic-driven stresses. , educational needs. some children's educational needs will be fulfilled more easily than others with the transition to learning at home. children with access to technology -personal devices and local internet connectivity -may make the transition to home-based or electronic learning more easily. children who usually receive learning accommodations in the classroom may find that their needs are not easily met with technology, further widening educational gaps between them and their peers. the so-called "summer slide" -the loss of educational progress -may be accelerated, and may persist, for as long as children find themselves homebound. children with autism or special needs who are unable to adhere to their routines may have particular difficulty during shelter-in-place regulations, and we can direct families to resources in their areas. pediatricians may be able to assist schools in finding children (e.g., over , in chicago) whom educators have been unable to be contact since the start of the pandemic. although all children are at risk for challenges during the pandemic, poor children are at higher risk. poverty makes shelter-at-home regulations more difficult. , the risks are both medical and non-medical. covid- infection and death rates are higher among people of color and those of low income. factors at the root of this disparity include: densely population living areas (where disease transmission is overall more prevalent), sub-optimal housing, and more difficulties meeting basic needs. , elderly family members living in multigenerational homes will be at greatest risk of severe illness. children with limited safe outdoor spaces and cramped living quarters may have adverse psychological effects while prolonged exposure to indoor pollutants and pests may exacerbate medical problems. , many children normally receive nutrition at school -with free or reduced-price breakfast and lunch -a provision that continues in many cities despite school closures. access to cleaning supplies, masks, and hand sanitizer may also be more challenging for families with limited financial means or private transportation. outside of our clinics, we can address the aforementioned situational vulnerabilities that put children at risk by advocating in the public policy arena. we can work for housing-related programs with mlps. we can advocate for employment protections, paid sick leave regulations, and economic stimulus for those most in need. we can similarly lend our voice to food access programs for children and families, partnering with those who distribute public benefits and with businesses and non-profits that bolster the underlying safety net. during the quarantine, many families have discovered that their homes are better-suited to sleep in rather than to live in continuously. online lessons have been challenging or impossible for children, because most families live in or -room homes where isolation and silence are impossible. over the longer term, we can advocate to city and building planners to design living spaces that allow easier social distancing in future pandemics with family-friendly areas for safe physical activity. we as pediatricians may not always be the ones in the lead, but we can always be in support, helping to prioritize fundamental needs for those most at-risk populations. , , because covid- is new, we do not yet understand the natural history or the factors that influence prognosis. we do not yet know the best treatments. everything is innovative. if we had more time, we could do more studies, but the rapid spread of the pandemic makes that impossible. we do not yet understand all of the ways the virus is transmitted, the best practices to slow or halt transmission, the range of illness manifestations, or the sensitivity and specificity of different diagnostic tests. we desperately need more research on all aspects of this new disease. the international medical community has adapted quickly to collect data and disseminate findings to help guide clinical practices. we are beginning to understand covid- 's clinical course. we are identifying complications afflicting certain children, like the kawasaki-disease-type sequelae after covid- infection now being called multisystem inflammatory syndrome in children. registries now enable sharing of data to better elucidate perinatal transmission and covid- illness in newborns. in the meantime, when we talk to families, we must be transparent about our knowledge gaps while still providing best evidence to parents when sharing decisions for the care of their children. guidance from professional organizations can support discussions with covid- positive mothers deliberating between post-delivery separation from newborns vs. cautious "rooming in" and directly breastfeeding vs. providing expressed breastmilk. screening, tracing, and asymptomatic transmission require elucidation to inform which practices will best prevent transmission of the virus in daycare, school, and sports settings so that we can help guide parents and administrators institute safe plans for reopening. the zeal for learning how to combat covid- will require temperance, however, with ethical principles that govern research in non-pandemic related areas. as investigators develop studies to test new treatments, they will need to clearly explain what is known, what is not known, the reasons why research is being done, the risks and benefits of participating in research, and the risks of being treated with therapies that have not been validated by rigorous clinical studies. there is no risk-free choice between participation in clinical research or opting for standard care when there is no standard of care. with either choice, parents may have unrealistic expectations about risks and benefits. doctors must guide them to understand the risks and benefits of either option. the ethics of research will become more complicated as candidate vaccines enter clinical trials. vaccine studies are technically and ethically complex. studies require large numbers of participants in order to assess both efficacy and safety. rushing development and endorsement of a covid- vaccine before establishing efficacy and safety could endanger not only recipients but also uptake of current safe vaccines. but holding off on vaccine use while completing studies will be stressful for both doctors and families. pediatricians will need to help design studies in ways that minimize risk and generate useful data as quickly as possible, and then help families understand the choices that they face. children have encountered and will continue to encounter unique challenges as the covid- pandemic continues. we cannot ignore discrepancies in social determinants of health. keeping families and communities safe, and our patients healthy is no small task. but we have an opportunity, if we pay close attention to the vulnerabilities exposed by this pandemic, to make conditions better for children than before the pandemic began. , , justice is a key value we need to be attentive to health disparities as we develop new programs to respond to covid- and covid-related health challenges. pediatricians have a fiduciary responsibility to each patient and family that they serve, but they also have a broader responsibility to the population of children in our communities. we must ensure that no subgroup is overlooked or left out. because some patients suffer unjustly due to situational vulnerabilities, fairness may require we focus more efforts toward those children at risk of greatest harms. transparency will mean making clear that despite uncertainty, our decisions and choices are reasoned, evidence-based, and strive for equity. maintaining rigorous ethical standards for research will produce knowledge we can confidently endorse. the positive downstream effects of early interventions on health are familiar to pediatricians. the challenges are daunting but they are not insurmountable. pediatricians can transform them into powerful opportunities for improving the lives of children. un policy brief: the impact of covid- on children ethics, poverty and children's vulnerability doctors fret over surgery backlog after immediate covid- crisis. the globe and mail effects of the covid- pandemic on routine pediatric vaccine ordering and administration -united states functional and morphological outcomes of pyeloplasty at different ages in prenatally diagnosed society of fetal urology grades - ureteropelvic junction obstruction: is it safe to wait? urology impact of wait time on outcome for inguinal hernia repair in infants mortality after groin hernia surgery a cross-sectional analysis of the bc children's hospital cleft palate program waitlist. plast surg (oakv) waiting for an operation: parents' perspectives impact of surgical waiting time on paediatric spinal deformity patients early developmental intervention programmes provided post hospital discharge to prevent motor and cognitive impairment in preterm infants does screening for and intervening with multiple health compromising behaviours and mental health disorders amongst young people attending primary care improve health outcomes? a systematic review use of electronic consultation system to improve access to care in pediatric hematology/oncology the impact of electronic consultation on a canadian tertiary care pediatric specialty referral system: a prospective singlecenter observational study initial assessment, treatment, and follow-up of minor pediatric burn wounds in four patients remotely: a preliminary communication computerized automatic diagnosis of innocent and pathologic murmurs in pediatrics: a pilot study utilizing lean principles to improve immunization administration efficiency in a pediatric mobile clinic program. pediatr qual saf the effect of a non-surgical orthopaedic physician on wait times to see a paediatric orthopaedic surgeon american dental association center for professional success covid- state mandates and recommendations redefining "medical care protecting the most vulnerable children from the impact of coronavirus: an agenda for action what are social determinants of health? partnering with families experiencing housing instability: considerations for interdisciplinary teams based in massachusetts and beyond hospital -food bank partnerships: a recipe for community health forging a pediatric primary care-community partnership to support food-insecure families a road map to address the social determinants of health through community collaboration prevalence and predictors of mental health distress post-katrina: findings from the gulf coast child and family health study abusive head trauma during a time of increased unemployment; a multicenter analysis increased incidence of inflicted traumatic brain injury in children after a natural disaster the great recession and the risk for child maltreatment calls to illinois' child abuse hotline dropped by nearly half amid coronavirus spread. here's why that's not good news. the southern illinoisan department of health & human services, administration for children and families, administration on children, youth and families child protection organizations seeing 'significant uptick' in predators using covid- as opportunity to exploit online. the globe and mail affective temperament, attachment style, and the psychological impact of the covid- outbreak: an early report on the italian general population co-parenting in the time of coronavirus: if you and your ex don't agree, what are your rights? the conversation tv watching and computer use in u.s. youth aged - screen media exposure and obesity in children and adolescents an investigation of children's posture and discomfort during computer use council on child abuse and neglect & committee on psychosocial aspects of child and family health. effective discipline to raise healthy children mental health in the coronavirus disease emergency-the italia response healthy children.org signs your teen may need more support in some illinois towns, internet shortfalls and lack of computers putting students at a disadvantage during an extended period away from the classroom. 'a poverty issue, pure and simple updates on coronavirus and the disabled community. forbes handle the autism spectrum condition during coronavirus (covid- ) stay at home period: ten tips for helping parents and caregivers of young children autism speaks covid- information and resources for families since cps shut, k kids went mia. chicago tribune covid- : vulnerability and the power of privilege in a pandemic covid- community stabilization and sustainability framework: an integration of the maslow hierarchy of needs and social determinants of health the covid- pandemic: a call to action to identify and address racial and ethnic disparities. j racial ethn health disparities health justice strategies to combat covid- : protecting vulnerable communities during a pandemic covid- : the painful price of ignoring health inequities mental health status among children in home confinement during the coronavirus disease outbreak in hubei province epub ahead of print indoor allergens and allergic respiratory disease housing code violation density associated with emergency department and hospital use by children with asthma feeding low-income children during the covid- pandemic covid- information and resources for families supporting equitable food access during national emergencies-the promise of online grocery shopping and food delivery services primary care pediatrics and public health: meeting the needs of today's children mitigating the impacts of the covid- pandemic response on at-risk children sars-cov- -induced kawasaki-like hyperinflammatory syndrome: a novel covid phenotype in children acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov- pandemic. circulation coronavirus infections in children including covid- clinical features, diagnosis, treatment and prevention options in children for lowering scientific standards adverse consequences of rushing a sars-cov- vaccine implications for public trust after hurricane floyd passed investigating the social determinants of disaster preparedness and recovery don't let disaster recovery perpetuate injustice this time must be different: disparities during the covid- the authors declare no conflicts of interest. abbreviations: covid- : coronavirus disease ; mlp: medical-legal partnership; ppe: personal protective equipment key: cord- - siubkz authors: rogers, amanda; lynch, kelly; toth, heather; weisgerber, michael title: patient and family centered (tele)rounds: the use of video conferencing to maintain family and resident involvement in rounds date: - - journal: acad pediatr doi: . /j.acap. . . sha: doc_id: cord_uid: siubkz nan problem: patient and family centered rounds (pfcr) is an invaluable element of the care of a hospitalized child that has been shown to improve patient outcomes and trainee education. , the covid- pandemic has led to social distancing requirements and personal protective equipment (ppe) shortages that caused many institutions to modify rounds. initial modifications within our institution included a movement away from bedside pfcr and toward table rounds with asynchronous discussions. there was a concern these modifications excluded patients/families and decreased resident involvement in clinical management/communication which could negatively impact patient care and resident education. there was therefore a need to identify ways to maintain core pfcr principles within the covid- limitations. modifications made based on feedback included ensuring teams were comfortable using technology, utilizing headphones to overcome difficulty hearing when wearing ppe helmets, and scripting that informed families about pfct. next steps/planned curricular adaptations: next steps include additional assessments of pfct including comparing the educational effectiveness and length of pfct to in-person pfcr. we are also identifying ways pfct could be utilized in the post covid era including involving off-site team members and families that are not able to physically join rounds. patient-and family-centered care and the pediatrician's role rounds today: a qualitative study of internal medicine and pediatrics resident perceptions key: cord- -fvrculi authors: riedijk, s. r.; niermeijer, m. f. n.; dooijes, d.; tibben, a. title: a decade of genetic counseling in frontotemporal dementia affected families: few counseling requests and much familial opposition to testing date: - - journal: j genet couns doi: . /s - - - sha: doc_id: cord_uid: fvrculi a decade of genetic counseling of frontotemporal dementia (ftd) affected families has generated two important observations. first, the uptake rate for presymptomatic testing for ftd is low in our department of clinical genetics at the erasmus medical center in the netherlands. second, ftd at-risk counselees reported substantial familial opposition to genetic testing, which is distinct from the attitude in huntington disease affected families. we hypothesize that the low acceptance for ftd genetic counseling is consequential to the familial opposition and explain this within the theoretical framework of separation-individuation. furthermore, we hypothesize that separation-individuation problems do not similarly influence the acceptance of hd genetic counseling, due to the educative role of the well-organised patient organization for hd in the netherlands. we offer counseling recommendations that serve to facilitate the individuation of the counselee with respect to the ftd genetic test. % of familial ftd series (rademakers et al. ; rademakers et al. ) . the mean age of onset of ftd with a mapt mutation is . ± . years (van swieten et al. ) and with a grn mutation it is . ± . years (rademakers et al. ; seelaar et al. ) . for both diseases, the penetrance is high, that is, - % of mutation carriers will become affected by years. the clinical spectrum seen with identical mutations may vary from alzheimer disease, lewy body disease, corticobasal syndrome, frontotemporal dementia, frontotemporal lobar dementia, ftd-als to primary progressive aphasia, etc. when the disease-causing gene is identified in a family, presymptomatic genetic testing becomes available. with the discovery of ftd causing genes (see for a review: van swieten ) gradually more families will be able to opt for genetic testing of patients, at-risk relatives and prenatal testing. however, the number of families tested for the less frequently observed mutations is still limited, which implies insufficient knowledge on the clinical variability of these mutations. in presymptomatic genetic testing, healthy relatives, usually siblings or children of identified ftd probands, are enabled to obtain testing for the mutation causing ftd in their family. family-specific and mutation-specific data on variability in age at onset and progression of the behavioral problems of ftd will be given, emphasizing the remaining uncertainties for newly identified gene carriers. the genetic counseling approach for ftd is similar to that for huntington's disease (hd) (brouwer-dudokdewit et al. ) and involves three steps: intake, blood withdrawal, and test result disclosure in two or three sessions depending on the extent of information and decision process prior to the first visit. the counselee is seen by a clinical geneticist and a psychosocial counselor at all visits. at intake, the clinical geneticist informs the counselee about ftd, the specific ftd related problems in the family, and the possible implications of testing. the geneticist aims to facilitate the counselee's decision-making about the choice for the test and its implications for his or her future. special attention is given to social support in-or outside the family and to possible problems of obtaining employment and life-or other insurance when an ftd carrier status is found. referral to specific information services is offered. at test disclosure the geneticist facilitates counselee expression of initial emotional reactions to the news and offers follow-up to support the counselee in coping with his or her new status, either as disease free or as a mutation carrier. the goal is adequate integration of the counselee's new status in daily living. psychosocial counselors, at intake, explore in-depth the motivation to seek testing, in order to raise awareness of the counselee's expectations with respect to testing. counselees are encouraged to think about what a favorable or an unfavorable test result would mean to them personally, in order to make a head start in psychological processing of any test outcome. how does the counselee expect to feel upon hearing that she or he is or is not a carrier of the gene? what would change? how will either outcome affect the bonds within the family? how will the counselee maintain quality of life awaiting a future of ftd? preparing for the outcome means anticipating a rather dramatic change: from uncertainty and hope towards certainty, with either a future with increasing distress, or liberated from the disease but possibly burdened for some period by guilt feelings towards affected relatives. in addition, the geneticist provides technical information on the genetics of ftd, the phenotype and penetrance, whereas the psychosocial counselor explores in-depth the psychological make-up and motivation of the counselee and the family dynamics with regard to the hereditary disorder, and discusses how this counselee may best adapt to either test outcome. furthermore, the psychosocial counselor provides follow-up, and if necessary, referral to psychotherapy. mutation analyses for presymptomatic testing for ftd in the netherlands were only performed in our rotterdam diagnostic laboratory until . the diagnostic laboratory of the free university in amsterdam has performed a few analyses since . nearly all counselees were seen at the department of clinical genetics of the erasmus medical centre in rotterdam. we made several important observations about clinical genetic testing for ftd. first, the uptake rate seemed to be quite low. after the identification of pathogenic mapt mutations in , members of three large families who were at % risk and > years of age, were informed of the discovery of the pathogenic genes and were offered genetic counseling (heutink et al. ) . we estimated that this information would also reach another adults at % risk in these families, and possibly more relatives at lower risk. only thirteen individuals from this potential group of people that were at % risk of being carriers requested genetic counseling between and . since we cannot be certain that the additional adults were actually reached, the acceptance of counseling ranges somewhere between and %. the counselees were on average years of age (range: - ), and of these counselees, pursued the test. five of these testees stated that resolving unbearable uncertainty with regard to carrying the mutation was an important motive for testing. since then, despite the discovery of other pathogenic ftd genes (bronner et al. ; seelaar et al. ), a mere additional individuals have been counseled for ftd in our center between . furthermore, during this decade ( we received only one request for prenatal diagnosis (pnd) in , and no requests for pre-implantation genetic diagnosis (pgd) for ftd. the low uptake for ftd presymptomatic testing is in great contrast with the % uptake rate for huntington's disease (hd) in the netherlands in (maat-kievit et al. ) , but it is comparable to low uptake rates for hd in other countries (tibben ) . in our clinical work we have made some characteristic observations about our ftd population that warrant special note. two of us (sr and at) have counseled all at-risk individuals who presented themselves in our department for ftd counseling since . all of these counselees reported experiencing clear opposition to genetic testing from their family members. healthy parents of counselees disapproved of testing and siblings attempted to dissuade them from testing; they were resentful about the counselees' wish to discuss the hereditary nature of ftd within the nuclear family. some counselees indicated that they consciously used the genetic test to liberate themselves from their oppressive and restrictive family. for them, genetic testing for ftd would either enable an independent, disease free life, or anticipation of a future disease without the additional burden of problematic family dynamics. family members play an important role in the decision whether or not to pursue genetic testing (bowles biesecker et al. ; mcdaniel ) . the low uptake for ftd genetic testing may result from a high prevalence of enmeshed family structures and its effects on separation-individuation processes. the concept of separation-individuation was originally formulated by margaret mahler and refers to an intra-psychic process that infants go through in early childhood in ending their symbiotic relationship with their mother (mahler ) . separation refers to the realization of self versus not self, and individuation pertains to the process of determining who and what the self is. a second separation-individuation phase begins in adolescence and is generally seen as a transition of major importance in the family life cycle (daniels ). therein, young adults gain their independence from their family of origin while continuing to have a function in the family. during this phase adolescents develop relationships with peers, or a partner that become equally important as the relationship with the parents (grootevant and cooper ) . generally, the second phase of separation-individuation is thought to start in adolescence, but it remains a lifetime task (white et al. ) . from this family systems perspective separationindividuation is seen as the interchange between individuality and connectedness between the individual and the nuclear family members. we consider the separation-individuation process to be of major importance in the clinical genetics setting. a counselee who is sufficiently individuated is able to distinguish his or her feelings and wishes from the feelings and wishes of the nuclear family. a desire to pursue genetic testing then, is a wish that has its origin in the counselee and the counselee is free to assume the responsibility for processing the information and emotions surrounding the test and to cope with the result. self-determination is combined with respectful acceptance of and disengagement from the feelings and reactions of the nuclear family. the counselee is able to tolerate that other family members deal with the genetic risk in her or his own way. when counselees have not adequately accomplished the separation-individuation task, relations with the nuclear family are either enmeshed or disrupted. in enmeshed families the counselees' motives for testing tend to be fused with the normative values of their family. these counselees may pursue testing because the nuclear family pushes them in that direction or, on the contrary, refrain from testing under familial pressure. in enmeshed families taboo and secrecy frequently surround the disease. genetic testing in such families may be instrumental allowing counselees to "escape" from family enmeshment. an unfavorable test result might excuse them from caregiver obligations, whereas a favorable outcome might empower them to build a new life. based on our decade of experience, ftd families seem to be particularly enmeshed. several processes threatening adequate separation-individuation in ftd families may promote such enmeshment. first, in dealing with illness families tend to become more cohesive to carry the burden together (rolland ) . such cohesion implies that in the face of a common threat families tend to stick together to support each other. to a certain degree family members will benefit from this cohesion. however, as olson's ( ) circumplex model of family functioning describes, when the cohesion becomes too strong individual family members have less space to cope in their unique way, and the family becomes enmeshed. enmeshed families become normative and exert pressure on members who deviate from this norm. such enmeshment interferes with adequate separation-individuation. second, goldman and colleagues ( ) noted that families affected by ftd seem to be psychologically damaged. psychological damage may occur as a consequence of having an ftd-affected parent; ftd patients have inhibition problems. their behavior is therefore a product of their impulses, which may cause them to be aggressive, promiscuous, unpredictable, etc. they lack disease insight, which results in their denial that anything is wrong. in addition, they become emotionally blunted and are unable to care about others. furthermore, their executive functions are damaged, and therefore their ability to organize and plan activities is impaired. clearly, ftd patients are unable to provide a stable and safe environment for their children in practical and emotional terms (fanos ) . one of the most damaging aspects of ftd for family members is that these symptoms occur early in the disease process. depending on the age of the children who are faced with a parent with ftd, they will be more or less prone to psychological damage. children in the preverbal phase are especially prone to develop an insecure attachment style as has been demonstrated in hd families (van der meer et al. ) . for these children, their parent's behavior will be particularly threatening. insecure attachment combined with traumatic experience will lead to dysfunction with respect to several developmental processes, including separation-individuation ( van gael ) . if the parent develops ftd when the child is in adolescence, the separation-individuation process is at-risk. for adequate separation-individuation it is important that adolescents are able to rely on a stable relationship with their parents. adolescents need the space to find out who they are and who they are not, but they also need to feel sufficient connectedness with their parents so that in this "letting go" process they do not lose their foundation (barber ; blos ) . adolescents with an ftd-affected parent may feel isolated because they are too embarrassed about their parent's behavior to bring friends home, and they may be unable to share their grief with their peers because generally adolescents do not have to deal with losing a parent. given this complex situation at home, it may be more difficult to allow peers to become equally important as the nuclear family members (carter and mcgoldrick ) . finally, it may be hypothesized that preoccupation with the future disease threatens adequate separation-individuation. in families affected by genetic disease young people may be preoccupied with the apprehension they will develop the disease in the future. this might be considered as "anticipatory loss" and a way to gain control over the future threat (rolland ) . a few of the counselees who presented for the genetic test for ftd described how they had grown up feeling they would be next. they had refrained from investing in significant relationships with persons outside of the nuclear family because they did not want to burden a potential partner with having to care for them once they developed ftd. in summary, the separation-individuation task for persons in ftd-affected families may thus be corrupted by at least three phenomena. these include: family enmeshment in response to a common threat, psychological damage due to growing up with an ftd-affected parent, and preoccupation with the possibility of becoming symptomatic for ftd in the future. a woman in her twenties, eva (pseudonym) presents herself for genetic counseling for ftd in our center. she is very ambivalent about the test. on the one hand she finds herself unable to deal with the uncertainty about her own genetic status. eva closely monitors her behavior and cognitive functions, searching for any symptoms of ftd, which would confirm her greatest fears. she has refrained from investing in a "real relationship" because she would not want to burden a potential partner with her possible ftd. she believes that in order to know which turn her future will take, she needs to learn whether or not she will develop ftd. on the other hand, eva worries that if she pursues testing, she is upsetting and betraying her healthy parent, her mother. her mother has taken care of her ftd-affected father for many years and she is terrified of losing her daughter to ftd as well. because of her fears she is unable to support eva's pursuit of genetic testing. on the contrary, the mother wants eva to support her by refraining from testing. eva is afraid of damaging her relationship with her mother because she realizes that if she has ftd, she will need her mother to take care of her in the future. eva lost her father to ftd in early adulthood, implying that her father had ftd during her adolescence. during this period she was ashamed of her father's behavior and found it difficult to understand and accept that his emotional bluntness was due to the disease and not a rejection of her as a person. because her mother had a hard time caring for her father and managing the household, she assisted her mother with many tasks. eva felt quite lonely because her friends were mostly going out and dating whereas she was continuously worrying about and feeling responsible for the situation at home. she felt so different from her peers that she was unable to invest in friendships. when her father was institutionalized she stayed with her mother because she did not want to abandon her. when finally her father died of ftd, eva allowed herself to fully consider that she too was at-risk of developing this disease, and her attention shifted from her parents to herself. based on her physical resemblance to her father she started to believe that there was a real chance that she would be the next person to develop ftd. now that her father had died, eva suddenly had to start building her own life. but ftd was looming behind every important decision she had to make. for five years she has found herself unable to make decisions and build a life of her own. this is why eva has presented for genetic counseling. however, her mother's disapproval is greatly upsetting her. eva believes that she will only be able to start living if she hears that she is not carrying the mutation, and that a negative test result would also greatly relieve her mother. in hd the strong impact of a devastating dementia on familial relationships was amply demonstrated (duisterhof and tibben ; duisterhof et al. ; vamos et al. ). members of hd affected families present more often with insecure attachment styles than the general population as a result of growing up with an affected parent ( van der meer et al. ) . insecure attachment is a risk factor for adult psychopathology (mickelson et al. ) the uptake rate for hd genetic counseling in the netherlands is relatively high, and it has been noted that a self-selected group of resourceful and psychologically "healthy" individuals presents for genetic counseling (decruyenaere et al. ; tibben ) . therefore our impression of hd counselees may be biased, making comparisons to ftd counselees difficult. still, among the small number of ftd counselees we have observed, familial opposition to testing has been particularly overt whereas we are no longer gathering similar impressions from hd counselees. the first generation of testees for hd did report experiencing similar familial opposition to testing (martindale ) . however, hd became a paradigm for genetic testing and a very active patient organization emerged. the amount of attention in the medical, clinical and research settings and in the media have paved the way for at-risk family members to seek genetic counseling. in particular, the hd patient organization is helpful to hd at-risk family members. the hd patient organization works closely with families and medical professionals, providing information and guidance regarding long term care and genetic counseling needs, based on data from longitudinal studies. the organization serves an educative function in that it offers guidance as to how families might deal with genetic testing. although we do not believe that a patient organization such as the one for hd in the netherlands will guide family members through the entire separation-individuation process, we do think it may provide some support for that process. even a highly enmeshed, damaged family that might at first not welcome genetic testing, might learn from the patient organization that individual members should be allowed to make their own choices with respect to genetic testing. in the netherlands, such a patient organization is lacking for ftd. given the absence of an educative patient organization, assisting counselees with individuation regarding genetic test wishes is a key task for the psychosocial counselor in the clinical genetics department. in ftd genetic counseling we recommend that the counselor address the degree of separation-individuation of the counselee and her or his wish for genetic testing. questions that may be helpful in assessing the degree of individuation regarding a test wish are: has the counselee confided in any family member that she or he wishes to pursue genetic testing? are the counselee's parents involved? how did siblings react? has the counselee experienced support from the nuclear family? is there openness or secrecy with regard to ftd in the nuclear family? how did the counselee reach the decision to come for genetic counseling? the answers to such questions will reveal the extent to which the counselee's wish for genetic testing has evolved in a sufficiently individuated manner. these questions will raise both the counselor's and the counselee's awareness of the context in which the wish for testing was shaped. if a wish for testing seems mostly reactive to family dynamics then this may be discussed as such. the counseling subsequently needs to address what consequences the genetic test may precipitate other than freeing oneself of the nuclear family, and whether the counselee is willing to accept these consequences as well. inherent to the wish to escape family enmeshment is the observation that mobilizing social support from the family may be particularly difficult for these individuals. occasionally the psychosocial counselor may be the counselee's sole source of support, and if that is the case, then more than the standard three sessions should be offered. genetic counseling is not psychotherapy. hence, we do not expect that geneticists will guide counselees completely through the process of adequate separation-individuation. however, addressing these issues might increase their awareness of the need for an independent decision with regard to testing. subsequently, adverse effects of deciding in favor of the test and of either test outcome may be minimized. if needed, referral for additional psychotherapy may be offered. our impressions are based on a small number of testing cases and their families. given the low uptake rate it is difficult to gather empirical data on this particular population. we have chosen to share our clinical impressions, and we invite others to provide their experience with genetic counseling and testing in ftd families. through this discussion we aim to contribute to enhanced genetic counseling, testing and enriched psychological support of individuals at-risk for ftd and their families. in summary, in our department of clinical genetics in rotterdam, a mere individuals at-risk for ftd have presented for genetic counseling over the last decade. we have observed that these counselees experienced pronounced familial opposition to genetic testing. we hypothesize that the low amount of requests for ftd genetic counseling is due to inadequate separation-individuation of the members of ftd affected families. in ftd families three processes may impair separation-individuation; ) family cohesion that becomes too strong in the face of a threatening illness, leading to enmeshment, ) growing up with an ftd-affected parent may lead to inadequate separation-individuation, and ) preoccupation with the future disease may cause at-risk individuals to refrain from separating from the nuclear family. furthermore, we hypothesize that separation-individuation problems do not similarly influence the acceptance of hd genetic counseling, due to the educative role of the well-organized patient organization for hd in the netherlands. geneticists should thus pay attention to the degree of separation-individuation evident in counselees' testing wishes. geneticists should also be aware that they may represent a major source of support for ftd counselees, 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dementia genetic basis of frontotemporal dementia phenotypic variation in frontotemporal dementia and parkinsonism linked to chromosome young adults and their parents: individuation to mutuality acknowledgements this paper was supported by the dutch research council (nwo: - - ).open access this article is distributed under the terms of the creative commons attribution noncommercial license (https:// doi.org/creativecommons.org/licenses/by-nc/ . /), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -n yqw authors: falicov, celia; niño, alba; d'urso, maria sol title: expanding possibilities: flexibility and solidarity with under resourced immigrant families during the covid‐ pandemic date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: n yqw the novel coronavirus has added new anxieties and forms of grieving to the myriad practical and emotional burdens already present in the lives of underserved and uninsured immigrant families and communities. in this article, we relate our experiences since the covid‐ crisis to the lessons we have learned over time as mental health professionals working with families in no‐cost, student‐managed community comprehensive health clinics in academic‐community partnerships. we compare and contrast the learnings of flexibility of time, space, procedures or attendance we acquired in this clinical community setting during regular times, with the new challenges families and therapists face, and the adaptations needed to continue to work with our clients in culturally responsive and empowering ways during the covid‐ pandemic. we describe families, students, professionals, promotoras (community links) and it support staff joining together in solidarity as the creative problem solvers of new possibilities when families do not have access to wifi, smart phones or computers, or suffer overcrowding and lack of privacy. we describe many anxieties related to economic insecurity or fear of facing death alone, but also how to visualize expanding possibilities in styles of parenting or types of emotional support among family members as elements of hope that may endure beyond these unprecedented tragic times of loss and uncertainty. serving underserved and vulnerable populations utilizing a humanistic transdisciplinary empowerment model with the community as teacher in the fields of medicine, pharmacy, dentistry, nursing, acupuncture, law, and social work. in addition to general primary care, specialty services of oncology, nephrology, neurology, gynecology, and mental health are provided by volunteer professionals supervising teams of students. there is also a legal clinic serviced by law students and volunteer attorneys that provides consultation, advice and referrals. the medical students acquire training in family medicine with the underserved through comprehensive patient care under the supervision of attending physicians, take didactic interactive courses related to underserved healthcare, learn community advocacy, and have ongoing opportunities for self-reflection (beck, ; wortis, beck, & donsky, ; beck et al., b) currently, there are four srfc clinics in different areas of the city of san diego providing services to a primarily a latinx population of mexican origin. latinx families account for a disproportionate share of uninsured and underinsured americans. lacking affordable and quality health and mental health care, and services that are culturally and contextually attuned, many latinx families are deprived of equal and just treatment (falicov, ; national council of la raza, ; ruiz, ) . three of the clinics offer mental health services. the clinics operate in spaces lent by religious or academic organizations and are open for around hours per day, one day per week, and on different weekdays depending on the location. the clinics have, for the most part, been at the same locations for many years. this evidences a particular relation between the clinics and the communities they serve. srfcs have built long-lasting partnerships of mutual respect and trust with local organizations to share physical and service resources. also, the individuals and families who attend the clinics rely on their consistency. although the covid- pandemic has affected virtually all areas of the world, manifestations are different depending on the location. in san diego, infection and mortality rates have been low compared to other regions of the country. despite recent acceleration, california still has a relatively low mortality rate ( per , people, compared to in new york city, and nationwide) (new york times, june , ) . within california, however, san diego county has one of the highest infection rates (number of positive cases per , people) and, as of june , there were deaths reported (new york times, june , ) . some factors that might have contributed to these rates in san diego are early adoption of shelter-in-place measures, and lower population density. despite these mitigating factors, disparities persist. the health and economic effects of the pandemic have impacted more severely the hispanic, black, and pacific islander communities in san diego county. hispanics account for % of the county population, but constitute % of cases. in addition, the infection rates for blacks and hispanics are respectively two and four times the rate for the white population in the area (city news service, june , ). these discrepancies can be partially explained by the overrepresentation of hispanics and blacks among essential workers and in jobs that require direct contact (e.g., food service, retail, childcare, transportation, and postal services). a study by the san diego association of governments (sandag, ) found hispanic and black families are three times more likely to live in the zip codes most affected by both and unemployment than their white counterparts. these areas are in the southern portion of san diego county, closer to the u.s.-mexico border, and are the places from which the srfcs receive most of their patients. the impact of covid- and its ramifications has also directly affected the provision of srfcs services. during covid- , the srfc physicians, pharmacy team, medical students, staff, volunteers, mental health team, social workers, and promotoras (experienced community members who are core staff and act as "trust bridges" to the community (beck, ) ) have come together to continue to provide health, mental health care and emotional support through telemedicine and delivery of medications and food to patients' homes. attending physicians, students, support personnel, and the pharmacy team are onsite. however, all patient visits are done by telehealth (phone or video) which has been both challenging and rewarding. our patients have been shown to be some of the most food insecure in the nation. students have created, in partnership with feeding america and the supplemental nutrition assistance program, a system where patients are screened for food insecurity and receive two bags of healthy food during medical visit (smith et al., ) . during covid- initially, families came to the clinics' this article is protected by copyright. all rights reserved parking lot while students and physicians brought medicine and food to their cars. now, for further protection, these are delivered to patients' homes by students . being part of the srfcs has always been inspirational and life-transformative . coping with medical students, physicians, social workers, interpreters, psychiatrists, psychotherapists and support staff in solidarity with each other and with patients have embraced new and innovative forms of patient care while developing clinical skills despite the challenges of social distancing. immigrant latinx patients are more likely to seek or accept mental health services within a community medical setting than independently seeking these services when they suffer depression, anxiety, trauma or relational conflicts (falicov, ; valdez et al., ; vega et al., ) . mental health distress may also manifest in different "idioms of distress" and somatic complaints such as headaches, stomach aches or fatigue that are brought up with health practitioners (falicov, a) the srfcs have had, almost from their inception in , psychiatry clinics with volunteer psychiatrists in teaching roles guiding medical students in diagnostic evaluation and psychotropic prescribing skills. there were also limited psychotherapy services provided. in , the first author, a bilingual bicultural psychologist and family therapist began her involvement with the clinic by volunteering to provide culturally and contextually attuned individual and family therapy to the medical patients in one of the clinic locations (beck, dominguez, & falicov, a) . after a few years of increasing referrals, the need for expansion of psychotherapy services became evident. she gradually recruited volunteer psychiatrists, psychologists and marriage and family therapists who provide psychotherapy in three of the four clinics today. this is how the second and third authors became involved in this project (falicov, dominguez, gonzalez, d'urso, abrams, & mcclish, ) . when conducting psychotherapy sessions, it becomes clear that for many families financial, housing, food or transportation needs are overwhelming stressors that must be addressed urgently (sousa & rodriguez, ) . in such cases, we coordinate care and advocacy with our social work team. the approach of the mental health team is based on a systems ecological orientation that respects cultural diversity without relying on ethnic stereotypes, and promotes empowerment by supporting strength-based rather than deficit-oriented approaches (falicov, ) . identifying strengths has empowering effects, and more so in situations of despair or hopelessness in families and communities who are economically marginalized and culturally or racially discriminated against. we center clients' voices as the experts on their needs and their preferred ways to address them. we favor a ground-up approach to learning from clients about their cultural preferences and their contextual stressors, endeavoring to move from professional-led to client-led solutions. in this respect, the mental health services share the srfcs philosophy inspired by tervalon and murray-garcia ( ) 's concept of cultural humility as a commitment to self-awareness by the providers in order to redress power imbalances and develop non-paternalistic partnerships. students and practitioners adhere to the premise that the patient or the client is the teacher/expert on their own lives. recent studies support the effectiveness of ground-up and shared-decision making approaches in the treatment of ethnic minorities (falicov, nakash, & alegría, in press; fraenkel, ; parra-cardona et al., ) . modifying existing mainstream psychological practices or finding innovations that consider culture and context can result in increased access, appeal and retention in mental health services (falicov, b; kanel, ) . the psychotherapy team shares strength-based, constructionist approaches to cultural diversity and social justice as a philosophical base for community services. within this umbrella, the conceptual frameworks of the therapists include family and community systems-oriented, structural, collaborative, narrative, solution-focused, emotion-focused therapy, cognitive-behavioral, relational psychoanalysis or experiential approaches. some therapists use these approaches flexibly, depending on the needs of the case. they adhere to the notion that it is possible to address social justice through a variety of approaches (mcdowell, knudson-martin & bermudez, ) . our priorities in selecting mental health volunteers are based on what we believe are healing common factors: professional excellence, passionate community concern and dedication, skills to listen collaboratively and respect local knowledge, appreciation for the ecology of families and, whenever possible, bilingual/bicultural skills. this article is protected by copyright. all rights reserved recently, medical students have been involved in psychotherapy sessions and report a better understanding of the process. they reported appreciating the collaborative nature of the sessions which has helped them modify their views of therapy as being practitioner-led and prescriptive. these exposures may increase students' cultural and contextual understandings of our population, and model reflective skills to use in their own practice as physicians. the current pandemic starkly confirmed the far-ranging inequalities that impinge on the survival of people of color. inequalities in access to the medical system cause a host of untreated or undertreated health conditions (diabetes, hypertension, heart disease, obesity) that decrease immune responses to infection. exposures and vulnerability to infection are intensified by dangerous work conditions and essential jobs, overcrowded housing, and neighborhoods that lack protective supplies and preventive information. these inequalities are further intensified for under resourced immigrants who lack health insurance, experience job instability, unstable housing, underemployment and underpayment, language and culture differences, and diverse beliefs about health and cure. the case examples illustrate how some of these intensified anxieties were addressed in therapy. lucía, , was followed up medically via telehealth. she told her physician that she had become depressed because her son, the main economic supporter of the family, had lost his job at a restaurant that closed due to social-distancing policies. because of this, the son had to move in with his mother and sister. in the initial video session, the therapist perceived that lucia´s depression was related to quarreling between her children. the therapist suggested inviting her children to the next video session to discuss their worries and find ways to support each other. lucía saw this as a way to help her grown children relieve their current distress. during the family session, it became clear that lucia conceived of her role in hierarchical terms as a parent bent on solving her offspring's problems with suggestions and advice. rather than appearing receptive, these attempts seemed to irritate her children. understanding the family interactions from a culturally sensitive, structural family therapy viewpoint, the therapist praised the mother for her maternal good intentions but created an age-appropriate boundary. the main therapy message reversed the idea that the mother needed to resolve their conflicts like she did when her children this article is protected by copyright. all rights reserved were younger. rather, the new losses of security and uncertainty brought about by the pandemic called for increased family cohesion and solidarity initiated by the young adults. covid- in immigrant family and community contexts may evoke new anxieties in the face of uncertainty, such as prospects of going to a hospital without health insurance, leaving a job because of illness, or asking for help from an already stressed social or family network. eugenia, a -year-old client who was attending the clinic for several health issues, and who had received psychotherapy for anxiety symptoms in the past, saw an exacerbation of her anxiety due to covid- . phone sessions were scheduled for psychotherapy. phone was preferred over video because it allowed eugenia the freedom to walk to an area in her home where she had privacy to discuss her concerns. video sessions were discarded because they would require using the only computer in the home, located in her daughters' room. eugenia knew that her physical conditions made her more vulnerable to complications related to the novel coronavirus. she was also aware that covid- treatment included isolation to avoid contagion. as a monolingual spanishspeaking immigrant relying on a reduced circle of love and help, the idea of being away from her family and possibly dying alone in a hospital in a strange land was devastating for eugenia, and led to intense fear and anxiety symptoms. her therapist (second author) engaged the collaboration of eugenia's physician via phone. with the information given by her physician, eugenia understood better the symptoms of covid- and the ways this disease propagates. this knowledge was empowering as eugenia felt better prepared to protect herself and her daughters, although this protectiveness, perhaps a cultural preference, made her still reluctant to share her fears with them in a family session. emotionally-focused interventions such as empathic reflections and emotional heightening gave eugenia the space to fully express her fears. from the perspective of this therapeutic model, our sense of felt security comes from our closest bonds. to increase eugenia's sense of felt security, these bonds were explored. eugenia expressed that her strength comes from the love and connection between her and her two adult daughters, who have always supported each other through thick and thin. eugenia also said that her faith in god was a source of comfort and hope, and that praying and "putting things in his hands" was something that filled her with peace when she felt afraid. interestingly, this was a new therapist for eugenia this article is protected by copyright. all rights reserved whom she only met on the phone. eugenia expressed how comforted she felt by the conversations with the therapist and how much she wished she could get to know her in person soon. immigrants suffer a series of traumas during the migration process: losses of family, language, and culture; unwelcoming reception by the host culture, and persistent economic vulnerability (falicov, ) . while immigration is a stressful process per se, the anomic conditions of isolation caused by fear of discrimination and detention, even for those immigrants with legal or quasi legal status, is an insidious psychological stressor in today's intense official persecutory anti-immigrant climate. most immigrants have lost many valuable relationships and cultural anchors. rebuilding social and family closeness, community supports, religious participation and other cultural rituals are protective elements against isolation, separation and suffering after immigration (falicov, ; sluzki, ) . during covid- , the need to maintain social distance precisely when physical and social presence are needed the most, blocks the protective in-person family and community elements for immigrant families, such as church going, or family or neighbor visiting. these experiences that lend connection, belonging and affirmation of identity become background rather than foreground because of the need to maintain social distance. physical connection may also be constructed culturally in non-verbal habitual ways. in most latinx groups, kissing, hugging or shaking hands are thought to be signs of affection and sociability often extended to people with whom one has a prolonged relationship, including psychotherapists, a cultural characteristic that has been dubbed "personalismo" (falicov, ) for this reason, we inquire about the effects of social distancing when talking with our clients electronically. also, replacing physical expressions of affection with caring words from a therapist during covid- may be comforting. these include "i hope you and yours stay healthy and safe", or "i hope you all don't worry too much", or even "i send hugs to all of you". a statement from a therapist to clients such as "know that you can always call on me" has more poignant implications at this time. we also regularly inquire about the place of religion, spirituality and faith in our latinx clients' lives, as we have learned from them about the powerful contribution of religious beliefs and practices to their well-being, particularly in hard times (falicov, a; falicov, ) . church-going and its social resources are drastically curtailed during covid- . however, silent and shared prayer this article is protected by copyright. all rights reserved or lighting candles as expressions of trust in god's will continue to be a central resource for our clients at this time. covid- stressors are not always the focus of the psychotherapy sessions. for some clients, the sessions continue to be largely focused on previous concerns, and even some new expanded possibilities opened by the sheltering-at-home situation. in fact, as practitioners inquire about possible concerns related to the pandemic, clients and promotoras have reported that some clients have found ways of adapting to the uncertainty of the situation without their lives getting dramatically worse. crowding and confinement, which may be stressful experiences for resourceful families during the pandemic, may be less onerous to our clients. enduring poverty, immigration, or language and transportation limitations, these families have adapted over time to a much more limited lifestyle situated almost exclusively in the smaller ecology of their neighborhood. also, it is possible that given their experience of frequently coping with multiple sources of adversity, clients have developed skills and family resiliencies (walsh, ) that are being put in practice as they face the effects of the pandemic. physicians' support. the care provided by the physicians and the students at the srfc clinic is a stellar example of knowing and caring for the whole person of each patient, their families and communities. the positive attitudes towards our services have ensured that we have frequent referrals for psychotherapy. studies have shown that the success of mental health services in primary care settings is dependent on physicians' attitudes (beacham et al., ) . attending physicians working with medical students at the clinic identify when a patient needs mental health services either because of coping with a difficult medical illness, because the patient's health complaints are insufficient to explain their degree of emotional distress, or by conducting regular screenings for depression and anxiety (soltani et al., ) . in solidarity, during covid- , physicians and students have continued to identify and promptly refer to us those patients in need of mental health attention. locating mental health services in the community. the health clinic where the first author originally provided psychotherapy services operated for many years in two empty classrooms and mobile trailers on the grounds of an inner-city public school and sometimes at a nearby community center. three of four clinics function in neighborhood churches. the physical location of the clinics in this article is protected by copyright. all rights reserved a church space with waiting rooms that have chairs close to each other invites social viewing and conversation among the patients. in one of the locations, the room has a table where patients, while waiting, can make crafts under the guidance of a talented community facilitator. next to it, shelves that offer water bottles, chips, cookies, coffee and sandwiches at minimal cost are handled by volunteer patients. medical students get energetically engaged with the patients in these spaces. the ambiance lends a collective sense of belonging, privacy and even ownership. many patients tell us that they have found in this environment an extension of "family", perhaps that is the reason why patients bring traditional home-made foods for the students, staff and other patients when possible. many describe these actions as their way to "give back" and it is nurturance appreciated by all. since covid- , this place of encounter has been temporarily lost. the new ways in which the community may be connecting and caring for each other are not evident to us, as we used to witness in the waiting rooms. knowing that the network experiences that happen face to face contribute to the clients' sense of community, we continue to brainstorm creative ways to provide spaces for collective connection. aspects of our professional community of care are recreated when our team of students, providers and staff make the effort to continuously check in with our patients and families using electronic means. patients may have more than one appointment per day with physicians and mental health or social work service. this somewhat evokes what a visit to the clinic pre-covid- was like. virtual coordination has been indispensable to smoothly deliver these combined services. this coordination often requires several staff involved: an it person, a student who enters the appointments in the electronic clinic records, a promotora who follows up via phone with the patient and several practitioners. these efforts rely on systems that have become more complex but had already been established before covid- . patients have reported that these efforts make them feel that they still have a community that cares and provides safety and security. flexibility of physical space for psychotherapy sessions. within the church-clinic settings, psychotherapy sessions are conducted in a variety of places according to availability. it could be sitting at a small table in the same room with other tables occupied by physicians, students and interpreters engaged in medical appointments, or in private all-purpose rooms that house file cabinets and supplies that need to be fetched by someone, briefly interrupting the session. the sanctuary of the accepted article church can be used too, where at one extreme there may be an acupuncture session in progress with patients in massage tables, and at the other extreme, near the altar, the psychotherapy session is taking place. the limitations in physical resources require providers to be flexible and adaptable. we find ourselves performing the balancing act of procuring the safety, confidentiality, and privacy necessary for conducting psychotherapy sessions while sharing the physical space with other clinicians. seating arrangements, screens and other physical barriers, along with softer voices aid in creating an atmosphere of privacy and confidentiality conducive to psychotherapy for our clients in the midst of space limitations. physical location was radically disrupted by covid- . the space that offered both the possibility of social interaction and the privacy to discuss one's concerns is no longer available. appointments need to be conducted now via telehealth with many barriers to be bridged. the flexibility gained by therapists functioning in a weekly changing space has become an invaluable asset in these changed circumstances. other forms of flexibility discussed below have always been needed and have become even more salient during this pandemic. clinical procedures have an implicit or explicit cultural expectation that the client will show up for scheduled appointments regularly, will be on time, will not overpass the allotted talk time when the end of the therapy hour approaches or will bring other family members as planned for family sessions. contrary to those mainstream cultural expectations of punctuality, regularity and prompt response to calls, many under resourced families are not always able to maintain a regular attendance and sometimes they only come sporadically. other times they just do not show up, without giving notice because they could not afford to renew their cell phone card, or their car broke down or they did not have money for public transportation. sometimes they do not bring another family member to the session as planned because they could not find their spouse or their adult son because he had just gotten an hourly job after months of searching. sometimes clients come two hours early because that is when they could get a ride. other times they come two hours late because a neighbor or relative could only bring them at that time. therapists at srfcs often try their best to see these clients, even if it means juggling schedules and delaying other clients, with their permission, rather than sending clients home, recognizing that they have tried hard to keep the this article is protected by copyright. all rights reserved session and it may take a long time before they can come again or bring family members to the session. among the pathways to reduce inequality, flexibility and procedural accommodation between provider and client are the foundation for a trusting relationship and a working alliance. we believe it is possible to conduct effective, helpful and even fairly orderly treatment in the face of these procedural differences. to accept these differences, the therapist needs to trust the client's motivation and question their own ingrained and often misguided beliefs that events, such as late arrivals, are psychologically motivated or signal resistance to treatment or a lack of commitment to change. the practitioner's trust on the clients' interest in psychotherapy is essential (brown, lopez, & lopez ) , they tend to use them only for calling and texting, the latter depending on the age group. studies show that among contemporary immigrants, family and community attachments continue intensely at long distance via phone, text and video (falicov, ) , and recently with the application what'sapp. unfortunately, these media are not hipaa compliant and cannot be used for clinical purposes, while those that are, like zoom, are not easily available without an email address or internet connection. this article is protected by copyright. all rights reserved the use of virtual technology is not entirely new to working with immigrant families at the free clinic. for therapeutic purposes, we have connected family members geographically dispersed using videoconferencing platforms nationally and binationally. virtual connections for family therapy have accelerated and expanded locally during covid- , making more visible technologies that were tangential to our work, but not totally invisible. electronic technology limitations intensify the perils of inequality and require focused attention to reduce it. the presence of an it person at srfc, fully dedicated to exploring the feasibility of setting up video calls with clients, while training students, practitioners, interpreters and clients in it use is indispensable to providing services during this pandemic. interpreters are valuable aids to explore and walk-through the feasibility of remote sessions. unencumbered by language barriers, they help clients figure out the best means of communication, time and place in the household. other barriers to virtual or even phone use arise: lack of time for essential workers or mothers schooling children, work situations with no privacy, and any area with poor reception. living in very small quarters greatly encumbers the privacy needed to have a therapy hour for individuals, couples and even families. interruptions of children or other home occupants occur. the presence of others may limit sharing private thoughts or information that may need to remain confidential. for that reason, therapists check at the beginning of the phone or video session whether the individual, couple or family members have the privacy and comfort to start the session. other times, sessions need to be momentarily put on hold while the client answers the relentless questions of a nearby child. sometimes, more than one person needs access to the same phone in the household. a visually impaired patient who is very lonely sheltering at home asked us to call him for counseling appointments in the mornings because it is the time that he is alone, and has access to the only phone family members share. in the afternoon others usually need to use the phone, but also, he is less lonely then as his spirit is lifted by the company of their voices. despite these barriers, the clinic's team and our client families continue to be the creative problem solvers that embody the free clinic philosophy by stretching the limits of the possible. for instance, a client may realize that a grandson who is a high school student, or the adult daughter of a neighbor could lend a computer and help to set a video session for telehealth. sometimes there are unexpected treatment insights of using video by seeing the home situation, perhaps the crowding, the this article is protected by copyright. all rights reserved warmth of grandmother and grandchild, or the level of noise. indeed, the video session can become a virtual home visit. similarly, phone sessions also offer opportunities to get a glimpse of family relationships. during a phone session, when a client was tearfully sharing the pain of a recent loss, the client's daughter brought her tissues, offered a hug, and whispered in her ear "you are not alone". although the client quickly apologized for the "interruption", this event opened new avenues in the session. client and therapist explored the ways the former experienced the love and support of her daughters during this painful time. relying on promotoras as community links. it is often the case that clinic patients do not have experience with mental health professionals or are not clear of why they were referred. as therapists, we are aware that as we are strangers, why should people open their hearts and minds to us? a promotora is a long-time resident of the community who deeply understands its challenges. she has the role of being a lay health facilitator and advisor. she is the confidant to whom families tell what ails them, what is happening in their lives, how going to church helps them, how worried they are about the son's vulnerability to gangs, or about a girlfriend they do not approve of, or about a husband who drinks too much. in the srfcs, the promotoras often began as patients, became volunteers, and have become the clinic's most treasured employees. because of their wisdom and community know-how, they are also invited to the university as valued teachers of the students and the faculty. a promotora is the meaningful link between the mental health team and the community. she often functions for us as a "patient consultant". not only is she supportive of our services and knows us well, she is the intermediary that introduces us to a patient by saying in her own words: "this is celia, or alba, or sol. she is one of our "consejeras" ("counselors"). you can talk with her, you are in good hands with her". this powerful introduction de-stigmatizes psychotherapy services and it paves the way for the client to feel more open and trusting of us. the door has been opened to allow us to be embraced as part of the circle of safety and the community of concern at the clinic. promotoras are effective in latinx communities because they are acquainted with their community social networks, cultural values, health and mental health needs. they are also able to communicate in a language and idioms that are appropriate and accessible to the needs of the this article is protected by copyright. all rights reserved community ( rhodes et al., ) . they are often seen as role models of resilience in the face of adversity and as displaying behaviors to be emulated (waitzkin et al., ; edelblute et al., ) . during covid- , with the best of intentions, the therapists of our clinics offered to expedite the process by booking their own appointments to relieve the promotora of this task. they soon discovered that clients sometimes did not answer the phone or returned calls, and sometimes did not comply with the scheduled appointment. why? the therapist often blocked their phone number or simply their phone numbers were not recognized, and the client, protective of their families, did not answer. the trusted person whose name and number were recognized and answered immediately was the community promotora. therefore, during covid- , in the absence of face-to-face contacts, the services of the promotora to set up and remind clients of their telehealth appointments has become even more essential. offering psychotherapy in spanish is another door that eases the relationship with latinx clients. language concordance is ideal when clients are primarily monolingual immigrants. spanish as the language of birth creates a powerful connection that overrides nationality among latinos. our bilingual therapists are immigrants from argentina, mexico, colombia and costa rica, yet their different nationalities are overridden by the emotional impact of a shared language. it is interesting that studies of cultural adaptations of mainstream interventions indicate that language is the most important factor linked to effectiveness of the intervention, over and above ethnic matching (griner & smith, ) . during the pandemic, having bilingual/bicultural therapists facilitates the therapeutic connection in circumstances when the lack of face-to-face contact can initially make the sessions feel distant or impersonal. using interpreters. we make use of excellent interpreters when the mental health professional does not speak spanish. a sensitive and warm interpreter, who uses accessible language and perhaps even decreases the distance and possible stigma of mental health treatment by engaging the client more fully in conversation is a great asset. fortunately, training programs for providing accurate and clinically relevant translation and interpretation have been developing nationwide (deangelis, ) . during covid- , we have continued to use interpreters to translate psychiatric evaluation interviews with medical students and non-spanish speaking psychiatrists. interpreters may be needed too in psychotherapy appointments conducted by a mental health practitioner and a student this article is protected by copyright. all rights reserved over several sessions. consequently, there may be several professionals involved in a phone or a video appointment with clients. interpretation during video or phone psychotherapy sessions is more taxing for all parties, but specially for interpreters because the length of sessions and the scarcity of non-verbal cues are more difficult than during medical appointments. a support and empowering group. an asset to the mental health services in srfcs is a community patient group called el grupo de empoderamiento y ayuda humanitaria (the group of empowerment and humanitarian help). a promotora began to facilitate these weekly meetings with a student many years ago. the group met continuously on a weekly basis until it was interrupted by social distancing restrictions during the covid- pandemic. this group provides an important resource of emotional healing for our clients. in fact, participants have described the group as "lifesaving," as "part of extended family" they could rely on at times of need, and as a space that offers acceptance and healing (beck et al., a) . this is a drop-in collaborative group for clinic patients of any gender, that is also open to health and mental health providers, social workers, students, and clinic staff. hierarchies are minimized as we all share current personal issues and learn from each other. the topics of discussion are usually abstract, such as compassion, forgiveness or self-care, or a mother's day celebration or a newspaper article with a human story, all of which do not demand selfdisclosure, but nonetheless the conversation becomes therapeutic or psychoeducational. the topics stimulate reference to personal narratives of traumatic events, difficulties with parenting, or couple's issues. health stressors of chronic illness and recurrent pain, or concerns about gangs or neighborhood safety are also brought up. at other times, the group engages in expressive art and crafts projects that lend a sense of useful participation when the products are sold by the women as their fund-raising contribution to the clinic, or the participants are happy to bring home a collage, gift earrings or crafts to their family members. the group ends with all participants standing up in a circle, holding hands and praying, a ritual that attests to the emotional power of religious devotion. for us, as family therapists, one of the important lessons about family systems changes is hearing how many clients of the group report significant family transformations which they attribute to bringing home what they have learned from the group . there is openness in the group to hear various members bring up ideas or projects. for example, just prior to covid- the third author engaged the group in a narrative technique of drawing and discussing their tree of life (denborough, ) . when one of the group members became silenced by her own memories of trauma, the other group members came to her aid by sharing the many positive ways that she had touched their lives. over the years, the participation of members of the mental health team in this group has contributed to self-referrals from the patients, who either approach us or the promotora for requests for advice or counseling for themselves or their families. conversely, our therapists, appreciating the group's mental health benefits, have also referred their clients to the group. during covid- , this face-to-face group is not in session as few patients can partake in a video group experience. because the deprivation of this source of support and empowerment is significant, we are attempting to find creative ways to re-establish it. one idea is to mail envelopes of art supplies with instructions for projects and include a paid envelope with the clinic's address for return by those who would like their craft shared or displayed. as covid- prevention restrictions relax, we are considering meeting in a park, as it occurred sporadically before, respecting social distance. it is possible that the bonds of friendship and reliance on each other that this group has provided over time are a form of increased social capital that may continue during covid- either by phone talks, or favors, or simply by knowing that those relationships exist and could be called on. we surmise from the interactions with clients that we have had so far during covid- that it is important for therapists to first show concern about the impact of the pandemic on the total family and on each member before developing a shared agenda for the session. expressing empathy for the pile-up of overwhelming stressors in these trying times is an important ground for connection. engaging in a sense of presence and understanding for the universal uncertainties we all share, while supporting those specific cultural values and rituals that are possible to maintain are important parts of healing conversations. but we also are attentive to openings for conversations about the learnings that come from immigrants' long familiarity with living with the uncertainty of shifting realities. these this article is protected by copyright. all rights reserved in the current changed life scenario, we witness the intensification of old and new anxieties and fears as we saw earlier in the cases of the families of eugenia and lucia, with current presentations that require new supports and strategies. nevertheless, new healing family developments are also reported, such as grown sons or daughters, including some who have been estranged, who have become more involved and concerned for their parents' daily needs and endeavor to protect them from the dangers of contagion. in some cases, mothers who are domestics temporarily unable to be employed, are spending more time with their children with the benefit of getting to know them more. we have been amazed at the creativity of latinx communities that celebrate quinceañeras ( -year-old traditional lavish birthday celebrations) or engage in funeral processions with caravans of decorated cars parading with their headlights on. an example of new developments and reflections evolving from this changed situation can be learned from the following psychotherapy case. a case example. the client is rocío, a latina woman in her early 's with two children, ages and . rocío receives help and financial support from the father of her youngest son; however, she is the primary caretaker for her children. rocío has been a patient of the clinic for several years for various medical issues, and has received mental health treatment at different times with more than one clinician. her mother and other family members have been patients in the same clinic, a situation that happens with some frequency and facilitates engaging in family therapy. six months ago, rocío started coming to therapy again due to new relationship challenges with close family members for which she was already finding her preferred ways of being when the pandemic started. due to shelter in place orders, rocío was unable to work in her job at a school cafeteria, had to stay at home with her children, became worried about her financial situation, and had to engage in long distance education with her children. the children were also anxious with the change in their routine, not seeing their friends and the confinement of their small apartment and not being able to play in the park. the therapist started online therapy having some sessions with rocio alone and other sessions with rocio and her children. at a time of social distancing when the only way to receive education, health or mental health services is virtually, a person who does not have wifi connection, a computer or has computer literacy is at a disadvantage. as is the case for most low-income immigrant families, rocío did not have this article is protected by copyright. all rights reserved internet access and had to go through the process of getting it in her home and learning about technology she had never been exposed to. her cousin, who already had internet, helped her by calling the company and arranging the internet installation. she had to wait several days to get the service set up and had to redistribute her financial means to pay for it monthly. to receive mental health and health care services from our clinic, rocío worked with medical students to set up a zoom account on her phone and learn how to use it. the third author worked with rocío on understanding the effects of covid- on her and her children's lives. the therapist learned different ways in which the uncertainty was creating a sense of fear and affecting rocío's sleep. from a narrative perspective, the therapist engaged with the client in the practice of "double-listening" (yuen, , white , paying attention to the stories of struggle dealing with the practical and emotional impact of covid- , and simultaneously listening for her and her family's abilities to respond to these new challenges. in the conversation, alternative stories emerged, where rocío spoke about how being at home was also having positive effects on her relationship with her kids, which surprised her. this opened a conversation where rocío shared that she noticed she was being more patient with her children, even during this session as they interrupted. as we explored this in detail, i asked rocío how she would name this new way of being with her children and rocío called it "amabilidad en el hogar" (kindness in the home). through the conversation we learned how kindness was showing up in big and small ways and its effects on each member in the family. this led to a conversation about the family legacy of amabilidad en el hogar as rocío shared that this was a value she had learned from her mother who had passed away a few years ago and was rocio's strong emotional support. rocío shared stories about her own upbringing and how amabilidad or kindness was taught and modeled by the women in her family. at the end of the conversation the therapist wrote rocío a letter, which is a common narrative practice, to document her initiative to stay close to "amabilidad en el hogar durante los tiempos de covid- " (kindness in the home during the times of covid- ). below is a fragment of the letter (spanish original and english translation) where the therapist underlines the client's preferred ways of being with her children and her hopes to stay close to amabilidad en el hogar during hard times in her life. the therapist also conveys how she has been moved in her personal life to think and practice amabilidad and thanks the client for the invitation to this article is protected by copyright. all rights reserved do so, making transparent how much she learns from her client and is transformed by their conversations and relationship. in the next session, the therapist met with rocío and her children where rocio shared her new discoveries with her children and they shared with her how they see her as happier and funnier and how they are enjoying their time with her more. ….cuando usted es mamá desde la amabilidad se acerca al tipo de persona que quiere ser y ha sido por mucho tiempo y también es otra forma de seguir honrando y recordando a su mamá. también lo bien que se siente al tratarse con amabilidad es algo que usted quiere recordar en estos tiempos difíciles, cuando la vida quizás nos invita a olvidar la amabilidad y a ocuparnos de otras cosas. fue un gusto como siempre hablar con usted rocío y aprender de las formas que usted está pudiendo ser como mamá, me invitan a mí a querer quedarme cerca de este valor de la amabilidad también, como persona y como mamá. muchas gracias por la invitación! con cariño, english translation of the fragment of the letter: when you are a mom from a place of kindness, you get closer to the type of person you want to be and have been for a long time and it is another way to continue honoring and remembering your mom. also, how good it feels when kindness is around is something you want to remember during these difficult times, when life invites us to forget about kindness and take care of other things. it was a pleasure as always to talk to you rocío and learn about the ways you are able to be as a mother, they invite me to want to stay close to kindness as well, as a person and as a mother. thank you very much for the invitation!! fondly, the continuation of no-cost mental health services to underserved and uninsured latinx immigrant families as part of an academic student managed community project during the covid- this article is protected by copyright. all rights reserved pandemic illustrates attempts to overcome unprecedented new procedural and clinical practice challenges in the conceptions of structures, responses, time and space for services. our team's overall goal is to reach this underserved population. flexibilities of time and space, using procedures and protocols that alter the standard professional approaches used with over-resourced families, are necessary to overcome multiple contextual constraints. rather than describing underresourced families as "hard-to-reach", we believe that our adaptations are a form of justice and empowerment that have become even more compelling under covid- 's new constraints. relying on our learnings about providing services to under resourced families and communities in regular times, during the covid- pandemic we expand the limits of what appears possible in a variety of new ways. sessions are conducted via telehealth for clients that never had wifi or used computers before, accommodating to time slots of relative quiet or task-free time in the household, substituting the presence and support usually given by now socially distanced family and friends with empathy, caring words and offers of phone support given by therapists. reliance on community helpers or promotoras as intermediaries between professional staff and our client families to facilitate setting up and confirming appointments or legitimizing mental health care needs is even more necessary during covid- . deep commitment towards our clients as well as solidarity of purpose among the various members of the health and mental health team have become even more essential during these extraordinary circumstances. we consider concrete acts of care and solidarity, such as home delivery of donated healthy food and medications by the medical students at the clinic an important aid towards the mental well-being of under resourced families at this time of isolation and deprivation. no doubt, covid- has created excruciating challenges for already overburdened immigrant families, but in their resilient ways, they also demonstrate new, often creative and inspiring ways of responding, be it in terms of celebrations, funerals or new ways of parenting their children, appreciating love bonds or caring for their elders. expanding the possibilities of our own flexibility and solidarity as a professional team embodies what we are learning from families whose resilience has helped them face with courage and dignity untold adversities and injustices over generations. focusing on the strengths of families, we attempt to create space for both, honoring people's complex this article is protected by copyright. all rights reserved experiences and reactions in these anguished times and co-creating ways of responding together in this process, visualizing elements of hope and positive changes that may endure. as we are finishing writing this paper, the country is shaken by the continuous police brutality against black communities. the local and national protests have heightened our latinx immigrant communities' fear of police violence, immigration agents and government attacks towards their families. our clinic students and the entire clinic's team have shown an outpouring of support and solidarity and have directed their attention to the effects of these stressors on the well-being of our clients. to respond to these current realities, a decision has been made in the srfc project to strengthen the anti-racism university curriculum in integrative health and mental health care for underserved families. community as client: reaching an underserved urban community and meeting unmet primary health care needs primary care medical provider attitudes regarding mental health and behavioral medicine in integrated and nonintegrated primary care practice settings the ucsd student-run free clinic project: transdisciplinary health professional education providing integrative health care for the latino community: learnings from a successful -year experience. presentation at collaborative family health care conference addressing the health needs of the underserved: a national faculty development program digital divide narrows for latinos as more spanish speakers and immigrants go online accepted article this article is protected by copyright. all rights reserved latinos getting covid- at four times the rate of whites in san diego county found in translation. monitor on collective narrative practice: responding to individuals, groups, and communities who have experienced trauma promotoras across the border: a pilot study addressing depression in mexican women impacted by migration latino families in therapy religion and spiritual traditions in immigrant families: significance for latino health and mental health on the wisdom and challenges of culturally attuned treatments for latinos: a commentary of evidence-based practices working with transnational immigrants: expanding meanings of family, community and culture ucsd student-run free clinic project volunteer mental health services. presentation at ucsd healthcare seminar centering the voice of the client: on becoming a collaborative practitioner with low income individuals and families engaging families as experts: collaborative program development inequity and the disproportionate impact of covid- on communities of color in the united states: the need for a trauma-informed social justice response fear of covid- and the mental health consequences in america culturally adapted mental health intervention: a meta-analytic review mental health needs of spanish-speaking latinos in southern california mental health effects of school closures during covid- . the lancet third-order thinking in family therapy: addressing social justice across family therapy practice critical disparities in latino mental health: transforming research into action coronavirus in the u.s.: latest map and case count queremos aprender": latino immigrants' call to integrate cultural adaptation with best practice knowledge in a parenting intervention lay health advisor interventions among hispanics/latinos: a qualitative systematic review accepted article this article is protected by copyright. all rights reserved coronavirus: a trigger for ocd and illness anxiety disorder? hispanic access to health/mental health services covid- impact on the san diego region: black and hispanic communities hardest hit migration and the disruption of the social network re-visioning family therapy: race, culture, and gender in clinical practice. nd implementation of a food insecurity screening and referral program in student-run free clinics the effect of involvement in a student-run free clinic project on attitudes toward the underserved and interest in primary care universal depression screening, diagnosis, management, and outcomes at a student-run free clinic the collaborative professional: towards empowering vulnerable families coronavirus disease case surveillance -united states accepted article this article is protected by copyright. all rights reserved cultural humility versus cultural competence: a critical distinction in defining physician training outcomes in multicultural education population estimates provider perspectives about latino patients: determinants of care and implications for treatment help seeking for mental health problems among mexican americans traumatic loss and major disasters: strengthening family and community resilience promotoras as mental health practitioners in primary care: a multi-method study of an intervention to address contextual sources of depression working with people who are suffering the consequences of multiple trauma: a narrative perspective health and the community pathways beyond despair: re-authoring lives of young people through narrative therapy accepted article key: cord- - lxy qrq authors: bolarinwa, obasanjo a.; olagunju, olalekan s.; olaniyan, akintayo t. title: factors associated with low contraceptive use amongst vulnerable mothers in south west state, nigeria date: - - journal: afr j prim health care fam med doi: . /phcfm.v i . sha: doc_id: cord_uid: lxy qrq background: young mothers tend to be more prone to high maternal and perinatal risks and are thus deemed vulnerable to adverse sexual and reproductive health rights (srhr) in terms of their right to choose contraceptives of their choice to enhance their maternal well-being and childbirth spacing should be well discussed. achieving sufficient srhr may be averted if the use of family planning by disadvantaged groups is not given required attention. aim: this study aimed to identify and analyse the factors associated with the low use of contraceptives amongst vulnerable women in the south west region in nigeria. setting: the study area was purposively chosen to capture contraceptive use amongst vulnerable women in osun state, nigeria. methods: a primary data collection was done in three senatorial districts of osun state, nigeria, with respondents each to give a total of respondents. collected data were analysed using univariate, bivariate and multivariate measures. results: the result showed a magnitude of association and relationship at both levels of analyses. living arrangements and family types were % and . %, respectively, associated with family planning use. in the same vein, living arrangement and family types were also statistically significant at p < . with an odds ratio of . ( % ci: . – . ) and an odds ratio of . ( % ci: . – . ) with family planning use, respectively. conclusion: we concluded that policies and interventions to accelerate and encourage contraceptives use amongst vulnerable mothers in south west, nigeria should be targeted at those whose husbands lived elsewhere and those whose husbands have more than one wife. an estimate of more than million women in the developing world were unable to access and use family planning or contraception (fp/c); this is also the cause of high fertility levels in african countries where nigeria is not excluded with a total fertility rate (tfr) of . per woman, and the annual population growth rate is . % with an estimated population of million. , , it is agreed that the increased spending on family planning has a compelling long-term interest in increasing human resources and family happiness. in order to reap the dividend from these investments and meet the third goal of the sustainable development goals (sdgs), the disadvantaged groups that are key to nigeria's population growth must be recognised. young mothers seem to be more exposed to high maternal and perinatal risks and are seen as being vulnerable. therefore, their sexual and reproductive health right (srhr) in terms of their right to the choice of contraception should be well addressed to improve their maternal and child health outcomes. it was also noted that most births in sub-saharan african countries take place in the union, and most of such deliveries are mistimed or unintended. this is more prominent in eastern & western africa where the unmet need for family planning is the highest. , , , , reduction in the non-use of contraceptives has a uniquely wide range of demographic, economic and environmental benefits, in addition to its well-documented health advantages for women and children, and this is because of its direct link to family sizes and population change. background: young mothers tend to be more prone to high maternal and perinatal risks and are thus deemed vulnerable to adverse sexual and reproductive health rights (srhr) in terms of their right to choose contraceptives of their choice to enhance their maternal well-being and childbirth spacing should be well discussed. achieving sufficient srhr may be averted if the use of family planning by disadvantaged groups is not given required attention. aim: this study aimed to identify and analyse the factors associated with the low use of contraceptives amongst vulnerable women in the south west region in nigeria. the study area was purposively chosen to capture contraceptive use amongst vulnerable women in osun state, nigeria. a primary data collection was done in three senatorial districts of osun state, nigeria, with respondents each to give a total of respondents. collected data were analysed using univariate, bivariate and multivariate measures. the result showed a magnitude of association and relationship at both levels of analyses. living arrangements and family types were % and . %, respectively, associated with family planning use. in the same vein, living arrangement and family types were also statistically significant at p < . with an odds ratio of . ( % ci: . - . ) and an odds ratio of . ( % ci: . - . ) with family planning use, respectively. we concluded that policies and interventions to accelerate and encourage contraceptives use amongst vulnerable mothers in south west, nigeria should be targeted at those whose husbands lived elsewhere and those whose husbands have more than one wife. keywords: family type; living arrangement; family planning; vulnerable; south west; nigeria. read online: scan this qr code with your smart phone or mobile device to read online. previous studies have noted that increasing contraceptive use in countries with high fertility rates has the potential of averting about % of all maternal deaths and % of childhood deaths. considering nigeria's maternal rate of per livebirths and child mortality rates of approximately per children, surviving up to months of age is particularly high compared with other regions that are economically developed or are on the path of economic development. this large variability of non-use of family planning is higher amongst young mothers in the south west region with an average unmet need for contraception at % compared with other regions regions with much lower unmet need for contraception, hence the young mothers could delay the progress of the country in harnessing or reaping its demographic dividend and adequate family planning use has full capacities of averting the high rate of maternal and child mortality. families can be classified on the basis of several dimensions; for example, on the basis of marriage type, families can be monogamous or polygamous which is also valid based on other classifications. it is estimated that about million couples, mainly in third world countries, do not use effective contraceptives, despite their desire to limit or space the birth of their children, with % of nigerian couples having unmet needs for contraceptives despite being sexually active. this has a lot to do with cultural and societal values the family accepts. in varying degrees, polygamy seems to be the norm in most african societies, with some % - % of women living in polygamous family settings. , there is also evidence of a close relationship between health, well-being and living arrangements. literature also argued the assertions that the decision-making on contraception comes within the domain of women but this rather depends on the type of union or how the living arrangement is structured. , despite significant global resources and investments that are being directed to nigeria to meet the current demand for family planning services, the level of contraceptive use amongst married or in-union women is lower compared with other developing countries. the gaps in attaining adequate sexual and reproductive health may be delayed if family planning use is not encouraged amongst vulnerable groups. hence, there are critical needs to examine the family type, living arrangement and contraceptive use amongst vulnerable mothers in south west, nigeria. this study aimed to identify and analyse the factors associated with the low use of contraceptives amongst vulnerable women in the south west region in nigeria. the study was quantitative research, which employed a cross-sectional descriptive survey design to elicit information on family planning use, family types and living arrangements amongst vulnerable mothers in osun state, nigeria using a structured questionnaire. osun state is an inland state in south-western nigeria. its capital is osogbo. it is bounded in the north by kwara state. the state enjoys a tropical climate characterised by two seasons: rainy season (march to october) and dry season (november to march). the study population comprised young mothers within the reproductive age of - years who already had a child in the last year. the study area was purposively chosen to capture contraceptives use and associated factors amongst this group. the data collection was done in three senatorial districts of osun state, nigeria. yamane's formula for estimating sample size was used to estimate sample size for this study, and the arrived estimated sample size was , including a % margin of error or non-response. respondents were interviewed in each senatorial district using a structured questionnaire and a multi-stage sampling procedure to give respondents altogether. the analysis was performed with stata software. the data were analysed adopting univariate, bivariate and multivariate measures. univariate analysis was used for the frequency distribution of selected socio-economic and demographic variables that are related to this study in the dataset displayed. a chi-square test and logistic regression analysis were employed to show an association, relationship and predict the likelihood between the independent variables, which are family type and living arrangement on the dependent variable, which is family planning use. the researcher ensured that the informed consent of the respondents was sought by explaining the purpose of the study; anonymity was maintained by not including their names, and confidentiality was assured. before administering the questionnaire, a copy of the research protocol was submitted for a full review to the research and ethics committee of institute of public health, obafemi awolowo university, ile-ife (iph, oau) and approval for this study was obtained with iphoau/ / . in contrast, the respondent's husbands socio-demographic characteristics show that majority of them were between ages and years with the mean age of years. the majority of the respondent's husbands have more than primary education (secondary . % and post-secondary . %). more than half of the respondent's husbands have their religion as christianity ( . %). almost all the respondent's husbands were employed ( . %). table presents the bivariate association and logistic relationship between living arrangement, family type and family planning used amongst women who already had a child in the last year. the result above showed a significant association between living arrangement and use of family planning in that % of women who were living with their husband and . % of women whose husbands were living elsewhere were using family planning. with regard to family type, the table shows that . % of women whose husbands had only one wife and . % of women whose husbands had more than one wife were using family planning. the table also reveals that there is a significant association between the use of family planning, living arrangements and family type as p < . . logistics regression analysis was carried out to assess the net effect of living arrangements and family type on the use of family planning. table also shows that women whose husbands live elsewhere are . less likely to use family planning with ( % ci: . - . ); in the same vein, women whose husbands had more than one wife were also . less likely to use family planning with ( % ci: . - . ). both living arrangements and family type were statistically significant in the study at p < . . this study also revealed that family type and the living arrangement were significantly associated with the use of family planning. the research shows that women whose husbands have more than one wife were less likely to use family planning compared with women whose husbands had only one wife. this may be due to marital conflict amongst wives with regard to the birth order or sex preference. also, women whose husbands were living elsewhere were less likely to use any method of family planning. this may be because women were not exposed to frequent sexual intercourse compared with women who were living with their husbands. the need to subscribe to contraceptives use amongst vulnerable young mothers who lived away from their husbands and who are in the polygamous union might not be there because of their infrequent sexual activities as the husbands are often not available. this study unveiled this peculiar group of non-family planning users to help scale up the adoption in south west state, nigeria. although the prevalence level of contraceptive use amongst sexually active men and women is low in nigeria, , , we studied variations by living arrangements and family types in the use of any family planning method amongst young mothers. the use of any family planning methods was high amongst young mothers living with their husbands and whose husbands have one wife. the family type was significantly associated with the use of family planning. women whose husbands were living elsewhere were less likely to use a method of family planning. also, the living arrangement was significantly associated with the use of family planning. young mothers whose husbands have more than one wife were less likely to use family planning. adoption of a family planning practice campaign that considers living arrangements and family types will further scale up contraceptive use amongst young women. , male partner influence on family planning and 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dna binding subuult of nf-kaplm-b is identical to factor kbfl and homologous to the rel oncogene product sequencins analyses and com~ of pmrainfluenza virus type- a and type- b np protein genes. virok>gie complete sequence of the gcnomic gna of o'nyon -nyong virus and its use in the constroctton of alphavir~ phylogenetic trecs molecular clouln s of the rinderpest virus matrix gene --comparative sequence analysis with other paramyxorirm~. vi~logy cautd~an p~ ancestry of a human endogenous retrovirus ~ determination of an epitope of the diffuse systemic sclerosis marker antisen dna topoisomerase-l: sequence $ mllagity with retroviral ~ protein suggests a possible cause for autoimmunity in systemic sclerosis. pro natlacad s i u& , : ~ . mcgeoch dj: pgotein sequence cota~lxs show that the 'psuedoprotesses' encoded by poxviruses and certain retrovirus~ belong to the deoxyoridine triphtmphate family ~sk life: ~es of comme//na yellow mottle vlrus's complete dna sequence, genomlc discontinuities and transcript su est that it is a pararetrovlnm i~l~titis c vllrll~ sborl~ amino acid sequence similarity with pe~tivirutu~ and flavivirus~ as well as members of two plant vlgus superggoupo mo~mann ti~ homology of cy~kine synthesis inhibitory factor (el- ) to the epstein-barr virus gene bcrfi nucleotlde sequence analysis of sa-omvv, a vlsna-related ovine lentivirus --phylo~-netic history of lentivirmms single copy seqoences in g~qgo dna retmmable a repetitive hnman aetrotrmmposon-llke family. y mo/e~/ , : re¢otnblnation resulting in unusual features in the polyomavlrus genome isolated from a murine tumor cell line sequence anal~is of rice dwarf elxytoreovirus genome sewments s , s , and s -comparison with the equivalent wound tumor virus segments ho~tu~ ~ s is a ehylngcueticellly distinct human endogenous reteovtgal rlement with structural mad sequence homology to simian sarcoma virus (ssv). vi~ologie identification of a novel -kl)a cell surface receptor common to pm~cee~flc polypeptide molybdenum hydroxylas~ ~ the amino acid seqoence of chicken hepatic solfite oxidase frequency of a]mloglnai h |lm~tn haemoglobins ~ by c ~ t trmmitions in cpg dlnucleotid~ evidence for conservation of ferritin sequences amon plants and animctbt and for a transit peptlde in soybean a -kda llpo~ortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation distinct fercedoxins from rhodobacter-capsulstus -complete amino acid sequences and molecular evolution n~ptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membgane cloning and characterization of a novel member of the cytochrome-p subfamily iva in rat prostate a directiy repeated sequence in the ~-globin promoter resulates transcription in murine efythroleukemla cells isolation and chamcterizatinn of the alkane-inducibie nadph-cytochrome-p- olf, idoreductsse gene from candida-tropicalls -identification of invarlant residues wlthin slmilmr amino acid sequences of direr'sent flavoproteins protein klnase-c inhibitor proteins -purification from sheep brain and sequence similarity to lipocortins and - - mci~ aveml~ b& sequence homology between purple acid phosphatases and phusphoprotein pho*phatsses --are phesphoprotcin phosphatatms metalloproteins collt~|nln~ oil~-bridged dinuclcar metal centers negative regulation of the human ~-globin ca~ne by transcriptional interference: role of an mu repetitive ~lement amino acid sequence of chicken catisequestrin deduced from c dna -comp~rison of caisequestrin and aspartactin caisequestrin, an intesccilular calciumbinding protein of skeletal muscle sarcoplssmic reticulm, is homolokous to ~, a putstive latminin-binding protein of the exteac¢llular matr~ bovsm~ ]prote~ c inhihl.gog with structugll and fun~ hotdoio~ou~ ]~-.gtl~ to hum~zn plum~ protein c inhibitor sequence of silkworm hemolymph antitrypsin deduced from its cdna nucleoude sequence --~on of its homology with ~.rplus. l b~cbem (tokyo) human mm~t cell tryptm~e multiple cdnas and genes reveal • multigene serlne protemje lmmlly howam> jc: msc ore. n k#on encoding protehm iteleted to the multidtog ite~letance family of tra~membt'mne tratmpofters m~, a tks~me-speclfi¢ b•tmment membrane protein, is a ia.minin.like protein commrvation of a cytoplasmic ~xy-termitml domain of couexin , a gap junctional protein, in mammal heart and brain the a~lba//a~ plasma membrane h+-a~ multigene ~ -genomle sequence and expression of • rd lsoform, f b/ /owra op#n of calliphora peripheral l~otoreceptors r - --homology with d~ rhl and po~tmnsi~domd processing evolution of rhodopsin supergene family --independent divergence of visual pibments in vertebrates and insects and po~ibly in mollusks ct~tpl¢ the g~ne~ amino acid ~'~m~me gene of sac~baromy~wcet~-v/~ae --nucleotide seque~tce, protein similarity with the other i~kers yeast amino acid petmme~mes, and nitrogen cataboht~ repreulon the -kda peroxlsomal membrane protein is a member of the mdr (p-glycoprotein)-related atp-bindin~g protein superfamlly a new clam of lym~o-real/vacuolar protein sorelng signais. l b~/chem complete amino acid sequence and homologies of human erythrocyte membrane protein band . . proc natl acad scd us a the primary structure of a halorhodopsin from n pbaraom~--structural, functional and evolutionary impnoations for bacterial rhod~ and haloghodopslns soluble lactose. blndln~ vertelmue lectlns: a ~ family the a regulatory subunit of the mltochondrlal fi-atpa~ complex is a heat shock protein. identification of two highly conserved amino acid sequences amon~ the ~x-subunits and molecular ~ sequence of h ilmlfl ~ l~ieat~ • novel gene family of integral membrane proteoglycans a protein with homology to the c-termimml relationaxip~ between/m~-nylate cycla~ and n•+,k+-ati~se lit pat pancgtmti¢ islets human na+ ,k+-ati~¢ genes ~ beta~ubunit gene family conmina at lcest one gene and one ~ evolution of the mltc cles~l genes of a new world lh'imate from ancestral homologues of human non-clessical genes the cdna sequence of mouse pllp- arid homololgy to hntman cd c~ll s~e antitpm and promot#ycen core unk proteins ~tjott of cdna encodin~ a hnman sperm membr~e protein related to a amyloid protebm ptwlflcstlo~ c~mu'actet~muon, and con with memb~ne carbonic anhydrase from human kidney hypermumbility of cpg dinucleoudes in the propcptide-enced/ng sequence of the human alb~tmi~ gene dystt'ophhl in electric or~n of to, pedo-~ homologous to that in h,ml~ muscle botste~ i~ homolosy of a yeast acun-binding protein to signal trmmductlon proteins slid myosin- the complete sequence of drosophila alpha-spectrin --conservation of structurml domahm between alplm-~ and alpl~t cttnin •~ettaatflon of a lqbrilisr collqwn gene ~ spruces reveais the etdy evolutionary appearance of two collqwn gene fmmilk~ the predicted amino acid sequence of ct-lnternexin is that of a novel neuronal lntegmedla~ ~ent protein otsen bl~ type xil collm~n. a larbe multidomnln molecule with partial homology to type ix cousllem / b/d aera amyioid protein in i~mni~l amyloidmfls (plmnlah type) ks homolollotm to gd$oilmb an ac, tht-i~h,.da~g protein. b/~bera b/q b~ res commun key ji~ ~ of a proline-rich cell wall protein gene ~ of soybesn. a ~ ana/ysis. j b/o/~em chicken liver evolutionary rehttinnships and impflcations for the resulation of phoophohpsse-a from snake venom to human secreted forms identification of a locality in snake venom a-ncurotoxins with a slsnlficant comlm*itinmd similarity to marine smdl ct-conotoxins: implications for evolution and structure activity al~ph[biml~ albmtm|nm ~s members of the albumin, alpha-l~toprotein. vitamin-d-binding protein mul~ flmily ~ni~on of the hnm~n llpoprotein lllmse gene and evolution of the llpase gene family e~'t~ion of cloned human reticulocyte - ipoxygenase and immunological evidence that -hpoxygetmses of different cell types are related identification of a protein alt~ inttaspecific evolution of a gene family coding for urinary proteins conservation between yeast and man of a protein a~ociated with i small nuclear rlbonucleoprotein stl~ctute and partial amino acid sequence of calf thymus dna topobmmaertt~-ii -coml~on with other type-h emmyme~ ol~nudeotide correlations between infector and hem genomes hint at evolutiotmry relationships. nu /e~ scot/~ik p& carotenoid desmurases fi, om ~ ~and nmoowo~craua are stru~ and l~n~'tinnally comerved and eonmin domains homolosons to flavoprotein dimdflde oxldoreductm~ deininger pi stt'uc~uee and vsrisbihty of recently inserted alu family members a novel neutrolphfl chemmtttactant generated duan an ln~ammmtory reaction in the l~mt peritoneal cal~lt~ tt~ t~t~o -l~tl'~t~tloil~ ~ amino acid seque~tce and structural relmtmmhip to interkukin-& b~ffx~m j the multlfimctinna -methylmllcyllc acid syn~ ge~e of ~~ ~ its ge~e structmm ieimive to tl~t of other po~lyketide symhase~. f.urj b/odaem mammalkm ublquitin carrier prmmtmh but not i~:i~k, ame ltdated to the -kda yeast , rad . bk~chem b/qohys res commun chambers gk: sequence. structure and evolution of the c.ene codin b for ~t-gi~erol- -phe~plmte ~rdrotfm~ in om,qt~ the cotaplete sequence of bogu/ktmm nenrotoxin type-#, and com~ with other clostrldhtl neugoto~hm if: a pamlly of cxam~fltutive c/bbp-llkc dna blndln~ proteins attenuate the il-l~t induced, ni~b mediated trans-activation of the ansiotemflnogen gene acute-phase response element different fort~ of ultmhithomx proteim generated by alternative spttcim~ are functionally equivalent evolution of collagen-iv genes from a -batm pair faton --a role for lntrmm ht gem~ evolution evolution of the insulin superfamlly tcetins are structoraily related sertoli cell proteim who~ ~on is tightly coupled to the iprtsence of germ cells ivarie r~ a bovine homolo s to the human myolletti c determination factor myf~ sequence conservation and ' proce~ing of transcripts proteiu sertne threonine phoephatmes -an expanding family coppes zl divergence of duplicate genes in three sciaenid species (perciformes) from the south co~t of uruguay coasfaneda m: rrs~j~o~a (mu-~--a~) repetitive dna seqmmce l~vointion in ~hically mstinct isolates. cor~ bnz~n physiol repetitive seq~ce involvement in the duplication and divergence of mouse lysozyme genes the structure of a subtermlnal nut/e/ a /ds res schoofs i~ h~ between amino acid sequenc~ of ~ v~'lt~tm'stte peptide hormones and peptides ~mlated fi-on~ invertebrate sources. corn# bm&.n mg~ol bun'nng s, ~us r& lqatelet gtycoprotetn nb-ma protein antssonim from snake venoms ---evidence for s fumlly of p~telet-~sgqpttlon lnhll~tol~ hikher plant orilgins and the whylogeny of gt~en allpte simihtrity between the t~ ~ sindln s proteins abf how big is the univet~ of e~otm worklwide diffegences in the ~ncideace of type ! diabetes are ammciated with amino acid variation at pos/tion of the hi~-dq ~ chain yeast general trtnscelptimt l~ctor gf! --sequence requirements for binding to dna mad evointhmky commrvttion. nudeg m/ds res concerted ]rv~ution of primate mplm smelllte dna. e'~kmce foe tm an~mt~ sequence sbm'ed by goal~ md human x ~e alpha ~ttdllte the nuchl~m~ sequence of etve ribommaal protein genea from the o/anene. of ~~ impacattom concem~ the mtytosene~ relationship bet~-en cyanelles and chloropluts wmslanoer l~ a new member of a secretory protein gene family in the dipteran c~t~onomot~ tentaus ~ a variant repeat stracture the ~r sequence ~ --die.inn on the x-chromosome and y-chromosome of a large set of closely related sequence~, most of wmda are i~eudogene~ ba~ttmo~e l~ cloning of the pso dna binding subutdt of nf-kapi~-b -homolo~" to gel and dortml l-~te two-monooxr~muse from m~ --clon~ nucleotide sequence, and primary structu~ homology within an enzyme family genetic hot~o~n~ty ~ acute and chronic acute forms of spinal muscular atrophy genetic variants of bovine ~-lactogiobulin --a novel wild.type ~-lacto#obulin w and ~ts primary sequence. b/or (~rn h tt e sey/er l~ltogh~ dna evolution in the olmcm species subgroup of drooophll~ f mot evot lovell-badge l~ a gene mapldng to the sex-determining gegion of the mouse y chromommae ~ a member of a novel ~ of zmbryonk~ly genes ~titmte , -dioxy~mm~ from p~.udomotm~ pustfi~mtion, characterization, ~md compm'tson of the f.mtymes from psemffmmm~m ta~o~k-ron/and aaammms~ spec~clties of the peptidyl prolyl cis-tratm isomeric activities of cydophmn and fk- bindh~ protein --evidence for the existence of a family of distinct enzymes. b~x/aem/ary mltochondrl~ dna evolution in primates -tt-atmltion gate has been extremely low in the lemug homeobox containing genes in the nematode ~enorbabd/f~ elk.gamin nucleic ac shdic add fateesses of ~ • voluttomu.y origins have serine active sites f~entlal arginlne residues dewact-rrer l~ the ltilm omal rna ~-quence of the s~t anemone anemom~s ssdcmta and its evolutionary intuition amomqg other eukaryotes inferred b'om s~l,.m.~ comlmrttmas of a heat shock g~ae in two nematorl~ the l~'/o multtgene family of ok~hag of cdna ~ for the ~ omin of human complement component ca~bi~una protein, seqaenoe homolo~ with thc a c~t~:~a~h proc natl acad s¢t usa highly conserved core domain and unique n terminus with presumptive regulatory moti~ in a hmman tata factor (l'lql~) [letter] identification cimractertzaflon of a novel member of the nerve growth fmctor/besln.dertved neurotrophic factor family ~ bind to s~dlfmme [eal(~-so )l~l-lcer ] and has a sequence homology with other pt'otelns that bind sulfated glycoconjut~tes anllllo acid seqmmce of clnnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicetn soluble and mtmo[~tle~ioc~ta~l h~ low-ml~n|ty adenomne binding protein (adenotin) --properties and homology with mtmmall~la and avian stress protelus. b~-/~om/stry edolatlon of complementary dna$ f~lcoding a cerebellum-enriched nuclear factor-i family that activates tt'anscription from the mouse m~.lin basic protein promoter ye~mt mltochondrlal dna polymet'ase is related to the family a dna polymerases nudeotide and deduced amino add sequence of a human cdna (nqo ) corresponding to a second membeg of the nad(p)h --quinone oxldoreductase gene family --extensive polymorphism at the nqo gene locus on chgomo~ome- . b/oc.heraistry ult~ sltnlltt'leles a~llolltll enzyme pterin binding sites as demonstrated by a monoeinnal amiidiotypic antibody blundell tl molecular anatomy: phylogenetic relationship* derived from three~limenslonal structure~ of proteins subfamily structure and evolution of the hnmtn . family of repetitive scquence~. f mot evo selmt~te mltochondrlal dna sequences are contiguous in htlmsa~ genol~ic dna l~t~lit~ within mmmm~lla~ sogl~tol deh~ --the prlmm'y structure of the human liver enzyme heterogeneous modifications of the l /alo ltrote~a of ibtegleuldn-~t cells are concentrated in a/,ti~hly r~qg~.titlv ~ amino-t~ vaults.ell rebofmcleoprotein structures are msl~ conserved among higher and lower e~tes rnas le~d support to the monophyletic nature of the ~erla lmmunoloslcal ~lmllmtties ~etween cytosolic and partictdate tissue trans#utamilsc. febs lat mans~ti x#tope m~w~zed by a protective m~aodonm antibody is identical to the sta~e-specific embryonic antlgen-l. proc naa acad sa o~ the murg gene of t-brucei contains multiple dom.l.m of extensive editinil and is hofaoin~m~ to a subultit of nadh dehy~ neparm-bindl~ nenrotrophtc x~tor (hbnf) and mk, member's of z new i~mily of homolosous~ developmentally l~ted proteitm pugmattion and strucrmml ~on of pttcentel nad + .mtked -hydroxyproma#andm dehydtoffmase ~ the primary structure reveals the enzyme to belon to the short-alcohol l)ehydrogena~ l~mlly. b/ochemistry structores and homologies of carbohydrate ~pho~ system ep~l~[ln, a ~o~a-gmjoclated mudn, is generated by a polymorphlc gene encodin splice variants with alternative amino termini a new member of the leucine zipper class of proteins that binds to the hia drct promoter. sc/ence attalysi~ of cdna for human ~ ajudgyrin i~dicltes a repeated structure with homology to tissue-differentiation a~td cell-cycle control protein the b subunlt of a rat hetefomeric ocaat-binding transcription factor shoes a striking sequence identity with the yeast hap transcription factor homology to mouse s-if and sequence similarity to yeast pt~ stgucttu'e and evolution of the small nuclear rna multigene family in primates: gene amplification under nat-¢wal selectinn? ident~catinn of an additional member of the proteln.tyrushle-phosp~ family --l*vidence f~ alternative spliclog in the tyrmine phosphzmme domain a ~le am~o acid difference dis~ishes the human and the rat sequences of statlmaln, a ubiquitous intracehular pho~phoproteln ~ with cell item comp~ison of the seve~le~ gene* of drosop~ffa t~'ff~ end ma ~ muty, an adenine ~ active on g-a mislmirs, has homology to ~t evolution of largesubunit iutna structuge --the ~cation of imvetbe~t d dommin amon mmjor phyiolpmetic groups discrepancy in diveqlenoe of the mltodtondrlal and nuclear genomes of m sensor/and y~ j mot evot ~ adenylate deamll~t~. a mt~flige~e fam~ in p..m~,n, and rats isolmion and structure of ceerol#m, itna,~le hat~ peptmes, from the smm~m, ~ mo~ comp a~a rmm~ i~ vmotocin ge~ of the teleom f.,xott intro~ botany. ~ hot~ ot'l~mization. b~hemioy the adb gene areal share features of sequence structure and nudeast~protected sites. m /cell bto/ the amino-acid sequence of multip/e lectins of the #.corn barnacle m~us-lgo~ and its homology with .animal ]~'tllls. bioclx'm btqobys acta amino add ~.-quence of mtmkey erythrocyte glycophorba mk. its amino acid ~'qu~'~icc ]f][~ a stri~tl~ homology with that of human glycophorin a flsp~r p& drtmophila proliferating cell nuclear antigen. structural and functional homology with its mammalian coonterpart phylogeny of n|trogen*me s~queac~ in ][~mnkla and other nlteogen-fixing ml~m$ vertebrate prot~mlne c~ne evolution. . sequence alignments and gene structure florin l~ a major styl~ matrix polypeptid~ (sp ) is a member of the f~thogenesia-reiated proteins superciass complete amino acid sequence of rat kidney ornithine aminoteat~fet-~e --identity with ijver omithine aminotransferme. l bnxl;em (tokyo) rlbonuclease p --function and variation. j b/o/~bem the primary strum of glycoprotein-m from bovine adrenal medullary granules --sequence similarity with bnmmn serum protein- , and rat sertoli cell giycoprotein- compm'ative ~quence/umlysis of m~mmantan f'a~or ix protaotegs the amino acid sequence of the b nman l~ia polymet'a~-h -kda subunit hrpb is highly cotmerved among eukaryotes phylogenetic conservation of atylsulfatases --cdna cloturing and expre~ion of hnman aryisul~t~e-b. j b/o/cbem c.oll/l~'vlltion and diversity in fatnllies of coated vemcle adaptlns cllaracterizaflon of petel porcine bone sialoproteins, soca'~ted phosphopgotein ! (sppi, osteopontin), bone siaioprotein, and a .kda glycoprotetn ~ demonstration that the -kda glycoprotein is derived from the carboxyl terminus of sppi characterization of matteuccin, the . s storag~ prote~ of the ostcich fern -evolutionary iteiatinnshlp to angiosperm seed storage ~ a new mmber of the glutamine-rlch protein gene family is characterized by the absence of internal lgepe~ts and the androgen control of its expression in the subm*ndlbuiar gland of pad novel insect n~ with homology to peptides of the vea'te~ tachykinin family identircation of a novel platelet-derived neutrophli-chcmaotgctic po~ with structural homology to piatelet-factor- a novel repeated dna sequoncc located in the intergenic regions of ba~tceial chromosomes. nuc eic.,k:ids res the proianlin storage protellx¢ of cere~ seeds ~ structure and evolution functional analysis of the '-terminal part of the balbiani ring gene by hlterspecies sequence comparison dr= mammaban ~yl phosphate symhetase (cp*) --cdna sequence and evolution of the cl m domain of the syrian hamster multifunctional protein cad mammalian dihydroorotase --nudeotide sequence, peptide sequences, and evolution of the imhydroorotsse domain of the multifunctinnal protein cad a receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins the control of flower morphogenesis in a~..ffd~um majusthe protein shows homoinff~ to transcription factors an element of symmetry in ytmst tata-box binding protein transcription factor-lid --consequence of an ancestra/ duplication? c-type natciuretic peptide (cnp): a new member of nateinretic peptide family identified in porcine brain evolution of antioxidant m~: ediol-dependent petoxidm~.s and thiol~ ~umong ptocaryotes towards the evolution of ribozymes alkyl hydroperoxide reductase from sa/mone/ta ~ur/um --sequence and homology to thinredoxin reductase and other fiavoprotein disuliide oxidoreducmses fc: nonuniform evolution of duplicated, developmentally controlled c~azrion genes in a sillumoth the fission yeast cutl + gene regulates spindle pole body duplication and has homolosy to the buddin structural homology b~ween the hnmmn fur gene product mad the sub---like protea~ encoded by ye~t/~x . nuc~ a¢/ds res nudeotide sequences and novel steuctut~ features of hnm=. and cimm~ lighter ~# primary stt~t~ and expression of a nuclear-coded subunit of complex-n n~ to protetm specified by the chtoropiast genome. b/ chera bnfhys r~ commun a novel gene member of the human giycophorin-a and glycophorin-b genc fatuily -molecular cloning and expression the x-chromosome of monotremes shares a highly conserved region with the eutherlan and marsupial x-o~romosomes despite the absence of x-chromosome ittactt~tion c~lract~tion and or~= nl~tion of dna sequences adjacent to the evidence for a new fmily of evolutionarily conserved homeobox genes elellatltlll and albolabrin purified peptides from viper venoms --homologies with the rgds domain of flbrinogen and yon willebrand pactor measurement of $~tiv~-site homology between potato and l~bbit muscle alpha-glum phosphoryiases through use of a iane~r free energy relationship white ~ weiss ~ the neuroflbromatosis typed gene encodes a protein related to gap the dna damage-inducible gcne-dinl of saocbarom q~ewcet~#.s/ae encodes a regulatory subunit of elbonucleotide reductase and is identical to gnr fhlgegprinting of ne~lr-homogeneous dna hgase-i and ligase-h from eh,m~n cells --similarity of their amp-binding domains control of m na st~mlity in • chnoc~qg.~um, by 'inverted ltepeats: effects of stem and loop mutations on degradation ofxtmba mlna/n vt~ nuc/e~ ac alternative messenger rna structures of the ciil-gene of bacteriophage ~. determine the rate of its tt'ansbttion initiation alternative mrna structures of the cm genc of bacta~ophage ~ detc:'mine the rate of its translation initiation. j mo/b~ / a model fog iina editing in klnetopiastid mltochondrla --guide rna molecules transcribed from max/circle dna provide the edited information elements and coding sequences. j mol bio , : - . chang c-y, ~ d-a, mohandas til chung b-c: stt~ctut~e, ~-quence, chromo~maal location, and evolution of the human fercedoxin gene family. dna cell b/o/ , : - key: cord- -urecb o authors: fraenkel, peter; cho, wonyoung l. title: reaching up, down, in, and around: couple and family coping during the corona virus pandemic date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: urecb o the worldwide corona virus (covid‐ ) has had profound effects on all aspects of life: physical health, the ability to travel locally or to more distant destinations, material and financial resources, and psychosocial wellbeing. couples, families, and communities and individual persons in those relationships have struggled to cope with emerging depression, anxiety, and trauma, and the rise of relational conflict. in this article, we suggest that the existential nature of the pandemic’s challenges require more than just the usual psychosocial interventions. we propose a taxonomy of responses to foster coping and resilience – “reaching up, down, in, and around”. “reaching up” includes accessing spiritual, religious, and ethical values. “reaching down” includes ideas and practices that foster a revised relationship with the earth and its resources, and that engage families to participate in activities that aid the earth’s recovery from decades of human‐caused damage. “reaching in” represents a turn towards experiences available in the mind and in shared minds in relationships that provide pleasure, excitement, joy, and peace, given that external sources of these emotions are of limited availability due to quarantine. “reaching around” involves reframing the mandate for “social distancing” as fostering social connection and support while maintaining physical distancing. the challenges for family therapists, whose practices are confined largely to online therapy, and who are struggling with the same fears and constraints as those persons they are attempting to help, are also discussed. the worldwide corona virus pandemic has presented extreme and unfamiliar challenges to couples, families, and communities, and the individuals living within these social groupings. much as the attacks on / shattered americans' and others' sense of safety and forced adoption of a "new normal" (fraenkel, a (fraenkel, , , the pandemic has prompted the entire world to revise this definition of "new normal" once again, this time centered on beliefs about the preservation of health and confidence that illnesses will more often than not be met with successful treatment. challenges include illness, death, and the fears thereof; employment and economic losses the growing evidence of heath care disparities between persons of differing social locations in terms of race, ethnicity, socioeconomic status, profession, and citizenship status; fears of losing housing, access to food and other basic needs; lack of consistent information about the level of risk of infection or guidance about best practices to reduce that risk; the need to quarantine with partners and family members despite conflicts that are usually mitigated somewhat by daily distance through going to work and school, and on the other hand, interminable separations between family members, friends, and community; a sense of foreboding, isolation, and lack of social support; and disruption of life plans and rituals such as birthdays, weddings, religious holidays, recreational activities, and funerals. it has also presented new and unusual challenges to family therapists dedicated to serving these couples, families, and communities, including the need to adapt care efforts to teletherapy; how to address not only psychological and relational problems but also the above-mentioned concrete needs and larger systems forces that create or accentuate psycho-relational difficulties; and how to address therapists' own personal challenges due to the same issues facing clients while maintaining a therapeutic, caretaking role. we suggest that the crisis cannot be met simply with traditional psychotherapeutic interventions, but rather, requires all persons to step back from conflict and draw upon philosophic, this article is protected by copyright. all rights reserved ethical, spiritual, and religious values (up); become better grounded in our relationship to the earth and to core needs for existence as opposed to strivings for material goods and the quest for new experiences (down); turn towards internal means of promoting engaging and pleasurable experiences (in); and use technologies to foster social support and connection with others while following requirements to maintain physical distancing (around). this taxonomy -reaching up, down, in, and around --as categories for particular activities, is of course based on common socially-constructed metaphors of location in "psychological geography", and is therefore somewhat arbitrary from an empirical perspective. for instance, describing spiritual and religious values as "higher" (and therefore, located up or above) is likely based on the trans-religious notion of god and heaven as existing "above" the earth, or perhaps based on a rank ordering from top to bottom, with such values being ranked highest. yet these values and their associated behaviors could be viewed as the basis or foundation of successful human existence, and therefore, situated below all other endeavors (and so, located down). likewise, concern for the state of the earth includes not only its surface, its oceans, and the animals and plants that inhabit it, but the sky above (up), or could be conceptualized as all around us. nevertheless, we believe this taxonomy has a kind of cultural "face validity" and we chose to employ it to organize our reflections on coping and resilience-building practices. although we hope that our taxonomy represents a novel and useful way to think about families meeting contemporary challenges, we build upon important related developments in the field. in terms of the coping approaches that constitute what we mean by "up", over the past two decades, family therapy has begun to address the need to incorporate fully the spiritual dimension of families' lives, both in general (walsh, ) and as a major active ingredient in family resilience (walsh, ) . indeed, walsh ( ) argues that the original systemic framework of "biopsychosocial" now must be recast as "bio-psycho-social-spiritual". some family and couple therapists outside the pastoral counseling profession and the field of counseling conducted explicitly within particular religious this article is protected by copyright. all rights reserved communities (for instance, premarital counseling in the jewish faith, christian counselors) have also turned to moral, spiritual, and religious principles to guide the conduct and goals of therapy (doherty, ; fishbane, ) , or have emphasized the importance of addressing particular religious ideas and practices central to a family's cultural traditions (beach, hurt, fincham, franklin, mcnair, & stanley, ; boyd-franklin & lockwood, ; elliot, ; falicov, ; fincham, ; marks & dollahite, ) . in terms of the attitudes and activities clustered under "down", although the systemicecological perspective in principle has always included the physical ecology and environment, only more recently has the field addressed in depth the link between family health and the health of the planet (laszloffy & twist, ) . the pandemic has raised concerns about the safety of our environment beyond the corona virus, and families are often unsure how to cope with these fears. in terms of the activities we categorize as "in", doherty ( doherty ( , has long described how couples and families are pulled apart by over-engagement in the quest for new experiences, especially extracurricular activities deemed by middle-to-upper-class parents to be essential to their children's development (and to building their cvs for college applications). the pandemic has all but eliminated access to these outside activities, leaving children, teens, and families at loose ends and often unable to generate sources of pleasure and endeavor among themselves to fill the time, other than increasing the already high levels of tv and internet activity (statista, ) . in terms of what we designate as activities that constitute the category of "around", research has long established the importance for families of social support and involvement in community (miller, ) , especially when children or adults suffer from health issues (varda & talmi, ) . given that families either are dealing with an ill member, or are worried about infection from the virus, connection this article is protected by copyright. all rights reserved with other families, neighbors, and community members is essential to coping with the stress of the pandemic. described initially to be "the great equalizer" (mein, ) -with ample coverage of privileged, high-profile white persons like prime minister boris johnson and newscaster chris cuomo --further studies have found that persons of color, especially african-descended and latinx persons, have significantly higher rates of infection and mortality than whites. national data from states plus the district of columbia found death rates for black americans " . as high as the rate for whites and . times as high for asians and latinos" (apm lab staff, ). put another way, the death rates per , people found blacks suffered . deaths, and native american groups . , whereas latinx, asians and whites have suffered lower rates - . , . and . deaths, respectively (apm lab staff, ). as mein ( ) succinctly summarizes, these unsurprising ethnic and racial differences have emerged during all pandemics over the past hundred years, due to a cluster of factors. racial and ethnic minorities are proportionally more likely to be below the poverty line, and to work in service industries, many of which have been deemed "essential services", requiring workers to show up or face dismissal. to maintain employment, they must be at the worksite (unable to work in the shelter of home) and must utilize public transportation, all increasing exposure risk. they are less likely to have health insurance, sick leave (paid or unpaid), and less likely to use sick leave or quit a job to protect themselves from illness due to their economic precariousness. they are also more likely to have preexisting this article is protected by copyright. all rights reserved conditions associated with greater rates of severe illness and death, such as diabetes, hypertension, coronary heart disease, and respiratory ailments. although of course it is low-income persons of color who suffer most from these disparities, it is also painful for persons of greater racial/ethnic and economic privilege who care about injustice to witness these inequities and feel they have little power to rectify them at least in the short run. one white upper-class italian ex-patriot client -whose country of origin has socialized medicine --spent several sessions talking about her sense of horror and rage about the socioeconomic conditions and political trends in the u.s. that have resulted in these disparities, and her sense of having little potential influence aside from donating some funds to organizations attempting to ameliorate current suffering. disparities aside, the virus's novelty means there is no reliable existing treatment or preventative vaccine. for most persons, having grown up in an era of significant medical advances even in treatment of diseases initially considered a likely death sentence (cancer, hiv/aids), this has altered the general assumption that reliable, effective health care is available for any condition. this has resulted in a significant increase in level of fear and sense of existential unpredictability in persons' lives. unemployment is at its highest level since the great depression. as of a may , report by the bureau of labor statistics, unemployment in april was . million people, with a rise in unemployment of . percent (bureau of labor statistics, ). although all industries are affected, losses in leisure and hospitality industries (restaurants, hotels) are particularly high, and these industries employ a disproportionate number of low-wage earners of color. as of may , approximately , , persons had applied for unemployment (department of labor, ). as was noted above, note that this article was written just before the murder of george floyd by a minneapolis police officer and the subsequent weeks of protests, in which persons from a wide range of socioeconomic and racial/ethnic backgrounds participated. many of the socioeconomic issues that resulted in differential illness and death rates have been named in these protests. this article is protected by copyright. all rights reserved financial instability and risk of illness are synergistic -those persons with less financial resources are more likely to stay in a job that puts them at risk than are those who have a buffer of savings, or paid leave. given the huge number of families now without income, there are grave concerns about their housing stability. consider new york city. the nyu furman center (nyu furman center, ), which studies housing, real estate, land use, and neighborhoods in the united states towards informing public policy, found that:  "out of almost . million households in new york city, almost , , (totaling nearly . million people) had at least one household member that worked in a vulnerable occupation, and that person earned about percent of the total income for the household.  in about , households, all earners worked in vulnerable occupations, making these households hyper-susceptible to income loss.  for households that earned less than $ , and had at least one worker in a vulnerable occupation in , the median household monthly income was about $ , and the median rent was about $ , .  hispanic workers in particular disproportionately worked in vulnerable occupations in . the potential negative impact for low-income people of color, who are predominantly renters, could be at a scale equivalent to the effects of the foreclosure crisis." (nyu furman center, ) how unemployed and precariously-housed families would cope with the very real prospect of inability to pay rent, mortgage foreclosures, and possible homelessness is beyond comprehension or feasible this article is protected by copyright. all rights reserved government planning. new york city currently has , families living in shelters, and the shelters are at capacity (coalition for the homeless, ). the effects of living through a pandemic include worsening of existing mental health issues, new onset of anxiety, fear, and depression, disrupted sleep and eating patterns, concern about becoming ill and about worsening of existing conditions. if ill from the virus, persons may experience guilt about not being able to function in one's usual roles, about receiving help from others, fears of not being able to receive proper medical treatment should the condition worsen, fear of death, or anxiety and depression about being alone with the illness (cdc, ; apa, ). many family members who are so far healthy but living in close quarters with an ill family member with little ability to create consistent physical distance have anxiety about seemingly imminent infection. and of course, families of persons who died from the virus are mourning this loss, if the family member dies while hospitalized, families are unable to be with them, and in many cases unable to hold funerals in the fashion prescribed by their religions (frayer, estrin & arraf, ), or even locate space in cemeteries to bury them, or in crematoriums to cremate them (feuer & rashbaum, ) . parents are suffering great levels of stress as a result of needing to care for their children / and aid in their online education and homework while attempting to fulfill job responsibilities. the harris poll conducted for apa between april and may found that the average stress level of adults was . (this compared to . in ); but for parents with children under years old, the average was . (compared to nonparent adults' average of . ), with % of parents rating their stress levels between and on a ten point scale. seventy percent ( %) of parents reported significant stress about meeting basic needs such as housing and food, as compared to % of nonparents; and % of parents rated access to health care as a stressor, compared to % of nonparents (apa, ). this article is protected by copyright. all rights reserved unfortunately, rather than coming together to cope as best as possible with these stressors, many couples are descending into their most destructive patterns. according to reports by the council on foreign relations (bettinger-lopez & bro, ), the who regional office for europe (kluge, ) , reuters (sigal, ramos miranda, martinez, & machicao, ) , and the un (un women. (n.d.) rates of domestic violence have skyrocketed globally, many due to "situational couple violence," in which conflicts that usually do not involve physical violence have escalated due to added stress and unrelenting proximity (university of nevada las vegas, ). couples on the brink of divorce --"last chance couples", in which partners may already have separated (fraenkel, ) --may be particularly at risk for increased conflict, because their commitment, cooperation, and collaboration levels are low, their communication skills are limited, and they are now quarantining together. once again, couples with more limited financial and housing resources, higher rates of unemployment, and at higher risk of contracting the corona virus, are at higher risk of conflict due to these enhanced stressors. in part as a response to the flood of information and specific advice from psychotherapists on the internet, which can feel overwhelming, generic, and unintegrated to disoriented families, we offer the following simple four-part framework of reaching up, down, in, and around as an organizing, flexible, culturally-adaptable guide for couples and families, as well as for individuals and communities. this pandemic and all its accompanying challenges present an existential challenge to couples and families (and societies more broadly) that exceeds simple psychological and relational techniquespractices such as good communication and problem-solving skills, creating a regular but flexible daily schedule that allows both partners to balance work obligations with parenting and domestic chores, mindfulness practices for soothing oneself and others, statements of mutual appreciation, and creating this article is protected by copyright. all rights reserved moments of pleasurable connection despite anxiety, fear, and mourning. although we strongly endorse these and other couple and family practices for highly distressed couples and families (fraenkel, (fraenkel, , , something more seems needed in the face of an unpredictable, largely uncontrollable, existential, and pervasive crisis like covid- . we suggest that couples and families need to reflect on their higher and broader values -whether derived from spirituality, formal religion, moral philosophy, or other sources of personal/humanitarian ethics. these values are often implicit and unexamined in how persons construct their lives (dreyfus, ) , but certain extreme conditions tend to force a reexamination of these underlying values and principles, so as to provide a psychosocial compass to guide traversing an unfamiliar life terrain. just as other crises have prompted reflection on the nature of being and human life, couples and families, as well as family therapists, need to engage their higher values to reflect on the following sorts of questions:  what are the essential components and features of a reasonably secure, satisfying, meaningful life? how much --money and material goods, novel and exciting experiences, influence over life outcomes --is enough? and what can we do without?  how can we go on living with a sense of hope and purpose in the face of a situation that may bring great reductions in our material wellbeing, and that may result in illness and death?  what is the source and nature of courage, of hope, and of faith in a better future, and how do we sustain those qualities and energy? this article is protected by copyright. all rights reserved inform building a new norm for our lives. reaching up is an integral part of this life reconstruction project, because it often prompts a kind of reflection that seeks a broader meaning, and thus creates space for new possibilities. reaching up can also shift the focus from individual and human-centered experience -what hernandez-wolfe ( ) has termed "anthropocentrism" --embedded in the assumed values of our cultural and societal norms, to adopting a wider framework that de-centers human experience and is more inclusive of ecosystems (discussed further in the section on reaching down). many religious, faith-based, and spiritual communities in western and non-western cultures provide such frameworks. many therapists feel uncomfortable asking couples and families to reflect on their spiritual, religious, and ethical beliefs. this is a time to venture into this important territory, because such beliefs may be crucial to promoting coping and resilience. walsh ( ) writes: spirituality is a powerful dimension of human experience involving transcendent beliefs and practices that foster meaning, well-being, and connectedness. a spiritual worldview orients individuals and families over the life course and across generations. it provides larger values and purpose, guides daily actions and relationships, and strengthens resilience in overcoming adversity. a growing body of research documents the powerful influence of personal faith, prayer, and meditation, and a faith community on physical, emotional, and relational well-being and on recovery from illness and trauma. spiritual distress or a spiritual void in one's life and relationships can contribute to suffering and block positive growth. yet, until recently, spirituality was regarded as "off limits" in clinical training and practice, leaving most therapists and counselors uncertain how to approach it, if at a. (walsh, , p. xi) . before rushing haphazardly to "adopt" the forgotten wisdom of certain religions or indigenous cultures, it is crucial to examine the worldviews (sire, ) at foundation of the family therapy field. this includes naming and re-examining the assumed secularism underpinning the theories and practices of mainstream family therapy, because therapists often interpret client(s) life experiences, relationships, and meaning-making through these non-spiritual lenses. furthermore, when conversations about spirituality, religion, and philosophy that de-centers human experiences are examined through the lens this article is protected by copyright. all rights reserved of secularism, what often results is stripping a rich, nuanced worldview into simply a coping strategy for people to turn to in times of crisis. i (wc) have heard well-meaning non-religious and non-spiritual therapists merely tolerate spirituality in their practice, presumably because it is the "culturallyappropriate, politically-correct" thing to do, and observe that "it is a nice value system that people lean on to cope with difficult circumstances." the assumption underpinning this stance is that spirituality or religious frameworks are created by humans, rather than through a relationship with a deity or spiritual power, as a coping mechanism (thus, a human-centered interpretation). this implies that non-spiritual reality is true. moreover, it invites the interpretation that those who need this imaginary framework to cope are intellectually compromised -an arrogant stance that can range from benign infantilization of religious and spiritual communities to belittling dismissal. we are not suggesting that we and all our clients must all become spiritual and religious. rather, i (wc), as a christian, would suggest that even non-religious and non-spiritual realities are based on a system of beliefs (e.g. poplin, ) . in this regard, traditional religious and spiritual frameworks are not too far out of reach from the secularism that underpins the practices and theories of family therapy. foundational to family systems theory is the premise that individuals' minds, experience, and behavior are intertwined with their relationships to others in their immediate and larger sociocultural contexts. all religious and spiritual beliefs also understand the individual as relational, but include relationships with non-human, spiritual beings such as god, or a universal energy. therefore, in therapeutic work with couples and families, and for families coping with the pandemic on their own, reaching up involves articulating the worldviews embedded in expectations about life and relationships (sire, ) , revisiting and possibly revising their relationships to higher values. this article is protected by copyright. all rights reserved (bynner, ) , he wrote: existence is beyond the power of words to define: terms may be used but are none of them absolute"….. if name be needed, wonder names them both: from wonder into wonder existence opens. most of all, in the spirit of wonderment, it is a time to step back from the daily relational grind and appreciate more deeply one's partner and kids; for kids and teens, to get to know one's parents through asking about their opinions, and stories from their childhood, and for all family members to enjoy each others' enjoyment -of music, ideas, art, books, sports, and other passions. research on the although taoism, or daoism, is often referred to as a religion, it actually has two branches, one philosophic,and the other religious (amore, hussain, & oxtoby, ). philosophic taoism, upon which the present comments are based, has no concept of god or deity, no belief in an afterlife, no prescribed rituals, no sacred text, and no established institution of religious leaders or devotees. taosim developed in the th century bce in part as a counterpoint and criticism of the dominant confucian religion of the time, with its emphasis on social hierarchy and respect for those higher in the hierarchy, as well as moral and emotional discipline that aligned with adhering to that hierarchy, religious taoism, which developed much later ( nd c, bce), developed all the typical beliefs, institutions, and rituals of a religion. this article is protected by copyright. all rights reserved relationship-strengthening effects of such positive psychology practices as inviting someone to lunch to tell them how much they have meant to you (seligman, ) , or gottman's ( ) research demonstrating the long-term positive impact of partners expressing admiration and appreciation for one another, support this activity. couple and family members can look at one another, listen to one another more fully and patiently, and be grateful that the other is not ill, or worse, dead from the corona virus. this can be a time to learn to respect gravity, to slow one's movements -physical, cognitive, and emotional. although the initial weeks of responding to the pandemic resulted in frantic gathering of food and toilet paper, and gathering needed work materials from the office, families and couples have come to recognize that the adverse conditions will not change so soon, and that they must slow the pace. it can be useful to suggest to couples that gravity has two meanings: it refers to the physical force that binds us to the earth, and it also is used to characterize some problems as serious. surely, this pandemic and its ramifications are grave, and it can be best to adopt a slowed-down means of discussing how best to meet new and old challenges. empirically-validated communication practices like the speaker-listener technique from the prevention and relationship enhancement program (prep; this article is protected by copyright. all rights reserved markman, stanley, & blumberg, ) -which couples sometimes balk at due to its initially artificial feel -can be introduced through this spiritual/philosophic perspective on the value of slowness. taoism also promotes a stance of choosing patient inaction or "going with the flow" over vigorous endeavor and attempts to assert control when "hard trying" is likely to result in frustration because of larger forces. laotzu (bynner, ) writes: because when a (person) is in turmoil how shall (they) find peace save by staying patient till the stream clears? how can a person's life keep its course if they will not let it flow? those who flow as life flows know they need no other force: they feel no wear, they feel no tear, they need no mending, no repair. various buddhist texts suggest a similar stance in the face of overwhelming, largely uncontrollable conditions. of course, families should continue to follow suggestions of science-based policies on limiting exposure to the virus. but couples and families need to cultivate greater acceptance of their inability to completely control their lives, to embrace uncertainty (chodron, ) and accept, even if painfully, the numerous cancellations of trips, family events, academic plans, and even employment stability if it is clear nothing can be done to alter the situation. finally, taoism suggests that formalized learning borne in the academy has its limits. he writes: leave off find learning! end the nuisance of saying yes to this and perhaps to that, what slightest use are they! if one man leads, another must follow, how silly that is and how false! this article is protected by copyright. all rights reserved professionals' recommendations for strengthening resilience, drawn from careful social science research and clinical practice, is only one set of knowledges that can be useful like other american products and practices touted to be better (and needed for advancement) than those created in the host countries, our professional knowledge may disrespectfully displace equal or even superior knowledge about coping with adversity, and lead persons in those countries to second-guess their own coping wisdom. indeed, we argue that this is a time to learn from such societies about longstanding skills about surviving and thriving despite adversity, developed by them due to limited material resources, political and economic oppression, and without western healthcare technologies and practices. when therapists introduce ideas or practices (such as mindfulness) drawn from a spiritual or religious tradition to couples and families, it is important to preface one's comments with a statement that one is not attempting to recruit the couple or family into a particular belief system. rather, these ideas and practices are viewed by the therapist as cultural products that the couple or family may find useful, in the same manner that the therapist might allude to music, film, art, poetry, or other cultural products that represent wisdom or that capture some aspect of their experience (fraenkel, ) . clients are asked to reflect on the ideas and practices introduced by the therapists, especially regarding whether these are helpful or not to them. likewise, when inquiring about clients' existing spiritual beliefs or religious faith, the therapist should be clear that she/he/they are not attempting to transform the therapy into a faith-based approach, but rather, is adopting a stance of respectful curiosity similar to how one would explore any other aspect of the clients' cultures of origin, or any aspect of their intersectional social locations (fraenkel, ). this article is protected by copyright. all rights reserved many of the root causes of climate change also increase the risk of pandemics. deforestation, which occurs mostly for agricultural purposes, is the largest cause of habitat loss worldwide. loss of habitat forces animals to migrate and potentially contact other animals or people and share germs. large livestock farms can also serve as a source for spillover of infections from animals to people. less demand for animal meat and more sustainable animal husbandry could decrease emerging infectious disease risk and lower greenhouse gas emissions….we have many reasons to take climate action to improve our health and reducing risks for infectious disease emergence is one of them. even without clear evidence of a link between disruptions in the global ecology and the pandemic, it has alerted the entire world to the uncontrollable forces of nature. one of us (pf) has noticed that since the beginning of the pandemic and quarantining, couples in therapy have, for the first time, described anxieties about the state of the planet and how global warming and other repercussions of environmental degradation may be related to the pandemic -despite the fact that damage to the natural world has been obvious and well covered in the media for several years, and even has resulted in new laws around recycling and forbidding use of single-use plastics (bottles, bags). sadly, this confirms that for many people, concern about humans' effects on the ecology only become salient when they experience the direct effects of negative environmental changes (swim et al., ). marshall ( hypothesizes that a combination of the brain's biologically-based cognitive responses of denial to overwhelming fear, coupled with an implicit social consensus not to discuss such overwhelming fears, has resulted in ignoring the ecological crisis. the proximal nature of the pandemic seems to be bursting through this denial. this article is protected by copyright. all rights reserved thus, in many ways, reaching down is an extension or corollary of reaching up, leading us to widen our perspectives and re-examine our expectations of life. the pandemic has halted the well-oiled machine of modern life in most countries and in our daily activities, centered around the human experience of production and consumption. this pause has put into question what we expect from our future, and consequently increased the general anxiety that comes with facing the unknown. it has also given us room to stop running ahead long enough to really take a good look around us -what is happening to the land; the oceans, lakes, and rivers; the sky; and all the nonhuman beings with whom we cohabit this planet. for many, it has provided time to reflect upon what the decades of industrialization and modernity has severed our lives from our ecosystem, which nevertheless is the direct source of our livelihood, food, and general wellbeing. social and physical distancing policies can be respected while spending time outdoors, close to the earth, and tending to nonhuman beings within and outside of our living spaces. this is easier for me not everyone is privileged to take leisurely walks during the pandemic. as we described above, there are pressing concerns including economic instability, job losses, food insecurities, and escalated relational conflicts that make taking a moment to walk through nature difficult. thus, it is essential that therapists encourage couples and families to explore and expand the possibilities within the constrained this article is protected by copyright. all rights reserved realities of their physical and economical contexts. to expand their sense of possibilities, families can start by drawing upon their surroundings to begin to redefine how they construe relational health and individual wellbeing. the pandemic has alerted us that reaching down to reconnect with our environment is not peripheral to the human experience. it is vital to our very survival on this planet. in addition to tending to the relationship with our surroundings, we suggest that couples and families can respond to their increased awareness of and anxiety about environmental disruption by learning more about their role in the environmental effects of human activities. we also suggest that couples and families turn to nature as a source of recreation and pleasure. our colleague, family psychologist dr. monica sesma, a mexican now living and working in calgary, in alberta, canada, where the weather is cold much of the year, has encouraged families to grow vegetables and other plants in their homes, and has observed the therapeutic effects of family members collaborating in these growing projects, as well as pride in providing themselves inexpensive, healthy food (fraenkel, sesma-vazquez, & freeman, ) . ron finley, living in the inner city of south los angeles, started a neighborhood movement to grow his own food on vacant lots, and during a ted talk observed that "gardening is the most therapeutic and defiant act you can do, especially in the inner city" (finley, ) . systemic therapists must consider helping clients reintegrate all that surrounds them. as hernandez-wolfe ( ) writes, "human survival depends on our effectively relating to everything that surrounds us" (p. ). it is important, especially in this current context of the pandemic that is quickly and permanently changing the modern lifestyle, that we invest in this relationship with our surroundings by reaching down and widen our meaning of what it means to be well. the dangers of the pandemic have driven individuals and families to "shelter at home" and stay confined to limited physical spaces for an extended period of time. for example, the requirement to this article is protected by copyright. all rights reserved shelter at home has disrupted the increasing pattern of families dining out (saksena, okrent, & hamrick, ) . by , the percentage of meals prepared and eaten out of the home rose to . %, up from % in . as noted earlier, children's and teens' extracurricular activities -sports, music, dance, and art lessons, volunteering, religious instruction, and informal time with friends has been all but eliminated. the most recent survey on screen time conducted in august (commonsensemedia, ) indicated teens spent an average of seven hours twenty-two minutes on phones or computers, and tweens, four hours and forty-four minutes -not including time spent for school or homework. with extracurricular activities on hold, parents are now facing childrens' and teens' intense demands for even more screen time (commonsensemedia, ) , which was already a major source of conflict between parents and kids (doherty, ; author, c) . as the themes of coping -reaching up, down, in, and around --are related, some guidance for families on how to generate novel forms of individual and collective entertainment have already been this article is protected by copyright. all rights reserved and their behavior towards one another. the partners noted, with an expression of genuine surprise, that they had never thought about that discrepancy, and went on to discuss in a helpful manner how to bring those values and their treatment of one another into better alignment. likewise, families can use being stuck together in quarantine as a time to engage in learning and reflecting on the global ecological crisis and planning changes in their patterns of consumption (reaching down). other "analog world" activities in the category of reaching in that avoid increasing online/screen time include reading to one another, playing games, singing and playing music (rather than just listening to professionally-produced music; goodchild, ) , writing stories and poems, making art (parents and children doing origami together is often quite pleasurable), building things with legos or blocks, creating skits or whole plays, including about the pandemic; exercising together, and doing mindfulness activities together like mindful breathing, walking, and qi gong, which creates a climate of calmness and compassion, useful in times of stress and frustration (fraenkel, ) . parents can also use this opportunity to get to know their children's musical and other tastes (books, video games) in more depth, as can couple partners. best of all, these activities are free, which is important in this time of economic precarity. gaston bachelard ( bachelard ( / described the way in which physical spaces become imbued with relational and other meanings. one of the assumptions that the pandemic has put into question is what the expected ratio of relatedness and autonomy is in relationships (kagitcibasi, ; kerr & bowen, ) . oftentimes, this expected ratio is assumed, socialized, and normed by the sociocultural context and intergenerational patterns within family units. broadly speaking, at least in the sociocultural context of the united states, the expected ratio is usually tipped in favor of more autonomy than relatedness. this can be seen in the way physical spaces in homes are often conceptualized -each child should have a private room, each partner is given a separate bathroom sink, and large spaces in the home is divided for breakfast, formal dining, entertaining, and family leisure. while not all have access to such physical this article is protected by copyright. all rights reserved spaces, this is what is largely expected and idealized. if these idealized floorplans of homes are symbolic of our expected levels of relatedness and autonomy, autonomy is valued and thus expected within relationships. those families whose economic means do not allow such individualized private spaces, or whose cultural traditions favor more shared spaces (for instance, parents and children sleeping together, siblings sharing a bedroom and study space) may see themselves as inferior to the largely white, middle-to-upper class assumption of physicalized separateness and autonomy; and therapists sharing this bias towards autonomy and privacy among family members may subtly communicate their concern or even disapproval for families members sharing spaces, as well as the ratio of time spent together versus apart. as available physical spaces become limited due to the public health crisis, the expected ratio of relatedness and autonomy needs to be revisited and adapted to the new reality. what are the expectations of the ratio for close relationships within the shared living space? by articulating the assumed ratio of relatedness and autonomy, the individuals within the unit become aware of their bias and the differing levels within their assumptions. this process then creates space for communication, adjustment, and attunement to one another within the relational unit that share living spaces in the new sociocultural context of the post-virus era. additionally, there is a starker distinction between those who are in-group (that is, a unit of relationships within a shared living space) and those who are out of this designated group. what is the expected ratio of relatedness and autonomy with those outside of this designated unit, both physically and emotionally? once expected and assumed norm of the ratio is identified, it is important to collaborate with the couples and families we are working with on what is pragmatic and reasonable in these current times when physical space and autonomy is limited. the work of adjusting to this new norm requires not only the adjustment of expectations, but our underlying definitions of healthy relationships and mental wellness. shifting both the relational norms of the client(s) and the norms of the therapist(s) is required this article is protected by copyright. all rights reserved to adjust to the new realities we are now living in. it requires the negotiation of new expectations, and the following work of growing each individual's capacity for more relatedness and less autonomy within the relational units. again, it is important to note that racial, ethnic, and cultural diversity provides a wealth of local knowledges (canagarajah, ) on what relationships with varying ratios of relatedness and autonomy looks like. we would do well to listen and learn from these communities. and again, before rushing haphazardly into adopting relational knowledge from other cultures that have been historically marginalized and discredited, it is critical to tread carefully with an ever-present suspicious eye on our own bias and attitudes embedded in our quest for knowledge. as couple and family researcher paula pietromonaco notes, "social distancing and staying home are key to reducing the transmission of covid- , but these safeguards disrupt couples' and families' routines" (association for psychological science, , n.p.). social support outside the couple relationship is well known as a protective factor, and is important to relieve stress and protect physical health (pietromonaco & collins, ) . one of the most unfortunate aspects of the government's and scientific community's suggestions for containing the virus' spread was utilizing the term "social distancing" (italics added). instead, we suggest rephrasing the safeguarding strategies as "physical distancing" while promoting social connectedness. although during "normal" times, communication technologies and social media can have deleterious effects on couple intimacy and family connectedness (fraenkel, b (fraenkel, , c turkle, ) , these technologies have become useful tools to fostering family and community social support. video chats and virtual conferences are being used to take the place of face-to-face meetings. although these virtual formats of connection cannot fully take the place of face-to-face connections, they seem to meet some of the needs of social support that physical distancing has created. these virtual formats have been used to replace social gatherings (e.g. this article is protected by copyright. all rights reserved video conferencing gatherings for birthday celebrations, using the video chat feature on smartphones to call extended family members). the same technology has also been utilized to replace professional and educational gatherings as well -both of us have held classes and conducted supervision online since the pandemic started, and i (pf) have seen couples and families online, without difficulty. indeed, online therapy eliminates for couples with young kids the challenge of arranging childcare, and commuting to and from sessions. as we work and socialize virtually from our living spaces, the various roles and identities in our lives are starting to blend and blur. for instance, clients have at least a limited view of our homes, which many therapists have found enhances emotional connection and the therapeutic alliance, and "humanizes" the therapist. as a therapist who integrates music and other arts and literature in couple and family therapy (fraenkel, ) , i (pf) have ready access to my instruments and books with which to make therapeutic points in more powerful ways than with regular "psycho-prose". and the fact that we are "all in this together" has strengthened the bond between therapists and clients. likewise, when we interface with co-workers for a work meeting, we are working from our living rooms, dining tables, or bedrooms. the others quarantined in the same living space may be sitting just off the screen or walking across the screen in the background. during virtual meetings, children may be making noises, vying for attention, or sleeping in the background. dogs are barking in the other room and cats are walking across the desk or demanding lap time, potentially disrupting but sometimes enhancing connection and a sense of humor and perspective. meetings may unpredictably be interrupted by deliveries or knocks on the door by neighbors. if in our pre-covid lives we had been able to limit the exposure of the various facets of our lives to specific groups (e.g. presenting only as a professional to co-workers, clients, and students, and as mother or father only to our children, other extended family members and friends), the intersections of our various roles as professionals, caretakers, life partners, and more are now being exposed to various communities. the sociocultural this article is protected by copyright. all rights reserved and professional norms of social connection and support need to broaden. that is, we must come to see and consider individuals in their intersections of multiple roles in various facet of the human life rather than a single-faceted relationship. we posit that the individuals, couples, and families with whom we work must rise to this challenge and start intentionally seeing one another more transparently, fully, and respectfully as having multiple roles and responsibilities, and as existing in multiple, overlapping social contexts. more than ever before, we need to be more deliberate in sustaining and nurturing our social connections --what john green ( ) describes in his ted talk as "imagining others more complexly" -and as noted earlier, in our connection to the ecosystem. we are suggesting that we reconsider our assumptions and norms around "boundaries" in relationships and widen the capacity of social connection from an individual, one-dimensional construction of others based on the main purpose of our relationship (work, partner, child, family, community) to construe persons as systemic beings -for instance, making room for our co-workers or employees to tend to a fussy baby in the middle of a work meeting, and having patience with them when they run off screen to stop their cats from knocking off objects on shelves. as systemic and relational therapists, we have the concepts to provide leadership in the mental health community, as well as in the communities we serve and in which we live. widening the capacity for social connection ultimately means to de-center the experience of an individual self to consider contexts in all our relationships. with this exponentially complex conceptualization of the other, it becomes impossible to know someone fully, in all of the intersections of their life. thus, rather than seeking to know a person fully, we must adopt the assumption that one cannot be fully known. this assumption that there is something more than meets the eye leaves room for consideration and curiosity. furthermore, it creates space and flexibility in the relationship to decenter the self and "give the benefit of the doubt" to the other, and requires higher values of compassion, grace, patience, humility, and generosity of time and attention. this article is protected by copyright. all rights reserved couple, family, and community approaches to coping and adjusting to the covid- pandemic require more than the usual panoply of techniques offered by family therapists. the pandemic and its sequelae, which will last well beyond the advent of vaccines and diminishment of the virus's spread, requires all of us around the world to critically examine our way of life. we need to revisit how we define and promote positive functioning and wellness at all levels of our bio-psycho-social-spiritual lives, the ecology, as well as in economic and political forces that remain biased in favor of persons in certain intersectional social locations and against others. now more than ever, systemic therapists, scholars, and researchers must provide leadership not just within the four walls of the office (or at present, on video screens) the virtual (literally and figuratively) explosion of advice from therapists and other professionals that has accompanied the coronavirus is well intentioned, but can leave distressed couples and families overwhelmed with information and suggested "best practices". we offer our simplified taxonomy -up, down, in, and around -as an easily memorable and culturally-flexible set of resilience principles and guideposts for our journey through the "psycho-relational geography" of coping. one of us (pf) was fortunate to study with one of the founders of the field, salvador minuchin, from whom he learned, among many other craft principles of therapy, the importance of "building intensity". for minuchin, "building intensity" meant the use of novel, memorable, engaging, short phrases and visual imagery (as well as novel relational enactments in session) to transmit a therapeutic message more powerfully than often does scientifically-accurate but plain, unimaginative, explanatory therapeutic prose (minuchin, reiter & borda, ) . we believe that powerful interventions must "capture the imagination" of couples and families (fraenkel, ) , and metaphorically speaking, "ring in their heads and conversations" like a clear bell. we suggest that the phrase up, down, in, and around represents just such a novel, salient therapeutic message. this article is protected by copyright. all rights reserved reaching up, down, in and around are interconnected categories of family reflection and activity which inform and borrow from one another. as an overarching "four-post" guide to foster coping and resilience, it represents various vantage points on the same call to widen our perspective on what phenomenological-existential philosopher martin heidegger called "dasien" or being-in-the-world (dreyfus, ) , what laotzu called the tao, or way of life (bynner, ) , and from a christian perspective is about rediscovering and adhering to god's original design for life (lewis, (lewis, / as systemic therapists, the assumed norms of relationships need to include considerations of bio-psycho-social-spiritual aspects of the human experience. by reaching up, down, in and around, we can sift through the assumptions and values that determine how we understand and study relational wellness, and begin to build a new normal better fitted to the drastic transformations to the world caused or exacerbated by the virus and its many sequelae. the color of coronavirus: covid- deaths by race and ethnicity in the pandemic effects on marriage and relationships the poetics of space the sound of a wild snail eating enhancing marital enrichment through spirituality: efficacy data for prayer focused relationship enhancement a double pandemic: domestic violence in the age of covid- . council on foreign relations spirituality and religion: implications for psychotherapy with african american families the way of life according to laotzu reconstructing local knowledge 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reality secular?: testing the assumptions of four global worldviews america's eating habits: food away from home authentic happiness: using the new positive psychology to realize your potential for lasting fulfillment accepted article this article is protected by copyright. all rights reserved another pandemic': in latin america, domestic abuse rises amid lockdown naming the elephant: worldview as a concept psychology & global climate change: addressing a multifaceted phenomenon and set of challenges. a report of the american psychological association task force on the interface between psychology & global climate change most popular leisure activities among adults in the united states as of coronavirus and climate change reclaiming conversation: the power of talk in the digital age covid- and ending violence against women and girls /issue-brief-covid- -and-ending-violence-against-women-and-girls-en love under lockdown: how couples can cope during covid- (interview with katherine hertlein, phd) the wiley handbook of existential therapy social connectedness in family social support networks: strengthening systems of care for children with special health care needs. egems the journal of electronic health data and methods (generating evidence & methods to improve patient outcomes spiritual resources in family therapy strengthening family resilience ( rd ed.) the earth shall weep: a history of native america this article is protected by copyright. all rights reserved key: cord- -yt uyqb authors: haynes, george; marshall, maria; lee, yoon; zuiker, virginia; jasper, cynthia r.; sydnor, sandra; valdivia, corinne; masuo, diane; niehm, linda; wiatt, renee title: family business research: reviewing the past, contemplating the future date: - - journal: j fam econ issues doi: . /s - - - sha: doc_id: cord_uid: yt uyqb this paper reviews articles published in the journal of family and economic issues (jfei) from to and considers future research opportunities. the jfei articles utilize theories and conceptual frameworks from several social science fields, to help readers understand the importance of including both the family and business in any discussion of family businesses. the literature review addresses four family business topical areas: ( ) household and business economics, ( ) business continuity and succession, ( ) managerial and adoption strategies, and ( ) values and goals. these jfei articles have focused on differentiating family businesses from other types of businesses; explaining the unique interface between the family and the business; and identifying the characteristics (i.e., demographics, adjustment strategies, continuity, capitals, and values and goals) for family business survival and success. in the future, family business researchers have the opportunity to better understand the impact of community resources and climate; more carefully assess the challenges of women, minorities, and immigrants; understand the role of cultural capital; and explore the impact of innovation during rapidly changing times, such as those created by the pandemic. this literature review addresses two important questions about family business research: ( ) what did we learn from the journal of family and economic issues (jfei) articles published over the past decade; and ( ) where do we go from here. the jfei articles have helped readers better understand family businesses by differentiating them from other types of businesses; explaining the unique interface between the family and the business; and identifying the characteristics (i.e., demographics, adjustment strategies, continuity, capitals, and values and goals) for family business survival and success. this jfei family business literature review was guided by an article by yilmazer and schrank ( ) . the reviews of published jfei family business articles were driven by the notion that an understanding of the family and the business is critical in the understanding of the sustainability of family businesses. looking forward, family business researchers have the opportunity to better understand the impact of community resources and climate; more carefully assess the challenges of women, minorities, and immigrants; understand the role of cultural capital; and explore the impact of innovation during rapidly changing times, such as those created by the pandemic. the guest editor assigned articles on family business published between and for this review. the scholars generating this research were from universities, government agencies and businesses throughout the us. ten of the twenty papers were authored by members of the north central region's agricultural experiment station project on family business. those research faculty members were from land grant institutions throughout the central and western us. nearly all of the authors were applied economists in agricultural economics, family or consumer economics, family business, or family resource management. the published jfei articles helped readers identify the attributes of the family and the business that are important for family business survival and success. while a substantial body of the family business literature has been generated by business faculty, this literature goes beyond the traditional business approach by integrating researchers from social sciences, including: family and social scientists, sociologists, psychologists, family economists, and others into the discussion. the strength of the jfei literature is the innovative ideas that have come from the research of scholars from several disciplines. for additional information on authors, disciplines, and affiliations see appendix table . the jfei family business articles made substantial contributions to research on family businesses by utilizing high-quality secondary data, innovative primary data, and established theoretical and conceptual frameworks. the family business articles published in jfei used eight high quality secondary datasets. they were: ) american family business survey, ) national family business survey, ) spatial hazards and losses data for the us, ) survey of consumer finances, ) national minority business owners survey, ) intergenerational family business survey, and ) survey of household finances in spain, and ) arthur anderson center for family business survey. in addition, primary data on family businesses, including farms, were used in studies in the united states, united kingdom, norway, india, and australia. nationally representative samples were available to researchers using the american family business survey, national family business survey, spatial hazards and losses data for the us, survey of consumer finances, national minority business owners survey and survey of household finances in spain. of the articles reviewed in this paper, were based on quantitative research followed by three on qualitative research, and two were literature reviews. fourteen established theoretical and conceptual models used by the authors, include the following: ) sustainable family business theory (stafford et al. ) , ) agricultural household model (singh et al. ) , ) general systems theory (von bertalanffy ), ) technology acceptance model (davis et al. ) , ) diffusion of innovations framework (rogers ) , ) strategic management framework (sharma et al. ) , ) social exchange theory (emerson ) , ) financial satisfaction framework (joo and grable ) , ) farming satisfaction model (coughenour and swanson ) , ) role theory (biddle ) , ) fundamental interpersonal relations orientations (schutz ) , ) dynamic capabilities approach (teece et al. ) , ) unified systems model (habbershon et al. ) , and ) family resource management (deacon and firebaugh ) . these papers have addressed several important concepts, including disruptions, survival and success, and adoption strategies. for instance, the concept of disruption covers changes in farm programs to substantial weather events, such as hurricane katrina; success covers objective measures, such as changes in profits to subjective measures, such as asking the respondent if they believed they were successful. adoption strategies consider responses to changes by family business owners, such as hiring additional help, an adjustment strategy, to adopting new technologies. while selected articles refer to households and others refer to families, these terms are synonymous in these studies. they refer to businesses owned or managed by family members related by birth, marriage, or adoption. for additional information on methods, theories, concepts, and frameworks see appendix table . the jfei family business articles summarize issues representing four topical areas: ( ) household and business economics, ( ) business continuity and succession, ( ) managerial and adoption strategies, and ( ) values and goals. the household and business economics topic covers cash flow problems, profit growth, perceived business success, savings behavior, survival duration, and ownership profitability, satisfaction, and perceived well-being. business continuity and succession topics include continuation commitment, daughter succession, and entrepreneurial skill succession. topics in the managerial and adoption strategy category include adjustment strategies, capital usage, and innovative technology adoption. the values and goals section addresses value orientation, goal orientation and performance, dynamic capabilities, and considerate exchange. the household and business economics literature recognized the importance of conducting holistic analyses when considering family business. a literature review by yilmazer and schrank ( ) provided an important foundation for examining resource flows between the family and business. this article carefully distinguished between bootstrapping from the small business finance literature and intermingling from the household and family firm literature in small and family-owned businesses. the authors concluded that both bodies of literature, taken individually, were not sufficiently comprehensive to fully capture the financial flows between the household and business and the factors that affect their utilization. the authors suggested that these two groups of research studies intersect and their merger would enable business and household finances to be more holistically understood for family businesses. the foundation provided by yilmazer and schrank ( ) was important in the development of a research study by mcdonald and marshall ( ) , which used sustainable family business theory (sfbt) and intergeneration family business survey data. mcdonald and marshall examined family business resource transfers and tensions to determine how the household and the business respond to cash flow problems. the authors found that cash flow problems increased resource tension in both the household and the business. when faced with family cash flow problems, the business did not transfer more funds to the household either because the business owner was unable or unwilling to transfer more funds to the household. when faced with business cash flow problems, family businesses responded by allocating less cash to the household. these results suggest that family businesses held on to financial resources to help the business, rather than the household. positive business or household cash flow depends on the generation of business profits or other financial resources. a study by lee et al. ( ) , using the sfbt and national family business survey, examined gender differences in profit growth. most importantly, this study found that female managers had higher levels of perceived business success and more profit growth than their male counterparts. for female managers, health status and business size were positively related to profit growth, while business liabilities and home-based location were negatively related to profit growth. even though female managers had higher profit growth, they had lower levels of human and financial capital than their male manager counterparts. family businesses depend on profit (and profit growth) and household savings to increase their net worth. the purpose of the study by remble et al. ( ) was to examine household saving behavior and the influence on a family-owned business. this study, using the survey on consumer finances and life-cycle earnings model, focused on the savings behavior of business-owning and nonbusiness-owning households. the study showed that business-owning households were more likely to save than nonbusiness-owning households. the results of this study indicate that household saving behavior was influenced by both characteristics of the respondent and the household. when considering different types of businesses, the authors found that farm and nonfarm business owning households were more likely to save than other households. when considering only businessowning households, the study revealed that farm households had significantly different and more positive saving behavior than nonfarm households. in addition, they found that having more human capital (e.g., formal education) or financial capital (e.g., possessing an individual retirement account, having an employer pension, or having health insurance) increased the likelihood of household savings among familyowned businesses. profit growth and savings are critically important for the long-term survival of family businesses. stafford et al. ( ) examined the factors affecting long-term survival of family businesses in the united states. they used the sfbt to guide the long-term survival model and employed the national family business survey and spatial hazard events and losses for the us databases. this study introduced important rigor into the survival analysis of family business by using a longitudinal sample of family businesses and controlling for community environment, family and business capital, management processes and disruptions external to the firm, such as a natural disaster. they found that family businesses that provided more income from their families, hired more temporary help, owned larger businesses, and had more experience had increased duration of survival. home-based businesses, female owners, and those using customer-centric management strategies had decreased duration of survival. most importantly, they found that businesses in more economically vulnerable rural areas and businesses owners who considered their business a "way of life" had increased duration of survival. both of these results planted seeds for future research as family businesses in economically vulnerable rural areas and those owners considering their business as a "way of life" were posited to have decreased, rather than increased duration of survival. this study provided further evidence that a family business does not make economic decisions in social isolation. stafford et al. ( ) emphasized that many factors other than objective measures, such as money, are important to survival of family businesses. subsequent research introduced other factors, such as relationships among owners (copreneurs), perceived financial solvency, and perceived resource well-being that were important for survival and success, especially for female owners and managers. the interface between the family and business becomes increasingly important when two or more family members are active in the business. mcdonald et al. ( ) , using the sfbt and intergenerational family business survey, examined small and medium-sized rural copreneurial businesses and the relationship between their business structure, relationship satisfaction, and profitability. in this case, the important resource is labor because two business owners work in the family business. this research showed that couples who self-select into working together as copreneurial business owners do so based on how satisfied they are with their relationship. the findings suggest that to have higher profitability, the copreneurial couple should place importance on maintaining relationship satisfaction within the business and in the family. while copreneurs are often men and women owners, gender of the owner is an important consideration in family business because women often have dual responsibilities for the business and household. the purpose of the study by archuleta et al. ( ) , using role theory and primary data from kansas farmers, was to explore factors that impacted farm women's perceptions of farm business financial satisfaction. the most important contributions were the following: being financially solvent, a subjective measure, was positively associated with farm business financial satisfaction, and occupying a decision-making role was negatively associated with farm business financial satisfaction. neither household income nor percentage of agriculture income, both objective measures, were significantly associated with farm business financial satisfaction. while an economic model would suggest that more money is better than less, archuleta et al. ( ) found that household income or percentage of farm income were not associated with financial satisfaction. in the same vein, another family business research study using the family resource management model and primary data from daycare providers, found that research on family child care providers had similar findings, namely that household income predicts little about how providers perceived their overall resource well-being (mimura et al. ) . they found that perceived resource well-being was related to more subjective measures, such as past and present economic situations, demands on time, contributions providers' family members make to support the business functions, and quality and availability of community resources, rather than more objective measures, such as household income. successful family businesses often choose to pass the family business onto the next generation. for some potential heirs, especially daughters, there are many challenges. the literature published by the jfei is largely literature reviews or data driven, rather than based on any theory or conceptual framework; however, these articles provided important insight into the succession of family businesses. mahto et al. ( ) , using the arthur anderson center for family business survey (massachusetts mutual insurance) survey of top executives, examined the factors that influence continuation commitment, or the family's commitment to continue the family business. they found that the top executive's age and generation (founder or successor), number of family owners, number of family meetings, and social identity were positively associated with the continuation commitment, while more education was negatively associated with the continuation commitment. in addition, the top executive's future performance expectation was positively associated with the continuation commitment. unless there is a continuation commitment from family it is unlikely that the business will continue in the same family. merchant et al. ( ) , examined business continuity using a sample of very small businesses in india. the aim of this study was to identify and validate factors that affected family business succession for small and medium-sized enterprises in india. the authors found that the two major drivers having the greatest positive impact on successful continuation for these indian family businesses were willingness and excitement of the potential successor to join the business and ability of the founder and successor to manage tensions. while many family business owners are concerned about succession of the business, small business owners may be specifically concerned about their children following in their footsteps. wang ( ) used a content analysis of previous literature to examine daughter succession in family businesses. this paper was driven by the notion that daughters are often forgotten or overlooked in family business succession. this paper presented various aspects of how gender inequality has been explained in the family business succession literature, and highlighted four areas of daughter succession in family business: exclusion of daughters, barriers to daughters' succession in family business, daughters' pathways to leadership and control, and the implications of the daughter option. the authors found that overlooking the potential of daughters as successors to a business would leave sub-optimal choices for successions, and also exclude daughters from involvement in the family business. family involvement in a business may encourage the involvement of a daughter or son. ferrando-latorre et al. ( ) examined relationships within spanish families to determine if children of entrepreneurs were more likely to become entrepreneurs. this study, using the survey of household finances in spain, found that children of entrepreneurial or self-employed parents were more likely to become entrepreneurs or self-employed than children of wage and salary employees. they found that transmitting entrepreneurial skills and values between generations was an important process in the transmission of entrepreneurship. family business survival and success depends on the ability of the family and business entities to respond to major shocks (for instance, natural disasters, change in farm programs, and other events beyond their control). adjustment strategies included financial choices, such as choosing risky or less risky investments, employment of family or business labor, and or using time resources (labor) or financial capital (money). in this set of articles, family structure was an important determinant of the adjustment strategies chosen. adoption strategies of the family and the business impacted by the tobacco buyout in and were studied by pushkarskaya and marshall ( ) utilizing two theoretical frameworks, i.e., agricultural household model (ahm) and sfbt, and primary data from kentucky tobacco producers. the results showed that family structure (single females, single males, older farm couples, younger farm couples, married older farmers living with younger family members, and married younger farmers with children) did have an impact on expenditure choices and adjustment strategies following a shock to the family business. for instance, younger, married farmers were more averse to starting a new venture compared to other household structures; couples with young children were less likely to choose the stock market for investment than an older couple living with their younger generation family; and the stock market was an investment choice for families who had experienced a family death. while changes in financial choices are important adoption strategies, adjusting behaviors in either the business or family system are important to accommodate the needs of the other systems when unusually heavy demands exist. the use of adjustment behaviors during high-demand times creates a resilience capacity that tends to automatically take effect when encountering a disruption. five adjustment types occur at the family-business intersection: ( ) reallocating personal time, ( ) obtaining additional help, ( ) adjusting family resources, ( ) adjusting business resources, and ( ) intertwining tasks (fitzgerald, winter, miller, and paul ) . lee et al. ( ) used the and national minority business owners surveys (nmbos) and sfbt to compare male and female minority business owners in their overall use of adjustment strategies. they found that minority female business owners were more likely to reallocate family resources to help with business tasks and were more likely to intertwine both family and business tasks than minority male business owners when demands were particularly great for the family or the firm. unlike in majority cultures, where male and female business owners may not differ in their use of adjustment strategies, in minority-owned small family firms, gender differences existed in adjustment strategy use. in particular, minority female business owners in this study were more likely to solicit unpaid help for their businesses and adjust family resources to meet business demands, than were minority male business owners. other adjustment strategies require changes in the use of other resources apart from time, labor, or money. glover ( ) examined how family farm businesses use different forms of capital (economic, social, cultural, and symbolic) to respond to critical changes in the family and business using bourdieu's theory of capital. in this study, based on case studies of farmers in the united kingdom, glover found that levels of economic capital were adversely affected when there is lack of contingency planning. however, both social and symbolic capital were important in assisting family members to cope and continue the family business. this study provided qualitative evidence that in adverse situations, business and family support one another to function smoothly and sustain the operation. innovative adjustment strategies include the adoption of new technologies. niehm et al. ( ) provided new insights regarding the benefits of integrated information technology (it) use for small family firms. this study was based on davis et al.'s ( ) technology acceptance model (tam) and rogers' ( ) diffusion of innovations framework, and utilized data from the national family business survey. this study found that prior it knowledge and the degree to which it was used in the business had the strongest impact on technology adoption, followed by community size, access, and affordability of related technologies. the findings suggested that family firms were able to successfully implement and gain advantages from it only after the business managers perceived its usefulness and developed technology capabilities. while survival and success are important to family businesses, the underlying value orientation and goals and family business dynamics are important in enabling family businesses to survive and succeed. an important study by distelberg and blow ( ) used the american family survey and a dual general systems theory and eco-systems perspective to explore the congruency of goals and values and how they influence perceptions of success; how the family business's resources and goals are appraised differently based on the family business's value orientation; and how unity in values supersedes value orientation regarding the functioning of the family business. they found that value orientations were not correlated with sales, satisfaction, or perceptions of success. however, they did find that value orientations have an effect on the value that owners assign to goals and resources. and, they found that individuals in a family business were more satisfied with their business when personal values were consistent with the values set for the business. following the lead of distelberg and blow ( ) , lee and marshall ( ) investigated the relationship between goal orientation and family business performance using the strategic management framework (sharma et al. ) and the and national family business surveys. more specifically, the authors were interested in determining the effect of goals on long-term success in family businesses. they found that two goals had a positive influence on longterm business performance: a positive reputation with customers and business growth. family business growth requires that the business remain competitive. duarte alonso et al. ( ) addressed three areas of need in family businesses, related to what family businesses do to adapt to a rapidly changing business environment, and the specific resources leveraged to remain competitive in this context. the authors studied the dynamic capabilities in the context of the family business, using the dynamic capabilities theoretical framework with primary data from australia. dynamic capabilities support capacities such as sensing new opportunities, seizing these opportunities by mobilizing resources, and transforming or reconfiguring business structures and assets for renewal. this study found that family businesses adapt to a changing business environment by embracing innovation to add value and efficiencies. in addition, to remain competitive, these firms leverage specific resources, such as unique firm attributes, an open culture, signature processes and idiosyncratic knowledge. adapting to a changing business environment assumes that family members are able to agree that changes are needed. successful succession of a family business requires family interactions. gezelius ( ) , using social exchange theory and a sample of norwegian farmers, clarified the concept of considerate exchange and extended the capacity of social exchange theory to explain interactions among close relatives of family-owned businesses. considerate exchange essential states "i care for you, because you care for me." this study found that considerate exchange enables family members to allocate scarce resources while sustaining and strengthening emotional ties that build well-being within the dynamics of the family and the business. in addition, this study suggests how considerate exchange may be useful to explain business dynamics and how exchange plays out, without damaging relationships. the jfei literature has added substantial breadth and depth to the family business literature; so what did we learn? based on suggestions by yilmazer and schrank ( ) , household and business economic research in the jfei explored the importance of cash flow, profitability, and savings in supporting family business well-being, satisfaction, success, and survival. business continuity and succession are important to many successful family businesses. the jfei literature examined the importance of family member commitment, inclusion of daughters (and sons) in the succession plan, and technical skills in successfully transitioning a business to the next generation. a successful business transition often requires the family businesses to quickly adopt new strategies to rapidly changing conditions. the jfei literature explored adjustment strategies employed by women and minority family business owners during hectic times; and, considered the importance of capital use and innovative technologies in managing the firm. successful management strategies are supported by individual, family, and business values and goals. the jfei literature assessed the importance of individual values and goals, dynamic capabilities of the business owner, and considerate exchange among family members in supporting a successful family business. most importantly, the jfei articles, utilizing theories and conceptual frameworks from several social science fields, have helped readers understand the importance of including both the family and business in any discussion of a family business. the jfei articles have examined family businesses through several theoretical lenses, most notably economics, sociology, family science, and psychology. these approaches have provided a broad base for understanding family business by recognizing the importance of considering the impacts on the family and business. while an extensive body of literature has been created over the past decade, significant research opportunities remain for jfei authors. previous studies have profiled family businesses and examined their short-and long-term survival and success. here are seven areas for substantive future research on family businesses: ( ) fitzgerald et al. ( ) , yilmazer and schrank ( ) , and mcdonald and marshall ( ) provide important foundations for examining resource flows between the family and the business. ample room exists to further study the implications of the financial interactions between households and businesses as both systems seek to adjust to these risks. ( ) stafford et al. ( ) found that family businesses in more vulnerable communities had a longer duration of survival. additional research is needed to investigate whether businesses in more vulnerable rural communities have higher attachment and continuation commitment than family businesses in less vulnerable urban communities. ( ) wang ( ) demonstrates how women are often overlooked in family business succession. more research is needed on women taking over family businesses and their overall role in the succession process. with a growing trend of women-owned small firms, spousal partnerships, and women in management roles, future studies could focus on how leadership style, demographics, communication, and business characteristics affect management and ownership succession. ( ) research has shown the benefits of considerate exchange and social exchange theory within family farms and how these interactions can cause better personal business relations (gezelius ) . additional studies of considerate exchange in other settings, such as in the us and other parts of the world and to nonfarm family businesses, would provide insight into incorporating considerate exchange into social exchange theory. ( ) following the lead of niehm et al. ( ) more research is needed to examine the specific forms of motivational, material, and skill access related to information technology to enhance the performance of family businesses. in addition, the impact of information technology resources on economic, social, symbolic, and other capital use warrants further research. ( ) lee et al. ( ) explored the use of adjustment strategies by minority owners. the opportunity exists to utilize the model developed by lee et al. to examine public policy issues, such as the impact of the pandemic on family businesses and access to economic stimulus programs. in addition, further development of theory relating to capitals, ethnic identity and subjective wellbeing of family businesses owned by people of color and recent immigrants in adapting to change is warranted (valdivia and flores ). ( ) pushkaraskaya and marshall ( ), archuletta et al. ( ) , and gezelius ( ) explored issues faced by agricultural producers. given the public policy interest in classifying other businesses, such as agricultural producers, as small business enterprises, additional research is needed to explore the heterogeneity among family-owned small businesses. in conclusion, jfei articles have the opportunity to bring family business research from throughout the world to the academic marketplace. while family business research may employ less familiar theoretical or conceptual constructs, interesting datasets and empirical analyses provided a glimpse into the challenges faced by family businesses. the research and information on family businesses in jfei offers a plethora of opportunities to future researchers and continues to educate the public on important family business matters. throughout our review of jfei family business research, much has been discovered, and the journal will continue to make substantive contributions to the literature by addressing the vexing challenges facing family businesses. funding no funding was provided for this literature review project. conflict of interest no conflict of interest or competing interests were reported by the authors. no primary data was collected using human subjects; hence, no informed consent statement was required for this literature review. this paper is a literature review study. the montana state university institutional review board has confirmed that this study is exempt; hence, no ethical approval is required. planning to exit tobacco farming ( / ), planned to use tobacco payment to expand the business ( / ), planned to start new firm ( / ), at least one adult family member was employed off-farm ( / ), planned to spend their tobacco payments mostly on household expenses ( / ), planned to pay off debts using tobacco checks ( / ), and planned to invest in the stock market ( family business managers' information technology (it) knowledge and use ( - ); perceived it ease of use ( - ); implementation and usefulness of it ( - ); and impact of it ( - ). all variables use likert scales. midwestern women's farm business roles and farm business financial satisfaction: an exploratory study recent development in role theory determinants of farmers' satisfactions with farming and with life: a replication and extension user acceptance of computer technology: a comparison of two theoretical models family resource management: principles and applications the role of values and unity in family businesses family businesses and adaptation: a dynamic capabilities approach social exchange theory intergenerational transmission of entrepreneurial activity in spanish families adjustment strategies in the family business: implications of gender and management role considerate exchange: exploring social exchange on family farms capital usage in adverse situations: applying bourdieu's theory of capital to family farm businesses a unified systems perspective of family firm performance an exploratory framework of the determinants of financial satisfaction goal orientation and performance of family businesses gender differences in perceived business success and profit growth among family business managers adjustment strategy use in minority family businesses: differences across gender continuation commitment: family's commitment to continue the family business family business responses to household and business cash-flow problems is working with your spouse good for business? the effect of working with your spouse on profit for rural businesses factors influencing family business continuity in indian small and medium enterprises (smes) resource wellbeing among family child care business owners technology adoption in small family-owned businesses: accessibility, perceived advantage, and information technology literacy family structure, policy shocks, and family business adjustment choices household saving behavior and the influence of family-owned business diffusion of innovations firo: a three dimensional theory of interpersonal behavior strategic management of the family business: past research and future challenges a survey of agricultural household models: recent findings and policy implications a research model of sustainable family businesses factors associated with long-term survival of family businesses: duration analysis dynamic capabilities and strategic management factors affecting the job satisfaction of latino/a immigrants in the midwest general systems theory: foundations, development, applications daughter exclusion in family business succession: a review of the literature the use of owner resources in small and family owned businesses: literature review and future research directions see tables and . key: cord- -jxkjn ld authors: andruske, cynthia lee; o'connor, deborah title: family care across diverse cultures: re-envisioning using a transnational lens date: - - journal: j aging stud doi: . /j.jaging. . sha: doc_id: cord_uid: jxkjn ld in an increasingly globalized world, the importance of developing a more culturally complex understanding of family care has been clearly identified. this study explored family care across three different cultural groups - chinese, south asian, and latin american - living in a metropolitan, pacific-west, canadian city. in-depth qualitative interviews were conducted with family members from one of the three family groups exploring how they practiced ‘care’ for their aging, often frail, relatives. the importance of conceptualizing family care as a transnational, collective undertaking emerged from the outset as critical for understanding care practices in all three cultural communities. three themes identified contributed to this conceptualization: the need to broaden the understanding of family care; the centrality of geographic mobility, and the need to rethink the location of aging and consider its relationship to mobility; and the use of technology by extended family networks to facilitate continuity and connection. an over-riding notion of ‘flow’ or fluid movement, rather than a fixed, static arrangement, emerged as critical for understanding family care. this perspective challenges the dominant approach to studying family care in gerontology that generally conceptualizes family care practice as one local primary caregiver, often female, with some support from other family members. understanding family care from a transnational lens builds support for the importance of a feminist ethics of care lens and has important implications for policy and service delivery practices. canada, like many other western countries, reflects an increasingly globalized world with almost a quarter of adults living in canada selfidentifying as an immigrant (stats canada, ) . of these, % are over age (stats canada, ) , and if they came after , they likely belong to a culturally diverse community, including % from asia and the pacific region and % from south and central america (citizenship and immigration, canada, ) . a majority of older immigrants have come to the country under canada's reunification policy and are more likely to be living with and/or supported by family for at least years after their arrival (mcdonald, ) . the need to understand the implications of this culturally diverse world on the aging experience (phillipson, ; torres, ) and the provision of family care (bryceson, ; chappell & hollander, ; keating & de jong gierveld, ; kirkland et al., ; roberto & anderson et al., ; guo, kim, & dong, ; pinquart & sorensen, ) and the differential uptake of support, both formal and informal (greenwood, habibi, smith, & manthorpe, ) . however, many questions remain. in particular, two gaps in this body of knowledge limit its usefulness. first, at a general level, the strong focus of the stress/burden model has tended to assume one fixed primary caregiver and resulted in an incomplete understanding of the role of the rest of the family in providing care and support (sims-gould & martin-mathews, ) . second, in considering ethnicity and/or culture in family care, this body of knowledge has rarely focused on finding a meaningful way to examine the significant role that immigration and acculturation may have in implicating family care practices (guo et al., ) . a second body of research, found largely outside of the gerontological literature, has focused more explicitly on the role of transnationalism and migration in family care. this body of work has explored intergenerational care across the life-span including a small but growing body of research examining care of aging parents. 'care' in this body of research has generally been extended to capture a diversity of caring arrangements and activities as proposed by finch ( ) and fisher and tronto ( ) . seminal work by a western australian research group (baldassar, baldock, & wilding, ; see also baldassar & merla, ) has been particularly influential as a foundation for considering transnationalism as it relates to family care in the field of aging. their work has explored how care for an aging parent is accomplished and negotiated between family members living far apart and in different countries. during one of their first ethnographic studies, they interviewed australian immigrant families -about two thirds were of european descent with the remainder from new zealand ( ), singapore ( ), iraq ( ), and afghanistan living in iran ( ). to capture the experiences of both the immigrant adult children and older parents in their home countries, the researchers interviewed the immigrants in australia and the parents and other family members still remaining in the home countries. interviews included family and immigration histories, types of communication used, travel, and reciprocal care for care receivers and care givers based on finch's ( ) typology of family care exchange (financial, emotional, personal, practical, and accommodation) . additionally, the researchers observed and recorded how families organized space, tools, and other artifacts used for communication exchanges . the work of this research group has made important contributions for understanding transnational care. in particular, they highlighted the diversity and complexity of how care arrangements were negotiated and managed, drawing attention to issues such as how obligation may be culturally mediated, the impact of the migration process on establishing care capacity, and the importance of technology in these care arrangements. a small literature base has built on this body of knowledge as it relates to aging family care especially in europe (see for example, kordasiewicz, radziwinowiczówna, & kloc-nowak, ; miette sagbakken, spilker, & ingebretsen, ; zechner, ) . findings continue to demonstrate the complexity of understanding these family processes, and miette sagbakken et al. ( ) concluded by recognizing the importance of considering both the availability of kin members but also their understanding of obligation and reciprocity and how that might reflect their embeddedness in more than one society. despite its promise, however, this body of literature is only rarely addressed in the broader gerontological work on family care and a 'substantial void' in the caregiving literature remains in relation to understanding cultural values and processes (lee, chaudhuri, & yoo, ) , especially in relation to examining the experiences of non-western immigrants (dhar, b; wilding & baldassar, ; zhou, zhou, , . this current study bridges these two, somewhat disparate, bodies of research. specifically, drawing on a broad understanding of care that is consistent with fisher and tronto ( ) ; also tronto, ) , the purpose of our study was to explore and compare the relationships between culture and informal family care practices across three diverse ethno-cultural groups: chinese, south asians, and latin americans living in vancouver, british columbia, canada. although transnationalism was not considered at the outset of the study, all three of these groups have unique immigration trajectories, with the latin americans representing a relatively recent group immigrating for political and economic reasons from south and central america while the south asians and chinese have much longer histories in canada. we explored the following questions: ) what are the cultural interpretations and meanings of caring for an older family member within select ethnic minority communities? ) how do cultural meanings of relationships influence the care process? this qualitative study drew on constructivist grounded theory (charmaz, (charmaz, , b charmaz, a) to explore the process and meanings of family care across non-western cultural groups. the constructivist approach is designed to facilitate development of a framework to better understand the processes individuals create around their experiences and ways of being in the world. in keeping with this, the interview was designed iteratively; the researchers began with a few broad questions in order to develop a deeper and richer exploration and understanding of concepts through reflection and theoretical interviewing. throughout the research process, it is important to acknowledge the reciprocity that exists between the participants and researchers as well as the expertise of the participants (charmaz, (charmaz, , b charmaz, a) . this also helps diffuse power imbalances between participants and researchers. adults self-identifying as chinese, south asian, and latin american, and providing unpaid, informal care or support to someone over the age of needing some level of assistance with activities of daily living, were invited to participate. drawing on the work of finch ( ) and fisher and tronto ( ; tronto, ) , we conceptualized care as a "multifaceted complex social phenomenon, entailing both emotional and material (instrumental) aspects" (kordasiewicz et al., , p. )this encapsulated notions of caring for, caring about, providing care, and caring with (fisher & tronto, ) -and left it up to participants to describe what they meant by providing informal care or support. participant recruitment initially took place through researchers' personal networks, ads, and organizations in each of the chinese, south asian, and latin american communities. as the study progressed, snowball sampling was utilized, and participants were purposefully selected to ensure cultural, gender, and relational diversity. the final number of adults included participants: nine ( ) chinese ( males and females) from china, hong kong, and burma. ten ( ) south asians ( males and females) originated from india, east africa, and england. the ten ( ) latin american participants ( males and females) immigrated from chile, colombia, argentina, el salvador, and venezuela. table provides an overview of the family members consenting to participate. with signed informed consent, up to three personal, semi-structured, audio taped interviews were conducted with each of the individuals in the language of their choice. to capture cultural meanings and connotations and ensure carers' comfort and ease of expression, all but four individuals chose to be interviewed in their native language (cantonese, hindi, punjab, or spanish). the interviews were designed as an iterative process where three experienced researchers (each fluent in the respective group's native language/s) began with broad questions focused on family members' experiences and perceptions of providing informal care or support to an older adult, and then developed a deeper and richer exploration of concepts through reflection and theoretical interviewing. topics included exploring how each defined giving care in terms of their own cultural beliefs and backgrounds, and how -from their perspectives -these influenced the assistance or support they provided. consistent with a constructivist grounded theory approach, interview questions were honed and refined throughout the data generation process to explore emerging concepts and understandings. follow up interviews, ranging between a couple of weeks to a month after the first interview per the accessibility and schedule of participants, were conducted, and for some a third meeting, focused on expanding and verifying insights. at the end of the final interview, a demographic questionnaire was completed orally with each participant to collect information about both the carer and care receiver (for example, age, education, health status, financial resources, time in canada, citizenship, and others). ethical approval was obtained through the ethical review board from the university of british columbia. table below provides a picture of care receivers as described by their relatives. each family was caring for or assisting at least one parent, spouse, or relative ranging in age from to . some care receivers were healthy and independent while others had severe health problems such as alzheimer's, heart problems, or other diseases. in keeping with canada's family reunification program (mcdonald, ) , of the older adults had immigrated to canada to reunite with their families. although only two latin americans still resided in south america, visiting their adult children only when economically possible and based on immigration policies, they planned on immigrating at a later date. furthermore, the care receivers had lived in canada for years or more and in some cases, even for to years ( ). also, most care receivers ( ) either lived with the carer or a family member. data analysis was a collaborative (team), iterative process using a strategy consistent with the constant comparative method. first, all taped interviews were translated verbatim, (to capture cultural meanings), into english by the respective research associate who conducted it. next, a three-stage process began with the research associate affiliated with each of the three groups analyzing several interviews and coding them. then, associates with the principal researcher in vancouver met to discuss the interviews to analyze meanings, initial codes, and compare these among the three cultural groups. using these team decisions and codes, a code book was developed. it was then used to conduct a line-by-line coding of each transcript. atlas-ti facilitated this coding process. once data was coded, the research team began looking for links among codes to develop categories, for creating broader themes as they began to emerge. these were then refined and developed through subsequent interviews with the participants and by returning to the data. data revealed a process of family care that was complex and dynamic across all three cultural groups. specifically, a sense of flow and fluidity created a picture of care as a shared phenomenon -across family members and geography -that was highly dependent upon communication technology. hence, a significant theme that emerged across all three groups was the importance of conceptualizing family care as a transnational, collective undertaking underpinned by three themes: broadening the conception of 'family care'; locating family care as transnational; and increasing reliance on technology. in this study, consistent with existing research dhwan, ; flores, hinton, barker, franz, & velasquez, ; guo, li, liu, & sum, ; hsueh, hu, & clarke-ekong, ; pharr, francis, terry, & clark, ) , family care emerged as a normative cultural expectation in all three groups. however, each labelled and described the concept differently. for example, for the chinese (ch) participants, this revolved around the language of filial piety and/or 'ganqing' based on a relational sense of moral duty and responsibility, resulting from confucianism. these values were generally volunteered spontaneously and explicitly: it is our "culture" to "look after" the elder. it's more emphasized in our education from when we were kids....although my parents never said: 'you have to take care of me,'… but from a young age when we were "brought up," you know "ganqing" [you feel you] have to take care of him/her… especially those with alzheimer's (an -ch daughter caring for mother, age , with alzheimer's). supporting this perception as a culturally expected norm, sing mei, caring for her chinese parents, noted: 'i see my other friends, they also doing the same thing to their parents, to take carce of their parents' (sing mei -ch daughter caring for parents, ages +). south asian (sa) participants also positioned family care as a normative cultural expectation, but they were more likely to draw upon a religious understanding: describing caring for seniors and others as part of their religion where caring for others is an ingrained culturally expected religious responsibility to serve god and, thus, others. for instance, depicting this link between 'seva' or religious service and 'farz' note: ⁎ the two chinese caregivers worked outside of canada longer than they resided inside of canada. ⁎⁎ originally, chinese agreed to participate. however, one male dropped out. ⁎⁎⁎ two of the south asians indicated that another person was also the "primary" caregiver with them. ⁎⁎⁎⁎ nr -= no response to request for age ⁎⁎⁎⁎⁎ one wife was also a daughter-in-law to one of the male la participants. of la americans, were only children. or cultural responsibility, kavya, a south asian wife, caring for her husband and living with their daughter stated: 'culture, we are so religious from inside that we are taught from the beginning culture and religions, they are the same, and they run parallel.' kavya explained further: our foundation is such right from the start; serving others is like our religion. it is like serving god when we look after someone who is sick. we are told from childhood to look after sick people. what our children are doing…is part of our culture. in our culture, we teach to share. we shared with them; they are returning it to us. (kavya -sa wife caring for husband, age +) although religion is an important underlying value for the latin american (la) participants in this study, they described and explained caring for elders, family, and friends as 'deber' or duty. here, "family obligation" was based on the notion of collective loving family relationships and loyalty to family cultural values that extended beyond duty to immediate family into the community of friends. sandra, a previously paid health care provider, recently retired to care for her aging chilean mother, captured latin american cultural caring as collective and communal: as a family member, there is the affective aspect. never would i leave a parent, a very good friend, godmother, or whatever, if they don't have a family member or a person close to them. i feel that it is my "deber" (duty and obligation) for feelings for sentiments. i feel it is my "deber" to take care of/be concerned with that person, pay attention to them. (sandra -la daughter caring for mother, age ) caring for elders as a social expectation of being a 'good' son, daughter, or family member in the kin relationship network, then, was consistent across all three cultural groups. these cultural expectations also extended to friend networks described by many participants from all three groups. moreover, participants explained this care was a reciprocal act embedded within the social fabric of the culture. subtle differences in the foundations for understanding these expectations emerged, however. caring obligations appeared to be linked explicitly to religion for south asian participants, but it seemed more related to the teachings of political philosopher, confucius, for the chinese whereas latin americans tended to describe caring as a more collective sense of family extending beyond blood relationships. importantly, across all three cultural groups, this notion of family care was communal, embedded in a loose definition of the extended family network that included friends, blood relatives, and relational family. to illustrate, in our study, carers did not define a primary caregiver, but they discussed caring in terms of networks and relationships. even only adult children like argentinian rossana pointed out that she had created networks from her family (husband, daughter, and grandchildren, among others) and paid companions as well as her -year-old mother's long-time friends to provide support and care. another argentinian, -year-old casimiro, an only adult son, drew on his partner and extended family, like his female cousin living in seattle to provide support and companionship for his aging mother living independently. casimiro commented: my cousin, andrea, who is like my sister, comes from seattle every two months, and sometimes she stays for a week with my mother to keep her company. when i am here at my breaking point, she comes. she takes over. she releases me. for example, she came last week, and she cooked for two weeks. she leaves things prepared. she cares for my mother all the weeks. she takes her; she brings her back. she gives me license… (casimiro -la son, only child, assisting mother, age ) thinking further about his support network, casimiro explained: when i have problems with her [my mother], andera, my cousin, and daniel, my partner, are the two people who assist me…ahhh. i have a very good friend of hers in the [spanish community] group also…. that woman is very, very good also, and she helps me too… yes…she knows her [my mother] very well. she is from ecuador, and she knows the latin culture very well, so she cheers me up. (casimiro -la son, only child, assisting mother, age ) care was often described in terms of friend networks that provided support. for example, yoyi, a colombian business woman and daughter-in-law noted: 'in my country, (laughs) if someone is sick, people arrive to care for them' (yoyi -la daughter-in-law coordinating care for mother-in-law, age ) while a sa daughter, aaliyah, commented, 'my white friends get very surprised that my friends come right away when i need them.' furthermore, aaliyah (sa) explained that friendship was much more: - = = - = diabetes = prostate = mothers = independently - = = glaucoma = hard of hearing ⁎⁎⁎ = father - = = south asians ( ⁎ ) ⁎⁎ = - = - = alzheimer's = mothers = with caregiver ⁎⁎⁎ - = = - = bedridden = fathers = independently - = = - = - = brain hemorrhage = wife = care home - = = - = no health issues reported = husbands = with cg's brother - = = - = mother-in-law += = + latin americans ( ⁎ ) = - = - = alzheimer's = mothers = with caregiver ⁎⁎⁎⁎⁎⁎⁎ - = = - = pace maker ⁎⁎⁎ = father = independently - = = - = diabetes = husband -nursing homes - = = - = prostate cancer = daughter-in-law - = = - = stroke = aunt = ⁎⁎⁎⁎⁎ = old age = visit c& live in south america = healthy note: ⁎ number of care receivers is greater because some caregivers are caring for more than one person. ⁎⁎ one south asian caregivers was also a care receiver. ⁎⁎⁎ chinese indicated health issues; chinese indicated health problems; latin americans reported illnesses ⁎⁎⁎⁎ although some of the south asians indicated that the cr lived with them, in actuality, a number would live part-time with others in the family assisting with care. ⁎⁎⁎⁎⁎ one cr was a canadian by birth but had lived outside of canada for a number of years. ⁎⁎⁎⁎⁎ one latin american cr was born in south american but resided in the us ⁎⁎⁎⁎⁎⁎⁎ some started out living independently and then lived with cg or returned to independent lving, or started out living with cg and then moved o a nursing home. i went to india after years, believe it or not, my friend kept my mother with her for a full month. who would do that? i can't imagine. my mother was very happy. if she [friend] hadn't, we couldn't have gone to india. we both had to go, me and my daughter, we couldn't have thought of it. we didn't worry at all. it is our culture. if there is anything, i know that my friends are here, i don't have family, but my friends will be there. (aaliyah -sa daughter caring for mother, age +) in essence, two types of friend networks emerged during conversations with participants: friends of carers (as pointed out above) and friends of care receivers. although care receiver friend networks may not have provided direct care for their aging friends, they offered different types of support. this 'care' was more supportive and reinforcing companionship. five of the la participants spoke directly to the importance of this support: for example, rossana, an only child, maintained: [my -year-old mother] has two friends, well, she had. she has another friend who telephones her every so often…..sometimes she goes to visit her, but only once a month, but my mother counts on that. my mother has a little book where she has everyone's telephone number written down, and she calls… she asks people to call for her because she can no longer dial because she has lost her dexterity, so someone dials for her, and so she can talk. (rossana -la daughter, only child, caring for mother, age ) the deeply embedded cultural values, perceptions, and actions held by these three ethnic groups regarding family care created tensions within the canadian context for some, especially for those individuals dealing with relatives suffering from alzheimer's or severe illness. for instance, a recently retired chinese daughter, an, whose mother with alzheimer's lived with her and her retired chinese husband recognized the stress of this tension as she talked about how she had visualized her retirement quite differently: …after i retired, i was hoping to live the life of a retiree, hoping to do some traveling and enjoying life in retirement, doing more activities. but because i have to take care of an elderly [ -year-old] person with "alzheimer," i can't do what i want to do personally. (an -ch daughter caring for mother, age , with alzheimer's) for an, even though caring for her aging mother was causing stress in her marriage, as a chinese wife and daughter, pointed out: this situation is -…because she's my mother, and she's very afraid of going into a seniors' home, and i can't -my heart myself -basically i'm chinese, so "until" she really cannot "manage" one day, i mean when i cannot "handle" her "at home," i cannot bear to send her away to a seniors' home. (an -ch daughter caring for mother, age , with alzheimer's) other participants from the three cultural groups articulated views about cultural tensions between the canadian context and embedded cultural values of their elders. according to abhijeet, a retired south asian son, caring for his -year-old mother, 'there is culture in us,…a -year culture…we brought with us [when we immigrated to canada], …still alive in us. we know, it is not for us, we know…elders from india, after , when their life is almost over, they cannot change' (abhijeet -sa son caring for mother, age ). sandra, the la retired health care worker, addressed the necessity of balancing canadian beliefs and way of life with her cultural values since she has a canadian born son and her -year-old chilean mother living with her. sandra was clear, however, that she would ensure that her chilean mother's cultural family caring beliefs would be respected and honored despite what others might say. the doctor told me, "…well… there are institutions where you can put your mother." i said, "forget it! forget it! my mother is never going to be in an institution either." because the doctor is also chilean, and he would say, "forget what i said. forget it." because i would just look at him, "do you really think my mother would accept going there?". (sandra -la daughter caring for her mother, age ) nevertheless, as a -year immigrant to canada, sandra's changing values were reflected in her views of her own future old age in canada. i say to my son, "forget it. i am canadian now. the day that i begin to fail, in front of my house, there is a nursing home, you don't have to do any more than cross the street, and you put me there" (laughs). because my son is canadian, and he's going to marry a canadian girl, so then i must accept that the canadian family is different so his wife might think, ya the woman [mother-in-law sandra] is very slow, and there [nursing home] she will be very calm. (sandra -la daughter caring for mother, age ) other participants expressed similar change s in their values the longer time they spent in canada. participants despite some accepting the changes in cultural values, abhijeet, retired south asian son and husband, a long-time resident ( years) of canada, expressed the tensions that emerged in all three groups in this way: my parents, my father, her [wife] parents, my grandfathers, these are their pictures. when they were aged, they were looked after. at that time, the culture was different. now, it has changed. people over here are westernized, who bothers? go and leave your mother there [nursing home]. we'll pay money there. your attachment?. (abhijeet -sa husband and son caring for wife, age , and mother, in summary, family, kin, and friend care networks often were bounded, at least loosely, along cultural lines in their beliefs, intentions, perspectives, and actions. participants across all three cultural groups in this study drew firmly upon cultural beliefs about family care to make sense of their actions and responsibilities. for many, cultural perspectives about family care helped them explain their own sense of responsibility. importantly, however, cultural values also set the foundation for caring arrangements that were much more communal and relational. the sense of responsibility, however, could create tensions as participants experienced perceived cultural clashes regarding the understanding and meaning of familial care. a surprising theme to emerge early in this study was related to the relevance of geography and mobility to the flow of care. care processes were fluid, often transcending national, international, and geographical boundaries. mobility involved all members of the care network, including the person being cared for. geographical flow of care often consisted of the care receiver being cared for or supported across multiple households and geographical borders -both within the immediate locality as well as across different countries. furthermore, individuals providing some type of support or 'care' varied in terms of the type of relationship (immediate family; extended family; or friend networks) and location. one description of the mobility of the flow and fluidity of care between households by the immediate family was explained by chaaya, a south asian daughter, sharing care of their mother with her sister: mummy, from the time she came from india, first she lived with me, and later my sister moved out, and now she lives with my sister. half the week she lives here, and the other half she lives with my sister, and she has a very flexible schedule, and we are happy to keep her with us. there is joy in the house, that there is an elder in the house. (chaaya -sa-daughter caring for mother, age ) while the movement of this flow of care often occurred with immediate families at the local level, particularly for frail care receivers, surprisingly, mobility of care receivers took place fluidly across transnational borders. it was not uncommon for older adults, particularly if health allowed, to spend time (often months) with family members either between different households locally or dispersed throughout north america and/or transnationally in a former country of origin. often, it was difficult to determine what country or geographic location was 'home' to the older adult care receiver given the frequency and regularity of these mobile relocations. although some chinese and south asian individuals were mobile locally and nationally, this transnational movement phenomenon tended to be more frequent among latin americans. what was clear in the data was the complexity of facilitating this geographical movement. to illustrate the intricate and complicated support arrangements created by extended families, margarita, chilean only daughter, caring for her -year-old mother later diagnosed with alzheimer's, described: i have a cousin who lives in los angeles. she is my mother's niece, and she called me one day and said, "i'm going to chile. do you want me to take my aunt?" i said, "yes," so that we bought her a ticket so that she could go to chile, and so that afterwards she would be able to go to brazil. i have a son in brazil. we bought her the ticket. i, my boyfriend, and my mother travelled to los angeles where my cousin lives, and from there my cousin took her to chile, and, initially, she was going to stay with her brother there, but it didn't work out, and before she left, we looked for another arrangement, and a distant relative, said that she could have her in the house, and for a reasonable amount of money so much so that she went there for a month, and after a month, she went to my son's house in brazil. (margarita -la only daughter caring for mother, age , with alzheimer's) as explained above, planning trips was extremely cumbersome. participants not only had to take into account the complicated nature of planning travel for their aging parents, but they needed to account for other factors influencing movement across international and national borders. for example, ignacio, the youngest son of three colombian brothers, explained that although his -year-old mother was independent and able-bodied, she is afraid of airplanes, so she cannot come alone. she is very frightened of airplanes, [and] she speaks absolutely nothing of english, or rather, for her to come alone is not an option either…" to counteract these issues, "we need to go there and bring her here, …" and then accompany her back when she returns to colombia. (ignacio -la son assisting mother, age ) ignacio pointed out the importance of revising immigration, airline, and health policies, and programs to facilitate travel for aging immigrant parents: 'if they [policymakers, healthcare professionals, airline owners, or others] would simplify the way to bring parents, that way they would not have to become permanent residents or citizens.' he described ways travel could be facilitated and more accessible for older as well as frail adults. ….a program,…, where someone could bring them [older adults], and one wouldn't need to go and bring them here, rather that there would be a system where someone could bring various at a time, accompanying them in the airplane and helping them with running around for immigration. that would exist a way in that they could have tickets, a discount on the air fares….in the future an easier and faster way would exist that if the children are here, and the parents are alone there to make the immigration process faster because the time that they are there alone, well, it could become a risk that they experience other inconveniences because they are alone. (ignacio -la youngest son assisting mother, age ) finally, not only was the older adult mobile, carers also travelled within or between cities nationally or internationally to help provide care for aging relatives. jasmine, a chinese canadian daughter, stated: [when i was] away in toronto for a brief period, about a bit more than a year, my sister came to live with her' [their mother]. …, my sister, my brother-in-law, and my father were with my mother. in the end, they left, went back to hong kong. in , i came back from toronto to be with my mother. (jasmine -ch daughter caring for mother, age +, with alzheimer's) a prevalent theme underpinning the data from all three ethnic family networks indicated the importance of technology for facilitating an approach to family care that promoted a geographically fluid caring experience. in its least sophisticated form, technology included regular telephone contact -both locally and transnationally -with other family members and the care receiver. however, it also involved taking advantage of 'newer' technologies such as email and skype. the importance of telephone connection was highlighted by many: 'comfort was just a phone call away,' noted chaaya, a counselor and sa daughter. it enabled sibling and kin caring for older adults to be in 'communication all the time, absolutely, all the time, so that, well, or rather directly with her and through my sisters…' (chaaya -sa daughter sharing care with her sister of their mother, age ). low cost international calling was relied upon extensively. regular phone contact -sometimes daily -was maintained between family networks. this ensured all members were involved in daily life and decision making for the senior and family. it also helped reduce stress for those currently providing hands-on care and ensured all family members had an understanding of the older adult's care needs. this allowed each family member to contribute to care decisions as they emerged daily based on each person's abilities and time to assist with and participate in the care and support process. meiling, -year-old chinese wife, caring for her -year-old husband with dementia e pointed out: our daughter came two years ago to see her daddy, but she has work and children and family, so they can't come every year….they have to wait for the right time, the children's summer holidays, to come over. so we rely on telephoning. they call , , or times a week to talk with us. it's good. at least with the phone call, you get to know the situation. you can talk…now, they ask how's life with daddy, how he's deteriorating, get to know his conditions, that can help me reduce my stress [laughs heartily]. (meiling -ch wife caring for husband, age , with dementia) illustrating the many ways phone calls were used strategically, one son, ignacio, − a physician working in canada as a researcher -telephoned not just to keep his mother in colombia close to him but also to assist in monitoring her health: in actuality, we do it [call] once a week, and i ask her how her health is, if she has had medical appointments. i always find out what happened during the medical appointment, what medicines, what exams they ask for, what prescriptions she is taking, how she is feeling. perhaps, her health is the most important thing that i am aware of in addition to that, her emotional state. (ignacio -la son assisting mother, age ) while not a replacement for actual physical contact and recognizing the complexity and financial cost associated with travel, phone calls were helpful. much of the calling was, not unexpectedly, between members of the family kin network, but the potential value of using telephone contact with health professionals strategically to keep family aware and involved, was also raised. thinking about his mother, ignacio suggested: -to find a way that the doctors there [in colombia] can communicate with the children here [in canada]…one could send them an email or tell them…a communication bridge to tell them how he found my mother, what needs to be done, because my mother goes to the doctor, and the doctor explains a mountain of things in medical terms that she is not going to understand. she goes home, and i ask her how it went, and she tells me fine. (ignacio -la son assisting mother, age ) frequently, telephone calls were used to share news, keep in touch, and communicate -often daily. usually, these calls dealt with the mundaneness of daily living and celebrations. some extreme examples demonstrated the importance of contact as a lifeline. for instance, one venezuelan niece, leila, described how a visa problem limited her ability to travel, so she had to rely on an international call to talk with her dying aunt. previously, leila had cared for her aunt for more than years in the us. devastated that she could not be by her aunt's side in her last moments, leila remembered saying as she listened over the phone to her aunt's labored breathing: little aunt, you are with god. never be afraid because god is with you. you will see that you are going to be super well. god loves you. i don't know how much. i love you so much, you know. (leila -la niece caring for aunt, age , with alzheimer's) leila recognized the importance of emotional caring by an extended family member as a fundamental aspect of care. the warmth of family caring could not be replaced by professional caregiving -and her simple use of telephone contact allowed leila to continue providing loving, heartfelt, emotional support, connection, and comfort. not being able to be there…, it was like disappearing from her life. before that…, we talked, and all, but……it was that i was there at every moment for her [when she had been caring for her aunt with alzheimer's on a daily basis in the us]. the woman [professional caregiver] she had was excellent, but it was not the same quality, or rather the love, or rather the quality of care could have been very special, very professional, and all, and…very affectionate, latina, …, quality, but nothing like the love of the family. (leila -la niece caring for aunt, age , with alzheimer's) despite its utility and importance for emotional support, comfort, and communication, technology had some downsides. even with reduced costs of international calling cards, daily international calls still remained expensive. moreover, telephone calls could be intrusive to one's personal life as explained by casimiro, argentinian son, an only child: if she [my mother] needed something, she would call me a number of times. i tell you that because if not, she becomes another of the things; this …is very interesting. with her, one must have balance because i visit her every day. she becomes very possessive, and afterwards, she thinks i have the obligation! at the same time, when i answer the telephone every time, but i do not answer one time, she says to me, "why didn't you answer the telephone!" …i say, "mami, i am working. i am doing business. sometimes, i cannot attend to you. leave me a message, and i will answer you." it is something very interesting. it is like an education…if i answer her immediately every time, sometimes when i do not answer, it is "oh, lala!" (laughs). (casimiro -la son, only child, assisting mother, age ) beyond the telephone, other types of technology -for example, skype -emerged as an important facet for facilitating more traditional views of collective caring and played a key role in uniting local, national, international, and transnational 'carers.' it brought family members at a distance closer together in terms of proximity, particularly for some latin american participants. a vivid illustration of how technology facilitated day-to-day involvement was offered by antonia. despite residing a continent away, this -year-old female doctoral candidate described the continued closeness between her two sisters living in venezuela, her mother, and herself in canada. antonia pointed out that through technology, she, as the youngest daughter, was in constant communication with her mother in order to be part of and share experiences with her mother's daily life: by…using skype, we also see each other. then, if she goes saturday to have lunch at my sister's, my nieces turn on the computer. i say, mami, get on so that i can show you the little flower that bloomed on the plant. then, i show her through the house with the computer showing her the little flower, and it is so wonderful…it feels wonderful because she knows… as if she had come to actually see how it is doing, what the house is like here, and if the plant, and if it grew or if it didn't grow. i show her how…i hung a few new pictures. then, through skype, i can see the paintings. this makes it easier. (antonia -la youngest daughter supporting mother, age ) technology played an important role in facilitating and fostering the fluidity and flow of care for cultivating a sense of closeness, proximity, and presence of family, kin, and friend networks caring for and involved in the life of the older person and family. as described by all three family cultural groups, technology spanned local, national, and geographical distance and transnational borders to bring comfort, support, presence, closeness, and involvement in decision making while allowing family, extended kin, and friends to participate in seniors' daily lives and experiences at a distance. recognizing the importance of culture within a context of informal family care, the research team for this study sought to better understand the process of family care within three ethnic communities in vancouver, bc, canada. a broad understanding of giving care was adopted, allowing participants to self-identify their role in the care process from their cultural perspectives. this, perhaps, set the stage for the importance of fundamentally challenging conventional ideas associated with the study of family care in north america that has often focused on one 'primary' caregiver. specifically, across all three cultural groups, our findings support an understanding of family care as truly occurring within broader family kin (and friendship) networks that tended to be culturally bounded, often loosely, but not geographically constrained. this meant that in all three cultural groups, but especially with the latin american participants, it was often difficult to name a 'primary' caregiver since even only children, like rossana and casimiro, created extended kin and friend support networks that aided in the provision of support and care. consistent with this approach, none of the family carers identified themselves as the 'primary' caregiver. the notion of a primary carer was further disrupted by the mobility and use of extended travel by both the older adult to live with other family members and/or by family members spending time with the older adult making it difficult to even determine the location where primary care was being provided. our participants described complex negotiated decisions made by kin networks as a collective family undertaking about how, when, where, why, and by whom care and support would be provided to aging seniors. care was fluid and flowing throughout individuals, networks, time, space, distances, and ages. this suggests to us the need to extend ideas about aging in place -a term that commonly directs policies and practice -to consider more specifically the notion of 'aging-across-place' (r. beard, personal communication, august , ) as a relevant concept for understanding care within culturally diverse, immigrant groups. these findings position family care using a lens that is far broader and relational than the narrow focus on the provision of instrumental care that has typically dominated understanding of family care within conventional gerontological literature. hence, our findings support the much more inclusive and complex approach to care offered by scholars, such as finch ( ) and tronto ( ) . tronto has developed the concept into an ethics of care framework. grounded in feminist relational theory, an ethics of care lens captures the reciprocity of care in that we are all interdependent, both receiving and giving care throughout our lives. in this framework, tronto identifies five principles to guide the provision of care: attentiveness, responsibility, competence, responsiveness, and solidarity. these correspond to five phases of care: 'caring about,' 'caring for,' 'care giving,' 'care receiving,' and 'care with.' these phases are fluid and can operate simultaneously and sometimes contradictorily, across different relationships. pragmatically, the phases and corresponding principles capture the complexity of the caring process and provide a useful lens for exploring relationships: purpose, needs, emotions, and power. this framework lens may be especially important for understanding the interface between the experiences of family and formal care, particularly within ethnically diverse communities where underutilization of formal supports has, in many cases, been identified as a problem. second, our findings raise questions about how place of residence may be re-interpreted and understood as a more fluid concept than is typically assumed with movement occurring at the local, national, and international levels. in raising these insights, the study contributes to the understanding of family care as a transnational global phenomenon and bridges two important bodies of research. as identified in the introduction, migration scholars introduced the importance of a transnational perspective to describe and conceptualize relationships individuals and communities develop and sustain across geographical distances and national borders (baldassar, ; horne & schweppe, ) . with few exceptions (baldassar, ; baldassar et al., ; merla, ) , this lens has rarely been applied to the study of aging and/or understanding family care of older adults (amin & ingman, ; horne & schweppe, ; näre, walsh, & baldassar, ; zechner, ) , especially related to non-western cultural groups (dhar, b; torres, ; wilding & baldassar, ; zhou, zhou, , . this lens did not initially inform our research. however, the importance of it emerged early on in the data generation phase, and, consistent with a grounded theory approach, was used to help question, refine, and develop understanding. for example, we began to explore the fluidity and flow of care, role of technology, and describe how some ethnic older adults experience this notion of aging across different geographical places. our research suggests that this lens has much to offer to the study of family care in gerontology and supports findings by baldassar et al. ( , baldassar, and merla, ) that family care does not need to be proximate in order to be effective. a transnational lens draws attention to a number of areas that require further exploration and development. first, consistent with the work of baldassar, nedflcu, merla, and wilding ( ) ; wilding, ; wilding & baldassar, ) , and others (see for example, ahlin, ; lee, lee et al., ) , the promises and challenges of information communication technology (ict) for facilitating a more holistic understanding and enactment of 'family care' emerged as a critical aspect of family care that requires further examination. our findings lend texture to wilding's ( ) suggestion that technologies blur lines of imagined proximity and physical separation as families creatively incorporate diverse types of technologies into their provision of care to meet cultural, social expectations, and health needs in these contexts that tend to be particular to specific points in time. it supports the value of ict for 'enacting everydayness' (ahlin, ) , p. as a key theme related to 'good care at a distance.' one powerful example of the blurring of distance and time in this study was the graduate student, antonia, having lunch with her mother daily via skype. an understanding of the importance of ict also highlights ways of re-visioning the provision of formal support. for example, family conferences using supportive technology may help ensure that the entire family -irrespective of geographic location -has the necessary information to provide quality care and support, particularly for ethnic collective cultures. ensuring both the availability and uptake of effective communication technology infrastructures and digital literacy for older adults and their families will be an important area for research and service development. this is especially urgent in these times of covid . our study also supports the works of bryceson ( ), wilding and baldassar ( ) , and zhou ( zhou ( , that focused on the need to better understand how structural issues may impede the process of family care. transnational care or 'distant care' has received little recognition in the area of policy development (baldassar & merla, ) , and yet, as identified by at least one participant, ignacio, in our study, policies and practices, for instance around travel, may deter families from being able to care and provide culturally appropriate support in the way that they want while residency requirements may act as barriers for use of health and social care support services. future research is needed to understand how diverse health and social policies in both the home country as well as the country of immigration impact family care ( lee et al., ) . finally, attention to the notion of care as fluid and flowing for both able-bodied as well as those who are frail or ill emerged from our study as an important finding. 'flow of care' related to the presentation of negotiated, reciprocal, and shared family collective informal care was based on ability, availability, and capacity to care, occurring in motion, fluidly, through time and space as the care receivers transitioned and moved geographically to be with family as they aged and/or family moved to be with them. in addition to being consistent with an ethics of care framework, this notion develops further the work related to the circulation of care being developed by baldassar and merla ( ) ; . the concept of care circulation offers a broad view on transnational care through its focus on 'the reciprocal, multidirectional, and asymmetrical exchange of care that fluctuates over the life course and within transnational family networks subject to the political, economic, cultural, and social contexts of both sending and receiving societies' (baldassar & merla, , p. ) . in some of their work, they have examined how the older adult contributes in this circulation of care: an important limitation of our study is that we did not explicitly explore the role of the older person in the family care process except in relation to receipt of care. further research is needed to broaden and deepen the picture of family care, addressing more explicitly the role of reciprocity within the process. through this study, we explored the process of providing informal care to a family member within three diverse, non-western cultural immigrant communities. findings highlight the importance of recognizing family care as more communal and geographically fluid, supported by the innovative use of technology. the study suggests the need for a reframing of our understandings of family care -for example broadening moving from a notion of 'aging in place' to one of aging across geographical locations -and the need for policies and practices that can accommodate a different way of providing family care. we need to reinterpret care as flowing freely and fluidly throughout family, kin, and friend networks of informal, collective, and communal care through space, time, and distances locally, nationally, transnationally, and globally. no conflict of interest existed by the authors. good care" with icts in indian transnational families eldercare in the transnational setting: insights from bangladeshi transnational families in the united states transnational families and aged care: the mobility of care and the migrancy of ageing de-demonizing distance in mobile family lives: co-presence, care circulation and polymedia as vibrant matter families caring across borders: migration, aging and transnational caregiving transnational families, migration and the circulation of care: understanding mobility and absence in family life ict-based co-presence in transnational families and communities: challenging the premise of face-to-face proximity in sustaining relationships comments about a concept in the text of a manuscript from the editor of the research 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reflections: 'care circulation' in an increasingly mobile world: further thoughts dementia and migration: family care patterns merging with public care services ageing in transnational contexts: transforming everyday practices and identities in later life culture, caregiving, and health: exploring the influence of culture on family caregiver experiences placing ethnicity at the centre of studies of later life: theoretical perspectives and empirical challenges differences between caregivers and non-caregivers in psychological health and physical health: a meta-analysisanalysis at the intersection of culture: ethnically diverse dementia caregivers' service use diverse family structures and the care of older persons theorizing in family gerontology: new opportunities for research and practice family caregiving or caregiving alone: who helps the helper? immigration and ethno-cultural diversity highlight tables. immigrant status and period of immigration, counts, both sexes, age ( years and over), canada, provinces and territories, census - % sample data expanding the gerontological imagination on ethnicity: conceptual and theoretical perspectives caring democracy: markets, equity and justice virtual' intimacies? families communicating across transnational contxts ageing, migration and new media: the significance of transnational care care of older persons in transnational settings space, time, and self: rethinking aging in the context of immigration and transnatonalism time, space and care: rethinking transnational care from a temporal perspective we would like to thank dr. daniel lai (formerly of the university of calgary and now the hong kong polytechnic university) and acknowledge his leadership for this project.we would like to thank research assistants sonia andhi, msw for her work with the south asian interviewees and dr. sing mei chan for her assistance with the chinese participants as well as their help during the research process. a special thank you goes to, vancouver public librarian marilyn macpherson for her help in locating some of the statistics canada data.we are especially grateful to the chinese, south asian, and latin american participants and their families for sharing their experiences of caring and supporting their elderly relatives. this research was made possible by a canadian institutes health research grant (cihr # -npi: dr. daniel lai) canada. this research was approved by the ethics committees from the university of calgary (calgary, ab) and the university of british columbia (vancouver, bc). key: cord- - ij fkrh authors: walsh, froma title: loss and resilience in the time of covid‐ : meaning making, hope, and transcendence date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: ij fkrh this article addresses the many complex and traumatic losses wrought by the covid‐ pandemic. in contrast to individually‐based, symptom‐focused grief work, a resilience‐oriented, systemic approach with complex losses contextualizes the distress and mobilizes relational resources to support positive adaptation. applying a family resilience framework to pandemic‐related losses, discussion focuses on the importance of shared belief systems in ( ) meaning‐making processes; ( ) a positive, hopeful outlook and active agency; and ( ) transcendent values and spiritual moorings for inspiration, transformation, and positive growth. practice guidelines are offered to facilitate adaptation and resilience. matriarch. a death is often experienced as a hole in the heart of a family that will never again feel intact. sudden deaths, most common in rapidly progressing, severe cases of covid- , are jolting experiences for families. a recovering loved one may suddenly take a turn for the worse. there is often extreme physical suffering before death, which is agonizing for loved ones, helpless on the sidelines and lacking treatment options. with quarantine restrictions, family members are unable to be at the bedside, to provide comfort and say their good-byes. additional heartache ensues when gatherings are prohibited for funeral and burial rituals that help families and their communities to honor the deceased, share grief, and provide mutual support (imber-black, ). my extended family experienced a heartbreaking death to coronavirus. in march, i received an anguished email from my cousin: she had been informed by her mother's nursing home, that her mother had contracted covid- , was in isolation and declining rapidly, but could receive no visitors. family members hovered outside the building, unable to be with her as she declined and died. they weren't allowed to see her body or to hold a funeral gathering. a week later, her daughter, who had visited her mother just before symptoms appeared, contracted the virus herself, was in quarantine, and worried about having spread it to other grieving family members. i was relieved to hear, a month later, that she was recovering from a mild case. but she and her siblings were deeply distressed over their mother's death and furious that the facility had not informed them that other residents had tested positive before her mother's diagnosis. they were wracked with remorse that they had let her go to a care facility and had not insisted upon taking her in to live with them. such heart-wrenching situations are all too common for families losing a loved one in this time of high contagion. the elderly and others with underlying medical conditions face heightened risk. with an unexpected loss, family members lack time to prepare emotionally or practically, to deal with unfinished business, or to say their goodbyes. grief can be complicated with regrets that it is too late to repair wounded bonds. in some cases, families and emergency care providers must make agonizing end-of-life decisions to forego or end life support efforts. strong disagreements or religious concerns can lead to long-lasting family distress. this article is protected by copyright. all rights reserved the isolating constraints of social distancing heighten awareness that our connections with others are vital to thrive. in traumatic experiences like a pandemic, when helplessness and confusion are common, we have an urgent need to turn to one another for support, comfort, and safety. separations are keenly felt. with high risks of severe illness and death for elders and those with chronic medical conditions, loved ones are fearful of bringing the virus to them. travel safety concerns limit visits by those living at a distance. elders miss out on the rapid developments of grandchildren and yearn for a hug, a kiss, the scent of a baby's breath. individuals in prolonged isolation, living alone or in care facilities, can suffer a sense of disconnection and loneliness, which increases risks for physical and mental decline, substance use, emotional despair, and death (caccioppo, cacioppo, & capitanio, ; killgore, cloonan, taylor, & dailey, ) . families need to sustain connections across distance: phone and internet contact, cards and letters, and children's drawings all offer vital lifelines. the severe economic shockwaves of the covid- pandemic have far reaching impact for financial security and wellbeing in families. job loss and the looming threat of prolonged unemployment, business closures, and uncertain economic recovery can be devastating, especially for lower-income families who lack savings and barely scrape by, paycheck to paycheck. the loss of essential income can have cascading effects with loss of homes, disruptive relocations, and persistent housing and food insecurity. an untimely death in the pandemic is especially heartbreaking for families. the loss of a child, even one in early adulthood, upends life cycle expectations and shatters hopes and dreams for all that might have been. in the rapid spread of the coronavirus, anticipatory loss (rolland, ) is a constant concern, with worry about one's own safety and the threatened loss of loved ones. dire forecasts of a prolonged economic recession generate deep anxieties about future livelihoods and retirement security. young adults, facing the loss of educational and job plans, fear the loss of life dreams: in pursuing careers, gaining financial independence, finding life partners, and starting a family. this article is protected by copyright. all rights reserved the loss of a sense of normalcy is widespread. life as we have known it has been derailed. life forward is on hold, the future uncertain, and the road ahead unclear. there is much talk about the "old normal" and the "new normal." yet, like the aftershocks of an earthquake, the ground keeps shifting, and nothing feels normal. these harrowing times take a mental, physical, and emotional toll. daily news reports increase a sense of overwhelm, with confusing and conflicting information and changing forecasts on what lies ahead. a cartoon depicts a couple in their living room, with flames rising up around them. as one partner sits on the sofa, trying to read a book, the other stands transfixed in front of the large screen tv watching the breaking news bulletin: "hell still on fire." in this unprecedented pandemic, there is a collective experience of shattered assumptions in our worldview: our taken-for-granted beliefs and expectations about our lives and our connections to our world (janoff-bulman, ) . the invisibility of the virus, its lethal potential, and the possible spread by non-symptomatic persons heighten fears of infection. the death of a loved one, and loss of physical contacts, life structures, and future life visions can shatter core beliefs and make our world seem unpredictable and unjust. as one father lamented, "everything i thought i knew is shaken." one global mental health specialist coined the term "covid cognitive cloud" to describe the disorganizing impact of the pandemic. ambiguities cloud our thinking and decision-making. who is trustworthy for leadership, information, and guidance? where and with whom are we safe? we feel trapped and angry at a loss of freedoms with lockdown and restrictions. paradoxically, we also feel unmoored and adrift, swept by strong currents in a perfect storm of extreme events beyond our comprehension and control. the impact of loss is compounded with situational risks, larger systemic/structural forces, and/or complex family dynamics. high risk situations and socio-economic disparities. the risk and pain of loss is intensified when loved ones are working on the front lines and in jobs with repeated exposure to the virus. it is this article is protected by copyright. all rights reserved heartbreaking for families of healthcare emergency workers who contract coronavirus while providing critical care, often lacking protective equipment, without respite from the overload of cases, and suffering emotionally when lives can't be saved. those who self-isolate to protect their own family members miss their support. socio-economic and racial disparities render disadvantaged and marginalized communities at higher risk for multiple losses in major disasters worldwide (norris, ) . in a pandemic, crowded living and conditions, job and environmental hazards, chronic medical conditions, and discrimination in disaster response heighten risks. blacks and latinx have been disproportionately affected by coronavirus across the united states and all age groups (oppel, gabeloff, et al., ) . stark disparities are seen in the highest death rates, particularly among low-paid workers and their family members. many employees. are caught between troubling options: going to work for a needed paycheck or losing their jobs and income if they stay home to keep themselves and loved ones safe. prolonged unemployment and financial insecurities have long-term effects, ambiguous loss. ambiguity surrounding risk and loss generate anxiety, depression, and conflict, interfering with adaptation (boss, ) . with covid- , ambiguities persist about how the virus is spread and whether a death was due to coronavirus. unclarity about the diagnosis, symptoms, severity can be an impediment in getting emergency care. family members may fault themselves for not having understood risks or acted to prevent a death and remain unclear about their future risks. unacknowledged and stigmatized losses. when losses are unacknowledged, hidden, or minimized, they leave families unsupported (doka, ) . the denial of the human tragedy of illness and deaths in the spread of covid- by national authorities renders their suffering invisible. the stigma of possible contagion surrounding a covid-related death fosters misinformation, secrecy, and estrangement, impairing social support as well as critical health and mental health care. reports are also emerging of a spike in suicides and addiction-related deaths, with concerns about further increases with long-term effects in the economy and vulnerable groups (gunnell, appleby, et al., ) . deaths by suicide or overdose are tormenting for families, who struggle to comprehend them and may need help with anger, blame, shame, or guilt over how they might have made a difference (walsh, in press ). as the first wave of the pandemic surges in many places, with a second this article is protected by copyright. all rights reserved wave expected, most families experience a roller-coaster course in efforts to cope and adapt. families can be overwhelmed by the emotional, relational, and functional impact of the many stresses in their lives. adaptation can be further complicated in highly conflicted, abusive, or estranged relationships or with reactivation of painful emotions around past trauma or loss (walsh & mcgoldrick, ) . the dominant anglo-american culture has fostered avoidance in facing death and loss, minimizing their impact, and encouraging people to quickly get "closure" and move on from losses and painful emotions (walsh & mcgoldrick, ) . some seek reassurance that death happens to others who are unfortunate or at fault, to assuage anxieties about their own risks. many are uncomfortable in responding to others' loss experiences and may distract attention or avoid contact. reflecting the cultural aversion, many therapists working with families have been hesitant in addressing significant losses, leaving grief to bereavement specialists and pastoral counselors. moreover, there's no safe professional boundary from emotional spill-over: therapists, as well as clients, are impacted by the pandemic and are dealing with losses, disruptions, and anxieties in both work and family spheres of life. like our clients, we are trying to hold it all together. in a larger cultural context and mental health field that favors brief solution-oriented approaches, therapists need to appreciate that loss is not a problem to solve. we can't bring back a deceased loved one or a livelihood or way of life that is gone. we can listen openheartedly to pain and suffering in families, facilitate their mutual support, and encourage active efforts for positive adaptation. the cultural ethos of the "rugged individual" fosters expectations for self-reliance and fierce independence in dealing with serious life challenges. vulnerability and dependence on others are shame laden, viewed as weakness and deficiency. associated cultural images of masculinity constrain accepted article many men's emotional expression and strain relational bonds. in couples, a distraught spouse may feel abandoned by an emotionally unavailable partner when mutual support is needed most. this ethos also encourages individuals to tough it out on their own: "i should be able to manage it all myself." "i don't want to ask for help or burden others." such expectations lead to burnout, especially for single parents, and leave no time to attend to emotional needs or find respite from pandemic-related stresses. vulnerability is part of the human condition. distress is normal in abnormal times.. although some families are more vulnerable in this pandemic, most face losses and upheaval. false assurances of invulnerability are foolhardy. acknowledgment of grief, suffering and hardship is a strength that can rally mutual support and collective efforts for recovery. we are relational beings. recognition of our essential interdependence is vital for our wellbeing and resilience. in turning to others for help, we can pay it back and pay it forward. mobilizing kin and social support, while challenging with social distancing restrictions, is crucial to build family and community resource teams. as a society, we are all going through this pandemic together. we need and depend on each other for our lives and our future. in this time of pandemic, there is much talk about widespread grief. it's important to clarify current research-based understandings of loss, and common misconceptions from earlier theories positing a single, universal model of "normal," or "healthy" grief. epidemiological and cross-cultural studies have found wide diversity in responses to loss, with variation in the timing, expression, and intensity of normal grief responses (walsh, in press) . in families, members may not be in sync, requiring respect for differences. grief and recovery processes do not follow an orderly stage sequence or timetable as proposed by kubler-ross and kessler ( ) . common reactions of shock and disbelief, anger, bargaining, sorrow, and acceptance are better seen as facets of grief, which ebb and flow over time. while usually decreasing in intensity, various facets can surface unexpectedly, particularly around nodal events. this article is protected by copyright. all rights reserved in the covid- pandemic. initial shock and disbelief are common, but unshakable denial becomes detrimental in not facing the reality that must be dealt with. in families, tolerance is needed for different reactions: one member may be consumed by sadness and yearning while another is enraged by the unfairness of a loss. a breadwinner may need to keep emotions under wraps to function at work. small children may show anxious clinging or need constant contact while adolescents may distance (walsh & mcgoldrick, ) . adaptation involves a dynamic oscillation in attention alternating between loss and restoration, focused at times on grief and at other times on emerging challenges . with pressing demands, many don't have the time and space to process complicated losses, which may find expression in substance use, relational conflict, or child-focused problems. many only seek counseling much later, after initial social support wanes and the full impact of loss-related challenges is felt. this will require pacing of interventions attuned to each family, weaving back and forth in attention to grief, coping efforts, and future directions. adaptation to loss does not mean full recovery or resolution in the sense of some complete, once and for all, getting over it. recovery is best seen in terms of adaptation over time, rather than a final outcome. many recover from coronavirus, yet some suffer long-term sequelae not yet understood. recovery from the economic effects of the pandemic may be partial, as will be recovery of aspects of past ways of life. efforts will be needed for both continuity and adaptive change. likewise, resilience in response to loss and other major disruptions does not mean "just bounce back," quickly rallying and moving on unscathed (walsh, b) . healing and resilience occur gradually over time. grief is a healing process: we don't get over grief--we go through it. resilience is forged through suffering and setbacks; it involves struggling well and integrating painful loss experiences into our life passage. the concept of resilience-the capacity to overcome adversity-is finding valuable application in situations of widespread disaster, collective trauma, and loss (landau, ; masten & motti-stefanidi, ; saul, ; walsh, ; b) . with advances in research, resilience is now this article is protected by copyright. all rights reserved understood as involving dynamic multilevel systemic processes over time. the response to a disaster by communities and larger systems can make the difference for individual and family wellbeing and resilience. for instance, abysmal failures in government response to hurricane katrina compounded widespread suffering and loss. in contrast, the coordinated response to the oklahoma city bombing tragedy by community leaders and agencies provided immediate support and fostered long-term positive adaptation (walsh, ; b) . family resilience refers to capacities in family functioning to withstand and rebound from disruptive life challenges in adversity. more than surviving loss and coping with disruptions, resilience involves positive adaptation: regaining the ability to thrive, with the potential for transformation and positive growth forged through the searing experience. a family resilience orientation is finding broad application in strengths-based, collaborative, systemic training, practice, and research (walsh, a (walsh, , b . a resilience-oriented approach with loss (a) contextualizes the distress; (b) attends to the challenges, suffering, and struggles of families, and (c) strengthens relational processes that support coping, adaptation, and growth. with a multisystemic lens, this approach draws on extended kin, social, community, sociocultural and spiritual resources, and strengthens larger systemic/structural supports. to help families forge resilience in response to pandemic-related losses and the myriad of challenges they face, therapists can usefully apply this author's family resilience framework. designed as a practice map to guide intervention with families facing extreme adversity, it has been applied to traumatic and complicated losses in communities and with widespread disaster (walsh, ; b) . the covid- pandemic is a perfect storm of stressors, involving acute crisis and loss events, disruptions in many aspects of life, and ongoing multistress challenges with evolving conditions. this situation is so extreme that families are experiencing the strains of grief and sadness over so much loss, fears for loved ones, and anxieties about the future. how a family deals with stress and loss is crucial; therapists can help families strengthen key transactional processes for mutual support and mobilize active efforts to overcome challenges. in gaining resilience, they strengthen bonds and resourcefulness in meeting future challenges. this article is protected by copyright. all rights reserved the walsh family resilience framework identified nine key processes--facilitative beliefs and practices--in three domains of family functioning: family belief systems, family organizational processes, and communication / problem-solving processes (walsh, (walsh, , b ). discussion in this paper focuses on the powerful influence of family belief systems in the covid- pandemic. shared facilitative beliefs are the heart and soul of family resilience. each family's belief system, rooted in multi-generational and sociocultural influences comes to the fore in times of crisis and loss, shaping members' experience and their pathways in adaptation. family resilience is fostered by shared beliefs ( ) to make meaning of the crisis and challenges; ( ) to (re)gain a positive, hopeful outlook that supports active agency, and ( ) for transcendence: to rise above suffering and hardship through larger values, spiritual beliefs and practices, and experiencing transformations in new priorities, a sense of purpose, and deeper bonds. core beliefs ground and orient families, providing a sense of reality, normalcy, meaning, or purpose in life. well-being is fostered by expectations that others can be trusted; that communities are safe; that life is orderly and events predictable; and that society is just. when the losses and upheavals in this pandemic shatter such assumptions, as noted above, there is a deep need to restore order, meaning, and purpose (janoff-bulman, ). meaning making and recovery involve a struggle to understand what has been lost, how to build new lives, and how to prevent future tragedy. meaning reconstruction is a central process in healing in response to trauma involving both death and non-death losses (neimeyer & sands, ) . it involves sense-making efforts over time, not simply a final stage in resolving grief, an "aha" moment when everything makes sense. in this pandemic, at first it is hard to understand what is happening, without previous experience to relate it to. as we grapple with the implications, we gradually try to come to terms with the situation, what can be known and the uncertainties that persist. in families, meaning-making processes involve shared attempts to make sense of the loss, put it in perspective to make it more bearable, and, over time, integrate it into personal and relational life passage (nadeau, ) . resilience is strengthened in helping families gradually forge a sense of coherence through shared efforts to make loss-related challenges comprehensible, manageable, and this article is protected by copyright. all rights reserved meaningful to tackle (antonovsky & sourani, ) . this requires dealing with ongoing negative implications, including the loss of hopes and dreams. contextualizing members' distress as common and understandable in their situation--normal in an abnormal times--can depathologize intense reactions and reduce blame, shame, and guilt. in the context of covid- , therapists need to explore both the factual circumstances of losses and the implications they hold for family members in their social and developmental contexts. commonly, they grapple with painful questions: "how did this happen?" "could it have been prevented?" "what will happen to us?" "what does it mean for our lives? such concerns persist when, for instance, the source of viral transmission, the development of vaccines and treatments, or the future of the economy remain unclear. causal attributions concerning blame, self-blame, and guilt can be strong when questions of failed responsibility or negligence arise, such a not following public health guidelines. meaning-making efforts and future planning are hampered by repeated unclear and inconsistent information by government authorities. frustrations may boil over in anger that more should have done to prevent widespread viral contagion and economic losses. systemic therapists can help family members to voice such concerns, come to terms with reasonable limits of control in the situation, and seek greater accountability and leadership by those in charge at local and national levels. families may struggle to envision a new sense of normality, identity, and relatedness to adapt to altered conditions. they can become trapped in helplessly waiting to hear what will happen next or in the future. a sense of active agency is vital for resilience: what can we do about it? what are our options? clinicians can support efforts to gain and share helpful information and become involved in community efforts. helping professionals are cautioned not to ascribe meaning to a family's unique experience. our role is not to provide meaning for those who are struggling, but to facilitate their meaningmaking process (frankl, ) . the multiple meanings of a particular loss evolve over time as they find expression in continuing patterns of interaction and are integrated with other life experiences. over time, adaptation involves weaving the painful experience and the resilience forged into the fabric of individual and collective identity and life passage. this article is protected by copyright. all rights reserved abundant research has found the importance of a positive outlook for resilience (walsh, b) . yet this should not be seen as relentless optimism and good cheer. in confronting significant challenges, with covid- , it is common to experience discouraging setbacks. sadness and nostalgic yearning are intensified when former lives can't be restored. many persons report that there were times when they didn't know if they could face another day, or felt that life no longer had meaning-but with the support--or needs--of others, they vowed to carry on. . family members' mutual encouragement bolsters active efforts to take initiative and to persevere. affirming individual and family strengths in the midst of difficulties can counter a sense of helplessness, failure, and despair as it reinforces shared pride, confidence, and a "can do" spirit. hope is most essential in times of overwhelm and despair, fueling energies and efforts to cope and rebuild lives. we hold onto hope in the midst of uncertainty. weingarten ( ) cautions us to practice reasonable hope and to avert false hopes. wishful thinking does not make a pandemic go away. flaskas ( ) notes the complex dynamics of hope and hopelessness within intimate relationships, embedded in family history, community, and social processes, which can support or undermine hope. in a couple, one partner may lose hope while the other holds hope for both. therapists can witness the coexistence of hope and hopelessness in a way that nurtures hope and yet emotionally holds both. in working with covid-related loss, we can help families reorient hope: as some hopes are lost, what can realistically be hoped for and worked toward? support may be needed to tolerate prolonged uncertainties and lengthy recovery processes, while holding hope in future possibilities with sustained efforts. as studies have found, resilience is fostered by focusing efforts to master the possible, accepting that which is beyond control, and coming to terms with what cannot be changed (walsh, a (walsh, , b . transcendent values and connections enable families to view losses and suffering beyond their immediate plight, cultural and spiritual connections are valuable resources to support adaptation, providing assistance to honor and grieve all that was lost and move forward with life (rosenblatt, ) . in the time of covid- , transcendent values and practices help families to endure and rise this article is protected by copyright. all rights reserved above losses and disruptions, by fostering meaning, harmony, connection, and purpose. they offer opportunities to reaffirm identity, relatedness, and core social values of caring and compassion for others. in times of loss and deep suffering, spiritual matters commonly come to the fore, whether based in religious or existential concerns (wright & bell, ) . clinicians are encouraged to attend to the spiritual dimension of experience to explore issues that constrain adaptation and to draw on spiritual resources that fit clients' preferences within and/or outside organized religion (walsh, b) . research has documented the positive effects of deep faith, belief in a higher power, prayer and meditative practices, and congregational support in times of crisis (koenig, ) . in this time of sequestering and social restrictions, connections with nature are important to nourish spirits--from soothing bonds with companion animals (walsh, ) , to the rhythm of waves on the shore, the songs of birds, and the hopeful renewal of life with new birth. the transcendent power of music and the creative arts fosters resilience, expressing unbearable sorrow and restoring the spirit to rise above adversity. in this prolonged period of angst, i find music most uplifting; it also connects me with my mother, a gifted musician, whom i lost too soon. times of great tragedy can bring out the best in the human spirit: ordinary people show extraordinary courage, compassion, and generosity in helping kin, neighbors, and strangers to recover and rebuild lives. for many, their spirituality is connected to a purposeful dedication to social justice or climate change activism. creativity is vital in our lives, as we need to invent new ways to overcome pandemic-related challenges. in some communities, individuals and multi-generational families are exploring safe ways to come together by creating "social pods"-contact clusters for interpersonal connection and practical support. mental health professionals are needing to transform ways of providing therapy when social distancing and face coverings constrain in-person "face-to-face" office sessions. therapists and clients are gaining new skills and comfort with telehealth therapy, despite the limitations. many notice a silver lining: increased access to therapy for those whose stress overload, incompatible schedules, disabilities, or distances from offices prevented in-person sessions. this article is protected by copyright. all rights reserved finding creative ways to celebrate important events, such as birthdays and graduations, can revitalize spirits and reconnect all with the rhythms of life. one young couple, saddened when plans for an elaborate wedding had to be cancelled, instead held a simple, yet deeply meaningful backyard ceremony, under a homemade wooden arch covered with a trellis of white blossoms. we witnessed the couple's joyful union via zoom, along with family members across two continents, snapping memorable photos with a screen-saving click. they look forward to a festive post-pandemic party. whatever our adverse situation, we can make the most of it, practicing the "art of the possible": "do all you can, with what you have, in the time you have, in the place you are." more than surviving loss or managing stressful conditions, family processes in resilience can yield personal and relational transformation and positive growth. in struggling through loss and hardship, in active coping efforts, and in reaching out to others, families tap resources that they may not have drawn on otherwise, and gain new perspective on life (walsh, b) . similarly, studies of posttraumatic growth have found that individuals often emerge from life-shattering losses with remarkable transformations: gaining appreciation of life and new priorities; warmer, closer relationships; enhanced personal strengths; recognition of new possibilities or paths in life; and deepened spirituality (tedeschi & calhoun, ) . the experience of suffering and loss often sparks compassionate actions to benefit others or address harmful conditions. clinicians can encourage families to find pride, dignity, and purpose from their darkest times through altruistic initiatives. many report stronger bonds forged through shared dedication, such as mobilizing community action coalitions or medical research funding (walsh, b) . bereaved families can find strength to surmount heartbreaking loss and go on in meaningful lives by bringing benefit to others from their own suffering. one african-american family lost their beloved matriarch to covid- . she had worked tirelessly as a home healthcare provider but never had the healthcare herself that she needed. when the family sought testing and care for her symptoms of coronavirus, their community lacked essential resources that might have saved her life. her children were devastated by her loss, but agreed that she wouldn't want this article is protected by copyright. all rights reserved them to become consumed by anger and grief. they vowed to do something meaningful to honor her life and her memory. as her son related, "we want something good to come out of our tragedy. we're taking up fierce advocacy for changes in our healthcare system so everyone gets quality care, no community is left behind, and no family will suffer as our has. she will be smiling down on us with pride." in the wake of loss, families cannot bring back a deceased loved one or recover all that was valued and lost, yet their suffering and struggle can yield new purpose and life priorities. many report that a major life challenge spurred them to reappraise their priorities and stimulated greater investment in meaningful pursuits. in the peak of covid- hospitalizations, as neighborhoods put up lawn signs thanking healthcare workers, a teenager in one family expressed outrage: "thanking them is nice, but we should value them by making sure they have the equipment they need and by paying them what they are worth!" she and her parents mobilized community members to lobby for changes. many become more keenly aware of urgent needs for children and families. in the pandemic, parents are juggling incompatible demands of jobs, housework, childcare, and home schooling, with planned school openings precarious and daycare resources unavailable. gender disparities are starkly revealed for women who provide essential income and carry most responsibilities for homemaking and childrearing. with the economy reopening, many parents are between a rock and a hard place: forced to give up jobs to attend to children's needs. the difficulties experienced in home-based learning sharpened awareness of the vital importance of quality education and the undervalued and underpaid role of teachers in our society. it also exposed the striking lack of access for remote learning in under-resourced, low-income and largely minority neighborhoods, setting children back from achieving their potential. transforming new insights into meaningful actions requires initiative, persistence, and creative solutions. we are living through time out of the ordinary. with our life course seemingly on hold and future forecasts cloudy, we cope by trying to "be here now," focused on getting through each day and week. while we are restricted in our social space, we need not be trapped in time in the "here and now." this article is protected by copyright. all rights reserved time out: as the initial overwhelm with covid-related loss and disruption eases and we contemplate a long haul, it affords the opportunity to reflect on our personal and collective lives and to re-appraise our values and aspirations (bruner, ) . a crisis can be a wake-up call, heightening attention to what matters and who matters. in thinking more deeply about the "old normal" and "new normal" we realize that many aspects of our pre-covid lives that were normalized need to be changed for the better. as we expand our vision beyond our personal struggles, we see needs for broader systemic changes with more urgency. reconnecting with the past: we can learn and grow stronger from the past. this is the time to deepen understandings and connections to our past, to the joys and sorrows experienced. we can encourage family members to share sweet memories to revive spirits and bonds in these hard times. we can reminisce together over photos, make scrapbooks and pass on keepsakes to cherish. using technology or old-school pen and paper, adults and their children can interview family elders and record their life stories: how grandparents fell in love; what their lives were like in their times. in hearing about experiences of crisis and challenge, it's important to draw out accounts of resilient responses alongside the difficulties. how did they and their families get through the great depression and world war ii? the courage, tenacity, and ingenuity in dealing with past loss and hardships can inspire current efforts. moreover, gaining perspective on elders' lived experience can increase compassion for their shortcomings and deepen bonds (walsh, b) . re-envisioning the future. in a pandemic that is novel, complex, and changing, long-term forecasts are hazy. we must learn to live with considerable uncertainty with flexibility to adapt to new developments. many joke about making plans a, b, c and beyond. we can't control everything that will happen, but we can dream and direct our energies toward our preferred vision. when future hopes and dreams are lost, therapists can help family members to reorient hope and envision new possibilities. linking the past, present, and future. when death ends a life, it does not end relationships. research finds that healthy adaptation to loss involves not a "letting go" or detachment, but rather a transformation from physical presence to continuing bonds (klass, ; stroebe, schut, & boerner, ) . these bonds can be sustained through spiritual connections, memories, stories, keepsakes, deeds, and legacies. in this time of covid- , families will need to find innovative ways to honor accepted article and sustain connections: through meaningful memorial events, whether virtual or postponed; in websites to share tributes and remembrances. in varied ways, family members can find meaning and resilience in "saying 'hullo' again" (white, ) and re-membering those who have been lost. where bonds were frayed, they can be healed. therapists can foster an evolutionary perspective that integrates painful experiences and yields meaning and hope for the future. present time / precious time. the pandemic sharpens our awareness of the fragility of life and jolts us not to take future time for granted. the inevitability of losing others becomes more salient: what would we regret --things unsaid, undone--if a loved one died, or as we faced our own impending death. loss and threatened loss can heighten appreciation of loved ones taken for granted and spur efforts to repair grievances in wounded bonds. time does not heal all wounds, but offers new perspectives, experiences, and connections that can help people forge new meaning and purpose in their lives. over time, we will need to integrate the pandemic experience into the chapters of our individual and shared lives, strengthening the relational connections that matter to us: with the families we were born into, those we choose, and our wider communities. there is no love--or life--without loss. we are all mourners now, trying to guide one another as we navigate our way forward and strive to make a better world out of tragedy. our resilience is relationally-based, nurtured and fortified through our interconnections. by facing our vulnerability and by supporting one another through the worst of times we are better able to overcome daunting challenges to live and love fully. resilience is commonly thought of as "bouncing back," like a spring, to our pre-crisis norm. however, when events of this magnitude occur, we cannot return to "normal" life as we knew it. as our world changes, we must change with it. in the wake of the / terrorist attacks, i suggested that a more apt metaphor for resilience might be "bouncing forward" to face an uncertain future (walsh, ) . this involves constructing a new sense of normalcy as we recalibrate our lives to face unanticipated challenges ahead. over the ages, individuals, families, and communities have shown that, in coming together, they could endure the worst forms of suffering and loss, and with this article is protected by copyright. all rights reserved time and concerted effort, rebuild and grow stronger. the painful experiences in this pandemic will require time and shared reflection for meaning making, questioning old assumptions and grappling with a fundamentally altered conception of ourselves and our interconnections with all others in our shared world. taking a systemic view, the pandemic and our response will generate reverberations we can't foresee or control. mastering these challenges will require great wisdom and humanity in the months and years ahead. family sense of coherence and family adaptation the denial of death loss, trauma, and human resilience: have we underestimated the human capacity to thrive after extremely aversive events? ambiguous loss actual minds / possible worlds the neuroendocrinology of social isolation disenfranchised grief holding hope and hopelessness: therapeutic engagements with the balance of hope man's search for meaning covid- pandemic, unemployment, and civil unrest: underlying deep racial and 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disaster the dual process model of coping with bereavement: a decade on continuing bonds in adaptation to bereavement: accepted article this article is protected by copyright. all rights reserved toward theoretical integration beyond the concept of recovery: growth and the experience of loss bouncing forward: resilience in the aftermath of traumatic loss and major disasters: strengthening family and community resilience human-animal bonds: i. the relational significance of companion animals spiritual resources in family therapy applying a family resilience framework in training, practice, and research: mastering the art of the possible strengthening family resilience ( rd ed.) complicated loss: fostering healing & resilience loss and the family: a systemic perspective living beyond loss: death in the family bereavement: a family life cycle perspective reasonable hope: construct, clinical applications, and supports saying hullo again: the incorporation of the lost relationship in the resolution of grief beliefs and illness: a model for healing the myths of coping with loss accepted article key: cord- - ekgb zx authors: hjálmsdóttir, andrea; bjarnadóttir, valgerður s. title: “i have turned into a foreman here at home.” families and work‐life balance in times of covid‐ in a gender equality paradise. date: - - journal: gend work organ doi: . /gwao. sha: doc_id: cord_uid: ekgb zx this article explores the gendered realities of work‐life balance in iceland during the covid‐ pandemic, in particular how these societal changes reflect and affect the gendered division of unpaid labor, such as childcare and household chores. the study draws on open ended real‐time diary entries, collected for two weeks during the peak of the pandemic in iceland. the entries represent the voices of mothers in heteronormative relationships. the findings imply that, during the pandemic, the mothers took on greater mental work than before. they also described intense emotional labor, as they tried to keep everyone calm and safe. the division of tasks at home lay on their shoulders, causing them stress and frustration. the findings suggest that, even in a country that has been at the top of the gender gap index for several years, an unprecedented situation like covid‐ can reveal and exaggerate strong gender norms and expectations towards mothers. this article is protected by copyright. all rights reserved. the covid- pandemic is not only a health emergency and economic hazard but has also resulted in dramatic changes in people's personal lives, and roles within families have been disrupted. during the pandemic, many countries have taken drastic measures to reduce the spread of the virus, such as social distancing, lockdowns, and closing schools, public institutions, and workplaces. children and adults alike have been forced to stay at home for a shorter or longer time and upturn their lives as the home became the school, the workplace, the playground, sports facility, and family sanctuary. unesco has estimated that more than % of the world's student population, or around , billion students, has been affected by either temporary school closings or restricted services (unesco, ) . this entails increased care responsibilities for parents across the world. even though the number of dual earner households has been increasing for the last decades, findings of several studies indicate that women still bear the burden of childrearing and household labor in industrialized countries (alon, doepke, olmstead-rumsey, & tertilt, ; carlson, petts, & pepin, ; friedman, ; knight & brinton, ; t. miller, ; schwanen, ) . it can therefore be assumed that they are more affected by the closing of schools than their male partners. in fact, several studies (alon et al., ; andrew et al., ; carlson et al., ) and media coverage (see e.g. ascher, ; c. c. miller, ; topping, ) on the impact of covid- on families have indicated complications and challenges, as this unprecedented situation appears to have revealed or exaggerated existing gender inequalities and divisions within families. some have even referred to this strange situation as the s was revisiting homelife (ferguson, ) , indicating a backlash in terms of gender equality and power positions in the home during these circumstances. during previous crises, women have been more likely to either reduce their working hours or temporarily step down from work (alon et al., ; andrew et al., ) . we still pressure for the last few months and that mothers have spent less time on paid work and more time on household responsibilities as compared to fathers during the pandemic (andrew et al., ; carlson et al., ; collins, landivar, ruppanner & scarborough, ; craig & churchill, ; hennekam & shymko, ; manzo & minello, ; qian & fuller, ) . studies have indicated that young children tend to seek help and attention by interrupting their mothers, and that the mothers in turn experience time as more fragmented (collins, ; collins et al., ; sullivan & gershuny, ) which can become a bigger challenge in lockdown as the one during covid- . since the lockdown, more mothers participating in andrew's et al. ( ) research have reduced their working hours and those who have stopped working do twice as much child care and household duties as their male partners who are still working. conversely, in families where the male partner has stopped working but not the female, the parents share childcare and household duties equally even though the mother works at least five hours of paid work a day. qian and fuller ( ) argue that the pandemic is far from being an equalizer when it comes to gender equality, as their research indicates a widening gender employment gap among canadian parents with young children. the pandemic has not only affected schools, as many companies and businesses have been forced to adopt to the circumstances with more working-from-home and telecommuting opportunities for their workers (alon et al., ) . juggling childcare and paid work has been very challenging for parents, but then again, this has meant increased flexibility for many employees, flexibility that has often been discussed as the solution to a better work-life balance, especially for women (gatrell, burnett, cooper, & sparrow, ; sullivan, ; wheatley, ) . however, there are various intricacies around the interactions of gender equality and work-life balance in normal times, which seem to have intensified during the this article is protected by copyright. all rights reserved. pandemic as the pressure on parents' time increases (e.g. andrew et al., ; carlson et al., ) . iceland has been considered a frontrunner, even among the other nordic countries, in gender equality (the world economic forum, ), which makes it a particularly interesting setting in this regard. we believe that times like the covid- pandemic provide a unique opportunity to explore and shed light on deeply entrenched and gendered social structures within the organization of the family. in fact, research has already pointed in that direction (auðardóttir & rúdólfsdóttir, ) . thus, the focus of this study is to look at how the societal changes reflect and affect the gendered division of labor, especially concerning the unpaid labor of childcare and household chores, from the perspectives of mothers in heterosexual relationships. this was done by collecting daily real-time diary entries from almost mothers for two weeks during the peak of the pandemic in iceland while severe restrictions were being followed. important steps towards gender equality have been taken in the western part of the world over the years, not least in the nordic countries. these include improved legal frameworks, rising female employment and educational levels, and improvement in fathers' involvement in childrearing (evertsson, ; eydal & gíslason, ; gíslason & símonardóttir, ; jóhannsdóttir & gíslason, ) . despite these steps, the gender pay gap remains unbridged, reflecting the persistent idea of male provider roles (petersen, penner, & høgsnes, ; snaevarr, ) . iceland's reputation as the most gender equal country in the world has been quite prominent in public discourse and in the media, both in iceland and around the world. this media discourse has portrayed iceland as a paradise for women, implying that gender equality has more or less been achieved in iceland (hertz, ; jakobsdóttir, ; kilpatrick, ; this article is protected by copyright. all rights reserved. tuttle, ), which has even been used for international branding purposes (einarsdóttir, ) . despite the importance of recognizing that the ranking of gender equality as practiced by the global gender gap index, among others, has its limitations and overlooks important institutional variables such as social norms and values (einarsdóttir, ) , certainly iceland is doing well in international comparisons. women's educational attainment in iceland has steadily increased over the last few decades (bjarnason & edvardsson, ) , and in the year , icelandic women had the highest labor ratio among the oecd countries at . %. the same applies to men's labor force participation of . % (oecd, ) . despite this active participation in the labor force, icelandic women have established families at relatively young ages and the average birthrate has been rather high up until very recently in comparison with other northern european countries (hognert et al., ; jónsson, ) . in iceland, as elsewhere, women work part-time jobs in higher numbers, and mothers reduce their labor participation following childbirth more often than do fathers (gíslason & símonardóttir, ) . regardless of international trends towards increased active female participation in the workforce, the labor market is still very gender divided, and the rates of gender segregation both in line of work and educational choices are striking (dinella, fulcher, & weisgram, ) . the same manifestation applies to iceland (snaevarr, ) . over the last few decades, the government of iceland has taken some important steps in making laws and policies to facilitate fathers' involvement in childrearing responsibilities. the most substantial step is probably an act on shared parental leave passed in , which gave parents nine months in total, "dividing the nine months so that three are sharable while each parent has three that are strictly non-transferable" (gíslason & símonardóttir, , p. ) , and was lengthened by a month on january , (act on maternity/paternity leave and parental leave no. / with amendments). in iceland, research has indicated that discourses on motherhood in this article is protected by copyright. all rights reserved. relation to breastfeeding imply more intensive mothering that starts when the children are very young. this is somehow in opposition to the governmental emphasis on gender equality that aim to get fathers more in involved in parenting (gíslason & símonardóttir, ) . despite all these advancements, there are some signs that these have been achieved at a cost and there are some cracks in icelandic's glossy image as the frontrunner of gender equality (einarsdóttir, ) . in recent years, media coverage about people experiencing burnout has been more common, especially among professions like nurses and elementary school teachers (halldórsdóttir, skúladóttir, sigursteinsdóttir, & agnarsdóttir, ; the icelandic nurses' association, ; the icelandic teachers union, n.d.) , which in iceland are typically female professions. it appears that people are increasingly experiencing stress in their everyday live, which, if prolonged, can result in both poor physical and mental health (jónsdóttir, ) . over the last few years, research results from iceland have indicated that conflicts between work and family are quite frequent among icelandic parents, even though they do not consider housework alone to be a great burden (Þórsdóttir, ) . family obligations and issues related to the care of children are more likely to be woven into the mothers' working hours than fathers' (hjálmsdóttir & einarsdóttir, ) . there are also indications that parents are more likely to express difficulties when it comes to everyday chores than are workers without children and that parents experience conflict in balancing work and family (eyjólfsdóttir, ; hjálmsdóttir & einarsdóttir, ; Þórsdóttir, ) . work-life balance refers to the ability of every individual, regardless of gender, to coordinate work and family obligations successfully. work, in this context, refers to paid labor performed outside the home (wheatley, ) . studies have found that, when parents manage to balance family and working life, they are more satisfied with their life, which positively this article is protected by copyright. all rights reserved. impacts their mental and physical health (haar et al., ) . successful work-life balance can, therefore, be considered to be an important public health issue (lunau, bambra, eikemo, van der wel, & dragano, ) . a growing number of people describe increased time pressure in their daily lives and experience time being a scarce resource for all the task in their daily schedules (fyhri & hjorthol, ) . time is gendered, and bryson and deery ( ) have claimed that gender inequalities are sustained by differences in the use and experience of time among men and women and "that 'time cultures' are bound up with power and control" (p. ). research has indicated that men have, on average, more control over their time outside work than women. more claims are laid on women's time from family members. they feel more rushed in their daily lives and are more likely to be expected to attend to household work. women are also more inclined to multitask than men (bryson, ; craig & brown, ; friedman, ; rafnsdóttir & heijstra, ; sullivan & gershuny, ) . for the last few decades, some countries have been changing their policies to improve the opportunity parents have to balance work and family (gatrell, burnett, cooper, & sparrow, ; sullivan, ; wheatley, ) . such policies are often based on more access to subsidized childcare or flexibility. work flexibility has been argued to be desirable and a step towards gender equality, since it has enabled people's work-life balance (gatrell et al., ; haar et al., ; sullivan, ; wheatley, ) . alon et al. ( ) predict that the somehow forced flexibility of many workplaces caused by covid- might last after the pandemic has run its course and be beneficial for both mothers and fathers. nevertheless, work related flexibility has both pros and cons and can even cause stress. the division between work and home can become more blurred when the employees bring their work home and take care of family matters during working hours (hjálmsdóttir & einarsdóttir, ; wheatley, ) . it has also been argued that not all professions offer an this article is protected by copyright. all rights reserved. opportunity to enjoy the of taking work home or having different working hours. such flexibility is often dependent on educational level, as well as being related to the gendered division of the labor market (pedulla & thébaud, ) . female dominated profession, like teachers and nurses, often have strict attendance obligations in their workplaces and less opportunity for work flexibility (pétursdóttir, ; wheatley, ) . men enjoy the opportunity to have flexible working hours or work from home more often, and flexibility can be more likely to have a negative effect on women's careers (friedman, ) . as such, seemingly supportive policies can have different consequences for men and women (pedulla & thébaud, ) . the structure of the family as an institution has changed in recent years, including the composition of families and the roles of the genders, and each family member now has more complex roles (júlíusdóttir, ) . starting a family and having children has turned out to have different effects on the lives of men and women, and it seems to be less beneficial for mothers. more families now rely on dual-earnings, and although the number of females working in paid labor has been on the increase, there is still a lack of active participation among men in the home. this applies to iceland and many other countries (gíslason, ; petersen, penner, & høgsnes, ) . having children and family relations maintain and support gendered positions and divisions of labor in public and private lives. petersen et al. ( ) underline how important it is to take such aspects into consideration when it comes to the positions of men and women on the labor market. t. miller ( ) claims that the reasons behind caring practices and their gendered performances "can be multiple and are interrelated, operating at the interpersonal and broader structural, political, policy and cultural levels" (p. ). research has indicated that social structures and prevailing attitudes can influence the gendered division of labor in relationships this article is protected by copyright. all rights reserved. (dotti sani, ; evertsson, ) . household labor has often been referred to as invisible work (hochschild & machung, ) , and the conceptualization of family work can be ambiguous since scholars often use different explanations of what such work actually entails (robertson, anderson, hall, & kim, ) . here, we follow these lines of thought and the three constructs of family work, commonly referred to in family work studies: housework, childcare, and emotional labor. emotional labor relates to activities relevant to the emotional wellbeing of other family members and giving them emotional support (curran, mcdaniel, pollitt, & totenhagen, ) . in an attempt to distinguish between emotional labor and mental work, robertson et al. ( , p. ) suggest mental work as the fourth construct of family work which "includes the invisible mental work related to managerial and family caregiving responsibilities", such as managing, monitoring, scheduling, knowing, and organizing the family life. mental work cannot be delegated to someone who does not belong to the family, and within families, mothers are much more likely to be household managers (ciciolla & luthar, ; curran et al., ; hjálmsdóttir & einarsdóttir, ; robertson et al., ) . this type of work often goes unnoticed by other family members along with the mental burden that such responsibilities require but impacts the mother's wellbeing with feeling of being rushed and strained in everyday life (ciciolla & luthar, ; craig & brown, ) . it has also been pointed out that it can be difficult to detect mental work since it is quite often closely connected with other activities related to the family (robertson et al., ) . in addition, many parents, especially mothers, experience work-family guilt when combining work and family, experiencing conflict between the tasks in the public and private spheres (borelli, nelson, river, birken, & moss-racusin, ) , which can add to the mental load of everyday life. this article is protected by copyright. all rights reserved. also, people had to ensure that they kept a distance of at least two meters between individuals. this entailed closing of swimming pools, gyms, pubs, and museums. however, no changes were made to the organization of schools (government of iceland, b) from the previous measures. due to these actions, those who possibly could work from home were encouraged to do so (sveinsdóttir, ) . health, ), including no more than children in the same group and groups not being allowed to interact. it was common for students to attend school every other day, for school days to be shorter and for meals to be available for a small part of the student body. parents were, in some cases, encouraged to let their children stay at home if they possibly could, while parents in occupations such as doctors, nurses, and police were identified as priority groups. this meant that they were somewhat less affected by school closures and restrictions. students in th to th grade ( -to -year-olds) had to study from home via distance education. this article is protected by copyright. all rights reserved. after-school care was closed; sports and other extra curriculum actives were cancelled, and children were encouraged to only meet with the kids in their small groups outside school (icelandic association of local authorities, ). as in other countries, all these measures had severe impact on families with children, even though the schools technically never closed, and lockdowns were not imposed. this is the context in which this study was conducted in march and april of . on may , , social distancing restrictions were eased, meaning that all children's activities were more-or-less back to normal (government of iceland, a) -at least for the time being. this article draws from a real-time diary study conducted during the ban on public gathering in iceland. the first week of the diary study started on march th , and the second week kan, ; kitterød & lyngstad, ) . for the purpose of this study, we only analyze and present findings from the open diary entries. according to bolger et al. ( ) , diary studies are well suited to capturing the experiences and particulars of the life of the participants. since this is a real-time study with a minimum of time lapse between the experience and reflections, the likelihood of retrospection is minimized. one of the benefits of real-time diary studies like this one is that events are reported in a natural, spontaneous context. by doing so, the data becomes richer and important contextual information and meanings are pieced together to include in the study. this article is protected by copyright. all rights reserved. the sample is self-controlled as it consists of individuals who responded to an advertisement that we posted in various large and active icelandic facebook groups, such as brask & brall (a sales group with around . members), and through our own extended networks. facebook is the most popular social media in iceland, used regularly by nearly all icelanders (facebook nation, ), which makes it a good forum for reaching a considerable part of the population. in all, parents participated in the study, seven male and female. in an effort to shed light on the everyday life of mothers during covid- , we analyzed the open diary entries from female participants in heteronormative relationships, or mothers. about half of them lived in the reykjavík metropolitan area (n = ) while the others were spread around the country. the number of children in the homes of these mothers varied from one to six, but the majority (n = ) of the mothers had two children. the educational level of the participants was rather high, as a majority of participants held a university degree, with bachelor's degrees and with master's degrees. twenty-eight were in paid labor, four were on parental leave, one was an independent laborer, one was a student, one was both studying and working, one was on sick leave, and one was on disability. in most of the cases, both parents primarily or solely worked from home during the time of the study, and most of them were working full-time the whole period, even though some worked reduced hours due to the pandemic. in all cases, the children could attend schools up to some extent, but with severe restrictions of many sorts. after providing informed consent, participants were asked to answer a questionnaire consisting of background questions. then, they received a daily questionnaire via microsoft forms for two weeks. the purpose of the questionnaire was twofold; to collect structured time-use data (fisher, et al., ) , and open-ended diary entries in which participants would this article is protected by copyright. all rights reserved. write an "old style" diary, reflecting on everyday life during covid- . in the diary entries, participants were asked to reflect on their day, the impact of covid- on their life, division of household duties and responsibilities, and other issues they wanted to share. it is important to consider the risk of failure in distinguishing participants' reports of atypical experiences related to or caused by a major event or general experiences (bolger et al., ) . therefore, participants were asked to reflect specifically on their experiences in the context of covid- . the total word count of the written reflections was around . words, which provided us with rich qualitative data. we analyzed the written reflections drawing on braun and clarke's ( ) phases of thematic analysis. the text was sorted by date and participant before we read it several times, added notes, and discussed the content together. then, we coded the text, applying an inductive approach. this means that the initial coding of the diary entries was open and emphasized understanding the participants' experiences without engaging too much with existing literature and theories. similar codes and text segments were then collated in order to identify repeated patterns of meaning across the data: stress, work-life balance, and division of household duties. participants were promised confidentiality and that measures would be taken to prevent identification. we provided participants with a random personal participant number to ensure their anonymity. information that could link participants' names to the number was deleted right after the data collection period. participants were able to withdraw from the study at any time, and some did for unknown reasons. due to the limited time for the study, we decided to use the most convenient way possible to share information about the research and recruit participants, facebook. that probably affected both the number of participants, as the window of time to recruit this article is protected by copyright. all rights reserved. participants was limited, and how homogeneous the group became, particularly in terms of educational level. analysis of the data generated two themes, presented in two sections. the first concerns the complexities of work-life balance in covid- times, particularly the gendered interactions of stress, work-life balance, and mental work. the second section specifically draws on the emotional labor performed by the women in the study, some of which is represented by how conscious the women were of the well-being of their family members. the diary entries quite clearly described complications and stressful situations as the women were trying to juggle their time between work duties and childcare. they described how strained they were and how their stress level was increasing, using words like overwhelmed, frustrated, tired, annoyed, and angry to describe their situations. below are a few diary entries from mothers who were all working - % that reflect this. in the following example, a mother of a -year-old working in mass media, who worked entirely from home as did her husband, described one of her days like this: "i'm a little anxious because of all this, the situation in society. then, i do not have the energy to do much, only the necessary things. the child wore pajamas the whole day." she mentioned how the whole situation made her feel anxious and drained her energy. this was true of many of the other women, like this mother of three ( , and ) who worked in a nursing home explained: "now we have spent more than a month in quarantine and home-schooling. it has started to take its toll mentally, and the day today was difficult. i was almost in tears." her husband was still working in his workplace while she had taken a leave for the first weeks of covid- . juggling home-schooling, childcare, and work created a lot of pressure on the mothers and some of them described the guilt they were experiencing from feeling that they could not keep this article is protected by copyright. all rights reserved. up with everything. the next example is from a mother who worked full time at her workplace. she had two children, and years old, and wrote about her experience in the follow way. i experienced a slight panic attack on the way home over juggling all these different duties, and i cried a little. i went to the grocery store to get some time for myself and shopped for my sister who is in quarantine . . . no one has energy to start putting the kids to bed, so they went to sleep too late. . . jesus, how the parental-fuse is short, and i feel guilty about that. as these examples show, the mothers experienced stress, a lack of energy, and even guilt. as during 'normal' times mothers are more likely to experience work-family guilt, as they feel guilty about not being the best while not spending enough time with their kids, despite being on the run all the time (borelli et al., ; hjálmsdóttir & einarsdóttir, ) . during covid- , this pattern seems to have intensified, supported by research from other contexts as well (e.g. hennekam & shymko, ) . the levels of guilt and how it affected them was addressed by more participants. this mother had two children ( and years old) and was working full time. she and her husband were both working from home. i feel as if i should be able to organize my time better. the day passes, and i have not had time to enjoy one cup of coffee in peace. i do not sit down, but still the apartment is in chaos, the children neglected, and work unfinished. these examples show how much time pressure these mothers were under, and how they experienced guilt over not being able to complete their tasks, neither work nor family related. studies have shown that parents are under significant time pressure in their daily lives (fyhri & hjorthol, ) , especially women (sullivan & gershuny, ) . this pressure seemingly this article is protected by copyright. all rights reserved. increased greatly during the pandemic, as other research has indicated as well (alon et al., ; andrew et al., ) . the above example also indicates a level of multitasking as did entries from several other mothers in the study. according to previous studies (e.g. bryson, ; craig & brown, ; friedman, ; rafnsdóttir & heijstra, ; sullivan & gershuny, ) , women multitask more often than men. the experiences of these women indicate that their perceived time pressure and increased need for multitasking laid heavily on their shoulders. towards the end of the study, when restrictions because of covid- were somewhat lifted, some mothers mentioned that they had just realized how much constraint was caused by having to erase the boundaries between work and family life. in the following diary entry, a mother with a six-month-old child, who worked as a manager in a half time job, explained how. i went to my workplace for the first time in weeks. it was so different. i do not think that i realized until yesterday how much constraint comes from working from home with a child at home. i cannot wait until i can return to my workplace every day and create these boundaries between private life and work. this description is interesting in the light of how flexibility and working from home has often been portrayed as the solution to work-life balance, especially for women, to improve parents' opportunities to better balance work with home life (gatrell et al., ; wheatley, ) . some of the other mothers also described how the boundaries between home and work had been blurred. these experiences indicate that working from home can be difficult for parents, particularly mothers, since they find their work time being interrupted by other duties. this has been documented in previous research (e.g. wheatley, ) . alon et al. ( ) predicted that changes in working practices adopted during covid- might be permanent, but we argue that it is important to consider that working from home and having this article is protected by copyright. all rights reserved. flexible working hours must be considered very carefully in favor of the working parents, bearing in mind gendered social structures. it was clear from the diaries that these unprecedented times revealed or intensified unequal divisions of duties at home, which made the mothers realize and reflect on their positions at home. a mother of two ( and years old), who was a teacher working full time but had started working from home, as well as her husband, said that: today, there was a little clash at home. i have noticed that i usually write the diary before dinner, and a lot of work awaits me afterwards. i usually put the kids to bed, bathe them, tidy up endlessly (usually in the evenings when they are asleep), read, and tuck them in. today, i threw a tantrum over this, . . . but we had a good conversation, and everyone agreed to contribute more . . . [my husband] agreed with me that he could be more present in these daily routines around the kids and home. this example shows how being responsible for the kids and home was on her shoulders, as well being responsible for taking action to change the balance. a few days later, the same woman explained how she was starting to realize how the situation affected the division of tasks, partially because her husband prioritized differently, e.g., around work or exercise, and also because the children asked her for help even though their father was also at home. we knew that the division of tasks is rather equal in our everyday life, but now that we are both working from home, it is obvious that he takes his space when he needs to attend to 'his' things, and i run, and i sprint from my work much more than he does. this example shows how the mother was easily interrupted with household responsibilities, which is in accord with other research findings that suggest that mother's time is more often fragmented (collins, ; collins et al., ; sullivan & gershuny, ) . according to this article is protected by copyright. all rights reserved. andrew's et al. ( ) study, mothers more frequently combined their paid work with other activities during the pandemic. this illustration also supports the notion of time being gendered (bryson & deery, ) , as she perceived that her husband had more control over his time to tend to matters unrelated to work or family. this is in accordance with previous studies on gendered control of time among parents (bryson, ; friedman, ) and new research conducted during covid- that indicate that unpaid work performed by mothers has increased during the pandemic (craig & churchill, ; manzo & minello, ) . the responsibility to divide duties at home lay on the mothers' shoulders, as they explained in several diary entries. this shows how mental work (robertson et.al., ) was central to their gendered realities. as one said, "everyone has to have certain duties in the home if domesticity is supposed to work without me losing my mind." this mother had two teenagers and was working full time from home while her husband worked in his workplace. another one, who had two children ( and years old) and was working full time, explained her situation in this way. it is not easy working from home with a two-year-old. i had to make sure that his father takes him to his parent's home, who were away, so that i could get some peace. then, i put him down to nap after lunch and had to make sure that father and son woke up at the right time . . . usually, i must make sure that things work . . . how are you supposed to be an employee, parent, leisure worker, cook, and a teacher all at once? this outlines quite well how she experiences the responsibility of managing the household. the father is a participant, but she is the manager and carries responsibilities that add to the mental burden of everyday life (ciciolla & luthar, ) , exacerbating to the mental draining women have felt during covid- (hennekam & shymko, ) . another this article is protected by copyright. all rights reserved. mother, with a two-year-old child, who worked full time from home along with her husband, similarly wrote that: i have turned into a foreman here at home. i am trying to get clearer oversight over what has to be done and activate my husband to prevent everything from becoming a mess, and i do not want to take care of it all by myself. so, i had a family meeting and put up a clear division of duties. this mother also wrote that, on an everyday basis, they did not have a clear division of tasks, but during covid- , it became necessary. this indicates that times of crisis can reveal deeply rooted norms and structures on gender roles within the home. the experience of another mother, who had three children ( , and years old), further supports this. she was a care worker and she and her husband were both working in their workplaces. i became tired today and reprimanded my husband. i take care of the management, division of tasks and responsibility for the children's education and practices. i feel like we are dangerously close to the gender development as it was before the middle of the last century. also, it is my responsibility to remind [him] of that this is not supposed to be like this, so that also adds to my basket of duties. all of these examples show how the situation during the pandemic revealed and exaggerated the mothers' roles as household managers (ciciolla & luthar, ; curran et al., ) . they planned and organized family life to make sure that everything worked. this is consistent with research from australia where mothers felt unsatisfied with the division of labor in their homes during the covid- (craig & churchill, ) . drawing on previous studies (e.g. craig & brown, ) , this invisible mental work became a burden for the women and clearly affected their everyday wellbeing. interestingly, this also added to their this article is protected by copyright. all rights reserved. duties, as they became somewhat responsible for getting other people in the household, particularly the fathers, to take on more responsibility to even the load. some of the women in the study described how they made an effort to hide their stress and anxiety from their children and other family members in order to ease the atmosphere and keep the family calm. in accordance with studies and theories of gendered aspects of emotional labor (ciciolla & luthar, ; craig & brown, ; robertson et al., ) , the women performed that kind of labor in addition to other duties. this is reflected in the words of a mother of two children, nine and ten, working full time mostly from home with a husband who mostly worked away from home. the days are getting really difficult, and i will take my first summer holiday tomorrow. the younger child is not happy about [the situation] and cries over everything that seems like adversity, even as little things like when she is asked to read or tidy up. the little patience i have is running out, but i try my best not to let her see it. the day after, the situation became worse, as the family was facing possible quarantine and they were waiting for further directions from a national team of contact tracers. she wrote this in her diary. now we possibly have to start days of quarantine. we will know tomorrow. at least we have to remain in quarantine for hours until the test results. i am pained by this situation, but i try to stay positive, especially with my husband and children. they cannot may not see [my] anxiety because then they become afraid. i continue to meditate and do yoga; everything will be ok. this article is protected by copyright. all rights reserved. as these diary entries show, this mother found it important to keep her anxiety to herself in order to keep the family calm. another mother with a five-and eight-yearold who worked in an elementary school was working full-time from home as did her husband. she described how difficult her day was, as one of her children cried a lot because she missed her friends so dearly. the day "was spent tending emotionally to the children." the women in the study had to devote time to emotional labor instead of work. another reflected on how she tried to calm the people around her. i am really focused on being well informed so that i can answer [questions] and calm elderly people and children around me. i am very cautious and try to follow up with my children on how to be careful without frightening them. one of the women explained how her husband was irritated because of the situation and tired because he was working shifts, so much so that he "exploded" at times. therefore, she made an effort to try to make sure that his irritation did not affect the children ( , and years old) too much. she was working % from home while he was working away from home. she explained. i take care of the children and the home every day, since he is asleep until he has to go to work or loafs around on the computer. everyone has a short fuse, but i make sure that i intervene and suggest a break, that everyone goes out, plays or when the children are starting to nag. it is difficult to be able to concentrate on work. this article is protected by copyright. all rights reserved. another example of the women's emotional labor included dealing with difficult thoughts and decisions related to the pandemic. a mother of two ( and years old) wrote that: despite a lot of physical resting lately, my mind has been spinning around worries and difficult decisions. should the children attend school or not? can i meet my father [who has heart problems] if i keep a m distance? is it necessary to disinfect all the groceries? according to curran et al. ( ) , this kind of work can be called emotional labor, as these women emphasize how they tend to the emotional wellbeing of other family members. this kind of labor was not limited to their children; it also applied to other relatives. for example, the emotional labor involved phone calls to parents or other relatives, sometimes several times a day. other studies have shown that this is often part of women's routines (ciciolla & luthar, ; robertson et al., ) . the months of covid- have been and are quite challenging for many families, and the drastic measures that have been taken to prevent its spread have meant severe changes to people's participation in everyday live and social contact (brooks et al., ) . in accordance with new research on the effect of covid- on everyday life (andrew et al., ; carlson et al., ; collins et. al., ; craig & churchill, ; manzo & minello, ; qian & fuller, ) , the time during the social restrictions was not easy. it is apparent from the diary entries of our participants that the period with the tightest restrictions was challenging for the mothers and their families, and they expressed feelings of frustration and being overwhelmed. despite advances in gender equality over the last decades, drastic events such as during the this article is protected by copyright. all rights reserved. covid- pandemic, can elicit situations that we do not necessarily pay attention to in our busy daily lives or even resist recognizing. in iceland, which has been portrayed as a "paradise for women" (jakobsdóttir, ) and which is considered a global frontrunner when comes to gender equality (the world economic forum, ), parents face challenges related to gendered realities, and gender equality has not been achieved regardless of what the dominant discourse may say. despite remarkably high labor participation, there are indications that women in iceland shoulder the greater burden of childcare and household labor (hjálmsdóttir & einarsdóttir, ; Þórsdóttir, ) , as elsewhere around the world (alon et al., ; knight & brinton, ; t. miller, ) . the diary entries of the mothers in the study demonstrates a gendered reality in which they experience burdens that seem to have escalated during the pandemic. it was stated in a media coverage that the covid- pandemic had brought back the s regarding gendered division of labor (ferguson, ) . the same phrase was used by one of our participants. some of the women wrote about how surprised they were about how much of the household chores and the childcare remained on their shoulders. despite some steps towards gender equality in the last few decades, there are few signs of a revolution, especially within the home. the focus on the struggle for gender equality has somehow been more on the public sphere, as reflected in the measures used for gender equality indexes that overlook the gendered division of labor in the home along with social norms and values (einarsdóttir, ) . one of the patterns identified in the reflections of the women in our study was how they seemed to be stunned by how uneven the division of labor turned out to be during the pandemic and how much time and energy they devoted to household chores and the management of the household, carrying out the mental work within the family. their experiences support the idea of time being gendered (bryson, ) , as they described how this article is protected by copyright. all rights reserved. their time was more restricted from childcare and household chores and how they prioritized their children's needs over work. when the families were pushed into the home due to lockdowns and social restrictions, women faced an uneven division of labor that they might have been too busy in our daily lives to observe or might have found difficult to acknowledge. we argue, based on this study as well as emerging findings from larger studies from different countries (andrew et al., ; collins et. al., ; craig & churchill, ; manzo & minello, ; qian & fuller, ) , that the situation caused by the pandemic brought to light pre-existing gendered performances and social structures, more than it caused drastic gendered division of labor in the home. in iceland, where the dominant discourses have centered on the country as a global leader in gender equality, the existing inequalities have been overlooked. our findings suggest that there is an uneven division of labor within icelandic homes as the mothers in the study bore the burdens of housework, childcare, emotional labor, and household mental work. if the aim is to close the gender gap both in the public and the private sphere, a focus on the gendered division of labor within the home is essential. the impact of covid- on gender equality. crc tr discussion paper series how are mothers and fathers balancing work and family under lockdown? 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qualitative sociology, - . advance online publication covid- and the gender gap in working hours. gender, work & organization, - . advance online publication feeling rushed: gendered time quality, work hours, nonstandard work schedules, and spousal crossover dual-earner parents' couples work and care during covid- . gender, work & organization, - . advance online publication gender, emotion work, and relationship quality: a daily diary study sex-typed personality traits and gender identity as predictors of young adults' career interests men's employment hours and time on domestic chores in european countries all that glitters is not gold: shrinking and bending gender equality in rankings and nation branding gender ideology and the sharing of housework and child care in sweden samraeming fjölskyldulífs og atvinnu: hvernig gengur starfsfólki á íslenskum vinnumarkaði að samraema fjölskyldulíf og atvinnu? (m.sc. dissertation) iceland magazine i feel like a s housewife': how lockdown has exposed the gender divide. the guardian exploring new ground for using the multinational time use study. iser working paper series still a "stalled revolution"? work/family experiences, hegemonic masculinity, and moving toward gender equality children's independent mobility to school, friends and leisure activities work-life balance and parenthood: a comparative review of definitions, equity and enrichment parents, perceptions and belonging: exploring flexible working among uk fathers and mothers faeðingar-og foreldraorlof á Íslandi: Þróun eftir lagasetninguna árið mothering and gender equality in iceland: irreconcilable opposites? iceland eases restrictions -all children's activities back to normal stricter measures enforced in iceland: ban on gatherings of more than people outcomes of work-life balance on job satisfaction, life satisfaction and mental health: a study across seven cultures tengsl streituvaldandi þátta í starfsumhverfi, svefns og stoðkerfisverkja hjá millistjórnendum í accepted article this article is protected by copyright. all rights reserved. heilbrigðisþjónustu [correlation between stressful factors in the working environment coping with the covid- crisis: force majeure and gender performativity. gender, work & organization why iceland is the best place in the world to be a women. the guardian mér finnst ég stundum eins og hamstur í hjóli the second shift: working parents and the revolution at home high birth rates despite easy access to contraception and abortion: a cross-sectional study icelandic association of local authorities how to build a paradise for women. a lesson from iceland vinnutengd streita. orsakir, úrraeði og ranghugmyndir [work related stress. causes, resources, and misbeliefs Ársrit um accepted article this article is protected by copyright childbearing trends in iceland, - : fertility timing, quantum, and gender preferences for children in a nordic context fjölskyldur -umbreytingar, samskipti og skilnaðarmál reykjavík: félagsvísindastofnun háskóla Íslands measuring housework participation: the gap between "stylised" questionnaire estimates and diary-based estimates iceland has become the first country to officially require gender pay equality diary versus questionnaire information on time spent on housework-the case of norway one egalitarianism or several? two decades of gender-role attitude change in europe a balancing act? work-life balance, health and well-being in european welfare states ) mothers, childcare duties, and remote working under covid- lockdown in italy: cultivating communities of care nearly half of men say they do most of the home schooling. percent of women agree. the new york times paternal and maternal gatekeeping? choreographing care auglýsing um takmörkun á skólastarfi vegna farsóttar labour force statisics can we finish the revolution? gender, work-family ideals, and institutional constraint from motherhood penalties to husband premia: the new challenge for gender equality and family policy, lessons from norway within the aura of gender equality: icelandic work cultures, gender relations and family responsibility covid- and the gender employment gap among parents of young children balancing work-family life in academia: the power of time mothers and mental labor: a phenomenological focus group study of family-related thinking work gender differences in chauffeuring children among dual-earner families launamunur karla og kvenna [the pay gap between men and women key figures, statistics bad mum guilt': the representation of 'work-life balance'in uk women's magazines speed-up society? evidence from the uk and time use diary surveys kórónuveiran: fyrirtaeki hvött til að þjálfa fólk í fjarvinnu [the coronavirus: companies encourage to train workers for distance work the directorate of health and the department of civil protection and emergency management the icelandic teachers union. (n.d.). streita og kulnun the global gender gap report working mothers interrupted more often than fathers in lockdown -study. the guardian one country is making sure all employers offer equal pay to women covid- educational disruption and response good to be home? time-use and satisfaction levels among home-based teleworkers vinna og heimilislíf reykjavík: félagsvísindastofnun háskóla Íslands this article is protected by copyright. all rights reserved. key: cord- -mvoq vln authors: nan title: autorenregister date: - - journal: med genet doi: . /s - - - sha: doc_id: cord_uid: mvoq vln nan complex mechanisms of dosage compensation regulate the mammalian x chromosome due to the presence of one copy in males (xy) and two in females (xx). x inactivation silences one x chromosome in females in early development, leading to specific epigenetic and structural changes. the inactive x chromosome becomes condensed and forms a bipartite structure within the nucleus, as we have shown by chromatin conformation analyses. specific long non-coding rnas are implicated in the formation of this unique structure. the inactive x chromosome is preferentially located near the lamina or the nucleolus. genes that escape x inactivation tend to be located at the periphery of the condensed inactive x chromosome. such genes are more highly expressed in females, and thus associated with sex-specific differences manifested even in early development. we have found that significant sex bias in gene expression are associated with escape from x inactivation in human tissues from normal males and females, and in tissues from individuals with sex chromosome aneuploidy, including turner or klinefelter individuals. institute for genomic medicine, columbia university medical center, new york, usa a central challenge in human disease genetics is the identification of pathogenic mutations. one key approach to distinguishing benign and pathogenic mutations is to use population genetic data to identify regions of the human genome under purifying selection. here i describe how the residual variation intolerance scoring framework has been applied to identifying pathogenic mutations in and outside protein encoding regions of the genome. next i report how these are related approaches are being used to identify pathogenic mutations in large-scale scale studies in epilepsy and other neurodevelopmental diseases. finally, i discuss how the identification of genetic causes of disease can inform treatment choices. scents indicate things, make promises, attract attention and stimulate imagination, feed anxieties and hopes: they are the salt in the atmospheric soup. we regard seeing and hearing as more important sensory functions, because they contribute more to conscious, cognitive processes of perception -but at moments of the greatest enjoyment we close our eyes and taste the scent, smell the taste. before the spirit and beauty of a person can fascinate us, our nose must become infatuated. the olfactory system in the nose acts as a window, monitoring environmental chemical information and convert chemical stimuli in electrical nerve impulses which are conducted along the olfactory sensory neuron to their glomerular target in the brain. olfactory receptors (ors) activation shows the distinguished (camp-based) transduction pathway for odorant perception. in buck and axel discovered the olfactory gene family, the largest gene family in the human genome, and postulated an exclusive expression in the olfactory epithelium. however, recent whole genome sequencing data from our and other labs show that ors have been found in every tissue of human body which was analyzed by next generation sequencing. the importance of such ectopic expression of ors is raised since the physiological function of some of ors was characterized. when identifying additional expression profiles and functions of or in non-olfactory tissue, there are limitations posed by the deorphanization of ors concerning the activated ligands and by the small number of antibodies available. in contrast to the olfactory sensory neurons which are believed to express all functional or genes (only one or type per cell), cells in non-olfactory tissues tend to express more than one individual or gene per cell. in addition, some of the signaling pathways in non-olfactory tissues seem to involve completely different components in comparison to the olfactory neurons. what is the functional role of these ectopically expressed olfactory receptors? evidences rapidly accumulate that ors participate in important cellular processes outside its primary sensorial organ where they function in odor detection and discrimination. in our lab the functional expression of the first was demonstrated in spermatozoa ( ) . in the meantime we could show the existence and function of ors in the cardiovascular system (heart, blood cells), the gastrointestinal system (small intestine, liver, pancreas), the genito-urinary system (kidney, testis, spermatozoa, prostate), the respiratory system (lung, smooth muscle cells), the skin (keratinocytes, melanocytes) and sensory organs (retina). interestingly we found a broad spectrum of important functions like cell-cell communication and recognition, tissue injury, repair and regeneration, cancer growth, progression and metastasis, nutrient sensing and muscle contraction. nevertheless the functional importance of ectopic ors is still not sufficiently understood. studies seeking to determine the function of ectopic ors are still in its infancy and require further intensive exploration. however, the potential of ors to serve a target for a wide range of clinical approaches is indeed given. this hold promises that the knowledge gained by future investigations would lead to deepen our understanding of or function in health and disease and may provide the basis for the development of applications in diagnosis and therapies in near future. enzyme replacement therapies have been developed over the last years for several of the lysosomal storage disorders (lsd's). the success of enzyme replacement therapy for gaucher disease paved the way for the development of similar treatments for the mucopolysaccharidoses, fabry and pompe disease and lately also for neuronopathic lysosomal storage disorders by intrathecal or intracerebral injections. in addition, small molecule approaches have been developed including substrate reduction therapies and chaperones, which can be used orally. while in gaucher disease enzyme as well as substrate reduction therapy results in reversibility of disease manifestations, with decreases in hepatosplenomegaly, normalization of blood counts and prevention of skeletal disease, this is unfortunately not the case for all patients affected with other lysosomal storage disorders. an important concept is the "window of opportunity for treatment" which is different for these disorders. for example, in fabry disease, early fibrosis fects are well defined, neither the specific mechanisms underlying neurological abnormalities nor the role of decreased cholesterol versus sterol precursor accumulation in disease pathogenesis have been clearly delineated. to identify cellular phenotypes and causative signaling pathways, we derived induced pluripotent stem cells (ipscs) from slos and lath subjects to model these diseases in vitro. slos subjects were known carriers of the most common dhcr mutations, including the intronic splice acceptor mutation c. - g>c and the missense mutation p.t m. while all ipscs demonstrated the expected biochemical defects due to dhcr or sc d mutations, cellular assays uncovered a defect in neural stem cell maintenance resulting in accelerated neuronal formation in slos ipscs. further molecular and biochemical analyses demonstrated inhibition of cholesterol-wnt interactions and loss of wnt/β-catenin activity mediated cellular phenotypes. however, this cellular phenotype was exclusive to slos, as lath ipscs did not exhibit a neural progenitor defect or inhibition of wnt/β-catenin activity. while this work demonstrates the utility of ipscs for modeling rare diseases and identifies signaling deficits potentially underlying slos phenotypes, questions remain regarding cellular and functional consequences, the specificity of lipid-wnt interactions, and the role of other disrupted signaling pathways in mediating developmental and functional deficits in these diseases. unpublished work using a variety of approaches will be discussed comparing the specific effects of cholesterol synthesis mutations on cell fate, functional activity, and lipid modulated signaling pathways to more precisely define the consequences of cholesterol synthesis defects and identify potential targets for patient therapy. induced pluripotent stem cell (ipsc) technology has become one of the major approaches for disease modeling since its first report in . the ability to reprogram cells from somatic into embryonic stem cell-like state and to differentiate them into desired cell types in the culture dish has allowed scientists to carry out the study of several diseases in cells such as neurons which, in the past, could not be isolated from living subjects. williams syndrome (ws), a genetic neurodevelopmental disorder where - genes are hemizygously deleted, is among those. despite cardiovascular abnormalities, its unique neurological phenotypes i. e. hypersociability is of our interest. for several decades, research on different neurological aspects of ws has been conducted in a variety of models such as patient-derived cell lines (lymphoblastoid cells and fibroblasts), post mortem tissue, and mouse models. however, the lack of physiologically relevant cell types such as neural progenitor cells (npcs) and neurons has left a critical gap in our knowledge the disease's cellular and molecular phenotypes. to fill this gap, we took the advantage of the reprogramming technology to capture the genomes of ws subjects in ipscs, which could be then differentiated into npcs and neurons, enabling evaluation of whether the captured genome with hemizygous deletion of those genes leads to relevant neuronal cellular phenotypes. dental pulp cells-derived ipscs of classical ws, rare ws and typical developing (td) subjects were neurally induced via dual-smad inhibition in order to generate npcs and neurons. we discovered that classical ws npcs exhibited increased apoptosis, and, therefore, doubling time, compared to td neurons. this could possibly contribute to the reduction in cortical surface area in classical ws individuals as assessed by magnetic resonance imaging. surprisingly, we found that rare ws npcs behaved similarly to td npcs rather than to classical ws npcs in terms of apoptosis. we confirmed that frizzled , which is deleted in the classical ws but not in our rare ws genome, is responsible for such phenotype via gain-and loss-of-function assays. moreover, classical ws neurons in general showed increased frequency of activity-dependent calcium transient compared to td neurons. finally, classical ws neurons acid alpha-oxidation, and ( .) glyoxylate detoxification. with respect to peroxisomal fatty acid oxidation peroxisomes catalyze the chain-shortening of certain fatty acids including very-long-chain fatty acids, but requires the active help of mitochondria to catalyze the degradation of acetyl-coa and the reoxidation of nadh as produced in peroxisomes. furthermore, with respect to ether phospholipid biosynthesis peroxisomes heavily rely on the endoplasmic reticulum to complete formation of ether phospholipids whereas fatty acid alpha-oxidation also requires the functional interplay between peroxisomes and mitochondria and the same is true for glyoxylate detoxification. recent evidence holds that the interaction between peroxisomes and the different subcellular organelles, including mitochondria and endoplasmic reticulum, is mediated by specific tethering protein complexes which bring organelles physically together thereby allowing metabolism to proceed smoothly. the importance of peroxisomes in metabolism is stressed by the existence of a large group of single peroxisomal enzyme deficiencies of which x-linked adrenoleukodystrophy is best known. our current state of knowledge with respect to the role of peroxisomes in metabolism and the peroxisomal enzyme deficiencies will be presented at the meeting. huntington's disease: rna-sequencing, small rna-sequencing, chip-sequencing and gwas data. department of neurology, boston university school of medicine, boston, ma , usa huntington's disease (hd) is a dominantly transmitted neurodegenerative disease of midlife onset. recently several different unbiased genome wide studies in hd have been performed. these analyses point to a variety of pathological pathways that are associated with important features of the disease, including age at onset, cag repeat size, and the extent of neuropathological involvement. genome wide association studies (gwas) have identified several regions of the genome that contain genes that are associated with the age at onset for hd. the strongest of these is located at q . for rs for which a very rare allele (maf = . %) is associated with an approximate -year younger age at onset for carriers of the minor allele. the same locus contains an independent effect for rs where a more common minor allele (maf = . %) is associated with a . year older age at onset for carriers of this allele. these single nucleotide polymorphisms are in the region of fan , mtmr and several other genes; some of which are not expressed in brain and are not likely candidates for hd modification. eqtl analysis has not resolved which gene may be implicated. other gwas implicated loci include rs at q . and rs at p . . we have sought to combine the information derived from multiple platforms to gain additional insight into the pathways that may be implicated in hd pathogenesis. in this strategy, we have performed mrna-sequencing, small rna-sequencing and chip-sequencing using the h k me mark for active transcription and the repressive mark h k me in human hd brain samples with gwas genotyping. while the striatum is most involved in hd, the extent of neurodegeneration in post-mortem tissue precludes meaningful comparison between disease and control samples, and consequently we studied prefrontal cortex (ba ). several common pathways were seen across these three platforms. mrna-sequencing and mirna-sequencing data identified altered transcriptional profiles implicating developmental pathways involving the hox genes and related homeo-box domain genes (e. g. pitx , pou f , etc.) . notably, micrornas located in hox gene clusters were among those most increased and levels of these correlate with pathological involvement in the striatum. these genes, associated with early embryonic development, are commonly silent in normal adult brain, and were among the most differentially expressed genes in hd brain. these prominent statistical effects are driven by the near total absence of expression in normal brain. pathways implicated in mrna-seq and chip-seq studies, included immune function and regulation of gene expression. these associations were very strong, indicating a large immune reactive response in the hd in the heart is related to unresponsive disease and unfortunately fibrosis may occur already in an early stage, sometimes even without prior hypertrophy. whether earlier intervention will be beneficial is largely unknown. and then: what is early? many unresolved questions exist at this stage, including the following: -what is the natural history and "point of no return" for the different lsd's? -what is the natural history and "point of no return" for subgroups of patients within one lsd's? -what are the long term complications: treatments change the phenotype rather than cure the disease -what is the influence of antibody generation on clinical effectiveness? -how do we manage the extreme costs of these products, especially in light of the many unsolved issues with respect to effectiveness? surprisingly, so far healthcare professionals, governments and industry have failed to systematically address these issues, resulting in insufficient knowledge for potentially lifesaving treatments. early conditional access, followed by a strict, transparent, independent, collaborative evaluation in addition to fair pricing should be explored. after the recent explosion in sequencing throughput, variant interpretation has quickly become the bottleneck in our effort to usher in the era of genomic medicine. while homozygosity for apparently pathogenic variants in the context of disease states is a well-established phenomenon, homozygosity can uncover many medically relevant aspects of the human variome that are difficult to study otherwise. for example, seemingly benign variants may prove pathogenic in the homozygous state. this includes variants with benign prediction using in silico tools as well as variants in dominant genes with no phenotype in carriers because they represent bona fide recessive inheritance. variants that are associated with one phenotype in compound heterozygous states may express themselves quite differently phenotypically when homozygous. furthermore, previously reported pathogenic variants can be challenged when their presence in homozygosity is associated with no abnormal phenotype, thus improving the specificity of the annotation of the morbid genome. homozygosity for lof variants is a special scenario that allows us to study naturally occurring human "knockouts", a powerful tool to study the physiological context of genes in humans. finally, homozygosity in the context of autozygosity provides a robust mapping tool that can greatly aid in the identification of relevant variants, especially those that exert their pathogenic effect in ways that defy detection by our usual algorithms. by expanding the spectrum of phenotypes that are studied, one can unlock the full potential of homozygosity to understand the medical relevance of the human variome in it its full range from embryonic lethal to essentially benign. helmholtz zentrum münchen gmbh, institut für experimentelle genetik, ingolstädter landstr. , neuherberg, germany the inheritance of epigenetic information in mammals across generations has been controversial. some reports provided initial evidence that a paternal high fat diet may propagate obesity and glucose intolerance in offspring, but potential confounders such as molecular factors present in seminal fluid, paternal-induced alterations in maternal care or transmission of microbiomes were not ruled out in these studies. we have shown in mice that a parental high fat diet (hfd) renders offspring derived via in vitro fertilization (f ) more susceptible to develop excessive overweight and type diabetes (t d) in a gender and parent-of-origin specific mode. female, but not male, offspring from obese parents became significantly more obese during a hfd challenge than female offspring from lean parents. body weight trajectories and distribution patterns of individual body weights in female offspring from one obese and one lean parent demonstrate that paternal and maternal germline propagate obesity in a roughly equitable and additive fashion, but likely different mode of action. in contrast, a more deteriorated state of hfd-induced insulin resistance was observed in both f genders, albeit predominantly inherited via the maternal germline. towards the identification of epigenetic information in sperm and oocyte from hfd and low fat diet fed parents, we are currently analyzing their transcriptome and methylome signatures. the status of this analysis will be presented. we report for the first time epigenetic inheritance of an acquired metabolic disorder via mammalian oocytes and sperms excluding confounding factors. such an epigenetic mode of inheritance may contribute to the observed pandemic increase in obesity and t d prevalence rates, especially in an environment where nutrition is abundant. brain which may be a major influence contributing to neurodegeneration. in many instances enrichment of h k me at transcription start sites was not accompanied by a corresponding increase in expression. the apparent inconsistency suggests that common regulatory mechanisms in the hd brain are disrupted and this may contribute to a complex interplay of factors contributing to the neurodegenerative process. often findings in human hd brain samples conflicted with those reported in hd transgenic mouse models, suggesting that one may wish to be cautious in interpreting the significance of either type of study in isolation. the causal pathogesis of huntington disease, new therapeutic approaches r. laufer senior vp discovery and product development global r&d, teva r&d product development mgmt. hatrufa st, netanya, israel huntington disease (hd) is an autosomal dominant neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, cognitive decline and death - years after motor onset. hd is uniquely caused by a polyglutamine encoding cag expansion in the huntingtin gene (htt), which allows for identification of pre-manifest mutation carriers as much as decades before onset and should facilitate development of disease modifying therapies. yet over years after identification of the hd mutation, available therapies offer only symptomatic relief and are fraught with side effects. development of safe small molecule therapies for hd has been hindered by difficulties identifying and validating tractable drug targets within the disorder's complex pathogenesis. teva pharmaceuticals is developing potential novel treatments based on a mechanistic understanding of disease pathways common to neurodegenerative diseases. the progress of these studies will be reviewed. rare complete gene knockouts in adult humans p. sulem statistics department, decode genetics, sturlugata , reykjavik, iceland loss-of-function mutations cause many mendelian diseases. here have create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. we sequenced the whole genomes of over , icelanders and imputed the sequence variants identified in this set into a total of chip-genotyped and phased icelanders. of the genotyped icelanders, around % are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (maf) below % in close to genes (complete knockouts). genes that are highly expressed in the brain are less often completely knocked out than other genes. homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of per , transmissions for variants with maf < %, % confidence interval (ci) = - ). we are currently systematically phenotyping such human complete knock out. this phenotyping lasts hours and attempts to cover most of the observable diversity in a non-invasive and cost efficient manner. i will demonstrate how using systematic phenotyping can advance the knowledge on individual gene knockout. we use results from in-house transcriptomics, existing animal models and complementary approaches to assess the observation in human. we will also discuss the scrutiny in other population in order to detect such complete knock-out. we will exemplify the impact of founder population and consanguinity in such an odissey. early onset and severe obesity can be inherited via loss of function mutations within the melanocortin pathway of hypothalamic body weight regulation. the most prominent player in this signalling pathway is the fat cell hormone leptin. leptin gene mutations were the first to be linked to monogenic early onset obesity. after binding of leptin to leptin receptors in the arcuate nucleus of the hypothalamus the neuropeptide msh is processed from the precursor pomc and acts as a ligand at the mc receptor. mutations in the leptin receptor gene, the pomc gene and the mc receptor gene were subsequently diagnosed in further patients with extreme early onset obesity. while leptin mutation patients can be treated with recombinant leptin -as shown already in the late s -all other monogenic obesity forms are leptin resistants, and additional leptin failed to decrease body weight. only recently pomc gene deficient patients were successfully treated with the msh-analogue setmelanotide (kühnen et al. ) . common severe obesity is defined by the lack of disease causing monogenic defects. a plethora of gwas identified a large number of snps in common obesity associated with the individual bmi but only to a low amount of not more then %. however, almost all these common obese patients are characterized by high leptin levels suggesting sufficient generation of leptin in the increased fat tissue and a state of leptin resistance. several new data concerning the contribution of epigenetic and genetic variants in the pomc gene locus argue for a role of the melanocortin pathway also in common obesity and imply, therefore, a potentially new treatment option also in common obesity based on msh-analogues. genome-wide association studies have highlighted the role of genetic associations with susceptibility to common inflammatory diseases, highlighting potential new insights into disease pathogenesis and opportunities for therapy. however understanding the functional basis of these associations and delivering translational utility remains a significant challenge to the field. non-coding regulatory genetic variants are most commonly implicated in such studies. recent work highlights how such variants are also major drivers of diversity in the immune response transcriptome. this talk will discuss approaches we are taking to try and establish functional links between immune phenotype-associated regulatory genomic and epigenomic variation, and specific modulated genes and pathways. i will describe insights from the application of expression quantitative trait (eqtl) mapping to define genomic modulators of the global transcriptomic response in different primary immune cell populations and to specific innate immune stimuli in health and disease. this work highlights the extent of local and distant context-specific eqtl, enabling resolution of immunoregulatory variants and the identification of specific modulated genes involving disease associated loci. examples will be described showing how mapping trans-regulatory loci can be a powerful approach for discovery and dissection of gene networks informative for disease. i will also show how we have applied analysis of the genetics of gene expression in patients with sepsis admitted to intensive care, revealing new insights into disease pathogenesis. further progress in this area will require characterisation of associated variants in the context-specific disease relevant epigenomic landscape in which they may act, requiring careful consideration of relevant immune cell types and environmental modulators to study, to-abstracts aktuellen stellungnahme der deutschen forschungsgemeinschaft sollen humane genomsequenzierungen die möglichkeit der rückmeldung von analyseergebnissen enthalten. als orientierung für einen verantwortungsvollen umgang mit dieser frage wird auf die projektgruppe eurat verwiesen. dennoch bleibt das problem der einordnung mitteilungswürdiger ergebnisse aus forscher-und probandensicht und der bereitstellung der für aufklärungs-und rückmeldungsalgorithmen erforderlichen ressourcen. darüber hinaus ist bis heute nicht geklärt, welche kommunikativen prozesse eine ausreichende basis für ein informiertes einverständnis darstellen. diese und weitere fragen möchten wir mit frau prof. dr. med. dr. phil. eva winkler (nationales centrum für tumorerkrankungen, heidelberg), herrn dr. phil. martin langanke (theologische fakultät, universität greifswald) und herrn pd dr. phil. peter burgard (universitätskinderklinik heidelberg) kontrovers diskutieren. die organisatoren werden in die fragestellung einführen, anschließend sind impulsreferate (ca. min) der referenten vorgesehen, bevor eine debatte mit dem publikum angeregt wird. fangerau, f. söhner (düsseldorf) in den er jahren erlebte die humangenetik wie eine reihe anderer medizinischer disziplinen auch eine erhebliche institutionelle ausweitung. in den er und er jahren wurde beispielsweise an den universitäten der brd das gros der humangenetischen lehrstühle, in den ausgehenden er und er jahren auch in der ddr, eingerichtet. ging vom symposium "genetik und gesellschaft" im rahmen des marburger "forum philippinum" die initiative aus, in der ganzen bundesrepublik genetische beratungsstellen zu gründen und damit die genetische forschung (wieder) medizinisch nutzbar zu machen. in dieser phase der etablierung der humangenetik auf akademischer und praktischer ebene setzt das geplante zeitzeugenprojekt ein. es will die entwicklung der humangenetik in ihrem selbstverständnis als quer-und als längsschnittfach (pfadenhauer : ) im deutschsprachigen raum ab den er jahren mit hilfe von expertengesprächen dokumentieren und analysieren. im forschungsprojekt sollen zwei komplexe von fragestellungen bearbeitet werden: ein wissenschaftshistorischer, in dem die entwicklung und anwendung von diagnostischen und therapeutischen techniken im mittelpunkt steht, und ein sozialhistorischer, in dem es um die etablierung und den ausbau der institutionen der humangenetik sowie um den verlauf der das fach betreffenden gesellschaftlichen debatten geht. neben der gründung von instituten und der fachgesellschaft sowie der normierung der ausbildung für ärztliche und naturwissenschaftlich ausgebildete humangenetiker sollen die funktion der historischen reflexion und bearbeitung der facheigenen nationalsozialistischen vergangenheit in den er jahren für die etablierung des fachs und der schwierige institutionelle trennungsprozess von der anthropologie mit ihren wirkungen auf das selbst-und fremdbild der humangenetik analysiert werden (weingart, kroll, bayertz ) . zusätzlich zur entwicklung in der brd sollen dabei auch die entwicklung des fachs in der ddr und mögliche deutsch-deutsche kooperationen zur sprache kommen (in jena befand sich z. b. in den er jahren das zentrale referenz-institut für genetische beratung in der ddr (vogel : ) . das projekt kann sich auf zahlreiche arbeiten zur geschichte der deutschen humangenetik stützen (vgl. kröner , cottebrune cottebrune , weingart ; bennike dysmorphism carrying a pathogenic variant in the ebf gene detected by whole-exome sequencing. five missense, two nonsense, one -bp duplication, and one splice-site variant in ebf were found; the mutation occurred de novo in eight individuals, and the missense variant c. c>t [p.(arg trp) ] was inherited by two affected siblings from their healthy mother who is a mosaic. ebf belongs to the early b-cell factor family (also known as olf, coe, or o/e) and encodes a transcription factor involved in neuronal differentiation and maturation. structural assessment predicts perturbing effects of the five amino acid substitutions on dna-binding of ebf . transient expression of ebf mutant proteins in hek t cells revealed mislocalization of all but one mutant in the cytoplasm in addition to nuclear localization. by transactivation assays, all ebf mutants showed significantly reduced or no ability to activate transcription of the reporter gene under control of the cdkn a promotor that corresponds well with loose association of ebf mutants with chromatin as demonstrated by in situ subcellular fractionation experiments. finally, rna-seq and chip-seq experiments demonstrate that ebf acts as a transcriptional regulator at cis-regulatory sequences and ebf mutant had reduced function due to partial disruption of the dna-binding domain. these findings demonstrate that ebf -mediated dysregulation of gene expression has profound effects on neuronal development in humans and add ebf to the growing list of genes in which mutations cause syndromic forms of intellectual disability. step, we performed wes in further unelucidated uhs cases and identified homozygous nonsense mutations in tgm (transglutaminase ) and in tchh (trichohyalin), respectively. elucidation of the molecular outcomes of the disease causing mutations by cell culture experiments of padi and tgm and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild type proteins. by immunofluorescence analysis, we could demonstrate a diffuse homogenous cytoplasmic distribution of the wt padi , whereas in the mutants the proteins were observed to form large aggregates throughout the cytoplasm. by use of human anti-citrullinated protein autoantibodies, we could show a strong labelling in the wt whereas the staining of the mutants was barely above background. in order to demonstrate the importance of padi in hair shaft formation, we generated padi knockout mice. electron microscopy observations revealed morphological alterations in hair coat of padi knockout mice. for tgm , we performed a transglutaminase activity assay. the analysis results revealed that the wt had a significantly higher transglutaminase activity in comparison to the truncated protein. here, we report for the first time the identification of uhs causative mutations located in the three genes padi , tgm and tchh. the two enzymes responsible for posttranslational protein modifications, and their target structural protein, are all involved in hair shaft formation through their sequential interactions. these findings provide valuable information regarding the pathophysiology of uhs and contribute to a better understanding of this protein interaction cascade. this could be of further value for cosmetics and pharmaceutics industries paving the way for development of novel products. deadenylases are best known for degrading the poly(a) tail during mrna decay. the deadenylase family has expanded throughout evolution and, in mammals, consists of mg + -dependent ' end ribonucleases with mostly unknown substrate specificity. pontocerebellar hypoplasia type (pch ) is a unique recessive syndrome characterized by neurodegeneration with ambiguous genitalia (mim% ). we studied human families with pch , uncovering biallelic, loss of function mutations in toe , which encodes an unconventional deadenylase. toe -morphant zebrafish displayed mid-and hind-brain degeneration, modeling pch-like structural defects in vivo. surprisingly, we found toe associated with incompletely processed small nuclear (sn)rnas of the spliceosome, which is responsible for pre-mrna splicing. these pre-snrnas contained ' genome-encoded tails often followed by post-transcriptionally added adenosines. human cells with reduced levels of toe accumulated ' end-extended pre-snr-nas, and immuno-isolated toe complex was sufficient for ' end maturation of snrnas. our findings reveal the cause of a neurodegenerative syndrome linked to snrna maturation and uncover a key factor involved in processing of snrna ' ends. the kidney maintains acid-base homeostasis and electrolyte balance through highly specialized cells. in the distal nephron acid secretion is mediated by type a intercalated cells (a-ics) , which contain v-type at-pase-rich vesicles that fuse with the apical plasma membrane on demand. intracellular bicarbonate generated by luminal h+ secretion is removed by the basolateral anion-exchanger ae . dysfunction of type a intercalated cells results in distal renal tubular acidosis (drta) and human mutations in v-atpase subunits and ae are causative for this condition. for the ae r h mutation a dominant-negative trafficking mechanism was proposed to explain ae -associated dominant drta based on studies in mdck monolayers. to test this hypothesis in vivo and to test potential rescue strategies correcting this mistargeting defect, we have generated a r h knock-in mouse strain, which corresponds to the most common dominant drta mutation in human ae , r h. heterozygous and homozygous r h knock-in mice displayed incomplete drta characterized by compensatory upregulation of the na+/hco -cotransporter, nbcn . as expected for the r h mutation, red blood cell ae -mediated anion-exchange activity and surface polypeptide expression were unchanged. surprisingly, basolateral targeting of the mutant ae in a-ics was preserved in contrast to previous studies in mdck cells. instead, we found ae expression in a-ics strongly reduced in a r h dosage-dependent manner. additional cell culture studies in two widely used immortalized renal cell lines verified that targeting and half-life time of mutant ae protein was indeed preserved. surprisingly, atpase expression was reduced and its plasma membrane targeting upon acid challenge compromised. ultrastructural analysis revealed a loss of apical vesicles in a-ics, while we observed lysosomal inclusions and multilamellar bodies. accumulation of p -and ubiquitin-positive material in a-ics of knock-in mice suggest a defect in the degradative pathway, which may ultimately lead to loss of a-ics. highlighting the expression of ae specifically in a-ics, type b intercalated cells were unaffected. we propose that reduced basolateral anion-exchange activity in a-ics inhibits trafficking and regulation of v-type atpase, compromising luminal h+ secretion and possibly also lysosomal acidification. our findings illustrate the considerable, context-dependent complexity of ae -related kidney disease. b. vona , d. liedtke , k. rak , , r. katana , l. jürgens , pr. senthilan , i. nanda , c. neuner , mah. hofrichter , l. schnapp , j. schröder , u. zechner , s. herms , , , p. hoffmann , t. müller , m. dittrich , , o. bartsch , pm. krawitz , e. klopocki , w. shehata-dieler , mc. göpfert , t. haaf although many genes have already been identified as causing non-syndromic hearing loss (nshl), diagnostic rates of approximately % among hearing impaired patients suggest that many more genes are remaining to be identified. nshl is the most common sensory deficit that has a prevalence between one and two per newborns. furthermore, it demonstrates classic genetic heterogeneity with as many as % of coding genes in the genome anticipated to be involved in non-syndromic forms of deafness. autosomal recessive ( - %) and autosomal dominant ( - %) forms dominate inheritance patterns of deafness; however, in a small fraction of cases, x-linked deafness ( - %) can be observed. whole exome sequencing of a german family with diagnostically unresolved nshl revealed a novel missense variant predicted as pathogenic in the gene ferm and pdz domains containing protein (frmpd ) on chromosome xp . . this gene, also known as preso , was first described as a regulator of dendritic spine morphogenesis. previous screening of pathogenic cnvs in array based comparative genomic hybridization among families with heterogeneous x-linked intellectual disability (xlid) showed duplication of xp . including part of frmpd which implicated the gene in xlid. interestingly, a segregating truncating and a de novo missense mutation in frmpd have associated this gene with xlid, a phenotype not observed in our family. mouse expression studies localize frmpd to spiral ganglion neuron peripheral dendrites of the developing cochlea. in addition, we analyzed frm-pd knockdown and loss-of-function zebrafish mutants for innervation and structural defects in the otic vesicle and lateral line neuromasts. posterior lateral line neuromasts are observed with reduced axonal outgrowth that is also likely reduced in the lateral line nerve. abnormal innervation is also apparent in the otic vesicle. fluorescent neuromast labeling marked a significant reduction of overall otic vesicle and lateral line neuromasts in mutants versus wild type zebrafish. scanning electron microscopy revealed a pronounced absence of kinocilia in posterior lateral line neuromasts of frmpd -/zebrafish. furthermore, adult frmpd mutants show significantly delayed acoustically evoked behavioural responses compared to wild type fish indicating hearing impairment. investigation of transgenic drosophila insertion mutants detected a mild auditory phenotype i. e. a reduction in mechanical amplification gain and associated reduction in antennal fluctuation power. our results associate frmpd with x-linked hearing loss and suggest mutations in this gene are correlated with pleiotropic effects abstracts has also been demonstrated to be an important pathobiochemical feature in rtt. to test whether common deficits in mtor signaling could be responsible for the molecular pathogenesis underlying both syndromes, we generated and studied a novel cdkl knockout (cdkl -/y) mouse model and performed in vitro experiments in human cells. in cdkl -/y knockout mice loss of cdkl is accompanied by reduced phosphorylation levels of critical components of the mtor signaling cascade. these findings point at a regulatory role of cdkl /cdkl on mtor activity and function. to gain further insights into the possible mechanism through which cdkl /pi k interaction could regulate mtor signaling, we used hek-t cells as cellular model. following knock-down of cdkl , the amount of pi k protein was significantly reduced compared to controls. to evaluate the contribution of our findings to pathogenesis, we performed rescue experiments in cdkl knock-down hek-t cells using wild-type and patient-specific mutant cdkl constructs. further experiments are ongoing to clarify the molecular mechanism by which cdkl regulates pi k protein level in the cells. inferring expressed genes by whole-genome sequencing of plasma dna medical university graz, graz, austria, university of technology, graz, austria the analysis of cell-free dna (cfdna) in plasma represents a rapidly advancing field in medicine. cfdna consists predominantly of nucleosome-protected dna shed into the bloodstream by cells undergoing apoptosis. we performed whole-genome sequencing of plasma dna and identified two discrete regions at transcription start sites (tsss) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. by employing machine learning for gene classification, we found that the plasma dna read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. in patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. we were able to determine the expressed isoform of genes with several tsss, as confirmed by rna-seq analysis of the matching primary tumor. our analyses provide functional information about cells releasing their dna into the circulation. institute of human genetics, fau-erlangen-nürnberg, erlangen, germany, institute of medical genetics, university of zurich, schlieren, switzerland rna-splicing is an important mechanism for eukaryotic gene expression and regulation. defective splicing significantly contributes to monogenic disease in humans. indeed, the mutational space for variants affecting splicing is larger than for coding variants. several computational methods have been developed to predict a variant's effect on splicing but lack predictive value outside the canonical splice sites and do not predict aberrant transcripts. thus the plethora of dna variants generated by recent advances in "next-generation" based sequencing (ngs) can be scored for a possible splicing effect, but a laborious wet-lab based confirmation and characterization is still required. rna-seq is widely used for quantification of gene expression and can be used to detect splicing events, but is limited for this use by the variable and often low read coverage of individual congenital anomalies of the kidneys and urinary tract (cakut) are the most common cause of chronic kidney disease in children. as cakut is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new cakut causing genes. using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (lifr) gene causing instability of the mrna in a patient presenting with bilateral cakut and requiring kidney transplantation at one year of age. lifr encodes a transmembrane receptor utilized by il- family cytokines, mainly by the leukemia inhibitory factor (lif). mutational analysis of further patients with severe cakut yielded two rare heterozygous lifr missense variants predicted to be pathogenic in three unrelated patients. lifr mutants showed decreased half-life and cell membrane localization resulting in reduced lif-stimulated stat phosphorylation. lifr showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying lifr variants. additionally, a form of cryptorchidism was detected in all lifr -/mice and the patient carrying the lifr frameshift mutation. altogether, we demonstrate heterozygous novel or rare lifr mutations in . % of cakut patients, and provide evidence that lifr deficiency and deactivating lifr mutations cause highly similar anomalies of the urogenital tract in mice and humans. loss of cdkl associated with deficient mammalian target of rapamycin (mtor) signaling in mice and human cells we and other groups have shown that mutations in the x-linked cyclin-dependent kinase-like (cdkl ) gene cause a severe neurodevelopmental disorder with clinical features including intellectual disability, early-onset intractable seizures and autism, that are closely related to those present in rett syndrome (rtt) patients. rtt is caused by mutations in the x-linked mecp gene. cdkl is a serine/threonine kinase and to date knowledge about its functional roles is scarce. we searched for cdkl interacting proteins by yeast-two hybrid screens. one of the candidates identified in these screens is a subunit of the phosphatidylinositol- , -biphosphate -kinase (pi k). the results obtained in yeast could be confirmed in vitro in mammalian cells and in mouse brain by immunoprecipitation experiments and by co-localization studies. pi k phosphorylates membrane lipids which act as docking sites to recruit targets upstream of mtor and thereby regulate among major cellular processes synaptic plasticity, which is the cellular basis for learning and memory. alteration of mtor signaling gene tubes for stabilizing rna immediately after drawing the samples. the subsequent rt-pcr analysis showed that of the variants located at potential splicing sites indeed affect splicing. thus of these variants could be classified as deleterious (iarc class ), while one chek variant could not be unequivocally classified as the rt-pcr analysis identified only % of the mutant transcript indicating continued usage of the constitutive splice acceptor site. this led to the classification as a probably hypomorphic allele. the variants in cdh and mlh did not affect splicing and were classified as benign (iarc class ). none of the rare synonymous and nonsynonymous exonic variants showed any effect on splicing. in conclusion, this analysis allowed the disambiguation of out vus at potential splice sites into a definite category (either iarc class or ). this work highlights the importance of computational splicing prediction and validation using rt-pcr of peripheral blood rna to assess the pathogenicity of vus. this in turn, allows more accurate genetic counseling and clinical management of affected families. gliomas present the major group of neoplasia in the central nervous system. they typically show invasive growth and high recurrence rate and are currently not curable. idh mutations are detected in nearly % of low grade gliomas and are considered to play a key role in low grade glioma development. while it is known that idh / mutation leads to high-levels of -hydroxyglutarate ( -hg) that functions as an oncometabolite, little is known about the influence of idh / mutations on energy metabolism and metabolic reprogramming in the tumor cells. since patient derived idh mutant cells do not grow in cell culture, previous studies from our group and others used transduced cell lines that overexpress idh . in order to develop in vitro models with reduced side effects, we used crispr/ cas to introduce the idh r h mutation in a patient derived glioblastoma cell line. the edited cells expressed idh r h in western blot and expression levels of idh were comparable to the expression in wild type cells. the mutation was stable in long time culture experiments, without signs of senescence. moreover we found elevated -hg levels, proving that the idh r h neoenzymatic function is present in our cell lines. thus, we were able to edit and culture genomic idh r h mutated glioma cells for the functional analysis of the idh r h mutation for the first time without the effects of overexpression models. edited idh r h cell lines showed extended doubling times compared to wildtype cells. measurement of krebs cycle metabolites using mass spectrometry revealed elevated glutamate levels. we found enhanced atp-levels that could be a consequence of decreased atp consumption. additionally, the cells showed reduced viability compared to wildtype cells when cultivated in glycolysis inhibiting media, pointing out the enhanced dependency on glycolysis in idh r h cells. these results indicate changes in tumor cell metabolism and energy household induced by the idh r h mutation. since we and others could show that idh r h can alter nad+ and nadph levels, we tested if the idh r h mutated cells are more susceptible to selective inhibition of nad/p regenerating enzymes. esirna-silencing of nampt specifically decreased cell viability in idh r h but not wildtype cells with a concomitant increase of dead cells. in conclusion, we developed genes. thus, a standardized ngs based approach to characterize potential splice variants is lacking. hence we investigated the utility of hybridization based gene-panel enrichment and ngs of cdna. based on results of computational simulation we selected twenty rna-samples of patients with a known pathogenic splice-site variant in an inherited cancer predisposition gene. these variants were previously characterized by rt-pcr in our lab or in the literature. after rrna depletion and dna digestion we performed first and second strand cdna synthesis followed by "tagmentation"-based library preparation, targeted enrichment using the trusight cancer panel and sequencing on an illumina miseq platform. a computational pipeline was established to enable automated detection of aberrant splicing events by implementing different alignment and splice-junction detection algorithms together with filtering against control data sets. we also considered variant calling for the detection of allelic imbalance and gene-level expression analysis in this data. breast and ovarian cancer (bc/oc) predisposition has been associated with a number of high-and low-penetrance susceptibility genes. advances in sequencing technology has made multigene testing a practical option when searching for genetic variants associated with risk for bc/oc. variants of uncertain significance (vus), though, represent a major problem. we now studied patients fulfilling criteria for brca and testing using the next generation sequencing based trusight sequencing cancer panel on a miseq platform (illumina). data was analyzed after remapping with bwa to hg (grch ) using seqnext software (jsi) for variants in known high and moderate penetrance susceptibility genes (brca / , atm, chek , palb , rad c, rad d, nbn, cdh , tp , mlh , msh , msh , pms ) . besides deleterious mutations we also identified vus. of these, variants ( each in brca , brca , palb , rad c, rad d, cdh , and mlh , and in chek and mlh , respectively) affect possible splicing sites. in addition, synonymous and nonsynonymous variants outside the splicing sites ( in brca , brca and cdh , respectively, in rad d) were not reported in exome variant server or exome aggregation consortium (exac) databases, so far. no families were available to study familial segregation. for all these variants a potential effect on splicing efficiency was predicted by three different computational algorithms (bdgp: splice site prediction by neural network, netgene server and the human splice finder (hsf . ) algorithm). we took advantage that these genes are ubiquitously expressed to investigate possible effects of these variants on mrna splicing using easily accessible peripheral blood. as mrna is notoriously unstable, we used pax-abstracts was evaluated for both the individual markers and their combinations derived from multiple algorithms. pronounced demethylation of all markers was observed at baseline among cases compared to controls. risk of developing lc increased with decreasing dna methylation levels, with adjusted ors ( % ci) of . ( . - . ), . ( . - . ) , and . ( . - . ), respectively, for participants in the lowest quartile of ahrr, p . , and f rl compared to participants in the highest quartiles of each site among controls. the individual markers exhibited similar accuracy in predicting lc incidence, with aucs ranging from . to . . combination of the markers did not improve the predictive performance (auc = . ). the individual markers or their combination outperformed self-reported smoking exposure particularly in light smokers. no variation in risk prediction was identified with respect to age, follow-up time, and histological subtypes. ahrr, p . , and f rl methylation in blood dna are predictive of lc development, which might be useful for identification of risk groups for further specific lc screening, such as ct examination. over the past decades the search for disease causing variants has been focusing exclusively on the coding genome. this highly selective approach has been extremely successful however, recent data have revealed the importance of the non-coding genome in fundamental processes such as gene regulation, d chromatin folding, and pinpointed its role in disease. in this study, we systematically investigate the cis-regulatory landscape of pitx , a homeodomain transcription factor that is exclusively expressed in the hindlimb. mutations and non-coding structural variations at the pitx locus have been shown to associate with a variety of congenital limb defects including club feet, polydactyly, and arm-to-leg transformation (liebenberg syndrome). we performed in vivo enhancer reporter essays in transgenic mice and identified several limb enhancer elements at the pitx locus; surprisingly they all showed both forelimb and hindlimb activity, although pitx is never expressed in the forelimb. capture hi-c experiments revealed a hindlimb-specific chromatin-organization at the pitx locus, which enables its promoter to contact several enhancers bearing a pan-limb activity only in the hindlimb. this tissue-specific chromatin folding plays a determinant role to refine the unspecific limb regulatory landscape toward a highly controlled and hindlimb delimited transcriptional output. to gain a better understanding of the pathology of pitx associated limb defects, we used crispr/cas to generate a set of deletions and inversions in the pitx cis-regulatory landscape in mice. genetic perturbations of the regulated d chromatin conformation lead to an ectopic forelimb expression of pitx , resulting in an arm-to-leg transformation in mice and in human patients respectively. our data further highlight the role of non-coding mutations affecting chromatin folding in congenital disease and give new insights into the regulation of pitx during development and the pathomechanism of associated limb defects. hoxb , a member of the embryonic homeobox transcription regulators, has been identified as the first susceptibility gene specific for prostate cancer (prca). the founder missense mutation g e, which likely originated from finland, can be found in most populations of european ancestry. we determined the frequency of hoxb g e for the german population, assessed in a cohort of unrelated cases, each with positive family history of prca, sporadic prca cases and in controls. additional affected relatives from prca families were included to explore association with aggressive disease in subgroups with high gleason score (> ), advanced tumor stage, or psa at diagnosis > ng/ml. carriers of g e were rare in controls ( . %) and showed increased frequencies in both sporadic ( . %) and familial prca cases ( . %). estimated risks were or = . (p = . ) and or = . (p = . ), respectively. the risk effect size increased with the number of affected individuals per pedigree: or = . (p < . ) for or more, and or = . (p < . ) for or more affected men. the strongest association with clinical features was observed between g e and advanced tumor stage (or = . ; p < . ). in conclusion, the observed frequency of hoxb g e mutation carriers in our study cohort was intermediate as compared to the common prevalence in scandinavia and the rare occurrence in mixed european populations from the us. the risk estimates of hoxb g e and the stronger effect sizes in families with increasing number of affected relatives were in line with a high penetrant germline predisposition. the association between g e status and tumor stage may be of greater interest for clinical practice, but needs further validation. the absolute penetrance of the hoxb g e mutation should be investigated in further studies in order to elucidate its suitability as a genetic predictor for prca. smoking-associated dna methylation markers predict lung cancer incidence homozygous smn loss causes spinal muscular atrophy (sma), the most common lethal genetic childhood motor neuron disease. smn encodes smn, a ubiquitously expressed housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. sma individuals harbor low smn expression from one to six smn copy genes, which is insufficient to functionally compensate for smn loss. however, rarely individuals with homozygous absence of smn and only three to four smn copies are fully asymptomatic, suggesting protection through genetic modifier(s). previously, we identified plastin (pls ) overexpression as an sma protective modifier in humans and showed that smn deficit impairs endocytosis, which is rescued by pls overexpression. here, we identify reduction of the neuronal calcium sensor neurocalcin delta (ncald) as a protective sma modifier in five asymptomatic smn -deleted individuals carrying only four smn copies. we demonstrate that ncald is a ca + -dependent negative regulator of endocytosis, as ncald knockdown improves endocytosis in sma models and ameliorates pharmacologically induced endocytosis defects in zebrafish. importantly, ncald knockdown effectively ameliorates sma-associated pathological defects across species, including worm, zebrafish and mouse. in conclusion, our study identifies a previously unknown protective sma modifier in humans, demonstrates modifier impact in three different sma animal models and suggests a potential combinatorial therapeutic strategy to efficiently treat sma. since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in sma and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies. mutations affecting coding or regulatory regions of smc cause dysregulation of condensins resulting in a phenotype reminiscent of cohesinopathies cornelia de lange syndrome (cdls) is a dominantly inherited malformation syndrome caused by mutations in genes encoding subunits (smc a, smc , rad ) or regulators (nipbl, hdac ) of the cohesin complex. this dna-bound complex regulates several chromatin-related processes such as chromosome segregation, dna-damage repair, transcription and chromatin structure. the project presented initially started with two children and their mother who showed clinical features reminiscent of cdls. while various sequencing approaches failed to identify the disease-causing mutation, a kb spanning deletion co-segregating with the phenotype was identified by array-cgh. besides the last exons of cylc , encoding a sperm head protein, no other genes were affected. subsequent in-silico analyses predicted the existence of a ~ kb tissue-specific regulatory element within this region, located approximately mb distant from the next protein-coding gene smc , which encodes a subunit of the cohesin-related condensin complex. significant reduction of smc expression was verified in patient's fibroblasts by qpcr analysis. accordingly, a strong dysregulation of smc was observed in hek and sh-sy y cells deficient for the putative kb regulatory element, which was deleted by crispr/cas genome editing. reporter gene assays further highlighted the functional relevance of the identified regulatory element in regulating the smc gene promoter. interestingly, we could prove on protein as well as on mrna level that alterations in smc expression are correlated with the dysregulation of other condensin subunits such as smc in patient's samples as well as in cris-pr/cas -generated cells. in a large exome sequencing project we have identified a smc frameshift mutation in an additional family with two patients who show clinical features overlapping with those seen in our initial family. quantitative pcr analyses in fibroblasts of both subjects also showed significant reduction of smc and smc expression, which is consistent with our findings in the first family. to further investigate whether alterations in condensin gene expression are specific for the dysregulation of smc , we have decreased smc levels in different cell types by sirna. quantitative protein as well as mrna analyses revealed reduced smc /smc expression. our data show for the first time the coordinated expression of different condensin subunits and its relevance for human disease. abstracts human pedigree. cardiac valves initially form through a process called endothelial-to-mesenchymal transition (emt) then subsequently elongate and mature during early juvenile life. expression analysis throughout embryonic and postnatal stages of adamts -/-mice revealed an expression in all cardiac valves after valve formation. high resolution, digital echocardiography showed that mice without adamts expression develop dysfunctional aortic valves early in life, reminiscent of the human phenotype. notably, the expression of adamts in the valve was restricted to valvular interstitial cells and not observed in endothelial cells. functional analysis using proteomic approaches suggest that the presence of ad-amts is necessary to maintain extracellular matrix remodelling during valve development and its maturation. not only do the lof mice fully recapitulate the human phenotype, they also highlight adamts as a novel marker for valvular interstitial cells to specifically target initial post-emt processes as well as serve as an important model to understand an ageing valve phenotype in humans. exome sequencing of bipolar disorder patients with rapid cycling implicates novel candidate genes in disease development bipolar disorder (bd) is a severe neuropsychiatric disorder characterized by recurrent episodes of mania and depression. bd has a lifetime prevalence of about % and a high heritability of about %. although recent genome-wide association studies identified the first susceptibility genes contributing to disease development, the cumulative impact of common alleles with small effect may only explain around % of the phenotypic variance (lee et al. ) . in consequence, rare variants of high penetrance have been suggested to additionally contribute to bd susceptibility. in the present study we focused on bd patients with rapid cycling (rc). rc is a course specifier of bd defined as having at least four recurrent episodes of acute illness within one year. since rc showed strong evidence for familiarity, we hypothesized that bd patients with rc might represent a more defined etiological subgroup and that rare variants of high penetrance might contribute to the development of rc in bd patients. we selected unrelated bd patients with rc of german origin and performed exome sequencing using the illumina hiseq platform. for data analysis, the varbank pipeline of the cologne center for genomics was used. we filtered for rare (minor allele frequency < . %), heterozygous and non-synonymous variants that were predicted to be possibly damaging or disease causing by at least of applied prediction tools. after these filtering steps, we identified a total of different genes which harbored rare functional variants in at least three independent patients. gene set analysis for these genes using consensuspathdb revealed decker , g. nuernberg , , d. hassel , g. a. rappold , mutations in the homeobox gene shox cause shox deficiency, the most frequent monogenic cause of short stature. the clinical severity of shox deficiency varies widely, ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia (léri-weill dyschondrosteosis, lwd). in rare cases, individuals with shox deficiency are asymptomatic. to elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with five affected individuals with lwd using whole genome linkage analysis and whole exome sequencing. the variant p.phe cys of the retinoic acid catabolizing enzyme cyp c co-segregated with the shox variant p.val ala in the five affected individuals, while the shox mutant alone was present in three asymptomatic individuals. two further independent lwd cases with shox deficiency and damaging cyp c variants were identified. the identified damaging variants in cyp c affected its catabolic activity, leading to an increased level of retinoic acid. we also provide evidence that high levels of retinoic acid significantly decrease shox expression in human primary chondrocytes and zebrafish embryos. individual morpholino knock-down of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. together our findings demonstrate that shox and cyp c act in a common molecular pathway controlling limb growth and describe cyp c as the first genetic modifier for shox deficiency. heart valve dysfunction in men and mice is caused by loss of function mutations in adamts , a novel marker for valvular interstitial cells on a global perspective defects of the cardiac valves are one of the most common heart abnormalities in humans, with a substantial number of them requiring surgical intervention at least once in their life. several mechanisms have been proposed ranging from acquired to developmental causes, but thus far the majority can not be explained on the molecular level. here we report on the identification of a unique human family affected by multiple dysfunctional cardiac valves early in life. genetic screening revealed a homozygous deletion of the first eight exons in ad-amts , a novel candidate gene for valvular heart defects. to investigate its role in heart valve development, we designed a transgenic mouse model that reconstitutes the loss of function (lof) in adamts found in the statistically analyzing de novo mutations identified in > , id patients highlighted ppm d as a candidate id gene. ppm d is a type c phosphatase that functions as a negative regulator of cell stress response pathways by mediating a feedback loop of p -p signaling, thereby contributing to growth inhibition and suppression of stress induced apoptosis. we identified patients with mild-moderate id and a de novo truncating ppm d mutation. deep-phenotyping of the patients revealed in addition to id overlap for behavioural problems (adhd and anxiety disorder), hypotonia, broad based gait, facial dysmorphisms and periods of fever and vomiting. ppm d is shown to be expressed during fetal (brain) development and in the adult brain. all mutations were located in the last, or penultimate exon, suggestive of escaping nonsense-mediated mrna decay. both ppm d expression analysis and cdna sequencing in patient ebv-lcls support the presence of a stable, but truncated transcript, consistent with this hypothesis. exposure of patient's cells to ionizing radiation resulted in normal p activation suggesting that p signaling is not affected by the truncated protein. however, a cell growth disadvantage was observed. thus, we show that de novo truncating ppm d mutations in the last and penultimate exon cause syndromic id which provides novel insights in the role of cell cycle checkpoint genes in neurodevelopmental disorders. de novo truncating variants in asxl are associated with a unique and recognizable clinical phenotype harvard stem cell institute, department of stem cell and regenerative enriched pathways (q < . ) including actin cytoskeleton and calcium ion binding. subsequently we applied the residual variation intolerance score (rvis) and identified genes which were ranked among the % most intolerant genes in the genome. these genes included the previously reported genome-wide significant bd risk genes syne and mll . in addition, we identified novel, promising candidate genes which have not previously been implicated in bd development such as ryanodine receptor (ryr , affected in six patients) and huntingtin (htt, patients). both genes are ranked among the . % most intolerant genes of the genome. ryr encodes a brain expressed intracellular cation channel that mediates the rapid release of ca + from the endoplasmic reticulum, thus making it a highly plausible candidate gene for contributing to rc. abnormal expansion of a trinucleotide repeat in the htt gene causes huntington disease which is a neurodegenerative disease characterized by motor, cognitive and psychiatric symptoms. the seven most promising genes are currently being followed up by resequencing in larger cohorts of independent bd cases (including patients with rc) and controls of european ancestry using the single molecule molecular inversion probes (smmips) technology. de novo truncating mutations in the last and penultimate exon of ppm d cause a novel intellectual disability syndrome abstracts with gα. signaling properties of g protein complexes carrying mutant gβ subunits were further analyzed by their ability to couple to dopamine d r receptors by real-time bioluminescence resonance energy transfer (bret) assays. these studies revealed altered functionality of the missense mutations r g, g v, a t, p s, p l, a t, and d g but not for l f, h r, and k q. in conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in gnb as a cause of gdd and provide functional evidence for a loss-of-function mechanism underlying the disease. comprehensive phenotyping and trio-exome analysis of children with neurodevelopmental disease whole exome sequencing (wes) has been proven as a powerful analytical tool to dissect the genetic basis of human hereditary disorders. here, we report on a prospective deep phenotyping and trio-wes study of children affected by previously undiagnosed and diverse complex neuropediatric disorders. all children underwent a standardised and comprehensive clinical work-up in a single centre that included detailed clinical evaluations by pediatricians and clinical geneticists, extensive laboratory and metabolic analyses, analyses of cerebrospinal fluid, mri of the brain and eeg, followed by trio-wes analysis. this systematic approach allowed to identify a pathogenic mutation in a known disease gene in altogether children ( %) and discovered a convincing candidate disease gene in additional children ( %). taken together, this translates into a successful genetic diagnosis of up to % in this cohort. in children with mutations in a known disease gene ( / = . %) the molecular diagnosis substantially influenced the clinical management and drug treatment. we further document an expansion of the phenotype in known disease entities in individuals. the extraordinary high gene discovery rate in our cohort emphasizes the potential of trio-wes even in a clinically inhomogeneous group of individuals with likely genetic disease. however, this requires a multidisciplinary approach including deep and sometimes reverse phenotyping, research-based interpretation of trio-wes identified genetic alterations, extensive review of the literature, use of several mutation prediction and protein-modelling tools, as well as openness and exchange of data with national and international researchers and clinicians working on similar diseases. exome sequencing of pooled dna samples for large-scale screening in individuals with sporadic intellectual disability b. popp, a. ekici, s. uebe, c. thiel, j. hoyer, a. wiesener, a. reis, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germany high throughput sequencing has enabled identification of many novel disease genes and empowered diagnostic testing for heterogeneous disorders, especially for intellectual disability (id) where more than genes have been implicated. due to this extreme heterogeneity gene panels are ineffective, and expensive exome or genome sequencing is necessary. furthermore, many affected individuals have to be sequenced to confirm candidate genes and to refine the phenotypic spectrum. we now explored if pooling strategies could satisfy the need for a genome-wide, simple, cheap and fast screening technology. the asxl genes (asxl , asxl and asxl ) participate in body patterning during embryogenesis and encode for proteins that are involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. germline de novo truncating variants in asxl and asxl have been respectively implicated in causing bohring-opitz and bainbridge-ropers syndromes, resulting in overlapping features of severe intellectual disability and dysmorphic features. to our knowledge, asxl has not yet been associated with a human mendelian disorder. in this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. all six had de novo truncating variants in asxl . a careful review en abled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia and developmental disabilities. although overlapping features with bohring-opitz syndrome and bainbridge-ropers syndromes exist, features that distinguish the asxl -associated condition from asxl -and asxl -related disorders are macrocephaly, absence of growth retardation and more variability in the degree of intellectual disabilities. we were also able to demonstrate with mrna studies that these variants are likely to exert a dominant negative effect, since both alleles are expressed in blood, with the mutated asxl transcripts escaping nonsense mediated decay. in conclusion, de novo truncating variants in asxl underlie a new neurodevelopmental syndrome, with a clinically recognizable phenotype. this work expands the germline disorders that are linked to the asxl genes. functional characterization of novel gnb mutations as a rare cause of global developmental delay over the past years, prioritization strategies that combined the molecular predictors of sequence variants from exomes and genomes of patients with rare mendelian disorders with computer-readable phenotype information became a highly effective method for detecting disease-causing mutations. the drawback of phenotype-based prioritization, however, is that they require a deep and comprehensive feature description to gain good performance. but in routine diagnostics, the naming of phenotypic features varies among clinicians, and sometimes a comprehensive phenotypic overview is not possible because of missing terminology. these gaps can be reduced by including a new layer of phenotypic information using facial recognition technology to detect dysmorphic features from two-dimensional photographs. automated image analysis is in principle able to identify any deviation from the norm and to quantify it objectively. we therefore developed an approach that combines facial dysmorphology novel analysis (fdna) technology with standard phenotypic and genomic features to identify pathogenic mutations in exome data. we have started collecting data from a diverse spectrum of patients with molecularly confirmed diagnoses in a multi-center study, and we present the current results. at the time of abstract submission more than patients from over contributing institutions were evaluated and used for simulation of a training set of exomes. automated facial recognition yields the correct diagnosis amongst the first ten suggested syndromes in more than two thirds of the cases and shows a high correlation with syndrome predictions that were based on expert annotated features. hereby, we could also confirm the diagnosis in cases with only subtle facial features. consequently, we used classical machine learning approaches to integrate scores based on the image analysis, phenotypic description and exome se-after initial evaluation of available computational methods by virtual pooling of exome data or simulated reads using different pooling fractions, we decided to exome sequence individuals with sporadic id in pools of samples each. this was suggested to be the optimal combination with a % detection rate. dna was mixed in equimolar concentrations and submitted for exome sequencing. read data was aligned to the human reference, and variants were called using a ploidy of . resulting variant calls in known id genes (sysid database) were then filtered for loss-of-function (lof) variants and for missense variants that were either previously reported as pathogenic or computationally predicted to be deleterious. furthermore, we screened id candidate genes and haploinsufficiency intolerant genes for lof variants. subsequently, sanger sequencing was used to determine the individual carrying each variant in the respective pool and to test segregation in the parents. this approach resulted in the identification of pathogenic variants (assumed or confirmed de novo) in known id genes (ahdc , ankrd , atp v b , cask, chd , kcnq , kmt a, kras, med l, rit , setd , tcf , wac, zbtb ), two pathogenic variants inherited from a symptomatic or healthy parent, respectively, (zmynd , ifih ), and a homozygous variant in the recessive trappc gene. this included loss-of-function and missense variants. additionally, we identified de novo variants in candidate genes. in our id cohort this resulted in a high mutation detection rate of %. thus, detection of rare variants from exome sequenced dna pools (pool-seq) is feasible and has a high detection rate similar other screening approaches. compared to affected-only exome sequencing this method can reduce costs by more than % with only marginal increase in sanger-sequencing costs and significantly speed up wet lab work with an acceptable increase in computational complexity. in contrast to targeted sequencing methods like molecular inversion probes or hybridization-based panels, our method has the advantage of allowing flexible re-analysis of the same data for new genes. in conclusion, we established exome pool-seq as a method for large-scale, cost-efficient and flexible sequencing in highly heterogeneous but well characterized disorders like id. three years of experience with targeted next-generation sequencing of developmental delay next-generation sequencing (ngs) has opened up new possibilities especially in the search for disease-causing mutations in disorders with common clinical features but a heterogeneous genetic background. the identification of the underlying genetic defect provides a clear diagnosis for patients more and more influencing their management and occasionally even their therapy, and it is the prerequisite for prenatal or preimplantation decisions in the affected family. ngs panels are used widely in clinical settings to identify genetic causes of various monogenic disease groups, such as intellectual disability (hu et al. ) , neurodevelopmental and neuromuscular disorders, among others. however, many new challenges have been introduced both at the technical level and at the bioinformatic level, with consequences including new requirements for interpretation of results, and for genetic counseling. we report on our experience with a targeted ngs panel comprising over brain related genes (mpimg- -test) in the routine clinical diagnostics of patients with syndromic and non-sydromic forms of developmental delay as well as patients with neuromuscular disorders. patients (age - ; mean ) with syndromic (s) or non-syndromic (ns) developmental delay or with neuromuscular symptoms (nm), seen at the genetic counselling unit of our institute, were analyzed with targeted exon enrichment and ngs. chromosomal re-arrangements and copy number variations were excluded in all patients previously by conven-it was recently shown that that clonal hematopoiesis can be driven by somatic point mutations. these acquired mutations occur with normal aging in up to % of older (> y) individuals ( ) ( ) ( ) and few reports in younger individuals. here we present a targeted re-sequencing assay that combines high throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smmip) ( ) . we have now analyzed dna from healthy blood donors from different age groups ( - ; - ; - ; - and - y) , with no previous diagnosis of cancer, for somatic mutation in loci. those loci included known drivers of clonal hematopoiesis ( ) ( ) ( ) and novel or candidate loci. the improved assay allows low-frequency variant detection with variant levels down to < . %. this improved sensitivity allowed the identification of somatic mutations in a limited set of loci in > % of old individuals, but also report those mutations in individuals of the youngest age group. most prevalent mutations include known hotspot mutations in dnmt a and asxl . here we show that somatic drivers of clonal hematopoiesis are more prevalent and occur in younger individuals than previously reported. these somatic events are age-related. however, the high prevalence and their occurrence in relatively young individuals implicates their origin as a common biological process involved in normal aging. a. m. nissen, j. graf, c. rapp, m. locher, a. laner, a. benet-pagès, e. holinski-feder medizinisch genetisches zentrum -mgz, munich, germany gene dosage abnormalities account for a significant proportion of pathogenic mutations in rare genetic disease related genes. in times of next generation sequencing (ngs), a single analysis approach to detect snvs and cnvs from the same data source would be of great benefit for routine diagnostics. however, cnv detection from exon-capture ngs data has no standard methods or quality measures so far. current bioinformatics tools depend solely on read depth which is systematically biased. we developed a novel approach based on: . utilization of five independent detection tools to increase sensitivity, . different reference sets for different kits and normalization against samples from the same sequencing run to improve robustness against workflow conditions, . definition of special quality thresholds for single exon events to minimize false negatives, . identification of reliable regions by assessment of capture efficiency using a reference set of cnv negative patients to minimize false positives. a cnv quence of the patients and could predict the pathogenic mutation among the top positions in a prioritized exome in more than % of the monogenic cases in our cohort. hence, our results show that computer-assisted facial recognition is not only a promising technology that could be applied in the routine diagnostic workflow, but also a technology that allows diagnosis in cases with non-typical clinical presentation and boosts the diagnostic yield in exome studies. the added value of rapid exome sequencing in critical clinical situations for critical clinical situations, turnaround times (tats) of exome sequencing need to be fast in order to have an impact on clinical decision making. we therefore set out to develop a fast exome sequencing approach (max. days). urgent exomes are preferably sequenced as trios to enable de novo analysis and assist data interpretation. dna library preparation is performed using the sureselect qxt protocol (v , agilent), and sequencing is done on a nextseq (illumina) with a high coverage ( - x). automated file handling allows rapid bwa mapping, gatk variant calling and annotation. a total of one-hundred samples have been sequenced until now using this rapid procedure: trio's, x mother and child, x parents plus children, and single cases. six trios with known aberrations were used for experimental setup. of the remaining families, in families (possible) pathogenic snvs were identified, of which some still need further follow up, whereas families remained negative after inspection of snvs and small indels. for cnv analysis, a trio based reference-free cnv approach is still under development. preliminary data show that all control cnvs ( kb- mb) are detected correctly, and retrospective cnv analyses of the other samples identified three possibly de novo cnvs that need further follow up. shorter tats days were already beneficial for some patients, i. e. an adult male suffering from myelofibrosis and autoinflammatory symptoms. a sting-like phenotype (= stimulator of ifn genes) was suspected, with a possible involvement of the jak/stat pathway. urgent exome sequencing was performed and results were available within days. interestingly, both a somatic variant in mpl (= trombopoetine receptor > myelofibrose) and a heterozygous variant in acp (trap, known immune dysregulation disorder) were identified, both fitting to the patients phenotype. based on these results the medication of the patient was changed, resulting in a substantial improvement of the patients constitution. in conclusion, we have implemented a rapid exome sequencing workflow for urgent cases. the rapid identification of pathogenic variants already had implications on patient treatment, underlying the added value of a fast genetic diagnosis. ement insertion, however, was spliced into the mrna of nabp , leading to a frameshift mutation and a premature stop codon, potentially altering or abolishing gene function. in summary, we have shown that transposon insertions, both common variants as well as rare or de novo variants, can be detected in wes data. such insertions in coding or regulatory regions of disease-relevant genes might therefore explain some of the cases in which no pathogenic coding mutation can be identified by wes. the influence of human genetic variation on epstein-barr virus sequence diversity: a genome-to-genome approach c. hammer , , a. loetscher , , em. zdobnov , genome-wide association studies (gwas) have identified common genetic polymorphisms that associate with clinical manifestation and immune response parameters of various infections. we here present an alternative approach, using variation in the virus sequence as phenotype, which is specific by nature and unique to genomic research in infectious diseases, for genome-to-genome (g g) association studies. building on the unprecedented possibility to combine large-scale human and viral genomic data, we explored interactions between human genetic variation and viral sequence diversity in individuals infected with epstein-barr virus (ebv). the major goal is the identification of key genetic players in the evolutionary 'arms race' between pathogen and host. ebv is the pathogenic agent of infectious mononucleosis and is associated with a broad spectrum of lymphoid and epithelial malignancies, including lymphomas and nasopharyngeal carcinomas. there is also evidence for a role of ebv in the pathoetiology of multiple sclerosis. its genome is approximately kbp long and encodes around proteins, not all of which have been definitely identified or characterized. it is known that high loads of ebv are present in patients with advanced human immunodeficiency virus (hiv)-induced immunodeficiency. we therefore selected immunosuppressed patients included in the swiss hiv cohort study (shcs) with low cd + t cell counts, and quantified ebv copy number in peripheral blood mononuclear cells (pbmcs). cell samples contained more than , viral copies in total and were subjected to target isolation and subsequent enrichment using the sureselect method by agilent biotechnologies, followed by illumina whole-genome sequencing. after data processing and quality control, variable amino acids were called as binary variables, resulting in > variable positions per individual in average. the same patients also underwent genome-wide genotyping to obtain host genetic variation, followed by imputation based on the haplotype reference consortium reference panel. the association analyses are currently ongoing, and we will present the results at the conference. we use logistic regression to test for association between host single nucleotide polymorphisms (snps) and binary ebv amino acid variants. bonferroni correction is applied for multiple testing correction on the sides of both host and pathogen. stratification is taken into consideration by including principal components (pcs) for the host, and phylogenetic pcs for the virus. this project will offer a global description of the adaptive forces acting on ebv during natural infection. we have shown before for hiv that a virus genome associates much more strongly with human genetic variants than clinical endpoints. the analysis of all signals resulting from the interaction between human and viral genomes has the potential to identify novel host defense mechanisms, which could serve as future diagnostic and therapeutic targets. is called in a reliable region if at least two out of five tools are concordant for the respective cnv. the pipeline shows a sensitivity of % and a precision of %. within routine gene panel diagnostics we analyzed a total of patients indicated to have rare mendelian diseases for snv and cnvs. in patients a cnv was detected in genes associated with the respective individual phenotype. interestingly, in several cases the cnv completed the patients report as it was detected in genes with a recessive mode of inheritance where previously only a heterozygous pathogenic snv was found. overall, with the additional analysis of cnvs we increased the diagnostic yield from % (class , single nucleotide events) to %. however, there are still issues in the detection of cnvs from ngs data for routine diagnostics. cnv pipelines are very prone to errors caused by enrichment inconsistencies compared to snv detection tools. the assessment of sensitivity and specificity is difficult due to the lack of datasets to validate cnv detection pipelines. originally, the analysis of cnvs was performed mainly in patients with mental retardation disorders, resulting in a paucity of cnv data linked to other mendelian diseases. moreover, the identification of the actual size and thus the assessment of pathogenicity of a cnv is difficult, because targeted ngs gene panels do not cover all genes. in conclusion, ngs data is a suitable data source for the simultaneous detection of snvs and cnvs for clinical diagnosis; however, with the current tools it is only applicable in accurately validated regions. identification of transposon insertions in whole-exome sequencing data s. lukassen, n. Übelmesser, ab. ekici, u. hüffmeier, ct. thiel, c. zweier, a. winterpacht humangenetisches institut, erlangen, germany % of the human genome consists of transposable element derived sequences, the most abundant of which are l and alu elements, followed by endogenous retroviruses. several hundred of these elements remain active, leading to insertion frequencies of up to one in live births for alu elements and posing a threat to genome integrity. while most studies on transposons employ whole-genome sequencing (wgs) or target-enrichment based sequencing approaches, the most commonly used form of diagnostic high-throughput sequencing is currently whole-exome sequencing (wes). we were therefore interested in investigating transposon insertions in wes data as a possible source of disease causing mutations. we developed a software to call non-reference transposon insertions from single-end wes datasets by split-read mapping and analyzed exomes this way. on average, non-reference insertions were identified in each exome, with an average of . sites per patient identified in < = . % of other patients. of these rare variants, % were deemed plausible by visual inspection. automated confidence calls of the software were concordant with visual inspection in % of cases. in % of cases a plausible insertion was awarded a lower score by the algorithm and in another % not called at all. in % of cases the automated call appeared to be falsely positive, in another % at the wrong position within the same bp window. laboratory validation of convincing insertions revealed a % true positive rate, leading to an estimated specificity of %. when performing calls for reference l insertions on exomes, % ( % - %) of known elements whose flanking regions were covered by at least two reads were correctly identified, leading to a sensitivity of %. we thus estimate the average number of non-reference transposon insertions in our wes dataset to be ( - ). % and . % of sites identified were associated with alu and l elements, respectively, with the majority of calls stemming from evolutionary young transposons still assumed to be active. . % of sites were located within the cds, . % in the utrs of genes, . % spanned an intron/ exon border, . % were intronic and . % of insertions were found in intergenic regions. we then chose insertions within intronic ( ) or utr ( ) regions for further analysis. seven were not detected in the mrna. one intronic alu el-abstracts ws - novel insights into male-pattern baldness pathobiology via integration of differential hair follicle mirna and mrna expression profiles with gwas data male pattern baldness (mbp) is a highly heritable condition and the most common form of hair loss in men. the phenotype is characterized by a distinct pattern of androgen-dependent progressive hair loss from the scalp that is restricted to hair follicles (hf) in the frontal and vertex scalp area. the molecular mechanisms that underlie this characteristic pattern and the differences in androgen-sensitivity between hf subpopulations in the frontal/vertex and the occipital scalp remain however elusive. to gain novel insights into the underlying biology and contributing genes and pathways, we systematically investigated for a differential expression (de) of mirna-and mrna-genes in hf samples from the frontal and occipital scalp area of healthy male donors. array-based genome-wide mirna and mrna profiling revealed expression of mirnas and , mrnas in human hf, of which mirnas ( %) and , mr-nas ( %) showed a de between hf subpopulations. the strongest de mirnas included mir- , mir- and mir- . among the strongest de mrnas were the wnt-signaling inhibitor dkk , the protein kinase pak and the retinoid acid receptor rora. a subsequent pathway-based analysis in mirpathdb revealed that de mirnas targeted numerous interesting pathways. among them the wnt-and mtor signaling pathway which have been implicated in the control of hair follicle cycling, a mechanism that is disturbed in mpb affected hf and other plausible candidate pathways such as estrogen, thyroid hormone signaling or epidermal growth factor binding which have not yet been implicated mpb pathobiology. to yield further evidence for an involvement of de mirnas and mr-nas in the developement of mpb, we subsequently integrated our expression data with association data from a large gwas meta-analysis on mpb (n = , ). of the de mirnas and mrnas, only mirna (mir- b) and mrnas were located within mb of one of genome-wide significant mpb risk loci. notably, the analysis revealed a co-localization of de mirna, de mrna, and nominally significant association signals (p < - ) at other genomic loci, pointing towards a role of these genomic regions in mpb pathogenesis. among them a locus on chromosome q . that comprises the genes encoding the ephrin-type-b receptor (ephb ) and the prostaglandin transporter slco a . interestingly, ephrins have been shown to be regulated by androgens and to play a role in hf formation, proliferation and hair cycling. and expression of prostaglandin d , which is transported by slco a , has been found to be upregulated in balding scalp where it inhibits hair growth. in summary, our systematic analysis of differential mirna and mrna expression and the subsequent integration with genetic association data identified novel potential risk loci for mpb and numerous candidate genes and pathways that are likely to play a role in mpb pathogenesis and emphasizes the importance of data integration of large-scale omic-analyses. palaeontological genomic analyses have shown that interbreeding between anatomically modern humans and neandertals occurred in europe and asia . - years ago. approximately . - % of the modern european and asian genome consists of introgressed dna from neandertals. some of these introgressed regions have been suggested to contribute to several traits and phenotypes including major depression and other mood disorders. in order to further assess the role of neandertal ancestry in cognition and the contribution of genetic risk for psychiatric disorders, we performed genome-wide analyses of neandertal alleles in publicly available psychiatric genomics consortium (pgc) gwas summary statistics with samples sizes ranging from about to individuals for the following phenotypes: educational attainment, attention deficit hyperactivity disorder (adhd), anorexia nervosa, anxiety disorders, autism spectrum disorder, bipolar disorder, major depressive disorder and schizophrenia. we estimated the proportion of heritability explained by snps in neandertal introgressed regions using stratified ld score regression (ldsc) and two sets of previously inferred neandertal introgressed regions. in a secondary analysis, we investigated whether specific functional annotations such as 'utr, promoter regions or histone marks within neandertal regions were significantly associated with selected phenotypes. we identified a modest enrichment of heritability in neandertal introgressed regions in anorexia nervosa, autism spectrum disorder, bipolar disorder and major depressive disorder, although none of the results were statistically significant. several functional annotations, such as h k me histone marks within neandertal introgressed regions, appeared significantly enriched for snps contributing to the heritability of anorexia nervosa and autism spectrum disorder. in bipolar disorder, dnasei digital genomic footprinting regions, h k ac histone marks and super enhancer regions within neandertal regions appeared particularly enriched for heritability. on the other hand, both sets of neandertal regions were slightly depleted of snps contributing to the heritability of schizophrenia. for example, one set of neandertal regions that contained % of all analysed snps only contributed to % of the variance of risk (standard error: . ; p-value: . × - ). in comparison to the rest of the genome, neandertal introgressed regions also contributed less to the heritability of educational attainment, adhd and anxiety disorders, although these findings were not statistically significant. to our knowledge this is the first study to systematically investigate the extent to which snps attributable to neandertal introgressed regions contribute to the heritability of several psychiatric/cognitive phenotypes. we are currently increasing our power to detect snp heritability in neandertal regions by applying the ldsc method to larger pgc datasets. harbor genes involved in the complement system, high density lipoprotein metabolism or extracellular matrix homeostasis. these pathways are known for their pleiotropic role in other conditions, such as cardiovascular disease, auto-immune diseases and cancer. here we aimed to investigate the extend of overlap between the genetic risk of various complex diseases and traits and the genetic risk for amd. methods: first, we catalogued , previously published, genome-wide significant variations associated with complex diseases or traits. next, we computed a genetic score by calculating the (weighted) sum of risk increasing alleles for each disease or trait. consequently, a higher genetic score indicates that an individual has more risk/trait increasing alleles of a given disease or trait. for each score, we computed the association with late stage amd using a dataset provided by the international amd genomics consortium (iamdgc) including , late stage amd cases and , controls. we also assessed the association of each variation individually with late stage amd risk in order to identify novel disease loci with strong evidence for pleiotropy. results: nineteen genetic scores of complex diseases and traits were significantly associated with amd risk (fdr < . ). most notably, all genetic scores related to autoimmunity were elevated in amd patients (p < . × - ), while scores related to cardiovascular disease were reduced in amd patients compared to controls (p < . × - ). we also found that the genetic scores of melanoma and related malignancies were higher in amd patients (p < . × - ). in addition, out of , variants, which were used to compute the genetic scores, were significantly associated with amd (fdr < . ), implicating novel, pleiotropic loci in amd risk. conclusion: our findings demonstrate a substantial overlap between the genetic risk of complex diseases/traits and the genetic risk for amd and provide evidence for novel, pleiotropic loci associated with amd. while our findings highlight common disease pathways that may facilitate to develop multi-use drug targets, they also challenge the notion that gene/genome manipulation could be applied in general terms to eradicate risk for a defined complex disease. worldwide genetic association study of exfoliation syndrome and glaucoma identifies common genetic variants at five new susceptibility loci exfoliation syndrome (pex), a complex systemic disorder of the extracellular matrix, is the commonest cause of secondary glaucoma in aging population and thus a major cause of blindness globally, affecting - million subjects worldwide. inside a large, international collaboration project a genome-wide association study (gwas) was carried out on , pex cases and , controls, recruited from countries across six inhabited continents, with replication in a further independent , cases and , controls from countries. significant association was observed at seven loci, of which two confirmed the already known associated loci at the genetic markers mapping to loxl -and cana a-gene, five are new (p < × - ). the five new loci map to chromosomes q (rs near flt -pomp-slc a , p = . × - ), q . (rs near tmem -arhgef , p = . × - ), p (rs at agpat , p = . × - ), p (rs at rbms , p = . × - ) and q (rs near sema a, p = . × - ). to determine the pathophysiological role of the three most significantly associated loci ( q , q . , p ), we investigated the expression and localization of the six related genes (flt , pomp, slc a , tmem , arhgef and ag- male-pattern baldness (mpb) is characterized by a progressive hair loss from the frontal and vertex scalp that affects ~ % of men at the age of years. epidemiological studies have shown positive associations between mpb and coronary heart disease (chd) and related phenotypes such as blood pressure (bp), diabetes (dm) or elevated blood lipid levels. the results however vary with regard to the associated pattern of hair loss (frontal or vertex) and the assessed endpoint measures for chd. and so far no study has investigated for a shared genetic determinant between the traits. using data from the heinz nixdorf recall study (n = , males) and a large meta-analysis on mpb (n = , ), we aimed at a systematic investigation of the association between mpb and chd on (i) an epidemiological and (ii) a genetic level. , men with vertex balding showed a higher bmi (β = . kg/m ), elevated fasting triglyceride (β = . mg/dl) and lower hdl-c levels (β = - . mg/dl). to assess the genetic overlap between mpb and chd, we created a risk score (rs) from mpb lead snps (p < × - ) and tested for association with chd and related traits phenotypes. no significant associations were observed. however, an age-stratified analysis revealed a % per allele risk increase for chd (hr = . , %ci: . ; . ) and a decrease in fasting triglyceride levels (β = - . ). we next used ld score regression analysis in to test for genome-wide genetic correlation between mpb and chd. the analysis revealed no significant correlations with cardiometabolic (n = ), lipid (n = ) or metabolic traits (n = ). finally, to investigate for a genetic overlap at single loci, we compared the mpb risk loci with reported gwas signals for chd. the analysis identified seven overlapping associations between mpb and bp (n = ); qt-interval length; atrial fibrillation; sudden cardiac arrest; and dm. for the majority of loci, the direction of effect differed between mpb and chd, opposing previous epidemiological findings. positive associations were identified between mpb and diastolic bp (fgf , q . ) and sudden cardiac arrest (atf , q . ). interestingly, fgf is known to stimulate cell growth and proliferation in multiple cell types, including cardiac myocytes and hair follicle (hf) cells, and atf is a hf expressed regulator of cell growth and differentiation that has been shown to prevent foam cell formation, which suggests that fgf and atf -signaling contribute to both traits. thus, our data support an association between mpb and chd related phenotypes and suggest that mpb deserves further evaluation as an additional risk factor for chd. pleiotropic effect of genetic variants associated with complex diseases and traits in age-related macular degeneration purpose: age-related macular degeneration (amd) is the leading cause of vision loss in western societies and is caused by both environmental and genetic risk factors. with regard to the latter, several associated risk loci abstracts otyping, results of which will be presented at the conference. of note, for nonsyndromic cleft lip with/without cleft palate (nscl/p), the most frequent form of orofacial clefting, risk loci have been detected by gwas so far, with some of them reaching (nearly) genome-wide or significant p-values in samples much smaller than cases. in the imputed nscpo dataset none of the presently known nscl/p risk loci showed a p-value < - . our data so far confirm previous molecular and epidemiological findings, that nscpo is genetically distinct from nscl/ p. furthermore, the results indicate that common variants alone might not contribute to the same extent to nscpo as compared to nscl/ p. the correlation between defects at specific imprinted loci and distinct imprinting disorder (id) was accepted for a long time. however, it is now put into question because of a growing number of patients with multilocus imprinting disturbances (mlid), i. e. the aberrant methylation at more than one imprinted locus. in particular, mlid is present in individuals with silver-russell syndrome (srs) and beckwith-wiedemann syndrome (bws), and it has meanwhile turned out that patients with opposite phenotypes can share common epimutation patterns. on the other hand, mlid always occurs as mosaicism and varies in different tissues of the same individual. interestingly, the majority of mlid carriers show only one specific id phenotype, though loci of other ids are affected in addition to the one specific for the phenotype. we become aware of a growing number of patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis for referral. amongst others, we detected the srs specific icr hypomethylation in p in two of our patients referred as bws. in the first case, the icr hypomethylation was detected only in lymphocytes but was not present in buccal swab dna. the patient only had a slight asymmetry, but showed normal growth and did not exhibit any other feature compatible with bws, nor with srs. the reason for the lack of clinical features is unclear, but is comparable to the observation in monozygotic, but clinically discordant srs and bws twins. here the unaffected twin often carries the epimutation only in lymphocytes whereas the affected one shows the alteration in additional tissues. a reason might be sharing of hematopoietic stem. it can be postulated that the patient presented here is born after an (undetected) twin pregnancy with early loss of the affected twin. in the second case, the initial diagnosis of bws was made due to asymmetry, though overgrowth or other features were not present. further clinical ascertainment did not confirm this diagnosis, but growth of the patient was in the lower percentiles, in concordance with the icr hypomethylation. these cases as well as further cases in our cohort confirm that there is an urgent need to provide detailed clinical data upon requesting molecular diagnostics for imprinting disorders. in fact, the growing number of patients with unexpected results complicates the interpretation and illustrates the broad phenotypic range, but also provides further insights in the etiology of ids and setting of imprinting marks pat ) by qrt-pcr, immunohistochemical-and western-blot analysis in genotyped ocular tissues of pex and control patients. all six genes displayed moderate mrna expression in all ocular tissues analysed, with highest levels in iris, ciliary body, and retina. however, only pomp showed a trend towards reduced expression in the presence of the rs risk allele, in both pex and control patients. in general, both mrna and protein expression of pomp and tmem were significantly reduced up to % (p < . ) in anterior segment tissues in pex eyes compared to controls. no differences in mrna and protein expression were detected for the remaining genes analysed. immunofluorescence analysis showed that pomp, a proteasome maturation protein, is ubiquitously expressed in most ocular cell types and that tmem , a transmembrane protein of unknown function, is primarily localized to endothelial cells of blood vessels and aqueous outflow structures. additionally, protein staining intensities for pomp and tmem were markedly reduced in anterior segment tissues of pex eyes compared to controls and co-localized to abnormal accumulation of pex material on ocular surfaces and in blood vessel walls. thus, at least two of the newly identified loci provide new biological insights into the pathology of pex syndrome/glaucoma and highlight a role for impaired proteasome function as well as vascular and trabecular endothelial dysfunction in the disease pathogenesis. nonsyndromic cleft palate only -evidence for a limited contribution of common variants in contrast to nonsyndromic cleft lip ± palate cleft palate only (cpo) is a common congenital malformation which might occur as part of a syndrome or in an isolated form, i. e., nonsyndromic cpo (nscpo). nscpo has a prevalence of : and is considered multifactorial with genetic as well as environmental factors contributing to the disorder. in a recent study we identified the first genome-wide significant locus for nscpo which has been independently confirmed in another study. in order to discover more nscpo risk loci we performed a genome-wide imputation study with gwas data from case-parent trios with european, asian and african ancestry which was retrieved from db-gap upon approved data access. notably, this gwas dataset had not yet been imputed, and we hypothesized that we can increase power to identify novel genetic associations by increasing the marker density and follow-up of suggestive findings by independent replication. genome-wide genotypes were imputed using impute based on genomes haplotypes, and snps were selected based on info-score > . and minor allele frequency > %. the imputation did not reveal any genome-wide significant snp, however, snps at loci showed p-values < - . loci with more than two variants below this threshold (n = ) were to be replicated using the massarray system (agena bioscience). three independent samples were used: two case/control replication cohorts from central europe ( cases, controls) and yemen ( cases, controls), and one european case-parent trio replication cohort (eurocran study; trios). in a first round we genotyped snps at eleven loci. one variant, rs at chr. q , showed p < . in the replication cohort and after combining replication and gwas data, resulted in a decrease of p-value from . × - to . × - . this indicates that this locus, which includes candidate genes such as igs , a known cell-adhesion molecule with yet unknown function in craniofacial development, might harbour a common risk variant with low effect size. we are currently performing a second round of gen-we report biallelic mutations in cad, encoding an enzyme of de novo pyrimidine biosynthesis, in four patients with developmental disability, epileptic encephalopathy, anaemia, and anisopoikilocytosis. two children died after a neurodegenerative disease course. treatment of two surviving children with oral uridine led to immediate cessation of seizures in both. a four-year-old girl, who was previously in minimal conscious state, started to communicate and walk with assistance after nine weeks of treatment. a three-year-old girl likewise showed developmental progress. blood smears normalised and anaemia resolved. our findings support the efficacy of uridine supplementation rendering cad deficiency a treatable neurometabolic disorder. delineation of the grin a phenotypic spectrum alterations of the n-methyl-d-aspartate (nmda) receptor subunit glu-n a, encoded by the gene grin a, have been associated with a spectrum of neurodevelopmental, speech and epilepsy disorders. we identified previously unreported patients with heterozygous pathogenic variants in grin a, including novel variants. after re-evaluation of all published grin a cases, previously reported patients met the acmg criteria for being pathogenic or likely pathogenic. thus, we are able to collectively review genotypes and phenotypes of individuals with grin a-related disorders. we show that the known phenotypic spectrum is expanded and ranges from near-normal development to severe and unspecific encephalopathy, comprising any disorder of speech development. furthermore, some patients do not display seizures. in contrast to previous reports, gri-n a missense variants cluster within the functionally most relevant domains. we are the first to describe genotype-phenotype correlations in grin a-related disorders, where carriers of pathogenic missense variants tend to have more severe neurodevelopmental phenotypes compared to carriers of truncating variants. the most severe end of the phenotypic spectrum was found to include novel features, such as infantile spasms and arthrogryposis and was associated with pathogenic variants in the pore-forming domain of grin a. the eponymic name galloway-mowat syndrome (gamos; omim ) has been coined for the association of early-onset nephrotic glomerulopathy, microcephaly with variable brain anomalies, and facultative diaphragmatic hernia. it is supposed to be inherited as an autosomal recessive trait and clinical as well as genetic heterogeneity has been suggested. in , wdr mutations were identified as a cause of gamos, but only a few cases have been reported to date. over the last years, we have collected dna samples and clinical data from unrelated families with one or more children affected by gamos or a gamos-like syndrome (glomerulopathy plus variable anomalies of brain morphology or function as inclusion criteria), including consanguineous families. in this cohort, we performed whole exome sequencing followed by targeted analysis by sanger and ngs multigene panel resequencing. in a total of families of this cohort ( %) the probable underlying genetic defect could be identified. in affected individuals from two consanguineous families, homozygous mutations of wdr could be found (vodopiutz et al., ) . thus, this gene accounted for only % of cases of our cohort. the affected child of another family had a novel homozygous mutation in arhgdia. this gene has previously been described in three families to cause early-onset steroid-resistant nephrotic syndrome (gupta et al., ; gee et al., ) , but there is some evidence that non-specific brain anomalies may also be part of the arhgdia-associated phenotype. fourteen and three index patients from unrelated families had mutations in one autosomal (osgep) and one x-linked gene (lage ), respectively, both encoding for components of the keops protein complex that has been implicated in transcription, telomere maintenance and chromosome segregation. no human phenotype has previously been assigned to mutations in this complex. notably, eight unrelated families with an identical mutation originated from the east asian population where the carrier frequency for this allele is . . in one consanguineous family with multiple affected children the disease segregated with a homozygous mutation in the sgpl gene encoding for sphingosine- -phosphate lyase. in four families, the kidney phenotype could be attributed to mutations in genes for non-syndromic nephrosis (nphs , plce , one novel gene), while the brain phenotype was apparently independent. in conclusion, the molecular genetic findings in this cohort confirmed that gamos is exceedingly heterogeneous, and still in almost half of the patients with a gamos-like phenotype the genetic cause remained unclear. on the basis of our findings we are now able to define new biologic mechanisms that are critically involved in both, brain development and integrity of the glomerular filtration barrier. genotype phenotype correlations are emerging. finally, we demonstrate that gamos can also be inherited as an x-linked trait. abstracts taminase gene (tgm ) is mutated in the majority of patients (around %), and its gene product, tgase , is therefore primarily targeted in our approach for protein substitution. patients with arci have an impaired skin barrier function, most of them are born with a collodion membrane and suffer subsequently from varying degrees of hyperkeratosis, erythema, transepidermal water loss and infections. the disease can be life threatening neonatally but lacks a causative therapy and is still only treated symptomatically. therefore, our aim is to develop a personalised, causative therapy where the defective protein is substituted topically via a nanocarrier. therapeutic, human tgase was synthesized in hek cells and assessed by western blot and flow cytometry analysis. enzyme activity was measured by in vitro assay. tgase was then coupled to a polyglycerol-based nanogel (dpg-ng) containing the thermoresponsive linker poly(n-isopropyl)acrylamide (pnipam), stabilising the enzyme as well as adding a thermal protein release trigger at °c, which is favorable for cutaneous applications. immunocytochemical stainings for tgase on monolayered basal keratinocytes that lack tgm expression confirmed the successful uptake of extrinsic tgase into the cells. further analysis over time showed that the enzyme was no longer detectable after h and consequently led us to define a treatment schedule for the following experiments. d full thickness skin models were used as in vitro system to determine barrier function and enzyme activity after treatment with varying concentrations of the dpg-ng/tgase complex. three different sets of skin models were used for these experiments: normal models mimicking the healthy skin with an intact barrier function, models where tgm was knocked down, and models made with arci patient cells with tgm mutations. franz cell tests on treated skin models lacking intrinsic tgase confirmed the impaired barrier activity in disease models, demonstrated an improved barrier function after repeated treatments with dpg-ng/ tgase and showed restored tgase activity using an in situ assay. furthermore, first toxicity tests using mtt revealed high biocompatibility of dpg-ng/tgase after treatment of d and d cell cultures. these findings are successful steps for an advanced topical drug delivery system and are a promising approach for causative therapeutic intervention in arci. after further optimization concerning protein dosage and thorough toxicity tests, we will adapt this system also for the use with other proteins involved in arci. pigmentation disorders (pds) comprise a large group of rare and heterogeneous disorders that are mainly characterized by various coloration abnormalities affecting single parts of the body or the complete integument. the large group of pds includes the autosomal dominant inherited hyperpigmentation disorder dowling-degos disease (ddd). ddd is genetically heterogeneous, and to date causal mutations in three genes, namely krt , pofut and poglut have been identified. after exclusion of mutations in these genes, we performed exome-and sanger-sequencing in six unrelated ddd-patients/families and identified six heterozygous truncating mutations in psenen encoding the presenilin enhancer protein . on closer examination of the histological sections, we came upon a novel feature that distinguished these individuals from previous ddd-cases by the presence of follicular hyperkeratosis. to assess the functional significance of psenen mutations in ddd pathogenesis, we performed mammalian cell culture based studies and knockdown experiments of psenen homolog psenen in zebrafish larvae (zfl). knockdown of psenen in zfl resulted in a phenotype with scattered pigmentation, which mimicked human ddd. in vivo-monitoring of pigment cells in the developing zfl suggested that disturbances in melanocyte migration and differentiation underlie ddd pathogenesis. interestingly, six of the psenen mutation carriers presented with co-morbid acne inversa (ai), an inflammatory hair follicle disorder. all individuals had a history of nicotine abuse and/or obesity, which are known trigger-factors for ai. although psenen mutations have been identified in a small subset (< %) of familial ai previously and the co-manifestation of ddd and ai has been reported for decades, our study is the first to demonstrate experimentally that mutations in psenen indeed can cause co-manifestation of ddd and ai, most likely triggered by predisposing factors for ai. thus, the present report describes a clinically and histopathologically novel ddd subphenotype in psenen mutation carriers, which is associated with an increased susceptibility to ai. protein substitution therapy for autosomal recessive congenital ichthyosis (arci) overall burkitt lymphoma showed a low genomic complexity with a low number of snvs and svs. however, the integration of cnas, snvs and svs allowed us to identify recurrently affected genes, which are involved predominately in the pi( ) kinase pathway, tonic bcr signaling, and cell cycle regulation, chromatin composition and germinal center development. burkitt lymphoma (bl) is the most common mature aggressive b-cell lymphoma in childhood. the genetic hallmark of bl is a chromosomal translocation involving the myc oncogene and one of the immunoglobulin loci leading to myc deregulation. three epidemiologic variants of bl are differentiated: endemic (ebl), which occurs predominantly in equatorial africa and is associated with ebv-infection, sporadic (sbl), which occurs in westernized countries and immunodeficiency-associated. in addition, burkitt leukemia (b-al) is differentiated from bl in cases with more than % of the bone marrow cells being lymphoma cells. another rare bl-variant is myc-positive precursor b-cell acute lymphoblastic leukemia coexpressing tdt and myc (tdt+bl). finally, we recently described a myc-negative variant which shows a typical alteration on chromosome (mnbll). the aim of the present study was to examine the epigenetic landscape of these bl variants. to this end, we analyzed the dna methylation of bl ( sbl, ebl, b-al, mnbll, tdt+bl) using the humanmethylation beadchip and contrasted the findings to diffuse-large b-cell lymphoma (dlbcl) and follicular lymphoma (fl). the majority of lymphoma were recruited in the framework of the icgc mmml-seq and mmml projects. the ebl were obtained from the nci ghana burkitt project. as controls, we used public available dna methylation data from b-cell burkitt lymphoma (bl), including its leukemic variant burkitt leukemia (b-al), is the most common type of pediatric b-cell lymphoma accounting for - % of new cases. its biological hallmark is the ig-myc translocation involving myc and mostly the immunoglobulin heavy (igh) locus or more rarely one of the immunoglobulin (ig) light chain loci. at the cytogenetic level the ig-myc translocation is the sole abnormality in around % of cases. overall, bl is characterized by a low genomic complexity. the aim of the present study was to analyze the genomic and transcriptomic landscape of pediatric/adolescent burkitt lymphoma by sequencing according to the guidelines of the international cancer genome consortium. a total of samples of bl/ b-al from pediatric/adolescent patients entered this sequencing study. all patients were treated in population-based prospective clinical trials. inclusion criteria were besides availability of suitable materials, consent to participate in the study and appropriate diagnosis: age at diagnosis (≤ years), the presence of ig-myc rearrangement detected by fish and/or whole genome sequence (wgs), absence of rearrangements of bcl or bcl genes. we performed wgs of tumor and matched control as well as transcriptome sequencing of the tumor cells according to the standards of the icgc (www.icgc.org). the pathognomonic ig-myc translocation was detected in of of the cases using wgs, but was observed in all cases by fish. an igh-myc juxtaposition was detected in patients and its variants igk-myc and igl-myc in and cases, respectively. we identified two different expression patterns of myc transcripts which were associated with the translocation breakpoint location. on the one hand the canonical myc transcript and on the other hand an alternative transcript with a transcription start site before the second exon. the latter produces an mrna which contains nucleotides not included in the canonical transcript but nevertheless it encodes the identical protein. the integration of single nucleotide variants (snv) and copy number aberrations (cna) identified a total of recurrently (≥ samples) mutated genes. myc, id , tp , ccnd , smarca , arid a, fbxo , ddx x were mutated in ≥ % of samples. in / ( %) cases, the id / abstracts ed. the annotation with the chromatin segmentation data of cd + t-cells from the blueprint project revealed enrichment of changes in methylation in distinct genomic regulatory elements in t-lgl. these differentially methylated functional regions were enriched for a set of transcription factor binding sites, known to be relevant in other lymphoid neoplasms. by bioinformatic analysis of methylation data and integration with gene expression data we identified hypermethylated and hypomethylated genes (e. g. bcl b, themis, zeb , hivep ) which point to candidate pathways potentially deregulated in the pathogenesis of t-lgl. conclusion: our study identified dna methylation changes in a set of candidate genes involved in various signaling pathways, which could potentially be used for diagnosis, prognosis and may become targets for novel treatment options. burkitt lymphoma (bl) is a mature aggressive b-cell lymphoma genetically characterized by a chromosomal translocation leading to ig-myc juxtaposition. treatment of bl is usually very successful particularly in children, with a cure rate of over % even among patients with advanced stage disease. however, the prognosis of the remaining patients experiencing disease progression and/or relapse is still very poor. bl has an overall low genomic complexity, thus secondary chromosomal changes in addition to the ig-myc translocation are rare. however, genomic complexity has been associated with aggressive disease and poor prognosis in various lymphomas including bl. because little is currently known about the underlying genetics of disease progression in bl we aimed at characterizing the molecular changes and characteristics that might lead to the relapse of bl. sequential tumor biopsies from initial diagnosis (id) and follow-up were available from a total of patients ( - years at id), which were divided into two groups: five patients experienced a relapse from their initial bl diagnosed - days after id (group ). in contrast, three patients developed twice a bl, i. e. presented with bl as secondary neoplasms diagnosed - years after id (group ). dna extracted from archival formalin-fixed, paraffin-embedded (ffpe) tissue was used to analyze genome-wide copy number alterations (cna) using the oncoscan® platform (affymetrix) and mutational landscape by whole exome sequencing (wes). analysis of the cna in the paired bl samples (group ) revealed an increase in genomic complexity in / pairs as in id a mean of cna was detected in contrast to . cna in relapse samples (p = . ). of note is that in all pairs, the relapse shared almost all cna which were present in id. wes analysis of group showed similar results in all analyzed pairs. in total, . % of mutations (median number of mutations = ) were shared in id and relapse. nevertheless, a considerable amount of mutations were unique in id and relapse with a median of ( . %) and ( . %) mutations, respectively. on the other hand, mutations detected in samples populations of various differential stages. furthermore, we investigated whole-genome bisulfite sequencing (wgbs) data of sbl and b-al in comparison to germinal center b-cell populations from healthy donors to decipher differentially methylated regions (dmr). these are defined as or more cpgs differentially methylated between two groups. unsupervised dna methylation analysis of bl, fl and dlbcl revealed that all bl variants cluster apart from the non-bl cases. thus, supporting on epigenetic level that all analyzed bl samples are bl variants. multigroup comparison (σ/σ max = . , q < e - ) separated the bl variants roughly in groups: ebl, ebv-positive sbl and all other bl variants. furthermore, this analysis revealed ebl to harbor a massive hypermethylation in comparison to all other bl variants. comparison of the dna methylation using the humanmethylation beadchip data of sbl and b-al revealed cpgs to be differentially methylated (σ/σ max = . , q < . ). in contrast, using the wgbs data of the same samples a total of dmrs could be identified which were mostly located in enhancer and polycomb target regions. in conclusion, we show that all analyzed bl variants share a similar dna methylation profile. interestingly, dmrs between sbl and b-al were mainly located in enhancer and polycomb regions. in contrast, ebl showed a massive hypermethylation in comparison to the other bl variants. thus, the differences identified by dna methylation analysis can improve the understanding of the biological and clinical differences of the bl variants. dürig , introduction: t-cell large granular lymphocytic leukemia (t-lgl) is a mature t-cell leukemia which often arises in the context of autoimmune disease. genetic changes like recurrent chromosomal aberrations are rare. recent studies identified somatic stat and tnfaip mutations in t-lgl cells. however, the molecular events driving leukemogenesis remain largely unknown. objectives: the goal of our study was to characterize the epigenetic basis of t-lgl to better understand leukemogenesis and potentially identify druggable pathways or diagnostic biomarkers for t-lgl. p. johansson , , l. klein-hitpass , g. castellano , k. kentouche , f. nicolau , i. oschlies , e. carrillo-de santa pau , m. przekopowitz , a. queiros , m. seifert , a. valencia , ij. martin-subero , em. murga penas , o. ammerpohl , u. dührsen , r. küppers , j. we analyzed the dna methylome of facs sorted tumor cells of t-lgl cases in comparison to benign αβ t-cell subsets. the infinium human methylation bead chip was used for analysis. we annotated our data with the publicly available chromatin segmentation data of cd + t-cells from the ihec/blueprint project. the expression levels of selected genes were tested by reverse transcription real-time pcr. results supervised analysis of t-lgl compared to benign cd + memory cells resulted in , cpg loci significantly (q < . ) differentially methylat- krawitz , , a. knaus , , m. jäger , , r. flöttmann , t. eggermann , b. hoechsmann , h. schrezenmeier paroxysmal nocturnal hemoglobinuria (pnh) is an acquired disorder of the blood-forming system. typically, affected hematopoietic stem cells (hscs) in pnh harbor a single somatic loss-of-function mutation in the x-linked piga gene. previously, a pnh patient with a different molecular etiology has been described and herein we report three more cases of this new subgroup: a predisposing germline mutation in pigt, which is an autosomal gene of the glycosylphosphatidylinositol (gpi)-anchor synthesis pathway, is followed by a second somatic hit. by means of deep sequencing and array-cgh, we observed acquired deletions of mb to mb on chromosome q in pnh cells that include pigt as well as a region that is commonly deleted in myeloproliferative neoplasms and myelodysplastic syndromes and that is known to be differentially methylated. this results in a complete loss of expression of certain genes at this locus which is also thought to contribute to the clonal expansion. the deficiency of gpi-anchored proteins on pnh cells results in a lack of the complement regulatory proteins cd and daf/cd on the cell surface and leaves them more vulnerable to the c b- membrane attack complex. in contrast to classical pnh without any fully synthesized gpi-anchors, pigt mutations impair the transamidase that links the substrate to the anchor and thus result in an accumulation of unbound gpi molecules. this difference in the pathophysiology can also be visualized in flow cytometric analysis of peripheral blood: while cd and cd surface levels are reduced in all pnh cells, the atypical pnh cells due to a transamidase deficiency can be discriminated by a specific antibody, t mab, that binds free gpi anchors. besides the classical pnh symptoms of anemia, thrombosis, and hemolysis, patients with pigt mutations also manifest with additional autoinflammatory symptoms, such as urticaria, fever, arthralgia and meningitis, and it is hypothesized that the free gpi-anchor that accumulates in affected cells is causally related to autoinflammation. based on these findings, we propose the new entity of atypical pnh. background: the prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. genetic variants explain only a minor part of this obesity epidemics induced by physical inactivity and over nutrition. epidemiological studies in humans and animal models of di-from patients with secondary neoplasm (group ) were mostly unique to id (= , . %) whereas only . % of all mutations were shared in id and secondary neoplasm samples (= ). furthermore there were no shared cna in the corresponding samples identified by oncoscan® analysis. to sum up, the oncoscan® and wes analysis, of the paired bl group ( ) provide strong evidence for a linear clonal evolution, meaning relapses may directly evolve from the previous lymphoma clone rather than a common precursor. in contrast, results obtained for patients with secondary neoplasm (group ) showed no indication for linear but rather for divergent evolution. thus, analysis of recurrent mutations shared in id and second neoplasm samples can provide important information about disease progression and are therefore subject of ongoing analysis. y. murakami , t. hirata , s. murata , t. kinoshita , m. kawamoto , s. murase , h. yoshimura , n. kohara , n. inoue , m. osato , j. nishimura , y. ueda , y. kanakura , p. m. in runx mutated aml the number of runx mutations, loss of the wild-type allele and the number and kind of additional mutations impact on prognosis a. stengel, w. kern, m. meggendorfer, k. perglerovà, t. haferlach, c. haferlach mll munich leukemia laboratory, munich, germany, mll , praha, czech republic aml with mutated runx show a distinct pattern of cytogenetic and molecular genetic abnormalities and an adverse prognosis. we analyzed the impact of multiple runx mutations and runx wild-type (wt) loss on associated genetic alterations and survival. for this, aml cases with runx mutations (mut) were split in ( ) runx wt loss (n = ), ( ) > runx mut (n = ) and runx mut (n = ). cases were selected for mutation analyses of genes. in cases with runx mut, + was frequently found, whereas in wt loss + was the most abundant trisomy (+ : % in runx mut vs. % in wt loss, p = . ; + : % vs. %, p < . ). cases with > runx mut showed an intermediate distribution (+ : %, + : %). missense mutations were the most abundant mutation type in wt loss cases ( % vs. %, p = . ), whereas in runx mut, frameshift mutations were found more frequently ( % vs. %, p = . ). in cases with > runx mut, both were observed at similar frequencies (missense: %, frameshift: %). mutation analyses of selected cases revealed additional molecular mutations. % of cases showed at least one runx -accompying mutation (range: - ). the median of accompanying mutations was n = in the total cohort and in cases with runx- mut and > runx mut, whereas it was n = in runx wt loss. srsf ( %), asxl ( %), dnmt a ( %), idh ( %), sf b ( %), tet ( %) and bcor ( %) were revealed as most frequently mutated genes. cases with runx wt loss showed a higher frequency of asxl mut compared to the other cases ( % vs. %, p = . ), while u af mut were absent from this group ( % vs. %, p = . ). median overall survival (os) in the total cohort was months. wt loss (os: months) and > runx mut ( months) showed an adverse impact on prognosis compared to runx mut ( months; p = . and p = . , respectively). mutations in asxl and kras and the presence of ≥ additional mutations also negatively impacted os ( vs. months, p = . ; vs. months, p < . ; vs. months, p = . ). in univariate cox regression analysis runx wt loss (hr = . ; p = . ), ≥ additional mutations (hr = . ; p = . ), asxl mut (hr = . ; p = . ) and kr-asmut (hr = . ; p = . ) had an adverse impact on os. multivariate cox regression analysis revealed an independent adverse effect on os for runx wt loss (hr = . ; p = . ) and krasmut (hr = . ; p = . ). for / cases we received samples during course of the disease. in none of these cases, an evidence for a runx germline mutation was found by analyzing the mutation loads, thus all runx mutations are somatically acquired. taken together, we found strong differences between the subgroups in regard of cytogenetic and molecular genetic aberrations as well as regarding prognosis. thus, not only the presence and number of runx mutations but also the conservation of an intact runx allele as well as the number and kind of additional mutations is biologically and clinically relevant. abstracts different chromatin states, where methylation is inversely correlated with active histone marks. using the hardy-weinberg law, we estimate that there are dmrs with a maf> . . we hypothesized that cis-acting dna polymorphisms could be responsible for the inter-individual variation of the dmrs methylation levels. we genotyped . million snps in the five donors and found that / ( %) dmrs have methylation levels highly correlated (> . ) with the genotype of at least one nearby snp (± kb window). this correlation was verified in / dmrs by targeted bisulfite sequencing in monocytes from individuals used for wgbs and from additional individuals. to validate our results in a larger population and possibly find correlating snps outside the ± kb window for the remaining dmrs, we performed genome-wide association studies (gwas) using snp genotypes and illumina k cpg methylation data from blood samples of individuals from the heinz nixdorf recall study. these methylation arrays encompass only cpgs contained in of our dmrs, showing that they fail to identify a great number of potentially important regions. we certified that for these cpgs, monocyte and whole blood dmrs methylation levels were correlated, and performed a gwas with ~ , snp for each of the cpgs. for / cpgs, the correlation peak was near the cpg position. for each gwas, the snp with lowest p-value (in most cases p < e - ) was designated as lead-snp. snps in high linkage disequilibrium (r > . ) to the lead-snps were located within the corresponding dmr or bp to ~ kb from it. many regions are bound by ctcf and other transcription factors. it is likely that snps affect the binding of these factors and thus the methylation state of the region. we conclude that these inter-individual differences in dna methylation are mainly driven by genetic factors. the dystonia (dyt ) protein thap recruits the histone deacetylase hdac to mediate gene repression sektion für funktionelle genetik am institut für humangenetik, universität zu lübeck, lübeck, germany, institut für neurogenetik, universität zu lübeck, lübeck, germany dystonia describes a heterogeneous group of neurological movement disorders characterized by contractions in various muscles resulting in abnormal postures, involuntary twisting and repetitive movements. dystonia (dyt ), a primary torsions dystonia that first has an impact on cranio-cervical muscles causing problems with speaking and eating, is caused by mutations in the thap gene (thanatos-associated domain-containing apoptosis-associated protein ). thap belongs to the family of thap proteins that are characterized by the presence of an evolutionarily conserved specific dna-binding thap zinc finger motif at their n-terminus. in humans thap family members are known, designated thap to thap . interestingly, most of the dyt -causing mutations affect this thap domain. while we have previously described thap -mediated repression of specific target genes, the molecular mechanisms how thap regulates promoter activity are rather unknown. it is known, that other members of the thap family such as thap and thap interact with the histone deacetylase hdac to mediate transcriptional repression. we have performed yeast-two-hybrid and gst pulldown assays to identify a specific interaction of thap with hdac . by the use of truncated thap fragments we were able to narrow down hdac binding to the n-terminal thap-domain. for further functional characterization we have decreased hdac levels by sirna treatment or chemical inhibition and used taqman analyses to quantify the effect on thap -target genes expression. thus, a significant increase of thap -target genes expression was detected in those cells treated with hdac sirna. to further investigate whether the observed increase in gene expression is due to alterations of histone acetylation within the promoter regions we performed chromatin immunoprecipitation (chip) assays followed by qpcr using antibodies specific for different acetylated n-terminal residues of histone as markers for transcriptional active promoters. by this, we detected an increased acetylation within the promoter regions of thap target genes that are dysregulated in cells treated with decreased hdac levels. et-induced obesity indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. possible adverse paternal effects are likely transmitted by the sperm to the next generation. to prove this hypothesis, we have systematically analyzed the effects of paternal obesity on the sperm epigenome and its implications for the next generation. results: to study the possible transmission of paternal bmi effects to the next generation, methylation levels of eight paternally expressed imprinted genes (peg , peg , peg , peg , peg , peg , nespas and igf ), two maternally expressed imprinted genes (meg and h ), and the obesity related gene hif a were quantified by bisulphite pyrosequencing in sperm of donors (undergoing ivf/icsi) and fetal cord blood (fcb) of resulting offspring (conceived by ivf/icsi with the same sperm samples). hif a showed a significant positive correlation between sperm methylation and paternal bmi. this effect on the sperm epigenome was replicated in an independent cohort of sperm samples. for hif a, paternal bmi also showed a significant positive correlation with fcb methylation. on the other hand, peg /nnat exhibited a significant negative correlation between paternal bmi and fcb methylation. in contrast to pyrosequencing, deep bisulphite sequencing (dbs) allows one to study dna methylation at the single molecule level and enables us to distinguish between maternal and paternal alleles in fcb samples with an informative snp. epimutations which are defined as alleles showing > % aberrantly (de)methylated cpg sites can also be identified with dbs. upon performing dbs on sperm samples, we observed a higher epimutation rate in the high bmi ( - ) group when compared to the low bmi ( - ) group across the four studied genes (peg , hif a, h and nespas). we are presently analyzing dbs data in selected cord blood samples with an informative snp to separately quantify methylation at the paternal and maternal alleles. it is important to decipher the methylation of the paternal allele when studying whether sperm methylation alterations are transmitted to the offspring. conclusions: our results suggest that male obesity is associated with modification of the sperm dna methylome, which may affect the epigenome (in fetal cord blood) of the next generation. allele-specific dna methylation occurs at functionally different regions: ) at imprinting control elements, ) on the silent x chromosome in females and ) across the genome and probably dependent on the dna sequence in cis. the latter is termed haplotype-dependent allele-specific methylation and may contribute to inter-individual phenotypic variation. in a previous study on monocyte to macrophage differentiation, we showed that dna methylation differences between individuals were greater than between the two cell types. to study the genetic basis of these inter-individual differences in dna methylation, we analysed the methylome obtained by whole genome bisulfite sequencing (wgbs) of monocytes from five unrelated donors. for identifying differentially methylated regions (dmrs), we created two synthetic methylomes: one with the highest methylation values of each cpg in the five samples and one with the lowest methylation values. defining a dmr as a region of at least cpgs with a methylation level difference of at least . , we identified dmrs, which cover cpgs and fall into cer, respectively. we show that ns-associated rit mutants intensified signal flux through the mek-erk pathway upon growth factor stimulation. by using heterologous expression systems, we identified the p -activated kinase (pak ) as novel effector of rit . we found that rit interacts with the rho gtpases cdc and rac , both of which are crucial upstream regulators of pak . disease-causing rit mutations enhance protein-protein interactions and uncouple complex formation from growth factors. expression of both wild-type rit and its mutant forms resulted in dissolution of stress fibers and paxillin-containing focal adhesions from the cell center and increased cell movement. we conclude that rit is a potent regulator of actin dynamics, and dysregulated rac /cdc -pak signaling controlling cell adhesion and migration may be one aspect of the molecular basis of ns. medical systems biology, tu dresden, germany, institute for clinical genetics, tu dresden, germany, cancer science institute of singapore, national university of singapore, singapore, institute of molecular biology, mainz, germany telomeres are short repetitive ttaggg sequences that cap the ends of chromosomes. these stretches of dna are covered by proteins and rnas which together protect the putative double strand break from dna repair mechanisms and facilitate replication. however, telomeres shorten with every cell division due to the end replication problem. the ribonucleoprotein telomerase counteracts this process by de novo elongation of telomeric repeats but its expression is mostly confined to the germ line and stem cells. even in the latter its activity is usually not sufficient to completely prevent telomere shortening. all cancer cells are also faced with this challenge and while the majority of cancer cells rely on telomerase, approximately % of cancers ensure sufficient telomere length via the recombination-based alternative lengthening of telomeres (alt) mechanism. to better understand telomere biology we aimed to identify novel telomeric factors by systematically screening for telomere-binding proteins in cell lines from different vertebrates. here, we identified and characterized zbtb , a zinc finger protein, as a novel direct telomere-binding protein across the vertebrate lineage. zbtb is directly binding to telomeric dna in vitro and it is localizing to telomeres in vivo via one specific zinc finger domain in both telomerase-and alt-positive cancer cells. interestingly, zbtb knock-out cells have longer telomeres, suggesting that zbtb limits telomere elongation. in addition, the combination of chipseq, rnaseq and proteome analysis revealed a transcription factor activity for a small, but specific set of target genes of zbtb , linking its telomeric functions to mitochondrial metabolism. in conclusion, zbtb is a novel direct telomere binding protein with transcription factor activity that acts as negative regulator of telomere length. our data show for the first time a functional interaction of the 'dystonia protein' thap with the histone deacetylase hdac and therefore give new insights into the molecular mechanisms of thap -mediated gene repression. interestingly, previous functional studies as well as structure analyses revealed that only a subset of the dyt -causing mutations affecting the n-terminal thap domain alter thap -binding to dna. in ongoing studies we want to investigate the consequences of dyt -causing mutations on thap -hdac complex formation and its relevance in the molecular pathology of dystonia. reproductive homeobox (rhox) genes are clustered on the x chromosome and share a unique amino acid helix-turn-helix dna binding homeodomain. they were identified in several species as having important roles in reproductive tissues, notably in the testis. the human rhox cluster is composed of three genes: rhoxf and two copies of rhoxf (rhoxf a, rhoxf b) which are referred to as rhoxf / b. rhox proteins are expressed exclusively by germ cells in human testis and aberrant rhox methylation is associated with several sperm parameters. because little is known about the molecular mechanism of rhox function in humans, the aim of the study was to identify target genes of human rhox proteins and to investigate the impact of rhox mutations on protein function. using gene expression profiling, we identified genes regulated by members of the human rhox gene cluster. some genes were uniquely regulated by rhoxf or rhoxf / b, while others were regulated by both of these transcription factors. several of these regulated genes encode proteins involved in processes relevant to spermatogenesis, e. g. stress protection and cell survival. one of the target genes of rhoxf / b is rhoxf , suggesting cross-regulation to enhance transcriptional responses. the potential role of rhox in human infertility was addressed by sequencing rhox in a group of patients with severe oligozoospermia. this revealed two mutations in rhoxf (c. g>a and c. c>t) and four in rhoxf / b (- c>g, c. g>a, c. c>t and c. g>a), of which only one (c. g>a) was found in a control group of men with normal sperm concentration. functional analysis demonstrated that c. g>a and c. g>a significantly impaired the ability of rhoxf / b to regulate downstream genes. molecular modelling suggested that these mutations alter rhoxf /f b protein conformation. by combining clinical data with in vitro functional analysis, we demonstrate how the x-linked rhox gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility. colorectal cancer (crc). here, we proposed the possible molecular mechanisms responsible for crc initiation, progression and invasion using a network biology approach. materials and methods: in order to investigate the underlying crc pathogenesis, the dataset gse consisting of normal tissues, stage i, stage ii, stage iii and stage iv of crc were obtained from gene expression omnibus (geo) and further examined. the differentially expressed genes (degs) were subjected to protein-protein interaction databases and a ppi network was constructed for each crc stage. topological analysis of resulted ppi networks revealed functional hub genes and involved in crc development. furthermore, the overlap genes between four studied crc stages were determined and deeply evaluated to identify deregulat ed biological networks during crc development. a standard real-time pcr was performed to validate the in silico findings utilizing sw and ncm cell lines. results: the most important hub genes (cdk for stage i, ubc for stage ii, esr for stage iii and atxn for stage iv) and sub-networks were identified in crc stages. moreover, several novel biomarkers were also introduced for each crc stage. gene ontology (go) and signaling pathway enrichment uncovered the important roles of wnt, mapk and jak-stat signaling pathways in regulation of crc pathogenesis. functional annotation of overlap genes revealed that cell cycle regulating genes are the most highly regulated genes during crc initiation, progression and invasion. in vitro analyses confirmed deregulation of atxn and cdk , two hub genes of stage iv, in metastatic colon sw cells compared to normal colon ncm cell line. our study provides a new insight into the distinct molecular mechanisms underlying the pathogenesis of crc. the functional hub genes, sub-networks, prioritizes key pathways and novel crc biomarkers were also provided that can be useful in therapeutic programs. targeted next-generation sequencing approaches as well as next-generation whole exome sequencing are becoming more widespread in routine molecular diagnostics for patients with ataxia. however, since ngs at present is not suitable to detect (trinucleotide) repeat expansions, a pre-ngs testing for common polyglutamine expansion scas seems mandatory. but also sca subtypes caused by expansions in non-coding regions of genes like sca , sca , sca , and sca as well as other ataxias known to be associated with repeat expansions like the fragile x-associated tremor ataxia syndrome (fxtas) should be taken into account before applying ngs-based diagnostics. in order to find an optimal diagnostic strategy in future more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia would be helpful. we therefore analyzed a cohort of patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for sca diagnostics and showed alleles in the normal range for the most common sca subtypes sca - , sca , sca , and sca . these patients were screened for expansions in sca , sca , sca , sca and fxtas as well as for the pathogenic hexanucleotide repeat in the c orf gene. no expanded repeats for sca , sca or sca were found in the analyzed patients. five patients with ataxia of unknown etiology showed sca cta/ctg combined alleles ( - ) that are discussed to be potentially pathogenic. one -year-old male patient with unclear dementia syndromes was diagnosed with a large ggggcc repeat expansion in c orf . and the analysis of the fmr gene identified one patient with a permutation (> cgg repeats) and seven patients poster *** = für den posterpreis nominiert preventive genetic counseling in neurogenetic disorders needs a better collaborative approach between genetic and neurology clinics -a report of four siblings with unverricht-lundborg disease: genetic counseling is the process of helping people to understand and adapt the medical, psychological and familial implications of genetic contributions to disease. for parents with a previous child or other family member with a known genetic syndrome expands options for preimplantation or prenatal diagnosis for the current or the future pregnancies. however, timely referral by health providers to genetic counselor and for discussing with couples regarding possible options is important. additionally, other factors such as personal decision making especially due to high price of some genetic services and uncertain results cause considerably delays to genetic testing. there are more than various types of inherited neurological disorders in which alterations in genes lead to an inherited condition such as huntington disease, inherited forms of alzheimer disease, ataxia, muscular dystrophies and epilepsies. the knowledge of the causative gene mutations in the affected individual is critical in the possible prenatal diagnosis in other members of the pedigree. therefore a multidisciplinary care team, including neurologist and genetic counselor for the conditions diagnosed as inherited neurological disorders is critical in prenatal setting and consideration of an effective management. here, our report of four siblings affected by a rare form of inherited epilepsy (unverricht-lundborg disease) with an autosomal recessive pattern highlights the importance of the needs for a better collaborative approach in the neurogenetic setting. in fact, the birth of four successive siblings affected by similar neurogenetics disorders in a specific family is showing the need for more attention to this important issue, especially in terms of intersectoral collaboration. poorebrahim hort: / differentially expressed transcription units). these differences in gene expression we detected did not correlate with dna methylation changes at the corresponding transcription regulatory sites. from our results we conclude that altered expression of imprinted genes indeed plays a role in tumorigenesis of germinal center derived b-cell lymphomas. however, the altered transcriptional regulation of these genes seems not to rely on the usual epigenetic mechanisms known from constitutional imprinting disorders. mf. abazari , h. bokharaie , m. asghari , v. poortahmasebi , h. askari , m. investigating the expression of genes associated with autism spectrum disorders to identify sex related differences s. berkel, a. eltokhi, g. rappold institute of human genetics, heidelberg university hospital, heidelberg, germany neurodevelopmental disorders such as autism, attention deficit and hyperactivity syndrome as well as language problems and learning difficulties have a higher prevalence in male individuals compared to females. autism is characterized by impairments in social interaction, communication deficits and restricted and repetitive behaviors. boys are more frequently affected than girls; the ratio of affected boys compared to girls is : for autism and : for asperger syndrome. in this study we aim to elucidate the reason for this gender difference by following up two hypotheses: ( ) risk genes for autism spectrum disorders (asd) might be expressed at different levels in males and females and ( ) asd risk genes might interact with sexually dimorphic pathways. first, we investigated the expression of genes associated with autism spectrum disorders, including the shank gene family, in the brain of male and female mice to identify sex-dependent differences. the rna expression levels were analyzed in five different brain regions (cortex, hippocampus, striatum, cerebellum, thalamus) at different developmental stages (e , e , p , p , p and adult) in male and female mice. we identified a sex dimorphic expression of shank and shank , but not of shank . due to the fact that early brain development is strongly influenced by sex hormones (estrogen, testosterone), we further investigated the influence of these hormones on shank expression in human neuroblastoma cells (sh-sy y) and primary mouse hippocampal neurons. a better understanding of the sex differences in the brain might help to explain the vulnerability for neuropsychiatric disorders like autism and paves the way to discover putative risk or protective factors for these disorders. imprinting defects in temple syndrome are caused by a failure in imprint establishment and/or maintenance j. beygo, c. mertel, g. gillessen-kaesbach, b. horsthemke, k. buiting institut für humangenetik, universitätsklinikum essen, universität duisburg-essen, essen, germany, institut für humangenetik, universität zu lübeck, lübeck, germany temple syndrome (ts ) is a rare imprinting disorder characterised by low birth weight and height, muscular hypotonia and feeding difficulties in the infant period, early puberty and short stature with small hands and feet and often truncal obesity. in a subset of patients with ts , the disease is caused by an imprinting defect (id) affecting the paternal allele of the imprinted region q . the id results in aberrant methylation of the three known differentially methylated regions (dmrs), the germline-derived primary dlk /meg intergenic (ig-)dmr (meg /dlk :ig-dmr), the postfertilization-derived, secondary dmr at the meg promoter (meg :tss-dmr), and the postfertilization-derived, secondary intragenic meg -dmr (meg :int -dmr). the meg /dlk :ig-dmr and the meg :tss-dmr are methylated on the paternal chromosome and hypomethylated in patients with ts and an imprinting defect. the meg :int -dmr is unmethylated on the paternal chromosome and hypermethylated in these patients. both the meg /dlk :ig-dmr and the with alleles in the grey zone ( to cgg repeats), thus suggesting that individuals with fmr repeat expansions in the gray zone may also present with neurological signs. bernhart , , , h. kretzmer , , r. wagener , c. mmml some genes are subject to the mechanism of imprinting, i. e. their expression depends on parental origin. they primarily function in the control of proliferation, fetal development and cellular differentiation. constitutional imprinting disorders are in part also associated with an increased tumor risk. loss of imprinting has been also described as somatic event in tumorigenesis. while this phenomenon has been broadly analyzed in solid tumors, data on alterations of imprinting in lymphatic neoplasms are largely missing. we analyzed the rna expression of transcription units/regions known or supposed to be subject to imprinting in two cohorts of normal b-cells and germinal center derived b-cell lymphomas. the first cohort (mmml) contains samples: burkitt lymphomas (bl), non-burkitt lymphomas (non-bl, including various subtypes like follicular and diffuse large b-cell lymphoma) and normal germinal center b-cell samples (gcbc, as controls). the second cohort (icgc mmml-seq) comprised samples with bl, non-bl and gcbc samples. gene expression was analyzed with affymetrix u a genechips in the mmml cohort and by rna sequencing in the icgc mmml-seq cohort. results of the transcriptional analyses in the icgc mmml-seq cohort were compared to the dna methylation available from a subset of the analyzed samples (kretzmer et al., nat genet, ) . of the transcription units sites, corresponding to transcription units, were present on the applied array used for the analysis of the mmml cohort. a two group comparison revealed significantly differentially expressed sites corresponding to transcription units between bl and non-bl including the plagl and peg genes. in total, and sites corresponding to and transcription units are differentially expressed between bl versus gcbc and non-bl versus gcbc, respectively. comparison of gene expression in the icgc cohort revealed differentially expressed sites corresponding to transcription units between bl and non-bl (overlap with mmml cohort: / differentially expressed transcription units), including again peg and plagl , differentially expressed sites corresponding to transcription units between bl and gcbc (overlap with mmml cohort: / differentially expressed transcription units) and differentially expressed sites corresponding to transcription units between non-bl and gcbc (overlap with mmml co-abstracts maintenance dna methylation of l promoters, spoc could function in targeting g a to l sequences. in conclusion our data implicate the epigenetic reader spoc in the suppression of line elements during germ cell development. s. bens , j. kolarova , m. kreuz , sh. characterization of the expression of the imprinted kcnk -gene in specific brain regions and the phenotypic analysis of kcnk knockout mice a kcnk /kcnk is a maternally expressed imprinted gene whose mutations are responsible for the maternally inherited birk-barel mental retardation dysmorphism syndrome. it encodes a member of the superfamily of k+channels with two pore-forming domains and is involved in the modulation of the resting membrane potential and excitability of neuronal cells. so far, only homozygous kcnk knockout mice with inactivation of both parental alleles were phenotypically characterized. these mice displayed cognitive deficits as well as a reduction of k+ leak current by %. in the light of maternal-specific imprinted expression of kcnk /kcnk and the maternal inheritance of the birk-barel mental retardation dysmorphism, a thorough phenotypic analysis of heterozygous kcnk knockout mice with inactivation of only the maternally inherited allele is also warranted. as first aim of our study, we characterized the parental allele-specific expression of kcnk in various regions of the mouse brain. quantification of allele-specific expression by pyrosequencing (quasep) method was performed for different brain areas from several developmental stages of (c bl/ xcast/ei) f hybrid mice. exclusive expression from the maternal kcnk allele was detected in the dentate gyrus, hippocampus, mesencephalon, medulla oblongata, thalamus and pons. biallelic expression with, however, a strong bias towards the maternal kcnk allele ( - % of the transcripts) was observed in the olfactory bulbs, cortex, cerebellum, striatum and olfactory tubercles. as the second aim of our study, the phenotypes of wildtype, heterozygous kcnk knockout mice with maternal inherited knockout allele (kcnk komat) and homozygous kcnk knockout mice (kcnk kohom) were comparatively examined in a behavioral test battery. due to the already known cognitive defects of kcnk kohom animals and especially the phenotype of the patients with birk-barel syndrome, it was assumed that kcnk komat and kcnk kohom animals show deficits in some of the tests. the spontaneous alternation in the y-maze test was significantly reduced by approximately - % in kcnk komat and kcnk kohom mice compared to wildtype mice indicating a clearly impaired working memory. in addition, kcnk komat and kcnk kohom mice displayed a reduced prepulse inhibition of startle response compared to wildtype mice indicating an impairment of sensomotoric gating, a process to filter out irrelevant information. acoustic startle response as a measure of anxiety levels was also significantly decreased, but only in kcnk kohom mice. our findings shall further elucidate the role of kcnk /kcnk in brain physiology and pathophysiology and open new avenues for treatment of cognitive dysfunctions in birk-barel syndrome. meg :tss-dmr act as imprinting control centres, although the meg / dlk :ig-dmr functions as an upstream regulator of the meg -dmr. so far, the function and regulation of the meg -dmr is unknown. the hypomethylation of the paternal allele in ts -id patients at the meg / dlk :ig-dmr and the meg :tss-dmr point to a failure in the establishment of the methylation imprint or to maintain the methylation imprint after fertilization. in this case, the incorrectly imprinted chromosome would be inherited from either the paternal grandfather or grandmother. to prove this assumption we are investigating the grandparental origin of the affected chromosome in our cohort of ten ts -id families by studying the parent-of-origin specific methylation of the three dmrs in combination with informative single nucleotide variants (snps). at the moment we have identified three families informative for the meg / dlk :ig-dmr, two families for the meg :tss-dmr and two families for the meg :int -dmr. so far we have obtained results in two families for the meg :tss-dmr. we found that in one case the allele harbouring the id was inherited from the paternal grandmother, but in the second case from the paternal grandfather, indicating that the id occurred after erasure of the parental methylation imprints. a complete lack of methylation observed in the majority of ts -id patients is therefore likely due to a problem in establishing methylation on the paternal chromosome, whereas in rare cases with methylation mosaicism, the id is probably due to a problem to maintain the paternal imprint after fertilization. bosch, s. lukassen, j. kaindl, j. schwarz, c. nelkenbrecher, a. herrmann, a. reichel, a. ekici, t. gramberg, t. stamminger, a. winterpacht humangenetisches institut, erlangen, germany, virologisches institut, erlangen, germany spoc /phf is a gene located on human chromosome region p . and mouse chromosome qe . the protein was first described in patients with epithelial ovarian cancer, where its expression correlated with tumour progression and reduced survival time. spoc is a reader of the epigenetic mark h k me / , dynamically associates with chromatin during mitosis and plays a role in chromosome condensation. spoc deficient mice show a pronounced hypoplasia of the testis with a progressive loss of germ cells. although loss of spoc leads to a significantly reduced chromatin condensation of the sex chromosomes in meiosis, the protein is not expressed in spermatocytes but in the undifferentiated precursor cells, the spermatogonial stem cells (sscs). here, we present chip-seq data of mouse testis tissue demonstrating that spoc strongly binds to evolutionary young l elements in undifferentiated spermatogonia. we show that in hek cells overexpression of spoc leads to repression of transposition activity of line-elements strongly indicating a role of spoc in l element suppression. the cell has developed several lines of defence against retrotransposition to maintain genomic integrity, including dna methylation. these defence mechanisms are most elaborate in spermatogonial stem cells since transposition events in these cells would have a dramatic impact on the next generation. therefore, the repression of retrotransposition is of fundamental importance for germ cell development and ultimately the quality of the gametes. moreover, we present medip results showing that the methylation levels of l sequences decrease upon spoc -knockout and demonstrate that the histone methyltransferase g a is strongly upregulated in preleptotene meiocytes of spoc -/mice. g a is expressed from spermatogonia until early meiosis where it regulates h k di-methylation and has been shown to be involved in the repression of l element in mouse spermatogonia. we are able to demonstrate that h k me levels are unaltered in spoc -/mice, suggesting a potential functional link between g a and spoc that does not affect the catalytic activity of g a. since g a can regulate de novo and medizinische genetik · lated to investigate ciliogenesis. data resulting from rnaseq experiments are analyzed by established informatics tools (tophat, cufflinks and derivatives thereof). we will show results from our work in progress and we hope to convince people to intensify rna analyses even in routine labs to uncover hidden mechanism and/or mutations impacting mrna splicing and thereby causing human disease. telomeres are located at the ends of chromosomes and have an essential role in the maintenance of genome stability. after each cell division, a small part of this specialized sequence is lost. when telomeres reach a critically reduced length, the cell either dies through apoptosis or enters a state of permanent cell cycle arrest. it has been demonstrated that telomere biology is directly linked to basic biological phenomena such as aging, tumorigenesis and maintenance of dna integrity. it is known that oxidative stress accelerates telomere shortening in cells, resulting in premature cell senescence. shorter leukocyte telomeres have been observed in type ii diabetes or degenerative disease like dementia and alzheimer disease as well in chromosomal instability syndrome, such as fanconi anemia (fa) and nijmegen breakage syndrome (nbs). any link between telomere length and inflammation has not yet been extensively studied in autoimmune diseases. accelerated length shortening might be related to autoimmune disease predisposition. yet the reasons for this shortening are likely manifold, including the individual genetic background, oxidative stress and chronic inflammation. in order to shed light on these relationships, we investigate genomic dna extracted from blood of patients diagnosed with multiple sclerosis and from patients with huntington disease. the samples were divided into age groups. stepone q-pcr was applied to detect the relative telomere length as a function of age. initially identified differences in telomere lengths still have to be confirmed in larger cohorts. background: intrauterine exposure to gestational diabetes mellitus (gdm) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. gdm-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these increased disease risks. to identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (fcbs) from gdm and control pregnancies. methods and results: using illumina's k methylation arrays and following correction for multiple testing, cpg sites ( of which are associated with genes) displayed significant methylation differences between gdm and control samples. three of four candidate genes, atp a , prkch, and slc a , from our methylation screen and one, hif a, from the literature were validated by bisulfite pyrosequencing. the gdm effect on fcb methylation was more pronounced in women with insulin-dependent gdm who had a more severe metabolic phenotype than women with dietetically treated gdm. however, the effect remained significant after adjustment for the maternal bmi and gestational week in a multivariate regression model. e. g. dekomien , , b. bellenberg , , n. trampe , , r. schneider , , c. prehn , , c. krogias , , r. kropatsch , , m. regensburger , , c. lukas , , r. gold , , human genetics, bochum, germany, ruhr-university bochum, germany, radiology, st. josef-hospital, bochum, germany, neurology, st. josef-hospital, bochum, germany, molecular neurology, erlangen, germany, university of erlangen, germany a pair of monozygotic -year-old twins suffering from hereditary spastic paraplegia (spg ) is described. patients underwent thorough clinical examination and magnetic resonance imaging (mri) and mr-spectroscopy (mrs) at tesla. genetic testing was performed by sanger sequencing and alternative splicing by rna analysis. clinically the patients presented a similar spectrum of symptoms with a higher level of disability in one of the patients. mri studies including morphometry and regional microstructural analysis by diffusion tensor imaging (dti) of the corpus callosum (cc) revealed marked thinning and corresponding increases of axial diffusivity (ad), radial diffusivity (rd) and apparent diffusion coefficient (adc) and reduction of the fractional anisotropy (fa) as compared to healthy controls in all cc sections, particularly in the anterior callosal body. there was marked supratentorial white matter reduction and to a lesser extent grey matter reduction in both patients. involvement of the cortico-spinal tracts was reflected by fa and rd alterations and cervical cord atrophy. the more strongly affected patient showed a higher degree of callosal microstructural damage and cervical cord atrophy. genetic testing of the spg gene revealed two mutations in compound heterozygous state, a known frameshift mutation as well as a novel synonymous exonic splice site mutation. this study shows similar but distinct clinical and imaging findings in monozygotic twins suffering from spg , suggesting individual downstream genetic effects. targeted next generation sequencing techniques tremendously improved our ability to identify sequence variants. however fixing disease causing mutation still lack behind because of several reasons: inappropriate gene specific data bank, insufficient prediction tools, incomplete analysis and others. in addition identified sequence variants are a mixture of severe disease causing mutations and a myriad of variants of unknown pathogenicity. in addition an unknown number of silent mutations, neutral polymorphism and sequence variants deeply buried in introns might severely influence splicing of the premature rna molecule. by solely analysis of the dna sequence this impact onto the integrity of the mrna is completely ignored. in order to catalog the mrna isoforms derived from genes of our interest we started to set up rnaseq technologies in our routine lab. to reduce the amount of data, to improve the power of analyses and to identify rare isoforms of transcripts we use targeted rnaseq to characterize the mrna molecules originating from those genes we are interested in (e. g.: hereditary breast cancer core genes ( genes), hereditary colon cancer ( genes), primary ciliary dyskinesia (pcd)( genes). genes involved in pcd offer the invaluable advantage that the respiratory epithelium where these genes are normally expressed can be sampled from the inferior turbinate of the nose by brush biopsy either from healthy probands or from patients suffering from pcd. in addition to direct preparation of rna from these cilia, cilia carrying cells or tissue can be cultured and manipu-abstracts long-range pcr and direct sequence analysis. the comparative analysis of parental haplotypes with the sequences flanking the deletion breakpoints in the patients revealed the absence of any de novo mutations in breakpoint-flanking regions of prs -mediated and prs -mediated type- nf deletions. we conclude that although nahr is a mutational mechanism causing large nf deletions, there is no evidence for a local mutagenic effect of these recombination events. hence it is unlikely that nahr underlying type- nf deletions involves error-prone translesion polymerases that would increase the de novo mutation rate in breakpoint flanking regions. furthermore, the detailed haplotype analysis of prs , a highly active nahr hotspot mediating the majority of large nf deletions, revealed that non-allelic homologous gene conversion (nahgc) between nf -repa and nf -repc, which results from non-crossover resolution of recombination intermediates, is the major driving force responsible for the haplotype diversity in this region. remarkably, the haplotype diversity patterns observed for nf -repa and nf -repc were markedly different indicating that during nahgc, nf -repa is disporportionately more often the donor sequence used to repair mismachtes in heteroduplex regions than nf -repc. we also noticed a correlation between haplotype diversity and the number of prdm a-allele binding sites suggesting that haplotype diversity and hence the nahgc rate within prs in nf-repa is regulated by prdm . heidelberg center for personalized oncology dkfz-hipo dkfz, heidelberg, germany dna methylation aberrations at differentially methylated region of imprinted genes interfere with the naturally parental-specific mono-allelic expression. that leads to a bi-allelic or absent expression of the imprinted gene, a cause of imprinting disorders (ids). we aimed at analyzing the genome wide dna methylation pattern of two patients with ids, namely transient neonatal diabetes mellitus (tndm) and multi locus imprinting disturbance (mlid), and their respective parents. the dna methylation profiles of these individuals were obtained by whole genome bisulfite sequencing (wgbs) on b cells sorted by magnetic cell isolation. the sequencing libraries were prepared as described in kretzmer et al. [ ] and sequenced on an illumina hiseq machine. wgbs data were processed with the methylctools toolkit. briefly, bisulphite-treated sequences were aligned with bwa-mem using a three-letter approach, and the methylation ratios were quantified for ~ . million out of . million cpg sites (coverage> ) genome-wide. quality control was performed to assess the quality of the dna methylation profiles and genetic fingerprinting was performed on the wgbs data confirming sample origin and family relationship. the wgbs data was further compared to already existing genome-wide human snp array . (snp array) and humanmethylation k bead array ( k) data, resulting in a good accordance with pearson's correlation coefficients > . . we detected an overall dna methylation level around % in all samples. already known dna methylation alterations, e. g. hypomethylation in plagl were validated by wgbs. by searching for differentially methylated regions (dmrs), defined as regions composed of at least five consecutive cpg loci showing a methylation difference between patient and corresponding parents above %, we identified dmrs in the mlid our study supports an association between maternal gdm and the epigenetic status of the exposed offspring. consistent with a multifactorial disease model, the observed fcb methylation changes are of small effect size but affect multiple genes/loci. the identified genes are primary candidates for transmitting gdm effects to the next generation. they also may provide useful biomarkers for the diagnosis and prognosis of adverse prenatal exposures and assessing the success of interventions during pregnancy. the nuclease hsnm b/apollo has a dual function in both dna-repair and maintenance of telomeres. as to the repair of dna interstrand crosslinks (icl), hsnm b/apollo is linked to the fanconi anemia (fa) pathway and cells depleted for hsnm b/apollo (sirna) resemble those from patients with fa. we have identified a single nucleotide polymorphism, rs , which is associated with quantitative differences in hsnm b/apollo expression (mrna). we analyze whether the differential expression relates to the degree of cellular sensitivity to the dna interstrand crosslinks inducing mutagen mitomycin c (mmc) and ionising radiation (ir), which induces, among other lesions, dna double strand breaks. all experiments are realized using lymphoblastoid cells derived from generally healthy donors. results of rt-pcr analysis of hsnm b/apollo expression and of the cell viability assays will be presented and discussed in the context of the potential usefulness of considering rs in predicting individual sensitivity to mutagens relevant in anti-cancer treatment. p-basepi- nahr events causing type- nf microdeletions are not associated with an increased mutation rate in breakpoint-flanking regions m. hillmer , , a. summerer , , v. f. mautner , , l. messiaen , , h. institute of human genetics, ulm, germany, university of ulm, ulm, germany, department of neurology, hamburg, germany, university hospital hamburg eppendorf, hamburg, germany, department of genetics, birmingham, usa, university of alabama at birmingham, birmingham, usa large deletions of the nf gene and its flanking regions are the most frequent recurrent mutations in patients with neurofibromatosis type (nf ). different types of large nf deletions have been identified which are distinguishable in terms of their size and breakpoint position. most frequent are type- nf deletions spanning . -mb and characterized by breakpoints located within the low-copy-repeats nf -repa and nf -repc which exhibit . % sequence homology within -kb. type- nf deletions are caused by non-allelic homologous recombination (nahr). two nahr hotspots have been identified termed prs and prs which encompass -kb and -kb, respectively. approximately % of all type- nf deletion breakpoints cluster within the prs and prs nahr hotspots. in this study, we analysed whether the nahr events causing type- nf deletions would be associated with an increased de novo mutation rate of sequences located in breakpoint-flanking regions. to do so, we sequenced the deletion breakpoint-flanking regions in the patients and compared these sequences with the homologous regions amplified from dna of the patients' parents who are not affected by nf . however, in the germline of these parents, the deletions were mediated by nahr and then transmitted to their offspring. the parental haplotypes within the prs or prs regions of nf -repa and nf -repc were analysed by in the alpl gene and inherited as an autosomal dominant trait can cause milder forms. so far detailed knowledge of the milder forms is lacking. patients with a mutation in the alpl gene were interviewed in a standardized questionaire concerning the different disease manifestations: teeth, bone fractures, pain of bones and muscles and quality of life. subgroups were formed with regard to the localization of the mutations in the three protein domains. patients with mutations clustering in the catalytic site of the molecule showed the most severe odontohypophosphatasia: one individual had premature primary tooth loss, % of patients showed adult tooth loss, % suffered from dental caries. the majority had the first manifestation before the age of . persons suffering from mutations in the two other domains reported a relatively high quality of live with low pain of muscles and bones. unexpectedly in all groups there was no significant difference in the portion of patients with bone fracture. conclusion: the clinical signs of dominant hpp are mostly unspecific. especially dental problems like severe adult teeth loss, an early manifestation of dental caries or enlarged pulp chambers can be a sign of odontohypophosphatasia and a dominant inherited mild hpp. mutations in the catalytic site of the alpl molecule are associated with a more severe odontohypophosphatasia. screening of non-neoplastic lymphatic tissues from children for the igh-myc fusion using a highly sensitive -color fish-assay burkitt lymphoma is a mature b-cell lymphoma which on the genetic level is characterized by the burkitt translocation t( ; )(q ;q ) juxtaposing the igh locus in q next to the myc locus in q . in a minor part of burkitt lymphomas, immunoglobulin light chain variants of the translocation result in overexpression of myc. despite being pathognomonic for burkitt lymphoma, the ig-myc juxtaposition alone is not sufficient on its own for a malignant transformation of the cell. other igh rearrangements like the igh-bcl fusion, typical for follicular lymphoma, were detected in a significant number of healthy individuals. for the igh-myc translocation, only scarce data in healthy individuals exist. this is most likely due to scattering of the breakpoints which are far more difficult to target by pcr than the igh-bcl translocation. therefore, we aimed at investigating if myc-translocation positive cells can also be detected in normal b-cell maturation. considering the epidemiology of burkitt lymphoma being the most common b-cell lymphoma in children, we focused on samples from young individuals. on the one hand, we analyzed non-neoplastic tissue specimen of bone marrow (n = ) (age range - , median age . years) and lymph nodes (n = )(age range - years, median age years). on the other hand, considering the typical clinical presentation of burkitt lymphoma, we included non-neoplastic tissue specimen containing peyer patches (n = )(age range hours- years, median age years). the specimen were analyzed using a four-color fluorescence in situ hybridisation (fish) assay with probes flanking the breakpoints on chromosomes and . in this setting, a positive result comprised the break on both chromosomes (seen as signal split for each locus) and fusion of the involved genes (leading to two different fusion signals). the assay was first validated on controls of cells with a normal male karyotype from healthy individuals as well as on five burkitt cell lines and each five ffpe embedded t( ; ) negative and positive tissues as negative and positive controls. the assay was then applied for the screening of a igh-myc fusion in the above mentioned paraffin-embedded tissues. successful hybridizations of overall , and ffpe sections from bone marrow, lymph nodes and peyer patches respectively could be obtained. trio and dmrs in the tndm trio. in the mlid trio / dmrs showed increased and dmrs decreased dna methylation in the patient's sample. of dmrs, are located in regions potentially associated with transcriptional regulation. further analysis revealed that / dmrs are associated with imprinted genes. in the tndm trio, we detected / dmrs to show hypermethylation in the patient compared to her parents and / dmrs with lower dna methylation. in these trio / dmrs are associated with regions potentially correlated to transcriptional regulation and dmrs with imprinted genes summarized, our results show that wgbs is a well suited and valid method for analyzing dna methylation. while the overall dna methylation levels does not differ between the analyzed patients and parents, a detailed analysis of smaller regions revealed the existence of respectively differentially methylated regions between the analyzed mlid and tndm patient and their parents. supported by bmbf through fkz: gm und gm and gm p-basepi- characteristic mutational profile in children of individuals exposed to ionizing radiation p. krawitz, h. manuel, a. knaus, g. hildebrandt, m. jäger, m. schubach, m. rodriguez de los santos, t. pantel, d. beule, s. mundlos, k. sperling institute for medical genetics and human genetics charite, berlin, germany the dna damaging effects of ionizing radiation are deliberately used in cancer therapy as well as feared in accidents related to nuclear technology. despite its influence on the exposed organism, irradiation was believed to have no major effect on succeeding generations, as irreparable dna damages were thought to result in cell death. recently, however, genome-wide mutation screenings in offsprings of male mice that were irradiated with high dosages showed an accumulation for certain de novo events. we therefore focused on these mutational classes in a small cohort of human individuals that were conceived while or after their fathers were exposed to high frequency radiation. in the whole genome sequences of such offsprings we could confirm de novo rates for single nucleotide variants in the order of - per base as previously reported. interestingly, however, we found de novo translocations of paternal origin as well as increased numbers of clustered de novo mutations that resemble the results from animal studies. this characteristic mutation profile might thus be used as an indicator of irradiation exposure in one of the individual's parents. from the upstream regular rb promoter. to test this hypothesis, we generated a genetic model carrying modifications in the rb promoter and in cpg using crispr/cas technology. data on the establishment of the model and first results will be presented. the gid/ctlh protein complex with its seven core protein members is conserved in all eukaryotic cells. in saccharomyces cerevisiae it functions as an ubiquitin ligase complex and regulates the metabolic switch from gluconeogenesis to glycolysis ( ) . recently, we could show that the vertebrate gid/ctlh complex also functions as an ubiquitin ligase, however substrates and exact function remain unknown ( ) . a growing number of components of the ubiquitin protein system (ups) are described to be regulators of ciliogenesis ( ). defects in such genes are considered to cause ciliopathies, genetic disorders with typical phenotypic variations in patients and model organisms ( ) . first data supports our hypothesis that the ctlh complex plays a major role in ciliogenesis, e. g. the ctlh subunit rmnd a localizes to the basal body which is a modified centriole of primary cilia in nih- t cells and rmnd knock down in xenopus laevis leads to defects in cilia formation of epidermal multiciliated cells. n. reich, m. sandbothe, r. buurman, b. schlegelberger, t. illig, b . skawran department of human genetics, hannover, germany background and aims: hepatocellular carcinoma (hcc) is characterized by genetic and epigenetic changes that lead to a deregulation of important tumor suppressors and oncogenes in a multistep process. one of these epigenetic changes is the elevated expression of histone-deacetylases (hdacs) which contribute to a transcriptional repression of certain genomic regions by remodeling the chromatin structure. thereby, not only the expression of tumor-relevant genes is affected, but also the expression of micrornas (mirnas). selected mirnas have been shown to play important roles in carcinogenesis. we aimed to identify mirnas deregulated by histone deacetylation and to understand their functional consequences in hcc tumorigenesis. methods: histone acetylation was induced by the global hdac inhibitor trichostatin a (tsa) in four hcc cell lines (hle, hlf, huh , hepg ) and two immortalized liver cell lines (thle- and thle- ) in order to identify differentially expressed mirnas and messenger rnas (mrnas) by global expression profiling. findings were validated by transfection of microrna mimics and sirna-mediated knockdown in hcc cell lines, quantitative pcr, western blotting and luciferase reporter assays. results: after hdac-inhibition, hsa-mir- - p was significantly upregulated. the mir- - p holds tumor suppressive potential and its expression is reduced in different types of tumors. one predicted target gene of mir- - p is the hepatoma-derived growth factor (hdgf). this mitogenic growth factor is highly expressed in a variety of cancers, for example in hcc, and its expression correlates with a poor prognosis, irrespective of the tumor type. hdgf is a multifunctional protein that is involved in several signaling pathways, contributing to proliferation and metastasis of cancer cells, induction of angiogenesis and inhibition of apoptosis. incubation of hcc cells with tsa or transfection with mir- - p reduced expression of hdgf. luciferase assays indicate a direct regulation of hdgf by mir- - p. moreover, expression of the death receptor fas, which is a potential downstream target of hdgf, is also regulated by the mir- - p. a translocation t( ; )(q ;q ) was not detectable in any of the investigated tissues. with the established assay we were able to provide a highly sensitive tool for the detection of the translocation t( ; )(q ;q ). however, we did not detect normal b-cells carrying this translocation. this does not exclude that such cells exist. alternatively, the growth advantage conferred by myc may promote the acquisition of secondary genetic changes. this may result in a rapid tumorigenesis, that if occurring these cells only present as full blown burkitt lymphoma. myotonic dystrophy: links to the nuclear envelope p. meinke, s. hintze, s. limmer, b. schoser friedrich-baur-institute, munich, germany myotonic dystrophies (dm) are slowly progressing multisystemic diseases with a predominant muscular dystrophy -making dm the most frequent muscular dystrophy in adulthood. dm is caused by heterozygous dna-repeat expansions in the dmpk gene (dm ) or the cnbp gene (dm ). the repeat-containing rna accumulates in ribonuclear foci and splicing factors are sequestered to these foci, resulting in abnormal regulation of alternative splicing. dm patients show overlapping phenotype presentations with progeroid laminopathies, which are caused by mutations in nuclear envelope proteins. in search for molecular signatures of this overlap, we found an enrichment of nucleoplasmic reticuli in dm and dm patient myoblasts. additional, we found an alternative splicing of the lmna gene -both effects that are associated with progeroid laminopathies. this implies possible shared pathomechanism between dm and progeroid laminopathies. retinoblastoma is a tumor of the retina occurring in young children up to the age of five. it is caused by biallelic inactivation of the tumor suppressor gene rb . we have shown that human rb is an imprinted gene and as such characterized by differential dna methylation of a cpg island (cpg ) in rb intron . cpg is not methylated on the paternal allele and acts as a promoter for the alternative rb transcript, rb -e b. it is argued that expression of rb -e b is causative of the observed skewing of regular rb expression in favor of the maternal allele. a true gametic differentially methylated region (gdmrs) is established in only one of the parental germ lines. it is supposed to be stable during early embryonic development and to be passed on to all daughter cells. we could show that cpg is free of methylation in human sperm. publicly available methylome data on oocytes revealed that cpg is fully methylated in human oocytes. these data are in agreement with cpg being a maternal methylated gdmr. we showed that the level of cpg methylation is percent in blood, as expected. however, in eight tissues of three individuals we observed a gain of methylation at cpg ranging from to percent in liver and skin, and increasing to to percent in the other tissues (heart, kidney, muscle, brain, lung and spleen). interestingly, the degree of methylation was lower in fetal tissue than in adult tissue, as determined for brain and muscle. we also observed gain of methylation at cpg in two human embryonic stem cell lines and induced pluripotent stem cells. this is consistent with the finding of complete methylation at cpg in eight different retinoblastoma cell lines. we therefore conclude that cpg is an unstable dmr. in oocytes, dna methylation of gdmrs is established by transcriptional read-through from an upstream promoter. therefore, we hypothesize that gain of dna methylation at cpg is caused by run-through transcription erozygous state (nomenclature according to hgvs; reference sequence nm_ . ). in silico-analysis by alamut (version . . ) predict the loss of the donor splice site of intron of the atm gene. cdna-analysis was performed and revealed the loss of exon of the atm by a complex activation of two kryptic splice sites. a premature stop codon was generated giving rise to a truncated protein that leads to a pathogenic variant. the results of the genetic analysis are discussed in the context of the clinical findings. identification of the underlying genetic causes of gastric cancer will give a better view of the mechanisms that contribute to the pathophysiology of the disease. gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, zentrum für diabetologie bergedorf, schwerpunktpraxis, hamburg, germany about - % of all pregnant women develop gestational diabetes mellitus (gdm) during their pregnancies and diabetes complicating pregnancy is associated with adverse maternal and perinatal outcomes, notably, risk of fetal macrosomia and neonatal hypoglycemia and development of diabetes after pregnancy. gdm is considered to result from interaction between genetic and environmental risk factors. the case of a -year old female german patient with a novel mutation in the pax gene (rare mody gene type ) as a cause of gestational diabetes mellitus is presented. we describe clinical, biochemical and genetic features of the patient, who developed gdm and gave birth to her child by cesarean section. mody genes type - were analyzed. sequencing the pax gene revealed a novel mutation in exon , pax ,c. delc, p.(leu cysfs* ); reference sequence nm_ . ), a deletion of a cytosine leading to a truncated, non-functional protein. to date, no small deletion has been detected in the pax gene. identification of the underlying genetic causes of gdm will give a better view of the mechanisms that contribute to the pathophysiology of the disease. furthermore, early identification may improve options to prevent gdm and complications for the mother and her child. the results of the genetic analysis are discussed in the context of the clinical findings. the modulation of dna methylation is highly flexible and plays an important role during cell differentiation. furthermore, the dna methylome alters considerably during aging. age related changes in the dna methylation of regulatory genes are assumed to have a major impact on carcinogenesis (teschendorff, ) . moreover, it was demonstrated that the chronological age of a human donor can be predicted with high accuracy by analyzing the dna methylation of a specific minor set of cpg loci which are aberrantly methylated during aging (horvath, ) . hence, we intended to investigate the effect of epimutations identified in different lymphoma entities in comparison with the influence of epigenetic changes in sequential b cell differentiation stages on the epigenetic age. the altered expression of the tumor suppressor mir- - p due to chromatin remodeling may play a fundamental role in hepatocarcinogenesis. we expect that histone deacetylation and putative target genes of epigenetically deregulated mir- - p can be targeted by new therapeutic agents. the microrna- family inhibits tgf-β-mediated liver cancer cell migration by targeting sox introduction: modulation of microrna expression is considered for treatment of hepatocellular carcinoma (hcc). therefore, we characterized the epigenetically regulated microrna- family (mir- a, mir- b, mir- c) with regards to its functional effects and target genes in hcc. methods: after transfection of mir- a, mir- b, and/or mir- c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. binding specificities, target genes, and regulated pathways of each microrna were identified by microarray analyses. target genes were validated by luciferase reporter assays and expression analyses in vitro. furthermore, target gene expression was analyzed in primary human hccs compared to normal liver tissue. results: tumor suppressive effects, binding specificities, target genes, and regulated pathways of mir- a and mir- b differed from those of mir- c. transfection of mir- a, mir- b, and/or mir- c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. importantly, mir- a, mir- b, and, to a lesser degree, mir- c directly targeted sox , which codes for a transcription factor involved in epithelial-mesenchymal transition and hcc metastasis, and thereby inhibited tgf-β-mediated cell migration. conclusions: this study provides detailed insights into the regulatory network of the epigenetically regulated microrna- family and, for the first time, describes distinct tumor suppressive effects and target specificities of mir- a, mir- b, and mir- c. our results indicate that particularly mir- a and mir- b may be considered for mirna replacement therapy to prevent hcc progression and metastasis. novel mutation in the atm gene and activaton of two kryptic splice sites in an year old female patient with gastric cancer gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, schwerpunktpraxis, hämatologie, onkologie und palliativmedizin, hamburg, germany, israelitisches krankenhaus, chirurgische klinik, hamburg, germany gastric cancer is a global public health concern, ranking as the third leading cause of cancer mortality. familial aggregation of gastric cancer is common in about % of the cases, and about half of these can be attributed to hereditary germline mutations. however, for most gastric cancer cases, whether genetic events contribute to cancer susceptibility remains unknown. here we present a case report of a patient with gastric cancer, a family history of breast cancer and a novel mutation leading to complex cryptic splicing in the atm gene. ngs panel sequencing and cnv/mlpa analysis of genes associated with gastric and breast cancer were performed. sequencing revealed a novel mutation in intron of the atm gene, atm,c. + g>a in an het-abstracts tions remained unidentified since positive deletion-junction pcr products could not be amplified. to identify the breakpoints of the deletions, we performed custom-designed microarray cgh analysis with a high resolution of probes located within and flanking the nahr hotspots prs and prs . the array analysis suggested that of the deletions exhibit breakpoints within prs , even although previously performed breakpoint-spanning pcrs with primers designed according to the reference sequence of the human genome have been negative in these cases. since prs exhibits high sequence diversity resulting from frequent nonallelic homologous recombination events without crossover, we surmised that haplotype diversity is responsible for the failure of the breakpoint-spanning pcrs performed with primers designed according to the reference sequence. therefore we characterized the haplotype diversity of prs in human individuals and designed deletion-junction pcr primers that facilitate the amplification of rare prs haplotypes. so far, we have identified the breakpoints of four of the type- nf deletions predicted to have been mediated by prs according to the array results. we are confident to identify further breakpoints by extending these analyses using primers suitable to amplify rare prs haplotypes. our findings indicate that the characterisation of nahr hotspots in terms of haplotype diversity is a premise to identify the breakpoints of nahr-mediated microdeletions by means of deletion-junction pcrs. p-basepi- *** array-based dna methylome analyses of primary lymphomas of the central nervous system ulm university, ulm, germany, university of cologne, cologne, germany, christian-albrechts-university kiel, kiel, germany, university hospital muenster, muenster, germany primary lymphomas of the central nervous system (pcnsl) are defined as diffuse large b-cell lymphomas (dlbcl) that are confined to the central nervous system (cns). although pcnsl cannot be distinguished from dlbcl by their morphology as well as their histology, they differ in prognostic outcome. the aim of the present study was to compare the epigenomic landscape of pcnsl and dlbcl. to this end, we analyzed the dna methylation of a total of pcnsl (cryopreserved or formalin fixed and paraffin embedded (ffpe)) using the infinium humanmethylation beadchip array (illumina) and contrasted these findings to dlbcl (kretzmer et al., ) . as controls, we used publicly available dna methylation data from a total of normal brain samples derived from different regions of the cns (gilbert et al., ; jaffe et al., ; kurscheid et al., ; mur et al., ; wockner et al., ) . after normalization of the data we performed thorough filtering and removed the random snps, all loci located on gonosomes, as well as those loci with a detection p-value > . in at least one of the samples, leading to , loci entering subsequent analyses. when comparing the dna methylation profiles of pcnsl versus dlbcl we identified differentially methylated loci (σ/σ max = . ; q < e- ). in order to remove those loci which represent a "brain signature", we compared dlbcl versus brain (σ/ σ max = . ; q < . ) based on the list of the previously identified loci. after removing this "brain signature", we ended up with loci that are differentially methylated between pcnsl and dlbcl. in a next step we wanted to make sure that the differences in methylation at these loci are not due to differences in starting material (cryopreserved versus ffpe) which is known to influence the outcome of the beadchip analysis. therefore, we compared the dna methylation profiles of five cryopreserved versus ffpe samples (derived from the same tissue samples) and identified differentially methylated loci (σ/σ max = . , q < . ). only five loci of both lists overlapped, which were subsequently removed from further analysis so that we ended up with a final list of loci which are differentially methylated between pcnsl and dlbcl. in order to analyze the biological implications of the differentially methylated loci we evaluated an enrichment of known functional groups (ku-additionally, our aim was to analyze whether entity-specific differences in the resetting of the epigenetic clock are generated during lymphomagenesis or derive from modified dna methylation in the germinal center b cells of origin. to address these issues, we performed dna methylation profiling (hum-anmethylation beadchip) of burkitt lymphoma samples (age - yrs), diffuse large b cell lymphoma samples (age - yrs) and follicular lymphoma samples (age - yrs) from the icgc mmml-seq and mmml-consortium (kretzmer et al., ) and the hematopathology section kiel as well as peripheral blood samples of healthy individuals ( - yrs) available from the same project and current publications of our group (kolarova et al., ; friemel et al., ) . in addition, we received b cell subpopulation samples ( - yrs) covering different stages of b cell differentiation that were measured in the same way (kulis et al., ; lee et al., ) . the epigenetic age of the samples was predicted using the "online age calculator" accessible at https://dnamage.genetics.ucla.edu and compared with the corresponding chronological age of the donors. in fact, the epigenetic age of peripheral blood samples of healthy donors was in high accordance with their chronological age (pearson's r . , p-value < . ) while the correlation between epigenetic and chronological age of sequential b cell differentiation stages was slightly lower (pearson's r . , p-value< . ). in contrast, the predicted epigenetic age of the burkitt lymphoma samples was significantly higher than the corresponding chronological age. this deviation may be interpreted as "epigenetic pre-aging". nevertheless, the epigenetic age of diffuse large b cell lymphomas and follicular lymphomas tended to be less affected. in conclusion, we found significant epigenetic pre-aging in burkitt lymphoma samples that seems to be induced during lymphomagenesis and does not derive from altered dna methylation patterns in the germinal center b cells of origin. moreover, no significant shift of the epigenetic age was observed for the other lymphoma entities, healthy blood samples and b cells of sequential differentiation stages. identification of type- nf deletion breakpoints mediated by rare prs haplotypes a. summerer , , m. hillmer , , v. f. mautner , , l. messiaen , , h. institute of human genetics, ulm, germany, university of ulm, ulm, germany, department of neurology, hamburg, germany, university hospital hamburg eppendorf, hamburg, germany, department of genetics, birmingham, usa, university of alabama at birmingham, birmingham, usa neurofibromatosis type (nf ) is a hereditary cancer syndrome with an incidence of in . in % of all nf patients, large deletions encompassing the nf gene and its flanking regions are causing the disease. the majority of all large nf deletions are of type- ; they encompass . -mb and are mediated by nonallelic homologous recombination (nahr) with crossover. the breakpoints of type- deletions are located within the lowcopy repeats nf -repa and nf -repc which exhibit high sequence homology to one another. previous studies suggested that type- deletion breakpoints cluster within the paralogous recombination sites prs and prs spanning -kb and -kb, respectively. in our present study, we investigated patients with type- nf deletions using long-range pcrs to detect breakpoints located within prs or prs . according to these analyses, ( %) of the breakpoints are located within prs and ( . %) in prs . however, ( . %) of the type- deletions were not positive for these deletion-junction pcrs. we surmised that some of these deletions may have breakpoints within the -kb region located between prs and prs . this -kb region also exhibits high sequence homologoy between the nf -reps which is a prerequisite for nahr. indeed, of the type- nf deletions exhibited breakpoints within this -kb region as determined by the analysis of seven overlapping deletion-junction pcrs. however, the breakpoints of dele-esophageal adenocarcinoma (ea) represents one of the most rapidly increasing cancer types in high-income countries. barrett's esophagus (be) is a premalignant precursor of ea and has an estimated prevalence of - % in the population. however, only . to . % of be patients develop ea. within an international consortium, we carried out a gwas meta-analysis in be patients, ae patients and . controls (gharahkhani et al., lancet oncology, ) . in a comprised be/ae analysis, we identified genome-wide significant risk loci, of which seven were previously unreported. the strongest associated new risk variant was identified for rs (p = . × - ), which maps within intron of the cftr gene. cftr encodes a protein that functions as a chloride channel and that is mutated in patients with cystic fibrosis (cf). mutations in cf lead to abnormal viscous secretions with altered chemical composition, resulting in dysfunction of the respiratory system and the gastrointestinal tract. the most common cf mutation is Δf , a deletion of three nucleotides in cftr that results in the loss of a single codon for phenylalanine on protein position . interestingly, cf patients show a highly increased incidence of gastroesophageal reflux, which represents the major risk factor for be and ae. in view of the phenotypic overlap for gastroesophageal reflux and cystic fibrosis, and for gastroesophageal reflux and both be and ae, combined with the association of cftr risk variants in patients with be and ae, it seems plausible that a common pathophysiological mechanism is triggered by cftr. in order to test this hypothesis, we analyzed the association of Δf in a european case-control cohort with be and ae patients. for this, we performed a genotyping assay of Δf in be patients, ae patients and controls. we could not observe a significant association (p = . ). this might be (i) due to insufficient sample power or (ii) due to the fact, that not Δf but other genetic variants at the locus might explain the underlying functional mechanism of the association. fine mapping of all genetic variation at the cftr locus and exten-lis et al., ). remarkably, cpg loci that are differentially methylated during normal b-cell maturation were significantly depleted. in turn we saw an enrichment of loci located in heterochromatin. in summary, we detected more than loci that are differentially methylated between pcnsl and dlbcl, which do not play a functional role in normal b-cell differentiation. replication study of gwas-identified genetic modifiers of age at huntington's disease onset although there is a strong correlation between cag repeat length and age at onset (ao) of motor symptoms, individual huntington disease (hd) patients may differ dramatically in onset age and disease manifestations despite similar cag repeat lengths. since the modifier variations described so far only account for a small fraction of the heritable contribution to the ao, the identification of loci and genes using genome-wide methods appears highly promising. against the background of incomplete understanding of the hd disease pathophysiology, the hypothesis-free approach of gwas offers an ideal starting point for the search of modifier genes. recently, a combined analysis of all gwa data to hd modifiers identified different loci with genome-wide significant signals for association to residual age at motor onset [gem-h. consortium]. interestingly, none of the most significant association signals and none of the trending snps in the european gwa analysis corresponded to any previously suggested candidate modifier genes. in order to be able to better assess these data, we tried to replicate the top ten associated gwas variants in a comprehensive cohort of german hd patients. we only found modest association with one of the top ranked snps (rs ), all remaining variations showed no correlation with the ao. this inconsistency highlights once again the difficulties of modifier searching in hd or any other monogenic disorder, which faces the same challenges as the genetic characterization of complex disorders. with an incidence of . - . / . malignant tumors of the thymus are a rather rare type of cancer. here we report the case of a man of german descent, who presented with a thymoma at age . in the pathological report the thymus tumor was described as an extremely unusual thymoma with partial loss of keratin and massive proliferation of myoid cells. it was subsumed to a primary thymic, partially epithelial neoplasia, resembling an uncommon b /b -thymoma. after the patient's death his widow looked for genetic advice concerning the risk of disease for her children. detailed personal and familial history brought up surprising information: thymoma was one of four cancers in our patient. he developed adenocarcinoma of the colon at age , squamous cell cancer of the nose/upper lip at years and in addition current cancer staging revealed a papillary renal cell carcinoma. according to family history his father and his uncle developed colon cancer with and years, the son of this uncle was diagnosed with colon cancer at age . this cousin of the propositus was referred to genetic counseling, because of msi-high-status and loss of mlh and psm in immunohistochemistry. he was found to have a deleterious mutation in exon of mlh gene (c. c>a, p.tyr stop) resulting in a premature termination of mlh -protein. our patient has never been tested for hnpcc. however a post mortem performed immunhistochemical examination of thymic cancer cells revealed an almost complete loss of mlh nuclear expression suggesting the presence of a mlh germline mutation and indicated hnpcc. considering the loss of mlh in tumor cells it is more than likely that the development of thymoma was the consequence of deficient dna mismatch-repair. there have been reports of rare tumors in hnpcc families in the last years (i. e. clear cell renal carcinoma and uterine sarcoma). pande et al. reported one case of thymoma in their registration of cancer occurrences in mutations carries from hnpcc families [ ] . our case emphasizes the importance of detailed family history and contributes to the discussion of widening the inclusion criteria for genetic counseling and testing for hnpcc. to this day the revised criteria of bethesda are used to identify families at risk. we propose that the established criteria have to be revised and rare tumors should be included. unknown partner genes in leukemias with rare translocations can be identified using targeted rna sequencing c. haferlach, n. nadarajah, m. meggendorfer, n. dicht, a. stengel, w. kern, t. haferlach mll, munic, germany in hematological malignancies fusion genes play an important role and function as therapeutic targets, impressively shown for e. g. bcr-abl and etv -pdgfrb. thus, the identification of fusion genes is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. the aim of this study was to test the value of targeted rna sequencing in a routine diagnostic work up. cases were selected harboring rearrangements of kmt a (n = ), runx (n = ), etv (n = ), pdgfrb (n = ), npm (n = ), rara (n = ) and jak (n = ) identified by chromosome banding (cba) and fish analyses. in none of the cases the partner gene could be identified using standard methods. targeted rna sequencing was performed using the trusight rna fusion panel (illumina, san diego, ca) consisting of probes covering genes known to be involved in gene fusions. this assay allows the capture of all targeted transcripts. sequencing was performed on nextseq (illumina, san diego, ca). analysis was performed with the rna-seq alignment app (basespace sequence hub) using star for alignment and manta for gene fusion calling with default parameters (illumina). sive functional analysis are needed to find the causal variant that explains how the cftr locus interferes with the pathomechanism of be and ae. a recent functional study indicated cftr as a tumor suppressor gene in murine and human intestinal cancer, providing further evidence for cftr as a true disease gene for be and ae. background: it has long been established that mutations in brca predispose for pancreatic adenocarcinoma with brca germline mutations identified in - % of familial pancreatic cancer cases. consequently, screening for pancreatic cancer has been recommended for mutation carriers with an affected first-degree relative since early detection has been shown to significantly improve -year survival from - % to %. for brca mutations, however, relevance in pancreatic tumorigenesis is still being discussed with several studies questioning an elevated risk of pancreatic cancer in families with brca mutations while others are suggesting that brca may also play an important role in predisposing to pancreatic cancer. clinical screening for pancreatic cancer commonly remains unavailable to brca mutation carriers and it has even been questioned whether brca should be analyzed in familial pancreatic cancer at all. clinical report: here we report on a year old woman with metastatic pancreatic cancer whose sister had died of pancreatic cancer at years of age. in this family we identified a pathogenic brca -germline mutation (brca : nm_ . :c. dupt,p.(leu phefs* )) by next-generation sequencing using a -gene panel. the index patients' tumor was available for genetic analysis and showed loss-of heterozygosity for brca . this strongly suggests the brca mutation to be causative of the pancreatic cancer development in this patient. when the family was first introduced to genetic counselling there was no evidence of breast-or ovarian cancer in any relatives. only after identification of the mutation did the index person reach out to distant family members and it was thereby revealed that a distant branch of the family had independently been counselled for hereditary breast and ovarian cancer. in this part of the family, however, there had not been any cases of pancreatic cancer. subsequent predictive testing was offered to healthy family members and further mutation carriers could be identified. two women were referred to breast cancer screening. additionally, the mutation was identified in a relative with recurrent metastatic breast cancer at the age of years. for her and the index patient parp-inhibition therapy thus became a possible further treatment option. conclusions: in conclusion we propose next-generation sequencing approaches including the analysis of brca to be used in familial pancreatic cancer. we also argue that brca mutation carriers with pancreatic cancer cases in their family should be offered the same screening program as brca mutation carriers. within the framework of a study this could allow for more precise risk stratification in the future. contralateral dcis is unknown. only the male breast carcinoma was herceptin receptor positive, all other breast carcinomas of the chek mutation carriers were her negative. among our chek positive families we noticed the association with chek mutation and female breast cancer. we observed a contralateral breast cancer, male breast cancer and other tumors in our families as well. the majority of the observed breast cancers was estrogen and progesterone receptor positive and herceptin negative. while benign uterine smooth muscle tumors are among the most frequent human symptomatic tumors, their malignant or borderline lesions are only rare findings. both lesions can show somatic copy number alterations, but their patterns differ, thus constituting helpful diagnostic tools. aimed at an advanced classification of the lesions we have performed molecular inversion probe array analyses of these tumors. besides complex patterns of genomic alterations seen in nearly all cases, two of the lesions presented with copy number neutral uniparental disomies i. e. normal copy numbers with an apparent monoallelic origin. in one case, an upd of part of the long arm of chromosome was detected in a uterine leiomyosarcoma. the tumor showed genetic heterogeneity with gains and losses. in addition, the . mb segment located at q . -q . was clearly of monoallelic origin throughout all cells investigated. all other genetic alterations were restricted only to part of the cells of the sample thus reflecting the presence of tumor cells as well as normal bystander cells which in general characterizes mutations that had arisen during tumor development. in contrast, the upd that was detected in all examined cells clearly suggests its germline occurrence. the second tumor was a leiomyoma-variant of the type with bizarre nuclei. again, besides gains and losses an apparent germline upd was found that covered a . mb segment on chromosomal segment q . . upd for even the whole arm of chromosome repeatedly has been reported not to coincide with phenotypic manifestations. nevertheless, the question arises whether or not the observed upds might be related to a familiar predisposition for uterine muscle tumors. of note, as a result of genome-wide association studies snps on q recurrently have been found to be significantly associated with fibroid development. triple negativity is an independent predictor of germline mutations in breast cancer predisposing genes breast cancer is the most common cancer in women. - % of all tumors are triple-negative breast cancers (tnbc) lacking expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor . so far, tnbc have been mainly associated with mutations in brca , although recent studies also found mutations in other breast cancer susceptibility genes. a brca / -centered perspective thus may ignore the significance of other predisposing genes, whose relevance appears obvious as dna damage repair by homologous recombination is a complex process involving many proteins. in / cases with rearrangements involving kmt a (n = ), runx (n = ), etv (n = ), pdfgrb (n = ), rara (n = ), npm (n = ) or jak (n = ) the partner genes were identified. these were in kmt a rearranged cases: mllt (n = ), mllt (n = ), itpr , flnc, asxl , dcp b, maml and arhgef . in runx translocated cases partner genes were plag (n = ), prdm , mecom, zfpm , man a , n amt , and kiaa l. prdm , mecom and zfpm have previously been described in the literature as runx partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements. the other identified partner genes have not been described so far. interestingly, prdm , mecom, zfpm and the newly identified plag are all members of the c h type zinc finger gene family. partner genes identified in etv rearranged cases were: abl , ccdc , clptm l, erg, foxo and cflar-as . wdr , zbtb , nfia and mprip were identified as partner genes of pdgfrb and rpp in an npm -translocated aml. in an all patient a jak -ppfibp fusion was identified leading to classification as a bcr-abl -like all. in an apl patient showing an ins( ; ) (q ;q q ) a zbtb -rara fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. further in a case with t( ; )(q ;q ) an irf bp -rara fusion was detected. conclusions: targeted rna sequencing was able to characterize rare gene fusions and provided the basis for the design of rt-pcr based assays for monitoring mrd. targetable genetic aberrations were identified, which were not detected by cba enabling more individualized treatment. targeted rna sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques. female carriers of a pathogenic mutation in the chek gene are reported to have a life time risk of about - % to develop breast cancer. there is evidence for increased risks for contralateral breast cancer, male breast carcinoma and other types of tumors. in addition to well-known mutation chek :c. del, other pathologic mutations are being identified in the gene due to the inclusion of the gene in most breast cancer gene panels for dna testing. between - the center for hereditary breast and ovarian cancer regensburg cares for families with pathogenic or probably pathogenic mutations in the chek gene affecting nine female patients and one male patient ( × c. del, × deletion of exon , and × variants considered as likely pathogenic: c. g> c, c. c> t, c. a> c). the mean age of diagnosis of breast cancer (both sexes) was . years (range - years). the patient with the deletion of exon was first diagnosed at years of age and developed a contralateral breast carcinoma (dcis) at years of age. the male patient was diagnosed with breast cancer at years of age and at years with a renal carcinoma. one patient was diagnosed with a papillary thyroid carcinoma at age years and developed breast cancer with years. in out of the families, breast carcinoma diagnosed with . years on average, was reported in the family history. in addition, there were additional malignancies such as prostatic carcinoma, thyroid carcinoma, colorectal cancer, gastric carcinoma, leukemia, cervical carcinoma and malignant melanoma. none of the affected family members was tested for the respective chek mutation. the tumors with an initial diagnosis at years and years were estrogen-receptor-negative and progesterone-receptor-negative. the other of the breast cancers were positive for the estrogen receptor, of the tumors were positive for the progesterone receptor. the receptor status of the abstracts it has been shown that in d culture hescs can be differentiated into neural retina containing organoids. we established this differentiation schedule and started comparative differentiation of wildtype h hescs and the rb null derivative (g , rb mt/mt ) into neural retina. during the first weeks of differentiation into neural retina organoids generated from the rb mt/mt hescs have a smaller diameter and thinner retina layer compared to wildtype organoids. however, during the time-course the mutant organoids began to catch up. thus, at later stages no difference in size and thickness could be observed anymore. comparative immunostainings of cryosections at d show no difference in expression of the markers pax and sox between the wildtype and mutant hescs. further comparative immunostainings for markers specific for neural retina like e. g. rx and vsx at d and d are ongoing and will be presented. exome sequencing identified potential causative candidate genes for unexplained cowden syndrome purpose: cowden syndrome (cs) is a cancer predisposition syndrome characterized by the occurrence of breast cancer, epithelial thyroid cancer, endometrial carcinoma and various other findings such as mucocutaneous lesions and macrocephaly. cs belongs to the pten hamartoma tumor syndrome (phts) primarily associated with germline mutations in pten. in recent years, germline mutations in additional genes (sdhb, sdhc, sdhd, pik ca, akt , sec b) have been described in few patients; however, to date, in - % of patients meeting clinical criteria for cs the underlying cause remains unclear. methods: to uncover predisposing causative genes, the exomes of clinically well characterized, mutation negative patients with suspected cs were sequenced (illumina hiseq) using leukocyte dna. assuming a monogenic disease model, the called variants were filtered for rare (minor allele frequency ≤ % for homozygous/compound heterozygous variants and ≤ . % for heterozygous variants according to dbsnp, evs, and exac), truncating (nonsense, frameshift, highly conserved splice sites), and missense germline variants (predicted to be pathogenic by at least / in-silico tools). for data analysis and variant filtering the gatk software and the cartagenia bench lab ngs software were applied. all candidate genes were included in a pathway analysis (ingenuity). in a first preliminary analysis, we focused on known cancer genes and genes interacting with pten. results: after stringent filtering steps, comparison with large datasets from population-based controls, and detailed manual inspection to exclude artifacts, genes were affected by presumed biallelic variants ( homozygous and putative compound-heterozygous), one of these is a known cancer gene (cbfa t ); in genes biallelic variants were found in - patients. heterozygous variants were found in genes in - patients, but none of these are known cancer genes. in genes, heterozygous truncating mutations occurred in only one patient, of these are cancer genes (msh , wrn, kdm a, pml) . the phenotype of the patient with a msh frameshift deletion fulfilled key features of cs (early-onset metachronous papillary thyroid cancer, breast cancer, endometrial and colorectal cancer), however, the tumor spectrum is partly compatible with lynch syndrome/ hnpcc. examination of the colorectal cancer demonstrated microsatellite instability and a loss of msh protein expression. the pathway analysis of the remaining candidate genes identified several interacting partners of pten (grhl , ehhadh, cstf ). conclusions: preliminary data indicate that exome sequencing might identify potentially relevant causative genes for cs, some of which are recurrently mutated. the present work-up consists of the inclusion of further non-cancer genes, validation of variants by sanger sequencing, testing of to determine the prevalence of mutations we performed panel-based germline mutation testing of high and low-moderate penetrance breast cancer susceptibility genes (brca , brca , atm, cdh , chek , nbn, palb , rad c, rad d and tp ) in consecutive individuals affected with tnbc unselected for age at diagnosis or breast and ovarian cancer family history. age at diagnosis ranged from to years with an average of . and a median of years. in women ( . %) we detected a pathogenic mutation, with a higher frequency ( . %) in the group manifesting cancer before years. deleterious brca mutations occurred in . % of tnbc patients, predominantly frameshifting ( / , . %). the most frequent, both among brca mutations and in total, were the founder mutations c. dupc and c. _ delag. deleterious brca mutations occurred in . % of patients, all but one (c. du-pa) being unique. while no mutations were found in cdh and tp , mutations ( %) were detected in one of the six other predisposition genes (palb , chek , atm, nbn, rad c, rad d). no individual presented more than one mutation. almost half of all deleterious mutations ( . %) were detected in very young women aged years or less. the median age at diagnosis was significantly younger for brca ( years) and brca ( . years) carriers compared to patients without a mutation (p = . e- ; mann-whitney) or compared to non-brca / mutation carriers (p = . ). in contrast, patients with non-brca / mutations were not significantly younger than mutation negative women (p = . ). interestingly, family history had an independent influence on age at diagnosis. taken as a whole, women with family history had a median age at diagnosis years earlier than those without (p = . ). this difference was lost in mutation carriers while it remained in cases without mutation. in summary, our data confirm and expand previous studies of a high frequency of germline mutations in genes associated with ineffective repair of dna damage by homologous recombination in women with tnbcs. many of these women would go untested with current restrictive criteria. in order that each patient receives therapies tailored to her genetic status, gene panel based mutation testing should be offered to all women diagnosed with tnbc, irrespective of age at diagnosis or family history. p-cancg- *** neural retina differentiation of hescs as an in vitro model for retinoblastoma d. kanber, m. hiber, d. lohmann, l. steenpass institute of human genetics, university hospital essen, university duisburg-essen, essen, germany retinoblastoma is the most common eye tumor of early childhood. inactivation of both alleles of the retinoblastoma gene (rb ) results in the development of retinoblastoma. our aim is to establish a human cell-based model for retinoblastoma. using the crispr/cas system we have generated human embryonic stem cells (hescs) carrying a mutation either on one or both rb alleles. all the detected mutations are located in exon of the rb gene and close to the splice donor site of this exon. analyses on dna, rna and protein level were performed for three mutant and one double-mutant clone. the following genotypes were identified by deep sequencing (nm_ . (rb _v )): clone c , c. _ del, heterozygous; clones c and g , c. _ del, heterozygous; clone g , c.[ _ del; c. _ dup] (complex mutation on one allele), homozygous (loss of heterozygosity). the mutations of all four clones result in a premature stop codon in exon . on rna level we detected expression of mutant rb transcripts reflecting the genotype in all clones and an additional mutant rb transcript with skipping of exon in three clones. as the heterozygous clones also showed expression of the wildtype rb transcript, rb protein (prb) could be detected for these clones (c , c , g ) by western blot analysis. however, the double-mutant clone g showed no expression of prb. so far, we have characterized heterozygous and one homozygous clone. another three double-mutant clones are under investigation. the recurrent germline missense mutation g e in the hoxb gene has been demonstrated to predispose to hereditary prostate cancer (prca), despite the underlying pathogenic mechanism is not yet understood. molecular examination of a first set of g e positive tumors sought for somatic characteristics, and suggested that oncogenic ets gene fusions may appear at unusually low frequencies as compared to the general prevalence of ets fusions in prca ( % vs approx. %). hypothesizing that hoxb could predispose to ets fusion negative prca, we have analyzed cases from three european ancestry populations (finland, germany and us) for the coincidence of hoxb g e and the most common ets fusion, tmprss :erg, in corresponding tumor samples. while the prevalence of tmprss :erg fusions was similar among the three study groups (range: . - . %), the frequency of g e genotypes differed markedly between us ( . %), german ( . %) and finnish samples ( . %). despite the expected frequency gradient among study populations, all subsamples showed a strong enrichment of g e mutation carriers among tmprss :erg fusion negative cases as compared to fusion positive cases (center adjusted or = . ; %ci = . - . ; p = . ). consistent with the previous study, the crude frequency of the tmprss :erg fusion in hoxb g e carriers was . % (range . % - . %). examination of disease characteristics highlighted age at diagnosis to be associated with tmprss :erg negative status (per year or = . , p = , ) and by trend, also with the presence of the g e germline variant (per year or = . , p = . ). within the subtype of tmprss :erg fusion negative carcinoma carriers of g e were diagnosed . years earlier as compared to non-carriers ( . ± . years versus . ± . years, p = . ). in conclusion, this study demonstrated a significant tumor subtype specific association for hoxb g e mutation carriers having a higher frequency of tmprss :erg fusion negative prca. meta-analyses from case control comparisons suggested that subtype specific risk of hoxb g e for tmprss :erg negative prca could be as high as or = . , as compared to or = . , when prca is regarded as one entity regardless of fusion status. finally, although tmprss :erg negative prca is usually known to be associated with later ages of diagnoses, hoxb mutations may indicate a subgroup of earlier onset cases within the fusion negative entity. relatives to determine the phase of assumed biallelic variants and segregation with the phenotype where applicable. with to cases in million inhabitants per year adrenocortical cancer (acc) is a rare disease. due to often late diagnosis and limited treatment options prognosis for patients are poor with a year overall survival rate of to %. though knowledge about molecular genetic events in acc increased over the last few years no reliable molecular prognostic factors, no effective targeted cancer therapy and no personalized treatment approach has emerged to date. that's why we intend to establish a reliable method to define a molecular signature of accs that could be used for a prognostic classification of adrenocortical cancers, for planning an individualized therapeutic approach and for the identification of known or potential targetable molecular events in the single patient. in a retrospective study dna from acc and matched blood samples is sequenced to detect somatic single nucleotide variants (snv), small insertions and deletions (indel) and copy number alterations (cnv). sequencing data are then compared to clinical data e. g. tumor stage, resection status, ki -index and time of progression free and overall survival to define molecular prognostic factors. target enrichment of genes that are known to be associated with different entities of cancer is performed with the human comprehensive cancer panel (qiagen) and sequenced on a nextseq (illumina). data are analysed with gensearchngs (phenosystems). znrf , a gene that was also described to be involved in the development and the progression of acc a few years ago, is sequenced separately with sanger and analysed with gensearch (phenosystems). to date tumor samples and matched blood samples from patients were analysed. one or more tumors comprise one or more snvs or small indels in of genes of the panel and in znrf . snvs and small indels are most often found in tp , ctnnb and znrf with frequencies of %, % and % respectively. in of genes cnvs -duplications and deletions -occur. cdk is duplicated in over % of the cohort. mdm gains are found in over %. one can also find three types of cn patterns: a quiet type with low number of copy alterations, a noisy one with high number of chromosomal breakages and a chromosomal one with high frequency of alterations of chromosomal arms. while no correlation between snvs and small indels and clinical outcome could be found so far, cn patterns of the accs seem to correlate with progression free survival and overall survival. patients with a noisy cn pattern have a shorter progression free and overall survival than patients with chromosomal and quiet type. though tendencies in the correlation of molecular markers and prognosis for patients suffering acc can be recognized, further samples need to be analysed to confirm the results. it is planned to sequence another tumor samples and matched blood samples for this retrospective study and to validate the results in a prospective study with another patients. expression of mir- a- p and mir- - p was analysed in serum samples by quantitative pcr. the cohort of gcnis patients consisted of patients with a solitary testicle, who had undergone orchiectomy for contralateral tgct, and patients with two testicles, one of which with gcnis, but no concurrent tgct. twenty men with non-malignant testicular disease served as controls. additionally, in situ hybridisation (ish) with a probe against mir- a- p was performed on four testicular biopsy specimens known to harbour gcnis. sequential step sections of the corresponding tissue blocks were analysed immunohistochemically, using oct antibody to visualise gcnis. the median expression value of mir- a- p in gcnis-patients was . (interquartile range [iqr] = . ) which is significantly higher than the median expression of . (iqr = . ) in controls. both of the two gcnis subgroups had significantly higher mir- a- p levels than controls, with a median expression of . (iqr = . ) and . (iqr = . ), respectively. regarding mir- - p expression, there were no significant differences between gcnis and controls. using a relative quantity of as a cut-off value, the mir- a- p was able to detect . % ( % confidence interval [ % ci] = . - . %) of gcnis, while only % ( % ci = . - . %) of the controls were positive. in the subgroup with previous tgct . % ( % ci = . - . %) of gcnis could be detected and in the subgroup without previous tumour the rate was . % ( % ci = . - . %). the detection rates for all gcnis and for both subgroups were significantly higher than for the controls. ish staining demonstrated the expression of mir- a- p in gcnis cells in two of the four cases. in conclusion, this study indicates a new and minimal-invasive way of diagnosing gcnis by measuring serum levels of mir a- p. this approach is endorsed by the demonstration of mir a- p in gcnis cells by ish staining. however, the sensitivity is still low and thus, the method certainly needs refinement possibly by applying a panel of additional mi-crornas. nonetheless, measuring serum levels of mir a- p may constitute a valuable aid in clinical assessment of men afflicted with high-risk factors of tgct. p-cancg- *** the mir- a- p is a highly specific and sensitive serum-based marker for the diagnosis and follow-up of testicular germ cell tumours testicular germ cell tumours (tgct) are a paradigm of curable malignancies. clinical management largely relies on measuring the serum biomarkers. inopportunely, the markers beta-hcg, afp and ldh are only elevated in about % of patients. therefore, micrornas of the clusters mir- - and mir- / were proposed as novel serum-based markers. we evaluated four of the candidate mirnas (mir- a- p, mir- - p, mir- - p and mir - p) with regard to their usefulness as tgct markers. overall, serum samples from tgct-patients and from controls were analysed using quantitative pcr. the first consecutive patients and controls were analysed for all four mirnas. after roc-analysis only the marker with the greatest discriminative power was studied further. the decline of mirna expression after orchiectomy was quantified in cases and in metastasized cases the marker was analysed repeatedly during the course of chemotherapy. additionally cases with relapsing disease were studied. the mir- a- p featured the highest discriminative power (area under the curve: . ; % confidence interval [ % ci]: . - . ). in the entire cohort, patients could be distinguished from controls with a sensitivity of . % ( % ci: . - . %) and a specificity of . % ( % ci: neuroendocrine tumor of the adrenal gland: an unusual manifestation of tsc c. müller-hofstede, j. horvath, b. dworniczak, p. wieacker institute of human genetics, university of münster, germany we report on a young woman asking for the recurrence risk of the neuroendocrine tumor of her mother deceased at the age of . her mother clinically presented because of therapy-resistent hypertonia, dyspnoe, progressive edema in the legs and face and a caput medusae. mri scan revealed a tumor ( × cm) in the right adrenal gland with lymph node metastases compressing the v. cava inferior and synchronous metastases in lung and liver. laboratory examinations showed highly elevated levels of cortisol and adrenocorticotropin (acth). cerebral mri was normal suggesting an ectopic acth secretion by a non-pituitary tumor. histologically, an undifferentiated, largely necrotic tumor was described so that the neuroendocrine nature of the tumor could not be proven. she died within three weeks after diagnosis. on suspicion of multiple endocrine neoplasia type we initially performed a sequence analysis of men on tumor dna by next generation sequencing without detection of a pathogenic mutation. thereupon the molecular genetic panel analysis (nf , ret, sdhb-d,tmem , tsc , tsc , vhl) uncovered the heterozygous mutation c. c>t (p.arg trp) in the gene tsc . this mutation is already described as pathogenic (hu et al. ). in the tumor dna the allele frequency of the normal allele mounted up to %, whereas the allele frequency of the mutant allele came to % pointing to a loss of heterozygosity (loh). the mutation was confirmed by sanger sequencing. taken all together, we assumed, that the mutation in the gene tsc represents a germline mutation. mutations in the suppressor genes tsc and tsc cause tuberous sclerosis, an autosomal-dominant disorder, resulting in hamartomatous tumors in the heart, brain, kidneys, skin and other organs. once in a while it is discussed whether neuroendocrine tumors (nets) represent a characteristic of tsc. there are some case reports describing nets in the context of tsc, but mainly in connection with nets of the pancreas (e. g. insulinoma) or the pituitary. to the best of our knowledge there exists only one case report of a bronchial carcinoid as a result of a germline mutation in tsc (dworakowska et al. ) and no description of net of the adrenal gland due to a mutation in tsc or tsc . ngs provides the opportunity of wide-spread testing, even post-mortem, in order to get clarification for the descendants. although in our case we could not distinguish if the mutation detected represents a germline mutation or a somatic mutation, we were able to offer a predictive testing to the daughter and other family members. we report on a rare case of net of the adrenal gland because of mutation in the gene tsc . this case illustrates that in the differential diagnosis of nets, tsc genes should also be considered. germ cell neoplasia in situ (gcnis) is the precursor lesion of testicular germ cell tumours (tgct). if detected clinically, this lesion may herald a pending tgct. unfortunately, the only way of diagnosing gcnis is by testicular biopsy and subsequent immunohistochemical examination. therefore, non-invasive methods of diagnosis are required. mirnas of the mir- - and mir- / cluster had been suggested as serum biomarkers of full-blown tgcts. we aimed to explore the utility of these mirnas for the detection of the pre-invasive stage of tgcts and we looked to the expression of two mirnas in serum samples of gcnis patients. psmc ip located on chromosome q is a putative tumor suppressor gene that encodes for the nuclear psmc interacting protein. the protein functions as coactivator of steroid hormone mediated gene expression and is important for rad and dmc -mediated homologous recombination during dna repair of double-strand breaks. recently germline variants in psmc ip, also known as gt , tbip, and hop , have been identified with low frequency in early onset familial breast and ovarian cancer (hboc) patients and in a patient with apparently sporadic early onset breast cancer. somatic variants in psmc ip are frequently observed in breast, ovarian, and fallopian tube cancers. in this study, we analyzed a cohort of brca / mutation-negative hboc (n = ) or early onset sporadic breast cancer patients (n = ) for variants in psmc ip. we identified seven different heterozygous variants in out of index patients: c.- g>a (rs ); c.- t>a (rs ); c.- a>t (rs ); c.- c>g (rs ); c. g>a p.(trp *); c. + g>c (rs ); c.* g>a. these variants were not listed or at very low frequency (< %) in the exac database. carriers of psmc ip germline variants were mostly ( / ) affected by early onset breast cancer (median age of onset years). for three out of seven different variants (c.- g>a, c. g>a, and c* a>g), a possible impact on psmc ip expression or function was observed. the stop mutation c. g>a p.(trp *) was found in two sisters, which were both diagnosed with unilateral breast cancer at age . the premature stop codon is located within the dna-binding domain of psmc ip and is predicted to induce nonsense-mediated mrna decay (nmd). remarkably, c.- g>a was already described in familial breast and ovarian cancer, and was found once in this study in a female that developed unilateral breast cancer at the age of years. the variant c.- g>a (rs ) was shown to induce a slightly, albeit significant decrease of reporter gene expression. the c. *g>a variant was identified in a woman diagnosed with unilateral breast cancer at the age of years. luciferase reporter assays indicated an impaired effect of c. *g>a on microrna binding. germline variants in psmc ip are present in breast and ovarian cancer families. whether mutated psmc ip is a new risk factor for early onset breast/ovarian cancer in families with hboc and/or apparently sporadic early onset breast cancer remains to be shown. . - . %) with this marker. in patients without metastases the mir- a- p expression declined significantly after surgery. in metastasized cases the levels dropped sharply after chemotherapy. all of the relapses had elevated mir-levels, and expression decreased upon chemotherapy. mir- a- p has significantly higher sensitivity than each one of the classical tgct markers and than a combined panel of beta-hcg, afp and ldh ( . % vs . %). in non-metastasised seminoma the mir- a- p expression depended significantly on tumour size. mir- a- p is highly sensitive and specific for tgct. it correlates with the stage of disease and with treatment effects and it therefore fulfils the prerequisites of a valuable serum-based biomarker. the significant association with tumour bulk in localised disease provides evidence for the tgct being the primary source of mir expression. the sensitivity of mir- a- p surpasses that of classical tgct markers by far, and thus it may become the new gold standard for serum diagnostics of tgct in the coming years. co-occurrence of radioulnar synostosis and amegakaryocytic thrombocytopenia (rusat) was initially described as an inherited thrombocytopenia syndrome that is caused by a mutation in hoxa . in three simplex patients, de novo missense mutations in mecom were reported as an alternative origin of the disease (rusat ). mecom, identified as a common ecotropic viral integration site (evi ) in murine myeloid leukemia, is known as a key transcriptional regulator in hematopoiesis and sporadic myeloid leukemia. we report here on a novel mecom mutation cys gly (uniprot q - ) identified by whole exome sequencing in a family with rusat, hearing impairment, hand dysmorphisms, and patellar hypoplasia in four patients spanning three generations. notably, two of four affected individuals in our family developed a myeloid malignancy. the novel mecom missense mutation cys gly affects a heavily conserved cysteine residue in c h -zinc finger motif in the c-terminal zinc finger domain of mecom. this residue is crucial for the tetrahedral coordination of a zinc ion stabilizing the zinc finger conformation and thus, is essential for dna binding of the c-terminal zinc finger domain. our findings reconfirm the causality of mecom mutations and indicate that mecom mutations also need to be considered in familial rusat patients. in addition, we report for the first time that mecom germline mutations targeting the c-terminal zinc finger domain are associated with an increased risk for myeloid malignancies. this extends the rusat -associated phenotype and proposes that mecom germline mutations can cause a genetic predisposition to myeloid malignancy. (z., b. and s., d. contributed equally to this work) many genes that harbor rare mutations which entail a medium or high risk for breast cancer (bc) belong to dna double strand repair and have been identified by linkage analysis or by sequencing of candidate genes in bc families. in addition, a considerable number of common but low risk germline variants have been found in genome wide association studies. however, these predisposing factors yet only explain a fraction of bc cases. with the intention to identify low frequency variants conferring an intermediate bc risk, we performed an association study using a candidate gene approach and testing dna repair capacity with the micronucleus test (mnt) as an additional second phenotype. rs in the slx /fancp gene showed an association with bc that was pronounced in younger cases and was confirmed in a verification cohort (combined analysis of , cases, controls: or = . ( . - . ) , p = . for cases ≤ years). genotyping additional snps and imputation revealed a specific european haplotype of ca. kb length that spans slx and adjacent genes. it is tagged by the observed mutual dependence of the two phenotypes allowed a considerably improved interpretation of the results: (i) the unknown causal variant on the haplotype can be assumed to be present mostly in cases, indicating a rare variant with a rather strong effect; (ii) using this information on the two phenotypes in the association between the mnt results and bc improved considerably the identification of the specific risk among cases (< years) who carried the haplotype. roc curves for bc depending on mnt results revealed that the stratification on carriers of the haplotype increased the auc from . (p = . ) to . (p = . ). both associations can be best explained by a risk variant carried by a fraction of the haplotypes that is enriched in early onset bc cases. slx is the only gene in the tagged region which can be functionally related to both associated phenotypes, while for the other genes no connection to bc or dna repair is reported. inherited dna repair mutations: are they modifiers of brca and brca penetrance and age at onset of hereditary breast and ovarian cancer? background: inherited mutations in brca and brca are the most common causes of hereditary breast and ovarian cancer (hboc). the risk of developing breast cancer by age in women carrying a brca mutation is - % and - % in brca carriers. however, mutations in brca and brca only explain about % of all hboc cases. the lifetime risk varies between families and even within affected individuals of the same family. the cause of this variability is unknown but it is hypothesized that additional mutations or rare variants in genes that are possibly interacting with brca / in different dna-repair pathways contribute to this phenomenon. methods: we obtained samples of patients positive for brca or brca mutations and an age-of-onset (aoo) of breast cancer below or above years of age from the german consortium for hereditary breast and ovarian cancer. panel sequencing was done to screen germline dna for mutations in genes involved in different dna-repair pathways. variants were classified into five classes according to a modified version of plon et al ( ) . only truncating mutations and known pathogenic missense mutations were considered pathogenic or likely pathogenic. results: the patient group with an early aoo ( women) had developed breast cancer at a mean age of . years (± . ) and the control group ( women) had developed breast cancer at a mean age of . years (± . ). a total of , variants were detected in all patients and of these ( . %; % ci, . %- . %) were presumed to be deleterious. mutations were found in genes other than brca and brca . mutations were mainly found in single-strand break repair (ssbr %), double-strand break repair (dsbr %) and checkpoint factors ( %). the rest were found in genes with other functions such as brca / interactors, centrosome formation, and signal transduction. the putative mutations were found in women of the control group ( . %; %ci, . %- . %) compared to women of the patient group ( . %; % ci, . %- . %). the incidence of germline mutations in dna-repair genes did not differ according to the age of onset (p = . ). prevalence of additional germline mutations in dsbr in patients ( %) was not significantly different from prevalence of dsbr mutations in controls . % (p = . ) conclusions: the preliminary results failed to show a difference in mutation load between the two cohorts of brca / carriers sorted by age of onset. larger studies are needed and may provide further insight into the role of mutation load in hboc age of onset of brca / carriers. objective: glioblastoma stem-like cells (gscs) carry stem cell features and therefore seem to be responsible for tumor initiating, maintaining, recurrence and chemo-and/or radiotherapy resisting. the current knowledge on genetic and transcriptomic characteristics of these cells especially in comparison to glioblastoma tissue is still limited. the aim of this study is to compare the genetic and genomic profile of glioblastoma tissue and gscs. thereby, differences in involved genes and affected pathways on dna level as well as on gene expression level are identified. material and methods: peripheral blood and tumor tissue were obtained from patients with glioblastoma. tumor tissue derived explant cell culture and serum-free culture were established. based on multi-parameter magnetic-activated cell sorting (macs) technique, cd and cd labeled cell subpopulations of gscs could be isolated. the tumor tissue, serum-free culture, and the isolated cell subpopulations as well as blood were analyzed by snp array and gene expression (excluding blood) in a paired design. for preliminary characterization of gscs in the serum-free culture we confirmed the stem cell features of gscs by the expression of nestin, sox , and cd (applying immunofluorescence staining). results: our results of snp array analyses showed genetic aberrations in all analyzed cellular entities (tumor tissue and cell subpopulations, e. g. gain of chromosome , loss of q . , loss of q . ->q . , and complete loss of chromosome ). furthermore, distinct genetic differences between the cell subpopulations and tumor tissue were observed (e. g. loss of chromosome and segmental uniparental disomy of p . ->p . , only in cancer stem-like cell subpopulations). in addition, we detected many possibly candidate cancer genes and pathways which may have an influence on tumorigenesis. gene expression analyses revealed strongest differences between fresh tumor tissue and serum-free culture based cells, where more than a third of investigated genes were affected. when contrasting fresh tumor tissue with stem cell marker positive serum-free cultured cells, , genes were upregulated in the stem cell marker positive cells, whereas , genes where upregulated in fresh tumor tissue. within these genes, strongest enriched pathways in stem cell marker positive cells included positive regulation of cell cycle and cancer-related pathways, whereas in fresh tumor tissue predominantly immune-related pathways were found, e. g. myeloid leukocyte activation, inflammatory response and phagocytosis. conclusion: differences between gscs and tumor tissue using snp array analyses and gene expression were detected. our results may help to get more information about the molecular pathomechanisms of glioblastoma. it still needs more investigations on the field of genetic and genomic analyses between gscs and glioblastoma tissue to identify novel potential targets for therapy development. background: fanconi anemia (fa) is a rare inherited chromosomal instability syndrome associated with bone marrow failure as well as myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). one-third of fa individuals exhibit bone marrow cytogenetic clones, notably gains of q and q and/or loss of / q, and % to % of fa patients developed mds/aml. in recent years, the application of high throughput technologies has revealed recurrent somatic mutations in genes implicated in myeloid malignancies. as additional genetic maladies facilitating mds/aml development in fa is lacking, we aimed to elucidate whether these mutations would be present in fa patients with mds/aml. methods: using illumina trusighttm myeloid sequencing panel (san diego, ca), we performed next-generation sequencing (ngs) on dna extracted from bone marrow specimens from fa mds/aml patients registered in the european working group of childhood mds. the sequencing panel targeted genes frequently mutated in hematologic neoplasms. results:. ten of the ( . %) evaluable patients had lesions ( to mutations per patient; missense, nonsense, insertion and duplication) in genes. the presence of a somatic mutation did not appear to correlate with complex karyotype or − / q. all affected genes occurred in isolation with exception of runx and kras. while of the mutations were pathogenic, were variance of unknown significance. mutations in genes involved in epigenetics (dna methylation, chromatin maintenance and cohesin complex; idh , tet , dnmt a, idh , ezh , rad and asxl ) and mutations in transcription factor genes (runx , ikzf and etv ) represented the most frequently affected genes. this was followed by mutations of genes encoding signaling molecules including the ras pathway (kras and ptpn ). altered runx was the most common lesion and occurred in individuals with aml, raeb or raeb-t. one patient with refractory anemia with ring sideroblasts (rars) had a mutation in the spliceosomal gene, sf b . conclusions: while the most common mutations encountered in sporadic cases of mds were in genes involved in rna splicing and epigenetics, these two broad categories of genes appeared to have less influence in our fa patients. most mutations were nonrecurring suggesting that there is no specific mutation pattern of these genes in fa-related mds/aml. however, runx mutations and also mutations involved in genes of the ras pathway appear to play a pathogenic role in fa mds/aml development. taken together, the data suggests that mutations in genes that cause clonal hematopoiesis in the population at large do not contribute significantly to fa hematopoietic clonal disease; however, particularly acquisition of runx and ras pathway alterations promote malignant myeloid disease progression. more extensive studies analyzing more patients are necessary to further define the secondary hits leading to fa myeloid disease. chromatin remodeling is a complex process shaping the nucleosome landscape, thereby regulating the accessibility of transcription factors to regulatory regions of target genes and ultimately managing gene expression. the swi/snf (switch/sucrose nonfermentable) complex remodels the nucleosome landscape in an atp-dependent manner and is divided into the two major subclasses brahma-associated factor (baf) and polybromo brahma-associated factor (pbaf) complex. somatic mutations in subunits of the swi/snf complex have been associated with different cancers, while germline mutations have been associated with autism spectrum disorder and the neurodevelopmental disorders coffin-siris (css) and nicolaides-baraitser syndromes (ncbrs). css is characterized by intellectual disability (id), coarsening of the face and hypoplasia or absence of the fifth finger-and/or toenails. so far, variants in five of the swi/snf subunit-encoding genes arid b, smarca , smarcb , arid a and smarce as well as variants in the transcription factor-encoding gene sox have been identified in css-affected individuals. arid is a member of the pbaf subcomplex, which until recently had not been linked to any neurodevelopmental phenotypes. in , mutations in the arid gene were associated with intellectual disability. in this study, we report on two individuals with private de novo arid frameshift mutations. both individuals present with css including id, coarsening of facial features, other recognizable facial dysmorphisms and hypoplasia of the fifth toenails. hence, this study identifies mutations in the arid gene as a novel and rare cause for css and enlarges the list of css-associated genes. the ubiquitin pathway is an enzymatic cascade including activating e , conjugating e , and ligating e enzymes, which governs protein degradation and sorting. it is crucial for many physiological processes. compromised function of members of the ubiquitin pathway leads to a wide range of human diseases, such as cancer, neurodegenerative diseases, and neurodevelopmental disorders. mutations in the thyroid hormone receptor interactor (trip ) gene (omim ), which encodes an e ligase in the ubiquitin pathway, have been associated with autism spectrum disorder (asd). in addition to autistic features, trip mutation carriers showed intellectual disability (id). more recently, trip was postulated as a novel candidate gene for intellectual disability in a meta-analysis of published id cohorts. however, detailed clinical information characterizing the phenotype of these individuals was not provided. in this study, we present seven novel individuals with private trip mutations including two splice site mutations, one nonsense mutation, three missense mutations, and one translocation case with a breakpoint in intron of the trip gene and clinically review four previously published cases. the trip mutation-positive individuals presented with mild to moderate id ( / ) or learning disability [intelligence quotient (iq) in one individual], asd ( / ) and some of them with unspecific craniofacial dysmorphism and other anomalies. in this study, we provide detailed clinical information of eleven trip mutation-positive individuals and thereby due to its heterogeneous etiology, primordial growth retardation is often a challenge for geneticists and clinicians in respect of diagnosis, therapy and prognosis. thus, pinpointing its genetic origin is required for a personalized treatment and prognosis. one syndrome mainly characterized by intrauterine and postnatal growth is silver-russell syndrome (srs), a clinically and molecularly heterogeneous disorder with a considerable overlap with other syndromes. in only % of patients with the characteristic srs phenotype the diagnosis can be confirmed molecularly, but % of cases remain without molecular diagnosis. in fact, in clinically less well characterized patients referred for diagnostic testing, the detection rate is less than %. however, systematic investigations on the contribution of mutations in genes which may be considered in the differential diagnoses of srs are still missing. we examined patients referred for molecular testing of srs but without molecular alterations associated with srs by ngs. a targeted ngs approach comprising genes implicated in the differential diagnoses of srs or suggested as srs candidate genes was performed. in patients fulfilling the criteria of srs accordingly to our recently developed clinical scoring system, disease-causing variants were found. these patients carried mutations in genes associated with bloom syndrome, mulibrey nanism, kbg syndrome, short syndromes or ig-f r-associated short stature, respectively. indeed, some of the differential diagnoses detected in our cohort have a major impact on clinical management, including cancer screening because of a high risk for tumor development. furthermore, we did not identify any pathogenic mutation in one of proposed srs candidate genes (e. g. mest, grb , copg ), thus raising the question whether these genes are indeed involved in the etiology of srs. we show that a (targeted) ngs approach is an important tool to identify the genetic cause in patients with unexplained growth retardation. furthermore, our data show (positive) clinical scoring in srs should not impede the consideration of differential diagnoses and other molecular causes. submicroscopic deletions of chromosome band p . have been reported in more than patients. common clinical features include intellectual disability/developmental delay, central obesity and behavioural difficulties. myt l became the main candidate gene for id and obesity since it is deleted or disrupted in all published patients. however, reports of deletions affecting only this gene and even more so of deleterious myt l sequence variants are very rare. to our knowledge, until now only two patients with de novo myt l point mutations have been reported. in the present study, we analysed a cohort of individuals with intellectual disability of unknown aetiology and their unaffected parents by whole exome sequencing. we identified de novo myt l sequence variants in two out of patients. patient carried a nonsense mutation (c. g>t, nm_ . ; gly *) whereas patient carried a direct splice site mutation (c. - a>g). according to prediction algorithms, both detected myt l variants are deleterious (patient : sift score , cadd score ; patient : cadd score . ). in addition, patient carried a de novo splice site variant in setd b. however, this variant is predicted to be benign (cadd score . ) as well as a known snv (rs , maf . ). a comprehensive clinical characterisation of the two patients yielded only mild or moderate intellectual disability, behavioural problems and muscular hypotonia as common clinical signs. surprisingly, obesity was only present in patient . postnatal tall stature and transient microcephaly were present in one patient each. this clinical picture is compared to the published phenotypes of patients with myt l point mutations, patients with microdeletions of only myt l and patients with larger p . deletions. with the reduced penetrance regarding obesity, the clinical picture of patients with myt l mutations is becoming more and more unspecific. the retina and anterior neural fold homeobox gene (rax) controls the embryonic eye development and is involved in human autosomal-recessive microphthalmia. so far only a few compound heterozygous mutations in rax have been described in microphthalmia patients. we report a first case of microphthalmia caused by a novel homozygous mutation in rax. the -month-old patient was born to consanguineous parents and presented with extreme microphthalmia, panhypopituitarism and developmental delay. mri of the brain showed bilateral agenesis of the anterior visual pathway and tractus opticus. ocular ultrasound confirmed bilateral anophthalmia. additionally, dysgenesis of the corpus callosum and an abnormal pituitary gland have been detected. the first child of these parents, who died shortly after birth, had also been diagnosed with bilateral anophthalmia. using panel diagnosis of the disease associated genome, we identified the homozygous pathogenic variant c. del, (p. fs) in rax. we performed a segregation analysis and confirmed that both parents are heterozygous for this variant. so far developmental delay and panhypo-expand the clinical spectrum of the trip gene in non-syndromic intellectual disability with or without asd. background: whole exome sequencing (wes) using next generation sequencing has proven to be a powerful tool in determining the underlying genetic cause of rare disorders. here, we show, that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering further disease-causing mutations. clinical report and genetic findings: we report follow-up data of a previously published consanguineous family with two children, a boy and a girl, suffering from severe encephalopathy, hypotonia, microcephaly and retinal dystrophy. wes had shown a homozygous intronic splice variant in pgap (c. - a>g;p.?) causative for the symptoms. both parents were heterozygous carrier for the pgap variant (granzow, paramasivam et al, mol cell probes ) . in the next pregnancy, the unborn child presented hydrops fetalis, omphalocele, short tubular bones and cystic kidneys. chorionic villus sampling showed the fetus to be homozygous for the pgap variant. however, neither of these symptoms fit with a pgap -associated disorder. additional wes of fetal dna and re-evaluation in the family showed a homozygous nonsense variant in ift (c.g t;p.e *) consistent with a diagnosis of mainzer-saldino syndrome (mss) which is characterised by the association of renal disease, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia, as a second diagnosis in the fetus. again, both parents were shown to be a heterozygous carrier for the ift variant. yet, as omphalocele was not accounted for by any of the identified conditions, a third genetic cause cannot entirely be excluded. alternatively, omphalocele may be a rare manifestation of mss, or be the result of a combination of both disorders. the couple opted for induced abortion. discussion: it is estimated that an individual carries multiple heterozygous variants for autosomal recessive disorders in his or her genome. especially in consanguineous families, this results in an elevated risk for children with more than one disorder. in recent publications of clinical exomes, double diagnoses have been reported in to % of investigated subjects. thus, the possibility of more than one causative gene should be carefully explored when working with wes and re-evaluation in case of additional clinical symptoms within a family should be considered. also, follow-up of families with rare genetic disorders may lead the clinical geneticist beyond the assumed single cause to multiple single gene disorders in the same family. conclusion: using wes, we have identified two independent single gene disorders in a consanguineous family demonstrating that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering multiple disease-causing mutations in one family. we present a case study using molecular cytogenetic approaches on a year old boy presenting with microcephaly-brachycephaly, macroglossia and absent speech development. the boy is the first child of healthy, consanguineous parents of pakistani origin. following an uncomplicated pregnancy, the hypotrophic newborn was delivered at weeks weighing g. in the third month of life, the baby had viral meningitis. regular pediatric follow-up revealed psychomotor delay with hypotonia. creatine kinase, lactate and fibroblast growth factor measured in serum were high. subsequent investigations at the age of months included brain mri, electroencephalogram and muscle biopsy that gave hints of a mitochondriopathy or potential neuropathy of axonal type. due to the suspicion of a complex mitochondriopathy, whole exome sequencing was performed using a sureselect human all exon kit (agilent, mb v ) on a hiseq (illumina). the analysis revealed a heterozygous microdeletion of kb on chromosome q . which was classified as an unclear variant (uv), ehmt -gen was not affected. re-examination of proband's dna using array cgh detected a larger kb heterozygous deletion in the q . region (arr[hg ] q . ( , , - , , ) x ), encompassing exon of ehmt (euchromatin histone methyltransferase ; transcript nm_ . ). haploinsufficiency of this gene results in kleefstra syndrome (omim ), a multisystem disorder due to either microdeletions in q . encompassing ehmt or intragenic point mutations. mlpa analysis (ehmt mpla-kit p ) of parental dna further indicated a de novo origin of the deletion in our proband. a similar deletion has been described previously in a case presenting with clinical features of kleefstra syndrome [ ] strengthening the importance to include the ' part of ehmt in sequencing as well as cnv screening. in summary, our study clearly shows that array cgh is a valuable complementary approach to ngs especially for poorly covered regions in ngs i. e. exon of many transcripts. we report on a three-generation family with variable manifestations including delayed and incomplete tooth eruption, early tooth loss due to short dental roots, acroosteolysis, osteoporosis, tendon ruptures, joint hypermobility, muscle weekness, glaucoma, neurological features, and psoriasis. after detection of elevated cd /siglec -expression on monocytes and an upregulation of interferon-stimulated gene transcripts, singleton-merten syndrome was diagnosed. the novel heterozygous mutation c. c>g (p.thr arg) in ifih was found in three affected family members. singleton-merten syndrome is a very rare autosomal dominant interferonopathy, so far described in not more than four families. until now, only two different gain-of-function mutations in ifih have been detected. mutations in ifih are also associated with aicardi-goutière syndrome and recently features of both conditions were found in the same family. our findings expand the mutational spectrum of singleton-merten syndrome and demonstrate the high intrafamiliar variability associated with mutations in ifih . pituitarism have not been described in association with rax mutations. therefore we conducted array comparative genomic hybridization and karyotyping in the index patient. both tests gave normal results. prenatal diagnosis by chorionic villus sampling in the next pregnancy excluded a homozygous carrier status for this rax mutation. mutations in the phf gene are associated with borjeson-forssman-lehmann syndrome (bfls), an x-linked intellectual disability disorder affecting mainly males. female carriers usually show no or mild clinical signs. however, recent studies described females with de novo phf gene defects (mutations, deletions) and severe phenotypes resembling coffin-siris syndrome [ , ] . here, we report on a girl with a maternally inherited phf mutation and a phenotype resembling those described previously in affected females. the mother had learning difficulties and mild dysmorphological features (hypertelorism, prominent forehead). when seen at age months, the proposita showed muscular hypotonia, was unable to sit and had limited head control (developmental delay months). dysmorphic features included scaphocephaly, hypertelorism, a small flat nose with anteverted nares, low set, prominent ears, a high, narrow palate, absent labia minora and linear skin pigmentation on the thighs. ophthalmologic investigation identified strabism convergens of the left eye, hyperopia and an excavated papilla with a pale optical nerve. ultrasound showed patent foramen ovale, tricuspid insufficiency and a unilateral incomplete duplication of the renal pelvis. karyotyping performed elsewhere was normal ( ,xx). results of a genome-wide snp array analysis (affymetrix cytoscan hd) were also normal. using a targeted ngs approach for syndromic and non-syndromic developmental delay encompassing over brain related genes (mpimg- -test), we identified a heterozygous lof-mutation c. c>t (p.gln *) in the phf gene (encoding phd finger protein ). in addition, a human androgen receptor (humara) assay using blood dna showed a highly skewed x-inactivation ( : ). segregation analysis indicated a maternal origin of the variant. the mother also had skewed x-inactivation in blood. her husband and other daughter tested normal for the c. c>t variant. here, we describe the first female patient with a maternally inherited phf mutation and a severe phenotype. the mild phenotype in the mother might be due to different patterns of x-inactivation or to (undetected) mosaicism. this report represents the th case of a severely affected female patient with a phf gene defect. findings support the assumption [ ] that the female phenotypes of bfls might be more common than previously estimated. toms typical for ada deficiency such as fever, livedo racemosa, abdominal colics, arthralgias, and raynaud's phenomenon were observed months later. cecr sequencing (nm_ . ) revealed two previously described pathogenic missense mutations: c. g>c, p.(gly ala) and c. g>a, p.(arg gln). compound heterozygosity was confirmed by parental analysis. to the best of our knowledge, this combination of mutations has not been described until now. the p.(arg gln) is considered as founder mutation in the dutch population, but first phenotype-genotype analyses did not allow further prediction of clinical outcomes. ada deficiency should be considered in patients with childhood stroke despite the absence of systemic inflammation and cerebral vasculitis. congenital hyperinsulinism (chi) has been described as heterogeneous entity caused by at least different genes. in , tein et al. first described a defective activity of l- -hydroxyacyl-coa dehydrogenase in a -year old patient with hypoketotic hypoglycemic encephalopathy. biochemical markers of l- -hydroxyacyl-coa dehydrogenase deficiency (schad) are high -oh-glutarate excretion in urine and c -oh-carnitine in plasma. the clinical presentation is very heterogeneous with regard to age of onset, severity of symptoms as well as response to medical treatment and leucine-sensitivity. in some patients even a near total pancreatectomy was performed. schad dependent hyperinsulinism (hhf ) is a rare autosomal recessive disorder that is caused by mutations in the gene hadh. here we describe patients from unrelated families out of a cohort of chi patients mainly from central europe. patients and are siblings from unrelated parents. the older brother manifested with hypoglycemic convulsions at the age of weeks. a subtotal pancreatectomy was performed in an outside academic hospital. in further course he developed epilepsy and has been treated with diazoxide and anticonvulsants. the nd child was born with hypoketotic hypoglycemia and chi was diagnosed in first days of life. diazoxide treatment stabilized blood glucose and both children were referred to our pediatric endocrinology at the age of years and months, respectively. mutational analysis revealed the homozygous variant c. - c>g within the region of the splice acceptor site in intron of the hadh gene in both affected children. this change is neither registered in exac nor described in the mutation databases hgmd or in the literature and was predicted to disrupt proper splicing. we then completed mutational analysis in unidentified patients of our chi cohort with diazoxide responsiveness and known or suspected consanguinity. patient was born to consanguineous parents and chi manifested in the girl at neonatal age with hypoketotic hypoglycemia. she was successfully treated with diaxozide. later, she developed convulsions and statomotoric developmental delay. the homozygous splice mutation c. + g>t in intron of hadh was detected in the child. the parents were identified as heterozygous carriers. in patient , a girl born to consanguineous turkish parents, chi manifested at the age of months with hypoglycemic seizures. she responded well to diazoxide treatment. a homozygous missense mutation (c. a>g; p.lys glu) in exon of hadh was detected in the patient and her parents were heterozygous carriers. hadh mutations in case and have been previously described in probands of turkish descent and appear to be founder mutations in the turkish population. in conclusion, we recommend hadh mutation analysis to be considered in chi children with unknown cause and known consanguineous pedigrees or originating from populations with higher prevalence of consanguinity. homozygous or compound heterozygous mutations in cecr (cat eye syndrome chromosome region, candidate ) have recently been identified to causing deficiency of adenosine deaminase (ada ; dada ) with childhood polyarteritis nodosa (pan) (omim # ). this inflammatory vasculitis affects the skin, and inner organs (predominantly kidneys and gastrointestinal tract) and also shows a high risk of ischemic stroke, brain hemorrhage as well as peripheral neuropathy. using whole-exome sequencing it was also found that the six adult patients (aged - ) described by sneddon in (sneddon syndrome, omim # ) likewise carried compound heterozygous cecr mutations. sneddon syndrome is characterized by a combination of dermatologic features (livedo racemosa) and ischemic brain infarctions. recently, clinical and genetic data of more than sixty ada patients have been reviewed and underlined the wide clinical variability in age at onset, clinical findings, outcome of neurological involvement, and additional hematological symptoms. typically, stroke has been reported to follow systemic inflammatory disease and predominantly affects posterior and central brain areas. here we describe one of the rare patients in whom acute mesencephalic stroke preceded systemic inflammation and presented as initial clinical symptom. symp- triploidy is a recurrent finding in prenatal diagnostics. in a small number of individuals, correction of triploidy has been suggested based on the finding of (mosaic) genome-wide uniparental disomy (upd). we here investigated uncultured und cultured amniotic cells (ac) and placental tissue from a fetus, in which ultrasound examination in the + th week of gestation revealed growth retardation, left diaphragmatic hernia with parts of stomach and bowel localized in the chest, dextrocardia, short nasal bone and single umbilical artery. these findings were confirmed at the + th week when the pregnancy was terminated. the pregnancy was conceived spontaneously by a -year-old mother and a -year-old father, both healthy and with uneventful family history. the parents were non-consanguineous and carry a normal karyotype. microsatellite analyses of uncultured ac obtained at initial presentation showed for chromosomes , and a pattern suggesting triploidy with only biallelic presentation. while y-chromosomal sequences were lacking the x-chromosome showed, unexpectedly a rather disomic pattern. metaphase yield on cultured ac was low but showed a mosaic karyotype ,xxx-,+ [ ]/ ,xxx[ ], which was confirmed for several chromosomes by interphase fish. remarkably, a triploid clone was cytogenetically not detected. thus, we performed further analyses using microsatellite markers, oncoscan technology and fish. these studies unraveled in uncultured ac a pattern suggestive of triploidy with the supernumerary chromosomal complement derived from a maternal isodisomy with the notable exception of the x chromosome. in cultured ac and placental tissue for all chromosomes, except x and , a diploid pattern was observed with alleles from both parents identical to those in the uncultured ac. trisomy x was confirmed in both tissues with the supernumerary chromosome x being of paternal isodisomic origin. the trisomy was seen only in cultured ac, and likely represents a pseudomosaic which nevertheless could not be proven due to insufficient yield of mitoses from the parallel cultures. finally, retrospective interphase fish on remnant uncultured ac showed two diploid clones, one disomic (approximately % of nuclei) and one trisomic ( %) for the x chromosome. the most likely explanation for the findings is a mosaicism for one diploid clone with genome-wide maternal isodisomy and a second diploid but bi-parental cell line with paternal trisomy x. given the identity of the (maternal) alleles in both clones our findings suggest that originally a triploid clone due to a maternal division error/inclusion of a polar body ii existed which underwent (erroneous) triploidy rescue resulting in one diploid biparental clone and one haploid clone of maternal origin that underwent haploid rescue resulting in genome-wide maternal isodisomy. the biparental clone with trisomy x either resulted from a sperm with two x-chromosomes or an erroneous x-duplication during trisomy rescue. since the introduction of non-invasive prenatal diagnosis (nipd, e. g. har-mony® test) in , this test is frequently demanded and routinely applied in prenatal centers and medical practices. mostly, nipd is intended to detect autosomal trisomies ( , , ), but also offers the possibility to analyze sex chromosomes. therefore, also sex chromosome aneuploidies (sca) (e. g. monosomy x (turner syndrome), triple x, xxy (klinefelter syndrome), xyy) are incidentally found. so far, in our prenatal center sca were detected in pregnancies by har-mony® test, consisting of three pregnancies with monosomy x (turner syndrome) and two pregnancies with klinefelter syndrome (xxy). triple x and xyy were detected one time each. of the three cases with suspected monosomy x, the diagnosis of turner syndrome could only be confirmed in one case. this fetus also had a hydrops at week + . for the other two fetuses, the chromosomal analysis of amniotic fluid revealed normal female karyotypes ( ,xx). in both cases with suspected klinefelter syndrome, this diagnosis could be disproved by amniocentesis (karyotype ,xy). in the pregnancies with assumed triple x and xyy, the true fetal karyotype was not further determined yet. from our experience, the rate of false positive results concerning the sex chromosome aneuploidies is noticeably higher than reported in two studies of nicolaides and hooks . this has to be strongly considered in the counselling of patients who wish to know the fetal sex by nipd. congenital myopathies and congenital muscular dystrophies: will genetic testing replace muscle biopsy in the near future? congenital myopathies and muscular dystrophies are a group of inherited neuromuscular diseases with early onset and broad genetic and histopathological overlap. the diagnostic approach has considerably changed with next generation sequencing methods available. here, we describe the diagnostic value of genetic and histological methods in a cohort of index patients and hence the efficacy of diagnostic procedures. of patients had a muscle biopsy as a first-tier approach. in of patients muscle biopsy was informative, leading to a classification in subgroups of cm or cmd. however, in only a few of these cases biopsy led to a specific diagnosis (e. g. merosin deficiency). in of patients genetic testing (candidate gene sequencing or ngs) was performed additionally to muscle biopsy as a second-tier diagnostic step, while patients of the whole cohort received genetic testing only. in almost two-thirds of these patients genetic testing identified known pathogenic or most likely pathogenic variations. these findings illustrate that genetic testing is superior to muscle biopsy in accurately diagnosing cm or cmd. in conclusion, we suggest that invasive muscle biopsy should be replaced by genetic testing as first-tier diagnostic procedure in patients with clinical signs of cm or cmd. nmd inhibition increases the amount of gaa-rna in patient's lymphocytes as well as in the cells of his parents. the residual function of the resulting protein has to be investigated. discussion and conclusion: rna analysis in lymphocytes with and without nmd inhibition is a simple method for analysing splice defects in all monogenic disorders with expression of the disease causing gene in lymphocytes. a further advantage for the patient is the use of blood cells instead of fibroblasts, because a skin biopsy can be avoided and analysis times are reduced. the exact characterization of pathogenic variants is an important aspect of diagnosis, prediction of disease severity and genetic counselling. in vitro nmd inhibition in lymphocytes of affected patients allows the characterization of splice defects. in the future successful inhibition of nmd in vitro might help to identify patients, who may profit from a therapeutic intervention with nmd inhibitors. even expression of a partial protein with low or no activity reduces the risk for the patient to develop antibodies hampering enzyme/protein replacement therapy. p-cling- q microdeletion syndrome: a family with short stature and silver-russel (srs)-like phenotype. introduction: the silver-russel syndrome (mim ), first described independently by silver and russell in , is a condition with intrauterine growth retardation, postnatal growth failure and other characteristic features, including relative macrocephaly (defined as a head circumference at birth ≥ . sd score (sds) above birth weight and/or length sds), prominent forehead, body asymmetry and feeding difficulties as recently defined in an international consensus statement. patients and methods: we report here on first degree relatives with a silver-russel syndrome phenotype who presented with prenatal-and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive. additional features such as dysmorphic facial features, periodically increased sweating, and scoliosis were present in one of the family members only, whereas learning problems and cardiac arrhythmia were present in one other. none of the patients had relative macrocephaly. high resolution array-cgh was performed to screen for cncs and mlpa to confirm the array-cgh result. results: no hypo-methylation of the imprinting center on p nor uniparental disomy of chromosome and were found in the index-patients. high-resolution array-cgh identified a q . microdeletion of . mb (arr[grch q . ( , , , )× ). the heterozygous loss was confirmed by mlpa in the index patient and the other two affected family members (i. e. her brother and mother). the deletion includes the genes hmga , llph, tmbim , irak , helb, grip , and the pseudogene rpsap . conclusion: to the best of our knowledge this is the first report on familial presentation of a silver-russel syndrome due to a microdeletion in q . . none of the patients had relative macrocephaly. supporting the hypothesis by takenouchi et al. that the causative gene for relative macrocephaly resides centromeric to hmga , the region centromeric of hmga is not included in the deletion in our family. spastic ataxia of charlevoix-saguenay (sacs) is an autosomal recessive neurodegenerative disorder and is caused by homozygous or compound heterozygous mutations in the sacs gene. first symptoms of sacs are walking difficulties due to unsteady gait. further typical clinical features include spasticity, ataxia, pyramidal tract signs, nystagmus and dysarthria. here, we report on a -year-old female patient who initially presented with disturbances in motor abilities including frequent falls and high arched foot. cranial mrt was normal while nerve conduction velocity was significantly reduced. the patient's parents did not show any clinical features. since no pmp duplication was detected we performed a gene panel including genes that are associated with hereditary motor and sensory neuropathies (hmsn) and related disorders by using targeted next generation sequencing. we identified the two heterozygous stop mutations c. t>a (p.leu ter) and c. dupt (p.leu phefs* ), located at the same position in the sacs gene. sanger sequencing did not enable us to properly display that there is a transversion and a duplication of the same nucleotide at two different alleles. this exemplifies that, in contrast to sanger sequencing, ngs can illustrate both alleles separately. to conclude, this case was only resolvable by ngs which makes this method appropriate for the detection of compound heterozygous mutations, especially in the rare event when two mutations occur at the same position. background: the precise identification and characterization of genetic variants in monogenic diseases has a wide influence on diagnosis and therapy. about % of pathogenic variants are splicing variants. due to the complex mechanism of splicing regulation it is difficult to predict the effects of variants on mrna splicing. possible consequences are exon skipping, intron retention, generation of novel splice sites or the utilization of a cryptic splice site. common consequences are a frame-shift and the generation of premature termination codon. this leads to rna degradation via the nonsense mediated decay (nmd) pathway. in a patient with the clinical symptoms of non-classical infantile pompe disease and a confirmed acid alpha-glucosidase (gaa) deficiency, we detected two novel, exonic variants in the gaa gene. both base pair exchanges suggested either an amino acid exchange or a splice defect as consequences. however, conventional investigation of the leucocyte mrna of the patient and his parents was inconclusive. degradation of the respective mutated rna by nmd was suspected. we developed an approach in order to characterize novel splicing mutations in a simple and non-invasive manner. material and method: isolated blood lymphocytes from patient and his parents were cultured in standard leucocyte medium supplemented with different concentrations of the nmd inhibitors ocadaic acid, anisomycin, and wortmannin for h. cells were harvested and rna was isolated. the reverse transcribed cdna was amplified in allele specific pcrs and qpcr assays. results: compared to the non-stimulated lymphocyte controls nonsense mediated rna decay was inhibited by anisomycin. the consequences of aberrant rna splicing were detectable: the maternal mutation results in exon skipping, the paternal mutation in intron retention. furthermore vascular ehlers-danlos syndrome (type iv) is considered to be an autosomal dominant disorder caused by heterozygous mutations in col a , which are missense or splice site variants in about % of cases. we here report on a three-year-old female of non-consanguineous parents born with bilateral clubfoot as well as dysmorphic facial features, joint laxity, and mild contractures of finger joints. developmental delay became evident. after trauma at years of age she developed brain haemorrhage. mri diagnosis at this age revealed an additional frontal aneurism as well as frontoparietal polymicrogyria. we identified novel compound heterozygous col a mutations: the nonsense mutation c. c>t (p.arg *) and the c. delc (p.pro leufs* ) frameshift mutation leading to a premature stop codon. further studies showed that the mutations were inherited from each parent who had no features for ehlers-danlos syndrome type iv. only two other families have been reported so far with recessive mutations of this gene and a severe vascular phenotype and polymicrogyria. biallelic mutations of col a seem to be accompanied with a significantly worse outcome compared with heterozygous mutations and polymicrogyria is an additional phenotypic feature. here we describe five patients with epidermolytic epidermal nevi in different degrees of severity with the mosaic mutation c. c>t (p.arg cys) in krt gene. the same mutation has previously been described in patients with ei (bygum et al. ) . we analyzed dna from peripheral blood and/or skin biopsies from affected and unaffected skin with next generation sequencing (ngs) and sanger sequencing methods. using ngs we found this mutation in blood in mosaic states ranging from % to %. the mosaic could only be confirmed by sanger sequencing in the patient with the highest mosaic frequency of %. in four of our patients we investigated skin biopsies from affected and unaffected skin. it is noteworthy, that only one of four patients showed the mutation in heterozygous state of % in the affected skin, whereas the other patients presented a mosaic state also in the affected skin. to exclude a recurrent sequencing artefact at this position, we examined control patients for this mosaic mutation using ngs. in none of these patients we found the same dna change. patients with epidermolytic epidermal nevi have a higher risk to have children with a full-blown ei phenotype. our results show the importance of ngs as the method of choice to explore the molecular genetic basis of epidermolytic epidermal nevi. strikingly, all our patients carry the same mosaic mutation c. c>t in krt . we suggest that this position is a hotspot for postzygotic mutations in krt . non-syndromic hearing loss (nshl), with presently around associated genes, is one of the most genetically heterogeneous disorders constituting nearly % of genetic deafness with a predominantly recessive inheritance pattern. thirty percent of hearing loss (hl) can be connected as a part of over distinct syndromes. next generation sequencing (ngs) technologies have revolutionized pathogenic variant identification. different strategies enhance pathogenic variant detection supporting detailed hl investigation to overcome the many ambiguities associated with clinical heterogeneity. detection of the disease causing variant in correlation with the phenotype can be challenging in small families, in situations with ambiguous clinical histories and allelic heterogeneity. using a clinical and whole exome sequencing approach, we tested over probands as part of a multicentre iranian and german genetics of hl study that included probands primarily with sporadic or dominant hl in a parent-child or parent-sibling trio context. the majority of these probands were pre-screened for defects in gjb and strc. libraries were prepared using trusight one and nextera rapid capture exome enrichment and sequenced using the miseq and nextseq desktop sequencers (illumina). analysis was performed using gensearchngs and an inhouse exome analysis pipeline. around % of cases were resolved from phenotype matching and segregation analysis. interestingly, the fraction of resolved cases was much higher in our iranian cohort (> %) compared to our german cohort (> %) which may be attributed in part to increased consanguinity in the iranian families. we observed likely disease causing variants in syndrome-associated genes including eya causing branchio-oto-renal syndrome, a phenotype that was retrospectively confirmed by acquisition of additional clinical information. with few exceptions, we observed a diverse collection of affected genes in probands from our german collected cohort. contrastingly, the iranian cohort revealed frequent mutations in myo a and otof. furthermore, co-segregation of variants in myo and tecta, with expected dominant hl phenotype, was a hindrance overcome by extensive segregation testing. familial locus heterogeneity was also observed by mutations in cib and slc a segregating in different branches of the same extended pedigree. success in the identification of disease causing variants in known hl genes is contingent upon analysis strategy, clinical information and opportunity for segregation testing. the ability to retrospectively connect an already apparent syndromic phenotype to a syndrome-associated gene without prior knowledge is a powerful application of comprehensive analysis that is not restricted to nshl genes. this work provides an improved understanding of population-specific genetic epidemiology of hereditary hl and highlights the challenges in defining genetic causes in a highly heterogeneous disorder such as hl. vere disproportionate microcephaly (- , sd), corneal clouding, myopia, teeth abnormalities and dysmorphism. panel diagnostic by next generation sequencing for primary microcephaly, including all known genes for seckel syndrome, was unremarkable. microarray analysis (affymetrix® cy-toscan hd) revealed a heterozygous kb deletion, spanning the plk gene. this deletion was confirmed by mlpa (multiplex ligation-dependent probe amplification) analysis. subsequent sequence analysis of the plk gene showed a variant of unknown significance on the second allele. in silico analysis of this variant indicated a significant decrease of the relative splice efficiency at the splice donor site. rt-pcr analysis confirmed altered splicing, resulting in a predominant loss of exon of the transcript and predicting truncation of the plk protein. interestingly, a residual wild-type transcript was also detectable in patient rna, implying that this variant effects splicing only partially. by analysis of the parents, the splice variant and the large deletion were proven to be compound heterozygous. discussion: up to now, only a few patients with plk mutations have been described in the literature. the phenotype comprises primary microcephaly, primordial dwarfism and chorioretinopathy (mccrp ). to our knowledge, we describe the first case of a plk heterozygous whole gene deletion and at least partial biallelic inactivation of the gene, therefore expanding the genetic background of this disorder. furthermore, we give a detailed phenotypic description of a further individual with plk alterations. the girl does not show retinopathy so far. while generalised retinopathy was discussed to be one of the most prominent distinctive features between mccrp and primary microcephaly/seckel syndrome, we consider plk rather to be a further candidate gene pointing towards seckel syndrome. additional investigations on centriole function in patient-derived cells are in progress. pathogenic variants of mitochondrial dna cause a wide range of severe congenital disorders with maternal inheritance and a high transmission risk for female carriers. we report on eight families with an index case presenting with the common pathogenic variant m. t>g (p.leu arg) in the mt-atp gene in virtually homoplasmic form. in five families the mutation was detectable in peripheral blood from the mother in heteroplasmic form. in three families with a sporadic case of leigh syndrome the mutation was not detectable in peripheral blood (or urinary or buccal cells) from the mother, possibly indicating a de novo event. furthermore, one family presented with a de novo nonsense mutation in the gene mt-atp , which was present in peripheral blood of the index case in about % and was not detectable in the mother or the unaffected sister. two female carriers with a heteroplasmy level of % asked for prenatal testing. both pregnancies showed an apparently homoplasmic load of the mutation. mutations in lztfl (bbs ) may be associated with a severe renal phenotype huntington's disease (hd) is a rare autosomal dominant neurodegenerative disorder caused by expanded cag repeats as diagnosed via direct dna analysis. for asymptomatic individuals, predictive testing (pt) can facilitate life planning and diminish uncertainty, but it is also associated with substantial social and psychological challenges. we present a prospective case series of counselees seeking predictive hd testing at the huntington centre north-rhine westphalia (bochum, germany) between and . the international protocol including several pre-test sessions was followed throughout. the aim of this study was to prospectively follow the decision-making process of individuals at risk in our centre and explore their experiences following the decision as well as the impacts of mutation test results by means of standardized questionnaires and a semi-standardized telephone interview one year after the initial counselling session. individuals participated in at least one of the three phases of the survey, including individuals for the telephone interview. in our cohort, almost all interviewees reported a balanced emotional state one year after initial counselling, regardless of the decision for or against the test. the most important motivations for a decision in favor of pt were the ability to plan private life and to eliminate uncertainty. the most important motivations against pt were the fear of an increasing risk for others (e. g. offspring) and the fear to obtain an unfavorable htt mutation result, followed by the considered, willful decision for "wanting to not know". furthermore, we identified evidence for gender-specific aspects in decision-making in line with and expanding our previous observations. this study represents one of the few comprehensive prospective evaluations regarding decision-making and coping strategies related to predictive testing for huntington's disease. we submit that gender-related aspects should be heeded in genetic counselling during the predictive testing and counselling processes. our findings could serve as a basis for more extended prospective evaluations with higher numbers of participants and longer follow-up intervals. institute of human genetics, heidelberg, germany, department of conservative dentistry, heidelberg, germany, cegat gmbh, center for genomics and transcriptomics, tübingen, germany background: plk (polo-like kinase ) has been designated as "master regulator" of centriole assembly. complete loss of plk is lethal in mice, whereas biallelic plk mutations with some retained function have been described in a few patients with microcephaly, growth failure and retinopathy (mccrp , omim # ). this is a heterogeneous entity overlapping with mcph (primary microcephaly) and seckel syndrome. during the last years several new genes have been discovered associated with this spectrum. clinical report and genetic findings: we report on a year old female patient with intellectual disability, primordial dwarfism (- , sd), most se-abstracts linked to rare autosomal recessive diseases with poor prognosis. we then compared couples and filtered for variants present in genes overlapping in both partners. putative pathogenic variants were tested for co-segregation in affected fetuses where material was available and in unaffected siblings. out of eleven couples of mediterranean and arabian ancestry (c: , nc: ) and two non-consanguineous couples of european ancestry, we found five cases ( / , %, c: , nc: ) with both parents being heterozygous carriers of rare potentially deleterious variants in one or more overlapping genes. in four of these couples the underlying genetic cause for pre-or early postnatal child death could be established, in two of the families the diagnosis was confirmed by homozygous detection of the parental variant in the available dna of the affected child. in a consanguineous couple with pathogenic variants for a severe autosomal recessive disorder identified in both parents, the molecular diagnosis for their child that had died at months of age could not be established. out of couples in whom no causative diagnosis could be achieved consented to undergo further wes analysis. identified variants are now used for preimplantation and prenatal diagnostics in all four families in which a causative diagnosis was established. our data show that ngs based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects in families that have experienced early child death and/or multiple miscarriages. k. komlósi , s. diederich , d. l. fend-guella , u. zechner , s. schweiger , o. bartsch recently an x-linked syndrome with maternally inherited or de novo mutations in taf was described with global developmental delay, intellectual disability (id), delayed speech, characteristic facial dysmorphology, generalized hypotonia and variable neurologic features (mrxs , mim: # , xlr). there have been only three publications of unrelated families, with single-nucleotide changes and with gene duplications including taf (kaya et al., ; o'rawe et al., ; hu et al., ) . we identified a german family in which two brothers ( and years) showed severe intellectual disability, absent speech and understanding, and hypotonia but different neurologic and behavioral phenotypes. besides severe id the older brother also had postnatal short stature (- sd), a severe lennox-gastaut epilepsy and a neurodegenerative course. the younger brother showed autistic behavior and lost his very limited skills at age to . years. both showed mild dysmorphic features (prominent supraorbital ridges, sagging cheeks, long philtrum, long face, thin upper lip, and high-arched palate), oropharyngeal dysphagia and generalized hypotonia. a gluteal crease with a sacral caudal remnant described as a characteristic feature was not seen in our case, and hearing impairment, microcephaly, dystonic movements or tremor were not observed either. the family history was highly suggestive of x-linked inheritance with an affected maternal uncle, a maternal aunt with multiple miscarriages, and two aunts with learning disability. since a previous analysis of known x-linked mental retardation genes had not revealed the cause in the older brother, we used a targeted ngs approach (mpimg -test: > brain related genes) for the analysis of the younger brother. following enrichment a bp paired end sequencing was carried out on an illumina miseq system with > % of target covered > -fold (hu et al, ) . only taf fitted the x-linked model and the phenotype. the unreported hemizygous sequence variant c. g>a (p.asp asn) in exon of taf was deemed pathogenic. it affected a highly conserved residue in the central "duf " domain, where / previously described mutations had clustered. segregation analysis confirmed hemizygosity in the older brother and heterozygosity in the mother with completely skewed x-inactivation ( : ). gonadism and learning disabilities. because of the delayed onset of symptoms, the diagnosis is often established during late childhood. however, in some cases renal morphological changes detected by ultrasound may resemble those usually seen in autosomal recessive polycystic kidney disease (arpkd). however, histologically bbs-kidneys differ distinctly from other polycystic disorders by cystic orientation, localisation, extension, structure and size. bbs genes have been identified to date. mutations in bbs , bbs , bbs and bbs have been found to cause an antenatal presentation of bbs that may in some aspects mimic meckel gruber syndrome (mks). there is increasing evidence that bbs, at least in some families, shows an oligogenic mode of inheritance with three mutations at two bbs loci. yet, only three patients in two families with bbs caused by mutations in lztfl (bbs ) have been reported. their diagnosis was established in childhood and all patients had mesoaxial polydactyly as a distinct manifestation. in contrast to previous lztfl cases, in our family the diagnosis of arpkd was suspected sonographically at weeks gestation (wg). pregnancy was terminated at wg. autopsy revealed postaxial polydactyly of both hands, enlarged spongy kidneys, hemivertebra t and some features of potter's sequence. histological examination of the kidneys showed multiple, not radially oriented thin walled cysts, internally lined by thickened pas-positive basement membranes and microcystic dilatation of collecting ducts. cystic changes were accentuated in the renal medulla. corticomedullar differentiation was mainly preserved. the tentative diagnosis was bbs. fetal dna was investigated using a next generation sequencing panel which included known bbs causing genes. hereby a heterozygous nonsense variant (np_ . : p.glu *) inherited from the mother and a heterozygous missense variant (p.glu lys) inherited from the father of the lztfl gene were identified. furthermore a maternally inherited heterozygous missense variant of unknown clinical significance in bbs was detected (np_ : p.pro ala). our case shows for the first time that mutations in lztfl can lead to a severe prenatal presentation of bbs due to profound renal manifestations with a kidney histology that is not considerably milder but distinct from that observed in mks. it is not clear to which extent the bbs variant may act as a disease modifier. this may challenge genetic counselling and prenatal diagnosis in a further pregnancy. furthermore our case shows that mesoaxial polydactyly is not always present in bbs patients with lztfl mutations and further studies are necessary to establish the frequency of mesoaxial polydactyly and other genotype phenotype correlations for bbs patients with lztfl mutations. p-cling- *** targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders k. komlósi, s. diederich, d. l. fend-guella, j. winter, o. bartsch, u. zechner, s. schweiger genetic childhood disorders leading to prenatal, neonatal or early childhood death are genetically heterogeneous. many follow autosomal recessive or x-linked modes of inheritance and bear specific challenges for genetic counselling and prenatal diagnostics. parents are carriers but unaffected and diseases are typically very rare but with recurrence risks of % in the same family. often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotype descriptions are insufficient and dna material of affected fetuses or children is not available for later analysis. a genetic diagnosis showing biallelic mutations or mutations on the x-chromosome in male fetuses or children is, however, the requirement for targeted carrier testing in parents, risk calculations, and prenatal and preimplantation diagnostics in further pregnancies. we employed targeted next-generation sequencing (ngs) for carrier screening of autosomal recessive lethal disorders in consanguineous (c) and non-consanguineous (nc) couples with one or more affected children. we searched for heterozygous variants (non-synonymous coding or splice variants as well as cnvs) in parents' dnas in a set of genes r. kropatsch , , j. preine , , jt. epplen , department of human genetics, ruhr university, bochum, germany, charcot-marie-tooth disease (cmt), also commonly called as hereditary motor sensory neuropathy, is the most common monogenetic disease of the peripheral nervous system with significant clinical and genetic heterogeneity. the main clinical manifestations of cmt include progressive distal muscle weakness and atrophy, impaired distal sensation, depressed tendon reflexes and high-arched feet. based upon electrophysiological and histopathological features cmt can be divided into predominantly demyelinating or axonal forms. an intermediate form also exists characterized by evidence of both, demyelinating and axonal, impairments. genetically cmt can be caused by mutations in over genes, including the dnm gene encoding dynamin protein, a large gtpase primarily involved in receptor-mediated endocytosis and membrane trafficking. only a small number of mutations in dnm causing cmt have been described so far. we report the case of a -year-old man presenting with backache, ataxic gait and distal muscle weakness of the lower limb considered to be a consequence of the pre-diagnosed disc prolapse years ago. over the past months bilateral progressive weakness of ankle dorsiflexion, foot drop and tingling paresthesia in stocking distribution have occurred. neurological examination disclosed depressed tendon reflexes of the upper and lower limbs. neurophysiologic investigations revealed an axonal sensible polyneuropathy with normal distal motor latencies and nerve conduction velocities. the sural nerve biopsy indicated single unmyelinated or thinly myelinated axons, loss of myelinated nerve fibers, numerous clusters of regenerating fibers without onion bulb formations suggesting an intermediate form of cmt. by using next generation sequencing (ngs) and a multi-gene panel, consisting of inherited neurological disease-associated genes, we identified a heterozygous missense mutation c. g>a, p.asp asn (rs ) in the dnm gene. this particular mutation is located in a highly conserved nucleotide region encoding the catalytic n-terminal gtpase domain. this evidence suggests a pathogenic phenotype caused by the described mutation, which is being underlined by the following facts: public genome database covering harmless variants of the human genome does not report it. additionally, the exac browser with exome sequencing data of > , unrelated individuals, lists the mutation and shows a low allele frequency of . , corresponding to one known heterozygous mutation carrier. other online prediction tools like the mutation taster, polyphen, sift and provean categorize it as pathogenic. in conclusion, the novel dnm mutation is responsible with high probability for the late-onset form of intermediate cmt of the investigated patient. heterozygous mutations in pcdh cause an x-linked female-limited form of an early infantile epilepsy (juberg-hellman syndrome). the phenotype of this syndrome is variable, ranging from benign focal epilepsy to severe, serial seizures, repeating up to more than times a day for several consecutive days. the intellectual outcome of affected patients ranges from normal to severe intellectual disability. psychiatric disturbances are frequent and manifest as autism, schizophrenia or aggressive behavior. neurological features such as ataxia may also be present. women with triple x syndrome usually show a normal physical development. cognitive deficits in vivo functional modeling of taf has already provided evidence for an effect on a neuronal phenotype. the phenotype in patients can be reminiscent of rett syndrome, but with milder regression and normal movements lacking a specific stereotypic pattern (no hand wringing). while severe neurodegeneration has been described in duplications, the present probands clearly showed developmental regression associated with a missense mutation. the analysis of further family members is pending. our case adds to the phenotypic spectrum of x-linked syndromic mental retardation type . targeted enrichment sequencing was successfully applied to identify greenberg dysplasia as cause of fatal anomalies in one fetus of a dizygotic twin pregnancy. p. m. kroisel , , b. csapo , , m. häusler , , l. michelitsch , , s. verheyen , , p. klaritsch , , k. wagner , institute of human genetics, medical university of graz, graz, austria, department of obstetrics and gynecology, gynecologist and obstetrician, weiz, austria in the first pregnancy of a year old kosovarian woman and her year old husband during the th week of gestation one of her dizygotic twins showed a severe skeletal dysplasia with all long bones extremely shortened and partially bended. the thorax was short and narrow. in addition a ventriculomegaly of the brain and an increased nuchal translucency was noticed. a very bad prognosis was expected and an achondrogenesis was suspected clinically. the other twin appeared to be normal. an amniocentesis was performed to potentially identify the genetic basis of the disorder. qf-pcr to rule out common trisomy's and cytogenetics revealed normal results and following a normal agilent k array cgh analysis as a next diagnostic step next generation sequencing by using the trusight one gene panel focusing on three genes including slc a , trip and col a was performed. since no pathogenic mutation was found by this approach, a more extended bioinformatics study was initiated. by filtering out common variants in the more than genes of the panel in our own database or in the exac-and genome databases our search was extended to genes with rare homozygous or compound heterozygous variants. by this strategy it was possible to reduce the potentially causative gene mutations dramatically and among those remaining genes for known very severe skeletal phenotypes just in the lbr gene the homozygous missense mutation c. a>g, p.asn asp was identified in our fetus. since this particular mutation is already known to be pathogenic leading to the lethal greenberg dysplasia (clayton et al., nucleus. ) the diagnosis could be achieved in the affected fetus of the pregnancy of our patient still before completion of the rd week of gestation. both parents were found to be heterozygous for this mutation in the lbr gene. recently it was shown that different mutations of the very same gene can also lead to less severe forms of bone dysplasia. the couple was informed about our results and possible consequences were discussed and offered. the couple however came to the decision not to draw any consequences. both fetuses especially the affected one were well documented sonographically including in a series of d images. in the th gestational week during a sonographic investigation the affected fetus did not show cardiac function and an oligohydramnios was found. since development of the second non affected fetus was still within the normal range, we hope the now single pregnancy will carry on normal until birth. from our finding we would propose that our chosen strategy is straightforward and can be applied in a wide range of pregnancies to identify various up to severe and fatal single gene disorders associated with sonographic anomalies within a few weeks which should provide substantial benefits for these families. after the working diagnosis ps had been established, molecular analyses regarding the recurrent akt mutation (p.glu lys) were performed by sanger sequencing in available affected tissue specimen of all three individuals. this revealed a high level of mosaic state for the akt mutation c. g>a, (p.glu lys) in affected tissues from bone and in meningiomas. re-evaluation of the ngs data from blood (individual i) confirmed the absence of that mutation in all reads, and no mutation was detected by sanger sequencing in dna from blood in individuals ii and iii. thus, a somatic mosaicism leading to a mild proteus phenotype could be confirmed as the underlying genetic cause in all three affected individuals. in conclusion, mild forms of proteus syndrome caused by the recurrent akt mutation in patients with limited regional involvement may be particularly difficult to diagnose and might be underdiagnosed. distal gne-myopathy: rare differential diagnosis of polyneuropathy here, we report a case of a -year-old patient with presumed polyneuropathy and elevated creatine kinase levels ( - u/l). clinical features included atrophic and bilateral paresis of lower legs of the frontal and rear compartment without high arched foot, while sensibility was not affected. additionally, a myopathic emg in m. tibialis anterior and a slight axonal damage in the motor neurography was detected. due to this overlapping neuromyological phenotype we performed a gene panel including genes associated with neuromuscular diseases using targeted next generation sequencing. gene panel analysis revealed the homozygous mutation c. c>t (p.arg trp) in the gne gene. this mutation is described in the literature as cause of a distal gne-myopathy and was also detected in an affected brother (ck u/l), having consanguineous parents. the current case emphasizes that a large gene panel analysis is recommended in case of an overlapping neuropathological and myopathological phenotype. c. landgraf, g. schmidt, s. morlot, b. schlegelberger, b. auber institute of human genetics, hannover medical school, hanover, germany ehlers-danlos syndrome (eds) is a heterogeneous group of connective tissue disorders. according to the villefranche classification, eds comprises six major types as well as some rare specific entities. one of these has been referred to as "eds, musculocontractural type" (mc-eds), "adducted thumb-clubfoot syndrome" or "eds, kosho type". first described in as an eds vi subtype, it recently was identified to be caused by biallelic changes in either the chst or dse genes, resulting in a loss of dermatan sulfate (ds) biosynthesis. characteristic symptoms are multiple congenital malformations such as contractures (club feet, adducted thumbs), visceral and ocular anomalies, generalized joint laxity, scoliosis, muscular hypotonia, fragile, hyperextensible and bruisable skin, as well as a typical craniofacial appearance. distinctive features include hypertelorism, down-slanting palpebral fissures, bluish sclerae, micro-corneae, short nose with hypoplastic columella and long philtrum, thin upper lip vermillion, small mouth, retrognathia, low-set and posteriorly-rotated ears. the psychomotor development is delayed. (chst ) and (dse) patients have been reported as yet. the patient, a -year-old woman using a wheelchair, had club feet, surgically corrected asd ii, muscular hypotonia, the characteristic face and hyperextensible skin with atrophic scars; particularly visible were those and learning disabilities are more common than in the general population and compared to siblings. their motor skills are likely to be somewhat impaired and coordination problems are frequent. in some patients psychological problems were described. furthermore, eeg abnormalities are occasionally observed, with clinical seizures present in up to % of patients. here we report on a -year-old girl with a ,xxx karyotype and early infantile, intractable epileptic seizures, beginning at the age of months. about three years later, she developed severe, serial seizures often related to febrile infectious diseases. in adolescence, the epileptic symptoms became less intense. she additionally showed autistic features, mental deficiencies, hypermobility of the joints and ataxia. array-cgh, fragile x-analysis as well as sanger sequencing and mlpa of the scn a and mecp genes revealed no additional abnormalities, besides the xxx karyotype. in the pcdh gene the heterozygous missense mutation c. a>g (p.asn ser) was identified, whereby the mutated allele seemed to appear in a : ratio compared to the wild type allele. this mutation has been previously described as disease causing. furthermore, three in silico prediction programs (sift, polyphen- , mutationtaster) classified the mutation as pathogenic. the patient's asymptomatic mother had a normal ,xx karyotype and was not a carrier of the pcdh mutation. pcdh -related epilepsy exhibits an unusual mode of inheritance in which only heterozygous females are affected and hemizygous males are asymptomatic carriers. random x-inactivation in the brain of females with pcdh mutations causes a cellular mosaicism, which likely accounts for the pathogenesis by altering the cell-cell-interactions ("cellular interference"). however, the precise mechanism is still unknown. hypothetically, the wide range of phenotypic expressions may be explained by partially skewed x-inactivation and thereby limitation of the cellular interference. hence, an unequal ratio of mutated to wild type cells should give a milder phenotype compared to the fifty-fifty situation. in contrast to this hypothesis, the phenotype in our patient was rather severe. nevertheless, we cannot exclude that the triple x status contributes additionally to the observed phenotypic expression. proteus syndrome (ps, omim ) is a highly variable disorder with asymmetric and disproportionate overgrowth of the body, connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations, caused by a somatic activating akt mutation. we report on three unrelated individuals (two adults and one year old boy) who showed similar clinical findings that not fulfilled the rigorous clinical criteria for ps (biesecker, ) . beside an asymmetric hyperostosis of the skull or facial bones, all three had an ocular dermoid. two individuals developed alveolar hyperostoses and intracranial calcifying meningiomas. only one individual showed skin changes. all three had normal feet and no vascular lesions. molecular analyses in individual i performed in blood revealed normal results for array karyotyping and no relevant variant in whole exome sequencing (trio approach). introduction: incontinentia pigmenti (ip) is a rare x-linked male lethal genodermatosis that affects the neuroectodermal tissue and is always associated with a bullous rash of the skin along blashko lines in female neonates. it is caused by mutations in ikbkg which encodes the regulatory subunit of the ikb kinase complex required for nf-kb activation. ikbkg has a pseudogene (ikpkgp) with identical exons to . the most frequent ip mutation is a recurrent exon _ deletion due to non-allelic homologous recombination with the pseudogene. here, we report a novel deletion of exons and in the ikbkg transcript recognized by rna analysis. patient: we investigated a yr old girl with typical erythematous rash after birth which resolved within - m. apart from a small hyperpigmented area around the right mammilla there were no skin alterations. she had few conically shaped teeth, normal nail and hair structure, no neurological manifestation and normal intelligence. only clinical sign were repeated vitreous hemorrhage of the left eye from age m. family history was negative. methods: analyses on genomic dna extracted from blood included testing for the common ikbkg exon _ deletion by long range pcr and mlpa (p -a , mrc holland), x inactivation analysis in the androgen receptor gene as described, and massively parallel sequencing (mpseq) of the ikbkg gene (trusightone, nextseq, illumina®; data analysis with nextgene/geneticist assistant [softgenetics®] and seqnext [jsi®]; reference sequence grch , hg ). rna was extracted from cultured blood lymphocytes, and the entire ikbkg transcript (nm_ . , exons) was sanger sequenced on cdna level (the a of the start codon is in exon ); the results were analyzed with sequencepilot (jsi). result: genomic dna analyses including mlpa of ikbkg and ikbkgp specific probes in our patient did not reveal a putative mutation. there was a completely skewed x-inactivation pattern. cdna sequencing of ikbkg demonstrated skipping of exon and (r. _ del) which is predicted to cause a frame shift starting from codon p.gln , a premature stop codon amino acids downstream (p.gln glyfs* ) and complete loss of protein function. the loss of exons and is most likely due to an intronic splice variant in intron ; investigations regarding the origin of this deletion are ongoing. conclusion: the presence of the high homologous pseudogene makes sequence analysis of ikbkg challenging. we report a deletion of exons and in the ikbkg transcript that required rna analysis for its identification. due to the skewed x-inactivation and typical clinical picture causality of the detected deletion is certain. the exact genomic cause of this alteration remains to be clarified. also in the era of mpseq, rna analysis may be necessary for detection of deep intronic mutations or the study of genes with homologous pseudogenes, as shown here in the case of ikbkg. primary congenital glaucoma (pcg) and early onset glaucomas are one of the major causes of of blindness in children and young adults' worldwide. both autosomal recessive and dominant inheritance have been described resulting from bowel surgeries due to colon perforation. her older sister who died aged following an acute abdomen had club feet and the typical facial appearance, while three healthy sisters seem to be unaffected. her parents are first cousins of turkish origin and do not show eds symptoms either. the two affected sisters had been diagnosed with a syndromic disorder that could represent a rare form of eds. however, neither a confirmation of the suspected diagnosis nor a classification had yet been achieved. due to the distinctive symptom complex and the presumed autosomal recessive inheritance pattern, we strongly suspected this to be a case of mc-eds. sequencing of the chst gene (reference sequence: lrg_ ) revealed a formerly undescribed homozygous variant (c. c>t; p.pro- leu). the variant changes the highly conserved pro residue which is located in the critical '-phospho- '-adenylyl sulfate binding site and can be classified as likely pathogenic (acmg standards and guidelines, richards et al., genetics in medicine ) . the parents are heterozygous carriers of this variant, respectively. this case represents a unique entity within the umbrella term eds and illustrates the importance of clinical assessment leading to a diagnosis confirmed by genetic analysis. the underlying genetic defect in patients with mitochondrial peo is either a primary mutation of the mitochondrial genome (single, large-scale mtdna deletion or mtdna point mutation) or recessively and dominantly-inherited mutations in nuclear genes involved in mtdna maintenance leading to clonally-expanded multiple mtdna deletions in muscle. the nuclear disease genes are largely implicated in the replication and stability of mtdna, and as such a pathogenic mutation leads to secondary instability of the mitochondrial genome. causal mtdna deletions can be found in a heteroplasmic (mixture of mutated and wild type mtdna) state. however, each tissue/cell has its own biochemical threshold of mutant mtdna load which needs to be exceeded before focal respiratory chain deficiency becomes evident. to investigate this, muscle biopsies of patients with genetically-and clinically-characterized mitochondrial disease of nuclear origin ( polg, twnk, rrm b and slc a (ant )) and healthy controls were analysed using quadruple oxphos immunohistochemistry, quantifying the biochemical phenotype in individual muscle fibres of patient muscle biopsies. this technique is based on quadruple immunofluorescence to detect structural components of complexes i (ndufb ) and iv (coxi), as well as porin (a marker of mitochondrial mass) and laminin (a cell membrane marker to define the boundaries of muscle fibres). further studies on / patients ( polg, rrm b, twnk, slc a (ant )) included the correlation of the biochemical deficiency with the mtdna abnormality in individual cells, following laser microcapture and determination of the size and level of clonally-expanded mtdna deletion within fibres by real-time pcr. our preliminary data from quadruple immunocytochemical studies show that the muscle biochemical phenotype is different in patients with multiple mtdna deletions compared to other mtdna mutations; work is continuing to determine the exact size and level of clonally-expanded mtdna deletion in individual muscle fibres and correlate this with the observed biochemical defects and disease thresholds. abstracts these families, and functional studies, together with phenotype descriptions in the literature, are essential for pathogenic grading. however, several difficulties remain, such as the huge size of the ttn gene (> kb) impeding functional studies, the wide spectrum of phenotypes and variants, the still small patient cohort, and often unspecific immunohistochemical abnormalities in muscle biopsies. the clinical evaluation of ttn variants thus presents a great challenge to the field of human genetics diagnostics. compound heterozygous variants in the qars gene (omim ) have been identified in only four patients with autosomal recessive progressive microcephaly with seizures and cerebral and cerebellar atrophy (mscca), to date. these patients showed severe developmental delay, progressive primary microcephaly, intractable seizures, hypomyelination or delayed myelination, thin corpus callosum, and small cerebellar vermis on brain imaging. here we report on two unrelated girls with progressive primary microcephaly, epilepsy and brain anomalies. trio exome analysis in each of the families revealed two different combinations of compound heterozygous variants in qars. all four variants are highly conserved throughout vertebrates, not reported in any database, yet, and in silico analysis predicted the variants as possibly damaging or deleterious. the first patient was born to non-consanguineous german parents. at birth, she was too short (− . sd) and mildly microcephalic (− . sd). she developed intractable seizures within the first hour of life. her growth continued to be mildly retarded (− . sd at age years) but microcephaly was progressive (− . sd at age years). she did not achieve any of the motor or cognitive developmental milestones, she did not have eye contact. the only interaction with her surrounding was a diffuse reaction to being touched. cranial mri showed no myelination of the supratentorial region, corpus callosum agenesis, simplified gyral pattern of frontal lobes, enlarged cerebral ventricles, and normal brain stem and cerebellum. trio-exome sequencing revealed the compound heterozygous qars variants c. c>t, p.(arg- cys) and c. c>t, p.(arg *). segregation analysis by sanger sequencing confirmed the heterozygous variants in the parents and two non affected sibling of the index patient. the second patient was initially evaluated at days of age when she exhibited myoclonic seizures, intrauterine growth retardation, microcephaly, and elevated lactic acid. at birth, she was microcephalic (hc cm) and microcephaly was progressive (− . sd at age months). cranial mri suggested undersulcation. she has required a gastrostomy feeding tube. trio-exome sequencing revealed the compound heterozygous qars variants c. g>a, p.(gly ser) and c. c>t, p.(arg trp). segregation was confirmed by sanger sequencing analysis. together with the four previously described patients we conclude that compound heterozygous variants in qars are associated with a primary and progressive microcephaly, early onset of intractable seizures and severe developmental delay. brain imaging in the neonate can show simplified gyral pattern as an early characteristic feature. overlapping phenotypes are seen in patients with epileptic encephalopathy, lissencephaly and primary microcephaly. application of ngs panels or exome technology will allow for early diagnosis and further collection of patients for better delineation of the phenotype. with involvement of several genes including cyp b , foxc , pitx , myoc and pax . however, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. to elucidate further genetic causes of these conditions we performed whole exome sequencing in a patient with pcg and retinal detachment and identified compound heterozygous variants in col a (p.met leu; p.ala- thr). targeted col a screening of additional patients detected three further heterozygous variants (p.arg *, p.gly ser and p.gly- val) in three distinct subjects: two of them were diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (oi), one patient had a diagnosis of pcg at age years. all five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main col a binding proteins: hsp and fibronectin. dominantly inherited mutations in col a are known causes of connective tissues disorders such as oi. these disorders are also associated with different ocular abnormalities, although the common pathology for both features is seldom recognized. our findings expand the role of col a mutations in different forms of early-onset glaucoma with and without signs of oi. thus, we suggest including col a mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with oi. the gene ttn encodes the largest known protein, titin, which plays a key role in structural, mechanical, developmental and regulatory functions of cardiac and skeletal muscles. accordingly, titinopathies are characterized by great clinical and genetic heterogeneity. the clinical spectrum ranges from severe phenotypes with cardiac involvement to pure myopathies at the milder end, including autosomal recessive and dominant inheritance patterns (chauveau et al. , hum mutat; : ) . next generation sequencing analysis identifies a large number of variants of unknown clinical significance; the potential clinical relevance of these variants cannot be assessed with certainty without further studies. three case reports highlight the difficulties in human genetics diagnostics concerning ttn. the first case is of a year-old woman with proximal muscle weakness, slightly elevated ck, scoliosis, and no family history. a heterozygous known pathogenic variant was identified in mex , associated with autosomal recessive congenital core myopathy combined with primary heart disease. additionally, an unknown variant was detected. both variants could be clinically relevant with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the second case is of a month-old finnish girl who presented with severe muscle hypotonia at birth and mental alertness with normal brain mri and eeg. congenital fiber-type disproportion was suspected. a homozygous frame-shift mutation in mex was identified which to our knowledge has not yet been described in the literature. this variant is likely of clinical relevance with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the third case is that of a year-old woman with suspected myofibrillar myopathy. a known pathogenic homozygous frame-shift mutation in mex was detected which is associated with autosomal recessive congenital myopathy with central nuclei. segregation analysis revealed that the healthy parents are heterozygous carriers of this variant. the clinical diagnosis of a ttn-associated disease could therefore be confirmed. ttn variants need to be assessed in combination with detailed clinical and muscle biopsy data. segregation analysis is necessary but not sufficient for the clinical grading of variants. identification of a variant in several independent families, segregation of the variant with disease phenotype in gous pathogenic smad variant c. dupt;p.(ala fs) (ncbi reference sequence nm_ . ). gastric as well as colonic cancer and polyposis was present in the paternal family history. conclusions: ts and other imprinting disorders are likely underdiagnosed, as the main clinical features (e. g. growth retardation, hypotonia) are distinct but unspecific. as exome sequencing becomes a more frequent diagnostic procedure, imprinting disorders caused by mutations in imprinting centers will presumably be diagnosed more often. methylation defects, however, will remain underdiagnosed, without a specific clinical differential diagnosis, which would guide to appropriate analysis of the methylation status. a bowel invagination in early childhood due to a single polyp can be a symptom of jps, especially in the context of a paternal history of polyposis and intestinal cancer; thus, family history should be carefully obtained. in the outpatient clinic, child psychiatrists as well as neurologists thoroughly work up phelan-mcdermid patients according to a standardized protocol by taking medical history, performing physical examination, and, if needed, organizing further supplementary examinations. in addition, a genetic analysis and hair/tissue sampling is performed. since its foundation, a steadily increasing number of so far patients from all over germany has been seen and treated. the outpatient clinic aims at facilitating and accelerating the diagnosis of phelan-mcdermid syndrome, improving medical support for affected patients of all ages, and, last but not least, fostering a better understanding of the causes and pathomechanisms leading to the symptoms of the disease. t. m. neuhann, l. neuhann, c. rapp, a. laner, a. benet-pages, e. holinski-feder medizinisch genetisches zentrum, münchen, germany congenital eye malformations, such as the microphthalmia-anophthalmia-coloboma (mac) spectrum, congenital cataracts, anterior segment dysgenesis (asd), and congenital glaucoma, affect more than : . newborns. the phenotypic spectrum of the aforementioned entities is highly variable and partially overlapping. eye malfomations are very heterogeneous; to date causative mutations have been described in more than next-generation-sequencing (ngs) technology has revolutionized genomic research and has transformed clinical diagnostics. ngs offers enormous potential for providing accurate diagnoses to individuals with previously unresolved syndromes. in the pediatric endocrine clinic, clinicians are often faced with the task of making a diagnosis in children with syndromic short stature. as there may be considerable clinical overlap between short stature syndromes, deriving a clinical diagnosis may prove challenging. furthermore, even if a clinical differential diagnosis is established, often several genes would need to be tested before a molecular diagnosis is made. as access to genetic testing is limited in algeria, we conducted a pilot study on algerian patients with syndromic short stature using a combination of two different ngs modalities, namely whole-exome-sequencing (wes) and mendeliome sequencing (trusight one sequencing panel). a molecular diagnosis could be established in / patients, making the diagnostic rate in this initial cohort %. as patients had novel mutations we could expand the mutational spectra of several genes, namely cul , npr , sos , vps b, and znf . we could thus substantiate the clinical utility of wes and the mendeliome in patients with a diverse array of syndromic short stature syndromes. chromosome harbours an imprinted locus at q . maternal uniparental disomy of chromosome , paternal deletions and paternal loss of methylation at the intergenic differentially methylated region (ig-dmr) and the somatic dmr within meg are associated with temple syndrome (ts , mim ). the phenotype of ts consists of pre-and postnatal growth retardation, early feeding problems and muscular hypotonia, joint laxity, motor developmental delay, premature puberty, and truncal obesity. juvenile polyposis syndrome (jps, mim ) is characterized by predisposition to hamartomatous polyps in the gastrointestinal (gi) tract, specifically in the stomach, small intestine, colon, and rectum, including the risk for gastrointestinal cancer. pathogenic variants in the bmpr a and smad gene are identified in about - % of affected families. we report on a family with two female children. the index patient, an -year-old girl, was diagnosed to have ts due to hypomethylation at the somatic dmr within meg with clinical features reminiscent of prader-willi syndrome in early childhood and milder clinical signs at further age (i. e. mild global development delay, muscular hypotonia, suspected central obesity, no prominent facial dysmorphisms). snp array-cgh analysis was unsuspicious and no deletion of the imprinting center was observed. thus, ts is caused by a sporadic imprinting defect in our patient. her -year-old sister was diagnosed with smad -associated jps after an episode of intestinal invagination due to a polyp, histologically diagnosed as peutz-jeghers polyp, in early infancy. sequencing identified a heterozy-abstracts coding vmat have only very recently been described as causal for brain dopamine-serotonin vesicular transport disease in two families with multiple affected children (rilstone et al., n engl j med , , - ; jacobsen et al., j inherit metab dis , , - ) . the index case presented here is a -year-old girl with severe mental retardation and a dystonic movement disorder. she is the tenth child of a consanguineous arabic couple and was initially referred to neuropaediatric examination at the age of four months due to recurrent oculogyric crises and muscular hypotonia. blood metabolic testing and cerebrospinal fluid (csf) analyses were inconclusive. notably, biogenic amines were within their normal ranges and the differential diagnosis of aromatic l-amino acid decarboxylase (aadc) deficiency could not be confirmed. conventional cytogenetics, subtelomeric screening, array-cgh and different ngs panel analyses did not identify a causative mutation. both parents and all eight living siblings are obviously unaffected. a brother with a known hypotonic movement disorder died at the age of three years due to prolonged seizures with hyperthermia and cerebral edema. by utilizing whole-exome sequencing, we identified a homozygous substitution in the slc a gene of the index case causing an amino acid change (c. c>a; p.pro his) in a conserved transmembrane domain of vesicular monoamine transporter (vmat ). homozygosity for this missense change could also be verified in a dna sample of her deceased brother. an obvious reduction in frequency of oculogyric crises was observed in our index case under therapy with pramipexole already within weeks after start of treatment. furthermore the patient shows less dystonic movements under therapy. the case presented here highlights the importance of considering brain dopamine-serotonin vesicular transport disease as differential diagnosis for early-onset extrapyramidal movement disorders combined with mental retardation even if neurotransmitters in csf are normal. for a large number of individuals with intellectual disability (id), the molecular basis of the disorder is still unknown. however, whole exome sequencing (wes) is providing more and more insights into the genetic landscape of id. in the present study, we performed trio-based wes in patients with unsolved id and additional clinical features, and identified homozygous cplx mutations in three patients with id from two unrelated families. all displayed marked developmental delay and migrating myoclonic epilepsy, and one showed a cerebellar cleft in addition. the encoded protein, complexin , is crucially involved in neuronal synaptic regulation, and homozygous cplx knockout mice have the earliest known onset of ataxia seen in a mouse model. recently, a homozygous truncating mutation in cplx was suggested to be causative for migrating epilepsy and structural brain abnormalities. id was not reported. the currently limited knowledge on cplx suggests that complete loss of complexin function may lead to a complex but variable clinical phenotype, and our findings encourage further investigations of cplx in patients with id, developmental delay and myoclonic epilepsy to unravel the phenotypic spectrum of carriers of biallelic cplx mutations. genes. due their heterogeneity, diagnostic testing for congenital eye malformations was limited in the pre-ngs era. we performed exome analysis in patients with congenital eye malformation (mac spectrum, asd, congenital cataract, congenital glaucoma). primarily, a gene panel comprising genes associated with eye malformations was evaluated. additionally the exome data was evaluated in selected patients as a second step. the panel analysis revealed pathogenic sequence variants in patients and genes (mab l , bcor, nhs, prss , cyp b , foxc , pitx , gcnt ). putatively causative sequence variants were identified additional patients. the diagnostic yield of the panel was highest in patients with non-syndromic microphthalmia/coloboma and congenital cataracts, and lowest in patients with syndromic mac spectrum (i. e. additional systemic features/malformations). ngs based panel testing is a strong diagnostic tool to determine the underlying causes of non-syndromic congenital eye malformations. due to the partially overlapping phenotypes and high heterogeneity it is more sensible to perform large gene panel analysis, as opposed to smaller single phenotype based panels. superactivity of phosphoribosyl¬pyrophosphate synthetase i (prpps) is a rare inborn error of purine metabolism that is characterized by increased levels of uric acid in blood and urine (omim ). the disorder is caused by gain-of-function mutations in the x-chromosomal gene prps . in male patients, disease manifestation is in early childhood. additional clinical characteristics include intellectual disability, hypotonia, ataxia and hearing loss. heterozygous female mutation carriers have a later age of onset and a less severe clinical course. only seven families with prps gainof-function mutations have been reported to date. we report on a -year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias and facial dysmorphisms. his mother also had hyperuricemia that was diagnosed at age years but was otherwise healthy. a novel prps missense mutation (c. g>c, p. leu phe) was detected in the proband and his mother. enzyme activity analyses confirmed superactivity of prpp synthetase. the family reported here broadens the clinical spectrum of prpps superactivity and indicates that this rare metabolic disorder is associated with a recognizable facial gestalt. homozygous and compound heterozygous mutations of the rnu at-ac gene are associated with mopd and roifman syndrome. mopd is characterized by severe microcephaly with brain malformations including abnormal gyral pattern, corpus callosum agenesis or hypoplasia, vermis hypoplasia and intracranial cysts, psychomotor retardation, short stature, skeletal dysplasia, dry skin, sparse hair, flexion contractures, round face with beaked nose and protruding eyes, and premature death with a majority of the patients who die before the age of months for unknown reasons. roifman syndrome was first described as a novel association of antibody deficiency, spondyloepiphyseal chondro-osseus dysplasia, retinal dystrophy, poor pre-and postnatal growth, cognitive delay and facial dysmorphism including long eyelashes, downslanting palpebral fissures, a long philtrum and a thin upper lip. all patients with roifman syndrome reported so far lack brain malformations. the rnu atac gene encodes a small nuclear rna (snrna), which is essential for minor intron splicing. homozygous (g. g>a, g. g>a) and compound heterozygous mutations (g. g>a;g. g>a, g. g>a;g g>a and g. c>t;g. g>a) have been described in mopd . all mutations involve the ' or ' stem loop of the u atac snrna. in contrast, all cases with roifman syndrome investigated so far showed compound heterozygous rnu atac mutations with one allele harboring a mutation in the mopd associated ' stem loop and the other allele showing a mutation in the stem ii site of the u atac snrna, which has not been involved in mopd , so far. thus, the different pattern of the mutations observed in mopd and roifman syndrome may contribute to the distinct features of both syndromes. however, our patient shows, that features of mopd , i. e., brain malformations, may also be present in patients who show roifman syndrome associated rnu atac mutations. this indicates that both syndromes may represent overlapping features of the clinical spectrum of rnu atac mutations. h. roth, h. stöhr, b. h. f. weber institute of human genetics, university of regensburg, germany introduction: inherited retinal degenerations comprise a genetically heterogeneous group of eye diseases with overlapping clinical presentations. up to now, more than genes have been associated with different forms of retinal dystrophies (rd) such as retinitis pigmentosa (rp) or cone-rod-dystrophies (crd) with mutations in and causative genes, respectively. here, we present the results from three patients with remarkable findings and discuss their implications for risk prediction and genetic counseling. methods: targeted next-generation sequencing (ngs) technology based on agilent custom designed gene panels (sureselect) has been established in our diagnostics department to identify causative mutations in a large patient cohort with approximately rd patients. high-throughput sequencing data are routinely analyzed with the clc biomedical workbench. classification of variants was based on bioinformatic analyses using alamut visual software, mutationtaster, sift and polyphen- prediction programs, allele frequencies, amino acid conservation and literature. results. ngs analysis revealed two patients with rp and one patient with crd, each of whom carry putative causative mutations in several rd genes. first, a male patient with a family history of crd, is a carrier of a nonsense mutation p.(arg ter) in rims and two likely pathogenic missense mutations in aipl (p.(tyr phe)) and guca a (p.(pro- leu)), each in a heterozygous situation. mutations in all three genes can cause adcrd. in addition, the patient carried a hemizygous nonsense mutation p.(glu *) in the x-chromosomal rpgr gene. secondly, a female patient with simplex rp was found to be homozygous for a frameshift-causing deletion p.(ser leufs* ) in the impg gene causing arrp. she also carried three heterozygous, likely pathogenic missense mutations in crx (p.(tyr cys)) causing adrp, in the x-chromosomal rpgr (p.(ala val)) and in ush a (p.(ile val)) associated m. s. reuter , a. riess , u. moog , t. a. briggs , , k. e. chandler background: disruptions of the foxp gene, encoding a forkhead transcription factor, are the first known monogenic cause of a speech and language disorder. so far, mainly chromosomal rearrangements such as translocations or larger deletions affecting foxp have been reported. intragenic deletions or convincingly pathogenic point mutations in foxp have up to date only been reported in three families. we thus aimed at a further characterization of the mutational and clinical spectrum. methods: chromosomal microarray testing, trio exome sequencing, multi gene panel sequencing and targeted sequencing of foxp were performed in individuals with variable developmental disorders, and speech and language deficits. results: we identified four different truncating mutations, two novel missense mutations within the forkhead domain and an intragenic deletion in foxp in fourteen individuals from eight unrelated families. mutations occurred de novo in four families and were inherited from an affected parent in the other four. all index patients presented with various manifestations of language and speech impairment. apart from two individuals with normal onset of speech, age of first words was between and years. articulation difficulties such as slurred speech, dyspraxia, stuttering or poor pronunciation were frequently noted. motor development was normal or only mildly delayed. mild cognitive impairment was reported for most individuals. conclusion: by identifying intragenic deletions or mutations in fourteen individuals from eight unrelated families with variable developmental delay/cognitive impairment and speech and language deficits, we considerably broaden the mutational and clinical spectrum associated with aberrations in foxp . h. rieder, f. beleggia, d. wieczorek we report on a -year-old boy with microcephaly, arachnoidal cysts, pachygyria, microgyria, and severe intellectual disability. he also had short stature including shortening and deformation of the femora, brachydactyly, and short ribs with costochondral dysplasia. he showed facial dysmorphism with narrow palpebral fissures, a short nose with a depressed nasal bridge, and a broad mouth with full lips. clinical laboratory investigations demonstrated persistently slightly elevated liver enzymes. exome sequencing revealed compound heterozygous mutations of the rnu at-ac gene, g. g>a;g. g>a, which has been described in an individual with roifman syndrome. we report on a seven-year-old girl, first child of non-consanguineous italian parents, with developmental delay, muscular hypotonia and distinctive craniofacial features (epicanthus inversus, ptosis, broad nasal bridge, mild retrognathia, low-set posteriorly rotated ears and malpositioned teeth in the mandible). because of the tentative diagnosis of blepharophimosis-ptosis-epicanthus inversus syndrome (bpes), conventional cytogenetic analysis, sanger sequencing and mlpa (multiplex ligation-dependent amplification) of foxl were initiated and showed unremarkable results. microarray-cgh revealed a kb microduplication of genetic material on q . : arr[hg ] q . ( _ )x encompassing the genes tubgcp , cyfip , nipa and nipa of maternal origin. patients with q . microduplication have been described to be affected by developmental delay, motor and/or expressive language delay, epilepsy, learning disabilities and/or behavioral problems. however, genotype phenotype correlation is complicated by incomplete penetrance. healthy and mildly affected carriers are reported in the literature. we speculate that the microduplication might contribute but does not fully explain the phenotype of our patient, in particular concerning the craniofacial features. subsequent trio whole-exome sequencing identified a de novo heterozygous mutation in setbp (c. t>a/ p.tyr *) leading to a premature stop codon and most probably resulting in a truncated and functionally impaired protein. mutations in the set binding protein gene (setbp ) on q . have been identified to cause schinzel-giedion syndrome (sgs, omim ), a rare autosomal dominant disorder characterized by postnatal growth failure, severe developmental delay, seizures, facial dysmorphism, genitourinary, skeletal, neurological, and cardiac defects. chromosomal deletions in q including setbp have been reported to cause a milder phenotype known as "autosomal dominant mental retardation- " (mrd , omim ). these observations suggest that the severe sgs phenotype might be the consequence of a gain-of-function or dominant-negative effect of the mutations and that setbp haploinsufficiency results in a different, milder phenotype. so far, the function of the set-bp protein is unknown. the presented case adds up to the yet small number of reported cases of mrd and thereby contributes to the clinical spectrum of setbp haploinsufficiency. this work was supported by "förderstiftung des uksh" (project number: _ ). the demand for genetic counseling had been constant, in germany, over many years. from to around . cases per year on the average and with minor fluctuations were reimbursed by the german sickness funds (public health insurance system; pabst and schmidtke, gendiagnostik in deutschland, bbaw, p. - , ) . in connection with the "genbin "-project, a new nationwide survey was initiated regarding with arrp. finally, in another female rp patient with no family history of rd, we detected a nonsense mutation p.(trp ter) and a likely pathogenic splice site change (c. + a>g) in the arrp gene eys, assuming compound heterozygosity. in this patient we also identified two heterozygous, likely pathogenic missense mutations in hmcn (p.(pro thr)) and cep (p.(arg cys)) underlying dominant and recessive forms of rd. in all three cases, specific mutation(s) could not be uniquely identified as causative. conclusion. results in the three rd cases emphasize that ngs can generate unexpected results that are difficult to interpret, particularly in the absence of segregation analysis and functional data on pathogenicity. the implications for genetic counselling and predictive testing will be discussed. smart qnipt study -detection of fetal trisomy based on methylation-specific quantitative real-time pcr m. sachse, s. werler, j. bonnet, u. neder, h. sperling, s. busche, s. grömminger, w. hofmann lifecodexx ag, konstanz, germany objectives: current non-invasive prenatal testing (nipt) methods for the detection of fetal trisomy (t ) are primarily based on next generation sequencing (ngs) strategies which are quite costly in clinical application and hence are limited to patients who can afford the testing. here, we describe the results of a blinded study with respect to the test accuracy of a newly developed nipt assay based on quantitative real-time pcr (qpcr) for prenatal testing of fetal trisomy (qnipt). methods: in the study maternal plasma samples were collected from , pregnant women and blinded by an independent contract research organization. after extraction of cell-free dna using qiasymphony instrument and methylation-specific digestion of dna samples a multiplex qpcr was performed. the primary qpcr data were finally evaluated with our ce marked data analysis software. results from this analysis and from confirmatory ngs testing were compared with nipt results using ngs. the study results of successfully analysed maternal plasma samples (n = ) demonstrated a positive percentage agreement (ppa; equates to sensitivity) of % (lower -sided % confidence interval of . %; n = / ) and a negative percentage agreement (npa; equates to specificity; n = / ) of % compared to ngs-based results. the negative predictive value (npv) for the novel qnipt and confirmatory ngs testing was % (lower -sided % confidence interval of . %). the average fetal fraction of the examined blood samples was . %. the qnipt assay provided reliable test results in blood samples with a fetal fraction below % and as low as . %. conclusion: our results suggest that the proprietary qnipt assay is a very reliable and robust method suitable for clinical routine in accordance with international medical associations. the assay represents a more cost-efficient solution over ngs testing and will also be able to provide results in the shortest possible time. while current nipt methods require a minimum fetal fraction of % in blood samples from singleton pregnancies, we could demonstrate in the study that our smart qnipt assay can be employed on blood samples with a fetal fraction of as low as . %. in summary, the application of smart qnipt could have the potential to become a nipt solution on a global scale for pregnant women of all ages and risk groups. further studies which aim to include the determination of trisomy and trisomy are currently underway. with respect to the developmental delay of our index patient, chromosome analysis and array-cgh were performed. a microduplication in p . (app. kb) of unknown significance and a microduplication in xq . (app. kb), which comprises the fmr -gene, were identified and shown to be of maternal origin (arr[hg ] p . ( , , , )x , xq . ( , , , )x ). fmr is associated with fragile x syndrome, which is one of the most common causes for x-linked mental retardation. cgg-trinucleotide repeat expansions in the ' untranslated region (> repeats) lead to aberrant hypermethylation of the fmr -promotor and silencing of fmr expression. in contrast, premutations ( - repeats) lead to a higher expression of fmr and cause a clinical syndrome that is characterised by late progressive cerebellar ataxia (fxtas). in line with this gain-of-function mechanism, we hypothesize that the xq . duplication, which could lead to an increased gene dosage of fmr , causes a fra(x)-/fxtas-like syndrome and explain the clinical findings in our family. vengoechea et al. described a patient with a similar duplication, who was affected by developmental retardation, epilepsy and hyperactivity. they discussed the microduplication, which arose de novo in their patient, as the cause for the boy's symptoms (vengoechea j. et al., eur j hum genet., nov; ( ): - ) . in conclusion, we assume a fmr -duplication syndrome in our family with variable expressivity and a different impact on male and female patients. to further prove this hypothesis, we are planning to perform a segregation-analysis within the whole family. background: congenital myasthenic syndromes (cms) are a genetically heterogenous group of disorders leading to weakness of skeletal muscles -especially ocular, bulbar and limb muscles -with onset mostly at birth or in early childhood. the severity of cms can vary significantly ranging from death in early childhood due to respiratory insufficiency to only mild muscle weakness in adulthood. more than genes that are highly expressed in the neuromuscular junctions are associated with cms. mutations in the chrne gene on chromosome p . are responsible for about one half of genetically solved cms cases. they can cause different subtypes of cms with either autosomal dominant or autosomal recessive inheritance. results: here, we report a -year-old boy who was born with bilateral eyelid ptosis and congenital vertical talus of the right foot that needed surgical correction. the boy displayed muscular hypotonia with a myopathic facial expression and delayed motor development. ophthalmologic examination revealed external ophthalmoplegia. a next generation sequencing based gene panel for congenital myopathies detected the homozygous frameshift mutation c. _ dup (p.leu profs* ) in the chrne gene in the boy. gene dosage analysis did not show an exonic deletion in the chrne gene. sanger sequencing confirmed the mutation in a heterozygous state in the boy's father. however, his mother did not carry the mutation in the chrne gene. conclusions: these results suggest the rare event of a (partial) paternal uniparental isodisomy of chromosome as cause of the homozygous c. _ dup (p.leu profs* ) in the chrne gene in the boy. further experiments are currently undertaken to confirm this hypothesis. the annual reimbursement frequencies of the relevant entries in the ebm fee schedule, , , and , for which only specialists in human genetics and subspecialists in medical genetics can account, from until . contrary to the findings in the earlier period the demand for genetic counseling has risen sharply: , (of a total of , ) cases in ; , ( , ) in ; , ( , ) in ; , ( , ) in ; , ( , ) in ; and , ( , ) in . we speculate that the temporal correlation of the rise of genetic counseling demand with the enactment of the german act on testing (february , ) is not coincidental. further factors that might contribute to the increase in demand are the ensuing guidelines of the german commission on genetic testing and cme activities related to attaining a qualification for genetic counseling for specialties other than human genetics. in the course of these activities the awareness for the importance of genetic counseling delivered by specialists in human genetics and subspecialists in medical genetics may have risen. acknowledgements: the "genbin "-project supported by the robert koch-institute through funding from the german federal ministry of health. we gratefully acknowledge the collaboration with dr. michael erhart, zentralinstitut der kassenärztlichen bundesvereinigung (zi-kbv), berlin, germany it is well known that duplications of down syndrome critical region (dscr) on chromosome q can cause down syndrome whereby the distinct phenotype is associated with the involved genes and the size of duplication. however, in literature are hardly any cases with mosaic duplications of dscr described. here we report on a year old boy with some clinical features of down syndrome including distinctive craniofacial dysmorphism, simian crease and sandal gap as well as delayed motor and speech development. no other organ abnormalities are known. conventional chromosome analysis showed no numerical or structural aberration whereas interphase fish analysis revealed three signals for dscr in approx. % of lymphocytes and in approx. % of buccal mucosa cells. array-cgh analysis on dna from peripheral blood confirmed a , mb duplication of chromosome q . q . . the duplication involves among others the gene dyrk a which is reported as a candidate gene for down syndrome. this case presents one of the smallest known duplications within dscr which causes even in a mosaic state a mild phenotype of down syndrome. -year-old boy was referred to our outpatient clinic due to global developmental delay mainly affecting his speech and his fine motor development. in addition, muscular hypotonia and an abnormal gait were reported by the referring paediatrician. his mother, his maternal grandmother, and numerous relatives are affected by gait ataxia. no causative mutation was detected in the maternal grandmother by means of a multi-gene panel for spinocerebellar ataxia encompassing genes. genetic testing for friedreich ataxia was also without pathological findings. cer, but the patient mother's grandfather had a cancer of unknown origin and died at the age of years. because of the suspicion of having a lynch syndrome an immunohistochemistry and microsatellite analysis have been performed on the tissue of the colorectal cancer and the hepatic metastasis. all four mmr proteins were properly expressed in immunohistochemistry in colorectal cancer. just the expression of mlh protein in the hepatic metastasis was focally weakened and inhomogeneous. the microsatellite markers bat , bat , d s (apc), d s and d s (mfd) were all stable, a a t-kras mutation was found. after performing a multi-gene panel (ngs, next-generation sequencing), a gross heterozygous deletion of exon in msh gene has been found in the cnv analysis of the ngs data. this mutation was confirmed with a mlpa and quantitative real time pcr analyses. furthermore, rna expression of msh was reduced to % in blood lymphocytes in comparison to control samples pointing to a potential role of msh loss in the patient's tumor development. we are observing more and more patients with probably pathogenic and pathogenic mutations in one of the mmr genes with normal immunohistochemistry and microsatellite analysis. therefore, we propose that the criteria for performing a molecular genetic analysis of hnpcc/lynch syndrome should be revised. exome sequencing reveals gata mutation in a patient with partial delta-storage pool deficiency and mild thrombocytopenia objectives: we report about a -year-old male patient of russian background with severe and frequent epistaxis and hematoma since infancy. he presented with mild thrombocytopenia and increased mean platelet volume. von willebrand's disease and subhemophilia had been excluded. previously, he was diagnosed with immune thrombocytopenic purpura. he never underwent elective surgery. his parents were asymptomatic. however, his -year-old daughter also suffers from severe bleeding symptoms (multiple, light red hematoma in consequence of minimal trauma). methods: whole exome sequencing (wes) was carried out for the patient, his asymptomatic wife, his symptomatic daughter and her asymptomatic -year-old brother. platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. lysates of gel-filtered platelets were analyzed for total granule p-selectin, cd and von willebrand factor (vwf) content by western blotting and for serotonin levels by elisa, respectively. results. platelet function and characterization of the patients granula suggested a delta-storage pool disease (spd). in most cases delta-spd occurs as part of a syndrome, e. g. combined with albinism, immunodeficiency or a thalassemic-like blood disorder. as the patient and his daughter did not show any conclusive phenotype, their dna was subjected to wes. exome sequencing revealed a not yet described gata -mutation close to two zinc finger domains (znf and znf ) in a highly conserved region of the gata gene in the -year old daughter (c. a>c, p.t p, heterozygous) and her father (c. a>c, p.t p, hemizygous). this mutation was absent in wildtype-controls but could also be demonstrated in the indexpatients' asymptomatic mother. only a few mutations are known to be located in this c-terminal region to date. mutations in gata may lead to different clinical presentations, depending on their location within gata (e. g. diamond-blakfan anemia (exon ), x-linked thrombocytopenia (znf ), transient myeloproliferative disorder (intron , exon , exon ) and acute megakaryoblastic leukemia (intron , exon , exon ) in case of down-syndrome). significantly increased hbf-levels (reference level: ≤ , %) in the affected family members of , % ( -year-old daugh- our proposita is a years old woman, who was transferred to our genetic counselling department for the suspicion of m. osler (hereditary hemorrhagic teleangiectasia, hht). during routine check up an anemia was diagnosed. a tumor search was initiated and unexpectedly the ct of the abdomen showed a suspect coin lesion of about . cm in diameter localized in the basal part of the right lung. further investigations revealed a pulmonary arterio-venous malformation which was hemodynamically relevant and already led to chronic right heart overload. a coil embolization was performed. retrospectively, medical history of the patient included episodes of severe epistaxis in childhood and a neurosurgical intervention for intracerebral bleeding at the age of years without permanent neurological deficits. during genetic counselling our proposita mentioned that her years old daughter also suffered from anemia due to multiple polyps of the colon. after polypectomy her hemoglobin values normalized. although histologically the polyps appeared as juvenile ones a mutation search in the apc-gene was initiated by the gastroenterologists without identifying a pathogenic mutation. combining the two pieces of information, we offered a mutation search in the smad gene and a pathogenic mutation c. c>t (p.arg cys) was found in both patients in heterozygosity. colonoscopy in the mother did not show juvenile polyps or gastrointestinal vascular malformations. vice versa, no cerebral or pulmonary arteriovenous malformations could be detected in the daughter. our family illustrates, that the same mutation within a family may phenotypically appear as different diseases. a careful taking of medical history and the knowledge of all relevant diagnostic findings (in this case e. g. the histology of the polyps) can enable the geneticist to offer a precise differential diagnosis leading to a well-directed molecular testing. to our opinion this is still relevant even in the era of ngs-based panels because the more precise the clinical diagnosis and the choice of the genes to analyse the less problems with unclassified variants will arise. the hereditary non-polyposis colon cancer (hnpcc, lynch syndrome) is caused by pathogenic germline mutations in mismatch repair genes (mlh , msh , msh and pms ) causing microsatellite instability (msi) and decreased or lost expression of the appropriate mismatch repair protein (mmr) in the immunohistochemistry (ihc) on tumor material. thus, ihc and msi testing help to identify the mmr gene, which most likely harbors a germline pathogenic variant. msi and ihc testing prior to germline analysis are specified in the s guidelines of hnpcc and if negative normally preclude further genetic analyses. here, we present a year old patient with a synchronous colonic (ceacum) and renal cancer at the age of years. at the time of the diagnosis hepatic metastases of the caecal adenocarcinoma have already been present. histologically, the colonic tumor was a poorly differentiated adenocarcinoma (pt pn) with lymphangiosis and haemangiosis carcinomatosa. the renal cancer showed histology of a moderately differentiated clear cell renal carcinoma. in the family history the year old sister and the parents are healthy. the twin sister of the patient's mother had a collateral breast cancer at the age of years and died two years later. the mother's grandparents had no canmedizinische genetik · p-cling- pitfalls in molecular genetic diagnostics a. tibelius, e. fey, k. hinderhofer institute of human genetics, heidelberg, germany probably every clinical laboratory geneticist may look back on at least one case in his career which has caused him quite a headache. this means those cases with completely unexpected and at a first glance implausible results which could be interpreted correctly only after intensive enquiry and additional testing. here, we report on three of such pitfall cases from our routine diagnostics. case : a -year-old woman, pregnant with monozygotic twins, was referred to prenatal cystic fibrosis diagnosis. she and her partner were carriers of mutations in the cftr gene ( + g>t and g x, respectively). prenatal molecular diagnosis demonstrated that both fetus had inherited only the paternal mutation. routinely, we performed maternal cell contamination analysis by comparing polymorphic microsatellite loci between the maternal and fetal dna. surprisingly, of tested microsatellite loci revealed a discrepancy between the maternal and fetal genotypes, meaning neither of both maternal alleles was present in fetal dnas. a potential confusion of samples was excluded. moreover, the presence of paternal mutation in fetal dnas indicated a correct genetic relationship between awaited children and the partner of pregnant woman. the only one plausible interpretation of the obtained result was a pregnancy by egg donation. afterwards, this suspicion was confirmed by the couple. case : we present a -year-old man with infertility resulting from azoospermia. conventional chromosomal analysis and an additional fish analysis using y probes indicated a ,xx karyotype with no detectable sry. in parallel, a molecular azf (azoospermia factors) diagnostic was performed by a standard multiplex pcr. by this method the absence of sry region and a deletion of regions azfb and azfc, was identified, explaining the observed azoospermia. interestingly, the pcr showed that the azfa region was still present in patient chromosomes, contradicting cytogenetic and fish results. thus, a complementary fish analysis was performed in order to reveal a low-grade y-mosaicism and sry material was detected in % of the cells (a result under the threshold level). based on this observation, pcr conditions for the azf diagnostic were modified and a very weak sry-specific pcr product detected. case : a molecular diagnostics for frax (fragile x syndrome) was requested for a -year-old boy with a slight delay in speech development. his brother was already molecular-genetically diagnosed as having frax. the analysis by southern blot hybridisation in the patient revealed a smear of methylated fragments characteristic for an expanded allele in the full mutation range. surprisingly, two fragments of normal length, methylated and non-methylated, could also be detected in patient's dna. a subsequent aneuploidy mlpa confirmed a supernumerary x-chromosome in the patient consistent with a klinefelter syndrome. these results were verified by an independent cytogenetic analysis. the syndrome of congenital symmetric circumferential skin creases (cscsc and cscsc ) replaces the old term michelin-tire-baby syndrome (mim ) and is characterized by congenital circumferential skin folds, primarily of the limbs, facial dysmorphism, cleft palate and intellectual disability. mutations in the β-tubulin encoding gene tubb or in the microtubule end binding family member mapre are the underlying genetic cause. ter) and , % ( -year-old indexpatient) suggested dyserythropoiesis, although thalassemic features of the blood count were lacking. conclusion: we describe a gata mutation as the cause of a delta-storage pool disorder. imbalanced x-chromosome inactivation might explain the different phenotypes of the gata mutation carriers and will be investigated through allele-quantification based on rna isolated from whole blood and from platelet rich plasma in case of the index patient and different family members. we saw three siblings (aged , , ; two female, one male) with variable signs of retinal disease. all three developed night blindness till the rd decade, furthermore near-sightedness and deterioration of the visual acuity to a various degree at age - . the clinical diagnosis was given as stargardt's disease, fundus flavimaculatus or unspecified retinal degeneration respectively. two more siblings (aged and ) and the mother (died age ) were clinically unaffected. the father (died age ) was reported to have had bad eyesight beginning in the th decade, his father similarly (no further information available). the three affected siblings have a total of five children (age - ), none of them showing clear signs of retinal disease up to now. considering the clinical diagnosis stargardt's disease we analysed the genes abca , elovl , prom and cngb by sanger sequencing. no pathogenic mutation could be detected. afterwards we performed next generation sequencing of several genes associated with retinal dystrophies and found a novel splice site mutation in the prph gene (mim # ). the mutation was confirmed in all three affected siblings by sanger sequencing. considering that mutation pathogenic we could re-diagnose our patients with patterned macular dystrophy type (mim # ). that disease is inherited in an autosomal dominant fashion, corresponding to the pattern of inheritance evident in our family. the etiology of epileptic encephalopathies, characterized by severe, early-onset seizures accompanied by developmental delay or regression, is highly heterogeneous. in recent years, next-generation sequencing approaches have led to the discovery of numerous causative genes; however the spectrum of associated phenotypes still needs to be further explored for many of these genes. we performed multi-gene panel analysis in a little boy of german non-consanguineous parents who showed severe early-onset infantile epileptic encephalopathy and almost absent neurological development. in this patient we identified a novel heterozygous missense mutation in the gabrg gene which was absent in the parents. in silico analyses strongly suggested a pathogenic relevance of this sequence variation which resides within a highly conserved region. so far, gabrg mutations have mainly been associated with milder types of epilepsy such as febrile seizures and childhood absence epilepsy. therefore, our findings extend the phenotypic spectrum associated with mutations in this gene at the severe end. referring on a case report by al marzouqi et al. ( ) , who reported on a girl with bilateral amastia in the context of skewed x-inactivation, we want to underline the importance of mlpa analyses in the case of negative sequencing of the eda gene, as whole exon duplication can be the cause of hypohidrotic ectodermal dysplasia. to our knowledge, the report of al marzouqi et al. has been the only case about this genetic alteration in the literature so far. novel pogz mutation in a patient with intellectual disability, microcephaly, strabismus and sensorineural hearing loss we report on a -year-old male patient with severe intellectual disability, microcephaly, sensorineural hearing loss, ocular abnormalities (strabismus and hyperopia), congenital heart defect (atrial septum defect and pulmonary stenosis) and minor facial abnormalities (thin upper lip, frontal upsweep). next-generation sequencing analysis revealed a novel heterozygous de novo mutation in pogz: c. _ del, p.(thr serfs* ) . the clinical problems of this patient are in accordance with the findings in the previously reported pogz mutation carriers. reports of additional patients with pogz mutations will be needed to establish detailed phenotype-genotype correlations of this novel and probably underdiagnosed syndrome. novel clinical and molecular aspects in two patients with kleefstra syndrome d. wand, b. seebauer, k. heinimann, i. filges medical genetics, university hospital, basel, schweiz the phenotype of kleefstra syndrome is clinically variable but characterized by facial dysmorphism, intellectual disability, childhood hypotonia and variably associated other malformations. haploinsufficiency of ehmt , caused by either heterozygous microdeletions in q . or sequence mutations in ehmt , has been identified to be the underlying causal mechanism. here we present two girls from two unrelated families with clinical signs of kleefstra syndrome. besides the main features such as facial dysmorphism, intellectual disability/developmental delay and childhood hypotonia, the years old girl presented with additional accelerated growth whereas the other girl, years old, showed failure to thrive. both children have no heart defect, renal or urogenital anomalies or severe respiratory infections. we identified two rare variants likely to be causal: a novel heterozygous splice site ehmt variant and a heterozygous microdeletion in chromosome q . , including exons an of the ehmt -gene . these patients broaden the spectrum of kleefstra -associated ehtm causes, contribute to novel aspects of genotype-phenotype correlations and a better understanding of the clinical variability of the disorder. here, we present two girls from two non-consanguines families showing clinical aspects of the syndrome of congenital symmetric circumferential skin creases. additional features are present in both girls. patient is the first child of healthy parents. she was born at + weeks of gestation with a weight of g, a length of cm and a head circumferences of cm. respiratory distress, a cleft palate, a heart defect (atrial septum defect), an anogenital malformation and facial dysmorphism were diagnosed after delivery, additionally to the skin creases phenotype. conventional karyotyping performed on blood lymphocyte cultures and cgh-array analysis showing normal results. patient is the second child of unrelated healthy parents. her older sister is healthy as well. because of an intrauterine growth retardation an amniocentesis with chromosomal analysis was performed, showing a normal karyotype of ,xx. patient was born at + weeks of gestation by caesarean section. the birth weight was g, the birth length was cm and the head circumferences was cm and the apgar score was / / . due to respiratory distress and hypoxia a tracheotomy was initiated. she also presented with cleft palate, feeding difficulties, a heart defect (atrial and ventricular septum defect), asplenia and facial dysmorphism. the skin phenotype was remarkably similar to that of patient with a prominent neck fold and skin creases mainly on the back part, but also at the limbs. in both children the skin folds gradually diminish over the time without any intervention like it was described for michelin-tire-baby syndrome/ cscsc and cscsc patients. for these reason, a disease-causing mutation in the genes mapre and tubb were excluded in both children. to identify a genetic cause, we performed a trio whole-exom sequencing, including the healthy parents and the affected child, in both families. a de novo stop mutation was detected in patient , while no promising results could be detected in patient . further studies, especially functional in vivo studies and analysis of further patients with a similar clinical presentation will answer the question if the above described phenotype is an expanded variant of the congenital symmetric circumferential skin creases or a unique new syndrome. clinical findings in a family with x-linked hypohidrotic ectodermal dysplasia due to a duplication of exon in the eda gene -a case report we want to summarize the phenotypic spectrum in one affected three generation family with x-linked hypohidrotic ectodermal dysplasia. we report on one strongly affected male and slightly affected female carriers, carrying a duplication of exon in the eda gene. reason for genetic assessment was the request of a female for evaluation of a possible risk for occurrence of the familial disorder in a further planned pregnancy. the woman reported an inability to breastfeed and she and her daughter showed conical teeth, a dry skin and sparse hair. the affected male showed absent deciduous teeth, hypodontia of permanent teeth, missing regulation of the temperature due to a lack of sweat glands, bilateral nipple hypoplasia, a dry and wrinkled skin, missing eye lashes, eyebrows and scalp hair and sparse body hair. the fingernails were inconspicuous, whereas the nails of the toes, particularly the nails of the hallux were yellowish and thickened. the affected male had an operation due to a gallstone and cysts were found in one kidney. there was no increased predisposition to infections. the suspected diagnosis of x-linked hypohidrotic ectodermal dysplasia was confirmed by genetic analysis of the eda gene (sequencing and mlpa analysis). a duplication of exon was detected in the affected male patient and was confirmed in the two mildly affected female relatives by realtime-pcr. terminator sequencing mix v . on an abi xl genetic analyzer (applied biosystems) and detected an insertion of bp between the exons and . we located the insertion's sequence in intron of the dmd gene and sequenced flanking sequences of gdna to find the underlying mutation causing the insertion. two hemizygous single nucleotide variants (snvs) surrounding the inserted fragment could be identified. the first variant (c. - a>c) is a common polymorphism (maf according to genomes project: . %) at the position of an existing acceptor splice site. the second variant (c. - a>g) is novel and creates a new cryptic donor splice site with high probability. these two cryptic splice sites create the formation of a bp pseudoexon which produces a frameshift and a premature stop codon (p.asp alafs*) in the dmd gene. in summary, we could genetically confirm the clinical diagnosis of a dystrophinopathy by two divs in dmd. although the insertion of the pseudoexon creates a premature stop codon the patient's clinical phenotype indicates a milder type of becker muscular dystrophy. this contradiction could be explained by the remaining existence of dmd wild type mrna most likely due to a not constantly active cryptic splice site. most interesting about the present case is the fact that a common snv facilitates the creation of a pseudoexon. this makes the region a potential hotspot for divs in the dmd gene which would be worthwhile further investigations. with more than % of all humans preferring to use their right hand, handedness is the most noticeable functional expression of cerebral lateralization in humans. however, the precise molecular mechanisms that regulate handedness and other related forms of cerebral lateralization remain largely elusive. therefore, the question which genetic, epigenetic and environmental factors contribute to human handedness is one of the central questions in research on lateral asymmetries. handedness is a complex, heritable trait, for which polygenic inheritance is assumed, meaning that a large number of genetic factors with a small additive effect contribute to the observed variance in hand preference. to date, genetic association studies have implicated only a few specific genes influencing handedness. particularly interesting is the association between the human androgen receptor (ar) gene and different aspects of handedness, since the interrelationships constitute a conceptual bridge between the theories that invoke testosterone as a factor in the development of cerebral asymmetries with theories proposing that the x chromosome contains a locus that influences the direction of hand preference. in an initial large association study in samples of healthy adults we already demonstrated that handedness in both sexes is associated with the ar cag-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. in addition, we have performed a second association study in an independently collected healthy cohort of more than test persons with comprehensive data on the handedness phenotype. we were able to replicate the association with longer cag repeats being related to a higher incidence of non-right-handedness, especially in females. since longer cag-repeat blocks have been linked to less efficient ar function, these results implicate that differences in ar signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization. dej. waschk, ac. tewes, p. wieacker, s. ledig institute of human genetics, münster, germany the mammalian female and male reproductive tracts develop from the paramesonephric ducts (müllerian ducts, md) and mesonephric ducts (wolffian ducts, wd), respectively. in the absence of testicular differentiation and anti-müllerian hormone, the wd regress and the md give rise to the fallopian tubes, uterus, cervix and the upper part of the vagina. disorders of normal md development in females can manifest as fusion anomalies of the uterus such as septate uterus, bicornuate uterus, unicornuate uterus and uterus didelphys, or more complex malformation patterns like mayer-rokitansky-küster-hauser syndrome (mrkh) or herlyn-werner-wunderlich syndrome. mrkh is characterized by congenital absence of the uterus and the upper two-thirds of the vagina in individuals with a normal female karyotype, most of whom show normal ovarian function. mrkh can further be associated with additional malformations e. g. of the kidneys and the skeletal system. herlyn-werner-wunderlich syndrome is characterized by uterus didelphys, obstructed hemivagina and ipsilateral renal agenesis. despite anomalies of the md occur quite frequently, the etiology of most cases with these disorders remains unknown. the homeodomain transcription factor emx (empty spiracles homeobox ) was found to be critical for urogenital and central nervous system development. previous studies showed that in emx mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. in order to elucidate whether mutations in emx are causative for md anomalies in humans, we performed sequence analyses of emx (genbank nm_ . ) in our study group of female patients with clinically characterized disorders of the md including patients with mrkh and cases with herlyn-werner-wunderlich syndrome. we found the heterozygous intronic mutation c. - c>a twice in this cohort. this variant has been described earlier (rs ) and is listed in the exome aggregation consortium exac variant with a minor allele frequency of . %. in silico analysis revealed no significant changes for the correct splicing of emx . we therefore consider this variant to be a benign polymorphism. we conclude that mutations in emx are not causative for disorders of the md in our cohort. a. zaum, w. kreß, a. gehrig, s. rost institute of human genetics, würzburg, germany dystrophinopathies are x-linked muscle diseases caused by mutations in the dmd gene (omim: ). due to the huge size of this gene, the detection of mutations is sometimes challenging. despite multiplex ligation-dependent probe amplification (mlpa) and sequencing of all exons, about % of patients do not show any mutations in coding regions and therefore remain without molecular diagnosis. we assume that the majority of these patients have deep intronic variations (div) which are not detectable by standard diagnostic techniques. the index patient analysed is a twelve-year-old boy who was by chance diagnosed with elevated ck levels (up to , iu/i) at eight weeks of age. today he is still able to walk without walking aids but needs assistance when climbing stairs. in , a muscle biopsy revealed complete absence of dystrophin which established the diagnosis of dmd. for the molecular diagnosis, standard diagnostics ascertained no causative mutation. therefore we decided to search for deep intronic mutations. we isolated mrna from muscle tissue of the patient and amplified overlapping cdna fragments using rt-pcr. the fragments were analysed by gel electrophoresis for size differences compared to an unaffected control. the cdna product comprising exons - revealed an augmented fragment size compared to the control and the expected product size (about bp instead of the expected bp). we sequenced the altered cdna product using bigdye recent research in psoriasis has identified pustular psoriatic manifestations as either mendelian traits or as major genetic risk factors in contrast to the numerous associated snps in classical plaque psoriasis vulgaris. autosomal recessive mutations in il rn have been identified in ~ - % of patients with generalized pustular psoriasis (gpp), a rare, severe pustular variant of psoriasis. in addition, heterozygous missense variants in card and ap s have been associated to pustular psoriasis as well and shown to be functionally relevant. in order discover how relevant those genes were in our large gpp cohort of patients recruited all over germany, we screened them for coding variants in il rn, card and ap s by sanger sequencing and for quantitative aberrations by mlpa. we identified homozygous or compound heterozygous il rn mutations in of gpp patients ( %) and single heterozygous mutations in patients ( %). the most common mutations were c. >c>t/ p.ser leu and c. c>t/ p.pro leu, present on %/ % of mutated alleles, respectively. we also identified three so far unreported mutations: c. c>a/ p.ser x, c. - delacct-tc/ p.thr _phe del and c. g>a/ p.val met. according to molecular modeling, c. c>a/ p.ser x resulted in a shortened, de-stabilized protein analogous to c. g>t/ p.glu x. the other two mutations were also predicted to result in destabilized, likely disease-relevant, loss-of-function proteins. heterozygous ap s mutations were detected in two gpp patients, both of whom carried additional homozygous or compound-heterozygous il- rn mutations. gpp patients were heterozygous carriers of rare missense variants in card ( %); of note, two of these patients carried additional mutations in il rn. our genotype-phenotype correlation revealed a similar gender distribution in carriers of il rn mutations and wildtype carriers, but a strong association between bi-allelic mutations in il rn and early age of manifestation (p = . x* - ). as in other autosomal recessively inherited mutations, the frequency of parental consanguinity was significantly higher in patients with two il rn mutations compared to non-carriers. overall, our genetic studies suggest a lower impact of variants in ap s and card in pustular psoriasis than of those in il rn. interestingly, the combination of il rn mutations with either ap s or card congenital prosopagnosia (cp), also known as developmental prosopagnosia or face blindness, describes the inability to recognize faces. cognitive functions such as intelligence as well as the sensory visual capabilities are usually not impaired but people with prosopagnosia are negatively affected in their social life because individuals with the disorder have difficulty in recognizing family members, close friends or colleagues. although the prevalence of cp is estimated at . % and it appears to run in families, the contexts, which genetic, epigenetic and environmental factors contribute to this trait, are largely unknown. therefore we started to establish a large, well-characterized cp cohort for genetic studies. we hypothesize that rare highly penetrant non-synonymous genetic variants could explain some cases of cp. as part of a larger genetic study of patients with cp, we performed family based whole-exome sequencing and targeted re-analysing on four individuals from two families with multiple affected members. by obtaining samples from affected and unaffected members of the same family, we hope to effectively identify de novo and inherited variations. variations are considered on the basis of allele frequency, mutation type, literature and mutation prediction tools, thus generating a list of candidate variations/genes for each patient that is amenable to interpretation and further analyses in the extended cohort. through this approach, we hope to identify causal variations/genes in families and isolated patients with cp. erythrokeratodermia variabilis (ekv) is a rare, autosomal dominant genetic skin disorder with a highly heterogeneous phenotype. to date, three causative genes (gjb , gjb and gja ) are described but further genetic heterogeneity is expected. card mutations are only described for psoriasis vulgaris, generalized pustular psoriasis and pityriasis rubra pilaris. for the first time, we present disease causing card mutations in patients with an "ekv-like" phenotype. it refers to one familial case with two affected individuals, with an autosomal dominant transmission from the mother to the daughter and one independent sporadic case. all patients present typical ekv symptoms. a rash of well-demarcated erythematous and scaly plaques interspersed with distinct islands of uninvolved skin or small reddish papules coalescing into large reticulated scaly plaques and palmoplantar keratoderma. to confirm the suspected diagnosis of ekv, we analyzed a custom designed multi-gene panel by next generation sequencing with genes associated to hereditary skin diseases (agilent haloplex technology). the sequencing results did not reveal any mutation in the genes gjb , gjb and gja , but we found two pathogenic mutations in card . in the familial case c. t>c p.leu pro (rs , enst . ) was detected, while the sporadic case carries c. t>c p.leu pro. we hypothesize that different genetic and environmental factors are involved in the evolution of the phenotype in patients with card mutations. our cases show that classification of unusual skin phenotypes can be challenging without genetic testing. therefore, gene panel sequencing is a cost-efficient and time-saving solution for solving difficult cases with sometimes unexpected genetic background. bipolar disorder (bd) is a complex psychiatric disorder affecting more than % of the world's population. the highly heritable disease is characterized by recurrent episodes of manic and depressive symptoms. as the cumulative impact of common alleles with small effect may only explain around % of the phenotypic variance for bd, rare variants of high penetrance have been suggested to contribute to bd susceptibility. in the present study we investigated individuals of large multiply affected families of european origin using whole exome sequencing (wes). in each family, two to five affected individuals with bd or recurrent major depression were selected for sequencing. wes was performed on the illumina hiseq platform and the varbank pipeline of the cologne center for genomics was used for data analysis. all identified variants shared within each family were filtered for a minor allele frequency < . % and potentially damaging effects predicted by at least four of five different bioinformatics tools. we identified a total of rare, segregating and functional variants implicating different genes, of which were brain expressed. subsequently, we applied the residual variation intolerance scores (rvis, petrovski et al., ) and identified genes that were ranked among the % most "intolerant" genes in the genome. gene enrichment analysis of these genes showed a significant enrichment for a total of pathways (p < . ) including neuron projection, axon development and cell adhesion. for follow up analyses, we prioritized genes that were either found in at least two unrelated families in the present study or that were previously reported in next generation sequencing or gwas studies of bd. in addition, we enclosed the genes that were predominantly driving the significant pathways in the above mentioned gene enrichment analysis. the different approaches of prioritization yielded candidate genes that are currently being followed up by resequencing in cohorts of about independent bd cases and controls of european ancestry. the candidate genes include cdh that encodes a calcium-dependent cell adhesion protein that may play an important role in the morphogenesis of neural cells during the development and maintenance of the brain. for resequencing we use the single molecule molecular inversion probes (smmips) technology that enables multiplex targeted resequencing in large cohorts. the smmips sequences were designed with an empirically variants in several patients indicated an oligogenic inheritance rather than a purely monogenic one. moreover, the oligogenic basis of this group of inflammatory diseases might currently be underestimated, as our study suggests that genetic risk factors other than il rn mutations remain to be identified in the majority of gpp patients. exome sequencing of multiply affected schizophrenia families provides new insights into the pathogenesis of the disorder schizophrenia (scz) is a multifactorial psychiatric disorder with a lifetime risk of ~ % and a heritability of about - %. analysing multiply affected families using whole exome sequencing (wes) is a very promising approach to identify new scz risk factors. in these families, individuals are affected with scz over several generations. it is likely, that in multiply affected families genetic variations with particularly strong effect co-segregate with the disorder and contribute to the development of the psychiatric symptoms. to our knowledge, the present study is the largest study analysing multiply affected scz families using wes worldwide so far. we included families with at least affected members each. from each family, - individuals were exome sequenced on an illumina hiseq and analysed using the varbank pipeline of the cologne center for genomics (http://varbank.ccg.uni-koeln.de) and the clc bio biomedical genomics workbench. we included rare (allele frequency ≤ . % in the exome aggregation consortium dataset) variants that were predicted to be pathogenic (combined annotation dependent depletion score ≥ ; cadd.gs.washington.edu), confirmed by sanger sequencing and co-segregating with the disorder. in total we identified potentially pathogenic mutations in ~ genes. a substantial proportion of these will not contribute to the pathogenesis of scz. in order to further tease out the most promising candidate genes we applied multiple strategies: (i) screening our mutations in independent patient and control cohorts through international cooperations (access to more than , scz patients), (ii) gene-based tests, (iii) pathway-and network-analyses, (iv) gene expression analyses and (v) sequencing of the candidate genes in , scz patients and , controls. analyses are ongoing and will be presented at the upcoming conference. abstracts and our evolution, we know very little about the different mutagenic processes in our germline. of particular interest are a handful of highly recurrent dnm associated with congenital disorders and/or rasopathies, that have been described as driver mutations expanding in the male germline. the mutation itself causes a change in the tyrosine kinase receptor/ras/ mapk pathway, which in turn confers the spermatogonial stem cell a proliferative advantage. selfish or driver mutations are quite common in cancer, but we still know very little about the selfish expansion in the male germline. the reason might be that mutations in the human germline are very rare, and it is rather difficulty to directly measure such rare events. most of our knowledge on germline mutagenesis comes from indirect sequence comparisons or whole genome sequencing of pedigree families, but it renders little information about individual mutagenic events. for this reason, we have adapted an ultrasensitive, next generation sequencing (uss) technology for the measurement of rare mutations to study the expansion of selfish genes in the male germline. as a proof-of-principle, we have sequenced at an extremely high coverage exon and of the fgfr gene in young and old sperm donors. we found an increased mutation frequency for the loci associated with achondroplasia and thanatophoric dysplasia ii in sperm of older donors. our results also show that we can distinguish ultra-rare mutations occurring at a frequency of one in hundred thousand wild type; thus, making this method ideal to discover potential driver dnm that might be expanding with paternal age. s. sivalingam , , a. j. forstner , , , s. herms , , , a. maaser , , c. s. reinbold , , t. andlauer , j. frank , h. dukal , d. schendel , , p. hoffmann , , , t. kircher , u. dannlowski , , a. krug , a. cichon , , , s. witt , m. rietschel , m. m. nöthen , introduction: affective disorders such as major depressive disorder (mdd) and bipolar disorder (bd) are genetically complex and heterogeneous disorders. both genetic and environmental risk factors contribute to the etiology of the diseases. however, the neurobiological correlates by which these risk factors influence the disease development are hardly understood. increasing evidence suggests that epigenetic modifications such as dna methylation have important implications on the development of psychiatric disorders including mdd and bd. several studies revealed that alterations in the dna methylation can modulate gene expression in response to the environment. to investigate this, genome-wide dna methylation analysis of female individuals from three extreme groups (genetic-, environmental risk and healthy controls) was performed. methods: for the genome-wide dna methylation analysis we selected: (i) individuals with genetic risk (at least one st degree relative with a life-time diagnosis of mdd or bd), (ii) individuals with environmental risk (maltreatment in the childhood trauma questionaire) and (iii) matched healthy controls. all individuals were of european origin. processing was done according to the manufacturer's protocol using the infinium methylationepic beadchip (illumina, san diego, ca, usa) covering more than . methylation sites at the life & brain center (bonn, germany). state-of-the-art data processing protocols, including correction for blood cell type heterogeneity, color correction, eliminating probes containing snps and cross reactive probes were used. after quality control trained design algorithm mipgen (boyle et al., ) and sequencing is currently performed on the illumina hiseq platform. our preliminary results strongly suggest that rare and highly-penetrant variants in neuronal and cell adhesion genes contribute to bd etiology. the results of resequencing of a large case/control sample will provide further evidence for an involvement of particular pathways. the use of zebrafish as model system to quantitatively assess the impact of risk variants in non-coding regions in vivo s. l. mehrem , , b. nagarajan , n. ishorst , , a. c. böhmer , , e. mangold , b. odermatt , k. u. ludwig , department of genomics, life & brain center, university of bonn, bonn, germany, institute of human genetics, university of bonn, bonn, germany, institute of anatomy, university of bonn, bonn, germany most human malformations occur early in embryonic development and are present immediately after birth. one common human birth defect is nonsyndromic cleft lip with or without cleft palate (nscl/p), affecting about in , newborns. nscl/p has a multifactorial background with a strong genetic component. recent genome-wide association studies identified several loci as risk factors for nscl/ p. notably, most of them map to non-coding regions and are expected to have a functional impact through regulatory mechanisms. given the early developmental time point of facial development and the resulting lack of accessible human tissue, follow-up analyses of risk variants are challenging. we are hypothesizing here that we might be able to quantitatively detect differences in regulatory activity between wildtype and risk variants located in predicted enhancers by using the zebrafish as model organism. the advantages of using the zebrafish are ( ) ex utero development, ( ) transparency of the fish, ( ) easy manipulation and ( ) relatively short generation times. we applied a dual-luciferase assay plasmid system which is based on a sequential measurement of two luciferases (firefly and sea pansy luciferase) in fish lysates upon injection of a single plasmid. this plasmid, which contains a minimal promoter (minp) and the enhancer region of interest, is microinjected into zebrafish eggs of one-cell stage. after three days, fish are collected, lysed and luciferase activity is measured using a luminometer. for our proof-of-principle analysis we analyzed an nscl/p risk locus on chromosome q (ludwig et al. ) . through database research one enhancer was predicted that contained two strongly associated risk variants. in vivo fluorescence analysis using egfp in zebrafish embryos revealed this enhancer to be active in craniofacial development, but qualitative differences in activity were not observed by eye. upon cloning of the enhancer in the dual-luciferase system, our injection results in vivo indicate that the system is working in principle. however, a high standard deviation between single replicate measurements was observed, probably due to variability in transfection efficiency. we therefore are planning to adapt the protocol in order to screen for positively injected fish embryos. we are currently investigating the functionality of these screening constructs in zebrafish embryos. results will be presented at the meeting. once successful, our approach represents a practical method to quantify the activity of regulatory elements in real time in vivo. this will be of particular importance in the functional follow-up of genetic findings in non-coding regions for the majority of birth defects. previously genome-wide association methods in patients with classic bladder exstrophy (cbe) found association with isl , a master control gene expressed in pericloacal mesenchyme. this study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. genotypes of markers obtained from cbe patients of australian, british, german ital-and normalization , cpg-sites were tested for genome-wide dna methylation by a linear regression model while accounting for biological as well technical covariates. results: the genome-wide dna methylation analysis of the three extreme groups revealed cpg sites (p < × - ) in the subgroup analysis "environmental risk vs. controls" and cpg sites (p < × - ) in the analysis "genetic risk vs. controls". in addition, we identified cpg sites (p < × - ) in the comparative analysis of "genetic risk vs. environmental risk". none of these cpgs showed significant differential dna methylation after correction for multiple testing. however the hierarchical clustering of the differentially methylated sites provided some evidence for differentially methylated patterns between the subgroups. discussion: our genome-wide dna methylation analysis of the extreme groups provided some evidence for differentially methylated cpg sites which unfortunately did not withstand correction for multiple testing. this may reflect at least in part that the sample size of the present study was too small to detect differentially methylated cpgs at the genome-wide level. a. tafazzoli , , t. vaitsiakhovich , l. pethukova , , , s. redler , , r. kruse , b. blaumeiser , m. böhm , g. lutz , h. wolff , , am. christiano , p. kokordelis , mm. nöthen , , rc. betz , alopecia areata (aa) is a common hair loss disorder that occurs in both sexes and all age groups. aa is thought to be a tissue-specific autoimmune disease directed against the hair follicle. both, gene-based and genome-wide association studies have identified more than susceptibility loci for aa; however, a large percentage of the overall heritable risk still awaits identification. to provide further insight into the immune related nature of aa, we and our us collaborators had each performed an immunochip-based analysis. we recently performed a meta-analysis, combining the data from both studies, and are now aiming to follow-up the best results in an additional german cohort by use of a sequenom assay to identify novel susceptibility loci. we conducted the meta-analysis by using data from the above mentioned two studies on illumina beadchip arrays including a total of , cases and , controls of central european origin. method of synthesis of regression slopes (msrs) was used for the analyses which are implemented in metainer software package. for follow-up step, we chose the most promising candidate snps. these will be examined with the sequenom massarray iplex platform in an independent aa sample comprising , cases and controls. by use of the meta-analysis combining data from the us and our sample, we identified novel loci with a suggestive p-value of pbecker-wu ≤ - (phet ≥ . ). among them, nfkb is the most significant locus (pbecker-wu = . × - ). in order to achieve genome-wide significance, we plan to follow-up the most promising snps in an independent german sample. we considered the most significant loci (lower p-becke-wu value) for the replication step. the experiments are ongoing and results will be presented at the meeting. despite the recent identification of susceptibility loci for aa, our understanding of the genetics of aa is incomplete. identification of new loci may provide novel insights into biological pathways and a better elucidation of disease pathophysiology. q . encompassing only one gene: kcnj . the duplication segregates with the phenotype in the family. kcnj belongs to the same subfamily of potassium channels as the known disease gene for cooks syndrome kcnj and both share several biological functions. recent data show that gain of function mutations in another potassium channel kcnh cause zimmermann-laband syndrome, a congenital malformation syndrome also associated with hypoplasia or aplasia of nails and terminal phalanges. therefore we propose that duplications of kcnj may also cause tissue specific misregulation resulting in digit and nail defects. taken together we show in a three generation pedigree that cooks syndrome is associated with a duplication of kcnj . our data further highlight the emerging role of potassium channels in congenital digit and limb anomalies. case report: deletion of the terminal short arm of chromosome (chromosome p deletion syndrome) without q-duplication with a familial history of a large pericentric inversion of chromosome b. gläser, n. hirt, e. botzenhart, j. fischer, m. leipoldt institut für humangenetik, universitätsklinikum freiburg, freiburg, germany we report on a male patient referred as a newborn with typical clinical features of chromosome p deletion syndrome. conventional karyotyping of lymphocyte cultures confirmed a deletion of the terminal short arm of chromosome with breakpoint in p . the size of the deletion ( mb) could be refined by microarray analysis and assigned to pos : - . parental cytogenetic investigations showed a normal karyotype in the mother whereas the father was revealed to be carrier of a large pericentric inversion of one chromosome . odd crossing-over in the inverted segment of a pericentric inversion in a parent can lead to unbalanced offspring caused by gametes with a terminal deletion of the p-arm together with a duplication of the q-arm or gametes with a duplication of the p-arm together with a deletion of the q-arm. in order to find out, whether the p-deletion in the child is the result of an independent event or if it is related to the structural chromosomal aberration of the father a microsatellite analysis is going to be performed p-cytog- a small supernumerary marker chromosome of the pericentric region of chromosome in a child with intellectual disability: case report and literature review b. hoffmann, g. gillessen-kaesbach, i. hüning institut für humangenetik, universität zu lübeck, lübeck, germany small supernumerary marker chromosomes (ssmc) are reported in . % of newborn infants. we report on a girl, which was born preterm at weeks of gestation via cesarean section due to pathological ctg. the pregnancy was complicated by gestational diabetes. she presented with muscular hypotonia, multiple hemangiomas, dysmorphic features and feeding problems. the body measurements were in normal range. the feeding problems made a tube feeding necessary until the age of four months. facial features consisted in epicanthus, high palate, broad nasal tip and broad nasal root. a brain mri showed periventricular leukomalacia and hypoplasia of corpus callosum. drug-resistant seizures with hypsarrhythmia started at the age of ten months. the affected girl was the only child of healthy non-consanguineous parents. the father also presented with a few small hemangiomas in the face. there was no history of intellectual diasability in the extended family. karyotyping showed ssmc in mosaicism. molecular characterization by array-cgh showed that the ssmc consists of pericentric chromosomal material derived from chromosome (arr [ h g ] p . p . ( , , - , , ) x ~ , q . q . ( , , - , , ) x ~ dn (grch /hg ). ian, spanish and swedish origin and , ethnically matched controls and from cbe case-parent trios from north america were analysed. only marker rs at the isl locus showed association (p = . × - ). a meta-analysis of rs of our previous and present study showed a p value of . × - . developmental biology models were used to clarify the location of isl activity in the forming urinary tract. genetic lineage analysis of isl -expressing cells by the lineage tracer mouse model showed isl -expressing cells in the urinary tract of mouse embryos at e . and distributed in the bladder at e . . expression of isl in zebrafish larvae staged hpf to hpf was detected in the developing pronephros region. our study supports isl as a major susceptibility gene for cbe and as a regulator of urinary tract development. to date, seven patients with interstitial deletions at chromosome q . -q . have been described in the literature. all patients reported had moderate to severe intellectual disability and a characteristic facial phenotype including blepharophimosis, telecanthus, epicanthus, flat malar region, and a thin upper lip vermillion. six of the seven patients had epileptic seizures. by analyzing the deletion's overlaps, two distinct critical regions have been suggested for the facial phenotype as well as for intellectual disability and seizures. here we present another patient with a de novo . mb deletion in q . -q . . the patient shows moderate mental retardation. he has an abnormal eeg, however, only one episode of clinical seizures has been observed so far. the facial gestalt resembles the typical dysmorphic features of the patients with q . -q . deletions reported previously. minor anomalies were short fingers and toes, and a single palmar crease. our report supports the assumption that deletions in q . -q . cause a distinctive and clinical recognizable microdeletion syndrome with characteristic facial features and intellectual disability. since the patient's deletion overlaps with most of the critical region for the dysmorphic phenotype but only with parts of the intellectual disability critical region, the molecular data presented here further narrow down the critical region for the intellectual disability seen in patients with q . -q . microdeletions. p-cytog- *** cooks syndrome is associated with a duplication of the potassium channel kcnj mundlos , , , d. horn , m. spielmann , , institute for medical genetics and human genetics, charité universitätsmedizin berlin, berlin, germany, max planck institute for molecular genetics, berlin, germany, northern ireland regional genetics service, belfast city hospital, belfast, ireland, berlin-brandenburg school for regenerative therapies -bsrt, berlin, germany cooks syndrome (mim ) is a rare autosomal dominant disorder classically characterized by onychodystrophy, and anonychia, with absence or hypoplasia of the distal phalanges of the hands and feet. large duplications including kcnj were shown to be causative for cooks syndrome. recently mouse studies revealed that tissue specific misregulation of kcnj , a potassium channel of the subfamily j, cause hypoplasia of nail beds and abnormal distal phalanges, thus resembling the cooks phenotype. here we report on a three generation pedigree with typical features of cooks syndrome that was negative for kcnj testing. we performed high resolution array-cgh and identified kb duplication on chromosome p-cytog- chromosome q . -q . deletion syndrome -an additional case with sensorineural hearing loss. s. leubner, c. hennig, a. junge mitteldeutscher praxisverbund humangenetik, dresden, germany the chromosome q . -q . deletion syndrome (mim # ) is a contiguous gene syndrome caused by a deletion encompassing the chromosome region q . -q . . initially, it has been described by ballif et al. ( ) . up to now, only a few cases with a microdeletion q . q . have been reported. most of them carry a microdeletion with recurrent breakpoints and similar size of about . mb. the common clinical features of cases with the chromosome q . -q . deletion syndrome comprise mild-to-moderate developmental delay, microcephaly, postnatal growth retardation, heart defects, limb anomalies, and hearing loss. we present an additional male patient with a . mb deletion of chromosome q . -q . , detected by array cgh (cytochip isca × k v . , illumina). our index patient shows typical symptoms of the chromosome q . -q . deletion syndrome like developmental retardation (in particular speech delay), a head circumference at the rd centile, postnatal growth retardation with a body height at the th centile, heart anomalies (right-sided aortic arch, patent ductus arteriosus), and sensorineural hearing loss on both sides. our data improve the characterization of the typical phenotype caused by a chromosome q . -q . deletion and reinforce the suspicion that this region might be associated with sensorineural hearing loss. partial, homozygous deletions of ahi gene causes joubert syndrome type d. meier, a. behnecke, jwg. janssen, u. moog, k. hinderhofer institute of human genetics, university hospital heidelberg, heidelberg, germany we describe a patient who is second child of consanguineous healthy parents from turkey. he was born after weeks of gestation with normal birth parameters ( g, cm, ofc cm). at the age of three months atypical eye movements became apparent. psychomotor development was delayed from the beginning, he presented with hypotonia and later developed ataxia. an abnormal breathing pattern was not noticed. an abnormal mri with hypoplasia of the cerebella vermis at the age of months and the clinical signs described above led to a clinical diagnosis of joubert syndrome. at that time no diagnostic testing was available. he returned to the outpatient clinic of the institute of human genetics at the age of years having developed retinopathy in the meantime. his height was below average (~ cm, <p ) but still within his family's range. aside from the known irregular eye movements he showed nail hypoplasia of the thumbs, and small hands and feet. he was working at a sheltered workshop. the year before, the pediatric department of the university hospital heidelberg had initiated testing of of the by then known genes causative for joubert with no mutation being found (nphp , tmem , cc d a, mks , rp-grip l, ofd- and mks ). the gene associated with joubert syndrome type (omim # ) was not included though. with the external mri not being available at that time we decided to first perform chromosome and array analyseis. the conventional chromosome analysis was without pathological findings. subsequent array analysis by affymetrix® cytoscan hd oligo/snp showed an kb homozygous deletion in q . within the ahi gene, the gene for joubert syndrome type . the deletion encompasses exon to which includes parts of the wd-repeats (tryptophan-aspartic acid (w-d) dipeptide) on the protein level. the homozygous deletion was confirmed by mlpa in the patient and both parents identified as heterozygous carriers. so far, alterations in ahi leading to joubert syndrome comprise homozygous point mutations or small heterozygous deletion/duplications in combination with a second pathogenic mutation. so we here present the first case of a large homozygous deletion in the ahi gene, being causative for joubert syndrome type . we conclude that in un-a review of the literature showed that the few patients with a ssmc of the pericentric regions of chromosome described in literature show early feeding problems, developmental delay, delay in acquired language, difficulties in social skills, difficulties in attention and activity levels or autistic-like behavior. the facial dysmorphic features were not specific. multiple hemangiomas have been reported in some cases. west syndrome or seizures have not been reported so far. the phenotype of our patient is consistent with the literature data, however there are no patients with a ssmc showing the same breakpoints reported so far. especially due to the mosaicism and the rare number of patients described in the literature, no precise clinical prognosis for this patient was possible. the generation of patient-specific induced pluripotent stem cells (ipscs) by reprogramming of adult somatic cells (e. g. skin fibroblasts) represents a novel technology for studying disease mechanisms. for generation of ipscs several reprogramming methods, including the use of integrating vectors and non-integrating vectors, have been developed. since genetic abnormalities can arise during the reprogramming process, genetic quality assurance is indispensable. the aim of this analysis was to compare the genetic stability of ipscs generated using either a dna-integrating or a dna-non-integrating reprogramming technique in order to reveal possible method-specific differences. we studied ipscs derived from patients with parkinson's disease (pd), the second most common neurodegenerative disease worldwide. to monitor the genetic stability, we investigated copy number variants (cnvs) in addition to conventional chromosome analyses in ipscs of ten pd patients and six healthy control individuals. using high-resolution chromosomal microarray analysis (cma), we monitored the formation of relevant cnvs during reprogramming to pluripotency by comparing fibroblasts and ip-scs of the respective individual. to date, fibroblast cultures of all selected probands as well as retroviral reprogrammed ipsc clones and eight sendai-viral reprogrammed ipsc clones were analyzed. aneuploidies were detected in three fibroblast cultures ( %), in five retroviral ipsc clones ( %), and in none of the sendai ipsc clones. de novo cnvs were identified in retroviral ipscs ( %) - clones showed one ( %), nine clones showed two ( %), four clones showed three ( %), and one clone showed four newly arisen cnvs ( %). in seven of eight sendai clones, de novo cnvs were detected ( %). two de novo cnvs were identified in three clones ( %), three cnvs in two clones ( %), four in one clone ( %), and even six de novo cnvs were detected in one clone ( %). additionally, a mosaic gain of the whole long arm of one chromosome was identified in one sendai clone. only of the retroviral reprogrammed clones ( %) as well as one sendai reprogrammed clone ( %) showed no newly arisen cnvs. the cnvs were between kb and . mb in length in retroviral clones and between kb and kb in sendai clones. because almost all cnvs contained genes, including genes involved in neurodevelopment and transcriptional regulation, a biological relevance seems highly probable and changes in cell phenotype cannot be excluded. since ipscs and cells derived thereof -independent of reprogramming methods -often contain aberrations not detected by standard chromosomal analysis, our findings highlight the importance of high resolution cma procedures. in spite of differences in the analyzed number of clones, our results indicate more and larger cnvs in sendai clones than in retroviral reprogrammed clones. supported by forips, bavarian research network induced pluripotent stem cells. abstracts mother's lymphocytes showed a balanced form of the aberrant karyotype found in her son. the healthy mother possesses the insertion ins( ; ) (q . ;q q ), the simultaneous resection of the q ? - q . material and generation of the additional ring chromosome with this material. during meiosis a normal chromosome , the derivative chromosome and the ring chromosome were passed on to the son leading to the unbalanced karyotype as described above. further analysis with pan-centromeric probes and immunohistochemistry with an antibody against cenp-a might confirm the existence of a neocentromere on the ring chromosome which was meiotically transmitted from the mother to the son. the mtor (mechanistic target of rapamycin) kinase is the most important regulator of local dendritic and presynaptic protein translation in the brain. it has been shown to fundamentally influence ampa receptor activity by shifting glua to glua balance and to control the synthesis of several proteins involved in synaptic function and plasticity. both, loss and gain of mtor activity lead to significant disturbances of brain homeostasis and neuronal function resulting in intellectual disability (id), epilepsy, autism and behavioural alterations. in order to analyse mechanisms of homeostatic regulation of mtor-dependent synaptic function we are using a heterozygous tsc knockout (ko) mouse model for tuberous sclerosis (ts). tsc (together with tsc ) is part of a complex that inhibits the mtor kinase. mutations in either of the two genes result in increased mtor activity and upregulated downstream signalling and are causative for autosomal dominant ts. it had been shown previously that mtor hyperactivity leads to a set point shift and significantly influences neuronal homeostasis by altering cell excitability and e/i balance in heterozygous tsc ko animals. according to these studies we hypothesize that this shift significantly leads to synaptic connectivity and plasticity as well as network alternations that result in progressive development of cognitive dysfunction, epilepsy, autism and behavioural alterations over time in ts. we have established a behaviour battery, which consists of a novel object recognition test (nort), a morris water maze test (mwm) and an evaluation of nest building and social interaction to study social behaviour and cognitive performance in heterozygous tsc ko mice in different age groups. interestingly we found that, while two months old animals do not show any alterations, three to four months old animals develop significant disturbances in social behaviour in the social interaction test and in nest building. furthermore, we could show significant changes in cognitive performance of seven to eight months old heterozygous tsc ko mice in the nort compared to three to four months old heterozygous tsc ko animals. these data show that the ts phenotype builds up on shifts of the e/i balance that develop at a very early stage. pharmacological intervention with e/i balance is a promising therapeutic strategy to prevent brain damage caused by congenital hyperexcitability and homeostatic dysfunction. clear cases of a joubert phenotype an array-cgh is recommended besides sequencing to clarify the molecular basis of the phenotype. recently, a new syndrome of intellectual disability (id) with dysmorphisms due to deletions within the chromosomal band q . harboring the tbl xr gene was described. additionally, a specific point mutation in tbl xr causes pierpont syndrome, a distinct mental retardation syndrome. we report four patients in which array-cgh analysis and real-time quantitative pcr of genomic dna revealed tbl xr microduplication. adjacent genes were not affected. the microduplication was verified as de novo by real-time pcr of parental dna in one patient. the other three cases occurred in two generations of a second, unrelated family. we compare the remarkably similar clinical findings in tbl xr microdeletion, point mutation and microduplication cases and expand the tbl xr -associated phenotypic spectrum. among others, intellectual disability, hearing loss and autism spectrum disorders are common features of tbl xr -associated diseases. our clinical observations add to the increasing evidence of tbl xr 's role in physiological brain development and simultaneously demonstrate that opposing genetic disease mechanisms affecting tbl xr can lead to similar id phenotypes. in conclusion, the observed phenotypic overlap indicates that this novel microduplication is a genomic sister-disorder to the q . microdeletion syndrome. neocentromere formation has been reported in more than small supernumerary marker chromosomes (ssmc). here we report the formation of a putative neocentromere within a supernumerary ring chromosome derived from chromosome in a five year old patient with generalized developmental delay, microcephaly and hexadactyly. chromosomal analysis (gtg-banding, resolution bands) of the boy showed an aberrant karyotype with a derivative chromosome demonstrating additional chromosome specific material inserted into the distal part of the long arm (ins( ; )(q . ;q q )), a simultaneous deletion of the chromosomal region q ? -q . on the same homolog and an additional supernumerary small marker chromosome, presumably a ring chromosome in all metaphases tested. in parallel, array-cgh was performed and revealed a gain of . mb in chromosomal region q -q . without a detectable imbalance of chromosome material within the limits of the method ( k array). fish analysis with wcp probes and telomeric probes for chromosomes and confirmed the interstitial insertion of chromosome material into the long arm of the derivative chromosome . the marker chromosome stained completely with a wcp probe but centromere probes for chromosomes and were both negative suggesting the formation of a neocentromere within region q ? q . . in total, the complex rearrangement resulted in a partial trisomy q in the boy as a presumable explanation for the phenotype. the mother has experienced one abortion and the parents have two more children who show a normal development so far. both parents were karyotyped. the analysis of the father was normal ( ,xy). konventional chromosomal analysis of the in flvcr have previously been linked to vision impairment and posterior column ataxia in humans, but not to hsan. using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the flvcr -mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. autosomal-dominant familial hypercholesterolemia (fh, omim ) is one of the most common genetic diseases with an estimated prevalence of : to : . three major genes have been associated with the disease: low density lipoprotein receptor (ldlr), apolipoprotein b (apob) and proprotein convertase subtilisin/kexin (pcsk ). we have recently published data on the mutation spectrum in these three genes in about fh patients from germany, however, this screen for mutations was negative in about % of the cases. stap (signal transducing adaptor family member ) was recently suggested to be the fourth fh gene. we therefore screened the stap gene for disease causing variants in a group of fh patients negative for mutations in the three established fh genes by direct dna sequencing (sanger). the newly identified variants will be presented including a phenotype-genotype correlation. phenotype: we present a -year-old girl with developmental delay, craniofacial anomalies, congenital eye anomalies ( . dpt), short stature and behavioral disorder with auto-aggressions. preliminary examinations (array-cgh analysis, karyotyping, diagnostic of metabolism, eeg, cmrt, sequencing of srcap-gene) revealed normal results. methods: in course of the midas-project, we sequenced the index patient and her healthy, non-consanguineous parents on a nextseq platform (illumina, san diego, ca, usa) to perform a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed with a sureselect human all exon kit v (agilent, santa clara, ca, usa), targeting coding exons and conserved splicing sites of over . genes. the library of the index patient was sequenced with a -fold mean coverage as -bp paired-end reads. % of the target region were covered -fold or higher. data analysis and variant evaluation was done using the clc genomic workbench . (qiagen, hilden, germany) and annotations from commercial as well as public databases. results: the trio analysis of whole-exome sequencing data from the index patient and her parents revealed variants. we identified the disease-causing de novo adnp mutation c. c>t (p.arg *, adnp), resulting in the introduction of a stop codon in exon / and truncation of the corresponding protein. both of the parents did not carry this mutation. adnp is part of the atp-dependent baf chromatin-remodeling p-monog- the challenge to insert costello syndrome causing hras mutations into human keratinocytes using the crispr/cas editing technology. l. brandenstein, k. kutsche, g. rosenberger university medical center hamburg-eppendorf, hamburg, germany germline missense mutations in the hras gene cause costello syndrome, a rare developmental disorder characterized by a typical facial gestalt, postnatal growth deficiency, intellectual disability, and predisposition to malignancies as well as skeletal, cardiac and dermatological abnormalities. the molecular pathophysiology caused by heterozygous hras gain of function mutations has been analysed in various tissues and cell types, however, up to date the molecular basis for cutaneous manifestations in costello syndrome is largely unknown. to address this question in an appropriate model system, permanent human keratinocyte (hacat) cells carrying costello syndrome-associated mutations in the endogenous hras gene should be generated by using the crispr/cas technology. double strand breaks induced by cas can be repaired in two ways: the error-prone non-homologous end-joining pathway for the generation of knockout models or the homology directed repair pathway, which allows precise editing. the latter enables the introduction of specific point mutations into a cell line by using a single stranded dna (ssdna) as repair template. however, we found that this is a very rare event and its efficiency depends on various factors including used cell line, selected guide rna, length and amount of ssdna and also cas variant. nonetheless, by using cas wildtype, we could insert the disease-associated c. g>t (p.g v) mutation into genomic hras both in hacat cells ( positive clones out of ) and hek cells ( positive clones out of ). in contrast, using the cas nickase protein variant that prevents off target effects, did not result in positive clones. taken together, in months of working with crispr/ cas we gradually gained experience with many problems and pitfalls of this technology and, finally, now we are able to introduce point mutations in cell lines. next, we will use the mutant hacat cell line to gain deeper insight into the function of hras for epidermal homeostasis and its deregulation in costello syndrome. ccdc could also play a role in prenatal development of the mouse retina and brain. embryonic stages of interest comprise <e at the initial development of the eye, e after formation of nasal pits and prominent brain growth, when brain vesicles are initially visible and e where the eye and nasal brain develops. this study aims at characterizing ccdc rna and ccdc protein expression during key steps of prenatal development in the wt mouse and respective ccdc reporter gene expression in the ccdc -/model. methods: ccdc rna expression was analyzed by quantitative ccdc qrt-pcr and ccdc protein expression was determined by sds page and western blot in eye and whole brain homogenates of the mouse at selected embryonic stages (<e , e and e ). in addition, µm sagittal cryosections of mouse embryos were hematoxylin and eosin stained and analyzed for ccdc reporter gene expression by x-gal staining or antibody staining followed by light or fluorescence microscopy. results: ccdc rna and ccdc protein is detected in the wt mouse at different developmental time points in the eye and brain tissue. accordingly, ccdc -/reporter gene expression is evident at prenatal developmental stages in cryosections of whole embryos. conclusion: expression studies of ccdc gene and products in the eye and brain in wt and ccdc -/mice in different developmental embryonic stages are of crucial interest, and the role of ccdc gene and products in prenatal development has to be evaluated in detail in further studies. nn. hauer, b. popp, e. schoeller, ab. ekici, a. reis, ct. thiel institute of human genetics friedrich-alexander-universität erlangen-nürnberg, erlangen, germany the application of next generation sequencing technologies (ngs) has been extremely successful in the identification of the underlying causes of genetic disorders. whereas, most variants are located in coding regions, variants within splice-sites that might lead to alternative splicing affecting protein function or causing mrna decay are less well characterized. even for canonical splice-sites, prediction of the splicing effects for an identified variant is cumbersome. this becomes even more apparent as ngs based exome and genome sequencing tremendously increase the number of variants potentially affecting splice-sites. in our study to identify the underlying causes of idiopathic short stature by whole exome sequencing in more than patients, we observed variants in consensus splice-sites defined as up to bases from the ' and bases from the ' end of an exon. we used several computational methods (splice site finder, maxentscan, nnsplice) to calculate their potential effect on splicing. these results were often inconclusive and did not predict the definite effect on transcripts. therefore we performed rtpcr amplification of the respective regions, electrophoretic evaluation of fragments and sanger sequencing. in case of a suspected alternative splicing the evaluation of the distinct resulting isoform, though, was often hampered by the overlap of sequences. this was especially severe when multiple naturally occurring alternative transcripts were present. to overcome these issues, we established a deep sequencing approach (targeted rtpcr-seq) to identify all present isoforms in the affected patients compared to controls. for this method the rtpcr fragments were normalized to generate an equimolar mixture. libraries for patients and controls, respectively, were generated by ligation of distinct index adaptors for both amplicon pools and subsequently sequenced on an illumina miseq platform. the resulting reads were aligned and visualized as sashimi plots. observed splice-junctions in patients were compared to the controls and public databases. we found concordant results between standard rtpcr evaluation and our rtpcr-seq approach for variants (alternative splicing for variants). for these confirmed variants the specific effect was more obviously ascertained by the ngs based approach. for variant only rtpcr-seq was able to separate multiple co-occurring distinct isoforms, both in controls complex, which is involved in the regulation of gene expression. an influence on neuronal cell differentiation is postulated. mutations in the adnp gene were previously reported in patients with autism and mild to severe intellectual disability. all reported adnp mutations are characterized by de novo mutations that are clustering in exon and severely disrupting the protein-coding sequence, either by the introduction of a stop codon or a frameshift induction. by additionally considering the clinical symptoms, we were able to diagnose the helsmoortel-van der aa-syndrome (hvdas; omim id: ). discussion: the challenge of next-generation sequencing (ngs) approaches in diagnostics is no longer the technical feasibility or costs but the medical interpretation of the large sequencing data sets. besides a robust genetic analysis with focus on disease-associated genes, family anamnesis and detailed information of clinical symptoms in a standardized scheme are important for a reliable diagnosis. here, we present one of over cases that will help to establish the multiple integration of data annotation software (midas), which will enable phenotype-genotype-correlations to enhance the validity of results of genetic analysis, minimize the risk of misinterpretation and enable faster diagnosis in routine patient care. peripheral neuropathies are a group of clinically and genetically heterogeneous disorders, mutations in a large number of genes have been described so far. among patients with charcot marie tooth disease (cmt)/hereditary motor and sensory neuropathies (hmsn), a common duplication of the pmp gene on chromosome p is by far the most frequent mutation; sequence variants in the genes gjb , mpz and mfn account for a considerable proportion of patients but all other associated genetic variants are extremely rare. deletions of the pmp gene lead to hereditary neuropathy with liability to pressure palsies (hnpp) but transition between the different phenotypes may be rather fluid in some instances. next generation sequencing (ngs) has revolutionized cmt diagnostics as a large number of genes can be investigated at a time but it also leads to challenging questions. use of ngs has certainly improved the diagnostic yield in cmt diagnostics and it has repeatedly resulted in unexpected findings. broad molecular testing has already disclosed overlaps between clinical conditions and more surprising results are yet to come. here we report our recent experiences with the diagnostics of neuropathies. our results, different testing strategies, implications for clinical management and genetic counselling will be discussed and possible further steps suggested. (gerding et al., hum mol genet., ) . retinal ccdc protein expression in the wildtype (wt) mouse demonstrates highest levels shortly after birth. moreover, ccdc protein can also be detected early postnatally in mouse brain tissue. therefore, there is strong evidence that p-monog- *** individuals with hereditary trichilemmal cysts present a combination of an inherited high-risk plcd allele (including two sequence variants) and additionally acquired somatic plcd variants in the cysts. trichilemmal cysts are benign tumors derived from the outer root sheath of the hair follicle. they occur most often solitary or multiple on the scalp but can also be found on other body parts. the cysts can develop sporadically or in an autosomal dominant mode of inheritance with incomplete penetrance. in , the hereditary cyst form was mapped to the tricy locus on chromosome p -p . in a familial genome-wide linkage study. we analyzed affected individuals from different tunisian families by whole-exome sequencing. the bioinformatic evaluation revealed no rare, disease causing mutations but the sequence variant c. c>t, p.s l (maf = . , rs , enst . ) in the phospholipase c delta (plcd ) gene within the tricy locus. furthermore, all five individuals present a second variant c. a>g, p.p p (maf = . , rs ) in the same gene. plcd is a member of the phospholipase c family. the enzyme is involved in calcium-dependent intracellular signal transduction and catalyzes the hydrolysis of phosphatidylinositol , -bisphosphate into the second messenger diacylglycerol and inositol triphosphate. homozygous knockout-mice have hair defects and show aberrant skin development with increased progression of skin tumors and intradermal hair-follicle derived cysts. in humans, plcd is highly expressed in the hair follicle but so far only nonsense mutations have been described causing hereditary leukonychia totalis without any skin or hair abnormalities. a segregation analysis of plcd in a tunisian family cohort and one german family ( families, individuals, affected) showed that all affected individuals contain the same two sequence variants. based on these results, we propose that plcd is responsible for the cyst phenotype. cdna sequencing from three different cysts revealed additional acquired somatic sequence variants in plcd . we found c. c>t p.s l in two cysts and the two variants c. c>t, p.s f and c. c>t p.s f in the third one. all three somatic variants are not described in the exac database and the genomes project. allele-specific rt-pcrs were performed with cyst cdna and we could show that the somatic variants are on the same allele as the inherited variants. the acquired somatic cyst sequence variants lie within or respectively near the c domain of the plcd protein. the c domain is involved in the calcium-dependent binding to membrane-integrated phospholipids. depletion of the domain leads to decreased membrane association and protein activity. we assume that the allele with the variants c. c>t and c. a>g is a risk factor for hereditary trichilemmal cysts and that additional acquired rare sequence variants are the genetic trigger for the development of the cysts. and patients. discordant results for variants proposed a higher specificity of the rtpcr-seq. thus, we established a simple amplicon based deep sequencing approach for standard rtpcr fragments to ascertain the effects of specific splice site variants. this technique has proven to be highly scalable, fast and efficient to analyze splice-site variants. p-monog- eif s mutations associated with severe x -linked intellectual disability syndrome mehmo mehmo (mim % ), is a rare x-linked syndrome characterized by profound intellectual disability, epileptic seizures, hypogonadism, hypogenitalism, microcephaly, and obesity. in steinmüller and colleagues described a large family with mehmo syndrome with five affected males in two generations and assigned the disease locus to the short arm of chromosome x (xp . - . ). we took advantage of massively parallel sequencing in four families with mehmo syndrome, including the family reported by steinmüller et al. to identify the underlying genetic cause if this severe disorder. we here show mehmo syndrome is associated with mutations in the x chromosome gene eif s . in three families we identified a c-terminal frameshift mutation (p.ile serfs) and in an unrelated boy who is less severely affected, we identified a novel maternally inherited missense mutation (p.ser arg) in eif s . eif s encodes the gamma subunit of the eukaryotic translation initiation factor (eif ). eif is essential for eukaryotic translation initiation and regulation of the integrated stress response (isr). subsequent studies in patient fibroblasts (p.ile serfs) showed increased isr activation due to the mutation and functional assays in yeast demonstrated that the p.ile serfs mutation impairs eif gamma function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the affected males with ile serfs mutation. our results suggest that more severe mutations in eif s cause the full mehmo syndrome, while less deleterious mutations are associated with a milder form of the syndrome with only a subset of the symptoms. introduction: larger structural genomic duplications or deletions (copy number variations = cnvs) are routinely detected by array comparative genomic hybridization (acgh). while acgh has been established as a robust and effective approach for cnv screening, it remains expensive and is limited by resolution. in addition, commercial mlpa testing today allows the identification of exon deletions or duplications for a limited number of core genes. more recently, massive parallel sequencing of multi gene panels (mgps) has been introduced as a fast and cost-effective tool in routine genetic diagnostic testing to identify causal intragenic sequence alterations not only in core genes but also in those with small contribution to the respective phenotypes. the obtained mgps data may also be bioinformatically assessed to detect exon deletions or duplications within the analyzed genes panels and thus can be expected to further improve the diagnostic yield. methods: more than patients were sequenced with phenotype specific gene panels on a miseq platform (illumina) and analyzed with our bioinformatic diagnostic workflow including a quantitative data assessment using our in house java based bioinformatic script to search for gene or exon deletions. detected deletions were confirmed by an independent method (e. g. mlpa, pcr amplification of the junction fragment or linkage analysis), if available. results: we here report details for six suspected deletions in seven patients detected by our in house bioinformatic workflow: heterozygous gene deletions of pafah b , spastin or arfgef , respectively; two heterozygous cftr deletions of exon and , one homozygous partial sftpb deletion and a homozygous ispd exon deletion. the complete gene deletions of pa-fah b and spastin as well as the cftr deletion of exon and could be confirmed by mlpa. for the partial sftpb deletion both breakpoints could be precisely located within the readout, allowing determining correct deletion size and design of primers to amplify the junction fragment. the homozygous deletion of exon in the ispd gene could be confirmed by pcr and linkage analysis. discussion: diagnostic multi gene panel sequencing after nextera enrichment allows sufficient homogeneity of the obtained patient and control data per target to quantitatively search for constitutional deletions covering two or more adjacent targets. combined data assessment considering the individual clinical data will not only further increase the diagnostic yield but can also be expected to further delineate the mutational spectrum for specific phenotypes by the simultaneous detection of clinically relevant sequence variants as well as cnvs. mgps sequence assessment may also allow to gain new insights into the genomic architecture and origin of target regions and haplotypes, involved in common structural variations. c. niemietz, v. sauer, l. fleischhauer, s. guttmann, s. reinartz groba, a. zibert, h. schmidt universitätsklinikum münster, klinik für transplantationsmedizin, münster, germany various types of somatic cells have been reprogrammed to induced pluripotent stem cells (ipsc) followed by differentiation into hepatocyte-like cells (hlc). recently, cells that shed from the renal epithelial system were shown to be a suitable and convenient source for ipsc generation. in the current study, urine-derived cells (ucs) were isolated from urine donations of patients having familial amyloid polyneuropathy (fap), a neurodegenerative disease caused by mutation of the transthyretin (ttr) gene and wilson disease (wd), a genetic disorder of atp b causing copper accumulation, predominantly in liver and brain. patient-specific hlcs were differentiated in order to study disease-specific mechanisms and to investigate the efficacy of novel compounds. for isolation of renal epithelial cells, urine of fap and wd patients was processed. ucs were reprogramed into ipscs using plasmids resulting in transient expression of factors sox , oct / , klf , and c-myc. after characterization of ipsc that expressed high levels of pluripotency markers, like oct and nanog, a -step hepatocyte differentiation protocol was performed. ipscs were subjected to a treatment with growth factors (activin a, wnt a, fgf , hgf) for days. the hepatic, patient-specific character of differentiated hlcs was assessed by functional analysis, gene expression profiling, genotype analysis, and immunostainings. therapeutic oligonucleotide efficacy targeting ttr was determined by immunocytochemistry, qrt-pcr and western blot analysis. ttr-stabilizing activity of tafamidis was investigated by means of thermal shift assay and western blot analysis. copper chelation by methanobactin was determined by atomic absorption spectroscopy. reprogramming of ucs resulted in stable ipsc lines with characteristic pluripotent marker expression. differentiated hlcs showed high similarity to human hepatocytes in terms of genetic profile and functional activity. small-interfering rnas (sirnas), antisense oligonucleotides (aso) (niemietz et al. , plosone ( ) :e ), and the ttr stabilizing compound tafamidis that are currently assessed in clinical studies were studied in hlcs derived from fap patients. a novel chelator was used to determine intracellular copper accumulation in hlcs derived from wd patients (lichtmannegger et al. , jci ( ): - ). fap-specific hlcs revealed differently expression of key regulators of the protein quality control (pqc) system. our results demonstrate that ipsc derived from urine are excellently suited to study hereditary liver diseases. hlcs could be investigated in the patient-specific genetic background. the efficacy of novel compounds was assessed and individual responses were monitored. involved in gene regulation. by parent-patient trio whole-exome sequencing, we were able to characterize the unterlying genetic cause in the patient, who has undergone multiple diangostic test before without receiving a diagnosis. discussion: although for many congenital syndromic diseases the disease-associated genes are known it remains difficult for physicians to interpret the highly variable phenotypes as well as their variable nomenclature in order to request the appropriate molecular analysis. here we present one of more than cases that will help to establish a database connecting specific phenotypes, using the human phenotype ontology terms, with the each corresponding disease-causing genes (midas). novel compound heterozygous nalcn variants in two brothers with muscular hypotonia and global development delay l. segebrecht , c. weiß , m. jäger , , t. zemojtel , , , d. horn , n. ehmke , institute of medical and human genetics, charité -universitätsmedizin berlin, berlin, germany, spz dpt. pediatric neurology, charité -universitätsmedizin berlin, berlin, germany, berlin-brandenburg center for regenerative therapies -bcrt, charité -universitätsmedizin berlin, berlin, germany, institute of bioorganic chemistry, polish academy of sciences, poznań, poland, labor berlin -charité vivantes gmbh, berlin, germany, berlin institute of health, berlin, germany we report on two brothers aged two and three years, with muscular hypotonia, global development delay, abnormal respiratory rhythm, mild facial dysmorphism, recurrent respiratory infections, and failure to thrive. sequencing of disease related genes identified compound heterozygosity for two novel mutations in nalcn: c. c>a (p.phe leu) and c. + t>c. nalcn encodes a voltage-independent, non-selective cation channel, which is involved in regulation of neuronal excitability. the missense variant c. c>a affects the highly conserved amino acid position phe which is located in segment s of domain iv in the pore-forming unit of nalcn. the variant c. + t>c alters the donor splice site in intron and is predicted to cause skipping of exon , resulting in loss of function of nalcn. biallelic mutations of nalcn are associated with infantile hypotonia, psychomotor retardation and characteristic facies (ihprf ), whereas heterozygous de novo mutations cause congenital contractures of the limbs and face, muscular hypotonia, and global developmental delay. the clinical features of our patients resemble mild ihprf , caused by a biallelic missense mutation in segment s of domain iv. it has been suggested that variants in or close to s and s of the pore-forming domains lead to the above mentioned autosomal dominant condition whereas variants in other regions or loss of function mutations result in autosomal recessive inheritance. this is the first report of a mutation in a s segment, inherited in autosomal recessive manner. our findings indicate that phenotype-genotype correlations in nalcn are more complex than suggested so far. phenotype: in a young undiagnosed patient with developmental delay, intellectual disability and craniofacial dysmorphic anomalies, whole-exome sequencing (wes) identified a de novo insertion in the gene arid b, known to cause the rare congenital coffin-siris syndrome and nicolaides-baraitser syndrome, respectively. methods: as part of the midas genotype-phenotype-correlation project, we sequenced the index patient and his healthy parents on a next-seq platform (illumina, san diego, ca, usa) and performed a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed using the sureselect human all exon kit v (agilent, santa clara, ca, usa) to target most of the over . genes. the libraries were sequenced to approximately -fold mean coverage as bp paired end reads. % of the target region was covered -fold or higher. data analysis and variant evaluation was performed using the clc genomic workbench . (qiagen, hilden, germany) and annotations from commercial as well as public databases (dbsnp, hgmd, clinvar, exac). results: we identified a de novo bp insertion in the arid b gene, causing a frameshift mutation that leads to the truncated protein. arid b is part of the atp-dependent chromatin remodeling baf-complex, which is abstracts tient affected by psoriasis carried the surrogate marker snp rs -a for the psors risk variant hla-cw haplotype homozygously; and the same snp was found heterozygous in his prp-affected father. neither the pathogenic variant in card , nor the risk variants for psoriasis described above were found in the healthy mother. whole exome sequencing revealed genetic variants, predicted to have serious consequences in further genes involved in the nf-κb as well as the notch pathway. these variants either segregate with prp or are present in the psoriasis affected individual only. the presence of an individual carrying the same card mutation as his prp-affected relatives but suffering from psoriasis instead strengthens the relation between prp and psoriasis, which has been repeatedly suggested in literature. we propose a balance between familial prp and psoriasis in the family investigated in this study and present genetic variants, which might influence this balance in addition to variants in card . wilson's disease (wd) is an autosomal recessive disease resulting from copper (cu) excess due to mutations in the atp b gene coding for a cu-transporting atpase. wd pathogenesis, however, can not only be explained by gene coding mutations since phenotypes exhibit strong variations despite the same exonic dna makeup in the gene. also in several patients with clinical wd symptoms no gene coding variants are detectable. our former studies revealed decreased liver atp b mrna expression in some wd patients. this decrease was not only observed in patients with nonsense atp b mutations leading to rapid mrna decay, but also in patients with missense mutations and also in some patients with suspected wd without atp b mutations. patients with low atp b expression presented with a more fulminant disease progression. however, we could not detect mutations in the atp b promoter region (c.- to atg) in those patients. there are possibly other deregulating mechanisms responsible for decreased atp b mrna expression. up to now, atp b transcriptional regulation is only poorly characterized. it is known, that four metal responsive elements (mre a, c, d and e) are located within the atp b promoter. gene regulation through mres is often metal-dependent. liver atp b mrna expression revealed also to increase under cu addition in several species by an unknown mechanism. up to now, only one atp b transcription factor (tf), the mrea binding ku protein, is known. the aim of our work was to further analyze the regulation of the atp b gene, especially through mres. to screen for tf-mre interactions and to narrow down the binding site of tf, we performed electrophoretic mobility shift assays (emsa) by incubating nuclear extracts of the liver cell line hle with probes corresponding to atp b mrec, d and e. to identify mre-binding tf matinspector analysis was performed. identified candidate tf were coexpressed with atp b promoter-driven reporter gene to evaluate their impact on reporter gene expression. one in the reporter assay positively tested tf was validated by different emsa experiments. further it was overexpressed with and without addition of metal ions in hle to investigate the impact on endogenous atp b expression. we showed that tf mtf is able to bind to mree within the atp b promoter and significantly increases atp b promoter driven reporter gene expression. mtf binding was primarily mediated by the first three bases of the mre consensus sequence. also for mrec and mred specific protein interaction could be shown and the protein binding site was narrowed down by emsa with protein identification still pending. fur-polyglutamine diseases is the formation of the so called neuronal intranuclear inclusion bodies (nii). as ataxin- is predominantly located in the cytoplasm, the formation of protein aggregates in the nucleus require a nucleocytoplasmic shuttling of ataxin- . we already demonstrated in vivo using transgenic mouse models that the toxicity of expanded ataxin- depends on its intracellular localization: while nuclear ataxin- gave rise to a strong phenotype with a high number of protein aggregates, purely cytoplasmic ataxin , however, even with a highly expanded polyglutamine repeat ( glutamines), was not able to induce a phenotype and even did not aggregate. we further identified and characterized intracellular transport signals (two nuclear export signals, nes, and one nuclear localization signal, nls) within the coding sequence of ataxin- . therefore, it is evident that proteins involved in the nucleocytoplasmic transport machinery recognize these localization signals, control the intracellular localization of ataxin- , thereby influence the toxicity and aggregation of ataxin- and, thus, the pathogenesis of sca . we now screened a library of transport proteins in order to identify the transport protein which is critically involved in the nucleocytoplasmic shuttling of ataxin- . we indeed identified a transport protein which modifies both the formation of aggregates and the intracellular localization of ataxin . while the overexpression of this protein moved ataxin- into the nucleus, its downregulation kept it out of the nucleus. we replicated this correlation in vivo in drosophila and observed in addition to this again a clear link between the intracellular localization of ataxin- and its toxicity i. e. its ability of induce neurodegeneration and a behavioral phenotype. likewise we even confirmed in a mouse model of sca the importance of the identified transport protein as its knockout largely prevented ataxin- from aggregating and alleviated behavioral and movement deficits. understanding the mechanisms behind the intracellular transport of ataxin- could give us clues into the pathogenic functions of expanded ataxin- and ways to mediate the progression of neuronal degeneration in sca . evidence for genetic factors outside card influencing the phenotype of a family with familial pityriasis rubra pilaris and psoriasis familial pityriasis rubra pilaris (prp) is an erythematous inflammatory skin disease caused by heterozygous activating mutations in card , a known activator of the nf-κb pathway. different genetic variants within card have been associated with psoriasis. the purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with prp in order to determine the mutation responsible for this severe skin disease in the three affected family members. a father, three of his adult children as well as the mother of one child affected by prp were investigated clinically. in addition we extracted genomic dna from the blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. clinical investigation confirmed that the father and two of his children were affected by familial prp, with the skin showing the characteristic pattern of prp, early onset and chronic course. a third child was unaffected by prp, suffered however from psoriasis. the mother of one child affected by prp showed no sign of skin disease. genetic investigation revealed a heterozygous missense mutation in exon of card , c.[ c>t], p. present in all investigated individuals with prp or psoriasis. the same mutation has been described before as being pathogenic in a different family with prp. regarding genetic variants associated with psoriasis, we found the risk alleles of three coding variants in card : rs , c. short stature is a common condition of great concern to patients and their families. in most cases it is genetic in origin but the underlying cause often remains elusive due to clinical and genetic heterogeneity. in an unbiased approach we carefully phenotyped patients and randomly selected for whole exome sequencing. sequence variants were analyzed for pathogenicity and the affected genes characterized regarding their functional relevance for growth. all patients received extensive clinical and endocrinological examinations, careful clinical genetic phenotypic evaluation followed by targeted diagnostic assessment for suspected diagnoses. we identified a known disease-cause in only % of patients, the most common causes being cnvs found in %, followed by syndromic monogenic causes in % and turner syndrome in %. whole exome sequencing identified additional mutations in known short stature associated genes ( ) in % of patients who manifested only part of the symptomatology precluding an early clinical diagnosis. here, heterozygous carriers of recessive skeletal dysplasia alleles (acan, npr ) were a surprisingly frequent cause of idiopathic short stature found in . % of cases. we next selected known short stature genes with mutations for pathway analyses of the affected proteins and found that % are involved in the main functional categories cartilage formation, chromatin modification and ras-mapk signaling. in addition we identified further strong candidate genes, of which seven had deleterious mutations in at least two families. interestingly, % of these candidate genes are involved in the main functional categories already identified for the known short stature associated genes further supporting their pathogenicity. finally, in % of the sequenced individuals our findings were of significant clinical relevance regarding preventive measures, symptomatic or even targeted treatment. besides evaluation for orthopedic or developmental issues especially screening for neoplasias (trim , ptpn , nf ), symptomatic treatment for chronic kidney disease (clcn ) and targeted treatment for severe hypertension (pde a) were of clinical relevance for the affected individuals. these results demonstrated that deep phenotyping combined with targeted genetic testing and whole exome sequencing is able to increase the diagnostic yield in short stature up to % with concomitant improvement in treatment and prevention. rigorous variant analysis considering phenotypic data further led us to the identification of further probable novel candidate genes. thermore, we found the endogenous atp b mrna expression to be significantly increased in hle after cu treatment or cu treatment and concurrent mtf overexpression. sole mtf overexpression did not alter atp b expression. we newly identified mtf to bind mree within the atp b promoter. its in vivo role in the pathogenesis of wd needs to be further elucidated. modifying genes have been identified for lung function in cystic fibrosis [ ] , disease severity [ ] and several comorbidities [ ] . within the european cf twin and sibling study, we focus on genes that modify the basic defect, assessed as defective chloride conductance in cftr-expressing epithelia, which we could describe by an association study on patient cohorts selected for informative endophenotypes among f del-cftr homozygous patients [ , , ] . as a first example, rs in fas modifies fas gene expression (p = . , data from intestinal biopsies [ ] ), cf disease phenotype (praw = . , comparing concordant mildly affected f del homozygous sib pairs and concordant severely affected sib pairs; praw = . , comparing unrelated f del homozygous index cases without residual chloride secretion by nasal potential difference measurement (npd) to patients with cftr-mediated chloride secretion; [ ] ) and alters binding affinity for the transcription factors nf-kbp , nf-kbp and hif a [ ] which govern the cellular response to infection and hypoxia. as a second example, the transcription factor ehf, derived as a positional candidate from a north american genome wide scan [ ] , is associated with npd-defined phenotypes (praw = . , comparing index cases with high response to amiloride in npd to index cases with low response to amiloride in npd; praw = . , comparing index cases without chloride secretion in intestinal current measurement (icm) to index cases with cftr-mediated residual chloride secretion in icm [ ] ) and affects the transcriptome of cf patients' intestinal biopsies in favor of a better processing of f del-cftr [ ] which we could confirm in epithelial cell lines as sirna provided against ehf results in a downregulation of mgat and mgat , both of which are key enzymes for the complex glycosylation of proteins such as cftr. these examples indicate that small, albeit carefully selected subpopulations facilitate the identification of genetic variants by an association study that can be validated in functional assays. furthermore, we suggest that while the selection of subsamples within a population with a rare disease such as cystic fibrosis results in a loss of power, findings obtained for more than one endophenotype are indicative for a true-positive finding of a modifying gene. finally, transcriptional regulation influences the cf basic defect. interference with these pathways may results in better f del-cftr maturation, leading to better cftr function in patient's tissue and thereby promoting health in cystic fibrosis. funding by: deutsches zentrum für lungenforschung dzl; mukoviszidose institut ggmbh abstracts at presentation at age years her head circumference was on the th centile, her height below the rd centile, and her weight on the rd centile. she showed a disproportional short stature and mild skeletal signs like clinodactyly of the ths fingers. minor facial dysmorphisms included an oval face, epicanthic folds, upslanting palpebral fissures and a bulbous nose. karyotype was normal and copy number variants were excluded by high-resolution cma. as no aetiological diagnosis could be made clinically we performed whole exome sequencing (wes) and detected a homozygous splice site mutation (c. + g>t) in pik c a (phosphatidylinositol- -phosphate -kinase catalytic subunit type alpha) (nm_ ). sequencing of rt-pcr products from cdna of patient's fibroblasts showed in-frame skipping of exon and , equally affecting all known isoforms. phosphatidylinositol -kinases (pi ks) are lipid kinases involved in a large set of biological processes, including membrane receptor signaling, cytoskeletal organization, and endocytic trafficking. pi kc a is ubiquitously expressed and has been proposed to play an important role in clathrin-mediated endocytosis and regulation of phosphatidylinositol -phosphate (ptdins p) levels. furthermore pik c a has been implicated in the biology of the primary cilium. the patient's distinct phenotype resembles the previously described phenotype in pik c a hypomorphic mice with pre-and postnatal growth retardation and a broad spectrum of renal abnormalities. the complete knockout of mouse pik c a showed embryonic lethality. ongoing studies on the exact consequences of the splicing defect will determine if and how much residual wild-type transcript is retained and if this is a hypomorphic variant. this case is to our knowledge the first description of a pik c a human phenotype. adaption of the crispr/cas genome editing system as a platform for the mutation of nipal as a representative of arci-associated genes in hela cells n. ballin, m-a. rauschendorf, j. fischer institute of human genetics, freiburg, germany autosomal recessive congenital ichthyosis (arci) is a rare genetic disorder with known disease-causing mutations in genes. functional implementations of identified mutations are in most cases still unknown, which is amongst others due to the limited amount of skin biopsies of arci-patients. thus, suitable cell culture models for the investigation of keratinocyte differentiation are highly needed. during the last years the prokaryotic clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated (cas) system has been turned into a potent tool in the field of genome engineering. the cas endonuclease is directed by a short guide rna (grna) to its target sequence, where it generates a dna double strand break (dsb). as cellular repair mechanisms often fail in reconstituting the original sequence, insertion/ deletion (indel) mutations occur potentially leading to a complete gene knock-out (ko). consequently, the crispr/cas system offers a simple rna-programmable tool for in vitro mutation of arci-associated genes. hence, this system was applied in hela cells to target nipa like domain containing (nipal ), the second most frequently mutated gene in arci patients. in this context, functional studies were used to validate nipal as suitable target in hela cells. successful application of the crispr/cas system lead to the generation of a new clonal hela cell line carrying a one basepair insertion in nipal exon (c. insg (ccds . ) ). this mutation was further characterized and potentially results in a complete ko of nipal . this system can now be used as a basis for targeting further arci-associated genes and for transferring the system into keratinocytes, which are the amyotrophic lateral sclerosis (als) is a late-onset progressive, neurodegenerative syndrome. most als cases are sporadic (≈ %). in familial forms, mutations in several different genes have been identified with a repeat expansion in c orf and mutations in sod being the most prevalent. no non-genetic cause of als has been identified. since there are no overt clinical or pathoanatomical differences between sporadic or familial cases, de novo mutations have been suggests in disease pathogenesis and two previous studies provided some evidence for this hypothesis. we present data of patient-parents trios from an international collaborative study. by whole exome sequencing we identified non-synonymous de novo mutations in the patients, however all of them occurred in different genes. there was no concordance between the mutated genes found in our trio set and the two earlier smaller trio studies. in silico analyses suggest that none of the here identified mutations are part of any of the previously postulated molecular pathways. also, gene-gene-interaction analyses failed to find an enrichment of interacting genes. lastly, we demonstrate that the de novo mutations in als patients in this and the two earlier studies are located in genes prone for de novo mutations in general. our results thus indicate that, in contrast to previous reports, de novo mutations do not seem to be a major contributor for als. disproportioned short stature and multiple anomalies in a patient with a homozygous pik c a mutation -a new ciliopathy? we present a year old female with disproportional growth retardation, eye and renal anomalies. she was born small for gestational age as the first child of healthy, consanguineous tunisian parents at weeks after an uneventful pregnancy ( g, cm). during her first year of life, there were severe feeding problems with regurgitation and she developed postnatal growth retardation. developmental milestones were normal. during the second year the girl developed strabismus in both eyes. later on, a cataracta polaris anterior, anomalies of the cornea, hyperopia and progressive retinal degeneration led to profound visual impairment. furthermore, a right kidney agenesis and a complex tubulopathy, as well as increased echogenicity of the single left kidney were diagnosed. r. brumm, m. schmuck, y. dinçer, s. schulz, s. wilson, i. rost, hg. klein, sh. eck center for human genetics and laboratory diagnostics dr. klein, dr. rost and colleagues, martinsried, germany next-generation sequencing may lead to a significant improvement in diagnostic yield for rare, heterogeneous disorders through the ability to simultaneously sequence all genes contributing to a certain indication at a cost and speed that is superior to traditional sequencing approaches. on the other hand, the practical implementation of ngs in a clinical diagnostic setting involves a variety of new challenges which need to be overcome. among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. especially the variant interpretation emerges as current bottleneck of the diagnostic workflow. here we present midas (multiple integration of data annotation software), a central software system for data integration in a diagnostic laboratory. the goal of midas is to construct a modular software system to integrate data from laboratory information management system (lims), data from the routine sanger sequencing workflow as well as ngs sequencing results and correlate the identified variants with the patients' phenotypical features to aid in variant interpretation and accelerate reporting. the phenotype is systematically recorded using the human phenotype ontology standard nomenclature. in particular, genotype-phenotype correlations identified in one patient are made available for all other cases, to aid the interpretation and build a comprehensive knowledge base. the midas software may thus serve as central information system all diagnostic patient data. midas is implemented in java using direct database access via jdbc, and javafx as graphical user interface. its architecture is designed modular including a dynamic module loader, a user management with ldab connection and basic search functionality. according to available modules the user management and search form are adjusted; granting access for module specific views. as an advantage of this architecture, other molecular diagnostic data, such as arraycgh or mlpa results can easily be integrated by implementing new modules. midas aims to aid molecular diagnostics by simplifying and accelerating data analysis and interpretation, improving patient care. midas is being developed as part of a prospective multicentric study including clinical, diagnostic and software development partners. a grant by the bavarian ministry of economy, media, energy and technology is used to fund this effort. institute of human genetics -medical faculty -rwth aachen university, aachen, germany, izkf aachen -rwth aachen university, aachen, germany with the implementation of next generation sequencing (ngs) based assays as key tool in dna-sequencing, conventional sanger-sequencing has become a standard method to verify single nucleotide polymorphisms (snps) of interest. however, designing specific pcr-and sanger-sequencing primers has always been a rather time consuming task in terms of searching the right genomic sequence, considering known snps that could result in allele-specific amplification, and converting selected primer-sequences to upload them to web-based services. to circumvent this, we developed optimus primer, a python script, that automatically designs respective primers. the script uses the database of the ucsc genome browser to download positional information of genomic sequence (refseq gene: hg ) and common snps (snp common: hg ) in the region of interest. the genomic sequence is downloaded via the ucsc primarily affected cell type in the skin of arci patients. furthermore, it is only a small step to expand the system from generating gene ko to gene editing allowing the introduction of patient-specific mutations in non-patient derived keratinocyte cells. hence, setting-up the crispr/cas system to target an arci-associated gene is an important starting point for future studies to investigate pathogenic effects of arci-causing mutations and to understand arci pathogenesis on a molecular level. in this context, the transfer of the established protocol into keratinocytes offers the possibility to generate d cell culture models of mutations in arci-associated genes and allows in vitro investigation of their implications on the differentiation process. this system would further represent an organotypic model of arci-disease with potential application in screening and identification of chemical compounds for the complementation of existing clinical therapies. background: telomeres cap and protect chromosome ends from degradation and fusion, and are therefore essential for maintaining chromosome stability and genomic integrity. they have a length of to kb (depending on age, sex and cell populations). due to the end-replication problem there is a continuous telomere loss of to bp/cell cycle and thus throughout lifetime. in laboratory practice different methods of telomere length measurement are used to identify patients with bone marrow failure syndromes (e. g. dyskeratosis congenita, aplastic anemia), hematological diseases, or other telomeropathies. each telomere length measurement method has its advantages and disadvantages regarding material required the complexity and feasibility of the method and other parameters. methods: in this study we compared and validated four different methods for telomere length measurement, i. e. southern blot analysis, quantitative pcr (qpcr), quantitative fluorescence in situ hybridization (t/ c-fish) and flow cytometry-fish (flowfish). whenever possible, edta and/or heparin blood samples were collected from a population of healthy individuals of different age groups (newborn - years). depending on the method dna (southern blot and qpcr), metaphases (t/ c-fish) or rather vital cells (flowfish) were analyzed. results: comparison and validation of the telomere length measurement methods allowed us to calculate percentiles for all age groups. percentile curves could be used in diagnostic to identify patients with short telomeres. all methods showed acceptable accuracy, but equally imply the necessity of validation and appropriate controls in each experiment. here, flowfish was the most precise, accurate and reproducible method compared to the other methods. discussion: our study emphasizes the influence of expertise and experience that is required in order to produce robust and reliable telomere length analyses. here, we provide advice on how to choose the appropriate method in general and for individual cases to safely discriminate between natural variability and pathological telomere shortening in individual cases. in the next generation sequencing age, the strongest challenges have shifted from genotyping to handling the myriad of variants detected. whole exome sequencing yields tens of thousands of coding and non-coding variants, a number increased to millions if the whole genome is sequenced. to master this mountain of data, computer-based identification of potential disease mutations is absolutely indispensable. however, current computational strategies are usually generated by computer scientists without integrating human geneticists into software development. this often leads to tools which fulfil their main purpose but are not ideally suited to the needs of human geneticists. to assess the relevance of a suggested disease mutation, geneticists need detailed information about the effect on the protein and about the gene or protein itself. to close this gap, we have developed mutationdistiller, a web-based tool for user-driven variant prioritisation based on biological disease properties. its analysis includes the potential role of a mutated gene in pathogenesis as well as the estimated effect of a variant on gene/protein function. thus, mu-tationdistiller allows human geneticists to use every piece of information they consider relevant. unlike similar tools, its input goes beyond the human phenotype ontology and can include complete diagnoses, biological pathways, gene expression, and gene function. potentially harmful variants are identified by mutationtaster, which provides a deleteriousness score together with data on the actual effect of the variants. mutationdistiller is not restricted to non-synonymous variants but can also handle intragenic non-coding or synonymus variants. moreover, the program incorporates the known modes of inheritance of disease genes and the genotype of the queried variants (including compound heterozygosity). the output page provides all information on one site: the core component is a concise overview table of the most likely disease genes and variants. here, the most crucial information such as the variants' effect on the protein, frequencies in polymorphism databases and known diseases caused by mutations within this gene and their mode of inheritance are listed. more detailed gene and disease information and hyperlinks to external data are provided below, hence offering a comprehensive overview of the available knowledge. this allows geneticists to draw their own conclusions without any tedious collection of relevant information from the internet. a beta-version of the program is freely available at http://www.mutationtaster.org. m. jäger , , m. schubach , t. zemojtel , k. reinert , d. m. church , p. n. robinson , , institute for medical and human genetics, charité, berlin, germany, bcrt, berlin, germany, institute for bioinformatics, department of mathematics and computer science, freie universität, berlin, germany, x genomics, pleasanton ca, united states, department of mathematics and computer science, freie universität, berlin, germany, the jackson laboratory, farmington ct, united states with the grch human genome release in the genome resource consortium extended the previous linear, "golden-path" paradigm of the human genome and introduced a more graph-like model in the sense of regions with alternate loci, representing common alterations in sequence and structure. in whole-genome sequencing (wgs) these stretches of sequence are largely but not entirely identical between the primary assembly and an its corresponding alternate locus can result in multiple variant calls against regions of the primary assembly. this results in characteristic and recognizable patterns of variant calls at positions that we term alignable scaffold-discrepant positions (asdps). we developed an algorithm (asdpex) that analyzes these patterns in the structurally variable regions of the current grch genome assembly. a heuristical approach then infers whether the pattern of variant calls of a sample contains sequences from the primary assembly, an alternative locus, or their heterozygous combination at each of these regions. api and common snps are annotated. in the following step primers are picked using primer (whitehead-institute). the target sequence is either the snp-containing exon in case of exons smaller than bp, or, the genomic region flanking the snp of interest. the positional information of the snp is gathered using the mutalyzer api (lumc). the last two bases of each primer are checked for all snps in the ' end (snp : hg ). the script will be integrated into a website for free easy access and usability (www.optimus-primer.com). the only information needed is the refseq id, the exon, and the cdna position (eg: nm_ . . :c. t>g). the script optimus primer will provide the user with primers in the widely used primer format. the users are also able to download the source from our website, and to run it on a local unix system as a command line tool. mutationtaster : moving towards a comprehensive evaluation of disease causing mutations o. ebner, jm. schwarz, d. hombach, m. schuelke, d. seelow charitè universitätsmedizin, berlin, germany mutationtaster is a free and user-oriented application for comprehensible evaluation of non-synonymous and synonymous as well as non-coding dna sequence variants. as + citations show, the software has been strongly embraced by the clinical and research community. however, its capacities for assessing the functional consequences of non-coding variants are still limited. while mutationtaster is able to predict the variant's effect on major intragenic regulatory features such as splice sites or polyadenylation signals, many other potential effects of intronic or utr variants are not covered by the software yet. variants in the non-coding region of the genome are frequent and thought to cause a substantial part of yet unsolved mendelian diseases. these variants can occur either in extragenic regulatory regions (see abstract on regulationspotter) or in the untranslated regions, including introns, of a gene. unfortunately, their effects are much harder to predict than those of non-synonymous variants. therefore, only few disease mutations outside the coding sequence have hitherto been found and experimentally confirmed. to close this gap, we are advancing the current version of mutationtaster to improve the analysis of intragenic non-coding variants. we do so by implementing additional tests for variants in the ' and 'utr to determine their effect on au-rich elements, microrna binding sites and the influence of secondary structure on gene expression. moreover, we plan to shift from the analysis of exclusively monogenic to complex diseases with cancer being the first disease model to be integrated into mutationtaster . already implemented enhancements of mutationtaster comprise the improved splice site analysis using maxentscan and the integration of mitochondrial polymorphisms from the human mitochondrial genome database. the addition of links to the integrative genomics viewer and the lof-metrics of the exac browser (exome aggregation consortium) streamline the usage of the mutationtaster results and enable researchers to get a detailed view of the relevant variants and associated genomic sequences. taken together, mutationtaster will offer much better capabilities to predict the disease-causing potential of intragenic variants than its predecessor. the software is freely available at http://www.mutationtaster.org neurocure exzellenzcluster, berlin, germany, neuropädiatrie charité-universitätsmedizin, berlin, germany, medizinische genetik charité-universitätsklinikum, berlin, germany, berliner institut für gesundheitsforschung bih, berlin, germany ifications, or genome-wide interactions. all variants which have the potential to influence one or several candidate genes are presented in a graphical interface and ranked according to their predicted effect on the target gene(s). instead of giving scores, we present the potentially affected regulatory features in an intuitive graphical matrix. the software can freely be used at http://www.mutationtaster.org p-techno- genecascade -a one-stop shop for finding disease mutations d. seelow , in the last years, our genecascade software suite for the elucidation of rare diseases has seen some major extensions, mainly for the discovery of non-coding mutations. the website contains a number of tools focusing on the different steps in studying genetic disorders. all applications are web-based, have easy-to-use interfaces and are aimed directly at human geneticists, without any need to install software, use the command line or to try to explain clinical features to it specialists. we think that software should adapt to the user -not the other way around. and, unlike other web shops, its use is completely free. homozygositymapper finds disease-linked regions in consanguineous families. users can upload genotypes from snp chips as well as wes and even wgs data. we display likely disease loci in intuitive graphical interfaces. the results can also be used in our 'downstream' applications such as genedistiller, mutationtaster, or mutationdistiller to identify the actual disease mutation. genedistiller provides a user-driven way to find the best candidate gene for a genetic disorder. users can specify various aspects of the patients' phenotypes and the results are presented in a comprehensive way without the need to manually query other internet resources to collect further information relevant to asses the disease potential. mutationtaster evaluates the disease potential of non-synonymous as well as of non-coding and synonymous dna variants within genes. it does not only display a prediction but also detailed information on the variants' likely effect on mrna and protein. it can either analyse single variants as found by sanger sequencing or complete vcf files from wes or wgs projects. regulationspotter lists information about potentially regulatory dna variants outside of genes. variants are ranked according to their predicted effect on gene regulation. in addition to a score, we provide a user-friendly summary of the functional effects a variant may have. mutationdistiller combines genedistiller and mutationtaster in a single application for convenient variant and gene prioritisation. it offers human geneticists much more freedom to enter the phenotype than similar applications and provides deeper information about the variant and the gene. cnvinspector is aimed at the study of copy number variants. users can upload their patients' (or cohorts') cnvs and compare them with their own healthy controls or public data stored in our database. we include deci-pher to indicate known diseases caused by cnvs at the same position. epossum examines the effect of dna variants on transcription factor binding. as tf binding site prediction is notoriously unreliable, we also give an indication of the statistical relevance of a result. the genecascade website can be accessed at http://www.mutationtaster.org. we investigate in-house wgs datasets and found that on average . ± . of the regions correspond to an alternate locus rather than the primary assembly sequence. filtering these genomes with our algorithm identified around variant calls per genome that colocalized with asdps. our findings suggest the potential of fully incorporating the resources of graph-like genome assemblies into variant calling. our algorithm already uses the information contained in the structurally variable regions of the grch genome assembly to avoid spurious variant calls in cases where samples contain an alternate locus rather than the corresponding segment of the primary assembly. the human phenotype ontology project provides three resources, the ontology of clinical features, disease-phenotype associations and algorithms that enable analysis of data that is described using hpo. this resource is being used for computational deep phenotyping and precision medicine. it enables clinical data integration for translational research. the hpo is being increasingly adopted in software, research projects and companies world-wide. we will discuss the progress and recent developments that the hpo project has made since . this will include the expansion of hpo for common (complex) diseases, novel algorithms for phenotype-driven analysis of genomic variation, cross-species mapping of phenotype data, translation of hpo into several languages and also the addition of a more patient-friendly terminology for hpo terms. millions of patients worldwide suffer from a rare genetic disease. to date, the omim database lists more than diseases with proven or suspected mendelian basis, but the molecular cause is known for less than %. although next generation sequencing has drastically facilitated the discovery of disease mutations, a substantial fraction of whole exome sequencing (wes) projects fail to identify the causal variant. this may be due to the fact that disease-causing mutations are not always located within the coding sequence. whole genome sequencing (wgs) could solve this problem, but we are not yet readily prepared to handle the vast amount of variants which are generated by this technique, as intuitive and reliable software solutions for variant evaluation are missing. the currently existing approaches are not well-suited for a diagnostic setting, because they output a battery of different scores whose interpretation is left to the users. however, human geneticists are specialists for the assessment of symptoms of genetic diseases -not for the analysis of numerical scores indicating different likelihoods of the occurrence of regulatory elements. this is especially true when it comes to the hundreds of thousands of extragenic variants found by wgs, for which effect predictions always face a high level of uncertainty. we consider it crucial to provide geneticists with the information they need to determine the significance of a variant, not to flood them with scores whose meanings are difficult to grasp. to facilitate the interpretation of extragenic variants, we are developing regulationspotter. in contrast to other approaches such as genomiser or cadd, we integrate as much knowledge as possible in a user-friendly fashion to pinpoint the variants which are most likely to disturb the expression of candidate genes. regulationspotter includes different data on regulatory dna elements such as dna methylation, transcription factor binding sites, histone mod-the human exon in the ' end of the murine huntingtin gene. by using a novel object recognition test with a h interval between sample and test phase we have found a profound deficiency of hippocampus dependent long-term memory in heterozygous transgenics. this phenotype was detected as early as weeks of age and is complementary to deficits that we have identified in the hdhcag mouse model previously. motor deficits as well as intranuclear aggregates are described at much later stages in both of these models. we have shown previously that in hd patients, mediated through mtor signaling, translation of mrna carrying expanded cag repeats is elevated (krauss et al., ) . we have also seen that the biguanid metformin antagonizes mtor signaling in neurons in-vitro and in-vivo (kickstein et al., ) . we show here that metformin, by interfering with the mtor kinase and its opposing phosphatase, pp a, regulates local protein synthesis in the brain and is able to suppress the production of disease making protein in early hd. furthermore metformin leads to a significant improvement of movement abilities in a c-elegans model for hd and to a rescue of early cognitive symptoms in the hdhcag animal model. these data suggest that metformin is a very promising candidate for early phase treatment of hd patients. allele-specific suppression of dominant-negative bestrophin mutations a. milenkovic, lm. braun, f. grassmann, b. h. f. weber institute of human genetics, regensburg, germany purpose: retinal pigment epithelium (rpe) differentiated from human induced pluripotent stem cells (hipsc) demonstrated degradation and mislocalization of mutant bestrophin (best ) protein in autosomal dominant best disease (bd). importantly, mutated alleles revealed a dominant-negative effect leading to an impairment of volume-regulated chloride transport, the basic function of the homo-pentameric best channel. here, our study aimed for a proof-of-concept to treat bd by selectively eliminating best mutant transcripts in patient-derived hipscs prior to rpe differentiation via the crispr/cas genome editing technology. methods: adult human dermal fibroblast were obtained from skin biopsies of bd patients and reprogrammed into hipscs. single guide rna sequences (sgrna) targeting best mutations were selected by the "optimised crispr design tool" (zhang lab, mit ) . editing efficiency and specificity of designed sgrnas were tested in hek cells using an established fluorescence-based assay. after transfection of hipscs the percentage of indel formation of on-and off-targets was determined by a pcr-based crispr/cas cleavage detection kit. cas -treated stem cell populations were analyzed for pluripotency and selected for full genome sequencing before differentiation to rpe cells. results: computational design of disease-causing best variants (n k, v m, s r, q r, a v, and i del) offered at least one sgrna with predicted high quality per mutant allele. the guide sequences were cloned into the cas -expressing plasmid px and co-transfected with pcag-egxxfp plasmids containing genomic fragments of ~ bp of either the mutated or wildtype sequence to the corresponding sgrna. as targeted cas cleavage results in reconstitution of the egfp expression cassette by homology dependent repair, the efficiency and specificity of cas cleavage was evaluated on a plate reader by quantifying egfp fluorescence after h of transfection. as a result, out of the sgrnas tested showed high allele-specificity and are now used for targeted genome editing in hipscs. conclusion: so far, there is no treatment for bd although the molecular pathology of best has recently been established. our proof-of-concept study aims to determine whether haploinsufficiency of normal best protein is sufficient to fully or partly restore cellular function in cells of primary bd pathology, namely the rpe. to this end, we will determine the degree of rescue (i. e. reconstitution of volume-regulated chloride conductance) by whole-cell patch-clamp analysis. if successful, our crispr/ cas -driven approach will be useful to treat other diseases with dominant negative effects of the mutated allele. p-therap- *** metformin rescues early cognitive symptoms in the hdhcag mouse model and is therefore a promising candidate for treatment of hd patients. huntington's disease (hd) is an autosomal dominant neurodegenerative disorder that is caused by an unstable glutamine (cag) trinucleotide repeat expansion within exon of the huntingtin gene and leads to cognitive decline and affects motor abilities. in the prodromal phase of the disease patients develop mood swings, personality changes and subtle cognitive impairment. close understanding of clinical signs and molecular mechanisms behind this early stage of hd is an important step for the development of a causal therapy. we have analysed a knock-in mouse model that carries cag repeats and single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation age-related clonal hematopoiesis associated with adverse outcomes clonal hematopoiesis and bloodcancer risk inferred from blood dna sequence age-related mutations associated with clonal hematopoietic expansion and malignancies cancer spectrum in dna mismatch repair gene mutation carriers: results from a hospital based lynch syndrome registry update on kleefstra syndrome characterization of a novel transcript of the ehmt gene reveals important diagnostic implications for kleefstra syndrome national institute of child health and human development abstracts p-monog- novel mutation in hoxc expand the mutation spectrum of pure hair and nail ectodermal dysplasia abstracts in conclusion, targeted sequencing represents an effective, fast, cost-efficient and flexible method, since new candidate genes can be added easily to the panel, for the sequential analysis in chh patients. furthermore, panel sequencing alleviates the uncovering of oligogenic inheritance in genetic traits like chh abstracts p-monog- neurodegeneration in the olfactory bulb and olfactory impairment in the ccdc -/-mouse model for retinal degeneration s. schreiber , e. petrasch-parwez one hallmark of sca and other mechanisms that govern ccdc -deficient degeneration, a detailed evaluation is performed in order to reveal processes that contribute to retinal degeneration during early postnatal development and adulthood. methods: the functional role of ccdc deficiency is investigated by two independent approaches: ) gene expression profiles at p and p are analyzed in retinal tissue of cccdc -/-and wildtype mice (genechip mouse gene . st array, affymetrix) followed by quantitative real-time pcr (qrt-pcr) and ) potential retinal interaction partners of ccdc protein are identified by yeast-two hybrid screening in the wildtype mouse and further analyzed by immunohistochemistry. results: using two screening methods (rna-expression profiles and protein interaction partners), our results indicate that ) the ccdc deficient mouse model reveals early changes in retinal rna gene expression already at p and the highest number of genes differential expressed at p . most expression differences were related to genes associated with the extracellular matrix. further genes are involved in retinal degeneration, angiogenesis, transcription factors and proteolysis. ) the ccdc protein interacts with the proteins eps (epidermal growth factor receptor kinase substrate ) and mpdz (multiple pdz-domain protein). both proteins are expressed in several retinal layers of the retina, confirmed by immunohistochemistry. moreover, mutations in the mpdz gene were already identified in patients with retinitis pigmentosa/leber congenital amaurosis. conclusion: expression profiles reveal expression changes at an early time point of retinal degeneration in the ccdc -/-mouse model enabling further studies on the role of genes and processes involved in early retinal degeneration. in addition, the interaction partners of ccdc , eps and mpdz, are the basis for further studies examining the pathways of retinal degeneration in the mammalian retina including man -and possibly contribute to future studies in man and human disease. ccdc null mutation causes retinal degeneration and dysfunction of medical and molecular genetics and skeletal dysplasia multidisciplinary unit hospital unit of genetics of neurodegenerative and metabolic diseases sel- , ws - , p-compl- ws - p-cling- , p-cling- ws - , ws - , p-cling- , p-cling- , p-cling- ws - , p-cling- , p-cling- , p-monog- p-cancg- p-cling- , p-cling- , p-cling- p-cling- , p-cling- ws - , ws - , p-cling- ws - , p-cling- p-cancg- , p-cancg- , p-cling- ws - medizinische genetik · ws - , p-cling- ws - , p-cling- ddd study . p-cling- ws - , ws - , p-cling- , p-compl- ws - , p-cling- , p-cling- distelmaier f. ws - ws - , p-basepi- , p-cancg- ws - , ws - , p-cling- , p-cling- , p-cling- ws - , p-cling- , p-monog- ws - ekici ab. ws - , p-monog- sel- p-cancg- , p-techno- plen , ws - golla a dfg-workshop göpfert mc sel- , sel- haferlach c. ws - haferlach t. ws - ws - , ws - , p-compl- , p-compl- p-cancg- , p-cancg- , p-cancg- gilissen c. ws - ws - , p-basepi- , p-cling- , p-cling- p-cling- , p-cling- , p-cling- , p-cling- ws - , p-cling- p-cling- , p-cling- , p-cling- ws - , p-compl- p-monog- medizinische genetik · ws - hüffmeier u. ws - ws - , p-cling- seq consortium ws - ws - , ws - , p-compl- p-cling- , p-cling- , p-cling- , p-cling- ws - , p-cancg- ws - , ws - , ws - , p-compl- p-cancg- , p-cancg- ws - ws - , p-cling- , p-cling- , p-cling- , p-cytog- ws - , ws - , p-compl- , p-compl- , p-compl- ws - , p-cling- p-cling- , p-cling- p-cling- , p-cling- p-cling- , p-cling- , p-cling- ws - , p-cancg- ws - , p-cling- ws - , ws - , p-cancg- khor cc. ws - sel- , ws - ws - medizinische genetik · p-cling- , p-cling- , p-cling- ws - , p-compl- , p-compl- ws - , p-cancg- ws - , p-cling- meggendorfer m. ws - ws - , p-cancg- p-cling- , p-cling- , p-cling- p-cling- , p-cling- ws - , p-cancg- ws - , ws - , p-monog- ws - ws - ws - , p-cling- pasutto f. ws - nanda i. ws - nöthen mm. ws - , ws - , ws - , ws - , ws - ws - ws - , ws - , ws - , ws - , p-cling- , p-compl- , p-compl- sel- medizinische genetik · p-cancg- schnapp l. ws - ws - , p-cancg- , p-cancg- , p-cling- ws - , p-basepi- , p-cling- p-cling- , p-cling- p-cancg- , p-cling- salaverria i. ws - ws - , p-compl- ws - , ws - , ws - , p-cancg- , p-cling- , p-cling- , p-cytog- , p-monog- , p-monog- p-cancg- , p-cancg- , p-cling- , p-cling- ws - sel- , ws - , p-compl- the international gamos consortium e ws - , ws - , ws - , p-cancg- , p-monog- , p-monog- sel- , ws - ws - , p-cytog- p-cancg- , p-cling- , p-cling- p-cancg- , p-cancg- , p-cling- stengel a. ws - p-cling- , p-cling- ws - , ws - , ws - , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- p-cancg- , p-cling- ws - ws - , p-cling- ws - witsch-baumgartner m. p-cling- p-cancg- p-cancg- wunderle m. ws - zechner u. ws - , ws - , p-basepi- , p-cling- , p-cling- , p-cling- ws - , p-cancg- , p-cling- edu , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- p-cling- , p-cling- , p-cling- wiesener a. ws - , p-monog- ws - , ws - , p-cancg- , p-cling- , p-compl- , p-monog- ws - ws - , p-cancg- autorenverzeichnis zenker m. ws - , ws - , p-cling- , p-cling- dialog mit shg, p-cling- ws - , ws - , p-cling- , p-monog- ac. koller , , j. strohmaier , ku. ludwig , , fc. degenhardt , m. wulff , , d. breuer , , l. winkler , , f. neukirch , , a. maaser , , a. forstner , , s. sivalingam , , a. reif , a. ramirez , w. maier , d. rujescu , i. giegling , h. thiele , p. nürnberg , , a. fischer congenital hypogonadotropic hypogonadism (chh) is a rare and clinically and genetically heterogeneous disorder. chh is characterized by incomplete or absent puberty caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (gnrh) neurons, disturbed secretion or action of gnrh, or both. chh is often associated with anosmia and is then termed kallmann syndrome (ks), as well as with other phenotypes like unilateral kidney agenesis, skeletal abnormalities, midline malformations, and hearing loss. x-linked, autosomal-dominant, and autosomal-recessive, as well as di-and oligogenic inheritance has been described for chh. in the meantime a multitude of genes has been reported to be associated with chh. actually, in fewer than % of the chh cases the underlying genetic cause can be identified. we analyzed a total of patients with chh ( female/ male) by using targeted sequencing of chh-associated genes kal , chd , fgf , fgfr , fshb, gnrh , gnrhr, hs st , kiss , kiss r, nsmf, prok , prokr , spry , tac and tacr and identified pathogenic mutations in chh patients of our cohort. mutations were detected in kal ( patients), tacr ( patients), prokr ( patient), hs st ( patient) and gnrhr ( patient). furthermore, we found in two patients with described pathogenic mutations (one patient with prokr mutation and the other with kal mutation, respectively) additional mutations in nsmf gene and tacr gene, respectively, suggesting digenic inheritance in these cases. pain is necessary to alert us to actual or potential tissue damage. specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (hsan). although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the flvcr (feline leukemia virus subgroup c receptor ) gene, which encodes a broadly expressed heme exporter. different flvcr isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. mutations institute of human genetics, bonn, germany, the rild institute, wonford, exeter, uk introduction: ectodermal dysplasias (eds) are a large group of heterogeneous genetic disorders characterized by abnormal development in ectoderm-derived tissues and organs including skin, hair, and nails. among the eds, pure hair and nail ectodermal dysplasia (phned) is a rare genodermatosis characterized by nail dystrophy and sparse or absent hair on the scalp. materials and methods: a family of iranian origin was enrolled in this study. two children from a consanguineous marriage are affected from phned. in addition, the father has alopecia areata (aa) but does not show any nail dysplasia. the mother is unaffected. the paternal and maternal grandfathers had nail dysplasia almost similar to the siblings but did not manifest any hair loss or aa. homozygosity mapping and genedistiller analysis were performed to identify candidate genes. sanger sequencing was used to localize the mutations. results: in total, we identified homozygous regions with almost candidate genes. among these genes were also krt and hoxc , already known to be related to the phenotype of our patients. therefore, we focused our additional analyses on krt and hoxc . sanger sequencing showed a so far unknown homozygous insertion of bp in exon of hoxc . conclusion: we identified an unknown mutation for phned which expands the spectrum of mutations for phned. the hair loss of the father seems rather be due to a distinct type of hair loss, namely aa, which is quite common in the general population. however, the nail dysplasia from both grandfathers is unclear and cannot be examined anymore. it still remains unclear if the nail dysplasia in the grandfathers was due to the same mutation in hoxc or is based on a different mutation. generation of a cell culture model for epidermodysplasia verruciformis by knock-out of ev , a novel gene involved in this genodermatosis e. imahorn , m. aushev , sj. de jong , e. jouanguy , , , jl. casanova , , , ph. itin , , j. reichelt , epidermodysplasia verruciformis (ev) is a rare hereditary skin disease leading susceptibility to certain types of cutaneous human papilloma viruses, mainly β-hpv, and a high risk for development of cutaneous squamous cell carcinoma. homozygous or compound heterozygous loss of function mutations either in tmc or in tmc have been described in several ev patients, but more than / of affected families do not have mutations in one of them. a third gene (ev ) has recently been identified to be mutated in ev patients without tmc or tmc mutation. investigations on the function of this gene may elucidate pathomechanisms of ev. we aim to generate an ev deficient model cell line to study the effects of ev despite the scarcity of patient material. detection of deletions during diagnostic massive parallel ngs gene panel sequencing neuronal differentiation at a later time point. a novel mutation c. a>g, pser gly in des gene in a family with catecholamine polymorphic ventricular tachycardia s. komatsuzaki, p. villavicencio lorini, k. hoffmann institute for human genetics, university hospital halle, germany background: myofibrillar myopathy (omim ) is a heterogeneous neuromuscular disease characterized by progressive muscle weakness, partially with cardiomyopathy and/or arrhythmia. pathohistological examinations show desmin-positive protein aggregations. the mutations in des, cryab, bag , myot, ldb , flnc, fhl , and dnajb have been identified in patients with myofibrillar myopathy. desmin is a intermediate filaments and composed of non-helical n-and terminal head domain and a central a-helical rod domain. till now more than mutations myofibrillar myopathy were identified in patients with and a genotype-phenotype was reported: mutations in rod domain tend to cause neuromuscular symptoms, and mutations in head domain is associated with cardiac symptoms. the head-domain is a serine rich domain and phosphorylated by protein kinase c. almost all reported mutations in head-domain in des are at serine residues (e. g. s i, s f, s f, s f, s y, s t). these findings suggested that the serine residues in head-domain could play an important role in biological function of desmin. here we report a novel mutation c. a>g, ps g in des in a family with catecholamine polymorphic ventricular tachycardia. case: a years old patient developed a syncope in a cold winter. the cardiological investigations revealed a diagnosis of catecholamine polymorphic ventricular tachycardia and the implantation of permanent pacemaker was indicated. his older brother, father and paternal uncle suffered from arrhythmia and all of them received a permanent pacemaker implantation at around years of age. his cousin was died at age of years due to sudden cardiac arrest. we performed a genetic analysis for ryanodin-rezeptor -gen and lamin a gene and no mutation in these gene was identified. next, we performed an exome sequencing. here a novel heterozygous mutation c. a>g, ps g in des was identified. this mutation was also identified in uncle of index patient. the c. a>g, ps g in des was not reported neither in human genome project, nor in exome aggregation consortium. a mutation at serine (p.s i) was previously reported in patients with myofibrillar myopathy with cardiac symptoms. based on these findings, it was strongly supposed that the p.s g in des gene is a causative mutation for myofibrillar myopathy with arrhythmia in our patients. discussion: recent studies suggest that the mutations in des influence not only on muscle stability and myocardial force generation, but also impaired ubiquitin proteasome system, which could be caused by aggregated desmine. generally, phosphorylation of serin and threonine in head-domain of desmin is related to disassembly of filaments. therefore the p.ser- gly mutation in des could cause altered phosphorylation of desmin. the functional abnormalities caused by pser gly in des gene need to be studied. gene dosage manipulation of the chromatin organizer ctcf in the nervous system of drosophila melanogaster results in neurological and morphological phenotypes e. konrad, a. gregor, m. brech, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germanythree-dimensional organization of eukaryotic genomes is crucial for temporal and spatial regulation of gene expression. architectural proteins, like the ccctc-binding factor ctcf are responsible for establishing and maintaining this organization. ctcf is involved in virtually all chromatin regulating processes including enhancer function, insulation, alterna-for this purpose we used the crispr/cas system to delete the ev coding sequence in an immortalized keratinocyte line. after isolation by facs, single cell clones have been expanded. we screened clones for deletion of the whole gene as well as for the expression of ev . three clones showed no detectable gdna sequence or expression of the ev gene. we found only one wildtype clone without deletion of ev or alterations near the cas cut sites. all clones have been characterized by a snp-array as well as sequencing of the knockout site and the most probable offsite targets. these ev deficient keratinocyte lines are the first cell culture model for ev. it will be a valuable tool to identify cellular pathomechanisms of the disease and allow insight into the control of β-hpv in the general population. hemizygous loss of function of single x-chromosomal genes is the most frequent cause for genetic intellectual disability (id). while a long list of gene mutations have so far been described to be responsible for the disease phenotype, little is known about the underlying neuronal mechanisms. reprogramming of somatic cells into stem cells (ipscs) followed by differentiation into neuronal precursors (npcs) is an important tool for translating research allowing an understanding of network dysfunction in id patients. opitz bbb/g syndrome (os) is characterized by a number of ventral midline defects combined with learning disability, developmental delay and intellectual disability. it is caused by mutations in the x-linked mid gene, which, as we have shown previously, regulates mtor dependent local protein synthesis. in a mouse model, loss of mid function leads to significant disturbance of axonal outgrowth. we have generated ips cells from several patients with os and from one mother that carries a loss of function mutation in the mid gene. by sorting cell clones after reprogramming we have established ips cell clones from this female carrier of a bp deletion in the mid gene (c. _ delctcc) either expressing from the mutated x-chromosome or the non-mutated x-chromosome and have shown that indeed the generated ipsc-clones express either % of the mutated or % of the wildtype mid . we determined this directly using an allele-specific mid -rt-pcr and indirectly by comparing the methylation of humara-alleles. comparison of mutation expressing ipsc-clones with non-mutation expressing ipsc-clones showed that the mid mutation results in significantly smaller cells with reduced s phosphorylation supporting aberrations in the mtor/pp a signaling cascade. when differentiating ipscs into neuronal precursor cells, significantly bigger embryoid bodies (ebs) can be detected in the mutation expressing clones, while the total eb number is higher for the non-mutated mid expressing clones. when further differentiated, ebs from all ipsc-clones form neuronal rosette structures expressing beta-iii-tubulin, with a lower rosette structure count in the mutated mid expressing cells. these data clearly hint a defect in neurogenesis in cells with hemizygous mid mutations. interestingly, while ipscs stably kept the x-incativation pattern of the original fibroblast, during differentiation x-inactivation was lost leading to biallelic expression from both x-chromosomes in the npcs pointing towards an as yet unknown reactivation mechanism of the inactive x-chromosome. we are currently analyzing x-inactivation throughout the differ- mutations in the aminoacyl-trna-synthetase genes sars and wars are associated with autosomal recessive intellectual disability l intellectual disability (id) is the common feature of a very heterogeneous group of disorders, which comprises a broad variety of syndromic and non-syndromic phenotypes. here we present mutations in two aminoacyl-trna synthetases that are associated with id in two independent iranian families. in the first family, we found a missense mutation (c. g>a, p.d n) in the cytoplasmic seryl-trna synthetase (sars) gene that affects the enzymatic core domain of the protein and impairs its enzymatic activity. this probably leads to reduced trnaser concentrations in the cytoplasm. in silico analyses predicted the mutant protein to be unstable. this prediction could be experimentally substantiated by results obtained through studies with ectopic mutant sars in transfected hek t cells. in the second family, we identified a compound heterozygous genotype of the mitochondrial tryptophanyl-trna synthetase (wars ) gene, consisting of a nonsense mutation (c. dela, p.ser alafs* ), which very likely leads to nonsense-mediated mrna decay, in combination with a missense mutation (c. t>g, p.w g). the p.w g mutation affects the mitochondrial localization signal of wars , leading to mislocalization of the mutant protein. thus, when taking aimp into account, which we have recently implicated in the aetiology of id as well, there are now three genes with a role in trna-aminoacylation that are associated with this condition. hence we propose that the functional integrity of t-rnas in general is an important constituent in the development and maintenance of human cognitive functions. dravet syndrome is a rare autosomal dominant genetic disorder with early-onset epileptic encephalopathy is mainly caused by different de novo mutations of the scn a gene encoding the type subunit of voltage-gated sodium channel. whole exome sequencing(wes) enables scanning a large number of genes which not only can confirm the diagnosis but also helpful in understading for possible relationship between clinical manifestations and mutaion. the authors investigate wes in a months term boy with hypotonia and convulsion of normal and relative parent. she had uncontrolled sizure without fever, and developmental delay from months.in treatment protocol anti covulsants changes to valorate, clonazepam and stiripentol as well. a deleterious novel heterozygous splice site mutation in scn a tive splicing, imprinting, v(d)j recombination, chromatin loop formation and defining topologically associated domains (tads). recently, we identified de novo mutations in ctcf in patients with a surprisingly mild phenotype of variable developmental delay or intellectual disability, mild short stature and microcephaly, and behavioural anomalies. apart from observing brain malformations and early lethality or learning deficits in two conditional knockout mouse models, little is known about the role of ctcf in neuronal development and preservation so far. therefore, we utilized the model organism drosophila melanogaster to further explore the role of ctcf in cns development and function. similar to observations in knockout mice, complete knockout or ubiquitous knockdown of ctcf is embryonic lethal in drosophila. we therefore utilized the uas/gal system to induce tissue specific knockdown or overexpression of ctcf in the fly nervous system. we first investigated development and morphology of the larval neuromuscular junctions (nmjs), an established model for synaptic development. while pan-neuronal overexpression of ctcf showed no morphological nmj alterations, pan-neuronal knockdown resulted in fewer nmj branches than in a specific control. additionally, we observed a reduced number of active zones in a hypomorphic mutant line compared to a wildtype control. using the negative gravitaxis assay to examine gross neurological function, we found a highly significant impairment of geotaxis behavior in flies with ctcf knockdown in neurons, motoneurons and muscle and in flies with overexpression of ctcf in glia cells, muscle and motoneurons. currently we are testing learning and memory behavior with the courtship conditioning paradigm. our findings of various neurological and morphological anomalies upon manipulation of ctcf dosage in the fly nervous system emphasize the role of ctcf in nervous system development and function and provide a basis to further study the molecular mechanisms underlying cognitive dysfunction caused by ctcf-deficiency. congenital or early-onset nystagmus (cn) is characterized by involuntary eye movements and shows enormous clinical and genetic heterogeneity. cn may be an ambiguous sign of many different diseases, including retinal dysfunction/degeneration, ocular/oculocutaneous albinism, and severe central nervous system disorders, such as pelizaeus-merzbacher or pelizaeus-merzbacher-like diseases (pmld). due to enormous heterogeneity found among the diseases leading to cn, whole exome sequencing (wes) and panel-based bioinformatics was considered as an approach to rapidly identify disease-associated genetic sequence variants. we have analyzed families with cn-affected patients. herein, we present three clinically different patients who were initially affected with cn, but developed further clinical symptoms of various severities. one of these patients showed features of retinal dysfunction, including night blindness and myopia, while two other patients developed severe phenotypes including mental retardation or pmld. wes identified four genetic variants in genes associated with cn. the first patient showed a hemizygous splice-donor variant (c. + g>a) in the calcium channel voltage-dependent alpha- f subunit (cacna f) gene, the second patient carried a hemizygous variant (c. g>a, p.r h) in the ferm domain-containing protein (frmd ) gene, and the third patient showed two heterozygous variants (c. c>g, p.y * and c. t>c, p.v a) in the gap junction protein gamma- (gjc ) gene. sanger sequencing confirmed the identified variants in the index patients and verified co-segregation in several family members. our results suggest a beneficial role of wes to identify the molecular causes of cn and to rapidly confirm an initially unclear clinical diagnosis. especially, patients with rare and severe disorders (e. g. pmld) will benefit from a wes analysis performed in the early stage of the disease. the ccdc -deficient (ccdc -/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al., hum mol genet., ) . in order to determine whether ccdc gene expression might also play a role outside the retina, this study aimed at characterizing ccdc protein expression during early postnatal development of the mouse brain. furthermore, morphological and behavioral impact of ccdc deficiency in the mouse brain was analyzed. methods: ccdc protein expression was determined by sds page and western blot in whole brain homogenates and in selected brain regions of interest (olfactory bulb, hippocampus, cortex, striatum, cerebellum, brain stem) during early postnatal development and in adult wildtype (wt) mice. in addition, cryosections of the ccdc -/-olfactory epithelium and bulb (during postnatal development) and the rostral migratory stream (in adult) were analyzed for ccdc reporter gene expression by x-gal staining. selected brain regions were additionally analyzed by electron microscopy. in order to correlate anatomical with behavioral data, olfactory performance was studied in aged ccdc -/-mice compared to ccdc +/+ controls by an olfactory habituation/dishabituation test (yang and crawley, curr protoc neurosci., ) , where olfactory exploration-time during the presentation of neutral and social odors is examined. results: ccdc protein was detected throughout the early postnatal development of the wt mouse brain, decreasing after birth. amongst analyzed brain regions, highest expression of ccdc protein was detected in the olfactory bulb exhibiting similar ccdc levels to retinal expression. accordingly, ccdc reporter gene expression was demonstrated in the mature olfactory bulb glomeruli, the adjacent olfactory epithelium and along the rostral migratory stream in the ccdc -/-mouse brain. interestingly, strong ccdc reporter gene expression in glomeruli of the ccdc -/-olfactory bulb was correlated with signs of degeneration in the ccdc -/-mouse, but not in controls. the degeneration was also reflected by olfactory impairment in ccdc -/-mice, which spent significantly less time for sniffing at initial presentation of unknown, neutral odors and barely responded to social odors. conclusion: besides the retina, ccdc protein plays a crucial role in the olfactory system as shown by its expression there as well as by ccdc deficiency resulting in neurodegeneration and alteration of olfaction-related behavior in the ccdc -/-mice. as impairment of the olfactory sense in multiple neurodegenerative disorders is a common finding, the ccdc -/mouse model is not only restricted to study retinal degeneration but possibly also degeneration of the central nervous system. background: the ccdc -deficient (ccdc -/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al. ) . in order to gain insights into the molecular disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. here we report a novel homozygous missense variant (c. g>a; p.g r) in the arl b gene, which we identified by whole exome sequencing of a trio from a consanguineous family with multiple affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. the same variant was also identified in a second family. we saw a striking difference in the severity of ataxia between affected male and female individuals in both families. functional analysis demonstrated that dihydrotestosterone treatment of sh-sy y cells induced a down regulation of arl b expression. both arl b and arl b-p.g r expression rescued the cilia length and shh defects displayed by arl bhennin (null) cells, indicating that the mutation did not disrupt either arl b function. in contrast, arl b-p.g r displayed a marked loss of arl guanine nucleotide-exchange factor activity, despite retention of its gtpase activities, highlighting the correlation between its loss of function as an arl guanine nucleotide-exchange factor and joubert syndrome. s. renner, a. busch, t. bierhals, j. butter, v. kolbe, g. rosenberger institute of human genetics, university medical center hamburg-eppendorf, hamburg, germany taad (thoracic aortic aneurysm and dissection) is a heterogeneous disease that often remains silent until a life-threatening complication occurs. it belongs to the connective tissue disorders and causes % of death in industrial countries. several disease genes have been already identified; however, about % of patients with taad-associated syndromes do not show a mutation in these genes. thus, further heterogeneity is obvious. since individual risk stratification and therapeutic options highly depend on the individually mutated gene, it is very important to identify more disease genes which are aimed to be found by whole exome sequencing (wes). many of the known disease genes encode for proteins that are important for the structure and stabilization of the extracellular matrix as well as for the contraction of vascular smooth muscle cells. one central pathway is the tgf-beta signaling which functions among other proteins via the tgf-beta receptor, its ligand and its downstream target smad / . our project plan includes (i) exome sequencing both in affected individuals within families as well as in sporadic patients, (ii) filtering of raw data and prioritization of sequence variants by using a bioinformatic in house pipeline, (iii) verification of novel putative disease genes in a cohort of mutation-negative patients with taad spectrum disease and (iv) functional analyses to gain deeper insight into the pathobiology of taad. in a first round of wes analysis and variant prioritization, we identified a highly conspicuous sequence variant in three family members with taad. screening of the respective gene in a large cohort of mutation negative patients revealed another variant in two siblings with taad. structural and functional considerations strongly support deleterious effects for both identified putative pathogenic missense variants that affect a novel cell cycle-and/or apoptosis regulating protein. functional analyses did not show an involvement of this protein in tgf-beta signaling. in ongoing experiments, we focus on mutation-induced consequences on cell proliferation, cell cycle progression and apoptosis. indeed, we found inhibitory effects of the missense variants on proliferation by affecting the cell cycle key protein cdkn a. we hypothesize that dysregulation of proliferation and/ or apoptosis of specific cells, e. g. smooth muscle cells, underlies taad.abstracts for screening. positively tested compounds were reanalyzed by whole-cell patch clamp recordings and cell volume measurements. results: the halide assay revealed reproducible halide permeability across wells and, as a control, reliably detected mdckii cells overexpressing wild type best by a decrease of yfp fluorescence to % following seconds iodide stimulation. cells harboring mutant best showed % of default yfp fluorescence after the same time interval. conclusion: the current study established an assay appropriate for high and small-scale compound screening targeting best localization and function. this assay will be used to screen for compounds in mutant cells lines best -t p and best -y n for their ability to improve trafficking to the pm or correcting protein folding to enhance ion permeability. background: neuropeptide y-y receptor (y receptor), an auto-receptor of neuropeptide y (npy) and attractive guanine nucleotide (g) protein-coupled receptor target, has been implicated as a potential therapeutic target for many clinical conditions, including epileptic seizure, depression, pain, and alcoholism. in huntington's disease (hd) patients and animal models of hd, npy-expressing striatal interneurons are selective preserved and increased with advancing disease. however, the potential role of y receptor in hd pathology remains under-explored. aims: to investigate whether activation of y receptor using npy and selective y r ligands could ameliorate behavioral deficits and neuropathology in r / mouse model of hd. methods/techniques: npy and selective y receptor agonist npy - were intranasally administered to r / mice, five days in a week, beginning from weeks of age until weeks of age. in the second study, r / mice received daily intraperitoneal administration of selective non-peptide y receptor antagonist (sf- ) to selectively block y receptor. results/outcome: intranasal application of npy showed significant increase in rotarod performance compared to the saline and sf- treated r / mice (*p < . and **p < . at and weeks of age respectively, n = ). however, treatment with npy - showed a clear trend towards increased rotarod performance at weeks of age compared to the saline and sf- treated r / mice but the difference did not reach significance. also, treatment with npy and npy - showed no significant effect on body weight loss in r / mice, contrasting with previous data obtained with single intracerebroventricular (icv) injection of npy in r / mice. furthermore, intranasal application npy or npy - led to decrease in mutant huntingtin (htt) aggregation and mediated increase in dopamine-and camp regulated phosphoprotein (darpp- ) and brain derived neurotrophic factor (bdnf) levels. additionally, we found that npy and npy - attenuate microglial activation, inducible nitric oxide synthase (inos) expression, and proinflammatory cytokines production in r / mice compared to the saline and sf- treated r / mice. conclusion: taken together, our findings suggest that targeting npy-y receptor might be a potential neuroprotective therapy for hd and other neurodegenerative diseases. best of both world's: a novel, rapid capture protocol that overcomes drawbacks associated with dna fragmentation in established methods j. seggewiß, c. ruckert, p. wieacker institute of human genetics, westfaelian wilhelms-university of muenster, muenster, germany rapid capture protocols are an attractive proposition for clinical sequencing labs, as they enable quicker sample-to-sequencing turnaround times. the fragmentation of input dna for the construction of pre-capture libraries is a bottleneck in established protocols. mechanical shearing is the gold standard, but is laborious using single-tube covaris instruments; and higher-throughput instrumentation is cost-prohibitive to many smaller labs. "tagmentation"-based methods (e. g. the nextera rapid capture system from lllumina, or agilent's sureselect qxt system) employ transposases for fast ond simple library construction. however, these protocols are associated with significant sequence bias, especially with low-quality ffpe samples and are extremely sensitive to dna input -thus requiring meticulous quantification of viscous, high-molecular weight dna. here we describe a newly-developed rapid capture protocol that combines the kapa hyperplus kit (kapa biosystems) with integrated, low-bios enzymatic fragmentation, and agilent's proven sureselect xt target enrichment technology. the streamlined method follows for the preparation of high-quality, sequencing-ready libraries in one working day. the novel enzymatic fragmentation reagent does not require careful quantification of input dna, yielding reproducible fragmentation profiles optimal for capture over the fold range-tested ( - ng) the single-tube kapa hyperplus protocol results in very efficient conversion of input dna to precapture library thereby decreasing duplication rates and increasing the complexity of the library going into the modified, min sureselect fast xt hybridization protocol. our protocol represents a significant improvement for fast routine diagnostics, where robust and reproducible pipelines ore needed to support timely treatment decisions. lmj. braun, a. milenkovic, bhf. weber institute of human genetics, regensburg, germany purpose: human bestrophin- (best ) is a chloride channel controlled by ca + and cell volume and is localized at the basolateral membrane of the retinal pigment epithelium (rpe). so far, there is no therapy for the best -associated diseases, of which the most common is best vitelliforme macular dystrophies (bvmd). in this study, we developed an assay applicable for high and small-scale compound screening targeting best localization and function. methods: to assess best channel function we developed a halide assay. briefly, mdckii cell lines were established, stably expressing wildtype best or bvmd-associated best mutants together with a yellow fluorescent protein (yfp)-based halide sensor. in polarized mdckii cells, wildtype best and best -r c localize regularly at the basolateral plasma membrane (pm) while best -l m and best -y n appear significantly reduced in quantity and grossly mislocalized to cytosolic compartments. cells were stimulated with extracellular addition of iodide known to pass the pm through anion cannels and, as a consequence, intracellularly quench yfp fluorescence. variations in yfp fluorescence levels as a marker for best function were recorded in well plates by a plate reader setup. a small-scale , compound library, commercially available as spectrum collection (microsource discovery systems, gaylordsville, usa) was used key: cord- -xcre zgh authors: harvey, bart j.; moloughney, brent w.; iglar, karl t. title: identifying public health competencies relevant to family medicine date: - - journal: am j prev med doi: . /j.amepre. . . sha: doc_id: cord_uid: xcre zgh public health situations faced by family physicians and other primary care practitioners, such as severe acute respiratory syndrome (sars) and more recently h n , have resulted in an increased interest to identify the public health competencies relevant to family medicine. at present there is no agreed-on set of public health competencies delineating the knowledge and skills that family physicians should possess to effectively face diverse public health challenges. using a multi-staged, iterative process that included a detailed literature review, the authors developed a set of public health competencies relevant to primary care, identifying competencies relevant across four levels, from “post-md” to “enhanced.” feedback from family medicine and public health educator–practitioners regarding the set of proposed “essential” competencies indicated the need for a more limited, feasible set of “priority” areas to be highlighted during residency training. this focused set of public health competencies has begun to guide relevant components of the university of toronto's family medicine residency program curriculum, including academic half-days; clinical experiences, especially identifying “teachable moments” during patient encounters; resident academic projects; and elective public health agency placements. these competencies will also be used to guide the development of a family medicine–public health primer and faculty development sessions to support family medicine faculty facilitating residents to achieve these competencies. once more fully implemented, an evaluation will be initiated to determine the degree to which these public health competencies are being achieved by family medicine graduates, especially whether they attained the knowledge, skills, and confidence necessary to effectively face diverse public health situations—from common to emergent. introduction e vents such as severe acute respiratory syndrome, and more recently h n , are examples of public health situations faced by family physicians and other primary care practitioners. these events, in conjunction with other public health issues such as vaccinepreventable infections and rising rates of obesity and the associated health challenges, have resulted in an increased interest in the interface between primary care and public health (e.g., the iom's consensus study ) and in better identifying public health competencies relevant to family medicine so that family physicians and other primary care practitioners might better appreciate the population-wide perspectives of public health issues. however, at present there is no agreed-on set of public health competencies that family physicians and other primary care practitioners should attain to better ensure they possess the necessary knowledge, skills, and confıdence to effectively face diverse public health situations-from common to emergent. using an iterative consultative process, the authors developed a set of public health competencies relevant to family medicine and primary care. this process began with planning for a -day, pan-canadian, family medicine-public health symposium that was held in march . this symposium arose out of discussions at the pan-canadian public health human resources task force concerning the role of primary care practitioners in canada's public health workforce. the primary goals of the symposium were to identify: ( ) the public health competencies that family medicine residents should possess at graduation; and ( ) the primary care competencies that community medicine residents should possess at graduation. to help address these goals, a presymposium discussion document was prepared by reviewing existing documents to develop an initial competency draft using a "multi-level competency model" developed by the university of toronto's department of family and community medicine. the resources accessed and reviewed to inform the preparation of the presymposium discussion document included: cation (acgme) statement regarding the community medicine objectives required in family medicine residency programs ; • the acgme statement regarding the requirements for residency programs in public health and general preventive medicine. a search of the literature, coupled with suggestions from individuals consulted, identifıed several other relevant and informative canadian, , american, [ ] [ ] [ ] [ ] [ ] british, - and australian - publications. these were also reviewed and assisted the development of a draft set of public health competencies relevant to family medicine and primary care. four levels of competencies were drafted, following the model developed in toronto's department of family & community medicine, which spans resident-readiness (expected of all md graduates), essential (expected of all family medicine graduates), enriched (achievable by most residents during family medicine residency training), and enhanced (achievable only through additional training after the family medicine residency). following the review of existing documents and preparation of the presymposium discussion document, the authors identifıed and used eight themes to guide the development of the draft set of competencies. these eight themes were: • disease prevention and health promotion; • infectious disease prevention and control; • emergency preparedness and response; • environmental health; • systems thinking, public health leadership, and management; • population health assessment; • policy, planning, communication, collaboration, and advocacy; • scholar and educator. a variety of family medicine and public health educatorpractitioner colleagues reviewed and provided feedback concerning the proposed draft set of competencies. the fırst group of reviewers was the program directors and residents who participated in the march pan-canadian family medicine-public health -day symposium. this group included residency directors (six family medicine, six community medicine) and residents (six family medicine, six community medicine) from across canada. the presymposium discussion document, which included a proposed set of draft competencies, was distributed to the participants for their review prior to the symposium. the draft competencies served as the major item for discussion by the participants during the symposium's plenary and small group discussions. symposium discussions enabled the authors to further revise the discussion document and proposed set of competencies. this revised draft was then sent to all symposium participants for additional review and feedback, which resulted in the proposal of a detailed set of public health competencies relevant to family medicine and primary care (appendix b, available online at www.ajpmonline.org). this detailed set of competencies was presented at the october family medicine forum, canada's annual family medicine conference. feedback received from conference participants suggested further revisions to the competency draft, particularly the need for a more limited, feasible set of "priority" areas to be focused on during residency training, especially because of the limited duration of family medicine residency training programs-being only years in canada and years in the u.s. as a result, the authors sought further feedback from interested family medicine and public health practitionereducators and residents from across canada to identify a more focused set of priority areas to be emphasized during residency training. these colleagues were asked to rankorder the public health competencies that they considered essential for any graduating family medicine resident. with this advice, the authors identifıed a priority list of eight competency areas: • the basic fundamentals of outbreak/emergency preparedness and management; • offıce infection control; • infectious disease reporting and management; • environmental health exposure reporting; • public health aspects of immunization; • public health programs applicable to primary care (e.g., maternal-child health); • addressing and managing one's practice as a population (e.g., developing practice health profıles and using quality assurance to increase the delivery of preventive services); • the principles and practices of screening (i.e., early detection and treatment) of diseases. public health is only one of competency domains in the university of toronto's family medicine residency program's recently developed competency-based curriculum (other domains include child health, women's health, and mental health). to ensure the overall coherence of the proposed sets of competencies, a full review was completed by a diverse group of family medicine leader-educators at a departmental retreat in april . a key fınding arising from this review was that only focused, essential competencies could be realistically identifıed within each of these domains. this resulted in fıve public health competencies being identifıed and approved for inclusion in the competency-based residency curriculum, each accompanied by one or more enabling subcompetencies. these fıve overarching public health competencies are: • demonstrate an effective approach to disease prevention and health promotion; • demonstrate an effective approach to infectious disease prevention and control, including outbreaks; • demonstrate an effective approach to environmental health issues; • develop and apply the knowledge and skills necessary to assess a population's health; • demonstrate an effective approach to public communication, collaboration, and advocacy. this more focused set, with the accompanying enabling competencies and a listing of suggested relevant topics for each, is included in appendix a. it should be noted, however, that the more extensive complete set of public health competencies relevant to family medicine (appendix b, available online at www.ajpmonline.org) is expected to continue to serve as the comprehensive listing to complement the more focused set of essential competencies and as the primary listing of the enriched and/or enhanced public health competencies relevant to family medicine and primary care. the focused set of public health competencies approved by the university of toronto for family medicine (ap-pendix a) has begun to guide its family medicine residency program curriculum, including resident seminars; family medicine clinical experiences, especially identifying teachable moments that occur during patient encounters; elective public health agency placements; and topics selected for the required resident research project (e.g., the prevalence of chlamydia, a profıle of those infected with h n influenza). these competencies will also be used to guide the development of a family medicine public health primer that would complement the association of faculties of medicine of canada's primer on population health developed for medical students, and faculty development sessions to support the family medicine faculty facilitating residents in their achievement of these competencies. once it is more fully implemented, an evaluation will be initiated to determine the degree to which these public health competencies are being achieved by family medicine graduates, especially whether they attained the knowledge, skills, and confıdence necessary to effectively face diverse public health situations-from common to emergent. it is anticipated that the experience gained through the implementation of the proposed set of competencies (and their evaluation), at the university of toronto and other family medicine residency programs where they are implemented, will provide evidence of the utility and suitability of the proposed competencies and of further revisions that might be warranted. publication of this article was supported by the cdc-aamc (association of american medical colleges) cooperative agreement number u cd . no fınancial disclosures were reported by the authors of this paper. college of family physicians of canada. the role of the family doctor in public health and emergency preparedness. mississauga on: college of family physicians of canada study: integrity primary care and public health medical council of canada. objectives for the qualifying examination royal college of physicians and surgeons of canada. objectives of training and specialty training requirements in public health and preventive medicine rcpsc core competencies for public health in canada. ottawa on: government of canada a set of minimum competencies for medical offıcers of health in canada. ottawa on: public health agency of canada moh% competencies% -% final% -% clean-% post-v ( ). pdf?nsnst_floodϭe c e a d b f beee ae accreditation council on graduate medical education. acgme program requirements for graduate medical education in family medicine accreditation council on graduate medical education. acgme program requirements for graduate medical education in preventive medicine college of family physicians of canada. priority topics and key features for assessment in family medicine. mississauga on: college of family physicians of canada designing a curriculum in disaster medicine for canadian medical schools healthcare worker competencies for disaster training public health and family medicine: an opportunity clinical prevention and population health: curriculum framework for health professions public health educational intervention in a family medicine residency training physicians for public health careers public health and primary care: partners in population health royal college of general practitioners. the rcgp-gp curriculum statements general practitioners with a special interest in public health; at last a way to deliver public health in primary care royal australian college of general practitioners. the new curriculum framework enhancing the population health capacity of general practice: an innovative training model for general practice registrars population health and public health training for australian rural general practice registrars: a six year program primer on population health: a new resource for students and clinicians references for appendix a . college of family physicians of canada. priority topics and key features for assessment in family medicine. mississauga on: college of family physicians of canada essential competencies:a. demonstrate an effective approach to disease prevention and health promotion a . assess patient needs for immunization (routine and highrisk patients). a . counsel individuals/families to receive immunizations appropriate to their age and risk status. a . anticipate, recognize, and report potential adverse events. a . assess patients for sociobehavioral risk factors and risk conditions a in relevant populations.a . provide evidence-informed brief contact interventions to reduce the risk of chronic diseases and injuries. a . counsel and reinforce protective behaviors. a a . counsel patients in defıned risk populations to receive recommended routine screening for cancer. a a . identify families at high risk for developmental and parenting challenges and refer them to public health or appropriate agency for follow-up (e.g., home visiting program, dental health program).b. demonstrate an effective approach to infectious disease prevention and control, including outbreaks b . demonstrate routine infection control practices for the care of all patients at all times, including recommended offıce infection control practices. b . demonstrate transmission-based precautions in conjunction with routine practices when patients are suspected or confırmed of being infected with transmissible or epidemiologically signifıcant organisms. b . recognize and report cases of notifıable diseases, conditions, and unusual diseases or patterns to public health authorities. b . coordinate management of individuals and families with broader public health investigation. a b . describe roles and responsibilities in preparing for and responding to infectious disease outbreaks, and other health emergencies and disasters. b . triage patients in an infectious disease outbreak, and other health emergencies and disasters.c. demonstrate an effective approach to environmental health issues c . recognize features of community health determinants, patient history, symptoms, and signs that trigger differential diagnoses that include exposure to an environmental health hazard. c . report potential cases to public health authorities for patients or populations who are likely being affected by an exposure to an environmental health hazard.d. develop and apply the knowledge and skills necessary to assess a population's health d . describe how the determinants of health a affect the health of one's patients and practice population. d . describe how a community's profıle of determinants of health a contributes to the occurrence of selected conditions. d . discuss a community's health needs considering underlying determinants of health, a evidence for effective interventions, and existing services. d . assess a practice population's status for clinical preventive services (e.g., immunization rates, cancer screening rates, and sociobehavioral risk factors and conditions). d . apply evidence-informed practice strategies to improve patient population coverage for preventive services (e.g., routine questions, chart/computer reminders/prompts, patient invitations/recalls). d . describe the complementary roles of family medicine and public health in achieving the prevention and control of conditions of public health interest.e. demonstrate an effective approach to public communication, collaboration, and advocacy e . communicate (when indicated) with individual patients, families, and the practice population regarding public health measures that concern their health and care. • education; • housing; • ses. supplementary data associated with this article can be found, in the online version, at doi: . /j.amepre. . . .did you know? you can listen to podcasts featuring kenny goldberg, health reporter for npr radio in san diego, as he interviews select ajpm authors. go to www.ajpmonline.org/content/podcast_collection to download the podcasts! key: cord- - ogcxd b authors: kaufman, aaron r.; hersh, eitan d. title: the political consequences of opioid overdoses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ogcxd b the united states suffered a dramatic and well-documented increase in drug-related deaths from to , primarily driven by prescription and non-prescription opioids, and concentrated in white and working-class areas. a growing body of research focuses on the causes, both medical and social, of this opioid crisis, but little work as yet on its larger ramifications. using novel public records of accidental opioid deaths linked to behavioral political outcomes, we present causal analyses showing that opioid overdoses have significant political ramifications. those close to opioid victims vote at lower rates than those less affected by the crisis, even compared to demographically-similar friends and family of other unexpected deaths. moreover, among those friends and family affected by opioids, republicans are % more likely to defect from the party than the statewide average republican, while democrats are no more likely to defect; independents are moderately more likely to register as democrats. these results illustrate an important research design for inferring the effects of tragic events and speak to the broad social and political consequences of what is becoming the largest public health crisis in modern united states history. for over-marketing and over-prescribing highly addictive opioids [ ] , while others point to social and economic determinants like poverty, unemployment, and limited economic mobility as important moderating factors of drug use and abuse. [ ] [ ] [ ] . while the public health effects of the opioid epidemic are clear, the social and political effects are largely unstudied, though a study from the beginning of the crisis estimates economic costs of opioid users as $ , canadian dollars per year [ ] . a broad literature in political science examines the consequences of traumatic events like terrorism [ ] , race riots [ ] , and war [ ] , largely finding politically mobilizing effects; there is good reason to suspect that the opioid crisis has produced complex political aftershocks as well. and while the effects of such shocks to individuals may be pronounced, they may also serve as drivers of local or national policy, as victims of such tragic events often become spokespeople in advocating for large policy changes. this paper applies a validated causal research design to study the political consequences of the opioid epidemic on the individuals closest to it-the friends and family of overdose victims. leveraging recent computational advantages and newly available large-scale government databases, we show that the opioid epidemic has significantly altered patterns of party identification and turnout, but with important heterogeneity across partisanship. in contrast to other studies examining the effects of tragedy on political participation, we find that friends and family of opioid overdose victims are less likely to turn out to vote than they were before tragedy struck, even compared to victims of premature cancer or a demographically-matched sample of registrants without familial opioid overdoses. moreover, opioid overdoses seem to change the beliefs and preferences of those affected: independents are far more likely to re-register as democrats, and republicans are nearly % more likely to defect to other parties, but democrats' party affiliations are unchanged. more broadly, we expand the literature on the persistent effects of shocks to political behavior by showing a context in which participation declines rather than increases, and where shifts in party identification are causally identified, offering new insights into possible long-term consequences of large public health crises. how might friends and family of opioid overdose victims respond politically? we examine two sets of outcomes: political engagement and partisan preference. prior research finds positive engagement effects for crime victimization [ ] , immigration [ ] , race riots [ ] and terrorist attacks [ ] , so we have reason to suspect that those affected by the opioid epidemic may mobilize to affect policy change. another line of research, however, suggests that sadness and depression might decrease voter mobilization [ ] , offering a contrasting hypothesis. on partisan preferences, too, prior research is split: whereas victims of terrorism become on average more conservative as national identity becomes more important [ ] , those proximate to race riots become more liberal, perhaps as they become more aware of the struggles of racial minorities [ ] . and in considering the psychological repercussions of opioid overdose, while anxiety may lead to more liberal preferences [ ] , fear may lead to more conservative preferences instead [ ] . our measure of partisan preference is the change in registered party affiliation, a strong behavioral indicator of preferences relative to survey reporting. finally, a large literature observes that apolitical events like sports victories, hurricanes, and shark attacks tend to disadvantage the incumbent party, whom voters blame for misfortunes [ ] , suggesting that overdose victims' families may defect from the democratic party during the democratic administrations, or defect to it during republican administrations; an equally large literature points out that events like overdoses may increase the salience of healthcare-related issues in the minds of voters, pushing them toward the party with the most ownership over the healthcare debate [ , ] . how do these conflicting hypotheses manifest in the friends and family of opioid overdose victims? our analysis strategy leverages panel data with voter behavior linked to accidental drug death records. we identify in public voter registration records the friends and family of opioid overdose victims, and compare their election turnout rates and party identification in the elections immediately before and after their loved one's death. opioid overdose victims are not a representative sample of the population, however, so any political differences we observe may be due to demographic differences instead. to avoid this inferential pitfall we identify a control group of individuals who prematurely died of cancer (which we define as years old or younger, corresponding to approximately the youngest % of cancer deaths [ ] ), and perform coarsened exact matching with mahalanobis distance [ ] to obtain a sample of individuals who died of cancer that look demographically identical to our sample of individuals who died of opioid overdoses. in this way, we causally identify the effect of having a loved one die of an opioid overdose as compared to another type of premature death. to threaten our causal identification, an unobserved variable would have to confound the difference between the change in party identification among opioid overdose victims' families and the change in turnout or party identification among cancer victims' families. (figs and ). this comparison separates out the effect of premature deaths from the effects of an opioid overdose death in particular, isolating the bundle of treatments distinctive to opioid deaths such as politicization, media attention, and stigmatization. we begin with a panel of two connecticut voter files: a voter file with , , individuals and a voter file with , , individuals; connecticut uses a unique voter id which we use to match registrants from to , producing , , matched individuals. the connecticut voter file contains party identification information as well as election turnout for as more than a decade of previous federal, statewide, and local elections. we merge these data with geography-based census measures from the national historical geographic information system (nhgis, see table ). the office of the chief medical examiner in connecticut makes free available a de-identified list of accidental opioid-related deaths; our version of this data contains , records. these records contain the location of the deceased individual, their age at death, their race, sex, town of residence, and the opioid(s) identified in their toxicology reports. we limit our analyses to those overdoses that occurred after the general election but before the primary election. to match these individuals to the voter file, we first re-identify them by searching for their dates, locations, and ages at death in publicly searchable obituary records. we take all uniquely identified opioid overdose obituaries and manually record their full names and precise birth dates. this results in , opioid deaths in our data set. we also identify the friends and family of these overdose victims by locating those individuals living at the same address as the overdose victim at the time of death, and find , such individuals. we exclude cases with more than registered voters living at the same address as they may not share a friend or family relationship with the victim. for our comparison sample, we search obituary records for premature cancer deaths that occurred after the general election but before the primary election, then manually the political consequences of opioid overdoses record their full names and precise birth dates. we find such deaths and, using the same procedure as for the overdose victims, we find household members. using coarsened exact matching (cem) with mahalanobis distance, we identify two samples of matched voters: one that looks demographically similar to the family members of cancer victims, and another that looks demographically similar to the family members of opioid overdose victims. by comparing political behaviors of these matched groups to the original samples of family members, we control for all observed (but not unobserved) factors that might confound the relationship between cancer or opioid overdose deaths and voter turnout. the variables in our matching procedure include race, age, sex, county, household occupancy, census block median household income, and prior turnout history. we summarize our data in table below. overdose deaths disproportionately fall on hispanics and african-americans, on men rather than women, and on the young more than the old, but otherwise resemble the population relatively well. unsurprisingly, cancer victims also have substantially more household members than opioid victims. however, we do observe that victims of overdose deaths and their families vote at lower rates than the statewide average. we first examine voter turnout (fig ) . the statewide turnout rate in the general election in connecticut was approximately % (top panel rightmost grey dot and dashed grey line); republicans voted at a higher rate (dashed red line). the overall turnout rate among the families of cancer victims (dark grey dot) is about percentage points lower from the overall turnout rate, while the rate among families of opioid victims is about percentage points lower. democrats among the families of both sets of victims vote at lower rates; both democrats and republicans among the families of both sets of victims vote approximately percentage points less than their copartisans who were unaffected, and to a lesser degree, less than the demographically-matched samples of copartisans. a different pattern emerges for primary election turnout: the friends and family of opioid overdose victims vote at a rate about percentage points lower than the overall rate, and both democrat and republican friends and family of opioid overdose victims vote at lower rates than the average, but cancer victim families of neither party vote at lower rates. turning to party affiliation (table ) , we examine the rates at which the friends and family of opioid overdose victims change parties relative to cancer victims' friends and family as well as the statewide average. statewide, both democrats and republicans left their party from to at a rate of . %; among democrats, both cancer and opioid overdose victims' friends and family leave the party at approximately the same rate- . % and . % compared to . % statewide. among republicans, however, both cancer and opioid overdose victims' friends and family leave the party at much higher rates-both . %-than the statewide average of . %. in comparison, demographically-matched control republicans left the party at rates of . % and . %, indicating a percentage point estimated treatment effect of premature death on republican party defection. this change is almost entirely due to republicans becoming independent ( . % and . %) rather than becoming democrats ( . % and . %). among independents, the friends and family of cancer victims join a major party at about the statewide rate, but independent friends and family of opioid overdose victims join the democratic party at a rate / higher than the statewide rate. to test the significance of these differences, we conduct chi-squared tests between pairs of columns represented as transition matrices. chi-squared tests indicate a statistically significant difference (p < . ) between the statewide and od families transition matrices and between the cancer families and od families transition matrices, but not between the statewide and cancer families transition matrices. as well, the difference between the od families and od matched control transition matrices is significant, but the difference between the cancer families and cancer matched control transition matrices is not. the opioid epidemic, much like covid- , represents a political conflict rooted in a public health crisis, and it is therefore important to examine its political ramifications. in this study we have shown that the opioid epidemic substantially affects the political behavior among the friends and family of its victims. while tragic deaths in general reduce turnout in the general election, only opioid epidemic deaths reduce turnout in the primary election compared to other premature deaths. moreover, turning to partisanship, republican friends and family were increasingly likely to defect from the gop, independents were increasingly likely to join the democratic party, both suggesting a liberalizing trend. the shifting partisan allegiances suggest that, in the face of opioid-driven tragedy, voters do not appear to hold the democratic party accountable for opioid overdoses; note that during this period of study, both the us presidency and the connecticut governorship were held by democrats. to the contrary, voters act as though they prefer more liberal policies toward managing the opioid crisis in the face of self-interest or socialization [ , ] , even if they may not know the differences between the parties' policies [ ] . we do note third key limitations of our results. first, our analysis is restricted to a single, unrepresentative state. secondly, we only find obituary records for . % of opioid overdose victims in the state, and that sample may be unrepresentative of opioid overdose victims as a whole. finally, by identifying cancer deaths through obituary records, we may have an unrepresentative sample of statewide cancer deaths. importantly, for this unrepresentativeness to bias our internal validity, cancer victims with and without obituaries that specifically mention cancer would need to be systematically different in a manner correlated with changing party registration. taken together, these three limitations restrict our ability to generalize our findings to other states and populations. however, if we could safely assume that the trends we observe in connecticut extrapolate to the entire us, based on results from [ ] we would estimate that the opioid overdose resulted in , fewer cast votes in the presidential election, nearly , additional democratic registrants, and an equal number of fewer republican registrants merely among the friends and family of its victims (see s text). extending this line of inquiry may involve interrogating whether this change in party affiliation is a policy-driven response to self-interest or a broader shift in outlook and emotional state in response to tragedy. finally, our results add important nuance extant literature on the politically mobilizing effects of dramatic events like terrorism and race riots, and future research in this field may also examine and characterize which types of shocks are mobilizing and which depress turnout, or which shocks tend to liberalize or conservatize. supporting information s text. (pdf) the opioid crisis: a comprehensive overview. current pain and headache reports contribution of opioid-involved poisoning to the change in life expectancy in the united states a time-release history of the opioid epidemic the role of science in addressing the opioid crisis the road to h.: narcotics, delinquency, and social policy opioid discussion in the twittersphere. substance use & misuse opioid-prescribing patterns of emergency physicians and risk of long-term use public health and international drug policy. the lancet opioid crisis: no easy fix to its social and economic determinants viewing addiction as a brain disease promotes social injustice social costs of untreated opioid dependence long-term effect of september on the political behavior of victims' families and neighbors can violent protest change local policy support? evidence from the aftermath of the los angeles riot. american political science review communication and political mobilization: digital media and the organization of anti-iraq war demonstrations in the us crime victimization and political participation revisiting lepanto: the political mobilization against islam in contemporary western europe. patterns of prejudice depression and political participation threat, anxiety, and support of antiterrorism policies. american journal of political science are all conservatives alike? a study of the psychological correlates of cultural and economic conservatism. the journal of psychology blind retrospection: why shark attacks are bad for democracy issue salience, issue ownership, and issue-based vote choice. electoral studies disputed ownership: parties, issues, and traits in the minds of voters. political behavior peer reviewed: heart disease and cancer deaths-trends and projections in the united states causal inference without balance checking: coarsened exact matching concentrated burdens: how self-interest and partisanship shape opinion on opioid treatment policy how should we wage the war on drugs? determinants of public preferences for drug control alternatives heuristics behaving badly: party cues and voter knowledge drug and opioid-involved overdose deaths-united states we thank sheng ha and two anonymous reviewers for invaluable feedback. we are indebted to neal beck, leo celi, brian libgober, luke miratrix, robert ward, and madeleine wolf for helpful comments during the preparation of this manuscript, and to obituarydata.com of generously providing access to national obituary data. all remaining errors are our own. this work was approved by the harvard university committee on the use of human subjects, irb protocol - ; it was designated "not human subjects research". the authors have declared that no competing interests exist. key: cord- - vn o ag authors: li, jing; ding, jiguang; chen, li; hong, liang; yu, xiaoqi; ye, enling; sun, gangqiang; zhang, binbin; zhang, xinxin; sun, qingfeng title: epidemiological and clinical characteristics of three family clusters of covid- transmitted by latent patients in china date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: vn o ag from january to february , three family clusters involving patients with coronavirus disease were identified in wenzhou, china. the epidemiological and clinical characteristics of the family cluster patients were analysed and compared with those of contemporaneous sporadic cases. the three index cases transmitted the infection to family members – days before illness onset. overall, of the sporadic cases and three of patients in the family clusters came back from wuhan ( . vs. . %, p< . ). in terms of epidemiological characters and clinical symptoms, no significant differences were observed between the family cluster and sporadic cases. however, the lymphocyte counts of sporadic cases were significantly lower than those of family cluster cases (( . ± . ) × ( )/l vs. ( . ± . ) × ( )/l, p = . ), and the proportion of hypoalbuminaemia was higher in sporadic cases ( / , . %) than in the family clusters ( / , . %) (p < . ). within the family cluster, the second- and third-generation cases had milder clinical manifestations, without severe conditions, compared with the index and first-generation cases, indicating that the virulence gradually decreased following passage through generations within the family clusters. close surveillance, timely recognition and isolation of the suspected or latent patient is crucial in preventing family cluster infection. since december , an epidemic of coronavirus disease (covid- ) , associated with severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in wuhan, hubei province, china [ , ] . sars-cov- has been characterised by high infectivity through human-to-human transmission and relatively low mortality [ ] . the mean r of sars-cov- is estimated to range from . to . [ , ] . as a result, the epidemic of covid- has rapidly spread to the whole country and worldwide. as of march , countries had been affected, and the cumulative number of confirmed cases had reached and in china and worldwide, with and deaths, respectively. currently, the epidemiological characteristics of covid- , especially transmission patterns, have not been well elucidated. the first family clustering study reported that five family members who travelled to wuhan from shenzhen were infected with sars-cov- , and when they returned to shenzhen, the additional family members who did not travel to wuhan became infected with the virus [ ] . the epidemiological and phylogenetic analysis indicates that sars-cov- can be transmitted person-to-person in hospital and family settings [ ] . subsequently, several studies also reported the family cluster transmission resulting in the infection of - family members [ ] [ ] [ ] [ ] [ ] , even during the incubation period. however, further investigation is required to understand the transmission patterns among family members. wenzhou is one of the regions with a high prevalence of infection outside hubei province, probably because of the close economic cooperation and convenient public transportation between two regions. in the early stage of the epidemic, over people came back to ruian, a county-level city under the administration of wenzhou. among these people, were diagnosed with covid- , and three family clusters of members were identified. in this study, we aimed to investigate the epidemiological and clinical characteristics of these three family clusters of covid- cases by comparing them with sporadic cases, which would provide insights for epidemic control in the context of the current serious situation worldwide. from january to february , covid- patients, who were all positive for the nucleic-acid test of sars-cov- and received isolation and treatment in the designated ruian people's hospital, were enrolled in the present retrospective study. among the patients, patients were identified to belong to three different families according to their family relationships and history of close contacts (figs and ) , while the other patients were sporadic cases. the epidemiological history, including exposure history, travelling vehicle, contact tracing, family member relationship, date of illness onset and date of admission and isolation, were collected in detail for each patient by two attending physicians. the transmission chain was carefully evaluated to clarify the relationship within the cluster members, according to the close contact history and exposure time. a close contact was defined as an act of sharing a meal, party, vehicle or living room with a confirmed or latently infected patient within days. an index case (g ) was defined as the original source of sars-cov- infection among the family. the patients who were infected by contact and exposure to the index case were defined as the first-generation cases (g ), and the patients who were infected by contact and exposure to the first-generation patients were defined as second-generation cases (g ) and so on. the patients without an infected family member were defined as sporadic cases. index case a, a -year-old female who came back from wuhan on january , had dinner with four family members on january . she developed symptoms of cough days later and was confirmed with covid- on february . the four family members, cases a - (g ), developed the disease − days after the dinner and were called firstgeneration cases of index case a (figs a and a) . index case b was a -year-old female who came back from wuhan on january . she developed a fever days later and was diagnosed with covid- on january . during the next two weeks, four family members, cases b , , and (g ), who had a close contact history with index case b were confirmed to be infected by sars-cov- sequentially. then, case b (g ), one of the four first-generation cases of index case b, transmitted the infection to five family members, cases b - and (g ), who were called second-generation cases. another first-generation case, b (g ), transmitted the infection to three family members, b - (g ). moreover, one of the second-generation cases, b (g ) transmitted the infection to cases b - (g ), the third-generation cases (figs b and b) . index case c (g ), a -year-old male, came back from wuhan and joined a party with four family members and two classmates on january . he developed illness with cough days later and was diagnosed with covid- on january . the six persons, cases c - (g ), who joined the party also developed an illness within the next weeks. case c (g ) was responsible for the second-generation infection to three family members, cases c - (g ), while case c (g ) was responsible for transmission to one family member, c (g ) (figs c and c) . all patients were diagnosed with covid- by real-time reverse transcriptase-polymerase chain reaction (rt-pcr) assay, according to the guideline for diagnosis and treatment for novel coronavirus pneumonia released by the national health commission of china ( th edition) [ ] . the rt-pcr tests for influenza a and b for all patients were negative. written informed consent, according to the declaration of helsinki, was obtained from each patient. this study was approved by the ethics committee of the ruian people's hospital (approval number: yj ). the clinical information of all enrolled patients was retrieved from the hospital history system, including the demographic data, laboratory test results, radiological results, treatment regimens, duration of treatment, duration of hospitalisation and treatment outcomes. the applications of intranasal oxygen inhalation and assisted mechanical ventilation along with comorbidities including hypertension, diabetes, chronic obstructive pulmonary disease, chronic kidney disease and malignant tumours were recorded. the patients were divided into different clinical types, according to the guidelines by the national health commission of china [ ] . patients who presented with classic symptoms and positive sars-cov- rna but without pneumonia lesions on computed tomography (ct) scan were defined as mild cases, and those with classic symptoms, positive sars-cov- rna and pneumonia lesions on ct scan were defined as common cases. in addition, patients who met the following criteria were defined as severe cases: ( ) respiratory distress, a respiratory rate (rr) ≥ beats/min; ( ) an oxygen saturation level less than % in resting state and ( ) a partial pressure of oxygen (pao )/oxygen concentration (fio ) ≤ mmhg ( mmhg = . kpa). sars-cov- rna was detected by rt-pcr assay with the taqman probes targeting orf ab, n and e genes, and expressed as the cycle threshold (ct) value (shanghai biogerm medical biotechnology co., ltd). the amplification products for genes with a ct value of less than were considered as positive. sputum samples or throat swab samples were taken for analysis at baseline and then every - days until hospital discharge. continuous variables were expressed as mean and standard deviation (sd) or median and interquartile range (iqr), and categorical variables were expressed as a number (%). the values were compared by student's t-tests, one-way anova or mann−whitney test, or kruskal−wallis as appropriate. all data analysis was performed with r software (version . . ) and empowerstates software (http:// www.empowerstats.com, x&y solutions, inc., boston, ma). a twosided p value of less than . was considered statistically significant. of all patients, were male, and were female. the mean age was . years old. the most common symptoms were table ) . of the sporadic cases, ( . %) returned from wuhan while three ( . %) of the family cluster cases returned from wuhan (p < . ). the family members were infected by the three index cases who were in the latent period - days before the onset of illness. interestingly, the time of illness onset of patient a , who had no travel history to wuhan or contact with other patients, was earlier than that of index case a. patient a (g ) developed severe pneumonia subsequently. the incubation period of sporadic cases ( . ( . - . ) days) was similar to that of the family cluster ( . ( . - . ) days) (p = . ). the time from symptom onset to hospitalisation, the time from symptom onset to diagnosis and the duration of hospitalisation were not significantly different between sporadic and family cluster cases (all p > . ) ( table ) . there was no significant difference in the frequency of common symptoms, including fever ( . vs. . %), cough ( . vs. . %) and sputum ( . vs. . %), between sporadic and family cluster cases (all p > . ). also, the proportions of mild, common and severe types were similar between sporadic ( . , . , and . %, respectively) and family cluster ( . , . and . %) cases (p = . ). however, the decrease of albumin was more frequent in sporadic cases ( . %) than in the family cluster cases ( . %) (p < . ). while the levels of alanine transaminase, aspartate aminotransferase and creatinine were not different between the two groups (all p > . ), the level of lymphocyte counts was significantly lower in sporadic cases (( . ± . ) × /l) than in the family cluster cases (( . ± . ) × /l) (p = . ). the viral load (ct value) was not different between the two groups (( . ± . ) vs. ( . ± . ), p = . ) ( table ). the imaging features of the pulmonary lesions on ct scan were not apparently different between the two groups (data not shown). jing li et al. among the family clusters, three index cases (g ) transmitted the infection to first-generation cases (g ), who transmitted the infection to second-generation cases (g ) and subsequently two third-generation cases (g ) (figs and ) . then, the epidemiological and clinical characteristics were compared between generations and (g + g ) and generations and (g + g ) cases. g + g cases were older than g + g cases ( . ( . - . ) vs. . ( . - . ) years, p = . ). the incubation time, the time from illness onset to hospitalisation, the time from illness onset to diagnosis and the duration of hospitalisation were not significantly different between the two groups (all p > . ). the proportion of mild cases in g + g cases ( / , . %) appeared to be larger than that in g + g cases ( / , . %) although the difference was not statistically significant (p = . ). the viral loads were not significantly different between g + g and g + g cases ( . ± . ) vs. ( . ± . ), p > . . the level of lymphocyte counts tended to be lower in g + g cases (( . ± . ) × /l) than in g + g cases (( . ± . ) × /l), but the difference was not statistically significant (p = . ) ( table ) . the differences of epidemiological and clinical characteristics between sporadic cases and generations of family clusters were determined. it was shown that the lymphocyte counts of sporadic cases were significantly lower than those of g + g cases (p = . ) but without a significant difference with g + g cases (p > . ). there were no differences in the incubation time, the time from illness onset to hospitalisation, the time from illness onset to diagnosis and the duration of hospitalisation among the three groups (p > . ) (tables and ). in this study, the epidemiological and clinical characteristics of three family clusters were investigated using sporadic patients as controls. such a specific population provides us an opportunity to analyse the relationship between transmission and the disease presentation in different settings where the sporadic patients were used as external controls to the family cluster patients. more patients in sporadic cases came back from wuhan than in the family cluster. all three index cases were latent patients without any symptoms at the time when they came back to ruian. this study revealed that sporadic cases had lower levels of albumin and lymphocyte counts than family cluster cases; otherwise, there were no significant differences in terms of other epidemiological characters and clinical features between the two groups. in addition, the lymphocyte counts in sporadic cases were lower than those in the cases of second and third generations family cluster cases although there was no difference in the lymphocyte counts among the different generations within family cluster. human coronavirus pneumonia is often associated with an elevated production of chemokines, which recruit massive inflammatory cell infiltration and release cytokines resulting in acute pulmonary injury [ ] . the decrease of lymphocyte counts and elevation of cytokines/chemokines are the hallmark of coronavirus-associated pneumonia and are associated with the severity of the pneumonia. recent studies on covid- have demonstrated that the lymphocyte counts in the peripheral blood are remarkably decreased in patients who are admitted in the intensive care unit (icu), compared with non-icu patients [ , ] . in addition, several studies on covid- or mers have shown that hypoalbuminemia is a frequent feature and associated with the severity of the pneumonia [ ] [ ] [ ] , probably owing to increased energy consumption or altered pulmonary vessel permeability. the finding in this study that the decrease of lymphocyte counts and hypoalbuminaemia in sporadic cases, compared with family cluster cases, indicates that there is an increased immune activation or dysfunction and thus more severe pulmonary inflammation in sporadic cases than in family cluster cases. within the family clusters, virus transmission through different generations and the clinical presentations were investigated. the age of second-and third-generation patients was younger than that of the index and first-generation patients. it has been reported that when hosts of different sexes or ages were encountered, the pathogen may change optimal exploitative strategy, leading to considerable variation of pathogen transmission and virulence [ ] . the trade-off between transmission and virulence would change in coordination with host immunity that is associated with age. sars-cov- seems prone to affect older males with comorbidities [ ] . in this study, two patients, a (g ) and b (g ), developed severe pneumonia in first generation, while no severe cases were observed in the second or third generation. more mild patients were found in the second or third generation, which are also noted to be younger in age, may be attributed to age. in addition to, it could be inferred that following the passage through several generations within the family cluster, the virulence of sars-cov- decreased gradually. epidemiological evidence from covid- family clusters has suggested that most index cases are asymptomatic carriers, mild patients or even latent patients [ , , ] , which is consistent with our observation. however, the characteristics of index cases with covid- are reportedly different from those in the mers family clusters, who have moderate or severe symptoms and are never asymptomatic carriers [ ] [ ] [ ] . the higher virulence and mortality in mers may explain the different characteristics of the index cases. as the viral load of sars-cov- detected in the asymptomatic patients is similar to that in the symptomatic patients [ ] , the asymptomatic index cases are capable of causing cluster infection in the family setting. a recent study with the largest sample so far in china showed that the median incubation period for covid- was days (iqr, − ), with the longest incubation period up to days, and only . % patients presented with a fever at admission [ ] , highlighting the importance of monitoring and isolating potential infected family members who have had an exposure history in the family setting. previous studies have demonstrated that the outbreak of sars-cov and mers-cov infections have resulted in large clusters of patients, most of which are associated with the nosocomial transmission, called super-spreader events [ ] [ ] [ ] . since the virological and clinical characteristics are similar among sars-cov, mers-cov and sars-cov- [ ] , it is worth noting the super-spreader events in covid- . as of march, the daily number of newly diagnosed patients had decreased to less than cases in china. currently, there are no specific antiviral agents or vaccines available for sars-cov- , which possesses a high infectivity, and thus advanced epidemiological surveillance and timely identification and isolation of suspected cases or individuals who had a close contact or exposure history remains a priority to prevent family cluster or superspread events. the chinese experience shows that intensive social interventions, including isolation are crucial in delaying and blocking the spread and subsequent outbreaks of the disease. in conclusion, family clusters of covid- can be caused by latent patients. the epidemiological and clinical symptoms are similar between the family cluster and sporadic patients, but the sporadic patients showed lower lymphocytes and hypoalbuminaemia. these findings indicate that close surveillance, timely identification and isolation of the suspected or latent cases is crucial in preventing family clusters or even super-spread events. clinical features of patients infected with novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak estimating the unreported number of novel coronavirus ( -ncov) cases in china in the first half of january : a datadriven modelling analysis of the early outbreak a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster asymptomatic cases in a family cluster with sars-cov- 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infected patients linked to viral loads and lung injury explosive school-based measles outbreak: intense exposure may have resulted in high risk, even among revaccinees epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case family cluster of middle east respiratory syndrome coronavirus infections sars-cov- viral load in upper respiratory specimens of infected patients clinical characteristics of coronavirus disease in china comparison of clinical course of patients with severe acute respiratory syndrome among the multiple generations of nosocomial transmission mers-cov outbreak in jeddah -a link to health care facilities the sars outbreak in a general hospital in tianjin, china -the case of super-spreader emerging threats from zoonotic coronaviruses − from sars and mers to -ncov acknowledgements. we thank all participating patients and acknowledge the support of a grant from ruian science and technology bureau (ms ). this project was supported by the medjaden academy & research foundation for young scientists (grant no. covid- -mja ). data availability statement. all data generated or analysed during this study are included in this paper. key: cord- -etod fgd authors: ritchey, katherine c.; foy, alice; mcardel, erin; gruenewald, david a. title: reinventing palliative care delivery in the era of covid- : how telemedicine can support end of life care date: - - journal: am j hosp palliat care doi: . / sha: doc_id: cord_uid: etod fgd telemedicine technology has become essential to healthcare delivery in the covid- era, but concerns remain regarding whether the intimacy and communication that is central to high-quality palliative care will be compromised by the use of this technology. we employed a business model approach to identify the need for system innovation in palliative care, and a quality improvement approach to structure the project. products from this project included a standard operating procedure for safe use of tablet computers for inpatient palliative care consultations and family visitations; tablet procurement with installation of video telehealth software; and training and education for clinical staff and other stakeholders. we describe a case illustrating the successful use of palliative care telehealth in the care of a covid- -positive patient at the end of life. successful use of video telehealth for palliative care involved overcoming inertia to the development of telehealth infrastructure and learning clinical video telehealth skills; and engaging front-line care staff and family members who were open to a trial of telehealth for communication. information gleaned from family about the patient as a person helped bedside staff to tailor care toward aspects meaningful to the patient and family and informed best practices to incorporate intimacy into future palliative video consultations and family visit. in the wake of the coronavirus pandemic, healthcare has suddenly and radically transformed. healthcare facilities and staff were by patients rapidly deteriorating and dying of covidrelated illness. skilled nursing facilities have been devastated, accounting for about a quarter of documented deaths from covid- in the united states. healthcare workers worldwide have faced unprecedented challenges, including accommodating to the need for physical distancing, supporting families who are prevented from seeing their critically ill loved ones, managing acute bereavement when patients die; and in some cases, working outside their usual scope of practice. , numerous heartbreaking stories have appeared in news media and professional publications about patients dying alone, with their loved ones denied permission to visit. [ ] [ ] [ ] [ ] [ ] palliative care has been highlighted as an essential part of the pandemic response. among the key roles served by palliative care teams (pct) are developing system-level pandemic response plans; serving on scarce resource allocation teams; identifying and addressing goals of care; supporting effective symptom management; providing psychosocial support and bereavement care for family members; and supporting other healthcare workers who are overwhelmed, stressed and traumatized by exposure to traumatic events they have witnessed. [ ] [ ] [ ] [ ] telemedicine technology was being used increasingly even before the covid- pandemic, but the pandemic has vaulted telemedicine into the role of critical healthcare infrastructure. during this time of social distancing and isolation, restoring meaningful human connection has required creativity and rapid innovation. there has been keen interest in the use of telehealth technology in palliative care, to improve communication between isolated patients and their families, and between patients and their care providers. , [ ] [ ] [ ] below, we share highlights of how this paradigm shift was brought to fruition in a single veterans affairs medical center and how the intimacy necessary for end-of-life care was maintained through the integration of telehealth technology into the work of the pct. we re-conceptualized the delivery of palliative care services as a business model innovation, to identify and implement the components required for adapting and sustaining high quality care at a time when traditional care was no longer possible, but the need for care was great. additionally, we structured our innovation using a quality improvement approach adapted from other commonly used models (table ) . [ ] [ ] [ ] restructuring the system of care delivery "business model" is a conceptual framework coined in the mid- 's and whose definition has evolved over the past several decades. simply defined, a "business model is a description of an organization and how that organization functions in achieving its goals (e.g. profitability, growth, social impact, . . . )". though healthcare is not a traditional "business," it may be helpful to apply a "business model" framework when restructuring the healthcare delivery system is necessary. when circumstances threaten the delivery of a service or good, restructuring requires critical appraisal of current functions and awareness that there will be a need for model evolution and/or innovation due to internal or external changes over time. in times of systemic disruption, innovation is required to overcome barriers and identify new strategies for a business to persevere (e.g. maintain healthcare service delivery). innovation may be defined as "the process of transforming one 'business model' (either entire or core components) to another." sustainable innovation involves analysis and planning with clear stages, actions, and pitfalls and an imperative to overcome inertia to change. , (table ) . the prerequisites to innovation include not only knowledge, expertise and experience, but also courage, creativity and fearless leadership. within misfortune or disappointment, it is often hard to embrace opportunities for growth and transformation. it is natural to hide behind fear, blame circumstances for failure, and resist change. finally, innovation requires teamwork. no one person can develop and implement changes in systems or models without engagement of key stakeholders and those who oversee or provide critical aspects of service delivery. teamwork involves diversity, simulating the innovative design process and fueling creativity for model re-design and implementation. the covid- pandemic posed a threat to essential palliative care services, including building connections between patients, families, and healthcare teams; mitigating isolation, loneliness, and fear; managing symptoms; determining care priorities in the face of life threatening illness; and promoting comfort, connectedness and dignity during the dying process. as in other medical centers, patient visitation was severely limited and was nearly entirely discontinued in the hospital-based skilled nursing facility ("community living center [clc]") where our inpatient hospice service resides. additionally, our ability to perform inpatient palliative care consultations was limited by the need to preserve ppe. our goal was to find ways to consult with patients, manage symptoms, and connect care providers, patients and families to allow for goals of care discussions, enhance the comfort of the environment, address isolation and loneliness, and minimize collective distress from veterans "dying alone." to identify barriers affecting delivery and quality of palliative care, we reviewed current literature, stories, testimonials and social media to assess the fears, concerns and lived experiences of families, patients and providers on the front lines of the pandemic. [ ] [ ] [ ] [ ] [ ] this provided a deeper appreciation for how the current pandemic has augmented moral distress surrounding isolation at the end of life (eol), including inability to make physical contact and say last "good-byes"; lack of funerals or collective celebrations of life or grieving and recognition of the uniqueness of individual lives amid massive loss of life; and the lack of other expressions of human connection, belonging and care. we noted the potential for tablet computers and video conferencing to bridge the isolation gap and support palliative care services, as well as the potential for telehealth visits near the eol to go terribly wrong. [ ] [ ] [ ] [ ] [ ] [ ] we identified people within the pct who could commit time and expertise in the following domains: telemedicine, quality improvement, patient care coordination, and eol and grief support services. as this group developed methods, plans and materials, we kept other pct members and facility stakeholders informed of our progress. before the pandemic, our pct had taken steps to receive telehealth training and begin video visits for outpatients, but competing institutional priorities delayed palliative care telehealth implementation. as the pandemic unfolded, there was a general embrace of rapid initiation and utilization of telehealth by the pct, patients, and the entire healthcare system. on march , , va also granted permission for va clinicians to use alternative non-public-facing video conferencing platforms during the public health emergency which increased the ease and availability of telehealth communications. the flexibility and availability of tablet computers and multiple video conferencing platforms resolved some initial barriers to telemedicine implementation. however, the processes for coordinating inpatient virtual consultations and family visits along with infection prevention procedures for tablet cleaning had not been specifically described by other va pcts or facilities. thus, we aimed to develop a formal standard operating procedure (sop) for tablet use for inpatient consultations and quickly train pct staff to facilitate telehealth encounters between providers, patients and families. we initiated discussions with providers across va and non-va systems and with local telemedicine champions to assess "best practices" regarding the use of tablets, video chat platforms, and procedures for safely transferring tablets in and out of patient rooms. we referenced internal va guides for training clinicians in the use of telehealth within va, and ensuring compliance with va approved video telecommunications procedures. , we reviewed existing protocols to ensure safe disinfection of tablets after use. , we also began to consider ways to "humanize" the care process during video chat sessions (table ) . we developed a task list to organize our activities and assign tasks among the workgroup members (table ). this included procuring tablets that operated through guest wi-fi services and completing "test calls" confirming our ability to perform telehealth encounters throughout the facility; training pct members in the use of va and non-va video conferencing platforms; and developing a facility-wide standard operating procedure (sop) for the safe use of tablet computers for clinical video telehealth consultations and patient-family visitations for both inpatient palliative care services and clc veterans (supplemental table ). we then engaged with stakeholders across our facility to inform and educate about our pct service adaptations to incorporate telehealth into inpatient eol care. our stakeholders included local telemedicine champions, medical directors and mr. l was a -year-old man with type diabetes mellitus who was admitted to the va with covid- pneumonia after leaving hospital a against medical advice. he was initially admitted to hospital a on april th, with fatigue and poor oral intake. a covid-pcr test obtained at admission was positive. his hospital course was complicated by superimposed bacterial pneumonia, delirium, malnutrition, and acute kidney injury. his family felt he was not receiving adequate care at hospital a. they felt nurses were rarely going into his room and they worried about his isolation and not receiving human touch. one of his family members risked exposure to covid to take mr. l out of hospital a and bring him to the va where they hoped he would receive care more to their liking. after va admission on may th, , his course was complicated by agitated delirium. he became increasingly tachycardic, tachypneic, and hypoxemic, and was transferred to the icu on may th with worsening renal function and hypotension. his respiratory status was tenuous on high-flow oxygen. his family wished to see him, but they had been exposed to covid- and face-to-face visitation was not possible. palliative care was consulted, and a telehealth family meeting was held with the pct using the family's smart phone to offer support and discuss who he was as a person, his condition and care goals. his family again described their distress about the absence of touch and caring they perceived at hospital a. they shared his legacy including his strong christian faith, his years of work with veteran service organizations and his love of restoring old harley motorcycles. he was a tinkerer who could fix anything, and an avid gardener and fisherman who took great pride in tying and designing his own flies. he loved to regale anyone who would listen with tall tales and could keep an audience spellbound. he especially missed his black labrador who had been his close companion since he was a puppy, but who had died months ago. his family believed he would not want to suffer in the hospital and that he would want to transition to a comfort measures only treatment plan. they were heartbroken not to be able to visit him in person, but willing to allow the pct to set up video visits using a tablet computer at mr. l's bedside and the family's own devices. roles and responsibilities were discussed in advance of the bedside video visits. each staff member was intentional about offering connection, compassionate care and touch, and checking back with family members to ensure their needs were being met. the pct nurse coordinator (a.f.) arranged for the tablet to be brought to the patient's room, scheduled times for each call, and obtained contact information for family members. the coordinator prepared the family regarding the patient's condition and what they might see in the icu room. the chaplain, icu nurse practitioner, and bedside nurse donned ppe and established a video link with each family member using the tablet. the chaplain sang hymns, read his favorite bible verses, and prayed with them. the nurse ensured that mr. l's physical symptoms were being addressed, and the nurse practitioner held the tablet so family could see the patient and the chaplain's spiritual care interventions and answer medical questions as needed. family and staff debriefs were used to evaluate the success of the palliative care telehealth program. the pct nurse coordinator debriefed with the family following the facetime visits. they were very pleased with how things went. family were told that staff would continue to say their names to him, talk with him about family memories they wished to have shared with him, and ensure he was touched regularly. although the circumstances were very sad, his family felt connected with him in his final hours and were able to say their goodbyes. in a staff debrief, participants found the experience surprisingly intimate and rewarding. this quality improvement project accomplished positive outcomes: the successful deployment of video telehealth technology for inpatient eol care; and the preservation of the intimacy and communication that is essential to palliative care. we found that implementing palliative care telehealth for inpatients involved building the infrastructure through protocol development, training, and physical infrastructure; and engaging the entire care team and family to explore non-traditional ways to visit with the patient. a second consideration was attention to rapport and trust building. we found that adjustments for communication by video were critical, in line with the reported experience of other , these included identifying and planning care around family and provider needs including family perceptions of a perceived lack of human connection and physical touch; feeling that the patient's basic needs were not being met; and the anguish of feeling socially isolated. by listening to the family, their concerns and perspectives, we found ways to adapt the video visits to be more human and personalized. this included demonstrating human touch and caring at the bedside during the video calls, coaching family as to what they would see, inquiring about legacy issues and other specific needs including spiritual care, and incorporating these into the video visit. during and after the video calls, bedside staff used this guidance from family to speak to the patient about specific family members, family stories, hobbies/ interests, offer music/entertainment the patient enjoyed, and offer spiritual care tailored to the patient's needs. staff performing bedside care reported a deeper connection and recognition of the person in the bed. his family valued the ability to see and talk with the patient virtually, and the opportunity to engage in life review and share stories of his legacy with healthcare staff. they expressed their gratitude in knowing the patient was being treated as a person. in the end, we found that the technological aspects of care receded into the background and the human aspects remained visible. and, we were fortunate that the technology worked well. given how much the incorporation of specific person-centered actions, storytelling and touch added to the experience for family and staff, we adapted our processes and trainings to formally include these components ( table ) . as the number of covid- patients has decreased in our region we are aware that subsequent waves of infections may yet occur, and it is imperative to maintain and improve our palliative care telehealth program. we continue to engage with stakeholders throughout the facility to inform and educate them about our virtual palliative care model adaptations, and how we can assist them to incorporate technology into eol-specific care. this involves on-the-spot and advance covid-specific training of bedside nurses on eol care, palliative care communication strategies, utilization of telehealth technology; facility-wide training in the use of our sop; and meetings with medical directors and nurse managers of covid-care and intensive care units regarding how to access the tablets and modifications in palliative care services during the pandemic. the pandemic was a powerful motivator for systemic change that propelled us to find new ways to provide caring and connectedness for patients at the eol and their families. our experience shows that although technology does not replace face-to-face encounters, it can offer meaningful connection. redefining the traditional palliative care model required humility and the courage to embrace change. we are learning to live with the fear that technology will fail and that some families, patients and staff will be disappointed if the virtual care alternative is not "as good as" the "gold standard," and are giving ourselves permission to make mistakes while we learn a new care model. by conceptualizing this work as a business model innovation and identifying the processes necessary to deliver personalized virtual palliative care within a quality improvement framework, we hope to provide others with a useful template for reinventing palliative care service delivery in other care systems. while we recognize that the applicability of our work to other care settings and patients may be limited by the focus on a single case at one va medical center, we extended its generalizability by grounding our approach in broadly accepted principles in business model innovation and quality improvement. in the spirit of learning together, we offer a few "take-home messages" in table . we hope that the model we developed, and the lessons learned will be durable and positive outcomes from the pandemic. table . take home messages. the technology is imperfect; have a backup plan (probably the telephone) video connections will not work well for everyone; be prepared to accept that not everyone's needs will be fully met. most people will appreciate that you tried "don't let the perfect be the enemy of the good" be flexible, creative and use the tools that are available: if you cannot get tablets, phones in every room are better than nothing. other alternatives: vocera devices; baby monitors; their personal smart phones to establish a connection when starting a palliative care telehealth project, develop a business plan and use a rapid-cycle quality improvement approach do not wait for someone else to get things started. start small, but do start nursing home care in crisis in the wake of understanding and addressing sources of anxiety among health care professionals during the covid- pandemic mental health needs of health care workers providing frontline covid- care coronavirus preys on what terrifies us: dying alone mourning in isolation': chaplain tries to comfort families of covid- patients. morning edition. published fact check: are coronavirus patients dying alone in hospitals? usa 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chief information officer ( ) disinfecting the ipad: evaluating effective methods disinfection of ipad to reduce contamination with clostridium difficile and methicillin-resistant staphylococcus aureus the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the authors received no financial support for the research, authorship, and/or publication of this article. katherine c. ritchey https://orcid.org/ - - - david a. gruenewald https://orcid.org/ - - - supplemental material for this article is available online. key: cord- -axr qu authors: rolland, john s. title: covid‐ pandemic: applying a multi‐systemic lens date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: axr qu the covid‐ pandemic has a pervasive effect on all aspects of family life. we can distinguish the collective societal and community effects of the global pandemic and the risk and disease impact for individuals and families. this paper draws on rolland’s family systems illness (fsi) model to describe some of the unique challenges through a multi‐systemic lens. highlighting the pattern of psychosocial issues of covid‐ over time, discussion emphasizes the evolving interplay of larger systems public health pandemic challenges and mitigation strategies with individual and family processes. the paper addresses issues of coping with myriad covid‐ uncertainties in the initial crisis wave and evolving phases of the pandemic in the context of individual and family development, pre‐existing illness or disability, and racial and socioeconomic disparities. the discussion offers recommendations for timely family oriented consultation and psychoeducation, and for healthcare clinician self‐care. the novel coronavirus- pandemic presents unique challenges for individuals and families. with the current lack of an effective treatment or a preventive vaccine, all aspects of life are affected (luttik et al, ) . stringent mitigation efforts, such as social distancing, household quarantine, facemasks, vigilant hand washing, and avoidance of public gatherings and transportation were instituted in the united states in march and are likely to continue in some form for the foreseeable future. the covid- pandemic has unmasked the stark reality and profound health and economic consequences of glaring socio-economic and healthcare disparities. risks for morbidity and mortality vary enormously by social location, in particular, race, social class, gender, age, ability, and geographic location (cdc, ; price-haywood et al, ) . with infection, the course, and recovery or fatality. with a pandemic individuals and families are continually dealing with developments at all levels. it is useful to inquire about and frame adaptation in those terms. most people focus on their local situation and risks in their daily lives, job, family, and social network -getting food, medicine, and other needs. this discussion will highlight the individual and family experience. also, this paper is written in the midst of the pandemic when some of the emerging issues, such as if and when of second or third waves/spikes of cases, the timing of a vaccine or economic recovery, and the ultimate scale of emerging mental health and psychosocial impact remain uncertain (amsalem, dixon, & neria, ; wanga et al, ) . how can we organize this complex covid- landscape in a manner helpful to families and clinical practice? the family systems-illness (fsi) model (rolland, ; ; ) provides a useful framework for assessment and treatment with families dealing with a range of serious and chronic illness and disability, including infectious disease. (see rolland [ ] for useful resources, practice guidelines and case examples). the fsi model is grounded in a strengths-orientation, viewing family relationships as a potential resource and emphasizing possibilities for resilience and growth (walsh, ) . seeing the family as the interactive focal point, this approach attends to the systemic interaction between an illness and family that evolves over time. the goodness of "fit" between the psychosocial demands of a particular condition over time and the family style of functioning and resources is a prime determinant of successful versus dysfunctional coping and adaptation. the fsi model focuses on three dimensions of experience: ) a psychosocial typology of this article is protected by copyright. all rights reserved chronic conditions; ) major time phases in their evolution; and ) key family system components. in addition to communication processes and organizational/structural patterns, particular emphasis includes: family and individual life course development in relation to the time phases of a disorder; multigenerational legacies related to illness and loss; and belief systems (including influences of culture, ethnicity, race, spirituality, and gender) (figure ). place figure about here covid- is a novel coronavirus infectious disease. to create a normative context for the pandemic experience, families can benefit from a psychosocial map as they navigate overwhelming challenges. first, the psychosocial map offers an understanding of covid- in systems terms, which explicates the expected (or uncertain) pattern of practical and emotional demands of this disease over the course of the pandemic. second, such maps are helpful for families in understanding systemic processes. in the fsi model, illness patterning can vary in terms of type of: ) onset (acute or gradual) ) course (resolves versus progressive, constant, relapsing) ) outcome (non-fatal to fatal) ) type and degree of disability or complications ) level of uncertainty about its trajectory. some diseases have clear-cut predictable patterns; such as als, which is gradual onset, progressive, disabling, and fatal. by contrast, covid- manifests in a variety of unpredictable ways. for most, it is not a chronic illness, but for some it has long-term health consequences. for some, it is fatal. despite significant risk differences related to social location, age, and health status, anyone can be infected and then can transmit covid- ; anyone may develop a severe case and may die. unlike other infectious diseases that run their course in known timeframes or are seasonal and/or regional, currently there is no known endpoint to the covid- pandemic. this uncertainty and indeterminate timeframe are particularly taxing in a cumulative way -pandemic fatigue. further, data is accumulating that recovery can be protracted and result in long-term medical complications - this article is protected by copyright. all rights reserved cardiovascular, pulmonary, and neuropsychiatric are among the most common (long et al, ; varatharaj et al, ) . in the united states, six in ten adults have a chronic illness (nccdphp, ) , which medically heightens their risk of getting covid- and increases the risk, to varying degrees, of a severe case and mortality. one in four adults have a chronic disability. the pandemic magnifies longstanding prejudices toward those with disability, stigmatizing and treating them as inferior. many in the disability community fear that their lives would be expendable in circumstances of a shortage of adequate covid- testing, icu beds, respirators, and response resources. ageism contributes to attitudes that older persons are expendable. compared with younger and disease-free individuals, older adults and those with preexisting chronic conditions must grapple with higher risk of complications and deciding their acceptable social risk as covid- restrictions are relaxed. individuals and families with an already chronically ill or disability-challenged member may be far less able to absorb the added caregiving burdens if either an affected member or a primary caregiver develops covid- . uncertainties and unknowns abound with covid- . perhaps the most challenging are the facts that transmission is invisible and many individuals that carry the disease, maybe even most, are asymptomatic, yet contagious. these disease characteristics heighten the experience of living with risk and anticipatory loss (rolland, ; ) . for example, the ambiguity of being at risk versus an asymptomatic carrier would increase fears of transmitting covid- to an older or chronically ill at risk family member. the importance of viral load (the concentration of virus in saliva) in covid- severity is established, but the length of exposure and the level of viral load needed for infection to occur are still ambiguous. further, the incubation period, although on average five days, can vary from two to fourteen days. technically, this means that if someone wants to visit an individual at higher risk beyond the socially distanced visit they should consider self-quarantine for fourteen days beforehand. for many families this is not feasible or realistic. now the discussion is about safe "social pods" visiting with others who are practicing safe protections. this article is protected by copyright. all rights reserved ) unpredictable course. some who are infected seem fine then progress rapidly to a lifethreatening state (see the case description below). some appear to be improving and then take a turn for the worse. this necessitates ongoing monitoring and vigilance by family members for a protracted period. ) uncertainty regarding long-term complications (e.g. cardiac, respiratory, cns). recovery has proven slow and ongoing complications may occur, even in those who were asymptomatic. this puts the affected member and family members into a protracted period of limbo. they don't know what will be the eventual new normal. key questions for families include: "what caregiving and role functions may need longer-term reevaluation?" and "what additional economic strains would this create?" larger collective public health uncertainties include: ) who has been infected? who has immunity and for how long? antibody status and degree and length of time of protection from antibodies are not yet well understood (robbiani, et al ) . ) how long until an effective and widely available vaccine emerges or until herd immunity (roughly % of total population has developed antibodies) negate the impact of the disease? despite some horrific initial spikes, such as new york city, the proportion of individuals that have been infected with covid- in these hot spots is only % of the population. ) incidence and fatality trends can improve in one region, but then spike in another region. a second or third wave of resurgence is very likely, unless widespread readily accessible testing and contact tracing are implemented, and the public adheres to ongoing and shifting precautions. as of july , we are seeing the dire consequence of too rapid state-level relaxation of restrictions and resulting widespread disregard of key precautions (e.g. mask usage). ) without strong national leadership, regulations, and guidelines, regional improvements and surges can occur independently. from a public health standpoint, often divergent and sometimes politically motivated state-based regulations are epidemiologically nonsensical. locally, individuals and their families lack clear, consistent information and guidelines. this chaotic process is both medically dangerous and psychologically exhausting. this article is protected by copyright. all rights reserved these myriad individual and population level uncertainties have significant impact on and implications for families. as this is written in july , the cumulative mental and physical health consequences of this "pressure cooker" existence are just beginning to emerge (killgore et al, ; wanga et al, ) . living well with illness uncertainties and threatened loss (rolland, ; ) entails acknowledging the possibility of loss, sustaining hope, and building flexibility into planning that can accommodate changing circumstances. similar to living with chronic illness, the metaphor that living with the uncertainties of the pandemic is a marathon not a sprint is apt and clinically useful. systemic racism has a profound impact on covid- susceptibility. july cdc data reveals that latinx and african-americans are three times as likely to become infected with covid- as their white neighbors throughout urban and rural regions of the united states and across all age groups. and, they are twice as likely to die from the disease as white people. native american people also experience similar disparities. lower income families, disproportionately african-american, latinx, and immigrant, rely on income from members working in job conditions at high risk of infection. crowded multigenerational living conditions makes social distancing difficult in households (cdc, ). living with racial residential segregation, at a distance from healthcare facilities and grocery stores, makes it harder to get needed care or garner supplies for stay-at-home precautions. lower income families' livelihoods require leaving home and taking public transportation daily to work in the service sector, factories, and/or other crowded conditions, where personal safety is often compromised for the sake of a paycheck needed for family survival. these families live with the daily fear that covid- will be brought into the home from work settings and travel to and from work. the ability of most whitecollar desk jobs to be accomplished remotely has provided double protection -preventing covid- infection and keeping one's job. this is a huge advantage over jobs requiring physical presence. dr. anthony fauci, the director of the national institute for allergy and infectious diseases, stated at a us congressional hearing (june , ) that the coronavirus has been a "double whammy" for african-americans, because they are more likely to be exposed to the disease by way of their employment in jobs that cannot be done remotely. second, they are more vulnerable to severe illness this article is protected by copyright. all rights reserved from the coronavirus because they have higher rates of underlying conditions like diabetes, high blood pressure, obesity and chronic lung disease (bolin & kurtz, ; cunningham et al, ) . these individuals often lack health insurance or are under-insured, and lack access to adequate basic health care, including paid family and medical leave (bartel et al, ) . this leaves them and their families extremely vulnerable to severe illness and economic ruin if covid- strikes. almost twothirds of us bankruptcies are linked to illness and unaffordable medical bills (himmelstein et al, ). the need for an "improved medicare for all" national healthcare system could never be more urgent. covid- is a risk to all family members and prevention requires an understanding of mitigation strategies within the household and in the community. this necessitates ongoing effective communication amongst family members of all ages. children coping with chronic illnesses, such as diabetes or asthma, need to learn about their illness and aspects of self-management from an early age. the covid- pandemic warrants the same mindset, although here healthy children (and adults) may be introduced for the first time to a "new reality" of personal disease risk. it is valuable to inquire whether anyone is protected in or excluded from such discussions (e.g. children; or aged, frail, or vulnerable family members) and why. adults may feel reticent to share with children given the uncertain nature of covid- , but communicating age-appropriate information about the disease supports psychological wellbeing for the child and the family (dalton, rapa, & stein, ) . the pandemic may reveal sharp differences between partners regarding beliefs about appropriate safety restrictions for children or each other (grose, ) . couples need to discuss these differences, which may warrant additional input from primary care providers. individual family members may process the same covid- information through very different historical, cultural, and political ideological filters, leading to conflicting views. it's crucial to understand beliefs about what can cause or prevent covid- and personal risk perception. research has repeatedly shown that the perception of risk is a more powerful behavioral motivator than actual risk (ferrer & klein, ) . given covid- 's highly transmissible nature, perceptions of relative invulnerability and disregarding precautions can put all family members at risk. teenagers this article is protected by copyright. all rights reserved and young adults often ignore social distancing and mask use out of a belief in personal invulnerability. it can be enormously helpful for families to hold regular family "meetings" to review daily life together during the pandemic and make any needed adjustments. where possible, tele-technology can enhance such pragmatic discussions by facilitating inclusion of family members living at a distance and separated by pandemic precautions. families at higher risk benefit from proactive discussions or consultations about role function flexibility and other potential extended family, community, or healthcare provider resources. this might include adult children (and their parent(s) conferring about shared responsibilities to protect an elder or tend to them if covid- strikes. another example is proactive discussions among parents and with their teenage children about any misguided sense of invulnerability. disagreements about appropriate safety precautions can occur among family members, between neighboring families, between states, and sadly often divide along political party lines. we need one data-driven and unified national public health mandate on precautions and adequate testing, which is separated from politicians and divergent political agendas. this would save countless lives. it must be well funded across socio-economic and minority group strata. compared to other countries strategies over time, us failings at a national level are increasingly and blatantly clear. a unified message would greatly help galvanize state, community, and family level buy-in and cohesion. although most contemporary couples and families strive toward flexible and equitable gender role relations, the influence of culturally based gender norms persist for caregiving roles. as a health crisis, covid- heightens the tendency to expect women to absorb the bulk of child and elder care and attend to the practical and emotional needs of an ill member. it is crucial to explore family expectations about caregiving roles. encourage flexibility and a shift from defining one female member as the caregiver to that of a collaborative caregiving team (walsh, ) . with the geographic dispersion of many families, members may need to accept not seeing loved ones for an indeterminate period. elders, especially those that are frail and chronically ill, this article is protected by copyright. all rights reserved consider that they may not see members living at a distance again. adult children and grandchildren might not see an older parent again. this possibility always is present, yet, is heightened with the pandemic. families can harness this fact to appreciate each other more fully, express caring, and use it as an opportunity to repair and heal relationship wounds. similar to many people living with the uncertainties of a life-threatening illness, the pandemic can stimulate a sense of urgency for family members to deal with unresolved relational issues. whether living near or far, adult sons and daughters have to weigh under what circumstances they would visit their elder parents. if their parents contract covid- or develop a different health crisis, would they accept the risk of seeing them and providing caregiving, thereby putting themselves and their own children at risk. conversely, adult children often fear bringing the infection to their older parents, who are at higher risk. these are profoundly difficult questions, warranting proactive family discussion and consultation with healthcare providers. adult children need to discuss these issues within their own nuclear family. a spouse or partner may have different views about acceptable risk as well as their own aging parents or kin to consider. as the pandemic continues to evolve, these discussions will need to be revisited -relating to new covid- data and to changing family circumstances, such as life cycle transitions or altered economic and health status. those living alone, especially seniors, are at increased mental health risk for anxiety, depression, even suicide during the pandemic (aronson, ) . they can experience despair that meaningful social contact and purpose will no longer occur within the timeframe of their remaining years. they typically lack the support and companionship provided by a spouse, partner, or sibling, for instance. other family members carry additional concerns about the vulnerability of an isolated member. social media and tele-technology options (e.g. zoom, facetime, skype) have provided ways for family members and friends to remain more connected. many families have actually had this option for several years, but are now discovering its utility for the first time. geographically dispersed members can gather for a weekly zoom meeting. grandparents can have a play date with grandchildren. despite the two-dimensional shortcomings and oft heard complaint that it's not the same as in-person, this technology enables clinicians to more easily convene family members for this article is protected by copyright. all rights reserved health and mental healthcare consultations and therapy. however, lower income families often lack the computer resources to avail themselves of this technology, adding another layer of disparity. long-term care facilities (ltcf) are a major challenge for safety during the pandemic. june data revealed that in most states a staggering - % of covid- related deaths occur in ltcf (d'adamo, yoshikawa, & ouslander, ). families may need to evaluate the relative safety of leaving a family member in a ltcf or bringing them to live with them. data shows that persons with cognitive impairment are four times more likely to die of covid- in a ltcf than other residents (cdc, ). lack of judgment, impulsiveness, and confusion with dementia all interfere with risk reduction behavior. those with dementia or intellectual disabilities may be particularly distraught and not comprehend why family members are not able to visit. a family caregiving for an elder with advancing alzheimer's disease, that is becoming unmanageable at home, may have to make a wrenching decision between their own limits and personally caring for a loved one. consultation with involved primary care, specialist, and family behavioral health providers can guide and support family decision-making. fortunately, use of technology through video conferencing allows persons who are hospitalized or in ltcf and their loved ones to sustain vital contact. this is especially important in situations of advanced chronic illness or covid- where death may ensue without loved ones physically present again. applying the fsi model time phases can help us understand two levels: ) the course of the covid- pandemic; and ) the experience living with personal covid- risk and infection over time. this section discusses the evolving challenges at both levels for individuals and families (rolland, ) . despite the uncertainty of the future pandemic unfolding, we can diagram a timeline for each level (figures & ) . the pandemic level phases (figure ) are intended to clarify psychosocial challenges over time rather than emphasize covid- biological or public health incidence and prevalence. core psychosocial themes in the evolving course of the covid- pandemic can be conceptualized into this article is protected by copyright. all rights reserved three broad phases. the initial crisis phase is characterized by a wave of cases with varying course, indeterminate length, and potential spikes. for some countries this initial phase/wave subsided within a few months. in the united states, as of july , with a rising spread of cases and regional spiking, it is unclear when the initial wave will end. in the next phase, a second wave is predicted with the potential for one or more following waves. in the final phase, the course of the pandemic spread diminishes as herd immunity is reached, with or without a vaccine. the pattern of the initial crisis wave has varied considerably in different regions. figure illustrates several general patterns in the initial crisis wave and recurrent wave(s). place figure about here with the current pandemic, the initial crisis phase involves adaptation to the societal risk for everyone. depending on social location, the entire population was presented with the medical risks of covid- as well as mitigation and prevention measures. families needed to reorganize family life, including roles and routines, and communicate about practical and emotional challenges of living with the pandemic. acknowledging risk is needed while maintaining hope. it is vital that families see the covid- pandemic as a shared challenge in "we" terms both at a community and family level. because this is a novel virus, our understanding of medical risks has been evolving as new and more population level data emerges. this changing landscape has been complicated by multiple scientific and political voices with different priorities and agendas. health care experts and policymakers often vehemently disagree about whether to prioritize disease prevention or immediate economic recovery strategies. the lack of a unified message in the face of a life-threatening illness is akin to multiple and divergent medical opinions that can leave families bewildered and frightened, often leading to opposing and conflicted viewpoints about what is needed. this pattern increases the likelihood that dysfunctional family patterns will emerge or pre-existing ones will be exacerbated. the economic consequences of the pandemic, stark witnessing and worsening of racial and socio-economic disparities, and the need for covid- -related health precautions are all simultaneously important. yet, immediate priorities, such as continued vigilant covid- illness prevention and back-to-work imperatives in the face of massive unemployment has collided both at accepted article the macro societal level and among family members. the relative weighing of these different risks and options can vary enormously depending on social location. more affluent families can prioritize covid- risk management because of a more secure economic base. most working and middle class families, who live with economic strains, cannot afford to prioritize health unless protected economically by continued government subsidies. with persistence of the initial wave many individuals and families have shifted from a crisis reactive mode to a longer-haul mind-set. where the initial wave has subsided, others may hold onto hope that there will be no second wave. whenever this psychosocial transition occurs, it involves fuller acceptance of the ongoing nature of the pandemic and living with covid- uncertainty/risk and threatened loss. it is a period when families may consider modifying precautions and reorganizing to adapt to a more protracted coping with the pandemic. the emotional strain of living with covid- risk can feel heightened as they experience the realities of living with an ongoing pandemic. as discussed below, the enormous variation in timing and unfolding of covid- new case trajectories locally, regionally, and nationally adds ambiguity to when this transition is salient for a particular family or community. as with the initial crisis phase/wave, one can conceptualize a more indeterminate length recurrent wave phase in the covid- pandemic. this is distinct from an infected individual living with a chronic phase of covid- . covid- is not the only threat to health and wellbeing. as aronson ( ) notes, the challenge for individuals and a society, is that two contradictory realities are simultaneously true. our approach to pandemic containment works, but our approach causes suffering, eroding physical and mental health, and causing economic dislocations. this affects everyone, but especially individuals with pre-existing chronic illness, disability, in later life, and/or subject to socio-economic and racial disparities. the key is to sustain daily structure and meaningful purpose. digital technology facilitates roles for elders, for instance, in connecting with grandchildren, volunteering to help the less advantaged, or political activism. increasingly with covid- , many families struggle with the ongoing challenges of being this article is protected by copyright. all rights reserved confined at close range for extended and open-ended time. in the united states, many parents needed to negotiate more flexible childrearing roles to accommodate having their children home / without going to school, having after school activities, and depending on usual daycare. with special needs children (e.g. autism, cerebral palsy, intellectual disability) or adults (e.g. serious mental disorders, acquired brain injury), families are managing alone without the usual specialized supportive and educational services. communities are exploring innovative ways to provide in-person education and specialized services. also, increased substance use and family violence are emerging as the pandemic continues (bradbury-jones & isham, ). primary care based health and mental health clinicians can touch base periodically with families at known risk and provide some measure of prevention and early intervention. different geographic regions vary in their approach to transition (re-entry) from the initial crisis phase/wave to a more protracted phase for living with ongoing covid- risk. state-by-state stages of re-entry are akin to recovery and rehabilitation from an illness, where "vital signs" are monitored to guide timing of graduation to the next stage of recovery or retreat to a previous one. these larger system rules and recommendations inform and guide families. it is essential for families to discuss this step-wise transition as newer longer-haul covid- guidelines become available. families need to consider how these guidelines fit with their own beliefs and priorities. distinctions for members at different ages (e.g. children, elders) warrant discussion. again, consultation with a healthcare provider can offer clearer guidance, for instance with decisions about a college student or young adult losing a job and returning home to live with parents. with the covid- pandemic, relative risk may wax and wane with the alternation of periods of "flattening the curve" and decline in new cases with periods of resurgence, higher risk, and the need to resume more intensive case suppression/mitigation public health measures. like relapsing illnesses, communities and families need to maintain some measure of vigilance and preparedness for both, not knowing "if" and "when" a resurgence/flare-up may occur (rolland, ) . unlike a condition that is always symptomatically present, here families are strained both by the potential fluctuation of transition between covid- quiescent and resurgence periods and the ongoing uncertainty of when a spike may occur. good spirits in resuming more normal routines may be dashed by a new wave of cases. hunkering-down again after a period of greater freedom can be this article is protected by copyright. all rights reserved discouraging and anxiety provoking. the psychological shift between these two ways of living is a particularly taxing feature for this pandemic as it is for relapsing chronic illnesses. this fluctuating pattern may exact a huge psychological toll over time. public health media and primary care based psychoeducation supplemented by offering periodic individual or family mental health consultation and brief treatment can be beneficial. when living with personal risk and actual infection, shown in figure , we can identify three phases: ) at risk, ) acute infection, which can be symptomatic (possibly fatal) or asymptomatic, and ) a long-term phase that can lead to recovery, may involve complications/chronicity, or can be fatal. living at risk has been integrated into earlier discussion. place figure about here if a family member becomes ill with covid- , especially an elder, precautions often require isolation and self-care without direct caregiving support from family members. in severe life threatening situations that require hospitalization, family members are restricted from direct contact with the ill member. and, if the member dies in a hospital or skilled care facility, it is typically without loved ones present. the inability to be there physically for the dying member's final hours, say goodbye, or have a traditional funeral and burial service can burden family members with myriad complex feelings, such as guilt and anger, that complicate grief reactions. in one recent case, bill, who is and in good health, with the covid- disease, the long-term phase for affected individuals and their families is influenced by an emerging wide range of complications and may be marked by constancy, progression, or episodic change. it is "the longer haul," involving day-to-day living with a potentially chronic condition and associated disruptions in lives and livelihood. salient family issues include: ( ) pacing and avoiding burnout; ( ) minimizing relationship imbalances between the affected individual and other family members; ( ) maximizing autonomy and preserving or redefining individual and family developmental goals within the constraints of covid- ; and ( ) sustaining connectedness in the face of threatened loss (rolland, ; ) . all major life course milestones and nodal events are impacted (rolland, ; ) . covid- risk has put a threatening cloud on the horizon of any event where family, friends, and community networks would gather. this includes: weddings, graduations, funerals and memorial services, family this article is protected by copyright. all rights reserved reunions, annual holiday or vacation traditions, and regular gatherings of community groups or religious congregations. improvisation and creative adapting of events and rituals are key responses fostering family and community resilience (imber-black, ). by example, one of our center trainees' wedding plans were modified to include only ten individuals present for the actual ceremony in their backyard, while other family and friends joined the ceremony via zoom. this adaptive response allowed family members, who live at a distance to join the celebration. while there are a number of important individual and family developmental phases, this discussion will highlight several especially salient ones. those over and/or with a chronic illness (e.g. heart disease, diabetes, obesity, respiratory conditions, immune system compromise) are at much higher risk with more stringent and ongoing restrictions. many families have organized to protect elders and members with chronic conditions, such as limit entering their home or having close range contact. the lack of physical contact, such as hugging, is particularly difficult, contributing to feelings of isolation and disconnection (killgore et al, ) . with all older clients and those with chronic illness, it is important for clinicians to encourage them to have updated advance directives, a health care proxy, and wills. with life-threatening conditions, like covid- , that can involve rapid progression, unresponsive state, or leave cognitive impairment, there is added incentive for preventive or early frank conversations. knowing family members' wishes concerning heroic medical efforts and life support can benefit everyone. despite the short-run challenge of having end-of-life discussions, it is important to keep in mind that many of the most wrenching end-of-life experiences for families occur when the wishes of a dying member are unknown or have been disregarded. since family members are typically not allowed to be present in the hospital with covid- , it is particularly important that a health care proxy has been designated and identified to the healthcare team (rolland, emanuel, & torke, ) . the proxy can be proactive regarding limits of heroic measures or other important cultural and religious preferences. and, the healthcare team benefits in knowing whom to contact in dire circumstances, where urgent life and death decisions may be necessary. this article is protected by copyright. all rights reserved with colleges managing remotely on-line and unemployment staggering among younger adults, future plans are impacted and some put indefinitely on hold. for financial reasons, many young adults are returning home to live with their families-of-origin concurrent with increased economic hardships at home. covid- anxieties, pandemic fatigue, and evolving public health restrictions; along with economic and co-habiting stresses can easily lead to or heighten family tensions. regular family "check-in" meetings can help manage these inevitable strains. covid- and its economic fallout are causing young adults to encounter huge uncertainties about future hopes and dreams. this may be a significant undercurrent strain for adolescents, young adults, and their parents that can benefit from open discussion. it is worthwhile to ask individuals and family members about prior illness or life crisis/adversity experiences that they can draw on when facing the covid- pandemic. areas of vulnerability and resilience are important to identify. for instance, a family history of an untimely illness or death by infection, the succumbing of a robust family member, or a family member suffering alone, can signify particular sensitivity in the context of covid- . a past experience of enduring well prolonged adversity (e.g. poverty) or an illness with high risk and uncertainty (e.g. heart disease) can inspire resilience in the face of covid- . as the covid- pandemic continues, the enormous toll on frontline healthcare workers is emerging as exhaustion, anxiety, depression, insomnia, trauma, and suicide (schechter, et al, ; wong, et al, ) . this toll profoundly and severely impacts their own family relationships. at times, the relentless and largely untreatable nature of covid- confronts the core identity of healthcare professionals to save and cure patients. although healthcare providers often make decisions amid uncertainty, "we typically focus on the patient's risk, not our own. in an infectious disease epidemic, our calculus must incorporate our own exposure risk -and how exposure would limit our ability to care for future patients" (rosenbaum, ) . the sense of helplessness and witnessing waves of death is deeply and cumulatively disturbing, and can lead to a sense of failure and moral injury (griffin et al, ) . this has been compounded by restrictions barring patients' family members' physical this article is protected by copyright. all rights reserved presence to provide comfort as their loved one's life is threatened and often in their final hours. this leaves clinicians aware that they may be the last person that the covid- patient interacts with. as one anesthesiologist put it, "i could be the last person some of these patients ever see, or the last voice they hear. a lot of people will never come off the ventilator. that's the reality of this virus. i force myself to think about that for a few seconds each time i walk into the icu to do an intubation" (saslow, ) . working with illness and loss typically stimulates concerns related to our own and our loved ones' physical vulnerability and mortality. this is especially true with covid- , where we are all at risk. a we-they mindset is impossible. there is an enormous need for debriefing with colleagues and the availability of individual and family mental health consultations. a multi-systemic lens and drawing on the family systems illness model can provide a psychosocial map to guide individuals, couples, and families meet the myriad challenges of covid- . as a worldwide crisis, the covid- pandemic gives us a sense of belonging that can promote solidarity and increase global empathy and caring for each other. the coronavirus disease (covid- ) outbreak and mental health: current risks and recommended actions for older 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virus risks medical bankruptcy in the united states accepted article this article is protected by copyright. all rights reserved rituals in contemporary couple and family therapy loneliness: a signature mental health concern in the era of covid- changing gender norms in families and society cardiovascular complications in covid- the covid- pandemic: a family affair hospitalization and mortality among black patients and white patients with covid- convergent antibody responses to sars-cov- in convalescent individuals anticipatory loss: a family systems developmental framework the expanded family life cycle: family and social perspectives helping couples and families navigate illness and disability: an integrated practice approach the family, chronic illness, and disability: an integrated practice model accepted article this article is protected by copyright. all rights reserved applying a family systems lens to proxy decision making in clinical practice and research the untold toll -the pandemic's effects on patients without covid- voices from the pandemic. the washington post psychological distress, coping behaviors, and preferences for support among new york healthcare workers during covid- pandemic neurological and neuropsychiatric complications of covid- in patients: a uk-wide surveillance study. the lancet psychiatry strengthening family resilience a longitudinal study on the mental health of general population during the covid- epidemic in china healing the healer: emergency health care workers' mental health during covid- key: cord- -ku tmjd authors: sabotič, jerica; kos, janko title: microbial and fungal protease inhibitors—current and potential applications date: - - journal: appl microbiol biotechnol doi: . /s - - -x sha: doc_id: cord_uid: ku tmjd proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. because of their essential roles, their proteolytic activity needs to be tightly regulated. therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. in medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. they can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. they are also valuable tools for simple and effective purification of proteases, using affinity chromatography. because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well. applications of protease inhibitors are intimately connected to the proteases they inhibit. so, as a preface to protease inhibitors, an overview of proteases with the modes of regulation of their proteolytic activity is provided. then, known microbial and fungal protease inhibitors are reviewed, with the emphasis on protein (tables and ) rather than small-molecule protease inhibitors (table ) . finally, their potential applications in the fields of medicine, crop protection and biotechnology are described, based on their target proteases. microorganisms (prokaryotes, yeasts and filamentous fungi) and higher fungi or mushrooms have been selected for review since protease inhibitors of microbial origin have already proven useful in many different applications. higher fungi have emerged as a valuable source of new protease inhibitors with unique characteristics only in the last decade and therefore offer great potential for future applications. proteases, also called peptidases or proteolytic enzymes, constitute a large group of enzymes that catalyse the hydrolysis of peptide bonds. cleavage of peptide bonds can be general, leading to complete degradation of protein substrates into their constituent amino acids, or it can be specific, leading to selective protein cleavage for post-translational modification and processing. peptidases that cleave peptide bonds at the termini of polypeptide chains are called exopeptidases, while endopeptidases cleave peptide bonds within the polypeptide chain. peptidases are classified according to their catalytic type into aspartic, cysteine, glutamic, serine and threonine peptidases, according to the main, functional amino acid residue at the active site. metallopeptidases, on the other hand, are those whose catalytic activity depends on the presence of a divalent metal ion bound within the active site. in the mer-ops database (http://merops.sanger.ac.uk/), peptidases are classified further into families, according to their sequence similarity, and into clans, according to their structural similarity. there are peptidase families assigned in the merops database (release . , july ) and clans, based on structural data (barrett ; rawlings et al. ) . peptidases are present in all living organisms, including viruses, bacteria, archaea, protists, fungi, plants and animals. serine peptidases form the most abundant class, followed by metallo-, cysteine, aspartic and threonine peptidases. there has been an explosive growth of the number of peptidase families observed in eukaryotic organisms, there being peptidases in bacterial genomes and half as many in archaeal genomes and from to peptidase genes in plant and mammal genomes. furthermore, there is a striking difference between the compositions of eubacterial and eukaryotic degradomes, (i.e. the complete set of proteases present in an organism). sixteen peptidase families constitute the core of the nearly ubiquitous peptidase families present in all living forms. additional peptidase families are widely distributed in eukaryotic organisms, while another ten are unique to higher metazoan organisms, performing mainly limited proteolysis in extracellular environments (page and di cera ; rawlings et al. ) . in addition to the merops database, information on proteases can be found in several other online databases, including the degradome database (http:// degradome.uniovi.es/) (quesada et al. ) and the proteolysis map (pmap) (http://www.proteolysis.org/) that comprises five α m a a, a a, c a, c a, c , m , m a, m b, m a, m b, s a, s b, s a a underlined families include protease inhibitors exclusively of microbial and/or fungal origin b × denotes the number of sequence homologues found in each group of organisms: × less than , ×× - , ××× - and ×××× more than (armstrong and quigley ; sottrup-jensen ) serine protease inhibitors ovomucoid (kazal-type) chymotrypsin (s ) and subtilisin (s ) families tight-binding, laskowski described in stramenopiles oomycetes (fungus-like microorganisms distantly related to fungi); e.g. involved in pathogenicity of phytophtora infestans (tian et al. ; rawlings and barrett ) aprotinin i chymotrypsin family (s ) broad inhibitory specificity (ascenzi et al. ; rawlings and barrett ) peptidase b inhibitor i subtilisin family (s ) bacterial inhibitors are propeptides of subtilisin-like proteases. fungal inhibitors are separate polypeptides, e.g. pleurotus ostreatus proteinase a inhibitor poia and yeast proteinase inhibitor yib they are potent but unstable inhibitors, gradually degraded by subtilisin (ascenzi et al. ; dohmae et al. ; kojima et al. ; kojima et al. ; kojima et al. ; rawlings and barrett ) marinostatin i proteases of family s (subtilisin) and certain proteases of family s (chymotrypsin) tight-binding, laskowski. structure stabilized by two internal ester bonds that are essential for their inhibitory activity exclusive to marine bacteria (kanaori et al. ; rawlings and barrett ) ecotin i chymotrypsin family (s ) tight-binding, laskowski for primary binding site. active as dimers, each monomer binds the protease at two binding sites ecotins from enterobacteria and parasites perform a protective role against host digestive proteases and target host proteases to facilitate colonization. structure enables inhibition of multiple proteases with the chymotrypsin fold (eggers et al. ; eschenlauer et al. ; rawlings and barrett ) streptomyces subtilisin inhibitor (ssi) family s (subtilisin, kexin), family s (trypsin, plasmin) and the metalloprotease griselysin (family m ) tight-binding, laskowski exclusive to bacterial actinomycetales order. they probably control endogenous proteases involved in proteolytic activation of transglutaminase (kantyka et al. ; taguchi et al. ; tsuyuki et al. ; rawlings and barrett ) carboxypeptidase y inhibitor i serine carboxypeptidase y (family s ) a defensive role against predatory insects has been shown, but a regulatory endogenous role is also possible (avanzo et al. ; odani et al. ; sabotič et al. ; rawlings and barrett ) human elastase- and endogenous aspergillus elastases (family s ) unknown homologues have been found in a few other ascomycete species and in proteobacteria, but none was characterized biochemically (okumura et al. ; okumura et al. ; rawlings and barrett ) serpin i chymotrypsin (s ) and subtilisin (s ) families. some members inhibit also cysteine proteases of papain (c ) and caspase (c ) families trapping. suicide inhibitors in which a rapid conformational change traps the cognate protease in a covalent complex the physiological role of microbial serpins has been proposed to be to protect the cellulosedegrading apparatus (cellulosome) against proteolytic degradation. the only fungal serpin (celpin), was characterized from the anaerobic fungus piromyces sp. e (kantyka et al. ; law et al. ; roberts et al. ; steenbakkers et al. ) cysteine protease inhibitors thyropin i papain-like proteases (family c ), and equistatin inhibitor unit inhibits an aspartic protease cathepsin d (family a ) tight-binding present in animals and in one bacterial pathogen (coxiella burnetii) that has presumably acquired the gene by lateral transfer (kantyka et al. ; rawlings and barrett ) survivin i caspases-aspartate-specific cysteine proteases (family c ) tight-binding, several mechanisms survivin plays a dual role as a mitotic regulator of cell division and as an inhibitor of caspase activation in the process of apoptosis. fungal homologues have been identified in ascomycete and a few basidiomycete genomes. the fission yeast homologue is a conserved chromosomal passenger protein (huang et al. ; luthringer et al. ; rawlings ) chagasin i protozoan and mammalian papain-like cysteine proteases (family c ) tight-binding parasitic chagasins are involved in regulating endogenous cysteine proteases essential for their life cycle. in bacteria and archaea, chagasins serve as endogenous regulators, and in some pathogenic species, they also serve a protective role against host proteases (kantyka et al. ; santos et al. ) clitocypin (i ) and macrocypin (i ); together named mycocypins i , i papain-like cysteine proteases (family c ) and legumain (family c ) and serine protease trypsin (family s ) tight-binding. mycocypins are small and exceptionally stable proteins. they have a β-trefoil fold formed by the core six-stranded β-barrel that supports loops which provide a versatile surface for the inhibition of several types of proteases unique to basidiomycetes. they probably have an endogenous regulatory role or a role in defence against pathogen infection and/or predation by pests. a defensive role for mycocypins is further supported by their high genetic variability and conformational stability as well as a broad inhibitory profile (brzin et al. ; kidrič et al. ; renko et al. ; sabotič et al. a; sabotič et al. ; sabotič et al. b the high specificity is the result of the structural stabilization of the ia inhibitor in complex with saccharopepsin since the unstructured inhibitor in solution forms an alpha helix upon interaction with the enzyme active site (green et al. ; phylip et al. ) different databases (cutdb, pathwaydb, proteasedb, substratedb and profiledb) (igarashi et al. ). the occurrence of proteases in all living organisms indicates their critical role in essential metabolic and regulatory functions in many biological processes. proteases are important in the production of nutrients for growth and proliferation. extracellular proteases catalyse the hydrolysis of proteins into smaller peptides and amino acids for subsequent absorption into cells, constituting a very important step in nitrogen metabolism. proteases perform critical regulatory functions in numerous physiological processes since they regulate the fate, localization and activity of many proteins, modulate protein-protein interactions and contribute to the generation, transduction and amplification of molecular signals. proteases are involved in a wide span of cellular and metabolic processes, including regulation of gene expression, dna replication, transport of proteins, cell growth and differentiation, cell cycle, heat shock response, sos response to dna damage, misfolded protein response, oxidative stress response and programmed cell death (lopez-otin and bond ; rao et al. ). furthermore, in plants, proteases are important in the build-up and breakdown of seed storage proteins during seed germination, protein remobilization upon organ senescence and in many developmental processes such as embryogenesis, chloroplast biogenesis, photomorphogenesis, hormone signalling, flower development, pollen-pistil interaction and local and systemic defence responses against pathogens and herbivores (simoes and faro ; salas et al. ; schaller ; van der hoorn ) . moreover, in animals, proteases are involved in tissue morphogenesis and remodelling, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, haemostasis, blood coagulation, inflammation, immunity, autophagy and senescence (lopez-otin and bond ). proteases of microbial and fungal origin offer a wide range of biotechnological applications. alkaline proteases have been used in the detergent industry for over years. proteases with elastolytic and keratinolytic activities have been used in the leather industry for de-hairing and baiting of skins and hides. the food industry uses proteases in cheese making, baking, preparation of various protein hydrolysates, meat tenderization and manufacturing protein-rich diets. in the pharmaceutical industry, proteases have found uses as therapeutic agents as well as additives in preparations of slow-release dosage forms. bioprocessing of used x-ray films for silver recovery involves the use of alkaline proteases. proteases allow potential applications for the management of wastes from various food processing industries and from household activities. in addition to industrial and medical applications, proteases are used in basic research; for example, proteases with very selective peptide bond cleavage are used in protein sequencing, unselective proteinase k is used in nucleic acid isolation, and trypsin is widely used in maintaining animal cell cultures (kumar and takagi ; rao et al. ) . there is also the downside to proteases as some are important virulence factors of many pathogenic bacteria, parasites and viruses. these proteases are involved in acquiring nutrients for growth and proliferation through host tissue degradation and evasion of host immune defences. in addition to colonizing and facilitating dissemination functions, they are also involved in evading the host immune system by interrupting the cascade pathways, disrupting the cytokine network, excising cell surface receptors and inactivating host protease inhibitors (maeda ; travis and potempa ; supuran et al. ) . because proteases play essential roles in life and death processes in all living organisms and because peptide bond hydrolysis is irreversible, anomalies in proteolytic activities lead to numerous pathological conditions, including cancer, neurodegenerative disorders and inflammatory and cardiovascular diseases, as well as bacterial, viral and parasitic diseases (lopez-otin and bond ; turk ) . activity of proteases is regulated on several levels, including regulation of gene expression at transcriptional and post-transcriptional levels, synthesis as inactive zymogens, blockade by endogenous inhibitory proteins, spatial and temporal compartmentalization, post-translational modification (glycosylation, phosphorylation, co-factor binding), proteolysis and degradation (lopez-otin and bond ). protein protease inhibitors constitute a very important mechanism for regulating proteolytic activity. they can be classified approximately according to the class of proteases they inhibit (for example, serine or cysteine protease inhibitors). however, those composed of multiple inhibitor units and pan-inhibitors (such as α -macroglobulin of family i ) that target proteases of different catalytic classes prevent unambiguous classification. a more detailed classification is included in the merops database (http://merops.sanger. ac.uk/), which follows a hierarchy similar to that for proteases. protease inhibitors are grouped into families based on sequence homology and into clans based on protein tertiary structure. in release . ( july ) of the mer-ops database, there are families of protease inhibitors, and those with available three-dimensional structural data have been assigned to different clans (rawlings ) . of the families, include members of microbial and fungal origin (tables and ). of these, seven families include members of exclusively bacterial origin (i , i , i , i , i , i , i ), and five families include members of exclusively fungal origin (i , i , i , i , i ). in addition to protein protease inhibitors, the merops database includes a list of small-molecule inhibitors that are well known and widely used. many of them have been synthesized in the laboratory; however, those that occur naturally (table ) have been isolated from bacteria and fungi (rawlings ) . there are two general mechanisms of protease inhibition, namely, irreversible "trapping" reactions and reversible tight-binding reactions. trapping reactions work only on endopeptidases and are the result of a conformational change of the inhibitor triggered by cleavage of an internal peptide bond by the host protease ( fig. a ). only three families utilize a trapping mechanism: i (serpins), i (α -macroglobulin) and i (viral caspase inhibitors). reversible tight-binding inhibition is widespread, the best known being the "standard canonical" or "laskowski mechanism", in which the inhibitor has a peptide bond that is cleaved by the peptidase active site in a substrate-like manner. the inhibitor is only slowly released due to the conformational stability of the stabilized loop that can mimic a substrate. this mechanism has been conclusively demonstrated only for inhibitors of serine proteases. other reversible tight-binding protease inhibitors physically block the protease active site by high-affinity binding to sites on either side of the active site (fig. b, c) . binding of an inhibitor to the active site can also be irreversible, when an electrophilic reactive group of the inhibitor forms a covalent bond with an amino acid residue in the enzyme active site. there are also some inhibitors that block the exosites, to which substrate binding occurs in addition to the active site in some proteases (christeller ; rawlings ; ). the families of protein protease inhibitors that include members of microbial and fungal origin are described in table , and information of their distribution in taxonomic groups is given in table . protease inhibitors are described in groups according to the catalytic class of protease they inhibit and following the merops inhibitor classification (rawlings ; rawlings and barrett ) . since prokaryote-derived protease inhibitors have been reviewed recently (kantyka et al. ), more information on protease inhibitors of fungal origin, including yeasts, filamentous fungi and mushrooms, is provided here. among the small-molecule protease inhibitors isolated from bacteria and fungi (table ) , there are several that show broad inhibitory specificity and inhibit proteases of different catalytic classes. several inhibit both serine and cysteine proteases (antipain, chymostatin, leupeptin), serine and metalloproteases (bestatin, puromycin), metallo-and cysteine proteases (amastatin) or metallo-, cysteine and serine proteases (bacitracin a). of the small-molecule cysteine protease inhibitors, the best known is e- ( -[l-n-(trans-epoxysuccinyl) leucyl] amino- -guanidinobutane), an irreversible inhibitor originally isolated from aspergillus japonicus (hanada et al. ) and routinely used as a class-specific cysteine protease inhibitor. a number of e- analogues have been synthesized in order to improve selectivity for a particular cysteine protease. several inhibitors are specific for metalloproteases and inhibit more than one protease family (e.g. phosphoramidon). the only natural small-molecule inhibitor of aspartic proteases is pepstatin, originally isolated from various species of actinomycetes (umezawa et al. ) , which inhibits several families of aspartic proteases. it is a hexa-peptide containing the unusual amino acid statine (rawlings ) . proteases play an important part in almost every biological process; therefore, unregulated activity often leads to disease. in this review, only excessive proteolysis will be addressed as it is the one that can be reversed by protease inhibitors. excessive proteolysis plays an important role in cancer and in cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. due to the obvious relevance of protease inhibitors, they have been studied extensively with the intent to develop therapeutic drugs (drag and salvesen ; turk ; haq et al. ) . proteases that have a potential as therapeutic targets are reviewed, according to their catalytic type, for each group of disease-causing organisms and for other human diseases. information on the availability of protease inhibitors for each protease described is provided, with the emphasis on those for which specific inhibitors have not yet been identified. proteolytic processing of virus polyprotein into structural and non-structural proteins is an essential part of the viral replication cycle, making the proteases an important antiviral drug target. several viral proteases have been studied as therapeutic targets. although proteases of any virus could be potential antiviral targets, viruses that cause chronic diseases (e.g. hiv, herpes virus) and those that could cause large-scale epidemics (e.g. sars coronavirus, dengue virus) have received most attention. several protease inhibitors acting against the human immunodeficiency virus (hiv- ) protease, a homodimeric aspartic protease, have been used in treating hiv- infection. fig. examples of protease inhibitors utilizing irreversible "trapping" reaction (a) and reversible tight-binding reactions (b and c). proteases are shown in light grey, their active site residues in black and inhibitors in dark grey. a serine protease trypsin in complex with serpin (family i ) (pdb id k o). the protease cleaves the reactive centre loop of serpin, which triggers a conformational change in the inhibitor and trapping of the protease in an inactive covalent complex. b cysteine protease cathepsin v in complex with clitocypin (family i ) (pdb id h s). the inhibitor binds to the protease active site cleft and obstructs access of substrate. c aspartic protease plasmepsin iv in complex with the small-molecule inhibitor pepstatin a (pdb id ls ). the inhibitor binds in the active site of the protease these are all low molecular weight peptidomimetic inhibitors whose design has been based on the structures of the compounds binding to the protease active site. in therapy, they are usually used in combination with inhibitors of reverse transcriptase (abbenante and fairlie ; anderson et al. ). in addition to a number of designed synthetic inhibitors, a potent peptidic inhibitor of hiv- protease of bacterial origin (atbi) has been found in an extremophilic bacillus sp. (dash and rao ; vathipadiekal et al. ) . other targeted viral proteases belong to the serine catalytic type. human cytomegalovirus (hcmv) is one of eight human herpes viruses and is widespread in populations worldwide, with infection rates of - %. it causes asymptomatic infections in healthy individuals but high morbidity and mortality in immunocompromised individuals. a few inhibitors of the cytomegalovirus protease have been described from bacterial (streptomyces) and fungal (cytonaema) origins (stoeva and efferth ; anderson et al. ). an additional target in antiviral therapy against cytomegalovirus is the proteasome, where the aim is to hinder the recruitment of host proteins by the virus for its replication. several synthetic and a few natural proteasome inhibitors (e.g. lactacystin from streptomyces lactacystinaeus) are known and have been reported to obstruct replication of several viruses, including influenza virus, herpes simplex virus type , paramyxovirus and rhabdoviruses, as well as cytomegalovirus (kaspari and bogner ). the serine proteases ns and ns of flaviviruses are targets for antiviral drug development against hepatitis c virus and dengue virus, with the former blood-transmitted virus causing various liver diseases (including cirrhosis and liver cancer) and the latter being a mosquitotransmitted disease causing dengue hemorrhagic fever. several structure-based designed low molecular weight inhibitors are in different phases of clinical evaluation (anderson et al. ; lange et al. ; tomlinson et al. ). the coronavirus associated with severe acute respiratory syndrome, sars, encodes a chymotrypsin-like cysteine protease m pro that is similar to picornavirus c protease. since the sars global outbreak, several strategies of structure-based design of low molecular weight protease inhibitors have been applied in the search for antiviral drugs against sars (anderson et al. ; sirois et al. ). the first to be considered were the protease inhibitors targeting the picornavirus c protease. the picornaviruses, which encode a c protease, are important human and animal pathogens such as poliovirus, hepatitis a virus, coxsackievirus, human rhinovirus and foot-and-mouth disease virus. inhibitors targeting the c protease of human rhinoviruses that cause common cold, as well as c proteases of other picornaviruses and coronaviruses, have been developed based on structural data, but none has yet successfully passed all the phases of clinical evaluation (neubauer et al. ; wang and liang ) . due to the rapid development of resistance in viruses, the search for novel strategies for developing inhibitors targeting different sites on proteases is encouraged, including the search for novel lead compounds from natural sources and structure-based drug development. bacterial pathogens employ an array of virulence factors that enable their colonization, evasion of host defences and dissemination. proteases are important virulence factors of many pathogenic bacteria, which play roles in acquiring nutrients by direct degradation of host tissue components. the even more important aim of disrupting host immune response and signalling cascades has been reviewed by potempa and pike ( ) . most currently available antibiotics target bacterial cell wall synthesis or protein synthesis. in the light of rapidly spreading antibiotic resistance, bacterial proteases are promising targets for the design of novel antibiotics. metalloproteases are most abundantly represented in primary and opportunistic pathogens, although all catalytic classes are found. these proteases are often associated with mobile genetic elements (plasmids, pathogenicity islands, integrated phages), and their expression is not constitutive but regulated through environmental or cellular signals (travis and potempa ; wladyka and pustelny ) . omptins (outer membrane proteins t) are aspartic proteases (family a ) found in several gram-negative bacteria, including the pathogenic species escherichia coli (ompt), yersinia pestis (pla), shigella flexneri, shigella dysenteriae (sopa), salmonella enterica (pgte), legionella pneumophila (lpa) and plant pathogens agrobacterium tumefaciens and erwinia pyrifoliae (plaa). omptins are bacterial virulence factors and, in addition to their proteolytic activity, possess adhesive and invasive activities. they modulate the coagulation system since they act as plasminogen activators (ompt, pla, pgte, lpa), inactivate tissue factor pathway inhibitor (ompt, pla, pgte), degrade thrombin-activatable fibrinolysis inhibitor (pla, pgte), degrade the complement system proteins (pla, pgte) and antimicrobial peptides (ompt, pla, pgte), and process autotransporters (ompt, sopa). lipopolysaccharide (lps) is required for their enzymatic activity. omptins have a unique catalytic mechanism that combines the elements of both serine and aspartic proteases, and partial inhibition by serine protease inhibitors has been reported (hritonenko and stathopoulos ; valls seron et al. ; yun et al. ). other than a weak substrate-based peptide inhibitor with a d-arg, shown to inhibit ompt (dekker et al. ) , and a colicin immunity protein shown to protect colicin e from degradation by ompt in escherichia coli (duche et al. ) , no specific inhibitors have been reported. no specific inhibitor has so far been found for the exfoliative toxin a, a glutamate-specific serine protease (family s ) produced by staphylococcus aureus, which is the causative agent in staphylococcal scalded skin syndrome. the target of the toxin is a transmembrane glycoprotein desmoglein- of the cadherin superfamily, which plays an important role in keratinocyte cell-cell adhesion (ladhani ) . immunoglobulin a proteases (iga proteases) are serine proteases (family s ) produced by several pathogenic bacteria, including species causing bacterial meningitis, haemophilus influenzae, neisseria meningitidis and streptococcus pneumoniae. the iga proteases enable colonization of human mucosal surfaces by cleaving the secretory iga antibodies, thus disrupting the specific immunity response. except for an early report on substrate analogue inhibitors (burton et al. ) and small synthetic peptide inhibitors (bachovchin et al. ) of neisseria gonorrhoaeae iga protease, there has been no further development of iga protease inhibitors (mistry and stockley ) . other immunomodulating serine proteases are c a peptidases from streptococci (family s )-those of group a (streptococcus pyogenes scpa) and group b (streptococcus agalaciae scpb) streptococci have been described in more detail. streptococcus pyogenes is the causative agent of pharyngitis and also causes rheumatic fever and skin infections, which can develop severe complications, including toxic shock syndrome. c a peptidases are important for streptococcal pathogenesis as they specifically cleave complement c a and therefore prevent the recruitment of phagocytic cells to the infection site (cheng et al. ; collin and olsen ) . antibodies raised against c a peptidase were used to inhibit c a peptidase in vivo (park and cleary ) , but no peptidase inhibitors have been described. serine proteases are important pathogenesis factors in bacteria involved in dental diseases. treponema denticola is a spirochete implicated in the progression of periodontal diseases. a serine protease, trepolisin (also called dentilisin), of family s is an important pathogenesis factor, a mediator of cytopathic effects by degrading host proteins, including extracellular matrix components and host protease inhibitors (sela ) . the broad-range inhibitor of serine proteases (families s and s ), chymostatin, inhibits trepolisin; however, no specific inhibitors have been described. another important oral cavity pathogen involved in periodontal disease, porphyromonas gingivalis, in addition to a few cysteine proteases (discussed further in the following), produces a serine protease, a prolyl tripeptidyl peptidase ptpa (family s ), which is involved in degrading host connective tissue, providing nutrients for bacterial growth (banbula et al. ) . a substrate-based specific inhibitor of ptpa has been developed (xu et al. ). bacterial type i signal peptidases spase (family s ) are serine proteases widespread among gram-negative and gram-positive bacteria and are membrane proteases required for processing newly synthesized secreted proteins. in addition to their essential role in bacterial viability, they are important antimicrobial drug targets as they are involved in the secretion of many virulence factors (paetzel et al. ) . synthetic penem inhibitors have been developed for inhibiting both gram-negative (escherichia coli) and grampositive (staphylococcus aureus, staphylococcus epidermidis) spases. the various penem derivatives display different degrees of activity against these pathogenic bacteria spases (allsop et al. ; harris et al. ). recently, substratebased peptide aldehydes have been shown to be promising inhibitors of escherichia coli and staphylococcus aureus spases (buzder-lantos et al. ). however, the in vitro inhibitory activity and in vivo antimicrobial activities of these inhibitors did not correlate well, so further optimization of and search for novel spase i inhibitors are expected. no specific inhibitors have been described for the streptococcal pyrogenic exotoxin b (speb, streptopain), a cysteine protease (family c ) produced by all strains of streptococcus pyogenes. it is a multifunctional protease and an important pathogenesis factor with several immunomodulating activities, including release of proinflammatory molecules, degradation of extracellular matrix, cleavage of igg in the hinge region and degradation of other immunoglobulins. in addition to class-specific cysteine protease inhibitors, a peptide derivative was shown to inhibit speb, as well as α -macroglobulin and an s-nitrosylated form of α -protease inhibitor (collin and olsen ) . ides (family c ) is another cysteine protease from streptococcus pyogenes that specifically cleaves igg, its only known substrate (vincents et al. ). in addition to specific, inhibitory igg antibodies (akesson et al. ) , synthetic reversible inhibitors were designed, with aldehyde compounds being the most promising; however, no specificity data are yet available for these inhibitors (berggren et al. ). staphylococcus aureus causes a range of diseases, from mild skin infections to life-threatening disorders, including septicaemia, endocarditis, toxic shock syndrome and pneumonia (lowy ) . it expresses several extracellular proteases with proposed roles in pathogenicity, including a serine protease v (sspa), cysteine proteases staphopains a and b (scpa, sspb) and a metalloprotease aureolysin (aur) (shaw et al. ) . staphopains a and b (family c ) are papain-like cysteine proteases that are co-expressed with their respective specific inhibitors staphostatins a and b (dubin ) . their role in pathogenicity has not been determined. staphopain b (sspb) is activated by the glutamyl peptidase sspa (v protease, family s ), which is expressed from the same operon (shaw et al. ). v protease modulates the surface protein profile and so influences the binding of fibronectin by staphylococcus aureus (mcgavin et al. ) . sortases are cysteine proteases (family c ) of grampositive bacteria that catalyse the covalent attachment of proteins to the cell wall peptidoglycan. they have been shown to contribute to the virulence of several important pathogens, including staphylococcus aureus, bacillus anthracis, listeria monocytogenes and streptococcus pneumoniae. therefore, they have been considered to be important targets for the development of novel antiinfective agents (suree et al. ; clancy et al. ) . staphylococcus aureus sortase (srta) has been at the focus of sortase inhibitor development due to the emergence of antibiotic-resistant strains and the need for novel antimicrobial strategies. several types of srta inhibitors have been investigated, including nonspecific sulfhydryl modifiers, peptide analogues of the sorting signal, compounds from plants and marine organisms, and synthetic small-molecule inhibitors. several inhibitors of srta have been described with varying strength and specificity; however, good in vitro inhibitory activity has not yet led to an effective in vivo sortase inhibitor (maresso and schneewind ; suree et al. ; clancy et al. ) . the cysteine protease clostripain (family c ) is a secreted protease of the anaerobic bacterium clostridium histolyticum, a causative agent of gas gangrene (clostridial myonecrosis). clostripain selectively hydrolyses arginyl bonds and constitutes an important clostridial virulence factor (jozwiak et al. ; manabe et al. ). in addition to oxidizing agents, thiol-blocking agents and heavy-metal ions that all inhibit clostripain, good reversible inhibitors have been described, namely, aziridine peptide esters, which are thus promising lead compounds for the development of specific clostripain inhibitors (schirmeister and peric ; barrett et al. ) . gingipains are extracellular cysteine proteases (family c ) produced by the oral pathogenic bacterium porphyromonas gingivalis, a major etiological bacterium of chronic periodontal disease. gingipains comprise two argininespecific cysteine proteases (rgpa and rgpb) and a lysinespecific cysteine protease (kgp). they constitute the major virulence factor of this periodontopathogenic bacterium as they are involved in multiple facets of its virulence and survival, including the destruction of periodontal tissues, disruption of the host immune system by inactivation of host proteinase inhibitors and deregulation of several proteinase cascades, as well as acquisition of nutrients required for bacterial growth and survival in the periodontal pocket (fitzpatrick et al. ; travis and potempa ) . due to their importance in pathogenesis, considerable efforts have been put into discovering or designing specific inhibitors of gingipains. tetracyclines, inhibitors of prokaryotic protein synthesis, have been shown to have, in addition to their antibiotic activity, inhibitory activity against cysteine proteases through binding to the proteinase outside the substrate binding site (imamura et al. ). peptidyl chloromethanes have been used as specific rgp and kgp inhibitors for their characterization (potempa et al. ) , and compound a was shown to attenuate porphyromonas gingivalis virulence through specific kgp inhibition (curtis et al. ) . based on histatin cleavage specificity, small peptide analogues were designed, which specifically inhibit rgp and kgp (kyt- and kyt- , respectively), which display attenuation of several virulence traits of porphyromonas gingivalis (kadowaki et al. ) . chlorhexidine, which has been used in oral healthcare preparations on account of its antimicrobial effects, also inhibits proteolytic activities, including those of gingipains. moreover, chlorhexidine inhibitory activity against r-gingipains was enhanced by the addition of zn(ii), which has also been used in human oral health care (cronan et al. ) . metalloproteases are important virulence factors of many primary and opportunistic pathogenic bacteria and cause major infectious diseases such as cholera, salmonellosis, legionnaires' disease, cystic fibrosis, botulism, tetanus and anthrax (miyoshi and shinoda ) . they have either direct roles in host interaction or indirect roles in processing other important virulence factors. therefore, much has been invested in the search for an effective protease inhibitor for use in treatment (jacobsen et al. ), but none has yet been developed, which would be used in clinic. metal chelators, including edta (ethylenediaminetetraacetic acid), egta (ethylene glycol-bis( -aminoethylether)-n, n,n′,n′-tetraacetic acid) and , -phenanthroline, inhibit metalloproteases in general. the ubiquitous presence of metalloproteases prevents the use of broad-spectrum inhibitors, and the search for potent and specific inhibitors of individual metalloproteases that could find clinical applications is important. the most studied bacterial metalloproteases are those of the thermolysin family (m ), including mpi protease of listeria monocytogenes, coccolysin of enterococcus faecalis, hemagglutinin/proteinase of vibrio cholerae and helicobycter pylori, pseudolysin of pseudomonas aeruginosa, aureolysin of staphylococcus aureus, legionella pneumophila protease and λ-toxin of clostridium perfringens. inhibitors of thermolysin family proteases are of bacterial origin, including those isolated from streptomyces, the small-molecule inhibitor phosphoramidon and protein inhibitor smpi (streptomyces metalloproteinase inhibitor) of family i . another family of inhibitors targeting bacterial thermolysins is family i of animal origin (adekoya and sylte ; supuran et al. ; rawlings and barrett ) . of the bacterial metalloproteases, the light chain domains that are zinc metalloproteases (family m ) of tetanus and botulinum neurotoxins (tent and bonts) from clostridium tetani and clostridium botulinum, respectively, have also been studied extensively. various β-aminothiols have been considered as selective bont and tent inhibitors and have been further developed into strong and selective pseudotripeptide inhibitors of bont/b (blommaert et al. ; supuran et al. ) . clostridium histolyticum collagenases and their homologues from vibrio (family m ) are very effective in connective tissue degradation and hydrolyse triple helical regions of collagen under physiological conditions. they are targeted for both therapy and diagnosis of clostridial infections, and several types of compounds have been found to inhibit them. however, in addition to bacterial collagenases, they also inhibit vertebrate collagenases (supuran et al. ; barla et al. ) . no potent and selective inhibitor has yet been found for metalloproteases of family m from pathogenic bacteria, including serralysin from serratia, pseudomonas and erwinia, aeruginolysin from pseudomonas aeruginosa, mirabilysin from proteus mirabilis and fragilysin from bacteroides fragilis. they are, however, inhibited by protein inhibitors of bacterial origin belonging to family i and hydroxamate inhibitors, including batimastat (supuran et al. ; rawlings and barrett ) . another group of medicinally important bacterial proteases for which specific inhibitors have not yet been described comprises the metalloexopeptidases, which belong to several merops families (m , m , m , m , m , m , m , m , m , m , m , m , m , m , m and m ). of the metallocarboxypeptidases (belonging to families m , m , m , m ) the zinc-containing d-ala-d-ala dipeptidase vanx (family m ) has been studied in view of its ability to mediate antibiotic resistance against vancomycin (crowder ) . similarly, the family m of membrane dipeptidases includes members that degrade β-lactam antibiotics. other carboxypeptidases, such as glutamate carboxypeptidases (family m ), have been studied with a view to clinical use in treating different types of cancer (holz et al. ) . bacterial metalloaminopeptidases, which perform essential cellular functions in protein synthesis and maintenance, have been studied as targets for novel antibiotics. a few reviews on inhibitors designed to inhibit bacterial aminopeptidases of families m (e.g. leucyl aminopeptidases or laps), m (alanyl aminopeptidase) and m (methionine aminopeptidases) cover the natural and designed compounds that could serve as lead compounds for inhibitors aimed at this group of proteases (mucha et al. ; holz et al. ; supuran et al. ; rawlings and barrett ) . bacterial metalloproteases that cleave immunoglobulin a (iga proteinases, families m and m ) constitute important colonization factors for several pathogenic bacteria (e.g. streptococcus, neisseria, haemophilus, clostridium, prevotella, capnocytophaga, bacteroides). no strong and specific inhibitor is available for these enzymes. the same is true also for another family of pathogenesis-related metalloproteases (family m ), including staphylolysin from pseudomonas aeruginosa and lysostaphin from staphylococcus simulans. in addition to their function in increasing virulence, staphylolysin and lysostaphin show bactericidal activity against staphylococcus aureus and have been studied with a view to their use in countering drug-resistant staphylococci (e.g. methicillin-and vancomycin-resistant staphylococcus aureus) (barequet et al. ; desbois and coote ) . the metalloprotease anthrax lethal factor lf (family m ) is a component of the anthrax toxin responsible for the major symptoms and death associated with bacillus anthracis infection (kim and yoon ). an increased interest in anthrax vaccination and treatment methods has been provoked by the use of bacillus anthracis spores as a bioweapon. lethal factor (lf) inhibitors would provide two-fold protection, namely, in preventing early spore protection in macrophages and, later, inhibiting lf disruption of signalling pathways through inactivation of mitogen-activated protein (map) kinase kinases. the search for an lf inhibitor to use in combination with antibiotic treatment is aimed at finding a selective and potent lf inhibitor (shoop et al. ; turk ), which would be non-(cyto) toxic and have good biological stability and bioavailability (johnson et al. ; kim et al. ; li et al. ). the predominant fungal diseases afflict immunocompromised patients and are caused by opportunistic pathogens candida sp. (e.g. candida albicans, candida glabrata, candida parapsilosis) and aspergillus sp. (e.g. aspergillus fumigatus, aspergillus niger, aspergillus nidulans, aspergillus calidoustus), followed by other ascomycetes of genus fusarium, basidiomycete genera malassezia, cryptococcus and trichosporon, and zygomycete genera rhizopus and mucor (boekhout et al. ). the importance of proteases in the pathogenicity of these opportunists is controversial; however aspartic, serine and metalloendopeptidases, as well as aminopeptidases, carboxypeptidases and dipeptidylpeptidases that are secreted by these species, have been proposed as the virulence factors that facilitate colonization and invasion by hydrolysis of host proteins or damage cells and molecules of the host defence system (segal ; yike ). the most studied are the secreted aspartic proteinases of candida albicans (saps) (naglik et al. ) . interestingly, the protease inhibitors targeted against the viral aspartic protease used in treating hiv infection also inhibited candida saps and reduced occurrence of candidiasis in these patients. secreted aspartic proteases are thus an important target for the development of new protease inhibitor based compounds for treating candidiasis (braga-silva and santos ; naglik et al. ; dash et al. ) . fungal proteases are also important fungal allergens, and most belong to the serine catalytic type. addition of protease inhibitor during aspergillus fumigatus and aspergillus niger protease and antigen sensitization attenuated allergic inflammation and hyper-responsiveness in an animal model (yike ) . secreted alkaline serine protease of aspergillus fumigatus was shown to help evade the host immune response by degrading human complement proteins, and it is therefore a good target for drug development (behnsen et al. ) . a network of proteolytic enzymes is very important for the survival of dermatophytes such as microsporum canis and trichophyton rubrum, the specialized pathogenic fungi that infect stratum corneum, nails or hair of healthy individuals. it includes the metalloendopeptidases, fungalysins (family m ), serine endopeptidases, subtilisins (family s a) and several exopeptidases (dipeptidyl peptidases (family s ), aminopeptidases (family m ) and carboxypeptidases (family s )) (monod ) . although proteases are only one of the several groups of virulence factors of pathogenic fungi, they aid in the invasion of tissues and evasion of immune responses. therefore, specific protease inhibitors aimed at them would constitute a valuable addition to the presently used antifungal drugs that target fungal cell wall and cell membrane integrity or dna replication-and to which many pathogenic strains have acquired resistance (marie and white ) . the classspecific (broad-spectrum) aspartic protease inhibitor pepstatin has been shown to inhibit adhesion of candida albicans and prevent invasion or mucosal tissue damage by inhibiting the saps (naglik et al. ) . recently, it has been shown that ergosterol production transcriptional regulator (sre ), which is activated by a metalloprotease stp , is essential for the survival of cryptococcus neoformans in the presence of antifungal drugs that inhibit sterol biosynthesis. therefore, regulators of the ergosterol pathway, including the metalloprotease stp , constitute promising targets for novel antifungal therapeutics to be used in combination with a sterol synthesis inhibitor for treating cryptococcosis in immunocompromised individuals (bien et al. ). protozoan parasitic diseases, including malaria, leishmaniasis and african and american trypanosomiasis, are some of the most important infectious diseases in the world, with high mortality and morbidity rates in developing countries. reasons for their persistence, despite prolonged use of antiparasitic drugs, include their toxic side effects and the increasing emergence of drug resistance. therefore, research over the past years has been focused on identifying new targets for antiparasite treatment and on developing substances suitable for human therapy. parasitic proteases constitute one of the very important druggable targets since they are key virulence factors due to their essential roles in cell metabolism and interaction with the host (zucca and savoia ; renslo and mckerrow ; mckerrow et al. ) . aspartic proteases plasmepsins (family a ), which are important for haemoglobin catabolism in parasites causing malaria (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae), have been targeted for the design of novel antimalarial drugs, i.e. selective protease inhibitors. although plasmepsin inhibitors (including the broad-spectrum pepstatin) have been shown to have antimalarial effects, the main challenge still remains to design an inhibitor that would be active against different plasmepsins i, ii and iv, and the histoaspartic protease hap involved in haemoglobin degradation, but also inactive against the homologous human aspartic proteases (cathepsins d and e) (zucca and savoia ; rosenthal ; ersmark et al. ; gil et al. ). in addition to plasmepsins, the cysteine proteases falcipains (family c ), metalloprotease falcilysin (family m ) and aminopeptidases have been targeted for development of new antimalarial protease inhibitors. a synergistic effect has been observed for their combined use (e.g. a cocktail of aspartic and cysteine protease inhibitor) (mckerrow et al. ; rosenthal ; zucca and savoia ; trenholme et al. ) . cysteine proteases similar to falcipain (family c ) have been associated with the pathogenesis of trypanosomiasescruzipain of trypanosoma cruzi (chagas disease) and brucipain or rhodesain of trypanosoma brucei (sleeping sickness). cruzipain (or cruzain) has been extensively studied and is considered to be a promising target for chemotherapy since it is critical for parasite viability in all stages of infection, especially for nutrition acquisition, tissue invasion and host immune response evasion (duschak and couto ). an irreversible inhibitor, k , was effective in pre-clinical models of chagas disease; however, a safer treatment would be achieved by utilizing a reversible and highly specific cruzipain inhibitor (beaulieu et al. ; mckerrow et al. ; brak et al. ). cysteine proteases have been implicated in the pathogenesis of leishmania species and are major virulence factors as they substantially modify the immune response. a surface metalloprotease gp or leishmanolysin (family m ) has been shown to be essential for establishing and maintaining the infection and would make a promising target for development of a selective protease inhibitor for antiparasitic chemotherapy (olivier and hassani ; yao ) . in addition to the computational design, development and optimization of a suitable protease inhibitor, based on the d structure of the target protease, the search for novel types of inhibitors from natural sources (pereira et al. ) , such as fungi and microbes, is important for identifying new lead compounds. serine proteases are also a neglected group of potentially targetable protozoan proteases involved in their pathogenicity, although several subtilisins (family s ) have been described from plasmodium falciparum and toxoplasma gondii, and two oligopeptidases (family s ) from trypanosoma cruzi (mckerrow et al. ) . in addition to protozoan parasites, helminths, which include parasitic roundworms (nematodes) and flatworms (trematodes and cestodes), also cause important parasitic infections. cysteine cathepsins (family c ), which are important for many aspects of the helminth-host relationship, are a potential target for developing antihelminthic drugs. cysteine protease inhibitors have been shown to impair fecundity of the liver fluke (fasciola hepatica) and blood fluke (schistosoma mansoni) in animal models. however, the similarity of host cathepsins, together with their importance, calls for the design of a very specific and selective protease inhibitor. alternatively, drug design could exploit bioavailability and pharmaco-kinetic and -dynamic properties to target parasitic proteases preferentially (robinson et al. ). in addition to proteases, parasite-derived protease inhibitors play important roles in manipulating the host immune system and establishing a niche for the successful feeding and reproduction of helminth parasites. therefore, protease inhibitors are also important targets for immunological control of helminth parasitic infections and make proteases that are insensitive to parasite-derived protease inhibitors valuable candidates for new types of antihelminthic therapy (knox ; stepek et al. ) . the ability of tumour cells to invade extracellular barriers and to metastasize to distant sites is associated with the activity of proteases (kos and lah ) . the major molecular mechanism, which involves the active role of various intra-and extracellular proteases, is the dissolution and remodelling of connective tissue and the basement membrane. it includes matrix metalloproteases (mmp), serine proteinases such as urokinase, tissue types of plasminogen activator (upa, tpa) and plasmin, aspartic proteinase cathepsin d and cysteine proteinases cathepsins b, h, s and l (schmitt et al. ). in addition to extracellular matrix remodelling, proteases regulate several other processes, leading to the progression of malignant disease, such as cell adhesion, migration, differentiation, proliferation and signalling of tumour cells. it is well accepted that tumourassociated proteolytic activity escapes the control of endogenous protease inhibitors and that treatment of patients with exogenous protease inhibitors may suppress the progression of the disease and improve the outcome of cancer patients. however, the treatment should specifically target the tumour-associated proteases that cause harmful actions and not those involved in the numerous physiological processes in cells and tissues. several protease inhibitors failed in clinical trials due to lack of specificity, resulting in severe side effects and/or lack of clinical benefit in treated patients (turk ; coussens et al. ) . new approaches to developing protease inhibitors applicable in therapeutic interventions include structure-based medicinal chemistry and development of molecular systems to deliver inhibitors to the site of action. among the small-molecule inhibitors of bacterial and fungal origin, peptidyl aldehydes such as leupeptin and antipain, hexapeptide pepstatin and epoxysuccinyl peptide e- and their analogues have been studied as anticancer agents. the thiol-protease specific inhibitor, e- , originally isolated from aspergillus japonicus (hanada et al. ) , has been studied extensively as a potential antitumour agent in cell culture and animal models. derivatives of e- , displaying selectivity between different cysteine proteases (frlan and gobec ) , represent the next step towards their application in treating cancer and other diseases. they were designed on the basis of the x-ray crystal structures of individual cathepsins, and the most studied were cathepsin b specific inhibitors ca- and ca- , cathepsin l specific inhibitors clik- and clik- , and cathepsin x specific inhibitor ams- . cathepsin s specific inhibitor clik- was designed on the basis of the structure of leupeptin and antipain (katunuma ) . antitumour activity was exhibited particularly by ca- , a specific inhibitor of the cysteine protease cathepsin b (johansson et al. ) , which appears to be crucial for tumour cell invasion (lah et al. ) . in animal models, it was shown that ca- reduces tumour growth, invasion and angiogenesis of many cancer types, including pancreatic cancer, melanoma and breast tumours, all tested on animal models. the cell permeability of epoxysuccinyl inhibitors was improved by esterification. the esters are less active than free acids; however, in cells, they are rapidly hydrolysed to their active form. better cell permeability was demonstrated for ethyl ester e- d and the methyl ester of ca- , which are also highly soluble and effective for prolonged periods (frlan and gobec ) . metalloproteases are an important group of proteases that have been considered extensively as targets for cancer therapy due to the many roles they play in carcinogenesis and tumour invasion, growth and dispersion. however, the diversity of endogenous metalloproteases and their numerous and versatile physiological roles have prevented the use of broad-spectrum metalloprotease inhibitors. much effort is invested in determining which metalloproteases to target and in designing highly selective and potent protease inhibitors based on the structural characteristics of individual target metalloproteases (coussens et al. ; bialas and kafarski ; dorman et al. ; overall and kleifeld ) . several proteases of the serine catalytic type have also been targeted for the design of specific protease inhibitors for use in cancer treatment, including the urokinase plasminogen activator and matriptase (abbenante and fairlie ; bialas and kafarski ; ulisse et al. ). the broad-range microbial inhibitors of serine, cysteine and threonine proteases, leupeptin and antipain, were also shown to inhibit malignant transformation (vaccari et al. ) or tumourigenesis (hozumi et al. ) . threonine catalytic type proteolysis is present in proteasomes, and several chemical classes of natural and synthetic proteasome inhibitors have been considered as anticancer agents because of their preferential antiproliferative and proapoptotic activity on cancer cells (kisselev and goldberg ; abbenante and fairlie ; bialas and kafarski ; cecarini et al. ; chen et al. ) . the aspartic protease inhibitor pepstatin has also been frequently tested as an antitumour agent since cathepsin d was identified as an important tumour promoting factor (greenbaum and sutherland ; benes et al. ). irregular function of proteases is associated with a variety of other diseases, representing targets for therapeutic application of all catalytic types of protease inhibitors. smallmolecule inhibitors of bacterial and fungal origin, described already in the previous section, particularly epoxysuccinyl inhibitors, have been reported as potential protective agents in autoimmune, neurodegenerative, antiinflammatory and cardiovascular diseases; osteoporosis; muscular dystrophy; diabetes and others. ca- and clik- were demonstrated to cause a switch between th and th t cell response due to the different roles of cysteine cathepsins b and l in antigen processing and presentation (katunuma ) . cathepsin s inhibitor clik- has been shown to suppress sj gren syndrome, an autoimmune disease associated with the processing of α-foldin by cathepsin s (saegusa et al. ) . the cathepsin x inhibitor ams- reduces the activation of integrin receptor lfa- (lymphocyte functionassociated antigen ), a molecule involved in t cell adhesion, proliferation and migration (jevnikar et al. ) . lfa- overexpression and activation by cathepsin x is typical of autoimmune diseases, particularly psoriasis. the same inhibitor significantly enhanced the proliferation of neuronal cells and neuritogenesis, preventing the processing of neurotrophic factor γ-enolase by cathepsin x (obermajer et al. ). epoxysuccinyl inhibitors have been tested in several animal models of neurodegenerative diseases. e- was shown to restore normal synaptic function in the app/ps mouse model of alzheimer's disease with overexpressed amyloid precursor protein (app) and presenilin (ps ) (trinchese et al. ). e- d and ca- me also reduced the accumulation of neurotoxic beta-amyloid peptides in brains, presumably inhibiting cathepsin b involved in processing the amyloid precursor protein (hook et al. ). aspartic proteases β-secretase (also known as memapsin- or bace and belonging to merops family a ) and γ-secretase (composed of two presenilins belonging to merops family a ) are important therapeutic targets for treating alzheimer's disease, and several compounds designed to reduce their activity are in clinical trials (de strooper et al. ) . another important target of aspartic proteases is renin (family a ), which is part of the complex renin-angiotensin-aldosterone system that regulates blood pressure and electrolyte balance. several inhibitors have been designed based on the renin structure and activity, and one of them, aliskiren, is the first non-peptide, orally administered, direct renin inhibitor available on the market for management of hypertension (nguyen et al. ; barrios and escobar ) . the very first protease inhibitor used in humans as a therapeutic agent, namely, an inhibitor of metalloprotease angiotensin-converting enzyme (ace), was also hypertension related. it is an important regulator of the reninangiotensin system, and ace inhibitors are used for treating hypertension, but are also implicated in other cardiovascular and renal diseases (abbenante and fairlie ; ondetti et al. ) . the inhibitors of collagenolytic enzymes, such as cathepsins l and k, prevent bone remodelling and can be useful in treatment of osteoporosis. several synthetic cathepsin k inhibitors are in different stages of clinical trials. in addition to osteoporosis, they are also considered for application in arthritis, atherosclerosis, obesity and cancer (bromme and lecaille ) . administration of cathepsin l inhibitor clik- significantly reduced invasion and metastasis formation in bones (katunuma ) . e d, a methyl ester of e , was tested for treating muscular dystrophy; however, its further development was stopped in phase iii due to insufficient effectiveness and hepatic injury in rats (fukushima et al. ). antipain, leupeptin and pepstatin have also been tested for treatment of muscular dystrophy; however, persuasive benefit was not demonstrated in animal models. specific inhibitors designed to target the serine aminopeptidase dipeptidyl peptidase iv are in clinical trials for management of type diabetes (abbenante and fairlie ; tahrani et al. ) . endogenous plant protease inhibitors constitute one of the plant defence strategies against pathogenic, parasitic and herbivorous organisms. they target the important proteolytic virulence factors of phytopathogenic bacteria, fungi, parasites and viruses, preventing their roles in nutrient acquisition and evasion of host defence. furthermore, they target digestive proteases of herbivorous pests (e.g. insects, mites, slugs), preventing the utilization of food-derived organic nitrogen for their growth and development (ryan ; haq et al. ) . since pests and pathogens depend on utilization of these proteases, there is a strong selection pressure operating to develop resistance to plant endogenous defensive protease inhibitors (haq et al. ; jongsma and beekwilder ) . therefore, the search for novel protease inhibitors with potential protective function is very important for the development of environmentally friendly pest and pathogen management strategies. in addition to investigations aimed at augmenting crop endogenous resistance by conventional breeding, there are several protease inhibitors of plant origin that have been used in the preparation of genetically modified crop plants with superior ability for biotic stress resistance (ferry and gatehouse ) . the use of protease inhibitors for insect pest management has gained most attention. insect pests cause major economic losses annually, of which lepidopteran (butterflies' and moths') larvae are considered the most destructive. agricultural pests causing significant economic impact also belong to orders coleoptera (beetles), diptera (true flies), hemiptera (e.g. aphids), orthoptera (e.g. locust) and thysanoptera (thrips). they cause either direct damage to crops by feeding or indirect damage by transmitting viral diseases or secondary microbial infections. the most notable for their destructive capacity are the migratory locust (locusta migratoria), several beetles, including colorado potato beetle (leptinotarsa decemlineata), boll weevil (anthonomus grandis), japanese beetle (popillia japonica), the western corn rootworm (diabrotica virgifera virgifera) and many species of aphids belonging to all families of the superfamily aphidoidea. different catalytic types of proteases provide the predominant proteolytic activity in different groups of insect pests. while serine proteases are predominant in digestive proteolysis in most insect species (e.g. lepidoptera, diptera), cysteine proteases predominate in hemiptera, coleoptera and thysanoptera. in addition, aspartic and metalloproteases complement protein digestion to different degrees in most insect orders (terra and ferreira ) . therefore, protease inhibitors targeting different groups of proteases have shown variable antinutritional effects when fed to different insect pests. the catalytic, classspecific, small-molecule protease inhibitors of microbial origin have often been used for proof-of-principle feeding experiments, but protein protease inhibitors were then employed to generate insect-resistant transgenic plants. these were predominantly of plant origin, with a few exceptions of animal-derived protease inhibitor genes (haq et al. ; dunaevsky et al. ; malone et al. ) . other important proteins that have been used for constructing pest-resistant transgenic crops are insecticidal proteins derived from bacillus thuringiensis (bt), and genetically modified maize and cotton varieties that express bt toxins have become an important component in agriculture worldwide and have reduced the use of pesticides and lowered production costs (ferry and gatehouse ; kumar et al. ) . the involvement of endogenous protease inhibitors in natural plant resistance against herbivores is probably the basis of the adaptation of lepidopteran and coleopteran species to ingestion of protease inhibitors that has been observed for several species (jongsma and beekwilder ; wu et al. ; bonade-bottino et al. ; lara et al. ) . they circumvent the antinutritional effect of the ingested protease inhibitors either by overexpression of native gut proteases or by production of insensitive proteases; some of which can degrade the ingested protease inhibitors (ferry and gatehouse ; jongsma and beekwilder ) . therefore, the pyramiding or stacking of different families of inhibitors to increase the spectrum of inhibitory activity has been shown to have synergistic effects, as well as combining protease inhibitor genes with genes of other insecticidal proteins, namely, lectins, bt or other bacterial toxins and α-amylase inhibitors (ferry and gatehouse ; schluter et al. ; christou et al. ; malone et al. ) . furthermore, the use of protease inhibitors of microbial and fungal origin could offer superior characteristics, such as stability, resistance to proteolytic degradation and diverse inhibitory patterns, for a more potent antinutritional or insecticidal effect. only a few examples of utilization of microbial small-molecule inhibitors as antinutritional agents are available, e.g. aminopeptidase inhibitors of actinomycetes amastatin and bestatin against the red flour beetle (tribolium castaneum) (oppert et al. ) , aspartic protease inhibitor pepstatin a from actinomycetes against the cowpea bruchid (callosobruchus maculatus) (amirhusin et al. ) , the serine and cysteine protease inhibitor leupeptin from actinomycetes against western corn rootworm (diabrotica virgifera) (kim and mullin ) and cysteine protease inhibitor e- from aspergillus japonicus against colorado potato beetle (leptinotarsa decemlineata) (bolter and latoszekgreen ) . the use of protein protease inhibitors is advantageous since transgenic plants expressing a pest resistance gene in a controlled manner represent a stable and cheap propagation source that would lower the amount of pesticides needed, making plant protection environment friendly. to our knowledge, the only examples of a protein protease inhibitor of microbial or fungal origin as an effective antinutritional agent are the cysteine protease inhibitors macrocypins (family i ) from the edible parasol mushroom (macrolepiota procera), which have been shown to be detrimental to the growth and development of colorado potato beetle larvae (istinič et al. ) . the high capacity for development of resistance to insecticidal proteins in major insect pests drives the search for effective protease inhibitors. novel protein protease inhibitors aimed at serine proteases would be useful for management of major agricultural pests such as beet armyworm (spodoptera exigua), cotton bollworm (helicoverpa armigera and helicoverpa zea) and tobacco hornworm (manduca sexta). a combination of serine and cysteine protease inhibitors would be useful against, e.g. boll weevil (anthonomus grandis), cowpea weevil (callosobruchus maculatus) and red flour beetle (tribolium castaneum), and cysteine protease inhibitors would, for example, be useful against the colorado potato beetle (leptinotarsa decemlineata), western corn rootworm (diabrotica virgifera), banana weevil (cosmopolites sordidus) and pea aphid (acyrtosiphon pisum). microorganisms that are generally recognized as safe and edible mushrooms offer a valuable source of such novel protease inhibitors that would also be acceptable for use in crops for human consumption. in addition to insect pests, other herbivores causing significant crop losses worldwide include mites and slugs. in both cases, cysteine proteases constitute the predominant digestive proteolytic activity. plant cystatins have been shown to be detrimental to mite development and reproductive performance in feeding trials on one of the major mite pests on agricultural crops, the two-spotted spider mite tetranychus urticae (acari: tetranychidae) (carrillo et al. ) . similarly, the growth of juvenile slugs deroceras reticulatum, the important agricultural and horticultural pest, was significantly reduced when fed with leaf tissue overexpressing a plant cysteine protease inhibitor (walker et al. ) . therefore, protease inhibitors of microbial and fungal origin have great potential for protecting plants from important mite pests and suppressing the growth rates of slug populations, in addition to their antinutritional characteristics for different insect pests. another advantage of the application of protease inhibitors in crop protection is that in addition to protection against herbivorous pests, they offer cumulative protection against nematodes and bacterial, fungal and viral pathogens (haq et al. ). the use of protease inhibitors is one of many strategies to protect plants from parasitic nematodes. their targets are intestinal proteases, mostly of the cysteine catalytic class. therefore, heterologously expressed plant cysteine protease inhibitorsphytocystatins (family i )-have been effective to different degrees against beet-cyst nematode (heterodera schachtii), root-knot nematode (meloidogyne incognita), potato cyst nematode (globodera pallida) and burrowing nematode (radopholus similis) (mccarter ; haq et al. ) . a plant-derived serine protease inhibitor offered satisfactory nematode resistance in transgenic wheat against the cereal cyst nematode (heterodera avenae) (vishnudasan et al. ; mccarter ). the antifungal effect of serine protease inhibitors has been determined with plant-derived protease inhibitors that target secreted proteases (mainly families s and s ) of phytopathogenic fungi involved in plant cell wall penetration by hyphae (wong et al. ). in addition, aspartic and cysteine proteases that are also fairly widespread (valueva and mosolov ) represent potential targets for protective protease inhibitors. similarly, phytopathogenic bacteria secrete proteases involved in pathogenesis, mainly aiding plant cell wall degradation. together with pectinases, cellulases and hemicellulases, they contribute to massive degeneration of plant tissue, for example, in wilt (e.g. ralstonia solanacearum) and soft-rot diseases (e.g. erwinia chrysanthemi, erwinia carotovora) (kunkel and chen ) . cysteine proteases belonging to families c , c , c , c and c , which have been implicated in the pathogenicity of many phytopathogens belonging to genera pseudomonas, xantomonas, ralstonia, erwinia and pantoea as effectors of the type iii secretion system, constitute potential targets for protective protease inhibitors (kunkel and chen ; mosolov and valueva ) . the importance of polyprotein processing in replication of viruses, especially those of families potyviridae and comoviridae, indicates that protease inhibitors could mediate resistance to plant viruses by inhibiting target viral proteases. indeed, a rice cystatin (oryzacystatin) expressed in tobacco plants conferred resistance to potato virus y (pvy) and to tobacco etch virus (tev) which correlated to increased inhibition of the target papain-like protease sudarshana et al. ). in addition to their protective role against biotic stress, endogenous plant protease inhibitors have been implicated in the control of proteolytic systems in plants under abiotic stresses with a dehydration component such as drought, increased salt concentration and freezing (vaseva et al. ) . it has been shown that the changes in metabolism triggered by water deficit involve active involvement of regulated proteolysis that assists the protective proteins (dehydrins and chaperones) in the cellular response to the increased levels of denatured, aggregated or oxidatively damaged proteins that accumulate during dehydration stress (brzin and kidrič ; benchabane et al. ; bray ; hoekstra et al. ; feller ) . overexpression of endogenous protease inhibitors increased resistance to drought stress, as shown for overexpression of the cysteine protease inhibitors cystatins atcys and atcys in the model plant arabidopsis thaliana ) and for overexpression of a serine protease inhibitor ocpi (oryza sativa chymotrypsin inhibitor ) in rice, with the latter showing improved drought resistance in terms of yield loss in the field (huang et al. ) . although protease inhibitors have great potential in protecting crops under abiotic stress conditions, detailed knowledge of the specific roles of different proteases in response to abiotic stress is needed before exogenous protease inhibitors can be used to manipulate and improve drought resistance in plants (vaseva et al. ) . small-molecule protease inhibitors are routinely used as buffer additives when preparing protein extracts, in order to prevent proteolytic degradation during protein purification procedures. broad-spectrum inhibitors that cover all the different catalytic classes are generally used, including pepstatin a for aspartic proteases, e- for cysteine proteases, chymostatin for serine proteases, antipain for cysteine and serine proteases, and leupeptin for cysteine, serine and threonine proteases; all of which were originally isolated from actinomycetes. for inhibition of serine proteases, synthetic protease inhibitors such as aebsf (pefabloc; -( -aminoethyl)benzenesulfonyl fluoride hydrochloride), the physiologically more stable derivative of pmsf (phenylmethanesulfonyl fluoride), are used. chelating agents such as edta and , -phenanthroline are generally used to inhibit metalloproteases and, for certain applications, inhibitors of actinomycete origin-phosphoramidon (m and m metallopeptidases inhibited) or bestatin (aminopeptidases inhibited). unwanted proteolytic degradation can cause severe reduction in yield of heterologously expressed proteins in various expression systems. therefore, natural or engineered protease-deficient strains are often used as hosts for bacterial (e.g. escherichia coli bl ), yeast (e.g. pichia pastoris smd , or ) and filamentous fungal (e.g. aspergillus niger prt pep) expression systems. however, these strains may not exhibit optimal bioprocessing characteristics due to lower fitness traits. alternative strategies are therefore used for preventing proteolytic degradation of recombinant proteins, including modification of the recombinant protein sequence to remove protease cleavage sites without affecting protein function, expression with a stabilizing fusion partner, optimization of cultivation conditions (ph, temperature, medium composition, bioprocess strategy) and use of protease inhibitors. for secreted recombinant proteins, small-molecule inhibitors can be added to the culture medium to inhibit the predominant secreted proteolytic activity of the host organism that is often of the serine and aspartic catalytic type. another strategy is coexpression of an appropriate protein protease inhibitor with the recombinant protein, which may, however, influence the yield or complicate the downstream purification procedures (potvin et al. ; sharma et al. ; rozkov and enfors ) . in addition to protection against proteolytic degradation of recombinant protein during the expression process, protease inhibitors as fusion partners have other advantages. one example is the use of the bacterial periplasmic serine protease inhibitor ecotin (family i ) as a vehicle for secretion to the periplasmic space of escherichia coli (paal et al. ). another example is the serine protease inhibitor from oyster mushroom, the pleurotus ostreatus proteinase a inhibitor (poia ) (family i ) that serves as an intramolecular chaperone enabling proper refolding of the fused subtilisin protease from inclusion bodies expressed in a heterologous bacterial expression system (kojima et al. ) . the strategy of using co-expression of protein protease inhibitors for reducing proteolytic degradation of heterologously expressed proteins has been successfully implemented in plant expression systems. preparation of a protease-deficient host is, in this case, not applicable because of the essential roles of proteases in growth and development. plant expression systems offer many advantages over bacterial and yeast expression systems (e.g. posttranslational modification capability) and over animal cell lines (e.g. lower cost and contamination risks) for largescale recombinant protein production, but have not yet been commercialized due to low levels of protein expression and of heterologous protein accumulation. the latter can be influenced by targeting the expression to specific organelles (e.g. endoplasmic reticulum) or tissues (e.g. seeds and tubers) or by co-expression with stabilizing fusion partners or protease inhibitors. co-expression of protective protease inhibitors does not have any adverse effects on plant growth and development, as described previously for examples of protease inhibitor expressing, insect-resistant transgenic plants. furthermore, the heterologously expressed protease inhibitors offer the added advantage of protection against proteolysis also ex planta, especially in the early steps of purification of crude protein extracts, thus minimizing the need for addition of protease inhibitors to the extraction buffers (rivard et al. ; desai et al. ; doran ; benchabane et al. ) . so far, only protease inhibitors of plant and animal origin have been used as co-expression partners; use of microbial or fungal protease inhibitors could offer superior protective characteristics. however, the properties of the protein of interest and of the selected host plant species influence the selection of an appropriate protective protease inhibitor, and the choice still has to be made on a case-by-case basis. another important biotechnological application of protease inhibitors is in protein purification, where they can be used as ligands in affinity chromatography. affinity chromatography is a simple one-step purification method of a molecule from a complex mixture based on specific and high affinity binding to a ligand immobilized on a solid support. reversible protease inhibitors of microbial origin are excellent ligands for purification of their cognate proteases by affinity chromatography. depending on the target protease, inhibitors with broad range or very specific inhibitory spectrum can be selected for a ligand. attention must be paid to the strength of the inhibitor as purification is not possible when the binding is too weak (no binding) or too strong (ineffective elution) (ge ) . the advantage of using microbial and fungal protease inhibitors is that many of them display unique inhibitory profiles and resistance to proteolytic cleavage, as well as high thermal and broad ph range stability, with the latter being very convenient since harsh conditions may be used for immobilization to the matrix as well as for the several cycles of elution steps, usually involving extreme change in ph and/or ionic strength. the broad-range inhibitor of pepsin-like aspartic proteases, pepstatin a, has been used for purification of aspartic proteases from several different sources, including higher fungi (sabotič et al. a) , plants (payie et al. ) and insect 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fulvum virulence protein avr inhibits host proteases required for basal defense the response of plants to drought stress-the role of dehydrins, chaperones, proteases and protease inhibitors in maintaining cellular protein function molecular cloning, over expression, and activity studies of a peptidic hiv- protease inhibitor: designed synthetic gene to functional recombinant peptide enzymatic characterization of the streptococcal endopeptidase, ides, reveals that it is a cysteine protease with strict specificity for igg cleavage due to exosite binding assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (pin ) gene new aspartic proteinase of ulysses retrotransposon from drosophila virilis transgenic arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug deroceras reticulatum (muller) picornaviral c protease inhibitors and the dual c protease/coronaviral c-like protease inhibitors regulation of bacterial protease activity proteins with antifungal properties and other medicinal applications from plants and mushrooms adaptation of helicoverpa armigera (lepidoptera: noctuidae) to a proteinase inhibitor expressed in transgenic tobacco novel inhibitor for prolyl tripeptidyl aminopeptidase from porphyromonas gingivalis and details of substrate-recognition mechanism major surface protease of trypanosomatids: one size fits all? fungal proteases and their pathophysiological effects proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins two cysteine proteinase inhibitors from arabidopsis thaliana, atcysa and atcysb, increasing the salt, drought, oxidation and cold tolerance current developments in the therapy of protozoan infections acknowledgements we are grateful to dr. jože brzin for numerous discussions and suggestions for the improvement of the review and to professor roger h. pain for critical reading of the manuscript and language editing. we would like to thank dr. miha renko for the assistance with fig. . key: cord- -ptrehipn authors: erdei, carmina; liu, cindy h. title: the downstream effects of covid- : a call for supporting family wellbeing in the nicu date: - - journal: j perinatol doi: . /s - - - sha: doc_id: cord_uid: ptrehipn parents of nicu infants are a vulnerable population from a psychological perspective, and often experience high levels of acute stress, depression, anxiety, and post-traumatic stress. the added burden of the current sars cov- disease (covid- ) pandemic is likely to exacerbate these issues, with potential implications for the wellbeing of infants and families in the short- and long-term. in this paper, we propose utilizing the stress contagion framework and consider how psychosocial stress can “spill over” into the parent-infant relationship domain, which can impact child development and family wellbeing longer term. as the effects of the pandemic will likely persist well beyond the acute stage, we offer advocacy points and general guidelines for healthcare professionals to consider in their quest to mitigate stress and build resilience in nicu families. the novel coronavirus sars-cov- disease (covid- ) pandemic has had a global impact resulting in stress on health systems worldwide. recent data from china, the first country to experience a large-scale outbreak, suggested that newborns remain susceptible to sars-cov- , although symptoms are milder and outcomes less severe compared to adults [ ] . transmission is thought to occur primarily horizontally by caregiver-to-infant postnatal transmission via respiratory droplets [ , ] . although newborns may be less impacted by covid- directly, there is concern that the pandemic and the necessary public health measures employed in response may negatively impact the psychosocial health of high-risk infants and their families in the neonatal intensive care unit (nicu). the impact of early adversity on infant brain development is well recognized [ ] , and recommendations are in place to mitigate these effects through nicu familycentered developmental care models [ , , ] . parents are critical to the health and wellbeing of their infants. from a psychological perspective, families of nicu infants are a vulnerable population, as they experience trauma due to separation from their infants alongside the stress regarding their medical conditions and related interventions [ ] . many nicu families experience altered perinatal parent mental health in the form of acute stress, anxiety, depression, and/or post-traumatic stress [ , ] . this is associated with an elevated risk for subsequent impaired child development and poorer family functioning [ , ] , which led to a recent call for enhanced mental health support for nicu families [ , ] . in this context, the current covid- pandemic acts as a sudden, new-onset major environmental stressor superimposed on preexisting high levels of nicu family psychological distress. a crucial public health measure in the fight against covid- is the social distancing imperative. this has resulted in difficulties with transportation and access to hospitals in some communities. within some units, it has also led to more limited family access to the nicu, challenging family-centered developmental care practices. this, in turn, may affect the ability for families to cope with pandemic-related stress, thus compounding the stress of having an infant in the nicu, and further impeding their confidence as primary caregivers. the parental experience of navigating the changes in the healthcare system, combined with potential new financial and other constraints during the covid- pandemic can spill over to how they engage and connect with their infant, which is referred to as stress contagion [ , ] . against this background, several issues can impact parental wellbeing in the nicu. we highlight two major sources of stress: when the interplay between these factors exceeds a family's capacity to manage stress, this may spill over to affect the complex relationship-building process that occurs after every birth, and is especially vulnerable for disruption in the nicu. nurturing experiences including breastfeeding, kangaroo mother's care, or parents reading/ talking with their infant which are routinely employed to mitigate stress may occur less frequently. parents might experience heightened anxiety and extreme worry about their infant's health in the pandemic era that is even higher than that typically experienced by parents in the nicu, an aspect that deserves further investigation. given the negative downstream effects that nicu stress contagion may have on child development and family functioning, professionals should consider advocacy and further action in their quest to foster family resilience in the covid- era. ( ) practice shared decision-making for every aspect of care. of particular note, this should include the psychosocial domain. healthcare providers should assess sources of stress and perceived risks from nicu parents, and partner with families to devise individualized care plans through shared decision-making. systematic assessment of nicu families psychosocial stress, and enhanced collaboration with mental health specialists will aid this goal. we note that disadvantaged families may disproportionately experience stress contagion, and additional supportive mechanisms may be necessary to meet each family's needs. issues unique to or exacerbated by the covid- pandemic include: • increased transmission of sars-cov- in family's community. • limited access to technology for virtual communication and/or care. • limited access to support systems in family's community. • exacerbation of preexisting mental health issues. • communication challenges for non-english speaking families. • poverty, housing, or food insecurity. • more limited ability to be present in the nicu if dependent on public transportation due to loss of income and/or travel restrictions. ( ) increase support to mitigate stress contagion. if infrastructure and model of care permits, nicus can temporarily consider increasing mental health support in the nicu during this stressful period, to facilitate development of family coping strategies and resilience. • emphasize basic wellness strategies: encourage parent wellbeing, healthy diet, sleep, exercise, virtual social connectedness, mindfulness, and self-compassion. • increase virtual and/or on-site social work and other mental health and psychosocial support, including behavioral health and virtual peer-topeer connection. • capitalize on the critical role that fathers/partners play both in the infants' care and support of mothers. • consider assessment of baseline nicu families mental health, as well as identification of pandemicrelated superimposed stressors unique to each family; evaluate which superimposed stressors offer opportunities for intervention. • optimize family-centered developmental care opportunities, support relationship-building within the family as much as safely possible, and prioritize interventions that meet the needs of the entire family unit. consider positive multisensory interventions facilitated by staff and technology where appropriate, when family presence is limited. ( ) prioritize stress mitigation for healthcare professionals, so they can themselves remain resilient and provide emotional support for nicu families [ ] . as the neonatal community has expeditiously employed novel strategies to meet the medical needs of infants affected by covid- , it is important to also consider the psychological short-and long-term effects of the pandemic on nicu families. the mental health aftermath of the pandemic will likely persist long after the acute phase, and it may disproportionately affect the vulnerable nicu population. as stress contagion in the nicu can negatively impact parent-infant relationships, strategies are needed to mitigate it in order to optimize child development and family resilience during this unparalleled period. lastly, innovation and discovery can often arise during crises. as the acute phase of the pandemic concludes, professionals might consider which covid- era interventions may prove beneficial in enhancing current family-centered developmental care practices in the nicu and are worth developing further. novel coronavirus infection in newborn babies under days in china perinatal aspects on the covid- pandemic: a practical resource for perinatal-neonatal specialists brain development and the role of experience in the early years the neonatal intensive parenting unit: an introduction guidelines for family-centered care in the neonatal, pediatric, and adult icu the newborn individualized developmental care and assessment program (nidcap) with kangaroo mother care (kmc): comprehensive care for preterm infants identifying mothers of very preterm infants at-risk for postpartum depression and anxiety before discharge very preterm birth influences parental mental health and family outcomes seven years after birth parent-child interaction, maternal depressive symptoms and preterm infant cognitive function a multilayered approach is needed in the nicu to support parents after the preterm birth of their infant the contagion of stress across multiple roles psychosocial stress contagion in children and families during the covid- pandemic amid covid- , self-care as important for providers as it is for patients acknowledgements we would like to thank dr. terrie inder, mbchb, md, chair of department of pediatric newborn medicine at brigham and women's hospital and mary ellen avery professor of pediatrics at harvard medical school, for her valuable consultation and feedback in the preparation of this paper. we are also deeply appreciative to the nunnally family and weinberg family for their generous support of the brigham and women's hospital nicu programs. conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - k v authors: happ, mary beth title: family caregiving for acute-critically ill older adults in the time of covid- date: - - journal: geriatr nurs doi: . /j.gerinurse. . . sha: doc_id: cord_uid: k v nan the idea for the column was spawned, as good ideas often are, over dinner and wine at a gerontological society of america annual meeting. as i recall, we lamented the relative dearth of interest in the science of geriatric acutecritical care at the conference and worried about the assumptions held by many students, funders, and colleagues that gerontological nursing focused exclusively on long-term care. and so, liz capezuti, sarah kagan, lorraine mion, and i joined together at the invitation (or was it challenge?) of editor-in-chief, barbara resnick, to address and amplify issues of significance in the care of acutecritically ill older adults. it is fitting that in this last year, we observed geriatric nursing's th anniversary with several columns that involved "taking stock" of acute-critical care for older people as well as review of progress on ageism, physical restraint use, and accommodations for patient communication impairment in acute-critical care. in the past years, scientific and practice initiatives addressed the hazards of hospitalization and immobility with strong evidence regarding the development and outcomes of iatrogenic "geriatric" syndromes, such as delirium, myopathy, falls, and cognitive/physical functional decline, during acute-critical illness. critical care rehabilitation is now a multidisciplinary specialty area promoting early intervention to prevent post-hospital functional decline. more adults are surviving critical illness [ ] and, thanks to curricular improvements and programs such as nurses improving care for healthsystem elders, [ ] more nurses have specialty knowledge about gerontological assessment and care principles. the anniversary year opened with the reminder that gerontological health care is no longer centered in the acute care hospital as it was years ago but has shifted to home and community settings. [ ] nine months later, we are in the midst of a global pandemic that disproportionately sickens and kills older adults while seriously straining the acute-critical care workforce and resources. vulnerable older patients enter acute-critical care settings without the supportive presence and advocacy of family caregivers. at the same time, the home setting has become a site for care across the continuum bolstered by telehealth for which family caregivers serve as technological support, interpreters, and implementers. gerontological nurses are uniquely qualified to support family caregivers as they interact with acute-critical care in remote and creative ways during the covid- pandemic. family caregiving for older adults admitted to acute -critical care covid-related family separation during acute-critical illness impacts older adult patients, family caregivers, and nurses on multiple levels and is likely to have long-term traumatic effects. [ ] restrictions on family visitation to prevent the spread of covid- infection vary depending on local infection rates, institutional policies, and patient condition (e.g., allowances for end-of-life visitation). information sharing, informed consent, treatment decision making, and end-of-life care may be impaired or inadequate in the absence of family caregivers. disability advocates criticize and challenge hospital visitation restrictions as discriminatory toward persons with disability and promote policies permitting the "entrance of a designated support person for a patient with a disability and permitting family members, serviceproviders or other individuals knowledgeable about the needs of the person with a disability to serve as a designated support person." [ ] please consider the following recommendations adapted from montauk and kuhl [ ] for how to support family caregivers during acute-critical illness: ( ) be present and authentic. montauk and kuhl [ ] advise that nurses should not be afraid to show their emotions in an empathic way (that not burdensome or oversharing). the goal here would be one of therapeutic use of self to provide human connection and caring that family caregivers may desperately need in a time of crisis, isolation, and, in some cases, grief. in which % of caregivers reported performing medical/nursing tasks. the study found that caregivers performing medical/nursing tasks had higher risk of emotional stress, physical strain, and high burden of care than those who without those responsibilities. in the midst of pandemic quarantine restrictions, family caregivers of home-dwelling older adults face limitations in assistance and respite as they take measures to protect older adults from infection while also managing or co-managing care for the older person's multiple chronic conditions and acute events. for example, a family who previously shared caregiving may now have limited caregiving to one individual due to daily exposures of members who work in essential services or travel restrictions. while more flexible work-from-home schedules are advantageous for some family caregivers, others have lost or relinquished jobs outside the home. clearly, the strain and sacrifice that family caregivers experience have multiplied during the pandemic. in addition, many community-based programs and settings for older adults (e.g., adult day programs, senior centers) that provide essential services to support family caregivers have limited services (e.g., lunch pick-up) or remain closed. evidence suggests that these adult caregivers, most of who are over years old, are also at risk for serious illness. a recent analysis of data from the understanding america study found higher proportions of poorer health during the pandemic across all outcomes among caregivers compared to noncaregivers. [ ] long-term caregivers reported the highest rates of poor health outcomes. psychological distress was most common, reported by . % of long-term caregivers vs . % of noncaregivers. somatic symptoms reported by long-term caregivers were also consistently much larger than noncaregivers ( %- % higher). [ ] health policy and programmatic initiatives are desperately needed to provide tangible support for family caregivers of frail older adults. geriatric nurses must make our voices heard about this issue in policy arenas now and until we realize an age-friendly future, free of the preconceptions, stereotypes, and discrimination we find in healthcare today. changes in hospital mortality for united states intensive care admissions from to a scoping review of the evidence about the nurses improving care for healthsystem elders (niche) program. the gerontologist taking stock of acute and critical care for older people: looking forward toward improvement covid-related family separation and trauma in the intensive care unit is your hospital communication policy discriminatory? online aarp and national alliance for caregiving. caregiving in the united states overburdened and underprepared: medical/ nursing task performance among informal caregivers in the united states caregivers mental health and somatic symptoms during covid- key: cord- - sabsrgy authors: quandt, sara a.; lamonto, natalie j.; mora, dana c.; talton, jennifer w.; laurienti, paul j.; arcury, thomas a. title: covid- pandemic among latinx farmworker and nonfarmworker families in north carolina: knowledge, risk perceptions, and preventive behaviors date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: sabsrgy ( ) background: the covid- pandemic poses substantial threats to latinx farmworkers and other immigrants in food production and processing. classified as essential, such workers cannot shelter at home. therefore, knowledge and preventive behaviors are important to reduce covid- spread in the community. ( ) methods: respondents for families with at least one farmworker (fwf) and comparable families with no farmworkers (nonfwf) in north carolina completed a telephone survey in may . the survey queried knowledge of covid- , perceptions of its severity, self-efficacy, and preventive behaviors. detailed data were collected to document household members’ social interaction and use of face coverings. ( ) results: knowledge of covid- and prevention methods was high in both groups, as was its perceived severity. nonfwf had higher self-efficacy for preventing infection. both groups claimed to practice preventive behaviors, though fwf emphasized social avoidance and nonfwf emphasized personal hygiene. detailed social interactions showed high rates of inter-personal contact at home, at work, and in the community with more mask use in nonfwf than fwf. ( ) conclusions: despite high levels of knowledge and perceived severity for covid- , these immigrant families were engaged in frequent interpersonal contact that could expose community members and themselves to covid- . the coronavirus pandemic has posed a substantial threat to immigrant farmworkers in the united states (usa) and other workers in the food production and processing system worldwide [ ] [ ] [ ] [ ] . such workers are deemed essential workers [ , ] and are unable to practice preventive measures such as sheltering at home and working from home that may be recommended to the general population. in addition, food system workers are often of low socioeconomic status, immigrant, minority, and undocumented so that they are excluded from some of the economic legal protections of workers in other industries [ ] . in regard to the pandemic, they are specifically excluded from the social safety net provided by the coronavirus aid, relief and economic security (cares) act [ ] . they also may not be reached by rapidly evolving public health messaging or provision of personal protective equipment intended to provide them with the knowledge and materials needed to protect themselves [ , ] . in the usa, many immigrant workers exist at the poverty threshold and lack health insurance and access to health care [ , ] , further diminishing their ability to protect their health in a pandemic [ , ] . such structural factors have been found to explain the uneven distribution of covid- in the usa population during the pandemic [ ] . substantial concern was expressed in the usa about latinx farmworkers' risk of covid- early in the pandemic [ , , ] . these workers often work seasonally, and the spring work season commenced within the first months of the pandemic. workers were considered to be at risk because close contact in crowded housing [ , ] and transportation used to reach the fields could increase rates of disease transmission [ , ] . within the fields, workers often work in close proximity picking row crops; and some equipment requires two or more workers to sit side by side, e.g., on mechanical setters as they plant seedlings. they also have limited access to water and other sanitation supplies [ ] . workers could then act as a vector to their larger communities by infecting other workers and family members. such patterns were observed by april , in immigrant worker populations in meat and poultry processing facilities [ ] , further increasing the concern for seasonal and migrant crop workers who would begin work in may and june in areas such as north carolina [ ] . public health directives about covid- in the usa changed rapidly over the first few months of the pandemic [ ] . early findings that coronavirus was stable on surfaces for hours or even days [ ] led to recommendations that focused on use of cleaning products to sanitize frequently touched surfaces such as doorknobs and countertops. these were subsequently downplayed as research and modeling of effects in other countries demonstrated the importance of droplet transmission of the virus, which could be reduced through physical distancing and use of face coverings such as masks [ , ] . similarly, some early claims for treatment and cures for covid- later proved false or were subject to hurried and incomplete evaluation [ ] . communication of these messages to the public, particularly to those who did not receive communications well in english, sometimes lagged behind scientific findings. taken together, the rapidly changing messages, coupled with public concern, and limited availability of up-to-date information in formats for those with limited english proficiency created a situation in the usa in which latinx workers such as farmworkers were likely to lack consistent and accurate information and, as a result, practice ineffective behaviors to protect themselves and prevent spreading disease to their social network. this study is guided by constructs from the health belief model (hbm) [ ] . the hbm tries to understand how knowledge and personal factors lead to actions to protect or promote health. in the hbm, perceptions of one's susceptibility to a disease and its perceived severity influence actions taken. individuals must perceive that they are susceptible, in this case, to covid- , and that contracting and spreading the disease would have serious consequences. in addition, self-efficacy, the belief in one's ability to take effective action in the situation of risk to health, influences whether or not one engages in health protective or promoting actions. this suggests that having a strong sense of self-efficacy in practicing protective measures to prevent contracting and spreading covid- will lead to engaging in such measures. in this study, we measure a number of these constructs, though we do not execute a full test of the hbm. interpretation of results is placed in the framework of structural vulnerability [ ] . this argues that one's health vulnerability is the product of one's place in the social hierarchy with its diverse set of power relationships, based on ethnicity and class. when applied to immigrant workers, factors such as occupation, documentation status, and access to government benefits provide context, and in fact, limit the choices, within which health behaviors understood within the hbm can occur. we report survey data collected in a narrow time window, may , from women in a sample of latinx farmworker families and a comparison group of latinx nonfarmworker families in north carolina, usa. the paper has three aims. in all cases, we will compare farmworker families and families with no worker engaged in farm work. first, we will describe the families' respondents' ( ) knowledge of coronavirus contagion and prevention, ( ) risk perceptions, and ( ) practices used for prevention and spread of covid- . second, we will describe household social interactions and protections taken, both outside of work and at work. third, we will use these data to identify specific risks for each group, as well as areas where policy changes can help mitigate the risk for covid- . the study reported here is part of a larger two-group, prospective study examining the health and cognitive effects of pesticide exposure in children in farmworker families. the larger study uses a comparative design, with a sample of families of latinx farmworkers with children and a sample of similar families but without any farmworker members. additional details of the study can be found elsewhere [ ] . the current study used a telephone survey to reach the mother of the children in these families in may , when no face-to-face contact between study staff and study participants was permitted by the institutional review board due to covid- -related health concerns for research participants. all procedures for both the original study and this covid- study were approved by the wake forest university institutional review board. the study received a certificate of confidentiality from the national institutes of health. inclusion criteria for the families were similar in both samples when recruited from march , to december ; they reflect the purpose of the larger study. each family had to have a child aged years at baseline who had completed the first grade in the usa. all children had to be from families that self-identified as latino or hispanic, and with household incomes below % of the usa federal poverty guideline. in the farmworker sample, the mother or her partner must have been employed in farm work on nonorganic farms during the past three years. in the nonfarmworker sample, adults could not have been employed in any industry that involves routine exposure to pesticides (e.g., farm work, landscaping, or pest control) in the previous three years. families in the nonfarmworker sample could not have lived adjacent to agricultural fields in the previous three years. exclusion criteria for both samples included children having life-threatening illnesses, prior history of neurological conditions, physical condition or development disorder that would not allow them to complete or would interfere with the results of neurobehavioral tests or mris (used in the larger main study), primary language other than spanish or english spoken in the home, or refusal of mother/guardian to complete the questionnaires. in the larger study, a total of children were recruited for the farmworker sample and children for the nonfarmworker sample. for the recruitment of the original sample, the community partner north carolina farmworkers project developed a list of farmworker families with an year old child and the locations where they lived. in addition, other community organizations that served farmworker families in the recruitment area were contacted. study personnel contacted the mothers. similarly, for the original nonfarmworker sample, local recruiters in winston-salem, nc, and community members developed a list. for both samples, mothers were contacted by a bilingual staff member who explained the overall study procedures, answered questions, and, if the mother agreed to participate, obtained signed informed consent from the mother and assent from the child. as recruitment progressed, community partners worked with the study team to balance the two samples on socioeconomic status. prior to the telephone survey, children in the farmworker sample and in the nonfarmworker sample withdrew, moved away from the study area, or were lost to follow-up. the remaining children represented farmworker families and nonfarmworker families, because some families had more than one child enrolled. for the telephone sample, families refused to participate and could not be reached, all in the nonfarmworker sample. a total of farmworker families and nonfarmworker families could be reached and agreed to participate. this sample of is used in this paper. data for this study were gathered from may to june , using a telephone survey. only interviews were conducted in june. interviewers were members of the larger study team who had usual interview contact with the mothers. each interviewer participated in an individualized televideo training after which the interviewer practiced completing the form and did an oral practice interview with the study manager. to recruit participants, interviewers called the last known telephone number for the mother in each family, explained the purpose and procedures for the study, and told the mother that she would receive a $ incentive for completing it at the next in-person study visit. if there was no answer, the interviewers tried at different times of day until the participant was reached or until at least unsuccessful calls had been made. if the mother agreed to participate, her informed consent was noted, and the interviewer proceeded to conduct a standardized interviewer-administered questionnaire in the language of the participant's choice using a tablet. data were entered in real time during the interviews using research electronic data capture (redcap). redcap is hosted at wake forest school of medicine through the clinical and translational science institute. the redcap system provides secure, web-based applications for a variety of types of research [ ] . data from these interviews were later merged with selected personal, family, and household variables collected in the main study questionnaires. questionnaire items relating to the coronavirus and covid- were adapted from existing studies (e.g., mcfadden et al. [ ] ), where available, or from questions recommended for covid- research by governmental and nongovernmental agencies. because of the need for rapid data collection, validation was limited to checks on face validity and interviewer reports of difficulties experienced by respondents during practice interviews. variables from the main study baseline questionnaire were used to create measures to describe the sample. these included the following measures for the mother: age, country of origin, educational attainment, and current occupation. group assignment of the family to the farm work or nonfarm work sample was also noted from the baseline questionnaire. current household size was obtained by querying the number of adults (persons years and older) and children living in the respondent's dwelling. knowledge of covid- was measured with a series of questions that asked the respondent to identify the correct answer from a series of statements for the definition of covid- , its transmission route, the definition of "close contact" for coronavirus, and availability of treatment and vaccine. a summary variable was created by summing the number ( - ) of items answered correctly. knowledge of behaviors that can prevent exposure to the coronavirus and its transmission was measured with a set of items in which the respondent was asked whether or not each could prevent exposure for self or others. the list contained items for which the correct response was positive (e.g., wear a face mask when out in public) and items for which the correct response was negative (e.g., take herbal supplements). the number of correct responses was summed to create a summary measure of questions answered correctly, with a range of to . perceptions of risk was measured with items containing statements about health risk to self and community from covid- . responses used a -point likert-type scale with values ranging from strongly agree to strongly disagree, which was collapsed to a -point scale for analysis with values (agree), (neutral), and (disagree). the two items concerning personal risk or self-efficacy were added to create a summary measure of self-efficacy with values to . this was divided into categories of low self-efficacy ( - ) and high self-efficacy ( ) ( ) . the cronbach's alpha for this scale was . . personal behaviors to protect health and prevent spread of the coronavirus in the past month were obtained by asking the respondent if they had never, sometimes, or always practiced each of behaviors. these included the positive behaviors in the knowledge items described above, as well as additional items (avoiding travel to areas infected with coronavirus; avoiding eating outside the home). these were summed with a possible range for the summary being to , with each behavior scored as (never), (sometimes), or (always). the next section of the questionnaire included questions asking about physical distancing and mask use for protection in order to overcome any social desirability [ ] that may have affected the previous self-reports of behavior. respondents were first asked how many adults had visited in the respondent's house in the past week. response options were none, or , or , and or more. those who had had visiting adults were asked how many visitors had worn masks during their visit, with the response options of all of them, some of them, and none of them. these questions were also asked about child visitors. respondents were also asked how many different houses, apartments, or trailers of others they had visited in the last week. response options were none, or , or , and or more. those who had visited other homes were asked how often they wore a mask during their visit, with the response options of all, some, or none of the time. similar questions were asked about the household children and the respondent's spouse/partner. respondents were asked how many people they worked with, defined as the number of persons with whom they worked closely enough to have a normal conversation for at least some of the work time. response options were none, or , or , and or more. mask use was queried for coworkers, with response options of all of them, some of them, and none of them wore masks at work. similar questions were asked for the spouse/partner at work. respondents were asked if their children had been cared for in the past week at a day care, pre-school, school, after school program, or at a relative or friend's house. any positive responses were followed by asking whether all, some, or no childcare workers wore masks and wore gloves. to obtain information on large social gatherings in the past week, respondents were asked if any household member had attended church, the approximate number of attendees, and if all, some, or none of the attendees wore masks. the same set of questions was asked about whether any household member had attended a party or other social event such as a cookout, baptism, quinceañera, wedding, or funeral in the past week. frequencies and percentages were calculated to examine the variables of interest by farmworker status and significant differences were examined using chi-square or fisher's exact tests as appropriate. all analyses were done using sas v . (sas institute, cary, nc, usa), and p-values < . are considered statistically significant. respondents ranged in age from to years (table ) . about % of both samples were born in mexico; spanish was the preferred language for most. years of formal education for the respondents ranged from to college graduate, with the median in both samples being ninth grade. their spouse/partners had slightly lower education; the medians for the farmworker and nonfarmworker samples were sixth and eighth grade, respectively. there were no significant differences between the two samples for these categorical variables. total household size ranged from to (median = ) and to (median = ) in the farmworker and nonfarmworker samples, respectively. for the farmworker sample, the number of adults in the household ranged from to , while the number of children ranged from (a respondent currently separated from her family) to . for the nonfarmworker samples, the ranges were to for adults and to for children. at baseline, farmworker families reported that the most common industry in which women worked was agriculture; for men, it was construction, followed by agriculture. for nonfarmworker families, most women were not in the labor force and the majority of men worked in construction. knowledge of the coronavirus was high ( table ). all individuals in both samples had heard of the virus, and none required an explanation of what it was. the farmworker sample had more correct answers than the nonfarmworker sample on three of the four remaining items. more in the farmworker sample knew that covid- was a respiratory disease caused by a viral infection ( % vs. . %; p < . ). for the item concerning treatment or vaccine for covid- , . % of the nonfarmworker sample did not know that there is currently no cure or a vaccine for covid- , compared to only . % of the farmworker sample (p < . ). overall, knowledge in the farmworker sample was significantly higher than in the nonfarmworker sample (p < . ), with . % of farmworker sample having a perfect score, compared to only . % of the nonfarmworker sample. knowledge of behaviors to prevent exposure to the coronavirus or spread of covid- was high in both samples (table ). for seven of the items, both samples had % correct responses. more in the farmworker sample knew that avoiding touching the face with unwashed hands was protective than in the nonfarmworker sample ( . % vs. . %; p < . ). the only other items for which the samples had different responses were three of the five in the list that were negative options (e.g., taking herbal supplements). for these, the nonfarmworker sample had significantly more correct responses for using herbal supplements ( . % vs. . %; p < . ). the farmworker sample had more correct responses for eating a balanced diet ( . % vs. . %; p < . ) and getting regular exercise ( . % vs. . %; p < . ). overall, the farmworker sample had somewhat better knowledge of prevention than did the nonfarmworker sample, but the difference was not significant (p = . ). the farmworker sample respondents perceived lower risk associated with covid- for themselves and their community on most items than did the nonfarmworker sample respondents (table ) . similarly, the farmworker sample perceived that they had lower ability to protect themselves from the coronavirus, with almost all responses ( . %) falling in the lower self-efficacy category, compared to . % of the nonfarmworker sample falling in the higher self-efficacy category (p < . ). for self-reported actual preventive behaviors, the farmworker sample was significantly more likely to report practicing three behaviors (avoiding travel to areas infected with coronavirus [p < . ], avoiding eating outside the home [p < . ], and avoiding close contact with people who were sick [p < . ]), while the nonfarmworker sample was significantly more likely to report practicing four behaviors (washing hands for s [p < . ], using surface disinfectants [p < . ], avoiding touching face with unwashed hands [p < . ], and covering cough with tissue [p < . ]) ( table ). the overall difference between the two samples was significant (p = . ). slightly fewer than half of farmworker families (n = ; . %) reported that they had had adult visitors at their home in the past week. of these, reported that none of the visitors had worn a mask. similarly, of these families ( . %) reported that children had visited in their home and none had worn masks. for nonfarmworker families, more had had adult visitors (n = ; . %), but some (n = ; . %) had worn masks. a lower proportion of the nonfarmworker families had had child visitors (n = ; . %), and some (n = ; . %) had worn masks. more farmworker than nonfarmworker family respondents reported visiting the homes of others in the past week (n = , . % vs. n = , . %). both categories of respondents reported visiting or other homes, except from farmworker families who reported visiting or . none of the respondents from farmworker families reported wearing masks when visiting; . % (n = ) of the nonfarmworker respondents reported ever wearing masks while visiting. twenty-seven respondents ( . %) from farmworker families reported that their children visited other homes in the past week, and none wore masks. they also reported that . % (n = ) of their spouse/partners visited other homes, and none ever wore masks. respondents from nonfarmworker families reported fewer children (n = ; . %) and spouse/partners (n = ; . %) visiting other houses, with one spouse/partner visiting five or more houses. about a third (n = ; . %) of spouses were reported to have worn masks, though several respondents did not know, and . % (n = ) reported their children had never worn masks while visiting other homes. among respondents in farmworker families, ( . %) reported working in the past week. most (n = ; . %) worked in places with five or more employees in close enough contact to have a normal conversation at least some of the time. these respondents reported that all (n = ; . %) or some (n = ; . %) wore masks in the workplace. almost all of their spouse/partners worked (n = ; . %); . % (n = ) worked in places with five or more employees in close contact, and some or all wore masks in . % (n = ) cases. about the same proportion of respondents in nonfarmworker families worked (n = ; . %), but fewer (n = ; . %) worked in places with five or more workers in close contact. most of these respondents reported that all (n = ; %) or some (n = ; . %) of coworkers wore masks. almost all (n = ; . %) spouses worked, though less than half (n = ; . %) worked in close contact with five or more workers. in about two-thirds of these worksites ( . %), some ( . %) or all ( . %) workers wore masks. during the time women were surveyed, schools were closed, and no children attended preschools or day care centers. seven ( . %) respondents in farmworker families reported that their children were cared for at a friend or relative's house and that none of the caregivers wore masks or gloves. four ( . %) respondents in nonfarmworker families reported similar childcare arrangements. however, half reported the caregiver wore masks and gloves. five ( . %) of the respondents in farmworker families reported that a household resident had attended church in the past week. total church attendance was estimated by the respondent at ( cases), ( case), and ( cases). all attendees wore masks in four of these church services, and none wore masks in the other. only one respondent among nonfarmworker families reported that a household member had attended church in the past week. attendance was about people and all reportedly wore masks. nine ( . %) respondents in farmworker families reported that a household member had attended a party or social event in the past week. estimates of total attendees ranged from to ; none wore masks. by comparison, three ( . %) respondents in nonfarmworker families reported someone had attended a party or social event. in two cases, attendance was estimated at ; the other was estimated at . no one wore masks at two of these events. this study was designed to describe the knowledge, perceived risk and susceptibility, and preventive behaviors reported by latinx immigrant farmworker and nonfarmworker families in north carolina during the first months of the covid- pandemic. these families are of particular concern because the rates of covid- nationally are elevated in minority populations. specifically in north carolina, on june , hispanics were reported to make up % of the state's population but % of the state's covid- cases [ ] . at the same time, several farmworker camps were listed as locations of covid- outbreaks by the state department of health and human services. the study found that levels of knowledge were extremely high among the latinx families surveyed, both farmworker and nonfarmworker. all respondents had heard of the pandemic and knew what covid- is and how it is transmitted. they had somewhat less accurate knowledge about the availability of a cure or vaccine; and women in farmworker families had, overall, slightly more accurate knowledge than did the women in nonfarmworker families. both samples had strong knowledge of the health behaviors that could protect against exposure to the coronavirus and contracting or transmitting covid- . in particular, they knew the primary public health messages promoted early in the pandemic. they were less accurate in differentiating these effective behaviors from ineffective behaviors that might be promoted for health risks other than covid- , such as exercising and consuming a balanced diet. although both groups perceived that covid- presents a serious risk to health, respondents in farmworker families were significantly less likely to affirm personal susceptibility (e.g., that they would avoid going to the hospital for another illness because of risk of contracting covid- there and that they were more likely than others to get . similarly, these women in farmworker families had lower self-efficacy concerning their ability to protect themselves. the two samples affirmed different patterns of health promoting behaviors. for the farmworker families, behaviors that entailed avoiding others (e.g., not traveling to areas infected with coronavirus, avoiding eating out, and avoiding close contact with sick individuals) were affirmed significantly more often than by the nonfarmworker families. the latter were more likely to affirm behaviors related to personal hygiene: hand washing, using disinfectants, avoiding touching the face, and covering coughs and sneezes. together, these findings give a sense that, while the women in farmworker families had somewhat better knowledge, they perceived less personal susceptibility to covid- . they had low confidence that they could protect themselves. this may be underlying the protective behaviors they reported. they avoided people and places that might be contaminated but did not subscribe to practicing personal hygiene behaviors. women in nonfarmworker families had greater confidence that they could protect themselves and they claimed to practice more personal hygiene behaviors. social desirability [ ] can bias the way individuals respond to lists of health behaviors. with knowledge of recommendations, they may tend to see themselves or want to portray themselves as more positive and compliant than they actually are. in order to investigate behaviors in detail and try to avoid social desirability bias, the telephone survey included a series of questions about social interactions by household members and wearing masks. complex question sequences are thought to reduce social desirability bias [ , ] . the focus on distancing and masks was considered important in light of the developing public health messages that identified the greater importance of maintaining physical distancing and protection against spreading infected droplets with masks, rather than practices such as disinfecting surfaces that had been promoted over mask use earlier in the pandemic [ ] . the responses to these questions contrasted sharply with the other reported protective behaviors. they showed a high level of social interaction beyond the immediate household for both farmworker and nonfarmworkers families, with both adults and children coming into the homes of respondents and members of the respondent's household visiting in the homes of others. there was virtually no mask wearing reported by farmworker family respondents, and only some use of masks reported by nonfarmworker respondents. household sizes reported in this study (median for farmworker and for nonfarmworker families) are considerably larger than the usa average of . people reported for [ ] , potentially creating large social networks of contacts. many of the adult household members were reported to be working outside the home and working in situations where they had close contact with other workers. these situations, plus the sheer number of adults in the household (up to six in farmworker families and four in nonfarmworker families), allows for the spread of infection through these interconnected households [ ] . mask use was reported to be common in the workplaces, though measures of the consistency or enforcement of mask use were not obtained. the respondents and their family members reported continuing to engage in social situations with large numbers in attendance. this occurred in both samples and was particularly common among the farmworker families. although masks appear to have been worn for church attendance, little mask wearing was reported for other types of social events. in total, these results indicate that, despite relatively high knowledge, strong perceptions of risk from covid- , and claims of avoiding situations where contracting or spreading infection might be likely, many of the farmworker families included here do not practice safe physical distancing measures as recommended; and their use of masks appears to be confined to work settings. the situation for the nonfarmworker families appears to be somewhat better, with greater mask wearing reported, particularly in large social gatherings. however, the social contact is still at levels that facilitate covid- spread. the inconsistency between women in farmworker families seeing themselves as avoiding situations for infection and their actual practices may be due to their living situations and to cultural values. most live in rural environments and few women drive [ ] , so they may perceive of themselves and their households as isolated from population centers. nonetheless, it is clear that interactions take place within and between households, which can exponentially raise the possibility of transmitting infection. this is in contrast to the nonfarmworker families who live in urban environments, many in multi-unit dwellings such as apartment buildings. they may correctly perceive less ability to socially isolate themselves and, so, give greater importance to personal hygiene measures to prevent infection. for these immigrant workers (from both farmworker and nonfarmworker families), living in close proximity to extended family members plus the cultural value of familismo [ ] likely affect interpretation of public health recommendations to maintain physical distance. many immigrant workers settle in the us with extended family from their home communities-siblings, cousins, parents, aunts, and uncles. this can provide considerable social and material support while living in a new environment and working in low wage jobs; family and household boundaries are likely more fluid than they are for other ethnic groups [ , ] . these relationships are supported and reinforced by familismo. this cultural construct includes strong identification with and loyalty to family, as well as respect for family members and placing family needs over one's own needs. time spent with one's immediate and extended family is valued. in such a context, wearing masks or refusing social interaction might be considered an affront. the result can be greater contacts and less physical distancing than public health recommendations intend, increasing the risk of coronavirus infection. while covid- is an emerging issue, findings from previous research with immigrant latinx populations support the findings in this study. for example, research with immigrant latinx women has produced results supporting the lower self-efficacy seen among the respondents from farmworker families. studies of hiv and cancer prevention behavior have found low self-efficacy in latinx farmworker women, which is sometimes amenable to change with intervention [ ] , though not always when cultural norms constrain health-promoting behavior [ ] . kilanowski [ ] , in a study of farmworker child nutrition, found self-efficacy for health behavior change was inversely related to acculturation, suggesting that self-efficacy may fall with greater time in the usa. none of the families in the current study are newly arrived immigrants because of the larger study eligibility criteria. other research with farmworkers has shown that they have low levels of perceived susceptibility to other health threats, most notably pesticides [ , ] . in these cases cultural values appear to promote these ideas of low susceptibility. the farmworker families included in this study are seasonal workers, meaning that they live in the area year round, and family members work seasonally in agriculture. they may not experience the extremely crowded barrack-style sleeping quarters, kitchens, and bathroom facilities of much of the grower-provided housing where migrant workers live [ ] . however, these seasonal worker families do have crowded housing [ , ] , and they face worksite hazards for infection in crowded transportation to the fields and while working in close quarters in some situations in the fields, as well as in greenhouses or packing facilities [ ] . they also often work alongside migrant workers who live in crowded conditions. although the respondents indicate mask usage, it is difficult to know how sustained that can be, considering the high levels of heat and humidity these workers endure in the fields [ ] . the contrast between what the respondents in this study know about covid- and their seemingly contradictory behavior can be viewed through the lens of structural vulnerability [ ] . the farmworker families, as well as many of the nonfarmworker families, include those who have been deemed essential workers. these include those in farm work, in construction, in building maintenance, and in food retail. as essential workers, they need to work in order to receive income. their jobs do not provide the luxury of working from home. as immigrants, most are ineligible for government benefits provided as part of the cares safety net [ ] . in the case of undocumented families, worry about the xenophobic climate [ ] may affect decisions to work, to seek medical care, and to complain about the lack of personal protective equipment. in short, these workers are not putting themselves and their communities at risk because they are uninformed about covid- . they know how dangerous it is, and, while cultural values and practices may lead to some excess exposure, they do know how to prevent covid- . one of the strengths of this study was the concentration of data collection in a short time during which changes in national information about prevention and state regulations were relatively stable. by may, reports of emerging research had started to establish the importance of physical distancing and mask use (although publications did not appear until june [ ] [ ] [ ] ), and the initial emphasis on hand hygiene and cleaning surfaces had been downplayed. within north carolina, all families in this study would have been subject to the same governmental orders. stay-at-home orders banning gatherings of > persons and closing schools, bars, gyms, playgrounds, and restaurants (except for take-out and delivery) were put in place in march. on april, school closure was extended for the rest of the academic year. although restaurant closure was loosened on may to % of capacity for indoor dining, most restaurants took longer to implement this and many still remained at take-out and delivery only well into the summer. gatherings were limited to people on march; although -person gatherings outside with social distancing were allowed on may, indoor gatherings were kept at with no special provisions for churches. this study did not collect data on information sources about covid- available to study participants. although both groups frequently get information from spanish language radio, the nonfarmworker families may have had greater access to public health signage and other local messages in an urban context than the farmworker families did in rural settings. other study limitations include the fact that behaviors were self-reported and not observed. the women interviewed also reported for others in the household. responses could not be anonymous because they were collected by interviewers that the women had known through participation in the larger study; this could have increased the social desirability in responses concerning behavior. small sample sizes prevent more detailed analyses of data. nevertheless, this study represents a unique opportunity to document the knowledge, perceptions, and behaviors of latinx immigrants in the usa during the early days of the covid- pandemic. in particular, farmworkers are often a hidden and difficult to reach population. this study demonstrates that even with a strong knowledge base, these farmworker families lack the self-efficacy to avoid the coronavirus and covid- . while they appear to believe that they are following public health recommendations on physical distancing and wearing masks, detailed data on their social interactions and use of personal protective equipment show that this is not the case. a comparison group of urban-dwelling latinx immigrants had greater self-efficacy, which might have led to the greater use of masks as personal protection reported by respondents in these nonfarmworker families. the transmission of a highly infectious virus like the coronavirus is facilitated by close contact among individuals in a population. the large household sizes, particularly large numbers of adults working in industries deemed essential, and weak adherence to personal protective equipment such as masks make the immigrant latinx population at risk for high rates of infection. it is likely that simple public health messages encouraging physical distancing and mask wearing may not protect the population in the context of structural barriers such as crowded housing and work in essential industries, coupled with strong cultural values placed on support of large extended families. specific actions beyond what is currently being taken by public health authorities may help improve the health-related behavior reported here and curb the spread of infection in this population. developing and disseminating culturally sensitive education to help families understand the extent of their social contact and the dangers it poses is essential. using adult educational approaches [ , ] that could include interactive exercises to demonstrate the potential spread of infection would likely be more effective than education based primarily on print materials in this low literacy population [ ] . the covid- pandemic has ravaged urban populations around the world, with high population density facilitating the spread of the disease. while one might, therefore, expect urban and rural conditions in the us to be markedly different, the findings here suggest that this may not be the case for latinx workers in essential rural industries. living 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of coping with social desirability bias: a review determinants of social desirability bias in sensitive surveys: a literature review the number of people in the average u.s. household is going up for the first time in over years nutritional strategies of latino farmworker families with preschool children: identifying leverage points for obesity prevention an anthropology of familismo: on narratives and description of mexican/immigrants educating hispanic women about cervical cancer prevention: feasibility of a promotora-led charla intervention in a farmworker community associations between gender norms and hiv self-efficacy among latina immigrants in a farmworker community patterns and correlates of nutrition among migrant farm-worker children applying pesticides without protective equipment in southern mexico farmworker and farmer perceptions of farmworker agricultural chemical exposure in north carolina the modern practice of adult education; the adult education company educational techniques for lifelong learners. principles of adult learning developing occupational safety and health training programs for immigrant workers: translating research to practice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors appreciate the support and participation of their community partner, the nc farmworkers project, and of student action with farmworkers. they also appreciate the valuable contributions of our community field interviewers in carrying out participant recruitment and data collection. they especially thank the mothers who participated in this study. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. key: cord- -jrsg pdk authors: hu, na; li, ying; he, su-shuang; wang, lei-lei; wei, yan-yan; yin, lu; chen, jing-xu title: impact of the family environment on the emotional state of medical staff during the covid- outbreak: the mediating effect of self-efficacy date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: jrsg pdk during the outbreak of the coronavirus disease (covid- ), the medical staff was facing severe work pressure, which led to a negative emotional state. the purpose of this study was to explore the relationship between the family environment and the emotional state of the medical staff members during the covid- outbreak. due to the importance of self-efficacy in regulating mental health, the mediating role of self-efficacy in the association between family environment and emotional state was also explored. a cross-sectional survey was performed, using an online questionnaire, on medical staff who participated in the epidemic prevention and control tasks during the covid- outbreak in beijing. family environment, self-efficacy, anxiety, and depressive symptoms were measured by the family environment scale-chinese version (fes-cv), the general self-efficacy scale (gses), the generalized anxiety disorder scale- (gad- ), and the patient health questionnaire- (phq- ), respectively. correlation analysis and mediating effect analysis were used to explore the relationships between them. first, a higher prevalence of anxiety ( %) and depressive ( %) symptoms were confirmed among the medical staff. second, the symptoms of anxiety and depression were negatively correlated with the dimensions of cohesion and expressiveness and positively correlated with the dimensions of conflict in the fes-cv scale. third, self-efficacy significantly mediated the association between the family environment and anxiety symptoms (p < . ) as well as the family environment and depressive symptoms (p < . ). these findings show that a negative family environment was the main predictor of symptoms of anxiety and depression in the medical staff during the covid- outbreak. furthermore, we found that self-efficacy played a critical mediating role between the family environment and the symptoms of anxiety and depression. our study also indicates that improvements in the family environment benefit the mental health care of the medical staff, and high self-efficacy enhances this effect. the outbreak of coronavirus disease continues to attract worldwide attention . to date, covid- cases have been confirmed in more than countries around the world, and it has become a public health emergency of international concern. many people who have directly faced this large-scale public crisis, especially the medical staff involved in the prevention and control of this epidemic, showed anxiety symptoms (huang et al., ) . they faced a high risk of getting infected at their workplace and the possibility of their family members at home getting infected through them (xiang et al., ) . medical staff working in a high-pressure environment suffer from psychological problems, especially anxiety and depression (kang et al., ; wang et al., ) . although guidelines on the care of the mental health issues in medical staff have been issued in china (kang et al., ) , some of the staff refused to receive mental health care . it has been reported that the incidence of anxiety and depression was high among the medical staff during the outbreak of covid- , with prevalence rates of anxiety and depressive symptoms being . % and . %, respectively (lai et al., ) . due to the stigma around mental health problems in china (bai et al., ) , many members of the medical staff community were reluctant to accept professional psychological help . however, they preferred to seek help from their family members. some medical staff was far away from their families because of the fear of infection, and they were reluctant to have close contact with them. others were isolated and cannot return home for a long time (raven et al., ) . it has shown that family support is very important for medical staff involved in epidemic prevention (mohindra et al., ) . however, there has been no research on how family members can help medical professionals suffering from mental health issues and what kind of family environment can be useful in relieving negative emotions in these individuals during an epidemic. the family, which is the basic social unit, may affect the mental health of family members (cheng et al., ) . in chinese culture, family relations are highly valued, and family is a very important support system (poulin et al., ) . it means that the importance of family is the core feature for most chinese people. previous studies have shown that the family environment can directly affect the emotions in family members, especially the dimension of cohesion (harris and zakowski, ; burnett et al., ) , expressiveness (luebbe and bell, ) , and conflict (yap et al., ; yap and jorm, ; fosco et al., ) . however, whether the family environment has an effect on the mental health of medical staff during the epidemic is still not clear. self-efficacy refers to an individual's judgment about his or her ability to complete a certain task successfully, and it reflects the self-confidence of an individual to cope with various difficulties and setbacks in life (tang et al., ) . previous research has shown a positive correlation between the family environment and self-efficacy (mahmoudi, ) . individuals with a better family environment were shown to be more likely to have higher levels of self-efficacy (hemati et al., ) . for example, individuals can freely communicate with family members, express more about themselves, and have a frequent high contact of intimacy with other family members, which will lead to a high level of selfefficacy (hemati et al., ) . studies show that increasing selfefficacy is an essential aspect of the psychological intervention to protect the mental health of individuals (caldwell et al., ) . it seems that the higher the self-efficacy in an individual, the better the mental health (sebastian, ; jiang et al., ) . high self-efficacy can improve an individual's mental health (bandura, ). however, a low level of self-efficacy harms a person's psychological well-being (cieslak et al., ; sachs-ericsson et al., ) . importantly, studies have shown that self-efficacy is one of the critical factors that modulate an individual's emotions, especially feelings of anxiety and depression (bandura et al., ; kanfer and zeiss, ; cybulski et al., ; schönfeld et al., ) . these studies suggested that higher levels of selfefficacy are associated with lower levels of feelings of anxiety and depression. during the outbreak of sars, medical staff with low self-efficacy often had a higher fear of the epidemic, which was positively correlated with their poor mental health status (ho et al., ) . self-efficacy can predict the significant difference in mental health during the epidemic. the lower the self-efficacy, the worse the mental health status (yıldırım and güler, ) . it has been found that lower psychological stress among dentists during the covid- epidemic is associated with being in a stable relationship and having a higher sense of self-efficacy (shacham et al., ) . it suggests that family relationships and self-efficacy during the epidemic may both affect the mental health of medical staff. the partial mediating role of self-efficacy in some psychological trait relationships has been supported by relevant research (haj-yahia et al., ) . indeed, it has been found that self-efficacy was a mediator for the association of daily stress and mental health (schönfeld et al., ) . however, there has been no study focusing on the relationship between family environment, self-efficacy, and the emotional state of medical professionals during an epidemic. the relationship between family environment, self-efficacy, and depression and anxiety, and whether the influence of family environment on anxiety and depression is regulated by selfefficacy needs to be further studied. therefore, the purpose of this study was to further explore the direct and indirect impact of the family environment on symptoms of anxiety and depression among the medical staff involved in controlling the epidemic. when examining the indirect effects, we took self-efficacy as an intermediary variable. mediating effect analysis was performed to explore the role of self-efficacy in the relationship between family environment and symptoms of anxiety and depression. based on our findings, we provide viable strategies for the family based psychological intervention of the medical staff during an epidemic that will help to improve our psychological crisis intervention system. this research was a cross-sectional study using the convenience sampling method to collect survey results through an online questionnaire. the online survey was conducted in beijing from february , to march , . inclusion criteria for the study were as follows: ( ) chinese, working in beijing; ( ) - years old; ( ) medical staff including doctor or nurse involved in covid- epidemic prevention and control; and ( ) has read and agreed to the online informed consent. the protocol of this study was approved by the ethics committee of the beijing huilongguan hospital. the family environment scale-chinese version (fes-cv) this scale was based on the family environment scale (fes) developed by moss (moos and moos, ) , which was translated into chinese by wang et al. (wang et al., ) . the scale has entries in total, including dimensions (cohesion, expressiveness, conflict, independence, achievement orientation, intellectual-cultural orientation, active-recreational orientation, moral-religious emphasis, organization, and control), with nine entries for each dimension. each entry has a true and false option. the higher the score of a particular dimension, the more prominent the characteristics of the family in that aspect. the scores of conflict and control were negatively correlated with the family environment, whereas others positively correlated with the family environment. this scale has been shown to have good structural, content, and external validity when applied to the chinese population (phillips et al., ) . in this study, we selected three dimensions of this scale that were closely related to emotions investigated in previous studies: cohesion, expressiveness, and conflict (harris and zakowski, ; burnett et al., ) . this scale was developed by german psychologist schwarzer (schwarzer, ) , which was translated and revised for the chinese version by wang et al. (wang et al., ) . the scale comprises of items with four answer options, and the answer options range from (not at all true) to (completely true). higher scores mean higher levels of self-efficacy; a score of . - . means low level, . - . means medium level, and . - . means a high level of self-efficacy. the revised scale has been shown to have excellent reliability and validity in the chinese population (wang et al., ) . the cronbach's alpha of this scale is . , the retest reliability is . , and the correlation coefficient between the items and the total scale score is . - . . this scale was developed by spitzer et al. (spitzer et al., ) . previous research demonstrated that the chinese version of gad- has good reliability and validity, and the sensitivity and specificity of this version were . and . , respectively (huang et al., ) . this scale is composed of seven items, and each item has a - points scale. the total score range is - points; - for no anxiety, - for mild anxiety, - for moderate anxiety, and more than for severe anxiety. the patient health questionnaire- (phq- ) the scale was developed based on the fourth edition of the diagnostic and statistical manual of mental disorders (kroenke et al., ) . we used the chinese version of this scale. this scale is composed of nine items, and each item has a - points scale. the symptom severity is determined by the total score, with - being mild, - being moderate, - being moderately severe, and - being severe. cronbach's alpha of the phq- in the chinese population is . , and the retest reliability is . , which indicates that this test has excellent reliability and validity (wang et al., ) . all of the analyses were performed using spss for windows . . we reported means and standard deviations for continuous variables showing normal distribution and frequencies and proportions for categorical variables. the original scores of the dimensions of conflict in fes-cv were not normally distributed and were presented as medians and quartiles. a chisquared test was used to test the relationship between the demographic data of the participants and their anxiety and depressive symptoms. the study participants were grouped based on whether they showed/did not show anxiety or depressive symptoms. the inter-group comparison of the scores of fes-cv and gses was carried out using the independent sample t-test and the independent sample kruskal-wallis test. spearman correlations were calculated to determine the relationships between the scores of the various scales. we categorized the family environment (cohesion, expressiveness, and conflict) as an independent variable, anxiety and depression symptoms as dependent variables, and self-efficacy as an intermediary variable. after controlling for demographic variables, the direct, indirect, and total effects of the family environment on the symptoms of anxiety and depression were examined. the mediation analysis was run on the process macro for spss (preacher and hayes, ), using , bootstrap samples for bias correction and to establish % confidence intervals. all of the tests were twotailed, and the significance level was set at p < . . the medical staff involved in this study mainly includes two groups. one is the staff of the hospital involved in treating patients infected with covid- ; the other is the staff at the isolation point, whose main task is nucleic acid testing and medical services for the quarantined personnel. a total of medical staff members completed the online questionnaire, out of which six individuals did not complete the basic information, and two took more than min to answer the questionnaire. thus, medical staff members participated in the study, of which ( %) were women, and ( %) were men, aged - , with an average age of . ± . . most of the participants were under ( %), had a bachelor's degree ( %), were married ( %), and were living with their families ( %). among these participants, ( %) had anxiety symptoms, and ( %) had depressive symptoms. the symptoms of anxiety and depression were closely related to the gender of the medical staff and whether they lived with their family members or not. the prevalence rate of anxiety and depression symptoms was higher in women (x = . , p = . ; x = . , p = . ) and in those who did not live with their families (x = . , p = . ; x = . , p = . ) ( table ) . we divided the participants into two groups: one group with anxiety symptoms (gad- score > ) and the other group without anxiety symptoms (gad- score ≤ ). we then investigated the significance of the differences in the scores of the two groups for the fes-cv and gses scales. next, the participants were divided into two groups according to whether they had depressive symptoms or not (depressive symptoms: phq- score > ; no depressive symptoms: phq- score ≤ ). like the anxiety symptoms, we investigated the significance of the difference between the scores of fes-cv and gses scales in the two groups. the results, which show the significant differences among groups, are shown in table . next, we used correlation analysis to determine the correlation between the scores of each scale. the results showed that there were statistically significant relationships between the anxiety and depressive symptoms of medical staff, their family environment, and their sense of self-efficacy. anxiety and depressive symptoms showed a significant positive correlation with the dimension of conflict in fes-cv scale (r = . , p < . ; r = . , p < . ), and a significant negative correlation with the dimension of cohesion (r = − . , p < . ; r = − . , p < . ), expressiveness (r = − . , p < . ; r = − . , p < . ), and self-efficacy (r = − . , p < . ). more specifically, individuals with bad family environments and low self-efficacy were more likely to show symptoms of anxiety and depression. besides, selfefficacy positively correlated with the dimension of cohesion and expressiveness and negatively associated with the dimension of conflict (table ) . after controlling for demographic variables, we examined the mediating effects of self-efficacy (figure ) . self-efficacy was significantly associated with the symptoms of anxiety and depression. it significantly mediated the association between the family environment and anxiety symptoms (β = − . ; % ci, − . to − . ; β = − . ; % ci, − . to − . ; and β = . ; % ci, . to . ). similarly, it mediated the association between family environment and the depressive symptoms (β = − . ; % ci, − . to − . ; β = − . ; % ci, − . to − . ; and β = . ; % ci, . to . ). when controlling for self-efficacy, the association between the family environment and anxiety symptoms were still significant (β = − . , p < . ; β = − . , p < . ; and β = . , p < . ) and similarly for family environment and depressive symptoms (β = − . , p < . ; β = − . , p < . ; β = . , p < . ). thus, self-efficacy partly mediated the relationship between the family environment and the symptoms of anxiety and depression. in the current study, we conducted an online questionnaire survey of some medical staff involved in covid- prevention and control in hospitals and isolation sites in beijing. we found that a considerable proportion of medical professionals had anxiety ( %) and depressive symptoms ( %), as noted in previous studies (huang et al., ; lai et al., ) . moreover, our results showed that the family environment of medical staff and their symptoms of anxiety and depression during the epidemic were closely related to self-efficacy, and self-efficacy partly mediated the relationship between the family environment and the symptoms of anxiety and depression. in this present study, we found that the self-efficacy of male medical staff was significantly higher than that of female medical staff (t = . , p = . ). however, the symptoms of anxiety (p < . ) and depression (p = . ) during covid- were significantly lower than that of female medical staff, which was consistent with previous research results. for example, it showed that female medical workers experience higher levels of anxiety, depression, and distress during covid- (lai et al., ) . it also reported that the self-efficacy of male medical staff is significantly higher than that of female medical staff (tang et al., ) . it should also be noted that medical staff who did not live with their families were more likely to have symptoms of anxiety and depression during the epidemic. thus, our study suggests that we should pay more attention to such medical staff and provide them psychological intervention. this observation also indicates that the family plays a certain role in regulating negative emotions. furthermore, our study showed a close relationship between the family environment of the medical staff and their symptoms of anxiety and depression. the medical staff members with low cohesion and expressiveness, as well as high conflict in the family environment, were more likely to show anxiety and depressive symptoms during the epidemic. notably, in this present study, the relationship between the family environment and the symptoms of anxiety and depression reveals that the family environment can, directly and indirectly, affect the emotions of medical staff during an epidemic. the family environment can significantly predict the emergence of anxiety and depressive symptoms directly, which is consistent with previous results. for example, some studies found that there was a correlation between cohesion in the family environment and depression in family members (burnett et al., ) ; families with high cohesion, which have high levels of family support and ties, likely reduce depression (park et al., ; cano et al., ) . in contrast, low family cohesion and conflict between parents increased the risk of depression and anxiety in family members (park et al., ; cano et al., ) . in families with a high degree of cohesion, individuals can get more psychological help and emotional support within the family (birgisdóttir et al., ) , so that the psychological pressure can be appropriately relieved. positive emotional expression within the family can prevent suppression of inner feelings and buffer internal conflicts, especially in the face of stressful events. in contrast, negative emotional expression and low emotional expression within the family are associated with higher anxiety and depression (luebbe and bell, ; park et al., ) . in a high-conflict family, family members are prone to conflict between each other, leading to anxiety. therefore, the results in this study support the hypothesis that the family environment can influence the emotional state of the family members and that a negative family environment is a psychological risk factor for the rising emotional distress of the medical staff during an epidemic. additionally, the influence of the medical staff 's family environment on their symptoms of anxiety and depression during the covid- epidemic is partly through the role of self-efficacy, which means self-efficacy plays a critical role in mediating the effect of family environment on symptoms of anxiety and depression. indeed, previous studies supported that self-efficacy had a protective effect on mental health (bandura, ) and played a vital role in the regulation of stress (bandura et al., ) . high self-efficacy was related to better psychological adjustment (bandura, ) and lower emotional distress (benight and harper, ) . individuals with high selfefficacy had positive expectations and beliefs, had successful experiences, generated positive emotions, and were more likely to seek psychological support to modulate their emotions when facing stressful situations (tsang et al., ) . a bad family environment can reduce an individual's self-confidence and ability (hemati et al., ) . self-efficacy is the embodiment of such confidence and ability (tang et al., ) . that is to say, the family environment affects self-efficacy by affecting people's self-confidence and ability, thus affecting individuals' behavioral patterns and emotional responses to stress (tsang et al., ) . for example, a medical worker with a good family environment has confidence in the success of the fight against the epidemic and also believes that he is capable of doing his job, which will ease his fear of the epidemic and anxiety about the high-risk work of infection. self-efficacy played a partial mediating role between the family environment and symptoms of anxiety and depression, indicating the existence of other variables between them. future studies should, therefore, include other relevant variables that are likely involved in the relationship between the family environment and negative emotions. this study indicates that adjusting self-efficacy is a meaningful way to regulate the anxiety and depressive symptoms of medical staff during an epidemic. because of the close correlation between the family environment and the symptoms of anxiety and depression of medical staff during an epidemic, we need to pay more attention to psychological assistance for medical staff from the perspective of their family situation. when providing psychological assistance to medical staff during the epidemic, we should not only focus on the medical staff but also care about their family members and family relations. by improving the family environment and increasing the active support of the family, their emotional problems can be effectively alleviated (mohindra et al., ) . the focus of the medical staff 's treatment of family relations should be to enhance the intimacy between family members, increase their interaction, encourage them to talk to each other, resolve the family conflicts in time, and create a good family atmosphere. based on the results of this study, we propose the following suggestions for medical staff. first, we suggest that medical staff should have time to communicate with their families and that they should be encouraged to share their feelings with family members and get their support and encouragement. for example, they should be encouraged to record their routines in the hospital and share them with their families . the hospital or isolation point shall provide relevant communication conditions and equipment for this purpose. second, during the epidemic period, the staff of the relevant departments of the hospital should be aware of the difficulties existing in the family of medical staff, and they should guide these staff members and help them solve those problems to avoid family conflicts. third, the family members of medical staff should be aware of the mental health issues of the staff member. family safety plays the most important role in reducing the pressure of medical staff during the epidemic . therefore, the staff members should stay connected with their families through wechat, sms, and other apps to understand their health status, which will help lessen the negative mental state of the medical staff during the covid- pandemic outbreak. these suggestions can bring medical staff closer to their families, have more emotional communication, and reduce family conflicts. with the implementation of these measures, the medical staff 's sense of self-efficacy will also be improved. besides, our results suggest that improving self-efficacy will help to alleviate the anxiety and depressive symptoms of medical staff during the covid- outbreak. manipulating self-efficacy is an important way to prevent mental health problems when dealing with stress (schönfeld et al., ) . previous studies have focused on the effects of self-efficacy on the mental health and work quality of medical staff (amiri et al., ; tang et al., ) , and it suggested that necessary interventions should be implemented to improve the self-efficacy of medical staff. in the prevention and control of covid- , medical staff is faced with two main difficulties. on the one hand, medical staff has heavy work tasks, great pressure, high risk of infection, and lack of support (spoorthy et al., ) . on the other hand, most of the medical staff are required to be isolated in hospitals or isolation points. their families will face more prominent problems (mohindra et al., ) . some positive motivation factors can boost morale and improve the self-efficacy of medical staff, such as family and social support, positive example, recognition, and appreciation from others, successful experience, self-identity (spoorthy et al., ) . positive feedback and encouragement from others could also effectively improve self-efficacy (bandura, ; zinken et al., ; brown et al., ) . the pre-job training, encouragement from colleagues and family, affirmation from patients and society, and sufficient material support were all helpful ways to improve the self-efficacy of medical staff during the covid- outbreak. medical staff in a good family environment can get better family support. the support reduces the sense of uneasiness caused by isolation, and improve self-efficacy, increase work confidence, improve work efficiency and quality, and reduce the negative emotions caused by epidemic infection. it has been reported that the mental health status of chinese medical staff is poor (zhou et al., ) , and they are exposed to immense workplace pressure and face complex doctor-patient relationships. the reason lies in the contradictions in the current medical system reform in china, such as the uneven distribution of medical resources (lu et al., ) , the disequilibrium between health care needs and medical development (zhou et al., ) , and the imperfection of the medical system (ta et al., ) . during an epidemic period, protecting the mental health of the medical staff would benefit their health as well as the control of the epidemic worldwide (kang et al., ) . the national health commission of china has published a national guideline of psychological crisis intervention for covid- , which is guided for the protection of the mental health of the medical staff (kang et al., ) . however, the family environment is particularly important to the mental health of the medical staff, and self-efficacy plays an important role in regulating the relationship between them. appropriate guidelines should be issued nationally to improve the family environment of the medical staff and for the improvement of their selfefficacy. there are some limitations to the current study that need to be addressed. first, there are limitations to the method of sampling. sampling bias may have occurred by using a convenient sampling method. second, we have a small sample size, and all participants are from beijing, so the research participants in this study may not be sufficiently representative of the population we are interested in studying, which may limit the conclusion of research results. third, online questionnaire surveys cannot observe the participants' answering process, there is the possibility of random answer and perfunctory answer, cannot guarantee the complete authenticity of data. fourth, we did not measure other potential confounding variables that may exist between the family environment and the emotional state of medical staff during the covid- outbreak. finally, the researchers are all medical staff, and the design of the survey may be more based on clinical observation. in the future, the research design can be combined with clinical observation and the existing theoretical framework. in the current study, we found that the anxiety and depressive symptoms of medical staff during the covid- outbreak was closely related to their family environment, and their self-efficacy regulated the relationship between them. this study provides a new direction for the psychological intervention in medical staff during the epidemic that mainly focuses on improving their family environment and their self-efficacy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. nh contributed to the manuscript writing. j-xc, yl, and s-sh contributed to the conception and designed the work. yl, l-lw, y-yw, and ly contributed to the critical revision of the article. all authors contributed to the article and approved the submitted version. study of the relationship between self-efficacy, general health and burnout among iranian health workers survey of stress reactions among health care workers involved with the sars outbreak self-efficacy in changing societies on the functional properties of perceived self-efficacy revisited 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a systematic review and metaanalysis parental factors associated with depression and anxiety in young people: a systematic review and meta-analysis covid- severity, self-efficacy, knowledge, preventive behaviors, and mental health in turkey determinate factors of mental health status in chinese medical staff: a cross-sectional study analysis system for selfefficacy training (asset). assessing treatment fidelity of self-management interventions key: cord- -tvy uo u authors: brock, rebecca l.; laifer, lauren m. title: family science in the context of the covid‐ pandemic: solutions and new directions date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: tvy uo u the coronavirus disease (covid‐ ) pandemic has precipitated substantial global disruption and will continue to pose major challenges. in recognition of the challenges currently faced by family scientists, we share our perspectives about conducting family research in the context of the covid‐ pandemic. there are two primary issues we address in this article. first, we present a range of potential solutions to challenges in research, resulting from the pandemic, and discuss strategies for preserving ongoing research efforts. we discuss approaches to scaling back existing protocols, share ideas for adapting lab‐based measures for online administration (e.g., using video chat platforms), and suggest strategies for addressing missing data and reduced sample size due to lower participation rates and funding restrictions. we also discuss the importance of measuring covid‐ relevant factors to use as controls or explore as moderators of primary hypotheses. second, we discuss how the covid‐ pandemic represents a scientifically important context for understanding how families adjust and adapt to change and adversity. increased stress precipitated by the pandemic, varying from acute stress associated with job loss to more chronic and enduring stress, will undoubtedly take a toll. we discuss ways that family scientists can contribute to pandemic‐related research to promote optimal family functioning and protect the health of family members. the novel coronavirus disease has profoundly impacted society at large. in addition to economic disruptions and significant burden placed on healthcare systems, the covid- pandemic has undermined -and will continue to undermine -the physical and mental health of individuals across the globe. as family scientists, we must navigate unforeseen challenges as we abruptly pivot to salvage ongoing research and make plans for the future of our research programs. moreover, we are uniquely positioned to investigate how to best build resiliency in families amidst large-scale systemic changes that can undermine family functioning. the primary aims of this article were to (a) present a range of potential solutions to problems threatening the rigor of ongoing research and (b) propose new directions in family science aimed at understanding how families adapt to change and adversity arising from the pandemic. one of the most pressing questions facing many family scientists is how to preserve the rigor of ongoing research that has been derailed by the pandemic (e.g., longitudinal studies put on hold, resulting in missed assessments during critical periods of development). there are numerous challenges to address that could require sweeping changes to research designs. in-person data collection might not be advisable when considering participant safety-or could be expressly prohibited at times-which limits the feasibility of certain methods commonly employed in family research (e.g., behavioral observations in controlled laboratory settings, neuropsychological testing, biological measures, in-person interventions or experimental manipulations). additionally, both researchers and participants might be coping with elevated stress and adversity while adapting to altered roles and routines. consequently, research participants might be less engaged in research or unwilling to participate altogether, thereby threatening the reliability and validity of scores and contributing to elevated rates of missing data. researchers may not have as much time or energy to devote to data collection efforts as in the past and may feel overburdened in anticipation of potential budget cuts or limits on extensions to grant funding in the upcoming months and years. another challenge faced by investigators is restricted access to offices, laboratory spaces, and on-site resources. as such, difficult decisions must be made about which aspects of carefully constructed research designs, if any, can be changed to accommodate new constraints and loss of resources. we this article is protected by copyright. all rights reserved now turn to a discussion of possible adaptations to consider for ongoing research, drawing largely from existing tools and approaches that are established in the literature. online data collection has become relatively mainstream, particularly for administering questionnaires. as such, a relatively straightforward adaptation to lab-based research involves asking participants to complete surveys from home rather than in the laboratory. of course, even this is not a simple transition, as it requires institutional review board approval, an appropriate device (e.g., smartphone) and reliable access to internet connection in participants' homes, and clear instructions to minimize confounds (e.g., asking participants to complete the survey in a private location with minimal distractions). however, research has demonstrated psychometric equivalence of paper-and-pencil and internet formats of questionnaires often used in family research (brock et al., (brock et al., , . as such, validity and reliability concerns associated with this shift in approach are minimal. for investigators who routinely use lab-based methods such as behavioral observation paradigms in carefully controlled environments, experimental manipulation, in-person interventions, or methods that require equipment that is not easily transported, new ways of measuring constructs of interest should be considered. of course, these changes will not be feasible in all circumstances. for example, neuroimaging in the home is not an option. but, by stepping back and thinking creatively, there is the potential for considerable innovation in family science. fortunately, many lab-based measures and protocols can be adapted for online administration. telehealth and video conferencing are increasingly used for clinical interventions and are viable options for investigators conducting treatment outcome research (e.g., arnberg et al., ; perle & nierenberg, ) . further, prior to the pandemic, researchers were already effectively transitioning to video chat platforms for administering assessment tools that require interactions with investigators (e.g., bridgers et al., ; sheskin & keil, ) . there are numerous benefits inherent to home-based assessments, including increased ecological validity, more diversity in samples, reduced barriers such as travel and parking costs, and enhanced participation rates. further, in the context of safety concerns related to covid- , participants might experience elevated stress and anxiety when attending in-person appointments, which could introduce significant confounds. further, it is unlikely that participants who are at greater risk of complications from contracting covid- (e.g., those who are immunocompromised or this article is protected by copyright. all rights reserved pregnant) will be receptive to attending lab appointments which could decrease generalizability of findings. as such, the benefits of remote assessments that allow for research participation from the comfort and safety of one's home are not trivial. of course, there are also drawbacks to consider when collecting data remotely and adapting lab-based protocols for use on video platforms. most notably, participants must have reliable internet access and a device that can be used with the required software. there are also extra steps that need to be taken in service of standardization and preserving the internal validity of scores derived from these approaches. for example, observational paradigms of family interactions can be implemented by stipulating where and for how long the interaction will take place, asking if the parent and child can be alone in a room together so that other family members do not interrupt or influence relationship dynamics, and standardizing the props used during interactions as much as possible (e.g., perhaps you mail or deliver a toy in advance of the interaction that the child can then keep as a gift). as is customary with lab-based paradigms, we carefully track and record any potential confounds (e.g., the presence of significant distractors, technology failures, etc.) that emerge and account for these in the data analysis stage; however, this becomes absolutely essential when altering study protocols midstudy. when analyzing data, it will also be important to control for where and when participants complete the study procedures (e.g., in the home post-covid versus in the lab pre-covid) if these changes are made in the context of an ongoing project. plan for higher rates of missing data and smaller sample sizes. careful planning goes into research to ensure (a) adequate power to test hypotheses and (b) maximum retention and participation rates. these considerations are particularly important to family scientists who often employ advanced quantitative techniques that require larger sample sizes. yet, in the context of the covid- pandemic, the reality is that we might not reach our original goals for recruitment and participation. fortunately, numerous scholars have laid valuable groundwork for navigating these challenges. planned missing data designs can help to guide this process (little & rhemtulla, ). an example with particular relevance to family science is the two-method missing design, an approach used when there is a gold standard measure of a construct (e.g., behavioral observations of family interactions) that cannot be administered to all participants due to time, money, resources, or, in the case of covid- , social distancing guidelines. consider whether there is a more feasible or this article is protected by copyright. all rights reserved inexpensive measure to administer (e.g., an online questionnaire) that is intended to assess the same construct. if so, a random subgroup of the sample will complete the more intensive, gold standard measure whereas the entire sample will complete the "inexpensive" measure. the gold standard measure completed by the subset of the sample enhances the validity of scores, and the inclusion of the inexpensive measure allows for a larger sample size and the corresponding benefits (e.g., power, generalizability). this design could be particularly useful if in-person data has already been collected from a subsample of a cohort using an intensive protocol (e.g., behavioral observations of parental responsiveness). if there is an inexpensive measure that was used with that subsample (e.g., a parenting questionnaire), and it is correlated with scores from the more intensive measure, the inexpensive measure can be administered remotely (e.g., online survey) with subsequent participants. in the context of longitudinal research, a wave missing design (little & rhemtulla, ) could help reduce participant burden and provide a solution for salvaging ongoing data collection that has been put on hold or now has insufficient funding. in these designs, some participants are intentionally omitted from certain waves; as such, not all participants are required to complete every time point of data collection. finally, a multiform design (little & rhemtulla, ) involves randomly assigning participants to complete different versions of a survey that contain different combinations of items rather than administering a full battery of questionnaires to everyone. this approach can minimize participant burden while still providing rigorous assessments of your study constructs by using otherwise lengthy questionnaires with strong psychometric properties. finally, in anticipation of reduced sample sizes, we must carefully consider which of our hypotheses will still be sufficiently powered (for a brief guide to power and a list of resources, see murray et al., ) . are there empirical questions we can adequately address with a smaller n? if not, are there analytic approaches that well-suited for smaller samples to which we can turn? for example, although bayesian methods are not currently mainstream in family science, they are effective in addressing small sample problems. specifically, bayesian methods may be better suited to producing accurate parameter estimates in smaller samples relative to more traditional frequentist methods. for a detailed discussion of considerations when implementing a bayesian analytic framework with small samples, please refer to mcneish ( ). this article is protected by copyright. all rights reserved recommendation for ongoing research is to routinely assess the numerous ways that families are impacted by the pandemic. these measures will provide important contextual information for including as controls in hypothesis testing and for determining whether study effects are altered (i.e., moderated) by features of the pandemic. for example, our lab has been conducting a large-scale longitudinal study of families who completed numerous waves of data collection prior to a national emergency being declared in the united states (where the research is being conducted) as a result of covid- . one of the primary goals of the study is to examine trajectories of change in family processes and the mental health of family members over several years, and identify factors predicting adaptive versus maladaptive trajectories. yet, the global nature of the pandemic-and corresponding stress and expected changes to family functioning-could greatly alter the nature of those trajectories if we include new data collected in the context of covid- . as such, we are asking parents to complete home surveys to assess the various ways they have been impacted as a family to measure potential confounds. we acknowledge that, in some ways, this recommendation contradicts our earlier suggestion to scale back protocols; however, accounting for potential confounds that could alter your results is so essential that making concessions in other elements of your study to accommodate these measures might be advisable. further, these covid- specific assessments do not necessarily need to be time-consuming or expensive. for example, the epidemic-pandemic impacts inventory (epii; grasso et al., ) is a relatively comprehensive measure of pandemic impacts that only takes - minutes to complete and assesses multiple domains of functioning (e.g., work and employment, education and training, home life, social activities, economic, emotional health and well-being, physical health problems, physical distancing and quarantine, infection history, positive change). if administering a questionnaire is not feasible, even a brief phone conversation with research participants about how they have been impacted by the pandemic could subsequently be used for a thematic analysis (braun & clarke, ) , and quantitative codes could be assigned to reflect degree of impact and adversity. we have presented several potential solutions for adapting to the current circumstances to preserve the quality of ongoing research. however, there might ultimately be certain research questions that are not well-suited to the current circumstances and, as such, it might be better this article is protected by copyright. all rights reserved to postpone data collection until life returns to a state of relative normalcy (e.g., routine access to facilities and resources is restored; social distancing guidelines are relaxed and research personnel and participants feel safe attending in-person appointments). if it is not feasible to collect data remotely, and you are navigating pauses in ongoing data collection, consider staying in touch with participants while things are on hold to keep them invested. something we have implemented in our research is maintaining a study website to keep participants in our longitudinal projects engaged, which has helped to minimize attrition. we share results as they are published, provide resources, and post answers to common questions. in the wake of the covid- pandemic, we adapted our website to include status updates on the study and a list of resources for helping families talk to their children about covid- and manage stress. finally, there might be ways to adapt and evolve a research program in new directions, pursuing empirical questions that had not been previously considered. indeed, we now turn to a discussion of how the covid- pandemic represents a scientifically meaningful context for studying families. covid- represents not only a global public health emergency, but also the beginning of a major mental health crisis (united nations, ). exposure to adversity and stress is a robust predictor of mental health difficulties across the lifespan (e.g., benjet et al., ; juster et al., ; mclaughlin, ) , and research examining causal pathways of risk and modifiable factors that interrupt those pathways (e.g., high quality support, access to resources) is vital to inform prevention and early intervention efforts. experimental manipulation is touted as the gold standard approach for examining causality; however, as family scientists, we abide by ethical standards that prohibit us from subjecting individuals to extreme levels of stress and adversity. in the absence of experimental manipulation, disaster-based research offers an alternate approach for understanding the impact of stress on the family system. because they emerge suddenly and are outside of human control, natural disasters approximate the randomization of a true experiment, with stress quasi-randomly "assigned" to individuals (king et al., ) . further, norris ( ) states that "disasters generate an array of individually and collectively experienced stressors of varying degrees of intensity that interact with accepted article multiple characteristics of the person and environment to produce diverse outcomes that evolve over time" (p. ). thus, the covid- pandemic represents a scientifically important context for elucidating temporal relations between stress and family functioning. before we explore potential avenues for research, it is important to acknowledge the factors that make the pandemic a unique context for studying the effects of stress and adversity. what is unique and significant about the covid- pandemic? relative to more isolated natural disasters (e.g., floods, tornadoes), the covid- pandemic will affect individuals across the world. as of july , , covid- had impacted nearly every country or region ( of ), with over million confirmed cases and half a million deaths worldwide (johns hopkins university, ; united nations, ). further, the pandemic has triggered a collective experience of acute stress and psychological distress. social distancing measures, albeit varied in scope, have been implemented in countries and territories to help prevent the spread of covid- (international energy agency, ). though certainly effective in minimizing the spread of the virus, these measures have also resulted in significant occupational, educational, and personal disruptions that warrant further attention. in particular, the covid- pandemic has prompted acute, unprecedented job loss and disruption. as of april , the international labour organization, a specialized agency of the united nations, reported that full or partial lockdown measures impacted % of the global workforce (international labour organization, ) . unemployment rates in the united states rose sharply to nearly % in april, reflecting the economic impact of the pandemic and efforts to contain it (u.s. bureau of labor statistics, ). lower socioeconomic status (ses) is associated with an increased risk for mental health problems across the lifespan (reiss, ; santiago et al., ) . in particular, childhood ses predicts long-term physical and mental health outcomes (cohen et al., ; conroy et al., ) . ses is also a robust predictor of relationship quality and stability, parenting, and child development (see conger et al., for a review). thus, as more and more individuals worldwide experience job disruption and financial instability, we should expect the economic challenges spurred by the covid- pandemic to exert a toll on both individual and family functioning (e.g., gilman et al., ; kingston, ) . further, certain populations might be at particular risk for adversity stemming from covid- . for example, black americans not only suffer from higher rates of this article is protected by copyright. all rights reserved unemployment, but they are also more likely to work in front-line jobs deemed essential (gould & wilson, ) . covid- has also disproportionately impacted black americans, and race is associated with mortality rates (ferdinand & nasser, ) . as such, underrepresented and marginalized populations warrant particular attention amidst the pandemic. beyond the acute stress and uncertainty associated with job disruption, the covid- pandemic has the potential to contribute to chronic stress burden (e.g., baum et al., ; lantz et al., ) . there are enduring sources of stress related to covid- that will vary across individuals and families, including fear of becoming infected or infecting others; inadequate supplies to meet basic needs; insufficient information from health and government officials; and the potential for stigma (brooks et al., ) . a robust body of literature demonstrates that stress and adversity impact family functioning (e.g., masarik & conger, ; randall & bodenmann, ; story & bradbury, ) . indeed, stress resulting from external (e.g., occupational) demands can spill over into the family system and undermine individual mental health, relationship satisfaction, and parenting behaviors (e.g., bakker & demerouti, ; bass et al., ; bolger et al., ; brock & lawrence, ) . for couples, stress experienced by one person may cross over to impact their partner. increased stress might also result in one person demonstrating increased family involvement to compensate for their partner's decreased involvement (nelson et al., ) . amidst the covid- pandemic, parents may be at increased risk for role strain as they adapt to changing -and potentially conflictingoccupational and family demands. for instance, some families may be navigating job loss and economic adversity, while others might have job security but are adjusting to new roles and expectations, such as working from home while providing childcare or homeschooling. for some families in dual parenting households, one parent may be leaving the home to work, potentially in a high-risk environment (e.g., hospitals, pharmacies), while the other parent navigates responsibilities at home. thus, the pandemic will generate high levels of between-subject variability in objective and subjective stress, as all families will experience adversity but to different degrees and in diverse ways. further, social distancing measures enacted to minimize the spread of the virus may interfere with important social connections that help us to regulate and cope with our emotions (van bavel et al., ; williams et al., ) . social distancing can also contribute to feelings of isolation and frustration (brooks et al., ) . in addition to the stress associated with major transitions and role this article is protected by copyright. all rights reserved changes, many families have been forced into proximity with one another for an extended period of time (van bavel et al., ) . in combination with elevated levels of stress, forced proximity may be detrimental to family functioning. indeed, research demonstrates that stress experienced by one partner often places a heavy burden on caregiving partners, who report poor marital adjustment and increased subjective distress (dekel et al., ) . the covid- pandemic may prevent family members from accessing external sources of social support that help mitigate distress (e.g., ergh et al., ; rodakowski et al., ) . in addition, partner support, which buffers the association between stress and mental health trajectories (brock et al., ) , may be compromised if both partners are experiencing high levels of subjective stress. conversely, research demonstrates that partners who boast strong support skills may experience greater relationship satisfaction, particularly during times of increased stress (brock & lawrence, ) . further, forced proximity may be beneficial for some families by increasing time spent together to establish family rituals that were not previously feasible due to competing demands. as such, it is important to identify the factors that contribute to adaptive versus maladaptive outcomes in the context of pandemic stress (e.g., communication, support processes, intrapersonal coping resources, division of labor, conflict management skills). further, the consequences of forced proximity of family members -and reduced connection with important social contacts outside of the household (e.g., grandparents, extended family members, friends, coworkers)represents a novel area of research worth closer attention. this might be of particular relevance among families with adolescent children given that adolescence is characterized by an increased need for autonomy and independence and, in turn, the potential for increased parent-child conflict (mcelhaney et al., ; steinberg, ) . finally, with no clear end in sight, the chronic nature of the covid- pandemic is particularly notable. indeed, longer durations of confinement are associated with poorer mental health, particularly trauma-related symptoms (brooks et al., ) . currently, there is a tremendous amount of ambiguity about the future, including which businesses will survive, future job prospects (e.g., for recent graduates), and how much the way we work and live will be altered for the long-term. this chronic uncertainty poses a significant risk to mental health and the family system more broadly (afifi et al., ) . finally, it has become apparent that there are diverse perspectives about the pandemic, the threat associated with the covid- disease, and what constitutes appropriate and this article is protected by copyright. all rights reserved reasonable safety measures. disagreements about the best way to proceed as a family could increase conflict, put strain on collective decision-making efforts, and undermine vital coping resources originating within the family (e.g., high quality support, a sense of belonging and shared values). though covid- is a biological disease by nature, mental health research has emerged as an immediate priority (holmes et al., ) , as evidenced by numerous calls for research by various funding agencies. in fact, the national institutes of health has devoted existing resources and emergency funding to support administrative supplements and competitive revisions that would allow active grants to investigate urgent research questions related to covid- , such as how remotely delivered stress management strategies impact mental and physical health outcomes, resilience to covid- infection, or symptom severity (not-at- - ). as family scientists, we are uniquely poised to contribute to pandemic-related research. drawing on past research and theory, we can investigate the specific impact of the stress and adversity arising from covid- on individual mental health and family functioning and identify modifiable risk and resiliency factors to target in interventions. leveraging ongoing longitudinal data collection will be vital to determine causal mechanisms associated with poor mental health outcomes (e.g., depression, anxiety) and family dysfunction (e.g., couple conflict, child neglect, decreased responsiveness during parenting). indeed, research utilizing existing cohorts, among whom mental health and family functioning have been previously assessed, offers a unique opportunity to examine the longitudinal impact of covid- and to identify a range of risk and protective factors that can be harnessed to promote family functioning. further, integrating quantitative and qualitative data, often staples in family science, allows researchers to explore the specific family processes and relationships that contribute to adaptive and maladaptive outcomes amidst the pandemic. in the absence of ongoing longitudinal studies, the covid- pandemic presents another avenue for family scientists to pursue new research on the impact of stress and adversity on family functioning. for example, prospective studies can be designed to study the immediate and long-term effects of exposure to varying levels of stress from the pandemic on family functioning, especially for high-risk populations. in sum, we have identified several key themes at the intersection of family science and the covid- pandemic. building off of these themes, there are a range of unique questions to consider this article is protected by copyright. all rights reserved in pursuit of a more nuanced understanding of family processes. some of these questions include, but are not limited to: how do stress and adversity impact couples in different relationship stages? are dating couples impacted in unique ways relative to more established, long-term couples? how has forced proximity and lack of access to social contacts outside of the family impacted sibling relationships? how are divorced and remarried family systems navigating custody agreements and shared responsibilities in the context of social distancing and travel restrictions? how are families impacted when a family member tests positive for covid- ? what if certain family members are at higher risk for complications resulting from covid- ? how are individuals working in essential jobs maintaining family relationships? what impact has this had on parenting? how has covid- uniquely affected pregnant women and their partners? how might this contribute to the intergenerational transmission of stress and psychopathology? in this article, we have presented (a) a series of potential solutions to problems arising when conducting ongoing research during the covid- pandemic and (b) ideas for new directions in research that explicitly address issues related to the experience of the pandemic for families. it is important to recognize that the time, energy, and resources available to devote to research will vary across investigators, and perhaps by region, as some areas of the world have been impacted by the pandemic more than others. thus, our intention was to present a series of potential solutions and ideas ranging from more intensive, time-consuming efforts to relatively small, but meaningful, steps that can be taken in family science in response to the pandemic. we also acknowledge that this is by no accepted article means an exhaustive list of strategies or approaches. we have shared some of the key considerations made in our own research, along with ideas shared by colleagues who are navigating similar challenges. we look forward to learning more about the new and innovative ways that family scientists respond to this crisis and move the field forward. the impact of uncertainty and communal coping on mental health following natural disasters internet-delivered psychological treatments for mood and anxiety disorders: a systematic review of their efficacy, safety, and cost-effectiveness the spillover-crossover model new frontiers in work and family research do job 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brief: covid- and the need for action on mental health using social and behavioural science to support covid- pandemic response key: cord- -ll xmk i authors: jenkins, louis s.; von pressentin, klaus b.; naidoo, kartik; schaefer, rachel title: the evolving role of family physicians during the coronavirus disease crisis: an appreciative reflection date: - - journal: afr j prim health care fam med doi: . /phcfm.v i . sha: doc_id: cord_uid: ll xmk i ten family physicians and family medicine registrars in a south african semi-rural training complex reflected on the coronavirus disease (covid- ) crisis during their quarterly training complex meeting. the crisis has become the disruptor that is placing pressure on the traditional roles of the family physician. the importance of preventative and promotive care in a community-oriented approach, being a capacity builder and leading the health team as a consultant have assumed new meanings. ten family physicians and family medicine registrars are in their first quarterly training complex meeting for . the setting is the garden route district in south africa, a semi-rural area with a population of people, of which % are dependent on six government hospitals for their health needs. the mood in the room is circumspect. many questions hang in cyberspace. we can all see and hear each other, but there is a difference. for the first time since we are meeting via videoconferencing, all sitting in front of computers, spaced out over km. since our last physical meeting in , the world has changed. an invisible enemy, the novel coronavirus, has infected more than . million people globally, with a case fatality rate of . %. the world is locked down; most businesses are closed; and people are keeping a social distance from each other, isolating themselves, washing hands for s, not touching their faces, wearing masks and avoiding hospitals. the elderly of the society and those with chronic diseases are at the highest risk of dying. five weeks of lockdown with almost no cars on the roads and no selling of alcohol have dramatically reduced mortality and morbidity from road accidents, murders and assaults. all routine outpatient clinics, elective surgery, outreach support and continuous medical education meetings have stopped. hospitals and clinics are preparing for the coronavirus disease (covid- ) crises, expecting to be overwhelmed. traditional roles have changed; for example, orthopaedic medical officers and ophthalmology specialists are screening people for acute respiratory symptoms, some joining nurses and community health workers. clinical rotations have changed; for example, ' month in theatre' has become ' week in emergency centre, followed by week in theatre'. student assignments and examinations have been deferred or cancelled. there is a complete relook at the way we teach and learn. the healthcare system is being redesigned. while 'disruption' is a negative term, it creates an environment for innovation, which is a positive term, well-described in technological industries. a disruption can be defined as an event in which a substantial share of agents belonging to a system is disrupted, typically requiring new skills and creating a pressure to change the value generation models of an organisation. the questions in the 'room' reflected a cautious concern for an uncertain future: • how will this crisis affect the registrar training programme, including my learning plans? ten family physicians and family medicine registrars in a south african semi-rural training complex reflected on the coronavirus disease (covid- ) crisis during their quarterly training complex meeting. the crisis has become the disruptor that is placing pressure on the traditional roles of the family physician. the importance of preventative and promotive care in a community-oriented approach, being a capacity builder and leading the health team as a consultant have assumed new meanings. the roles of the family physician in south africa and the contribution to district health services have been well described. , apart from clinical competence, the family physician needs to be a consultant, capacity-builder, clinical governance leader, champion of community-oriented primary care (copc) and clinical trainer. , researchers in a recent harvard business review highlighted four traps that may prevent leaders in a crisis from balancing the management of the present with leading beyond the crisis. family physicians, having a diverse and adaptive skill set, are regarded as expert generalists. our roles make us leaders in our clinical and community contexts and, therefore, vulnerable to falling into these traps (see figure ). for example, over-centralising the response, in an attempt to control the crisis, can reduce capacity-building, missing an ideal opportunity to build capacity in others. in this example, our challenge is not to over-centralise, but remain 'centred' to our purpose and leadership role as family physicians. the solution is to adopt the cognitive aids depicted in figure (lower half). despite the roles of the family physician being broad, inclusive and adaptive, the covid- crisis imposes challenges to exercise these roles. there are restrictions on movement, social distancing, de-escalation of elective procedures and minimisation of patient-clinician contact. time for regular activities is reduced as we are called upon to assist in many contexts and capacities, from training copc, community-oriented primary care. cleaners to advising joint operations committees (joc). we therefore need to use every opportunity to exercise our roles. figure (left side) presents the 'traditional' model of how a family physician might exercise his or her roles during the covid- crisis. in contrast, the right side presents an example of an activity that the family physician is called upon to assist with, in this case, optimising personal protective equipment (ppe) use. this shows how the various 'roles' can be exercised from this one activity. the idea is to facilitate family physicians in exercising roles through activity rather than through ourselves. this enables agility and responsiveness during the crisis but still enables the expert generalist to use this crisis as an opportunity to strengthen the health system. coronavirus disease has made us realise how important understanding human behaviour is, something we still have not figured out with the tuberculosis epidemic. it has created a pressure to change our focus from routine activities to thinking critically about real health needs of individuals in communities, as well as staff health and safety. we need to innovate to prevent infection, promote health (including mental health), work closer with many more sectors of society, including social services, community volunteers, business sector, manage resources better and cooperate with the private health sector. a 'new normal' is setting in. a shift is happening from reactive curative care towards a more promotive, preventative community-based approach. perhaps this is the time to shift from just measuring the leading causes of death to also include the leading causes of life. health workers as well as people in communities need a sense of agency (power to do), to have hope (believe in solutions), to connect more (with self, others and environment) and to appreciate intergenerativity (realising that everyone is part of a continuum of those that went before and those that are coming after). family physicians, with their skills set of clinical competence across all clinical disciplines, leadership skills of communication and collaboration within teams and viewing patients as part of families in communities, are ideally placed to respond appropriately to this crisis. the roles of the family physician have been disrupted by an invisible enemy. preventative care in a patient-centred, community-oriented approach is of critical importance, now more than ever. the governance of resources, such as masks, gloves and medicines, has global implications. the isolation, uncertainty and fears of patients and colleagues necessitate diligent self-care, mental healthcare and a renewed focus on caring for each other. the skills to reflect, communicate and share within our teams are being sharpened like never before. covid- updated situation reports trauma centre's caseload down % as driving reduces a disruption framework the contribution of family physicians to district health services: a national position paper for south africa: forum strengthening the district health system through family physicians. s afr health rev [serial online] are you leading through the crisis … or managing the response? world health organization statement -transition to a 'new normal' during the covid- pandemic must be guided by public health principles the leading causes of life: an integrative idea the authors would like to thank the whole team of family medicine, including drs elmari de klerk-green, paul kapp, covid, coronavirus disease; icu, intensive care unit; fam med, family medicine; opd, outpatients department; ppe, personal protective equipment; ipc, infection prevention and control; joc, joint operations committee; copc, community-oriented primary care. this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data sharing is not applicable to this article as no new data were created or analysed in this study. the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. herman kruger, hayden poulter, kobus venter and milton groenewald, for their support. they would also like to thank the clinical and medical managers, including drs andries brink, jaco botma, charles dreyer, paul rüschenbaum and zilla north, for their support. the authors have declared that no competing interests exist. l.s.j. conceptualised the study. k.b.v.p., r.s. and k.n. contributed towards subsequent drafts. all authors contributed to and approved the final version of the manuscript. this article followed all ethical standards for a research without direct contact with human or animal subjects. key: cord- - i jjys authors: myers, jeffrey l; mulder, lotte title: frontline workers in the backrooms of covid- : caring for the living and the dead date: - - journal: am j clin pathol doi: . /ajcp/aqaa sha: doc_id: cord_uid: i jjys objectives: to review the response to the coronavirus disease (covid- ) pandemic in a forensics center that integrates an academic department of pathology with multiple regional county medical examiners’ offices. methods: faculty and staff were asked to volunteer stories, data, and photographs describing their activities from march through may . the information was assembled into a narrative summary. results: increased deaths challenged capacity limits in a hospital morgue and a large urban medical examiner’s office (meo) successfully managed by forensic teams and monitored by an institutional command center. autopsies of suspected and proven cases of covid- were performed in both facilities. severe acute respiratory syndrome coronavirus (sars-cov- ) testing of decedents was performed in a meo serving a large urban area. scene investigators worked directly with families to meet needs unique to a pandemic. artful photographs of decedent’s hands and/or tattoos were offered to those unable to have in-person viewings. pathologists and social workers were available to families of the deceased and created novel solutions to facilitate the grieving process. conclusions: forensic pathology is important to successfully navigating emerging diseases like the covid- pandemic. direct conversations with families are common in forensic pathology and serve as a model for patient- and family-centered care. in the midst of a pandemic unprecedented in modern times, the story of forensic pathology-its pivotal role in our collective response, including direct interactions with the families of those who perished-has rarely been told. when we think of forensic pathology, we often think of crime shows and pathologists determining the cause of death. what those outside the field may miss is that forensic pathologists are strong patient advocates, especially with the families of the deceased with whom they have impactful conversations that help them understand and more effectively process their loss. the first case of coronavirus disease (covid- ), the illness caused by severe acute respiratory syndrome coronavirus (sars-cov- ), was reported in the united states in late january . in the nearly months that have passed at the time of this writing, the united states has seen . million confirmed cases and over , deaths, , of them in michigan. we've seen pictures and read stories of health care workers, such as first responders, nurses, emergency department and critical care physicians, respiratory therapists, and others, who are justly celebrated as medicine's frontline in our war with a novel coronavirus. laboratories have also been • investment in a cross-functional, interdisciplinary forensics center that combines an academic medical center and regional medical examiners' office is important for navigating a pandemic. • forensic pathology is an essential public health service for recognizing emerging diseases, defining novel diseases, and providing objective data to inform responses to current and future pandemics. • through interdisciplinary support of families, including direct communications with pathologists and laboratory scientists, forensic pathology is a model for patient-and family-centered care. in the news, their importance suddenly brought to the forefront. they were celebrated as essential for protecting public health while simultaneously vilified for being slow to respond to the demand for the diagnostic testing that underpins medical decision making, and the serologic testing considered essential by many as we transition to recovery. pathologists and laboratory professionals understood the challenges and supply chain barriers but their explanations could not be heard amidst the maelstrom of public perception and the political turbulence swirling in the wake of divided national, state, and local leaders. through all of this, the forensics teams-pathologists, investigators, laboratory professionals, photographers, social workers, autopsy and pathologist assistants, and administrative staff-have been at the frontline of pathology's largest covid- front, contributing to the public welfare and the science of sars-cov- while caring for the dead and the living with a measure of compassion and kindness that few understand or know. our purpose is to tell a piece of that story as it occurred in southeast michigan. in a unique forensics center was created at michigan medicine to integrate a hospital autopsy service with a federation of regional county medical examiners' offices. facilities include the morgue at university hospital in ann arbor, which functions as the medical examiner's office for washtenaw and livingston counties, and the wayne county medical examiner's office (wcmeo) in detroit, which also functions as the medical examiner office for monroe county. all participating staff and faculty are employees of michigan medicine and all faculty are appointed in the university of michigan medical school ❚image ❚ and ❚image ❚. as government-funded offices they serve the public health needs of their jurisdictions. as a critical section in an academic department they fully participate in all missions and have access to the infrastructure of a large academic medical center and a public research university. during the covid- pandemic, the forensic pathology program at the university of michigan proved critical to supporting the counties they serve and families of the covid- patients and others who died at home and in hospitals. the pandemic had a profound impact on hospital-based morgues and medical examiner offices in hot spots across the country. some hospitals, including facilities that had retreated from providing autopsy services, struggled to house, manage, and release the decedents entrusted to their care. as a consequence, bodies were sometimes warehoused in makeshift morgues in institutions that were unable to provide the security, cooling, and privacy expected by those who loved and cared for them in life. in michigan, the april caseload at the wcmeo was % higher than april (carl schmidt, personal communication); in washtenaw county cases increased by over % from april to april (allecia wilson, personal communication) compared to the average caseload for the same period of time in the preceding years. morgue census and the rate at which bodies were being released at each of michigan medicine's facilities became a standing metric for the command center overseeing covid- -related operations. despite substantial increases in the number of bodies being managed in michigan's facilities, all decedents were treated with respect and dignity by experienced and dedicated autopsy personnel whose volunteerism mirrored the dedication and commitment of other frontline health care workers. forensic pathology plays an essential role in public health as a first-line defense in recognizing emerging disease, defining the pathology and pathophysiology of novel diseases, and providing objective data that inform current and future responses. this goes beyond simply managing morgue inventory and includes autopsies as unique learning opportunities for a disease that was unknown to us until it arrived early in . dr stephen geller, emeritus chair of pathology at cedars-sinai medical center, commented in a covid- autopsy listserv on march , , that "the pathobiology of covid- and its effects in the body above and beyond pulmonary system involvement remains to be understood. every autopsy performed expands the opportunity to learn more." michigan medicine's director of autopsy and forensic services and chief medical examiner for washtenaw and livingston counties, dr allecia wilson, stated, "this embodies the essence of why we perform autopsies and why we are called in on issues that pose a public health threat. it's the one thing we do better than anyone else." michigan medicine's autopsy and forensic services developed procedures and protocols that ensured use of ppe appropriate to the threat level ❚image ❚, and implemented other measures, such as limiting the number of people in attendance for covid- or suspected covid- autopsies, to mitigate the risk to pathologists and staff ❚image ❚. autopsy cases from michigan and elsewhere are yielding insights and observations that are expanding our understanding of the pathology and pathophysiology of covid- , including the features that it holds in common with other respiratory viruses and the unique findings that set it apart. largescale testing of nonhospitalized decedents underway at wcmeo ❚image ❚ is likely to yield information about the prevalence of this virus in populations who die of other causes, information that may prove critical to better understanding the potential value of large-scale testing in communities and organizations as we anticipate a return to whatever becomes our new normal. forensics programs are teaching us about the complex impact of pandemics on our communities. much of the global focus is on the direct effects of sars-cov- infection, but the increased volume in large urban offices like wcmeo reflects the increase in potentially preventable deaths among those with urgent medical needs that are unrelated to covid- . anecdotal estimates are that medical examiner offices have seen a % increase in non-covid- related deaths, an increase driven primarily by adults dying at home (leigh hlavaty, personal communication). this reflects the experience reported in the united states and elsewhere that patients with medical emergencies are avoiding emergency departments and hospitals. , in california's san joaquin county a drop in visits to the emergency department was counterbalanced by a % increase in cardiac arrests reported in the field by emergency medical services in march . all of these patients tested negative for covid- , and most were declared dead at the scene. individuals in michigan medicine's county forensic services who died of conditions other than sars-cov- ❚image ❚ a large team at the wayne county medical examiner's office in detroit is responsible for autopsies each day and comprises a combination of michigan medicine staff (including pathologists' assistants), trainees, and a faculty member who functions as officer of the day. other faculty participate as demand requires. the team is pictured here in may . ❚image ❚ photograph of autopsy teams at the wayne county medical examiner's office in detroit working on cases with powered air-purifying respirators in may . infection are a heterogeneous group with chronic and acute conditions for which care was either not sought or deferred out of fear that the risk of covid- was worse. many were older adults with chronic conditions such as cardiovascular disease. others suffered from acute conditions for which mortality rates are generally low in populations with access to modern medical care. some likely knew the gravity of their circumstances but did not seek care for fear of becoming infected. a few reached out to providers overburdened with covid- related demands who either could not respond or triaged their care based on pandemic-driven paradigms. for example, a young mother suffering abdominal pain after a normal vaginal delivery was advised to first try home remedies given the risk of infection in acute care settings. she died from sepsis. in another case a young man was advised to manage his nausea and abdominal pain at home given what seemed a greater risk of covid- infection were he to seek the care that might be standard in more normal times. he died following rupture of his appendix. a young inmate died in custody following a ruptured appendix after refusing care for abdominal pain for several days. according to a fellow inmate, she did not want to learn that she had covid- only to be placed in isolation. dr milad webb, assistant medical examiner at wayne county and assistant professor of pathology, wrote of the experience: "in , its unthinkable that young people would be dying of such manageable conditions. but that is what the fear of covid has done." those with greater expertise in epidemiology and public health may rightly indicate that these are the inevitable costs to avoid far greater loss of life from curves not flattened and hospitals overwhelmed by a surge in viral infections. and yet as forensics teams across the country collect the data, it is impossible to shake the suspicion that these costs are disproportionately borne by those with less. this is consistent with evidence published by others that the pandemic disproportionally impacts minority groups, including african americans, asians, and native americans. [ ] [ ] [ ] race and income were the main factors that determined fatality rates for covid- in those who perished in new york. forensic experiences like michigan's may better inform battle plans that redirect a portion of the firepower brought to the covid- war to the unrelated skirmishes and ambushes that many were left to fight on their own. from a more holistic point of view, covid- may prove a trigger for redesigning a health care system that seeks to flatten all curves, including the disparities that separate so many of our citizens from a level of access and opportunity to which others have grown accustomed. scene investigators from michigan's forensics teams were often the first to enter the homes of those who died outside of an acute or chronic care facility. the families ❚image ❚ nasopharyngeal swabs were routinely collected for sars-cov- testing on a large number of decedents at wayne county medical examiner's office including external inspections and autopsy cases. ❚image ❚ photograph of dr allecia wilson (left) and autopsy assistant, monique micallef, preparing to do a covid- autopsy at michigan medicine's university hospital in a negative pressure isolation room designed for this purpose. only individuals participated in each covid- autopsy, with no access allowed to others during the case. of those who died in the course of the pandemic, whether from covid- or unrelated illnesses, struggled with a complex emotional landscape that compounded the grief sparked by catastrophic loss. the families commonly expressed frustration, anger, guilt, regret, and feelings of abandonment by a system that prioritized public health and covid- care above their own needs. families were grateful that their loved ones were treated with dignity and respect by michigan's forensics teams, especially on those occasions when other responders refused to enter their homes. for decedents received at wcmeo for whom there was no specific indication for autopsy (eg, suspected homicide), external exams, nasopharyngeal swabs for covid- testing (image ), and toxicology were performed regardless of whether infection with sars-cov- was suspected. all decedents were treated the same. when families inquired about autopsies they were pleased to learn that their loved one had been examined with care and dignity, and that the examination included covid- testing. families repeatedly expressed gratitude for getting the testing that they could not get in life, and for turnaround times that rivaled those being advertised in acute care settings. when they called the michigan's medical examiner's offices, they got to speak with a physician who took the time to answer their questions, often already armed with the test results in which they were interested. michigan's faculty recount multiple conversations with family members who felt isolated from organized medicine until finally getting through to someone who took the time to listen and to respond, even if that occurred only after the loss of their loved one. and that someone was a pathologist. friends and families of those who died in hospitals and chronic care facilities with strict visitation policies wrestled with other frustrations and regrets at not being present for one of life's most heart-wrenching moments. the autopsy and forensics service at michigan medicine is tightly integrated with social work programs including the office of decedent affairs (oda), which is staffed by program managers for adult and medical examiner services and for children's and women's bereavement. early on, oda recognized how the pandemic and the response were impacting families experiencing deaths in the hospital and in the community. providers already overwhelmed with covid- care found themselves doing unfamiliar work addressing end-of-life needs of patients and families at an unprecedented scale. as a consequence, quality for postmortem paperwork suffered and families experienced additional distress as body disposition was delayed, making a tragic time more challenging. incorrect autopsy-related documentation predates the pandemic but the impact of these operational gaps was magnified by a surge in hospital deaths. in response, oda staff created several novel communication vehicles that were intended to speak directly to the needs identified by families and providers. key information from the postmortem policy was summarized in easy to read -to -page handouts. a single page summary of best practices for end-oflife and after-death care was crafted using a standardized situation, background, assessment, recommendation (sbar) format and circulated to michigan's covid- command center. these proactive measures improved process with the hope of mitigating some small piece of the complex grieving process that surrounds loss of human life. in-person viewings for hospital and medical examiner families at the ann arbor campus were suspended during the pandemic, which proved to be one of the greatest covid- challenges imposed on our autopsy and forensic service. viewings are typically supported by oda and are especially important as the last opportunity to spend time with the deceased for families pursuing direct cremation. families with limited means are overrepresented in this group. many of these families expressed pain and guilt for not being able to say a final goodbye to their loved one when they could not afford to have the body embalmed. grief therapists contend that often the first step to processing a loss is accepting the reality of the death. viewings are a therapeutic intervention that promotes dealing with the reality. this can be especially effective when professionals are trained to clinically bear witness to another's pain while offering empathic support, psychological first aide, and important information for planning next steps. as part of our forensics teams, our social workers and oda program managers are trained to sit with a person's pain while not talking them out of their feelings and experience. our autopsy and forensics team quickly substituted photographs for in-person viewings to comfort families grieving the loss of a loved one. prior to the pandemic, michigan's autopsy and forensics supervisor and biomedical photographer, lisa neal, understood the power of tasteful, professional photography in preserving a memory for those who are left behind. she initiated this service through collaboration with oda and the medical examiner's office, and expanded the program during the pandemic when in-person viewings were suspended. families identified by oda and/or the medical examiner's office were given the opportunity to receive a subtly artful photograph of their loved one's hands or tattoo ❚image ❚. high-resolution photographs of fingerprints for memorial jewelry making and hand or footprint impressions were also available upon request. it is hard to measure the impact of this simple measure. one grieving father replying to an email sent in response to his request for photographs said, "having these helps [me] considerably." oda developed a bereavement outreach program to add a layer of support for families impacted by hospital visiting restrictions, suspension of viewings, social distancing rules, and executive orders that limited funeral options. the goals were to expedite arrangements for disposition of decedents and to provide support to families who have experienced the death of a loved one. the program consists of telephone calls to grieving families made by clinical social workers from throughout the health system who volunteered their time. the first call is made within days of the death and is focused on acknowledging feelings, providing concrete information, encouraging expedited funeral arrangements, and helping to identify support systems. the second call is made to weeks after the death and is focused on providing empathic support, assessing coping, and providing referrals for additional community grief supports. if oda staff is unable to reach the family by phone, a letter is sent to provide local grief support resources and connect them to the oda. redesigning health care systems should involve structures to support and facilitate conversations between patients, their families, and pathologists. all patients deserve dignity and respect, whether alive or deceased; when a patient dies that respect is due their families who serve as the custodians of their wishes and shepherds of their dignity. direct conversations with pathologists empower patients and their families; it fosters a deeper sense of empathy and allows families to start processing their grief. , when processing loss, hearing about a loved one's death from the pathologist who determined the cause by examining them with compassion and dignity provides the framework for families to begin acceptance. conversations with members of a dedicated and well-trained forensics team allows the family to experience the mutual respect fundamental to patient-and family-centered care. pathologists and laboratory professionals have additional opportunities to expand their advocacy for patients and families as covid- continues to percolate across our country. diagnostic and serologic testing for sars-cov- has driven an unprecedented level of interest among not only patients and their families but also providers, public health experts, government officials, business leaders, universities, and health care executives regarding laboratory testing strategies for disease managing and safeguarding public health. providing patients and families direct explanations of specific laboratory test results and why certain tests were performed over others is especially important given the complexities of covid testing including the differences between nucleic acid testing for diagnosis and serologic testing for other purposes. in much the same way, when a patient dies-covid related or not-findings and test results can be explained in ways important for more fully understanding the cause of death and the relative contribution of various factors in individuals with complex medical problems. such information can be crucial in helping family members move forward. the most important aspect of communication in the forensics context is empathy, which includes creating and holding the space for people to process their many emotions that accompany loss. forensic pathology is an eloquent example of the bridging of science and emotional intelligence. it is with awareness of others and experience with responding to and managing their emotions that the skills and responsibilities unique to a cross-functional interdisciplinary forensic program empowers and provides support for families. the covid- pandemic proved a pivotal moment for those who often work in the backrooms of health care to be present at the frontlines working directly with families to first understand and then respond to their unique needs. forensic teams do this days of the year, modeling for all of us the skills, experiences, and purpose essential to navigating patient advocacy in the midst of public health calamities like the covid- pandemic of . ❚image ❚ black-and-white photograph of a decedent's folded hands taken at the request of her family. this service predated the pandemic but proved especially impactful for those unable to do in-person viewings given covid- related restrictions on visitation in michigan medicine's hospitals. families were offered the opportunity to receive professional photographs of their loved one's hands or tattoo. the forensic pathologist as patient advocate. lablogatory do not stay at home: we are ready for you. nejm catalyst where are all the patients? addressing covid- fear to encourage sick patients to seek emergency care. nejm catalyst evidence mounts on the disproportionate effect of covid- on ethnic minorities new york city coronavirus map and case count navajo nation surpasses new york state for the highest covid- rate in the us. cnn it's time to talk: the lifechanging impact of patient-pathologist interactions. the pathologist working directly with families to understand and respond to their needs in a time of personal loss and national catastrophe is the unique province of an autopsy and forensics program. in southeast michigan this is a collaborative initiative between a department of pathology in a cross-functional academic medical center, multiple county health departments, and a governmentowned facility in a large urban center. each contributes in unique ways to a robust interdisciplinary program that steps up to deliver on expectations for public health, education, and research, while also delivering compassionate care for those who are the direct or indirect casualties of a pandemic unlike anything most have experienced in their lifetimes. the effects of this pandemic will be with us always, and include a legacy that speaks in real terms to the importance of continuing our investments in the infrastructure fundamental to our public health and welfare. covid- also showcased forensic pathology as an exemplar for what it means to be a patient-and familycentered discipline.corresponding author: jeffrey l. myers, md; myerjeff@med. umich.edu. key: cord- - yzrcfe authors: hart, joanna l.; turnbull, alison e.; oppenheim, ian m.; courtright, katherine r. title: family-centered care during the covid- era date: - - journal: j pain symptom manage doi: . /j.jpainsymman. . . sha: doc_id: cord_uid: yzrcfe family support is more, not less, important during crisis. however, during the covid- pandemic, maintaining public safety necessitates restricting the physical presence of families for hospitalized patients. in response, health systems must rapidly adapt family-centric procedures and tools to circumvent restrictions on physical presence. strategies for maintaining family integrity must acknowledge clinicians’ limited time and attention to devote to learning new skills. internet-based solutions can facilitate the routine, predictable, and structured communication which is central to family-centered care. but the reliance on technology may compromise patient privacy and exacerbate racial, socioeconomic, and geographic disparities for populations that lack access to reliable internet access, devices or technological literacy. we provide a toolbox of strategies for supporting family-centered inpatient care during physical distancing responsive to the current clinical climate. innovations in the implementation of family involvement during hospitalizations may lead to long-term progress in the delivery of family-centered care. family-centered care is threatened during the covid- pandemic. the participation of family members in a manner that allows families, patients, and the healthcare team to collaborate is the core of family-centered care. strategies for delivering family-centered care typically include open family presence at the bedside; regular, structured communication with family members; and multidisciplinary support. these prepare family members for decision-making and caregiving roles, with the goal of reducing family members' experiences of anxiety, depression, and post-traumatic stress following hospitalization. family-centered care is desired by patients and families, may improve their outcomes, and may also reduce burnout and moral distress among clinicians. large-scale disasters intensify stressors and basic human needs to feel safe, connected, calm, useful, and hopeful. yet, infectious diseases outbreaks make proximity dangerous. physical, or social, distancing is the principal mitigation strategy used to reduce transmission in the covid- pandemic, with a profound impact on the delivery of family-centered inpatient care. health systems must severely restricting or eliminating family presence for all patients, to protect the health of patients, family members, and workers. restrictions on family presence should not undermine adherence to the principles of familycentered care. defining patients' goals of care is a priority during the pandemic and typically necessitates family engagement. therefore, it is essential to rapidly adapt family-centric procedures and tools to circumvent restrictions on physical presence. we present a framework for family-centered care in the context of covid- and provide a toolbox of strategies to implement in the inpatient setting. the goals of family-centered care during physical distancing remain the same and are focused on facilitating ( ) respect for the role of family members as care partners, ( ) collaboration between family members and the healthcare team, and ( ) maintenance of family integrity. the pandemic necessitates that efforts to meet these goals adapt to a rapidly changing clinical culture. family-centered care has primarily relied on family members' physical presence at the bedside to promote trust, communication, involvement in caretaking, and shared decisionmaking. the term "visitation" is replaced by "family presence" in the family-centered care paradigm. during the covid- pandemic, family presence must be supported in non-physical ways to achieve the goals of family-centered care. in this pandemic, as in prior infectious outbreaks, governments, health systems, and individual clinicians change their typical practices to focus on public health rather than individuals' outcomes. clinicians may also be performing unfamiliar duties, including learning new clinical procedures and providing care in novel spaces with newly formed teams. family-centered care strategies in this context must acknowledge the changed ethical perspective and clinicians' limited time, attention, and effort to devote to learning and assimilation. strategies to support family presence during physical distancing rely heavily on existing patient or family smartphones and computers, stable internet access, and technological literacy. these strategies are likely to cause differential access to family-centered care. in the united states, where a majority of the population reports use of the internet or a smartphone, there are wide racial and socioeconomic disparities in access to computers and broadband internet. older americans are less likely than younger groups to use the internet regularly. fewer than twothirds of homes in rural areas of the united states report home broadband internet connection. urban areas face similar areas of internet inequality along socioeconomic lines. therefore, use of technology-heavy family-centered care strategies requires assessing individual families' access to these resources and devising ways to overcome these potential barriers to avoid worsening existing health disparities. despite the need for physical distancing, permitting limited family presence at the bedside may be necessary for the protection and safety of the patient or to maintain family integrity. for example, physical family presence should be supported when possible for pediatric patients, laboring or post-partum patients, and people with severe neurocognitive disability or who are nearing the end of life. exceptions allowing for physical presence should be clearly defined and communicated to clinicians, families and patients. exceptions should be adjudicated in a transparent and equitable manner, preferably through a centralized system, to avoid discrimination in family access and additional strain on the clinical team. these processes should also aim to decrease the bedside clinical team's moral distress and "avoid conflicts of commitments," aligned with the recommended practices for resource allocation decisions. the united states is permitting use of technologies that may not be fully compliant with the health insurance portability and accountability act privacy, security, and breach notification rules (hipaa rules) during the covid- public health emergency. this enables communication and provision of telehealth through existing commercial platforms. clinicians using technologies that do not follow hipaa rules should disclose to patients and families that they may compromise patient privacy. use of hipaa rules-compliant platforms and security features on all platforms should be prioritized to protect patient privacy. the delivery of family-centered care begins at entry to the health system. the patient and family should receive an explanation of any restrictive policies that limit the physical presence of family members. as families often have limited face-to-face contact at the point of entry, a publicfacing website should provide additional information. the explanation of the policy should include rationale and use language and tone that seeks to defuse and avoid conflict. the publicfacing material should also empower patients and families to anticipate and prepare for next steps. the website should also link to community resources, free or low-cost public internet programs, and information about the health system's preferred communication platforms. finally, hospitals should provide a mechanism for delivery of essential items to the patient, such as glasses, phone chargers, and advance directives. delivery of family-centered care may require reinterpreting or reinventing roles within the multidisciplinary team as clinical staff become a scarce resource. medical, nursing, or social work students removed from clinical rotations may be able to provide skilled support while advancing their own education and skills. students can virtually visit families and patients to promote coping strategies, coordinate engagement efforts, and streamline communication with the clinical team. additionally, the health system should leverage partnerships with community organizations to collaboratively assist family members. proactive outreach to community partners about policies limiting family presence may alleviate health system stress as the need for supportive care increases. for example, local faith leaders may be equipped to provide virtual pastoral care support. separation near the end of a patient's life is particularly tragic. conduct conversations explaining transition to comfort-focused care via multi-user videoconferencing, including multiple distanced family members, translators, and longitudinal clinicians as appropriate. when possible allow physical presence, even if very limited, and maximize family presence using strategies in table . involve supportive care teams for the patient and family, including palliative care, pastoral care, and behavioral health, recognizing that these service lines are also likely to experience strain. family-centered care is more, not less, important during a pandemic. physical distancing requires nimble adaptation of standard practices. innovative approaches that involve family members in inpatient care during the covid- pandemic may lead to long-lasting progress in, rather than regression from, the standards of family-centered care the healthcare community has recently achieved. guidelines for family-centered care in the neonatal, pediatric, and adult icu how can careproviders most help patients during a disaster? ethics of outbreaks position statement. part : family-centered care scientific and ethical basis for social-distancing interventions against covid- a heart-wrenching thing': hospital bans on visits devastate families. the new york times the importance of addressing advance care planning and decisions about do-not-resuscitate orders during novel coronavirus (covid- ) charts on internet use around the world as countries grapple with covid- . pew research center digital gap between rural and nonrural america persists. pew research center the digital access and equity report in baltimore city . robert w. deutsch foundation dealing with "difficult" patients and families: making a case for trauma-informed care in the intensive care unit a framework for rationing ventilators and critical care beds during the covid- pandemic transfer out of intensive care: an evidence-based literature review on the importance of nonverbal communication in the physician-patient interaction serious illness care program covid- response toolkit medical student education in the time of covid- key: cord- -c wu tt authors: dickerson, victoria c. title: the “flip”—sustaining complexity and multiplicity post‐quarantine date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: c wu tt this article describes poststructural ways of responding to the pandemic by looking for openings or gaps within more traditional ways of interacting with dilemmas. the author situates herself within a “positive deviance “ epistemology, which looks for what already works rather than getting captured by the current problem. we “flip” what are the usual ways of responding and explore three different programs that illustrate inventive and assets‐based approaches. linking‐lives storython, created specifically to utilize our covid shelter‐in‐place experience; re‐authoring teaching: creating a collaboratory, begun years ago to meet the needs of a wide‐spread community; and witness‐to‐witness, a current response to contemporary disastrous situations—all have utilized technology to open possibilities for those who are helped as well as those who are helpers. the article shows how each program has specifically flipped more traditional ways of responding, but also how this “flip” employs a practice of “disciplined improvisation.” each program has a built‐in structure that depends on technology to make it work; each has a disciplined approach that allows the helpers to improvise to meet the needs of the receivers. it is this “flip,” this way of thinking, that can sustain us and our work in times of great complexity and multiplicity. this article is protected by copyright. all rights reserved in early march i sat in a crowded restaurant having brunch with a colleague; we knew about a "coronavirus," but we were unconcerned. five days earlier i had been teaching a graduate family therapy seminar when another colleague and i asked the students how worried they were about the coronavirus; they said, "not very." the colleague in question had checked with the provost about whether or not she should continue to commute by air from her home city to teach in person. the response was, "definitely." a week later, the principals of the sports world started shutting down sporting events. then, one-by-one: broadway went dark, disneyworld/disneyland closed, businesses were shuttered, schools were locked, restaurants and bars turned off the lights; "it was the day the music died." during previous plagues people were told to "leave home and don't come back;" now we were told to "stay home and don't come out." tests were in short supply, people were getting infected, hospitals were overrun, protective gear was unavailable, patients died, healthcare workers died, morgues were overrun. airports were eerily quiet, freeways were mostly vacant, the air became clearer. we were told to "shelter in place," that it would lead to "flattening the curve." we found ourselves with not enough toilet paper, no sanitizer available, and only "essential" forays out were allowed. soon protective screens, gloves, masks, six-feet social distancing-all were required. our time was standing still, reminding us: tomorrow, and tomorrow, and tomorrow, creeps in this petty pace from day to day, to the last syllable of recorded time; and all our yesterdays have lighted fools the way to dusty death. (macbeth) with the rearranging of routine and inventing new rituals, with an unknown ending and not much to look forward to, we attempt to reconnect to our reflexive skills and create new meaning. we are stuck in time. it collapses; one day bleeds into the next; goals are amorphous, timeliness has little meaning. no one really knows what "going forward" might mean. time, as we have come to know it, is dramatically changed by quarantine. in einstein's dreams, by alan lightman ( ) , each chapter is an alternate world in which time is differently constructed. this article is protected by copyright. all rights reserved "while people brood, time skips ahead without looking back" (p. ). we are experiencing an alternate existence with a precarious definition of time. we are in a position of predictable unpredictability, where we cannot imagine the future and struggle to connect to our history. in these unknown, uncertain times, most of the therapy and education worlds surrendered to some form of telecommunication, which begged for understanding and meaning. when the coronavirus brought the whole world to its knees, we scurried to find multiple platforms to manage our communication options as we redesigned how we interacted with others as therapists and healers. isolated from wider human contact, sheltered in place, we needed to find ways to connect. many systems: schools, health concerns, psychotherapy, rituals, friends and neighbors, were reduced to video conferencing. this paper draws from a poststructural and systemic approach to posit a perspective from which we can learn and be proactive for an unknown future. many questions come to mind when considering a poststructural epistemology and systemic family therapy in the context of covid- . how was the pandemic altering our lives, our homes, our learning, and our relationships? is it possible to "zoom out"-take a wide-angle lens view? instead of focusing on what doesn't work with the technology with which we're stuck, is it possible to pay attention to how we can proactively use what has been given to us? by attending to a "creative" and "enlightening" quarantine, might we find openings and possibilities instead of a stuck-in-place distress and possibly a passive stance? we explore what it might be possible to accomplish, questioning whatever the path might be, charting a future that "flips" what we think we ought to do. in the days and weeks that followed those early days of march, there was a multiplicity of responses. fortunately, the technology was there; we had already predicted that there would be times (not predicting all the time) when our interaction would need to occur via a plethora of platforms and apps ready for use: go to meeting, zoom, skype, doxy, google hangouts, facebook, and many others. in fact, an entire industry had been built to offer online/teletherapy, as well as online learning centers, to potential users. teletherapy is how most therapists are meeting their clients. some therapists are finding teletherapy very useful, especially those practicing from theories that operate in a directive or systematized way, a structural epistemology, where there are more prescribed ways of offering a this article is protected by copyright. all rights reserved way forward (e.g., cbt, dbt, act). (see also dickerson, .) these therapies have been shown to be effective in person but also seem to adapt somewhat easily to an online presence. however, many have negative views of video options. video calls were accused of "messing with our minds" (hinde, ) , including all the pitfalls that occur when one can't be face-to-face. in a new york times article at the end of april, "why zoom is terrible," the author (murphy, ) writes about how our perception is distorted because of the loss of the ability to see the intricacy of facial expressions. this leads to an inability to "mirror," which is a major way that we recognize emotion and thus can establish empathy and connection. this reflects what rick moody ( ), a creative writing teacher at brown university, writes when he suggests that compassion occurs when humans can be in a room, feeling and expressing something, with others listening. he comments on his concern about how "humanness" can be lost through a zoom call. before the pandemic we were easily captured by a "business-as-usual" model: the therapy office, the agency consultation rooms, the hospital meeting spaces; all of which were inperson, face-to-face. this is what we had come to expect, unquestioning, accepting it as a settled certainty. then-facial cues, body positioning, subtle looks, ability to talk over, ease of talking after, hanging out-all taken for granted by us, invisible, unnoticed, until they were seen by seemingly being lost in zoom or other video platforms. we began to recognize what had been unremarkable to us previously: what it means to interact as clinician and client in a therapy room, in a physical space designed specifically for the sharing of experience, for personal communication and understanding. some of us who are family therapists, teachers of family therapy, social workers, and frontline workers find ourselves in the uncomfortable position of how to help those in our care. in a new yorker article, adam gopnik ( ) mused about the reality of "the empty couch." he echoes much of what many therapists realized when their clients were not coming to their office. most never questioned where they do therapy. we have a therapy office, which we furnish and decorate according to our tastes. we manage the space corresponding to the needs of our clients. we take for granted our ability to establish empathy, to create and foster relationships. the space we're in holds the interactions. this article is protected by copyright. all rights reserved when i began to use zoom for therapy sessions for some clients who were requesting remote meetings, i saw it as a poor substitute. working as a family and couple therapist from a poststructural perspective, i believed that being in the room with the people we are "seeing" is a preferable process. i noticed that, using zoom for therapy, i was less focused. my preference, as a narrative therapist, is to be curious, ask questions, try to understand the client's experience, getting closer to what they meant. i observed that i tended to short circuit the curiosity process and jump to therapist statement. i was less and less satisfied with my performance and wondered what the client was discerning. poststructural therapists tend to emphasize "local, interpretive, and idiographic accounts" (dickerson, , p. ) . as a result, zoom therapy conversations felt thinner than the in vivo meetings-accessing the client's human experience seemed difficult. i developed an implicit "rule" that i would only set up a zoom session if i had previously worked with clients in my therapy office, so these sessions were an exception to an in-person experience. then i got a request for remote therapy with someone i hadn't met, so i bent my rule and started meeting online. even then, i made it a point to request a meeting in person when it became possible. the shift this provided was remarkable. the flat screen video became a d human being-i suddenly had height and weight, hair style, eye glances, enhanced facial expressions, body movement, all coordinated with vocal volume and tone, intimations and inuendoes. i wasn't conversing with a ghost; i was speaking with a lively flesh-and-blood person. this experience sent me back to the belief that "meeting in person" is superior to online therapy. this article is protected by copyright. all rights reserved during the pandemic video conference platforms have been the only viable option to inperson meetings. whatever criticisms have arisen about its faults, and/or its possibilities, what do we know about the complexities this moment affords? focusing specifically on our interactions with others from a position of a therapeutic relationship, it does allow us to connect. if we expect the video call to be the same as "in person," we will be gravely disappointed, and yet, when it's the only option, how do we make it work? some writers are currently looking at the "advantages" of a zoom liaison over an inperson connection, reflecting new "intimacies"-rooms where the calls occur, family members flowing by, cats walking across the screen, dogs barking, lawns being mowed, and people calling across the street to each other. one author asked if there was a "kind of leveling that takes place" (gottlieb, ) . changes imposed to shelter-in place have called attention to what had been undetectable to us beforehand: what it means to interact as clinician and client in a therapy room. we notice how technology shifts the experience, especially when it seems all that is available to us. can this noticing help us see other possibilities, previously invisible? what does the technology give us to sustain our connectedness, its multiple uses, advantages, and opportunities? how are relationships formed and sustained over time? what is lost? what can we learn? how do we change our future? a poststructural perspective allows us to garner the learnings available from this experience, think about possibilities, and access the creativity available to us for going forward. the pandemic bends and shapes the various ways we engage in providing therapeutic services to a variety of populations, cultures, and geographies. complexities in technology have become dominant, modalities that existed but had been under explored. how might the effects of the pandemic be changing our thinking? we can change, because we "have" to, and/or we can think about how we want to be with the change, how to manage it, and how to use the change in a way that is sustainable. family and couple therapy is systems work, and systems work is about a way of thinking, an epistemology. it often requires a wider lens, a larger systems umbrella. evan imber-black this article is protected by copyright. all rights reserved ( ) in what now seems prescient, wrote families and larger systems, that calls out for us to embrace thinking about the complexity of a larger system interaction. the particular technology slice called teletherapy may not lend itself well to a poststructural epistemology. obviously, poststructural therapists use teletherapy. the question is: how can we use it to assist us? perhaps the simplest response is that it allows us a type of connection. however, as mentioned above, the concern with teletherapy is a loss of a richer possibility for empathy, given the "thinness" of the experience. poststructural couple and family therapists' work is centered in possibilities. we explore openings, look for gaps, and ponder how we can embrace what might be new or different. by entering into those possible openings, ways of being and acting became more visible. the work of therapy becomes one of collaboratively reauthoring a preferred narrative from having explored those openings. arvind singhal ( singhal ( , talks about "positive deviance." which, "flips," our usual problem-based method and uses an assets-based approach toward social change. is there a way to think about our current dilemma of pandemic/quarantine, our need to rely on technology in our systems work, that could help us see a way forward from a "positive deviance" understanding? positive deviance begins not with the question of how to solve this problem, but how has this problem already been solved, and who has solved it. it is similar to the quest for openings and gaps, as it attends to possibilities. can we take a positive deviance approach toward video therapy? can we "flip the question"-use an assets-based understanding? from a problem-based method, we might try to see how we can either "tweak" the video therapy approach to make it work better for us, or simply accept it as imperfect option to an in-person meeting. if we are focusing primarily on our work with clients, there likely is no "flip." that would require our noticing or creating a video therapy that worked extremely well-one possibility would be to create a d model from a flat screen. because we have not yet seen that, we are instead noticing what forms of technology do work well. understanding positive deviance as a similar epistemology to a poststructural one, we want to utilize what technology is well suited to do, not what we think we can bend and shape it this article is protected by copyright. all rights reserved to do. this takes us to the realm of working in larger systems-with groups of people, online, utilizing the best tools at hand. this is in the interest of the "flip"-not how can we solve the problem of "zoom" therapy often seeming inadequate, but what do we already know about what technology can achieve? making use of what technology provides us comes from the position of the "flip." what was happening in the field of family therapy that was not only attending to our quarantine situation but was actually creatively utilizing it? we began to notice events that were embracing what covid- created. what often goes unnoticed and is worth noting is "the inadvertent reconstruction of access to resources created by an 'online-only' world, that tends to privilege middle-class, english-speaking, us-centric, and (what seems to be) often urban communities" (zamani, personal communication, , june ). when we discuss the use of technology, we are omitting those who do not have access: marginalized populations, usually the underclass, and often non-white. a way to "flip" this lack of access is worth considering. an immediate and particularly ingenious endeavor was the creation of a "storython" (https://tinyurl.com/yaz rvar), imagined and produced by stephen gaddis of the narrative therapy initiative in salem, massachusetts (https://www.narrativetherapyinitiative.org). gaddis thought that a good use of our pandemic time would be to put together a -hour global story telling event, a wave of stories beginning in new zealand, traveling across the globe. it would cross china, western europe, spend a good deal of time in the united states, and end up in mexico city. gaddis was intentional in wanting to open space for those whose lives were less known, rather than privileging what he calls "the narrative elite." one of his values is to bring in voices from the margins to make visible the imagination, ingenuity, and sustainability accessible around the world, but which often goes unnoticed. he believes we can see the possibilities available if we allow ourselves to be open to difference. in this way, he follows michael white's "de-centered and influential" (white, ) . gaddis took advantage of time, geography, and living links around the globe to speak out for the common experience we are all sharing. pilar hernandez-wolfe and navid zamani and i collaborated to create a structure for one of the hours, pilar and navid each interviewing their guest storyteller, and then the three co-hosts acting as a reflecting team/outsider witness group. the major question posed to our this article is protected by copyright. all rights reserved guests/storytellers was: how has your life become more or less complex since covid? how has this complexity impacted all your relationships? our intention was to extend our understanding of how the pandemic / quarantine has impacted us in multiple complex ways. we were also interested in how people "improvised" to relate to the situation. this curiosity was based on our own reflection that what we were creating was an "improvisation" as a way to respond to the state of affairs that encompassed us. pilar invited claudia, a young indigenous peruvian woman, musician, storyteller, and therapist, who opened the hour with sounds from her flute. as an immigrant she spoke of her continuing commitment to the life of her community, the belief that "we must prevail" because "we have to." her words spoke directly to our quarantine experience, reminding us that during this time of slowing down we have the opportunity to see openings that might otherwise be invisible to us. she also commented that slowing down or resting is an advantage not available to everyone. navid's guest, nidya, identifies as a "dreamer." as such, her constant experience is one of "no relaxing," always on alert; and counter to the "rest" that claudia identifies as necessary to see openings, niyda says she lives in a "sea of uncertainty." her openings become available to her as she connects with her extended family and her commitment to community. gaddis "flipped" our experience of the pandemic/quarantine from what many were experiencing as a problem to noticing and utilizing ways to honor the lives of all of us stuck at home with creative ideas as our companions. from a positive deviance approach, he knew what technology could do and he imagined a process that could create a wave of possibility around the globe. in the particular hour i was privileged to help design, we were embraced by stories of the possibilities of learning (and knowing) how to respond to stress and danger and the need to prevail. in the narrative therapy community was shocked with the untimely death of michael white. there were multiple responses to this loss: most in an endeavor to immortalize his remarkable and unprecedented approach to working with people. one response focused on our looking through the wider lens (e.g., using technology) to gather together the narrative community in the wake of the loss of its founder and leader. peggy sax, in vermont, who wrote ( ), envisioned using the ideas from that this article is protected by copyright. all rights reserved book to create an online study group to engage narrative practitioners worldwide. her vision captured the support of david epston, narrative therapy's co-founder, and years later there is a rich and robust website (https://reauthoringteaching.com). sax used the evolving technology in intentional ways, to create possibilities to connect and sustain us. she began by offering an online study group and over time utilized platforms that offered connections in real time. now her site includes a "collab salon" not only with webinars and online courses, but also with customized consultation, a youtube channel, an upcoming resource library, and more. her commitment is to "use the internet in productive and socially responsible ways." talking with sax recently, she remarked how quickly a dozen years have passed, during which time she developed an incredible team of talented people and now has a rich and extended online community. her vision was realized beyond her wildest dream. how does this happen? one can see the thoughtfulness and intentionality she used to allow technology to help us, to be mindful of what it can accomplish, and to capture its reach to gather together like-minded people. her "flip" was twofold: one was, by allowing technology to create a way for the narrative community to connect to each other-those who previously were mostly sustained by the worldwide traveling of michael white and david epston. it was also, however, a "flip" of the "how" in the use of the internet-more subtle but perhaps more powerful, focusing on technology taking us "places no one has gone before." what sax and her collab team did was catch up with technology and use it instead of allowing it to use us. the complexity is not so much in the content, but in the process. this flip reflects the over years ago remark from marshall mcluhan ( ) : "the medium is the message" (p. ). witness to witness (w w) is a program initiated by kaethe weingarten, drawing on her witnessing model ( , , ) . it is contemporary, timely, and complex with multiple this article is protected by copyright. all rights reserved possibilities, beginning its life in mid- in response to a current and grievous need. i believe it is a centerpiece and model of how to flip what might be a more usual response. witness to witness is a concept that offers the support of "witnesses," who specifically take on the role of "helping the helpers," or "witnessing the witnesses." it begins with partnering volunteers (clinicians) with the many frontline workers. "witness to witness" could not have come at a better time, with clinicians, attorneys, and frontline workers responding to the problems that immigrants, detainees, asylum seekers, and all those affected by a virulent racism, were experiencing at our southern border. the witnessing of violence often has a traumatic response, meaning that the helpers also need help. the support of others, someone who can listen without judgment, is paramount, and often family and friends are ill-equipped to take on that role. this program is featured elsewhere in this issue (weingarten, ) , so i will not describe it in detail here. (see also https://www.migrantclinician.org/witness-to-witness .) it is especially geared toward our present experience with news every day of more injustice and harm perpetrated on the most vulnerable. these problems certainly have a history but have been exacerbated and accelerated by the current administration. at the time of this writing, multiple cities across the us are protesting the death of black persons as the visible perpetration of systemic racism. as one clinician stated: we keep thinking it can't get any worse, and every day something worse happens. the w w program continues to develop and grow with four components responding to the needs that arise. as the coronavirus began to take hold, w w pivoted toward offering services specifically tailored to those involved in frontline work, currently by webinar and peer support groups. each component of this program occurs "virtually," by phone, video call, webinar, facilitated group. w w was initiated to operate in ways that could reach people who could not be available for in-person consultation. with these components persons who most could benefit from the vision and goals of w w have access. since that original work in of partnering clinicians with frontline workers, the program quickly expanded, now having just celebrated its two-year anniversary. as a consummate example of "flipping" the usual reaction, this approach notices that those of us who help also need help. other responses might be to send in more workers or to offer a variety of services to prevent burnout. w w is an excellent example of our existing use of this article is protected by copyright. all rights reserved technology to serve those who might otherwise not be served. the question wasn't: what should we do to address the problem? it was: how can we act in ways we already know to be helpful? it acknowledges the distress that has brought to the fore and that so many are experiencingpeople with differences: immigrants, racial disparities, class divisions, and more recently covid- . it is a program that is fluid and adaptable to meet whatever need becomes critical. madsen ( ) speaks of "disciplined improvisation" as a response to what he calls the "predictably unpredictable." this phrase reflects what it is that family therapists, social workers, and others on the frontline do to deal with the "messiness" that we often experience when we work with people where there are multiple stressors. madsen flipped the usual notion that it is therapists who do all the healing work with families when he began to write for the frontline workers in applications of family-centered services (e.g., systems of care, wraparound, family-driven care, the recovery movement, and family group conferencing, among others). he suggests that the heart of family-centered approaches is a shift (a flip) in how services are provided to families. (see "taking it to the streets," .) each of the three inventive creations described above not only flipped ordinary ways of looking at problems to noticing possibilities for extraordinary ways, they also shared a way of doing things in common. they all knew how to improvise to meet the needs of the times, to make music. this article is protected by copyright. all rights reserved positive deviance looks at what works with a group of people for whom something ought not to be working and applies it to others. or, to put it another way, by noticing "uncommon behaviors" that a community might already be practicing in response to a given problemagainst all odds-a positive deviance approach applies those practices to other situations. it is a sustainable approach relying on collective intelligence. singhal ( ) tells us that the global record for solving complex problems is dismal; his antidote is for us to "flip our questions." instead of asking, "how can we solve this problem?" we ask, "who has already solved this problem?" or-to question who has acted their way into a new way of thinking, rather than presume we can think our way into a new way of acting. it is about asking the right questions. no one really knows what "going forward" might mean in the time of pandemic / quarantine. what happens when we zoom out, get the larger view, what does it show us? we can construct a new meaning for time. we move away from the day-to-day changes. we have the privilege of being able to "rest," to "slow down" in order to contemplate openings. we have the possibility of experiencing wonderment. what can we see that was previously cut off by the borders? what do we see inside the scape that was lost in the previous view? how can we access our creativity? can we see things that we've seen before but that now have more meaning? what might pop up if we did? and what might have viability across another time? who are we serving and how might we serve them better? how might we, from a poststructural / positive deviance perspective, garner our assets and note how we might skillfully respond? what if we thought of all of the approaches described here as a direction, a way to organize our thinking about what the quarantine has wrought? our first step would be to notice the need, secondly to access what we know to be our assets, thirdly to understand and appreciate the tools we need to address the issue. from a positive deviance perspective, we would flip the question, asking: what do we know about how this problem has already been solved? often, the answer to that question lies in the margins, initially outside our view. the "flip" discussed here is one way of widening our lens, allowing us to see possibilities. by utilizing our experience and expertise as systems thinkers, we can, in a disciplined way, improvise a response to the situation. we need to be nimble, able to think this article is protected by copyright. all rights reserved outside the box, and ready for the unexpected and unpredictable. a poststructural, positive deviance way of thinking, a disciplined improvisational way of acting, and a witnessing of what we have before us. from these positions, from "the flip," we can best sustain the complexity and multiplicity of our times-post-quarantine. recently told me how much better her mood and her partner's mood was after a good night's sleep, i asked her how she thought about that. she immediately described what happened when they didn't get a good night's sleep and how they tried to figure that out and solve it. i asked if she knew what allowed her to actually have a good night's sleep, rather than how to solve it when she didn't. it opened up an entirely new conversation about what she might do to replicate the conditions for a "good night's sleep." i flipped the question. in his invitational email, gaddis wrote: i would like to humbly invite you to help me create a small global conversation. searching for a relational response to the introduction of the coronavirus, i thought it might be nice to make space for stories from across the planet to be shared and respectfully witnessed. not even stories really just some small meaningful moment. i'm calling this event, "the linking-lives storython." i'm imagining a gentle ocean swell that meanders slowly across the world, lifting up local knowledges along the way. my hope is these small stories can offer people moments of resonance, connection, and inspiration, and a chance to get to know one another and build community. see claudia cuentas, https://www.claudiacuentas.com. as an example, weingarten led a webinar in mid-june on supporting a friend, family member or colleague who was suffering due to the pandemic. however, with the onslaught of protests in the wake of the death of george floyd, her first question was, "are you suffering from covid, racism or both?" (this webinar is available at https://youtu.be/ib havpl_ac.) positioning oneself within an epistemology: refining our thinking about integrative approaches the advance of poststructuralism and its influence on family therapy the new theatrics of remote therapy the surprising intimacy of online therapy sessions during the pandemic the psychology behind videocalls-and why they mess with our minds families and larger systems einstein's dreams. pantheon collaborative therapy for multi-stressed families collaborative helping: a practice framework for family-centered services collaborative helping maps: a tool to guide thinking and action in family-centered services taking it to the streets: family therapy and family-centered services collaborative helping: a strengths framework for homebased services accepted article this article is protected by copyright. all rights reserved understanding media: the extensions of man why zoom is terrible missives from my locked down friends humanism: remote. the new yorker re-authoring teaching: creating a collaboratory deviance, social complexity, and generative relationships american family therapy academy th annual meeting arvind singhal: the flip, solving complex problems with mental somersaults witnessing, wonder, and hope common shock: witnessing violence every day reasonable hope. family process family process therapeutic posture. michael white workshop notes this article is protected by copyright. all rights reserved key: cord- - bo ooy authors: sano, yoshie; mammen, sheila; houghten, myah title: well-being and stability among low-income families: a -year review of research date: - - journal: j fam econ issues doi: . /s - - - sha: doc_id: cord_uid: bo ooy scholarship on families in poverty, in the last decade, documented various struggles and challenges faced by low-income families and expanded our understanding of their complicated life circumstances embedded within the contexts of community, culture, and policies. the research articles published in the journal of family and economic issues during this time, that highlighted poverty, focused primarily on three topic areas: economic security, family life issues, and food security. overall, findings conclude that family well-being and stability cannot be promoted without the consideration of environmental factors. they depend on the interaction among individual (e.g., increased human capital), family (e.g., positive co-parental relationship), community (e.g., affordable childcare), and policy changes (e.g., realistic welfare-to-work programs). collectively, the articles have provided a road map for future research directions. family well-being, essential to the smooth functioning of communities and societies, is hindered when there is high incidence of poverty. poverty rate in the us hovered around % prior to the enactment of the personal responsibility and work opportunity reconciliation act (prowra) of (u.s. census a). following welfare reform, the poverty rate started to decline (to a low of . % in ) (u.s. census a), although scholars have questioned if prowra is the cause of this decline. uncertainties in the economy, including the great recession, caused the poverty rate to climb again and remain at around % until . with the fading effects of the recession, the us poverty rate was at . % in early , right before the current coronavirus pandemic. one group that is most vulnerable to poverty, however, are female-headed households, who consistently comprise % of all households living in poverty. other vulnerable groups include non-whites [poverty rate in , blacks: %; hispanics: %; native americans: %] (kaiser family foundation ); rural communities [poverty rate in , non-metro: %; metro: %] (economic research service ); and children [poverty rate in , %; i.e. in every children] (us census b). family well-being is a multidimensional concept that refers to a family's subjective sense of overall welfare, taking into account the physical and emotional health of family members as well as their interconnectedness, which in turn results in family stability (a sense of consistency, predictability, and continuity). there are many components that contribute to the well-being of families such as income sufficiency, food security, stable family environment, mental and physical health security, safe housing and communities, employment opportunities, and adequate transportation. these components, taken as a whole, provide the necessary foundation for the well-being of families. for low-income families, in particular, the lack of some or all of these dimensions can be severely detrimental to their well-being since this could lead to poverty. such a direct link between lack of well-being and poverty can ultimately lead to family instability. in this paper, we will review select research findings of the past decade published in the journal of family and economic issues from to that have increased our understanding of low-income families living in poverty. each study employed a unique approach to its particular topic. some studies utilized large secondary datasets including both metropolitan and non-metropolitan residents while others collected their own data from a smaller sample generated by non-probability sampling. however, all studies focused on low-income families in the united states with the exception of one study that examined poverty-related social policy in columbia. the papers, while highly diverse, all illustrated the strengths and challenges faced by individuals and families living with limited resources. our review was carried out in multiple stages. first, each author independently reviewed the articles, and then the authors qualitatively compared and contrasted the main themes that emerged from these articles. in the last step, the authors identified three specific dimensions of wellbeing : economic security, family life, and food security. our objective was not to provide a comprehensive summary of all poverty-related issues addressed in these articles but, rather, to synthesize the research findings along these three dimensions to see how they have contributed to the current knowledge base regarding low-income families and to provide a path for future research in order to improve family well-being and stability. in the last decade, research on the economic security of lowincome families has centered around poverty dynamics, the effectiveness of welfare-to-work programs, employment issues, the earned income tax credit, and banking behavior. mammen et al. ( ) developed the economic well-being continuum (ewc) as a comprehensive measure to describe the circumstances of low-income families in eight specific dimensions (child care, employability, food security, health care security, housing security, transportation, reliance of assistance programs, and capabilities) and establish their level of economic functioning (persistently poor, struggling, and getting by). when certain life circumstances and trigger events experienced by low-income mothers, which contributed to their entry into and exit from poverty, were examined with the ewc, the authors found that family health issues and changes in mothers' intimate relationships acted as significant trigger events that established or altered the economic functioning of the families. we believe that what mitigated families' hardships was their support networks. prawitz et al. ( ) reported on the centrality of locus of control among low-income individuals who expressed less financial distress and more hopefulness when locus of control was more internal to them. when low-income individuals were able to make financial adjustments, however, they had more financial distress, accompanied with more hopefulness, possibly implying that while the current situation may be bleak, their adaptive responses may have fostered hopefulness that things would improve. one of the goals of prwora was to enable recipients of temporary assistance for needy families (tanf) to exit the program and enter the job market. the transition from welfare to work, however, was not as effective when lowincome individuals were trained only through labor force attachment (lfa) programs. kim ( kim ( , found that former tanf recipients were more likely to obtain employment when lfa programs were combined with human capital development (hcd) programs as participation in hcd programs were related to longer employment durations and lower probability of tanf re-entry. participants in welfare-to-work programs, who succeeded leaving assistance and obtaining employment, disclosed low wages; informal labor market activity; notable levels of unmet needs; and continued government, community, and social support use (livermore et al. ) . those with higher earnings and regular nonmonetary help from family and friends were likely to have more needs met; those who had fewer needs met reported lower wages, had more young children, used government support programs (including childcare subsidies), and engaged in informal labor market activity (davis et al. ; grobe et al. ; livermore et al. ) . an important way to exit poverty and attain economic security is through employment. unfortunately, many lowincome mothers, especially rural low-income mothers, face daunting challenges to remain employed. son and bauer ( ) reported that mothers who were able to remain in the same job did so because they utilized their limited resources and developed strategies to combine work and family life. these strategies included utilizing social support network for childcare and other household activities as well as relying, where possible, on flexibility at work such as non-standard work hours and supportive supervisors. one way that low-income mothers were more likely to be employed, and especially employed full-time, was if they were provided state childcare subsidy (davis et al. ) and the receipt of childcare subsidy was tied to their employment (grobe et al. ) . high level of job instability (job loss, major reduction in work hours), however, created a greater likelihood of losing the childcare subsidy. while job changes per se was not related to loss of childcare subsidy, parents required the subsidy to remain employed. the eitc program, initiated in , is the largest federal assistance program targeted towards working poor families in order to supplement their household wages and to offset their social security taxes (mammen et al. ) . despite the many benefits of the eitc, a substantial portion of working families, especially in rural communities, do not participate in the program. mammen et al. ( ) found that, among rural low-income women, the eitc non-participants were more likely to be hispanic, be less educated, have larger families, perceive their income as being inadequate, live in more rural counties, and possess little understanding of the eitc. participating rural working mothers, on the other hand, were more likely to be single, food secure, and satisfied with life. one important element of the eitc program is the frequency with which the tax credit payments are received by the working families: lumpsum, periodic, or monthly. kramer et al. ( ) reported that periodic eitc payment recipients experienced significantly lower levels of perceived financial stress. this relationship was partly mediated by less need to borrow money, lower levels of food insecurity, and fewer unpaid bills. therefore, periodic eitc payments may enhance the positive association between the eitc and financial well-being of families. having a bank account is more likely to enable low-income families to build assets and to offset unexpected financial expenditures. according to the federal deposit insurance commission (fdic), among households with incomes less than $ , , % of them were unbanked in (federal deposit insurance corporation ). grinstein-weiss et al. ( ) found that low-income households who did not have a bank account (unbanked) were more likely to be younger, black, unpartnered, have more children, and have less income. they were also less likely to have attended college and less likely to be employed full-time. banked participants, however, were more likely to have better saving performance in individual development accounts (ida) programs and lower risks of dropping out the ida programs. according to rao and malapit ( ) , for female-headed households, having an additional child increased their likelihood to be underbanked or unbanked. such financial behavior is more prevalent among female-headed households compared to couples or male-headed households, likely due to the opportunity cost of time for women and the intimidation they feel, perhaps, based on their lack of banking sophistication. family is where individuals seek rest and support, take nutrition, promote good health and, perhaps, most importantly, raise the next generation. in this section we will discuss findings from the last decade on work-family balance, parenting dynamics, and child well-being and poverty. many rural low-income families face daunting challenges to balance work and family life. katras et al. ( ) found low-income families were able to juggle the demands of work and family life if they had access to resources such as informal social support, could manage both work and family time, and were in jobs that supported work and family life. difficulties regarding availability of resources or inflexibility in employment created problems in work and family life balance (katras et al. ) . as mentioned previously, low-income mothers relied on informal support for childcare and household tasks. they also depended on sympathetic supervisors who provided flexible work hours (son and bauer ) . work-family life balance that working mothers try to achieve can be easily sabotaged by housing instability. kull et al. ( ) reported that higher residential mobility was associated with changes in employment status and relationships, experiences of intimate partner violence, as well as private-market rentals, substandard housing, and bad neighborhoods. in their study of unmarried couples who coparented children, jamison et al. ( ) documented that the difficulties of living in poverty, combined with the demands of parenting young children, can create stress and chaos. parents who were successful in coparenting were those who were able to manage their limited resources well. jamison et al. concluded that the best way of assisting low-income couples manage day-to-day stress is by providing them with adequate resources as well as information on how to use these resources effectively. traditionally, poverty research has focused on lowincome mothers. myers ( ) , however, studied how lowincome fathers defined responsible fatherhood. previous findings on middle-class fathers have emphasized the importance of breadwinning and childcare rearing roles (schoppe-sullivan and fagon ). low-income fathers, who did not provide finances or primary care, on the other hand, did not consider responsible fatherhood to include provision for either of these two functions. instead they defined responsible fatherhood as spending time in non-caregiving activities, voluntarily distancing themselves from a child when it is in the child's interest to do so, acknowledging paternity in non-legal settings, spending money on presents, engaging in fun activities, attending to special needs, keeping abreast of what is going on in the child's home, and ensuring that they are not absent from the child's life (myers ) . the association between poverty and negative child outcomes has been well-established. children growing up in poverty are more likely to experience negative health outcomes, poor academic performance, higher dropout rates, and behavioral issues compared to children in middle-and upper-income households (brooks-gunn and duncan ). focusing on three economic indicators (income, material hardship, and non-liquid assets), kainz et al. ( ) found an association among them and variations in -month old children's social and cognitive development. poverty status, measured by income-to-needs ratio, was related to lower cognitive skills while the presence of non-liquid assets was associated with higher cognitive skills. greater material hardship was correlated with more social problems for these children. investing in children's education produces positive child outcomes (chaudry and wimer ) . child subsidy programs expand childcare options for low-income parents. de marco and vernon-feagans ( ) found that parents who received child subsidies tended to choose center-based care. they concluded that childcare, regardless of type, was of higher quality when these families received child subsidies. okech ( ) , whose focus was on parents' decision to enroll in preschool children's college education accounts, found that decisions were influenced by parental education level as well as parents' participation in information sessions about the account. another indicator of child well-being is good health. according to valluri et al. ( ) , low-income mothers chose healthcare visits for themselves and their child simultaneously. pediatric visits increased with new medical conditions and greater number of chronic conditions among children, and maternal healthcare use increased with higher maternal depression scores, chronic conditions, new medical conditions, more children, more pediatric visits, prenatal/ post-partum needs, and having health insurance coverage. maternal health visits, on the other hand, decreased with maternal depression, pregnancy, being latina or black, having more children, and if mothers were covered through private health insurance. consumption of nutritious food is necessary for a healthy, productive life for both adults and children. having enough food at home contributes to an enhanced sense of family well-being. in this section, we will discuss findings related to the measurement of food insecurity, factors influencing food insecurity, and food-related assistance programs. balistreri ( ) argued that the commonly used measure of food security ( -item u.s. household food security survey) only captures the prevalence of food insecurity, not its depth or severity. he has, instead, proposed the food insecurity index (fii) to assess the degree of food insecurity. using the fii, balistreri found that low-income households without children experienced the most rapid increases in the depth and severity of food insecurity since the great recession until . although white non-hispanic households, with or without children, had lower food insecurity prevalence rates, they experienced steeper increases in both depth and severity throughout the last decade. finally, black non-hispanic households, with and without children, were most likely to suffer food insecurity. guo ( ) documented that, regardless of socio-economic status, family food security is related to household assets. this is because the interaction between household assets and income loss buffered changes in food consumption patterns. further, regardless of household income level, the risk of food insecurity increased, when faced with liquidity constraint and asset inadequacy (chang et al. ). this relationship was strongest among low-income families. financial constraint was found to be an exogenous factor in the determination of food insecurity. food insecurity also resulted partly from the interaction between unstable income and nonstandard work schedules (multiple jobs, part-time, varied hours). while this association differed across household types, it was most pronounced in maleand female-headed households, and weakest among married couples (coleman-jensen ). the above findings, taken together, implies that food insecurity should be considered in the broader context of asset building and work environment. the food security of latino immigrant families in rural communities was influenced by multiple ecological layers. this included family characteristics (higher literacy and life skills), community conditions (state of the local economy, embrace of diversity, affordable housing, and access to health care), cultural values (familism), as well as federal immigration policy (sano et al. ). the rapidly expanding growth among latino families in rural areas of the us requires that attention be paid to the food security needs of this mostly vulnerable population (hanson ) . in rural colombia, conditional cash transfers (cct) increased the perception of food insecurity and subjective poverty among marginalized families (morales-martínez and gori-maia ). the conditionalities (families' commitment to education, good health, and proper nutrition) imposed on the beneficiary families reduced their dissatisfaction with health and education. in and , metro and non-metro households had relatively similar levels of food insecurity. yet, nielsen et al. ( ) reported that a higher proportion of non-metro households received government food assistance (supplemental nutrition assistance program [snap], special supplemental nutrition program for women, infants and children [wic], free and/or reduced school meals, and related local and/or federal programs) compared to metro households. after the great recession, when government resources were expanded, this assistance gap widened even further. nonetheless, according to chang et al. ( ) , participation in snap and wic programs increased fruit and vegetable consumption significantly among disadvantaged families. other factors such as exercise habits, family support, and willingness to adopt a healthy lifestyle played a bigger role in increasing consumption of fruits and vegetables. for some families, however, nutrition knowledge seemed to decrease actual intake of the same. in a study that identified nonfood needs of low-income households who patronized food pantries, fiese et al. ( ) classified product needs into three categories: products for survival (water, food, medicine), products to keep the household together (soap, toilet paper, hygiene products), and products to "make do" (paper plates, dish soap, household cleaning supplies). when households went without these products, it resulted in stress, personal degradation, and in illegal activities. the research findings from jfei articles presented above have identified multiple challenges and have suggested future research directions to improve the well-being and stability of vulnerable families. taken together, the findings imply that family economic functioning depends on the interaction among individual, family, and contextual factors (e.g., social network, culture, policies). additionally, emphasizing employment alone, without consideration of factors such as childcare (availability, accessibility, affordability) or jobs (availability, flexibility), is not adequate to successfully enable welfare recipients to exit the program. governmental and institutional support also play an important role in the economic security of low-income families, such as participation in the eitc, for those who are eligible, and in the banking sector. in order to balance work and family life, which would contribute to family well-being, working poor mothers require informal social support, especially for childcare and household tasks. in addition to effective resource management skills, it is important for low-income mothers to have a reliable co-parent who is more likely to decrease day-to-day stress and chaos in the household. even those low-income fathers, unable to provide finances and primary care, may provide support in non-traditional ways, thereby, contributing to family stability. utilizing available resources such as childcare subsidies, college savings programs, or local financial institutions enhance child well-being. food security is another important aspect of family wellbeing. new measures combined with traditional approaches should be used to capture the true extent of the depth and severity of food insecurity. multidimensional in nature, food insecurity is impacted, not only by income, but also by • low-income individuals expressed less financial distress and more hopefulness when locus of control was more internal to them. when able to make financial adjustments, they had more financial distress, accompanied with more hopefulness (prawitz et al. ) • what are other life circumstances and trigger events that affect changes in economic functioning of low-income families, including size and duration of its impact? • how will these changes be mitigated in vulnerable families by individual's agency such as internal locus of control and hopefulness? • what are changes in short-term as well as long-term subjective financial well-being when financial education is introduced to low-income consumers? • are low-income consumers motivated to continue to improve their financial behaviors if they are provided optimism and hope for a better financial future? effectiveness of welfare-to-work programs • when transitioning from welfare to work, former tanf recipients were more likely to obtain employment and remain employed when they participated in labor force attachment (lfa) programs along with human capital development (hcd) programs (kim (kim , • successful welfare-to-work participants revealed low wages, informal labor market activity, unmet needs, and continued use of government, community, and social support (kim (kim , • those who had more needs met had higher earnings and regular nonmonetary help from their social network while those with fewer needs met had more young children and relied on government support programs (davis et al. ; grobe et al. ; livermore et al. ) • would an expansion of educational opportunities for tanf recipients, along with modified work requirements, contribute to more successful and sustainable welfare-to-work transitions? • do state differences and variations in tanf rules (work exemptions, sanctions, time limits, earnings disregards, family caps, time to work requirements) make significant differences in positive employment outcomes and better economic outcomes for families? • what is a realistic measure of what it means to be successful in welfareto-work programs for women to become self-sufficient and truly meet their needs? employment issues • rural low-income mothers remained in the same job because they utilized their limited resources effectively and developed strategies to combine work and family life including using social support network and supportive supervisors as well as flextime (son and bauer ) • low-income mothers were more likely to be employed, especially full-time, if they were provided state childcare subsidy tied to their employment (davis et al. ; grobe et al. ) • high level of job instability created a greater likelihood of losing the childcare subsidy (grobe et al. ) • what are the differences between low-income mothers who maintain continuous employment and those who are employed intermittently? • what are strategies to incentivize (a) employers to provide flexible work policies and (b) communities to establish community-based support system for limited-resource parents of young children? • what is the impact on employment stability when states decouple lowincome parents receiving childcare subsidy and work requirements? • how is child subsidy loss related to families' choices for childcare arrangements and how do childcare subsidy rules vary from state to state? the earned income tax credit (eitc) • the eitc non-participants among rural low-income women were more likely to be hispanic, be less educated, have larger families, perceive their income as being inadequate, live in more rural counties, and possess little understanding of the eitc (mammen et al. ) • participating rural working mothers, on the other hand, were more likely to be single, food secure, and satisfied with life eitc (mammen et al. ) • periodic eitc payment recipients experienced significantly lower levels of perceived financial stress (less need to borrow money, lower levels of food insecurity, and fewer unpaid bills) compared to those who received it in a lumpsum (kramer et al. ; mammen et al. ) • what are the multiplier effects of the eitc on communities, particularly rural communities? • what incentives would increase eitc program participation among eligible working poor families? • what is the optimal frequency of the eitc payments (one lumpsum, periodic payments, or monthly payments) which would reduce family financial stress and increase overall economic security? household assets, food management knowledge and skills, cultural values, community resources, as well as federal policies. this is particularly true for racial/ethnic minorities and rural immigrant families. the articles from the journal of family and economic issues, that are reviewed here, suggest strategies for improved family well-being and increased stability. these strategies incorporate the true needs of low-income families with a variety of support systems at the individual (e.g., increase human capital), family (e.g., positive co-parental relationship), community (e.g., affordable childcare), and policy (e.g., realistic welfare-to-work programs) levels. the findings of these studies have provided a road map for future research directions. in this section, we will present a general direction for future research; detailed research recommendations, tied to specific findings, can be found in table (economic security), table (family life issues), and table (food security). future research should examine life circumstances and trigger events that may affect changes in families' economic functioning including the size and duration of its impact. recent examples of trigger events that could cause a cascading effect on low-income families include natural disasters, the opioid crisis, technological displacement of jobs, and the novel coronavirus pandemic. research should also look at how such events may be mitigated in vulnerable families by individuals' agencies such as internal locus of control, hopefulness, and financial literacy. the evaluation of current welfare programs and policies strongly suggest that future research must explore the impact of variations of state welfare policies including work requirements, strategies to incentivize employers to provide flexible work policies, and community-based support systems for parents of young children. scholars should also explore low-income families' attitudes, knowledge, and decision-making processes in the area of finances including their reluctance to participate in the banking sector and, for those who qualify, in the eitc program. at the same time, scholars should also not neglect to identify disincentives created by financial institutions that stand in the way of families participating in the banking system. previous research has established that work-family balance is vital for low-income mothers to obtain and maintain their employment in order to promote family well-being. future research should focus on strategies to incentivize employers to provide flexible work policies and to establish community-based support systems. this current pandemic has created a loss of employment opportunities and loss of income especially for low-income working families; future • having an additional child increased the likelihood of female-headed households to be underbanked or unbanked compared to couples or male-headed households (rao and malapit ) • what specific steps could increase the likelihood of low-income households' engagement in banking activity including savings and participation in ida programs and other mainstream financial services? • what are the demand side (opportunity cost of time to women and intimidation based on lack of banking knowledge) and supply side (institutional bias against women and the perceived need for greater monitoring of them) factors that contribute to gender disparity in banking behavior? table summary of findings and suggested areas of future research questions on family life issues among low-income families topic findings suggested areas of future research questions work and family balance • rural low-income families were able to juggle the demands of work and family life if they had access to resources such as informal social support, could manage both work and family time, and were in jobs that supported work and family life (katras et al. ) • low-income mothers relied on informal support for childcare and household tasks and depended on sympathetic supervisors who provided flexible work hours (katras et al. ; son and bauer ) • higher residential mobility was associated with changes in employment status and relationships, experiences of intimate partner violence, as well as private-market rentals, substandard housing, and bad neighborhoods, all of which can lead to work-family life imbalance (kull et al. ) • what are factors that will enable low-income mothers to successfully maintain work family balance? • what strategies would incentivize employers to provide flexible work policies and establish community-based support system for limited-resource parents? • how does a residential move affect family well-being and what is the role of housing subsidies on family well-being in the context of residential mobility? parenting dynamics • difficulties of living in poverty, combined with the demands of parenting young children, can create stress and chaos among unmarried couples who coparent children (jamison et al. ) • since successful coparents managed their limited resources well, the best way to assist them cope day-to-day stress is by providing adequate resources and information on how to use them effectively (jamison et al. ) • low-income fathers, who did not provide finances or primary care, defined responsible fatherhood as spending time in non-caregiving activities, voluntarily distancing themselves from a child when it's in the child's interest, acknowledging paternity in non-legal settings, spending money on presents, engaging in fun activities, and attending to special needs, keeping abreast of what's going on in the child's home, and ensuring that they are not absent from the child's life (myers ) • what is the special dynamic among unmarried, limited-resource coparents and the unique challenges that they face including their cohabiting status and the presence of other children and stepchildren? • what are the social contexts (i.e. upbringing) and the exposure of fathers to a variety of paternal responsibility that are related to their fatherhood ideologies in comparison to mainstream fatherhood ideology? child well-being and poverty • among young children ( months), poverty status was related to lower cognitive skills; presence of non-liquid assets was associated with higher cognitive skills; greater material hardship was correlated with more social problems (kainz et al. ) • low-income parents who received child subsidies tend to choose centerbased care and childcare, regardless of type, are of higher quality with child subsidies (de marco and vernon-feagans ) • decision to enroll in preschool children's college education accounts were influenced by parental education level and their participation in information sessions about the account (okech ) • low-income mothers chose healthcare visits for themselves and their child simultaneously (valluri et al. ) • what is the interaction among income, material hardship, and non-liquid assets and how does this impact the physical, cognitive, and socio-emotional development of children? what is the result of using the same measures on older children from low-income families and children from other socio-economic status? • what is the role of isolation in the availability of childcare programs? what is the association between childcare quality and stigma surrounding receipt of welfare benefits among families who do not apply for this benefit? • what is the long-term outcome of childcare subsidies on children? what are the factors that influence parent's decision to participate in children's college education accounts? • how do different types of health insurance coverage affect mother's joint decision-making for pediatric and maternal healthcare usage? table summary of findings and suggested areas of future research questions on food insecurity among low-income families topic findings suggested areas of future research questions measurement of food insecurity • the food insecurity index, proposed to assess the degree of food insecurity, found that low-income households without children experienced the most rapid increases in the depth and severity of food insecurity (balistreri ) • although white non-hispanic households, with or without children, had lower food insecurity prevalence rates, they experienced steeper increases in both depth and severity throughout the last decade while black non-hispanic households, with and without children, were most likely to suffer food insecurity (balistreri ) • what is the depth and severity of food insecurity among vulnerable populations, particularly among those without children? • what are factors related to the degree of severity of food insecurity, beyond the commonly used socio-economic indicators such as food purchasing patterns and childhood food experiences and cultural variations in these factors? factors leading to food insecurity • regardless of socio-economic status, family food security was related to household assets. when faced with liquidity constraint and asset inadequacy, risk of food insecurity increased among low-income families, regardless of household income level (chang et al. ; guo ) • food insecurity, resulted partly from the interaction between unstable income and nonstandard work schedules, was most pronounced in maleand female-headed households and weakest among married couples (coleman-jensen ) • food security of rural latino immigrant families was influenced by family characteristics (higher literacy and life skills), community environment (state of the local economy, embrace of diversity, affordable housing, and access to health care), cultural values (familism), as well as federal immigration policy (sano et al. ) • conditional cash transfers in rural colombia increased the perception of food insecurity and subjective poverty among marginalized families (morales-martínez and gori-maia ) • what is the role of economic volatility, market conditions, and policy changes in understanding the relationship between their family finances and food insecurity? • how can community-based resources (food banks) mitigate the severity of food insecurity? • how is the work form of household heads related to food insecurity, particularly among those who recently exited welfare to enter nonstandard work arrangements? • what is the effect of documentation status and immigration policy changes on food insecurity among low-income immigrant families? • is the perception of food insecurity and subjective poverty long lasting with conditional cash transfers and what are the psychological effects of this on beneficiary parents and their children when compared to non-beneficiaries? food-related assistance programs • compared to metro households, a higher proportion of non-metro households received government food assistance (snap, wic, free and/ or reduced school meals, and related local and/or federal programs). this assistance gap widened even further, after the recession, when government resources were expanded (nielsen et al. ) • although participation in snap and wic programs increased fruit and vegetable consumption significantly among disadvantaged families, exercise habits, family support, and willingness to adopt a healthy lifestyle played a bigger role in increasing consumption of fruits and vegetables. for some families, nutrition knowledge seemed to decrease actual intake of the same (chang et al. ) • when households went without certain products considered survival (water, food), to keep the household together (soap, toilet paper), and products to "make do" (paper plates, dish soap), it resulted in stress, personal degradation, and in illegal activities (fiese et al. ) • resources in food-related assistance programs are not always allocated in a cost-benefit manner (nielsen se al. ; chang et al. ) • why does urban-rural disparity occur among food insecure families when attempting to access food? what are possible strategies that would enable food-insecure metro families to access food? • what is the impact of food assistance programs on the medium-term and long-term patterns of fruit and vegetable consumption among low-income families? • what is the impact of family income and food budgeting on families' dietary habits as well as parental modeling and family food environment on fruit and vegetable intake? • using qualitative studies to develop a more nuanced understanding, what are the nonfood needs of low-income households? research should, therefore, evaluate the meaning of work flexibility to include off-site work and job sharing. positive child development is embedded in family and social contexts. to prevent generational poverty, future lines of inquiry should go beyond mothers' perspectives alone to include multiple voices of other family members such as co-parents (especially fathers), older and step-children, and grandparents. additionally, research should focus on the impact of parental decisions regarding childcare enrollment and healthcare visits on the long-term outcome of children. finally, the association between receipt of governmental assistance and the stigma experienced by low-income families, particularly among rural families, would be another important area of study. future research must investigate the role of economic volatility, market conditions, and policy changes in understanding the relationship between family finances and employment of low-income families and food insecurity. for poor immigrant families, the effect of documentation status and immigration policy changes on food insecurity cannot be understated and, to capture the nuances of their food needs, qualitative and mixed-methods studies would be preferred. future studies should also incorporate geographical information to identify reasons why urban-rural disparity occurs among food insecure families when attempting to access food and possible strategies that would enable food-insecure metro families to access food. it is equally important to assess family income and food budgeting on families' dietary habits as well as parental modeling and family food environment on healthy food behavior. 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 to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. a decade of change: measuring the extent, depth and severity of food insecurity the effects of poverty on children household finance and food insecurity do snap and wic programs encourage more fruit and vegetable intake? a household survey in the 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s li v nan potentially long-lasting threat to the family unit (hart, turnbull, oppenheim, & courtright, ) , particularly given that in times of disaster, it is known that the basic needs of humans to feel safe, connected, useful and helpful are intensified (howe, ) . a family-centred approach to care is an important feature of nursing care, grounded in recognition of the family as a social unit connected not just by blood (international council of nurses, ) and the mutually-beneficial partnerships that form between family and clinicians (grant & johnson, ) . during the covid- pandemic, family-centred care is more, not less important (hart et al., ) . the role of family members as part of the care team and finding ways for family involvement and collaboration is imperative, as are strategies that work to protect the integrity of the family unit in spite of ipc restrictions (hart et al., ) . when a patient is dying, avoiding separation from their family is a priority (hart et al., ) . dying is a time of intimacy and poignancy for family; one that is remembered in detail (donnelly & dickson, ) . family members of dying patients want to stay close (slatyer, pienaar, williams, proctor, & hewitt, ) , to keenly observe, to protect and provide comfort for the dying person (donnelly & battley, ) , and to have an opportunity to say their farewells (mossin & landmark, ) . when covid- ipc measures mean family presence is not possible, opting for other strategies that address family members' need to be close to the dying person should be considered. suggestions include the use of mobile phones and other electronic devises to enable video calls (hart et al., ) , or even encouraging families to visit from the other side of an external window (wilson, ) may also provide some comfort. even when innovative solutions are found to facilitate patients and their families connecting, albeit virtually, patients and their families may only have minutes to share final messages and say goodbye (goldstein & weiser, ) . when circumstances mean that even virtual visits or connections are not possible, it may be up to nurses to share the patient's final moments (natarajan, ) . aside from these additional actions to support family presence, necessary because of covid- , other components of end-of-life care, deemed most important for dying patients and families must continue. effective communication, shared decision-making and receiving expert care has been identified as highly important for dying patients and their families (virdun, luckett, davidson, & phillips, ) . receiving good physical care, which includes being kept clean and having symptoms managed was also important (virdun et al., ) . addressing the cultural and religious needs of patients and families before and after death and the provision of immediate grief and bereavement support for families is also essential this article is protected by copyright. all rights reserved. accepted article (raymond, lee, & bloomer, ) , and exemplify the respectful and compassionate care nurses and other health professionals are known for (virdun et al., ) . another aspect to consider is the potential impact that having to restrict family visits could have on nurses. the potential for compassion fatigue in nurses working in critical care and palliative care settings has been widely studied, identifying that stressful workplace situations was among factors increasing the likelihood of compassion fatigue (alharbi, jackson, & usher, ) . evidence of the adverse impact of working on the covid- frontline are starting to emerge (alharbi, jackson, & usher, ) , particularly in relation to the emotional toll of attempting to facilitate family connections to say goodbyes (natarajan, ) . interestingly, wu et al. ( ) surveyed frontline workers in china, and surprisingly showed that nurses and medical practitioners working on a ward dedicated to covid- patients had a lower frequency of burnout than those working on their usual wards, suggesting that this may be due to feeling a deeper sense of personal achievement when seeing that the care provided had a positive impact on patients and the pandemic. whether this sense of personal achievement remains when having to deny visits to a family member, needs to be investigated alongside other potential factors. while it may be too early in the covid- pandemic to realise the potential impact of compassion fatigue and burnout on nurses, evidence collected following the severe acute respiratory syndrome (sars) epidemic (maunder et al., ) showed that healthcare workers that cared for patients with sars had higher levels of burnout, psychological distress and post-traumatic stress than other healthcare workers. from what we know about factors that may increase risks of compassion fatigue and burnout, having to limit visits for family members of critical ill and dying patients is likely to also have a negative impact on nurses by increasing their feelings of providing inadequate family-centred care. nurses have been at the forefront of the response to the covid- pandemic and are facing extraordinary circumstances, where mortality is high, and patients have sometimes been dying alone. restricting family visits, while a necessary measure to decrease the spread of covid- to vulnerable patients and the community, is likely to have negative effects on families and nurses alike and these impacts need to be carefully considered. in an effort to promote patient and family needs and observe public health measures, a focus on the use of technology to improve communication and enhance patient and family connection may assist to avoid these negative consequences. given that nurses are known for their ability to provide family-centred care, nurses should lead this work during the covid- pandemic. family visits could be facilitated by educating and supporting a designated family member in the compassion fatigue in critical care nurses. an integrative review of the literature the potential for covid- to contribute to compassion fatigue in critical care nurses position statement. acccn and acipc position statement on facilitating next-of-kin presence for patients dying from covid- in the icu. surrey hills: australian college of critical care nurses (acccn) relatives' experience of the moment of death in a tertiary referral hospital relatives' matched with staff's experience of the moment of death in a tertiary referral hospital / / ). 'i cried multiple times': doctors say final goodbyes for patients who can't. the new yor times advancing the practice of patient-and family-centered care: the central role of nursing leadership critical care utilization for the covid- outbreak in lombardy, italy: early experience and forecast during an emergency response a heart-wrenching thing: hospital bans on visits devastate families. the new york times family-centered care during the covid- era how can careproviders most help patients during a disaster? the icn code of ethics for nurses life in the pandemic: some reflections on nursing in the context of covid- long-term psychological and occupational effects of providing hospital healthcare during sars outbreak being present in hospital when the patient is dying -a grounded theory study of spouses experiences coronavirus: 'i spend the final moments with dying patients understanding the bereavement care roles of nurses within acute care: a systematic review presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area finding privacy from a public death: a qualitative exploration of how a dedicated space for end-of-life care in an acute hospital impacts on dying patients and their families dying in the hospital setting: a systematic review of quantitative studies identifying the elements of end-of-life care that patients and their families rank as being most important visiting loved ones through the window who director-general's opening remarks at the media briefing on covid- - rational use of personal protective equipment for coronavirus disease (covid- ) and considerations during severe shortages. interim guidance a comparison of burnout frequency among oncology physicians and nurses working on the frontline and usual wards during the covid- epidemic in wuhan key: cord- -my nioko authors: foster, carolyn c.; steltzer, michelle; snyder, amanda; alden, carrie; helner, khrystyna; schinasi, dana a.; bohling, katie; allen, kiona title: integrated multimodality telemedicine to enhance in-home care of infants during the interstage period date: - - journal: pediatr cardiol doi: . /s - - - sha: doc_id: cord_uid: my nioko performing interstage home monitoring using digital platforms (teleihm) is becoming commonplace but, when used alone, may still require frequent travel for in-person care. we evaluated the acceptability, feasibility, and added value of integrating teleihm with synchronous telemedicine video visits (vvs) and asynchronous video/photo sharing (v/p) during the interstage period. we conducted a descriptive program evaluation of patient-families receiving integrated multimodality telemedicine (teleihm + vv + v/p) interstage care from / / to / / . first, provider focus groups were conducted to develop a program logic model. second, patient characteristics and clinical course were reviewed and analyzed with univariate statistics. third, semi-structured qualitative interviews of family caregivers’ experiences were assessed using applied thematic analysis. within the study period, patients received teleihm + vv + v/p care, of which were still interstage and died. about half ( %) of patients were female and % were a racial/ethnic minority. median age was days old (iqr , ) at interstage start, with a median of total days (iqr , ). a total of vvs were conducted with a median vvs (iqr , ) per patient. parents sent a median pictures (iqr – , range – ). qualitatively, families reported an adjustment period to teleihm, but engaged favorably with telemedicine overall. families felt reassured by the oversight routine telemedicine provided and identified logistical and clinical value to vvs above teleihm alone, while acknowledging trade-offs with in-person care. integration of multimodality telemedicine is a feasible and acceptable approach to enhance in-home care during the interstage period. infants with single ventricle physiology (svp) are medically fragile patients who require close surveillance between staged surgical palliations. interstage home monitoring (ihm) of objective clinical parameters, such as daily weight, oxygen saturation, and enteral intake, has become a standard care strategy to minimize the morbidity and mortality of infants with svp and other complex heart conditions between palliative interventions. [ ] [ ] [ ] "teleihm," which is the implementation of ihm using a remote patient monitoring digital platform, [ ] has become increasingly widespread in the pediatric cardiology setting. while teleihm proactively tracks objective clinical parameters pertinent to a patient's clinical status, teleihm alone often excludes routine collection of contextual or subjective information (e.g., family perspective of how infant is doing, infant appearance) and by design typically occurs separately in time than other interactions with the health care team (i.e., asynchronously). some platforms also allow for video/photo sharing (v/p), where a family can send a provider a photo or short video of their child, which also typically occurs in an asynchronous manner. so, while these tools provide important care delivery options, home surveillance based on asynchronous teleihm and v/p sharing alone may still result in frequent patient travel for routine in-person evaluations or necessitate family-provider phone calls to discuss or clarify asynchronously collected information. video visits (vvs)-defined as real-time (i.e., synchronous) secure video conferencing between a health care provider and a patient and/or family caregiver [ ] -provide another technological method to connect providers with family caregivers of affected infants. while vvs have become more common since the onset of the covid- pandemic, the feasibility and acceptability of using these vvs along with teleihm and v/p sharing as a scheduled method to enrich routine home surveillance during the interstage period remain mostly unexplored in the literature. to improve this gap in the literature, we used program evaluation to explore our institution's experience with integrating three telemedicine modalities (teleihm, vv, and v/p sharing) during the interstage period. program evaluation is an established public health method used to describe a program's activities in a systematic way and assess its perceived value by end-users, in this case by patients and their families. [ , ] our goal in this manuscript was to use program evaluation to describe the acceptability, feasibility, and perceived added value of telemedicine, including its perceived impacts on patient care and the family experience. we conducted a descriptive evaluation of our single ventricle program's use of three telemedicine modalities (teleihm, vv, and v/p sharing) from / / - / / using the centers for disease control and prevention (cdc) framework for program evaluation. [ ] we used the cdc's mixed-methods approach of creating a logic model, engaging stakeholders through provider focus groups and family interviews to understand their experience of the program, and gathering evidence to summarize the key elements of the program (e.g., patient chart review) [ , ] . the work was determined to be exempt by our institutional review board as a program evaluation. focus groups were conducted with the interstage program providers and staff to develop a program logic model and care model diagram. [ , [ ] [ ] [ ] the logic model synthesized the program's elements into an outline of what was needed to make the program function. a care model diagram was then created to further describe the integration of telemedicine with in-person care. data were extracted from the electronic health record to describe patient characteristics and clinical course. collected data included basic patient demographics (e.g., age, sex, race/ethnicity), baseline cardiac physiology, type and dates of first and second procedure including discharge and admission dates, and feeding method. the total number of vvs, in-person clinic visits, emergency department (ed) visits, and planned and unplanned hospitalizations were totaled. teleihm data entry and adherence as well as use of v/p sharing were directly extracted from the teleihm platform (locushealth ®, charlottesville, va). to evaluate the feasibility of teleihm, adherence was defined as the total number of days of teleihm data entry by the family (numerator) divided the total interstage days (denominator) times . data were analyzed with univariate statistics using stata (stata statistical software: release . college station, tx: statacorp llc.). results were presented using the median as the measure of central tendency given the non-normal distributions of most data. qualitative semi-structured interviews were conducted with participating primary family caregivers (i.e., parents) to assess the acceptability, feasibility, and program value from the family perspective. qualitative interviews are a method in medical research and program evaluation to "explore the experiences of participants and the meanings they attribute to them." [ , ] participating family caregivers had to be legal guardians and proficient in either english or spanish; temporary guardians of patients who were currently in custody of the state were excluded. applied thematic analysis, a method to inductively identify themes expressed by interview participants, [ ] was conducted using dedoose software (v. . . , los angeles, ca). interviews were analyzed until the themes captured the majority of the data to describe the families' experience with the program. [ ] . the single ventricle program is part of the larger heart center within a freestanding quaternary care children's hospital in the midwest. patients with a range of cardiac anatomy and physiology are followed in the program, and they typically include: . infants with either single ventricle physiology such as hypoplastic left heart syndrome, tricuspid atresia, or double inlet left ventricle prior to bidirectional glenn procedure; or . infants with complex biventricular physiology requiring staged palliation with either ductal stent or aortopulmonary shunt placement prior to an eventual biventricular repair. patients are referred to the program at the time of diagnosis and followed throughout the "interstage period," defined as the time from first discharge home to either bidirectional glenn procedure or definitive biventricular repair. [ ] care for patients during the interstage period is coordinated through the single ventricle program's high acuity clinic. the clinic is staffed by a dedicated registered nurse (rn) coordinator (a.s.), advanced practice nurse (apn) (m.s.), and four physicians including a dedicated medical director (k.a.). since november , all patients with single ventricle physiology whose parents were proficient in english or spanish were trained in ihm prior to hospital discharge and sent home with home monitoring equipment, including a scale, pulse oximeter, and binder. once home, the team's dedicated rn coordinator and/or apn coordinated the ihm with oversight by the interstage cardiology attendings. in from july onward, families were assigned a tablet (ipad ® cupertino, ca) to conduct vvs via a secure videoconference application (polycom® realpresence® system, santa cruz, ca). in september , the program transitioned to teleihm; so in lieu of a binder, the tablets were also loaded with the teleihm platform (locushealth ®, charlottesville, va), which collects and displays information (e.g., heart rate, oxygen saturations, weight) entered by parents, provides v/p sharing, and offers access to pre-written materials (e.g., instructions, references) in both english and spanish. additionally, the tablets were loaded with supplementary applications, including an application to access their infant's electronic health record (mychart © epic systems corporation, verona, wi), applications for meditation and stress relief (headspace © headspace, inc; calm ©, calm.com, inc.), an application providing educational material on congenital heart conditions (heartpedia ©, cincinnati children's hospital medical center), and an informational pop-up on cardiac catheterization written by the care team. for limited english proficiency families, translation was provided by an interpreter who was placed on speaker phone by the cardiac provider during the vvs. the integrated multimodality telemedicine care model for infants during the interstage period is shown in fig. demonstrating the high-level integration of vvs with teleihm and v/p sharing for patients followed in the interstage program. monthly clinic visits are conducted in-person with the cardiology providers and developmental team (speech, feeding, physical and occupational therapy) and typically include additional testing (e.g., echocardiogram, electrocardiogram). these in-person visits are supplemented with remote telemedicine care comprised of daily teleihm data collection plus scheduled weekly vvs with the team's apn or rn coordinator. for teleihm, parents are required to document multiple daily elements that covered weight; heart rate; oxygen saturation; oxygen flow (if present); intake (feeding information, e.g., concentration, amount); outputs (stool, urine, and emesis); time to collect and input ihm, and presence/absence of parental concern. all are catalogued daily except for heart rate and oxygen saturation, which are documented twice per day. optional data entry elements include topics such as breastfeeding and administration of synagis. family caregivers can additionally conduct asynchronous v/p sharing with the care team, on an as-needed basis. vvs are arranged weekly but are typically skipped during a week in which an in-person visit occurs. additional vvs are scheduled more frequently if additional follow-up is desired. if available, therapists (e.g. speech/feeding) or nutritionists join the apn or rn coordinator in the vvs session. following the end of the interstage period, the use of routine, scheduled vvs is determined on a per patient basis as needed and based on any protocols in place at the time of transition. teleihm with v/p sharing may also be utilized after the interstage period, if patients are experiencing significant heart failure, are medically fragile, or in the setting of ongoing, complex feeding regimens (e.g., nasogastric tube feed weaning). the program logic model is shown in fig. demonstrating program inputs and activities as well as intended outcomes and goal impact. the teleihm operates as a unique partnership between the apn (m.s.) and rn coordinator (a.s), who is specially trained and focused on interstage care, allowing her to independently monitor and triage asynchronous v/p data and conduct synchronous vvs with families. if concerning data findings arise, the rn coordinator has direct and immediate access to apn and physician staff. the apn and rn coordinators' activities are divided between "off camera" activities and "on camera" interactions with families. "off camera" activities include telemedicine training, teleihm and v/p data review, and care coordination. during the actual "on camera" vvs, the rn or apn coordinator obtains an interim history, discusses teleihm data, shares study or lab results, conducts a visual assessment of the patient, solicits family understanding, and reinforces and counsels on any new adjustments to the care plan. the providers document a patient's overall appearance, work of breathing, color, presence of facial swelling or secretions, incision appearance, placement of nasogastric tube and oxygen nasal cannula, as applicable. providers also observe feedings. physician team members are available demonstrates the programs' inputs and activities as well as intended outcomes and goal impact on-demand during vvs to address any acute concerns and help determine next steps in management and assessment as needed. physician team members also routinely review teleihm for all patients weekly and as needed to identify abnormal values or trends and coordinated regularly with the patient's other cardiologist(s) to maintain inpatient/outpatient continuity of information. together the team's on and off camera activities are integrated with the in-person care visits, with the goal of maximizing patient growth and development while minimizing morbidity and mortality in a family-centered manner. a key facilitator to program development was the institution's willingness to support dedicated rn and apn coordinator time to conduct the non-billable vvs prior to covid- -related reimbursement. the integration of vvs into care was also made easier by the care and education processes already in place for teleihm, including family training before discharge. vvs, in turn, supported the family's adherence to teleihm by keeping families engaged in the teleihm data entry process during weekly vvs. program barriers included limited therapist co-participation in vvs (e.g., feeding therapy) due to their inability to bill directly for the encounter. the inability to easily integrate language interpretation into the vvs for families with limited english proficiency remains an ongoing barrier, both due to platform interface challenges and scheduling constraints. lower health or technology literacy of family caregivers, which required additional education and training, was also noted. families who had no or low wifi access were provided data-enabled tablets paid for by institutional funds. a working billing model for the teleihm and v/p sharing care application, which is currently supported by institutional funds, remains an additional ongoing limitation. at the end of the evaluation period, infants had received the fully integrated multimodal telemedicine (tel-eihm + vv + v/p) care, of which had completed the interstage period, were currently receiving interstage care, and had died. participating infant characteristics are shown in table . overall, % of infants were female and % were a racial or ethnic minority. median age at discharge from st surgery (start of interstage) was days old (iqr , ). overall the median number of interstage days was (iqr , ). of the infants who died: one died during a planned cardiac procedure and the other three died after immediate triage from home to an ed and/or inpatient setting. participating families reported that teleihm data collection and entry took a median min (iqr , ). adherence-wise, families entered teleihm on a median . % (iqr . %, . %) of the interstage days. a total of vvs were conducted with a median vvs (iqr , ) per infant. this equated to a median . vvs (iqr , . ) per month during the monitoring interstate period, excluding hospitalized time. a total of in-person clinic visits occurred (median (iqr , ) per patient), which equated to a median of ( . , . ) in-person clinic visits per patient per month. a speech therapist joined in the care of patients over visits. physical therapists joined one patient for a visit. during the same period, patients ( %) experienced an ed visit (total , range - /patient). twenty-five patients ( %) had a planned hospitalization (total , range - / patient) and patients ( %) experienced an unplanned hospitalization (total , range - /patient). overall, patients were hospitalized for a total of days during the interstage monitoring period with a median of (iqr , ) hospitalization days per patient. most (n = , %) families used asynchronous picture sharing. however, picture sharing use ranged widely from to pictures shared per patient; with a median of (iqr - ) pictures sent per patient. fewer families (n = , %) used asynchronous video sharing; with a range of - videos shared per patient. out of thirty patient-families who were initially approached for an interview, families participated while actively declined, passively refused (i.e., did not return call or email), and were not reached. two caregivers chose to participate within the same family ( mother and father); otherwise, only one family caregiver participated per child. all of the family participants were female (mothers), except the one male (father) who joined as an additional interviewee. of the mothers, ( %) were non-hispanic white and the remaining majority were a racial or ethnic minority. three spoke spanish. mean age was years old (sd . ) and ( %) had less than a college degree (i.e., high school or trade school). all had experience with commercial video conferencing software (e.g., facetime, skype) prior to enrollment but only two had previous experience with virtual healthcare prior to their child's interstage period. eleven families reported experiencing a technological problem during vv care, but most problems were short-lived. one family had more long-standing problems with wifi access due to their more rural locality. another family had to swap out their tablet for a new one. vv scheduling was distributed evenly between morning and afternoon, with participants reporting timing was often based on a patient's naptime. four general themes summarizing the family experience with the integrated multimodality telemedicine care are shown in table with illustrative quotes: ) adjustment period, ) ease of use, ) reassurance provided by remote care, and ) tailoring of care to patient/family. first, almost all families reported an adjustment period with the telemedicine activities, where they had to "get into a routine" with the daily teleihm data collection and entry. some reported adjusting quickly and reported it as "easy" and even "enjoyed" entering teleihm data noting that they "liked being able to see the trends," especially weight gain. a few families, however, found the daily teleihm "time consuming and kind of stressful" and "a tad overwhelming," primarily due to the amount of total care tasks they had to complete for their child while still recognizing the teleihm was "for a good reason." a few noted that this adjustment to the teleihm occurred in conjunction with an emotional adjustment to being discharged home after sometimes long hospital stays, with parents noting that the vvs provided continuity with their in-person stay. despite this adjustment, however, almost all interviewed families felt the telemedicine applications were "easy" to use. a few caregivers expressed stress and anxiety with the application interfaces during their first few uses. however, none of these challenges were sufficient enough for families to deter use and most issues resolved with some early trial and error. some specific user-interface suggestions did arise for more "tech-savvy" families, most commonly the request for a notification/ or alarm for both teleihm and vvs to remind parent about an upcoming data entry or a visit, respectively. one parent requested fewer "taps" to enter in data and the ability to view the rate of change in the data in addition to absolute values. another wanted to customize some of her own data entry. one user noted that she would have liked to enter weight in pounds, not just kilograms. one family wished they had been offered the option of -way viewing to enable both parents to join in vvs when one parent was at work outside the home. families described the v/p sharing as a helpful adjunct to the more formal, scheduled vvs. they reported primarily using the photo function when they had specific questions about a rash, incision, or other skin finding. they also used this function for "social" and "fun" reasons, such as showing the child in a "cute" pose or outfit or on a milestone like a birthday, as a way to connect to the provider team. some described liking the v/p sharing option because they knew it was sent and they "didn't have to wait until our [scheduled vv] call," but some of the families did not use the v/p sharing at all or only used it once or twice. lastly, more than half of families reported positively engaging with supplementary applications;, but rarely described consistent long-term use. family caregivers did, however, consistently describe that the integrated telemedicine care provided them with "peace of mind," "confidence," "reassurance," and/or "comfort" that the care team was making sure that they were not "missing anything" in their care at home which kept their "mind at ease." parents emphasized that consistent and routine communication through vvs and teleihm provided reassurance that someone was keeping track of how their child was doing at home. two even specifically noted that they felt that it eased the loss of bedside nursing care to which they had become accustomed while inpatient. show [the providers] when she was eating by mouth like with a spoon or i show them like when she was like i took a picture like a birthday picture …just multiple like things like you know milestones or like pictures that we were proud of… like when they weren't around, like when they were busy or have other calls … [v/p sharing] was easy to just you know reach out even when they're busy, so like it was still convenient in a way."-parent "i [shared] [my daughter] in her [holiday] outfit kind of thing, but i also took pictures …like you know different colors of her incision as she went through her healing process, you know just different things like that. she had some issues come up with her incision. she was slow to heal, so i had to take pictures and upload those."-parent reassurance provided by remote care integrated remote care provided "peace of mind" for families "actually it made me feel like more comfortable with bringing her home knowing that in the beginning it was a weekly [vv] and the fact that we had to log all of that [teleihm], even though it was overwhelming it made me feel more comfortable with the situation because i felt like we weren't alone in you know trying to handle her medical issues, so yeah, it was actually kind of comforting having that."-parent "i personally loved [the vvs]. i loved being able to touch base with our team every week. that was wonderful. and if i ever had any concerns, i knew that i was gonna be able to touch base with them and i mean just with like the complex needs of my son's care. like it was great to like always have the piece of mind to know that like i'm gonna get to touch base with them…-parent "i think it made me feel, i thought it made me feel a lot more comfortable just bringing him home. everyone was, people were really accessible …i just thought it was helpful." -parent tailoring care to patient need and family backgrounds or user preferences the family interviews also highlighted the need to tailor telemedicine care to the patient's medical needs and the family literacy, preferences, and personalities. for example, a few families perceived that the frequency of teleihm data collection and scheduled vvs could have decreased over time, noting it could often feel "repetitive," and some parents reported different ability or preferences with the actual teleihm data collection or measurement, such as feeling confident in how well they quantified oral intake or in using a pulse oximeter. one mother did not "pay attention" to the teleihm data between visits and relied on vvs to discuss it, while others described expending a lot of time and emotional energy on data collection and review. the specific "value-add" of vvs is summarized in table with illustrative quotes grouped by: ) logistical and financial value; ) clinical value; and ) limitations/ other considerations. families reported a range of logistical advantages to vvs that included not just their time, but the cost of driving, stress of parking, and avoidance of foul weather exposure. family caregivers recognized clinical value of vvs, including the avoidance of exposure to contagious illness and the ability of providers to visualize their child (e.g., breathing pattern). families also felt that the vvs facilitated the opportunity to evaluate the teleihm trends, including distinguishing any inconsistencies in the teleihm data. notably, this scheduled, summative discussion during vvs was reported as helpful both by family caregivers who described themselves as engaged heavily in the teleihm (i.e., reviewed the teleihm data trends actively on their own) and those who reported that they simply entered in data and only really reacted to alerts for out-of-range datum. lastly, families reported that the use of vvs instead of in-person visits reinforced the infant's normal biological sleep and feeding patterns, because they did not have to disrupt these patterns to travel to the clinic. family caregivers did recognize some limitations to vvs and its impact on their overall care experience. most prominently, they recognized that having most of their visits virtually resulted in a much longer in-person visit in order to accommodate all the imaging and other hands-on evaluations that were not done in intervening weeks. when asked whether they would prefer fewer vvs in lieu of shorter inperson visits, all but one family preferred to continue to limit the number of longer in-person visits noting that the illustrative quotes from family caregivers regarding their experience with the integration of video visits, interstage home monitoring, and video/ audiopicture sharing for their children illustrative quotes desire to tailor data collection and visit frequency over time "i feel like once we got into our routine that it was very easy to log all of our daily information into the ipad. as far as our telehealth calls, i felt like [the providers] were always available to us… but i didn't feel like they were necessary, you know as we got further out. … i felt like you know we had them i think once a month and that seemed to be very sufficient."-parent "i just didn't feel like things changed day to day that much and i already had so much to do that it was just one more thing to do and a lot of the times it was at the bottom of my list because there's a lot more important things for me to do in my opinion… i don't know if overwhelming is the right word, but it was hard for me to do … it was kind of, it was like very repetitive."-parent preferences and ability gathering measurements "sometimes it's hard to remember [how much a child ate] after a whole day, so i think like if you just had to put it in almost after each feeding it would be a more accurate reading…"-parent "when i would take his oxygen, he would move his leg a lot. and sometimes he would move and i couldn't take his oxygen. but then i would grab his leg or he would stop and everything was good."-parent variable technology literacy and engagement "well yeah, i had never done this before so i was afraid, right? what if i don't do it right? this one time i did make a mistake, i didn't do the password right and i locked the [tablet] . and my niece helped me and we unlocked it so we could continue to talk to the nurses."-parent "i didn't really pay attention to the [teleihm] data that much unless something was like too alarming for me, but i think they were connected, yeah, because i think at [the hospital] they would look at the data and then let me know if something was off. they would let me know during the telehealth conference."parent "i'm a very organized person when it comes to like having to have a schedule,.. like something was going on, it was detailed down to like the hour. … it took [the apn] to say you need to calm down, it's okay if you do mid-day. it doesn't have to be perfectly exact as we would like for it to be, but if you can do it midday, it's okay. just make sure you get a night one too."-parent table summary of perceived added value and limitations of video visits during the interstage period logistical and financial value travel, parking, and wait time avoided "i thought it was really convenient. we're in the suburbs of [major urban city] but it still takes us like h to get there and that was a really convenient way … to be able to get any changes to her treatment …i thought it was really helpful."-parent "i mean the calls, the video call seems way more efficient i guess because going in, seems like we always were stuck in a waiting room for almost like more than an hour usually between all the different visits we would do in a day. so a lot less wasted time traveling and parking, waiting." -parent travel cost avoided(e.g. gas, parking, missed work) "… my family as a whole, like the cost, like you pay, you have to pay to park, everything is a little more expensive because it's downtown,.. there was something that was concerning throughout the week that i wasn't comfortable with or you know i had to you know make some changes or i wanted them to see it you know for themselves, especially since i was doing both breastfeeding with the baby and feeding the baby through the tube, i wanted them to see how she was reacting and i think that helped because there were times where we were during the call that she was you know actively eating and they could see you know how everything was kinda going. … [and] like her breathing, like is she breathing okay?"-parent "you know with my daughter being a newborn, we would get her on the screen you know, just so that they could see her and kind of visualize you know here's what we're talking about with color or a change in this or her incision or something like that."-parent facilitates discussion regarding teleihm data trends, for family both more or less engaged in the teleihm data "i really like that you can see any progress or like regression that was made on the [tablet]…[the vv's] was actually one of the ways that they were able to see that she needed another surgery months earlier than they had originally thought."-parent "i think the whole thing helped to keep everybody on the right track, so it was nice. you know having the [tablet] was amazing in the fact that i knew with confidence that i could log any information in there and then when we came for our visits, they could pull up all the graphs and the charts and the information and we often referred back to the data that was put in by my husband and i."-parent disruption of infant biological patterns avoided (i.e. naps, scheduled feed "i think we mostly did [vvs] at : . it was a time that just worked out between naps …"-parent "[in-person visits] would be a hassle because i had to put her on her feeding tube, like she was on a certain schedule. sometimes the schedule would overlap right over like i was driving and i would have to feed her and she was one of those babies that was always throwing up throughout her feedings. …this was more convenient overall for us, for our family it just worked better."-parent limitations/other considerations total time cost to them would still be less. the families did also recognize that the vvs were limited in their ability to perform every aspect of the physical exam, necessitating the option of some in-person care. lastly, all three spanishspeaking families reported phone interpretation as lower quality than in-person or video interpretation. this evaluation found that the integration of scheduled, routine vvs with remote home monitoring (teleihm) through a multidisciplinary advanced practice model can feasibly enhance the care of in infants during the interstage period with overall high family acceptability. the death of four infants during the interstage period is consistent with the published norms for this population, but also highlights the high-risk nature of this cohort. [ ] [ ] [ ] [ ] while one infant died during a planned cardiac procedure, the other were outpatients who were rapidly triaged from home to higherlevel care based using information obtained in part from the program's telemedicine modalities. these are infants that might otherwise have died at home due to unrecognized subtle clinical changes. while the use of vvs has long been described in adult health care [ , ] and a range of pediatric populations [ ] , this evaluation provides new, detailed information about the specific experience for infants during the interstage period and their families. this work also highlights the roles of the apn and rn coordinator in facilitating the use of integrated telemedicine between in-person visits. as our logic model shows, these care providers conduct a diverse range of activities both on and off camera that streamline the coordination of care between the home, clinic, and hospital setting. given that this program was in place prior to the covid- pandemic, care to patients was minimally disrupted when stay-at-home orders were announced and outpatient clinics had to adjust to new care restrictions. with the ensuing executive order that relaxed telemedicine regulations and expanded reimbursement for vvs, the program was then able to start billing for visits. while it is unknown at this time whether reimbursement for vv care will continue in the post-covid era, the general expansion of vvs across institutions nationally [ , ] may launch news models of care for patients, including outside of the interstage period. one consideration from our study is how patients during the interstage period require care that is similar or different to other high-risk cardiac populations (e.g., early posttransplant, status post ventricular assist device, pulmonary hypertension) and how this model could be applied more broadly. as our findings show, family costs regarding the time and effort required for remote data collection (teleihm) and provider time interpreting data must be weighed against the utility of the clinical information obtained between visits. these costs should be balanced against the costs of in-person health care appointments and both medical and family costs such as transportation and missed workdays. additional consideration must be given to the strain and disruption of travel on the infants themselves, which is compounded by a child who may be on nasogastric feedings or require frequent medication dosing. these patient and family costs of care should not be underestimated given the increasing evidence that they are quantifiable barriers to health care access and optimal health outcomes, especially for disadvantaged groups including those with lower health literacy and/or limited english proficiency. [ ] [ ] [ ] as the care of children with cardiac conditions continues to evolve, we should be vigilant to develop care models that do not necessarily remove but rather optimize in-person care. these considerations may be particularly beneficial for chronically ill patients with relatively rare diseases who may receive care at regional children's hospitals far from home. expansion of integrated telemedicine modalities should also consider the individual needs of families including their technology preferences, health literacy, and access to digital platforms (e.g., wifi access, digital internet). published reports during the expansion of telemedicine during covid- have highlighted the concern for inequities in the use of telemedicine-based care [ , ] , and programs should address these barriers during design and implementation. our findings suggest that the initial training of families, adjustment period at home, and access to cellular data or wifi are key areas of support. as with any retrospective chart review, not all relevant data are available which limits the completeness of the evaluation and our sample size was relatively small. while this evaluation incorporates the viewpoints of spanish-speaking families that are often left out of many evaluations, families and patients who were non-english/spanish speaking were excluded from interviews. given historical changes within the program and the fact that this program was rolled out to all patients, we did not have a pre or concurrent group with which to compare clinical outcomes. therefore, future work should include rigorous evaluation of the application of integrated telemedicine models on interstage duration and unplanned health care and other clinical outcomes, including family stress, using quasi-experimental design. also, given that families still recognize the value of the physical exam, the use of peripheral telemedicine devices such as digital stethoscopes to supplement vvs may also provide additional reassurance to families and clinical information to care providers between in-person visits. this evaluation provides a transportable framework for the use of integrated scheduled vvs with teleihm and v/p sharing as one potential solution to decreasing familial and patient cost of in-person evaluations in children during the interstage period. our evaluation showed that scheduled vvs enrich teleihm care from the family perspective, by providing reassurance and aiding in clinical care through visualization of the patient and connection with the family. home surveillance program prevents interstage mortality after the norwood procedure association of interstage home monitoring with mortality, readmissions, and weight gain: a multicenter study from the national pediatric cardiology quality improvement collaborative interstage home monitoring for infants with single ventricle heart disease: education and management harnessing teams and technology to improve outcomes in infants with single ventricle about telemedicine: what is telemedicine? framework for program evaluation in public health summary of the framework for program evaluation morbidity and mortality weekly report recommendations and reports consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups evaluative criteria for qualitative research in health care: controversies and recommendations qualitative interviews in medical research applied thematic analysis. sage, thousand oaks . guest g ( ) how many interviews are enough? an experiment with data saturation and variability comparison of shunt types in the norwood procedure for single-ventricle lesions evolving strategies and improving outcomes of the modified norwood procedure: a -year single-institution experience patients at risk for low systemic oxygen delivery after the norwood procedure risk factors for interstage death after stage reconstruction of hypoplastic left heart syndrome and variants telehomecare: virtual visits from the patient home. home health care management & practice reducing the cost of frequent hospital admissions for congestive heart failure: a randomized trial of a home telecare intervention telemedicine: pediatric applications telehealth transformation: covid- and the rise of virtual care covid- transforms health care through telemedicine: evidence from the field transportation barriers to health care in the united states: findings from the national health interview survey, - caring for children and families with limited english proficiency: current challenges and an agenda for the future health literacy and child health outcomes: a systematic review of the literature a call for addressing barriers to telemedicine: health disparities during the covid- pandemic access to telemedicine-are we doing all that we can during the covid- pandemic? acknowledgements we thank luis morales for his assistance with interviewing spanish-speaking families for this project.authors' contributions dr. foster conceptualized the evaluation, oversaw data collection, conducted data coding and analysis, drafted the initial manuscript, and edited the final manuscript. ms. steltzer, ms. snyder, and dr. allen conceptualized the evaluation, aided in data collection, participated in data analysis, and reviewed and edited the manuscript. ms. helner conducted data collection, participated in data coding and analysis, and reviewed and edited the manuscript. ms. alden, dr. schinasi, and ms. bohling aided in conceptualizing the evaluation, participated in data review, and reviewed and edited the manuscript. all authors approved the final manuscript as written.funding none. conflict of interest the authors declare that they have no conflicts of interest. key: cord- -zqmychmr authors: stall, nathan m.; johnstone, jennie; mcgeer, allison j.; dhuper, misha; dunning, julie; sinha, samir k. title: finding the right balance: an evidence-informed guidance document to support the re-opening of canadian nursing homes to family caregivers and visitors during the covid- pandemic date: - - journal: j am med dir assoc doi: . /j.jamda. . . sha: doc_id: cord_uid: zqmychmr abstract during the first few months of the covid- pandemic, canadian nursing homes implemented strict no-visitor policies to reduce the risk of introducing covid- in these settings. there are now growing concerns that the risks associated with restricted access to family caregivers and visitors have started to outweigh the potential benefits associated with preventing covid- infections. many residents have sustained severe and potentially irreversible physical, functional, cognitive, and mental health declines. as canada emerges from its first wave of the pandemic, nursing homes across the country have cautiously started to reopen these settings, yet there is broad criticism that emerging visitor policies are overly restrictive, inequitable and potentially harmful. we reviewed the nursing home visitor policies for canada’s ten provinces and three territories as well as international policies and reports on the topic to develop evidence-informed, data-driven and expert-reviewed guidance for the re-opening of canadian nursing homes to family caregivers and visitors. for nursing homes. , as the community prevalence of covid- continues to decrease in canada, and regions across the country begin phased re-openings, experts and advocates have grown increasingly concerned that subsequent visiting policies and family caregiver access to nursing home settings remain overly restrictive, causing substantial and potentially irreversible harm to the health and wellbeing of residents. a more balanced approach is needed that both prevents the introduction of covid- into nursing homes, but also allows family caregivers and visitors to provide much needed contact, support and care to residents, to maintain their overall health and wellbeing. we reviewed the emerging nursing home visitor policies issued by canada's ten provincial and three territorial governments (see appendix ) as well as international policies and guidance on the topic in order to recommend, evidence-informed and data driven guidance to support a balanced, risk-mitigated re-opening of canadian nursing homes to family caregivers and visitors. while this guidance is specific to nursing homes, many of the guiding principles and planning assumptions presented in this document could be applied to other congregate settings such as retirement homes and group homes. these efforts should be executed with the support and input of family caregivers, existing resident and family councils as well as from nursing home medical directors, administrators, involved primary care and specialist providers, and local ipac and public health leadership. we also recognize that reopening nursing homes will require additional resources including government funding for personal protective equipment (ppe), covid- testing, and addressing chronic staffing shortages to support visitor protocols. importantly, homes must ensure that existing care resources are not reduced to support this implementation, which could negatively impact resident care, especially for those residents who do not have family caregivers or visitors. family caregiver: is any person whom the resident and/or substitute decision-maker identifies and designates as their family caregiver. as essential partners in care, they can support feeding, mobility, personal hygiene, cognitive stimulation, communication, meaningful connection, relational continuity, and assistance in decision-making. essential support worker: is a person performing essential support services (e.g., food delivery, inspector, maintenance, or personal care or health care services such as phlebotomy or medical imaging). general visitor: is neither a family caregiver nor an essential support worker and is "visiting" primarily for social reasons. in reviewing the literature, consulting with national and international experts (see acknowledgements), and hearing from both residents, and their family caregivers and visitors through various forums, we have identified six core principles and planning assumptions as foundational and fundamental to any current and future guidelines. these recommendations focus on family caregivers and general visitors rather than essential support workers and nursing home staff, and are made with the acknowledgement that the approach to visiting may need to be dynamic based on the community prevalence of covid- . . policies must differentiate between "family caregivers" and "general visitors". residents, determine who is essential to support them in their care. it is imperative that visitor policies identify and distinguish "family caregivers" from "general visitors" who are visiting primarily for social reasons. while socialization is certainly important, family caregivers as partners in care should be prioritized to support resident health and wellbeing. family caregivers are those individuals who assume essential caregiving responsibilities for a spouse, family member, or friend who needs help because of limitations in their physical, mental, or cognitive functioning, and are essential to meeting the needs of residents especially in the face of chronic staffing shortages. [ ] [ ] [ ] [ ] [ ] [ ] family caregivers also help ensure that all residents receive culturally safe and appropriate care, especially for lgbtq s+ and indigenous residents and/or those with language barriers. importantly, while the term family caregiver is widely used, it is important to recognize that approximately % of all caregivers are not related to their care recipients, including some who may be privately hired. the importance of identifying family caregivers is that they are not accessing the nursing home primarily for social reasons, but rather to provide services and care such as assistance with feeding, medical decision-making, and management of responsive behaviours among residents living with dementia. while the definition of family caregiver has been operationalized in various ways, in a resident- centred and caregiver-partnered long-term care system, residents must have the sole authority and autonomy to determine who is essential to support them in their care; substitute decision makers should make this determination for incapable residents. this differs from approaches such as those used in australia that have relied on identifying family caregivers as those individuals with a clearly established and regular pattern of involvement in contributing to the care and support of residents prior to the covid- pandemic. this definition fails to recognize that some individuals may be willing and able-or need to-assume caregiving responsibilities to assist with special care needs and staffing shortages that have been further aggravated during the covid- pandemic, or provide care which they may not have been able to previously. it also fails to recognize that as conditions change during a pandemic, so too might a resident's desire or need for support change, and their ability to designate family caregivers must be flexible, consistent with their ongoing right to choose. it also fails to address that limiting or eliminating congregate dining and recreational activities during the covid- pandemic may now necessitate that those who were once "general visitors" become "family caregivers" to better address unmet resident needs. other definitions being proposed also violate the principles of resident-centred and caregiver- partnered care, including those that identify family caregivers as those individuals providing services that would otherwise require a private duty caregiver; this definition could be open to interpretation and a source of disagreement between nursing homes, residents and their families. , given there are both diverging definitions and interpretations of who constitutes a family caregiver, residents, substitute decision makers and their families must retain the authority and autonomy to designate their own family caregivers and this should be clearly documented in the resident's care plan and record. initially, each resident should be supported in allowing the reintroduction of at least two family caregivers, and these individuals should receive a caregiver identification card or badge. , strict blanket 'no visitor' policies were enacted early on during the pandemic with the recognition that visitors were potential vectors for the introduction of covid- infection into nursing homes and transmission back into the wider community. when these policies were implemented, nursing homes were more vulnerable to covid- outbreaks for several reasons: ) the extent of asymptomatic transmission and atypical presentations of covid- were not fully appreciated, ) access to timely and comprehensive covid- testing was limited, impairing homes ability to identify outbreaks, and determine scale and scope, including symptomatic and asymptomatic cases, ) many homes had not fully adopted robust ipac approaches including universal masking of staff and enabling them to work at only one healthcare setting, and ) access to ppe was more limited. - now that many homes are working to address these deficiencies, it is essential that we also focus on the considerable detrimental effects of the ongoing lockdown of nursing homes and restricted access to family caregivers and general visitors. , many residents have experienced severe and potentially irreversible functional and cognitive declines, deteriorations in physical and mental health, severe loneliness and social isolation, worsening of responsive behaviours and increased use of psychotropic medications and physical restraints. [ ] [ ] [ ] [ ] worse, many residents have died alone without family present to support end-of-life needs. while virtual visiting was implemented to try and meet the psychosocial needs of residents, it is no substitute for family caregivers who prior to the lockdown were providing substantial care and support for many residents. these negative outcomes have raised concerns that the risks associated with ongoing blanket visitor restrictions outweigh the benefits associated with preventing covid- outbreaks in nursing homes, particularly in canadians jurisdictions with low rates of community transmission. additionally, these restrictions may be violating the autonomy of residents and their right to make informed and risk-based decisions which prioritize their access to visitors over the risks of them contracting covid- . in ontario, the long-term care homes act recognizes the right of every resident to "receive visitors of his or her choice…without interference", which is legally required and enforceable under contract as set out in the act. there are also several active legal challenges across the country arguing that fundamental resident and human rights are being violated. visitor policies must prioritize equity over equality, recognizing that a "one size fits all" approach is neither optimal nor practical. whereas equality would mean giving all nursing home residents the same access to visitors, equity means giving nursing home residents the right amount of access they need to maintain their health and wellbeing. importantly, visitor policies must not prioritize the convenience of the nursing homes over the best interests of their residents in receiving the care and support of family caregivers and visitors. nursing homes must reserve the right to create and implement visitor screening protocols consistent with local public health guidance and procedures for visits that maintain the safety and wellbeing of all residents and staff members. however, blanket implementation of policies must be avoided, and instead policies uniquely supporting family caregivers and general visitors must be both flexible and compassionate, recognizing that some of the new conditions and procedures surrounding visiting may not work for all residents, family caregivers and visitors. , , this includes providing flexibility around the timing of visits (e.g., some visitors may have work and other caregiving duties), the location of visits (e.g., some residents and/or visitors may not be able to tolerate outdoor visits because of inclement weather and/or bedbound status), the length or frequency of visits (e.g., as some visitors may be traveling long distances, longer visits should be considered), absolute restrictions on physical contact (e.g., some residents with cognitive impairment and/or behavioural issues may neither be able to understand nor comply with physical distancing). it is imperative that individual homes, with the support of local health authorities and public health units, collect and report data on covid- cases as it relates to reopening. in canada, the national institute on ageing long-term care covid- tracker could support this (https://ltc- covid -tracker.ca). it is recognized that many decisions about balancing different risks to residents, staff, family caregivers and visitors to nursing homes are difficult. however, it is also true that it is less difficult to impose restrictions than it is to remove them. public health and governmental authorities should also be actively working to use modelling and evidence to remove visitor restrictions as quickly as possible as regional community prevalence declines. in order to find the right balance between infection prevention and supporting resident health and wellbeing, the six core principles and planning assumptions described in this guidance document were used to create recommended, evidence-informed, and expert-reviewed visitor policies for family caregivers (table ) and general visitors ( table ) to nursing homes. all authors meet the international committee of medical journal editors (icmje) criteria for authorship and have no competing interests, financial or otherwise. recommended policy . defining an "family caregiver" • residents, substitute decision makers and their families must retain the authority and autonomy to determine who is essential to support them in their care and designate their own family caregivers. • governments, public health authorities and homes must not define who is a family caregiver, especially on the basis of either an individual's caregiving involvement and role prior to the pandemic or by identifying those individuals providing services that would otherwise require a private duty caregiver. • a resident may designate at least two family caregivers. • similar to guidance from alberta health services, a resident may identify a temporary replacement family caregiver if the primary designated family caregivers are unable to perform their roles for a period of time; the intent is not for designates to change regularly or multiple times but to enable a replacement, when required. . allowable number of family caregivers in the nursing home at one time • one family caregiver per resident should be allowed in the home at a time. • under extenuating circumstances (i.e., end-of-life), this allowable number should be flexible. • as essential partners in care, family caregivers should have access to areas both outside and inside the home (similar to staff members) but must maintain physical distancing from other residents and staff. they should be provided with an individualized caregiver identification and/or badge, and must abide by all ipac and ppe requirements and procedures concerning staff members of the home. , • in order to promote relational continuity and meet the ongoing needs of residents, family caregivers should still have access to the home during a covid- outbreak, as long as the following conditions are met: -the family caregiver attests that they understand and appreciate they are entering a home under outbreak and that they may be at increased risk of covid- infection -they must be trained in ipac procedures and the proper use of ppe and abide by all outbreak-related policies that apply to staff members of the home. • no restrictions as long as it does not negatively impact the care of other residents or the ability of other family caregivers to provide care and support. • as partners in care, family caregivers should be subjected to the same covid- screening requirements as nursing home staff. if asymptomatic covid- testing is recommended, family caregivers should be provided with the same access to testing as staff members of the home. • as partners in care, family caregivers should receive an orientation and be educated and trained to follow the same ipac and ppe requirements and procedures as staff members of the home, including remaining masked at all times. the ottawa hospital has designed a ppe training video specifically for family caregivers: www.youtube.com/watch?v=gkayc wcn c&feature=youtu.be • homes must maintain ample ppe supply to enable family caregivers' participation in care. • failure of family caregivers to comply with these procedures could be grounds for loss of their rights to participate in care as family caregivers, which should be appealable. • outdoors: similar to guidance from the saskatchewan health authority, outdoor visits can include more than one visitor at a time, provided that physical distancing can be maintained. additionally, family members from the same household and/or bubble should not have to physically distance from one another. • indoors: one visitor per resident in the home at a time. similar to guidance from the british columbia centre for disease control, a visitor who is a child may be accompanied by one parent, guardian or family member. • outdoor visits should be prioritized, when possible and feasible, to both minimize the risk of covid- transmission and to maximize the number of possible visitors. provinces like manitoba plan to construct outdoor, all-season visiting shelters. • when outdoor visits are not feasible for either the resident or the visitor (e.g. for cognitive, psychiatric or physical reasons), the home must provide an indoor alternative which provides ample open space for physical distancing and adequate ventilation. • exceptional circumstances may sometimes necessitate the visitor meeting the resident in their room, but this should be a last resort if none of the previously noted alternative options are deemed feasible. • if the home goes into covid- outbreak status, general visits may need to be temporarily suspended (if advised by the local public health authority), but if the outbreak does not involve the entire home, consideration should be given to suspending visits only on the floor or unit under outbreak. virtual visits must be upscaled during suspensions of in-persons visits. • as per the ontario ministry of long-term care, visits should be at least minutes/visit and residents should have access to visitors at a minimum of once per week. • visitors must pass an active screening questionnaire (which may include an on-site temperature check) but there should be no requirement for covid- testing for outdoor and physically distanced visits. if exceptional circumstances necessitate a visitor entering the resident's room, they should be subject to the same screening and testing requirements as family caregivers. • visitors must remain masked (cloth or surgical/procedure for outdoor visits and surgical/procedure for indoor visits) at all times and maintain at least metres of physical distance from the resident they are visiting. visitors should be encouraged to bring their own cloth masks for outdoor visits, but appearing without a mask should not be a barrier to visiting. • if masking of visitors causes distress to the resident (e.g. for cognitive or mental health reasons) or poses difficulties with either recognizing (e.g. cognitive impairment) or understanding the resident (e.g. hearing-impaired residents who rely on lipreading) a face shield which wraps around the chin or a transparent mask can be considered as alternatives. • consideration may be given to allowing brief hugs and handholding while maintaining as much distance as possible between the faces of the resident and visitor, and ensuring the availability of alcohol-based hand sanitizer for prompt and effective hand hygiene both immediately before and after these encounters. • homes must maintain ample ppe supply to enable resident visits. • failure of visitors to comply with procedures could be grounds for a loss of visiting rights, which should be appealable. • visitors must have access to bathrooms (an accessible outdoor sheltered bathroom or designated indoor bathroom). • outdoor visiting must occur in weather protected settings (e.g. a shaded area with hydration for hot weather, a sheltered area for rain, or a heated area for colder weather). • residents designated as being "critically ill" or at "end-of-life" (< -day life expectancy) should be provided with the same level of access that would be rendered to a family caregiver. if visitors need to enter the home under these circumstances, they should be subject to the same conditions and procedures as "family caregivers". ipac guidance will be provided. . end-of-life considerations: -designated essential visitor is permitted to visit "as much as required". -there is no limit on the number of different individuals who can visit overall, but visits must be coordinated with the care team and the site. -up to two designated family/ support persons at a time are allowed to visit as long as physical distancing can be maintained between the family/support persons. *may leave the home for medically necessary care or treatment. yes, defined as: -visits considered paramount to resident care and well being, such as assistance with feeding, communication, personal care, emotional support or mobility. -existing registered volunteers providing services as described above only. yes, defined as: -"caregivers who provide or would like to provide significant assistance and support to a loved one to meet their needs and contribute to their integrity and well-being. assistance and support may include: helping with meals; supervising and being attentive to the person's overall condition; providing support with various daily or recreational activities; assistance with walking; providing moral support or comfort". -a significant caregiver…residents must have received support from the person before visiting restrictions were put in place due to covid . -visitors are only allowed in chslds, intermediate or family-type resources (sapa program) or private seniors' homes without a covid- outbreak. a visitor is anyone who wants to visit the person in the home and who does not meet the criteria to be identified as a caregiver. . may designate more than one essential family caregiver. . a maximum of two essential family caregivers from the same household can be in the home at a time. . yes, as of june , . . no limit on frequency or on length of time. . self-monitoring of symptoms. no testing requirement. most sign a consent form stating that "their decision was informed and voluntary, with full knowledge of the associated risks and knowing that they could become infected during their visits or even infect their loved one." . must remain continuously masked and wear ppe as required. will be given a face shield. training of visitors and procedural masks must be available in sufficient quantity for visits to be allowed. . compassionate visits will be permitted when death is imminent ( - hours). a maximum of two visitors are allowed at one time. pandemic experience in the long-term care sector: how does canada compare with other countries? family presence in older adult care: a statement regarding family caregivers and the provision of essential care infection prevention and control for covid- : interim guidance for long term care homes better together: re-integration of family caregivers as essential partners in care in a time of covid- easing lockdowns in care homes during covid- : risks and risk reduction. ltccovid, international long-term care policy network ontario ministries of health and long-term care. covid- directive # for long-term care homes under the long-term care homes act challenges, solutions and future directions in the evaluation of service innovations in health care and public health words matter: the language of family caregiving families caring for an aging america nothing informal about family caregiving we should care more about caregivers considerations on caring for caregivers in an aging society why canada needs to better care for its working caregivers essential family caregivers in long-term care during the covid- pandemic registered nurses' association of ontario. person-and family-centred care the change foundation. the caregiver identification (id) program and family presence policy the ontario caregiver organization. partners in care: a resource for welcoming back caregivers to hospitals and long-term care epidemiology of covid- in a long-term care facility in king county, washington presymptomatic sars-cov- infections and transmission in a skilled nursing facility typically atypical: covid- presenting as a fall in an older adult a hospital partnership with a nursing home experiencing a covid- outbreak: description of a multiphase emergency response in toronto groupe de recherche et d'etude des maladies infectieuses -paris s-e. severe acute respiratory syndrome coronavirus (sars-cov- )-related deaths in french long-term care facilities: the "confinement disease" is probably more deleterious than the coronavirus disease- (covid- ) itself detrimental effects of confinement and isolation on the cognitive and psychological health of people living with dementia during covid- : emerging evidence international long-term care policy network loneliness and isolation in long-term care and the covid- pandemic nursing homes or besieged castles: covid- in northern italy loneliness and social isolation during the covid- pandemic ontario government warned that isolating seniors is elder abuse and violates the charter legal warnings issued to long-term care homes who continue to isolate seniors one person per resident. . not specified. . not specified. . must be screened upon entry and includes temperature checks must be supported by staff in appropriately using ppe maximum of two visitors at a time outdoor visits only in designated areas on the grounds must wear a non-medical mask and maintain physical distancing. must follow ipac guidelines. may remove mask once at the designated visiting area if physical distancing can be maintained and if needed for effective communication documents/covid- -management-in-long-term-care-facilities-directive.pdf indoors and outdoors self-monitoring of symptoms must remain continuously masked in the home and wear ppe as required. training of visitors and procedural masks must be available in sufficient quantity for visits to be allowed we gratefully acknowledge the numerous experts who reviewed and commented on this guidance -their names are listed below. none of them received compensation for their contributions. *residents allowed outside as long as they are physically distancing.yes, defined as:-where the resident's quality of life and/or care needs cannot be met without the assistance of the "designated essential visitor".-may be a family member, friend, religious and spiritual advisor or paid caregiver. appendix : nursing home visitor policies for canada's ten provinces and three territories (as of july , ) . creative solutions:-some homes to set up large, -sided plexiglass cube that will shield residents from their visitors to allow for enhanced communication; -plans to add disposable gloves that would 'poke through' the plexiglass allowing families to hug; -large, marquis-style tents that will be put up in the gardens and available in the rain or shine. key: cord- - rws authors: bonal, xavier; gonzález, sheila title: the impact of lockdown on the learning gap: family and school divisions in times of crisis date: - - journal: int rev educ doi: . /s - - -z sha: doc_id: cord_uid: rws the lockdown of schools in spain to confront the effects of covid- caused an enormous impact at both societal and educational levels. schools and families had to react rapidly to a new teaching and learning scenario without the benefit of previous planning or government guidelines. in this context, some schools were better able to adapt to the new circumstances than others. likewise, the structure and size of families’ economic, social and cultural capital produced significant differences in the learning opportunities for children from different backgrounds. this article assesses the impact of the school lockdown on the learning gap between children from different social backgrounds in catalonia. based on , responses to an online survey administered between and march to families with children aged between and , the authors’ analysis shows that learning opportunities varied significantly. middle-class families were able to maintain higher standards of education quality in a critical context, while children from socially disadvantaged families had few learning opportunities both in terms of time and learning experiences (schoolwork and maintenance of after-school activities). results differed by type of school (public/private) where students were enrolled, family economic, social and cultural capital, and family living conditions. in the final part of the article, the authors highlight the importance of the role of the school in ensuring learning opportunities for children from low socioeconomic backgrounds, and they discuss some policy implications of their findings. l'impact du confinement sur les écarts en matière d'apprentissage : disparités familiales et scolaires en période de crise -la fermeture des écoles en espagne durant le confinement pour faire face aux effets de la covid- a eu d'immenses répercussions sur les plans sociétal et éducatif. les écoles et les familles ont rapidement réagi au nouveau scénario de l'enseignement et de l'apprentissage sans pouvoir s'appuyer sur une planification préalable ou des directives gouvernementales. dans ce contexte, certaines écoles ont réussi mieux que d'autres à s'adapter à ces nouvelles circonstances. de même, la structure et la taille du capital économique, social et culturel des familles a montré qu'en fonction de leur milieu, les enfants avaient des possibilités d'apprendre très inégales. cet article évalue l'impact de la fermeture des écoles en espagne durant le confinement sur les écarts en matière d'apprentissage chez des enfants de différents milieux sociaux en catalogne. s'appuyant sur réponses à une enquête en ligne menée entre le et le mars auprès de familles avec des enfants âgés de trois à dix-huit ans, l'analyse des auteurs révèle de considérables disparités concernant les possibilités d'apprendre. les familles de la classe moyenne ont réussi à maintenir un niveau d'éducation élevé dans cette situation critique, tandis que dans les familles défavorisées sur le plan social, les possibilités des enfants étaient restreintes, tant en termes de temps que d'expériences éducatives (devoirs et maintien d'activités extrascolaires). les résultats étaient différents en fonction du type d'établissement (public/privé) où les élèves étaient inscrits, de la situation économique de la famille, du capital social et culturel de cette dernière et de ses conditions de vie. dans la dernière partie de l'article, les auteurs soulignent l'importance du rôle de l'école pour garantir la possibilité d'apprendre aux enfants de milieux socioéconomiquement faibles. ils abordent en outre un certain nombre de conséquences qu'entraînent leurs constatations pour les politiques en matière d'éducation. el impacto del cierre escolar en la brecha de aprendizaje: divisiones familiares y escolares en tiempos de crisis -el cierre de escuelas en españa para hacer frente a los efectos de la covid- causó un enorme impacto tanto a nivel social como educativo. escuelas y familias tuvieron que reaccionar rápidamente a un nuevo escenario de enseñanza y aprendizaje sin contar con planificación previa o con directrices gubernamentales. en este contexto, algunas escuelas fueron capaces de adaptarse mejor a las nuevas circunstancias que otras. asimismo, la estructura y el tamaño del capital económico, social y cultural de las familias produjeron diferencias significativas en las oportunidades de aprendizaje de los niños y niñas de diferentes orígenes. este artículo evalúa el impacto del cierre de las escuelas en la brecha de aprendizaje entre el alumnado de diferentes orígenes sociales en cataluña. sobre la base de . respuestas a una encuesta en línea realizada entre el y el de marzo de a familias con hijos e hijas de entre y años, el análisis muestra que las oportunidades de aprendizaje varían significativamente. las familias de clase media pudieron mantener niveles más altos de calidad educativa en un contexto crítico, mientras que los niños de familias socialmente desfavorecidas tuvieron pocas oportunidades de aprendizaje, tanto en términos de tiempo como de experiencias de aprendizaje (tareas reducing physical contact has been the most common strategy adopted by governments to reduce the spread of covid- . it has led most countries around the world to close their schools for periods of time. the lockdown of schools in spain, one day before the declaration of a state of alarm on march , has had an enormous impact at both societal and educational levels. schools and families had to rapidly adjust to a new teaching and learning scenario without the benefit of previous planning or guidelines from the spanish ministry of education or regional departments of education. in this context, some schools were better able to adapt to the new circumstances than others. likewise, the structure and size of families' economic, social and cultural capital (resources) produced significant differences in the learning opportunities for children from different socioeconomic backgrounds. while the covid- pandemic has no precedent in terms of effects on the economy and social life, schools have previously experienced periods of closure. for example, large-scale outbreaks other diseases (such as ebola and influenza), teacher strikes, natural disasters and violent conflicts have already forced schools to stop their activity in the past. researchers and international organisations have studied the effects of school closures on students' learning and found a measurable loss in the acquisition of basic skills, particularly for the most disadvantaged children (quinn et al. ; cattaneo et al. ) . it is highly likely that the current pandemic will have a dramatic long-term impact on students' competencies and increase existing education inequalities. in this article we reflect on how school closure produces unequal learning opportunities in terms of both formal and non-formal education for children and youth from different socioeconomic backgrounds attending different types of schools in catalonia. during the covid- catalan lockdown, the instructional time received by students from different social backgrounds has been unequal, as has the educational value of the activities developed at home in non-school time. we investigated what learning opportunities were available to students from different backgrounds by administering an online survey during the second week of the state of alarm (i.e. between and march ). hypothesising that exposure to learning (both formal and non-formal) among children from lower-income households was likely to be lower than for their peers from higher-income families, we expected this to irremediably increase the existing learning gap between them. based on the results of our survey, this article reflects on how students' social background and conditions of confinement were associated with their learning opportunities. these unequal opportunities, in turn, are likely to exacerbate existing inequalities in skills acquisition and academic performance. we begin our article with a review of existing evidence on learning losses and their unequal impact on different groups of students due to past periods of school shutdown. the next section provides information on the context in which we conducted our own survey and the methodology we used to analyse the data. we then present our key findings regarding inequalities in formal and non-formal education, while the final section draws conclusions and discusses some policy implications of our findings. although it has only been a few months since schools in many countries around the world closed due to the covid- pandemic, research on the educational effects from that closure has already been notably rich. obviously, most of these studies have measured the short-term effects of the lockdown. it is certainly too early to know whether these effects can be considered a learning loss or just a temporary effect as a result of disuse that can be easily regained with practice (coe et al. ) . beyond this recent evidence, several previous studies based on student absenteeeism and past school closures have focused on the impacts of being out of school on learning outcomes (abadzi ; eef ) . these research studies can help us understand the potential effects of current school closures on learning and the mechanisms by which educational inequalities occur. some of the reasons causing schools to reduce or even stop their activity in the past range from summer school holidays, student or teacher absenteeism and teacher strikes to violent conflicts. extant research evidence based on these experiences identifies the existence of a general loss of learning connected to school absence, which can be more or less severe depending on students' previous performance, family characteristics, age, and education pathway, among other factors. school closure, even when distance (remote) learning is offered, usually implies a reduction in instructional time and, by consequence, also a decline in learning time. past studies have consistently identified a positive relationship between learning time and student achievement -along with other educational outcomes (abadzi ; hanushek ; scheerens ) . therefore, some studies have used instructional time as a proxy for "opportunities to learn". variables that measure exposure to curriculum seem to show a greater effect on learning than variables related to teacher behaviour or school climate. students' prolonged and repeated exposure to stimuli and feedback has been identified as a key educational resource. even though evidence is not always conclusive (cattaneo et al. ) , most research suggests that students with a migrant background, and those who are socioeconomically disadvantaged, will be slower learners. thus, these students are likely to benefit more from an increase in learning time than their peers (dahmann ; gromada and shewbridge ; huebener et al. ). consequently, not going to school reduces learning opportunities for all, but particularly for students from low-income backgrounds and less-skilled children. student (or teacher) absenteeism can also be understood as a lack of instructional time. student absenteeism results in poorer academic achievement, gaps in skills development, abilities and behaviours necessary for educational success, and an increased likelihood of school dropout. effects go beyond the school environment: absence has also been connected to risky behaviours such as smoking, juvenile delinquency, alcoholism, drug use, risky sexual behaviours or unwanted pregnancies, as well as other effects in adulthood (e.g. unemployment, alcoholism) which diminish life opportunities for young absentees (abadzi ; coe et al. ; gonzález motos ) . in spain, as in other european countries, the school lockdown between march and june seamlessly merged with the beginning of the summer holidays, resulting in students' absence from school lasting six months. past studies comparing students' learning progress during the school year with the summer break have consistently shown that achievement tends to slow or decline over the summer holidays. although there is no agreement on the magnitude of the learning loss during holidays (von hippel ), it is clear that it is steeper for mathematics than reading, and it is especially acute for upper-grade students . moreover, research points out that this loss does not affect all students equally; during the summer period, educational inequalities between socially advantaged and disadvantaged children increase (alegre ) . beyond the evidence related to breaks in the ordinary school year (e.g. absenteeism, summer break, instructional time), other research studies have analysed the effects on students' learning caused by external factors. for example, the literature on weather-related school closures (e.g. as a result of heavy snowfall or hurricanes) also contributes to a better understanding of the potential unexpected consequences of disruptive closures. most of the evidence is based on small samples, is geographically specific and refers to short break periods. however, in all cases, studies have found a clear effect of each day of school cancellation on achievement, especially among students attending less-resourced schools or coming from low-income families (goodman ; marcotte and hemelt ) . teacher strikes have also forced a sudden cancellation of classes. strikes affect student learning outcomes (mainly in terms of achievement and grade repetition) by reducing the time that children attend school, but they also affect the quality of schooling and increase the likelihood that students may engage in risky behaviour (jaume and willén ). in addition, research has found heterogeneous (uneven) effects, with children from vulnerable families being most affected by school disruptions (jaume and willén ; belot and webbink ). the main difference between school closures examined by previous studies and the current lockdown is that this time schools have managed to maintain some contact with students, mostly through online instruction. however, despite the efforts to support remote learning, many students have not had access to it. existing data about the digital gap and surveys conducted during the lockdown indicate that teachers have been unable to contact a significant number of students, mainly because of lack of an internet connection or adequate devices to engage in distance learning ; van lancker and parolin ). even when students have been able to connect, remote learning during school closures seems to have widened the existing attainment gap between students from different socioeconomic backgrounds (coe et al. ) . for instance, a recent study undertaken by researchers from brown and harvard universities into the use of an online mathematics programme (zearn) before and during lockdown in the united states (chetty et al. ) shows a sharp decline of student progress in mathematics in classrooms located in low-income zip (postal) code areas, while in high-income zip code areass no changes were observed (goldstein ) . before the covid- pandemic, in-school learning had already been proven to be more effective than distance learning. furthermore, student outcomes resulting from online learning have been shown to be poorer, on average, than outcomes resulting from face-to-face instruction (heppen et al. ) . conditions for effective remote learning (good internet connection, and clear explanations, scaffolding and feedback from teachers) are not easy to accomplish. the combination of a digital gap with teacher inexperience in providing high-quality distance learning makes it difficult to improve students' learning opportunities . moreover, there is now a significant risk that vulnerable students have less access to quality teaching than their peers, widening the attainment gap due to the school lockdown (coe et al. ; kay et al. ) . if it has already been proven that students from low-income families experience more interruptions and disruptions of their instructional time under normal school conditions (abadzi ; alegre and benito ) , then poor distance learning can be understood as a new obstacle to effective learning. apart from school organisation or distance learning strategies developed by teachers, parental engagement in children's learning, practised in some families alongside school attendance, becomes more important when school is replaced by at-home instruction. educational studies have demonstrated that parental involvement and the quality of learning at home improve academic outcomes. research in this field has consistently observed greater academic achievement among students whose parents are actively involved in the educational process. it has also found a clear relationship between engagement in the learning process and parental background in terms of social class and ethnicity (oreopoulos et al. ) . unequal parental capacities to help children with their homework and different uses of family time have been extensively documented (meyer et al. ; mora and escardíbul ) . therefore, family reactions to school lockdown are likely to produce significant differences in the learning opportunities for children from different types of social background (burgess and sievertsen ) . regardless of their causes and mechanisms, previous crises have had a more intense and negative effect on student learning outcomes in contexts with higher proportions of disadvantaged families (borse et al. ; chen et al. ; iqbal et al. ; sadique et al. ; shores and steinberg ) . previous research also suggests that the learning loss can not only have a limited short-term effect, but may in fact result in cumulative losses (abadzi ). likewise, other researchers suggest that the current school lockdowns will widen the learning gap between vulnerable children and their peers, possibly even reversing the progress made during the last decade to narrow this gap (coe et al. ; van lancker and parolin ). on march , the catalan government ordered the closure of all schools and colleges (universities) due to the covid- pandemic. more than one million students in compulsory education were asked to stay at home. during the first two weeks of confinement, the catalan department of education advised schools not to provide new curriculum content, in an attempt to contain the adverse effects of the technological gap on educational inequalities. despite the department of education's order, some schools reacted rapidly to the new circumstances and switched to remote learning strategies. other schools stopped their activities entirely. between these two extremes, there were a range of reactions and responses. between and march, after two weeks of school closure and before the spanish government ordered a more restricted lockdown, we (the authors of this article) conducted an online survey with families whose children were aged between and . the survey was addressed to families with children enrolled in preschool ( - years old), primary school ( - years old), lower secondary education ( - years old) and post-secondary academic or vocational education ( - years old). organised into two main sets of questions, our survey included items, with a variable number of subquestions that branched out to adapt to respondents' particular contexts. the first group of items characterised the different social, spatial and technological conditions in which families had to respond to the school lockdown (such as the size of their home, the availability of outdoor spaces, access to the internet and to digital devices). the second set of questions was designed to assess children's learning opportunities in (a) formal education (contact with school teachers, frequency and types of school tasks, feedback provided by schools), (b) informal education (uses of time, family activities, support to carry out school tasks) and (c) non-formal education practices from home (participation in after-school activities, types of activities and forms of participation). in addition, our survey included variables to characterise respondents' social and economic conditions, such as family structure, parental education attainment, income, ethnic origin, gender and work status. to test the survey, we conducted a brief pilot. due to the exceptional circumstances, the pilot was implemented using an informal strategy: we asked families with children of different ages and on different school pathways to complete the survey. after the pilot was completed, we distributed the final version of the survey through various online communication channels (twitter, facebook and whats app). this strategy helped us to improve the survey's visibility among different social groups as well as reducing sampling bias linked to the use of social networks (blank ) . we also mobilised strategic contacts in the most deprived areas of catalonia to reach those families less likely to answer online surveys. in total, , people clicked the link to the survey, approximately , started to answer it and more than , families completed it. after excluding some respondents for reasons like incomplete answers, children's ages outside our survey's scope, families not living in catalonia, the final sample included information from , families (with a total of , children aged between and ). as expected, high-income and highly educated parents were over-represented. due to the lockdown and the urgent need for data collection, we were unable to complement our online survey with other strategies such as telephone calls or face-toface interviews. we opted instead to use non-response weighting to bring the sample closer to the population distribution. to do this, we weighted the sample by the level of parental education attainment, as a key variable of social and cultural differentiation, and as a reliable and accurate source of census data. table shows both the weighted and unweighted samples. of our survey respondents, % were female, . % lived in heterosexual biparental households, . % lived in single-parent households and . % were part of a same-sex couple. more than half of the households ( %) were composed of four members, three-member households represented % of the sample, and % of the households had five or more members. sample distribution according to school variables accurately reflects the distribution between the public and private school sectors. in catalonia, the proportion of children in compulsory schooling who attend public schools is % (this was . % in our sample), while % of students attend private subsidised schools ( . % in our sample), and only % are enrolled in private independent schools ( . % in our sample). our sample was also well balanced regarding children's ages, with a slight over-representation of younger students. for our data analysis, we split information across three databases according to different units of analysis. first, a "household database" included information about family members living together during the confinement. second, a "student database" collected information about the learning activities of children aged between and . third, the "after-school activities database" contained information about the extracurricular activities children were involved in before and after the lockdown. we also constructed some subsamples to carry out specific analyses. in this article, we present some data based on subsamples by children's age or educational level. in this section, we present and discuss the data from our survey regarding social differences in the learning conditions of catalan children and the impact of these conditions on learning opportunities during lockdown. inequalities in families' economic, social and cultural capital impacted on student learning opportunities by different means, including school responses to the lockdown, access to digital facilities and the level of parental learning support. this section reviews how these factors are drivers of inequalities in the three domains of learning: formal, non-formal and informal. learning conditions at home differ on the basis of a number of variables. for example, the amount of physical space and access to technological devices both have an impact on the learning conditions of children. our survey included questions about the size of the household and the outdoor spaces available, to serve as indicators of the physical conditions of confinement. the responses showed that most homes of confined families had a balcony or other outdoor space/s. however, there were significant differences among residents depending on the size of the municipality (families living in cities had less access to outdoor spaces) and other social indicators, such as parental education attainment. for instance, % of families with an adult who had completed compulsory education did not have outdoor spaces, while this was only the case for % of households in which at least one adult held a university degree. likewise, % of households in the poorest income quintile (q ) had less than square metres of space, reducing to % in the case of the richest income quintile (q ). students also had different internet connectivity conditions and unequal opportunities to access technological devices to carry out their schoolwork. since we could only implement an online survey, we were unable to grasp the full extent of the actual digital divide. data from the catalan department of education estimate that there are currently approximately , school children without access to an internet connection (vallespín ) , which would situate the digital gap at between % and % of all children in the catalan education system. our survey revealed that . % of our sample had only a mobile phone internet connection or no connection at all. most families in our survey had access to a high-speed internet connection. however, there were important differences regarding the "possibilities of use" of technology. for example, . % of respondents declared that they had access to a single device at home or only had access to a mobile phone. we calculated the number of devices per person and found that in % of cases there was less than one device per person. the digital divide can also be expressed in terms of access to devices based on the composition of the household. in our survey, % of households with two children had only one device available; % of households with three children had two or fewer devices available; and % of households with three children had three or fewer devices available. access to technology depends on the level of family income. while % of surveyed families in q (the lowest income quintile) had access to only one digital device, for families in q (the highest quintile), the corresponding figure was only %. furthermore, while % of families in q had access to four or more devices, this applied to % of families in q . taking into account the size of the household, % of families in q did not have access to one device per person. among families in q , the number of households with less than one device per person reduced to %. figure shows the proportion of households for each income quintile that had access to one or more device per person. differences are sorted by income, with an inequality factor of . between q and q . in addition, the unequal impact of the crisis on parents' working situation also altered the social and psychological conditions that ensured an adequate learning process. our survey revealed that before lockdown, % of adults were unemployed, while % were working full-time. when we asked about the impact of the covid- crisis on their working situation, these figures changed dramatically: % of respondents indicated that they had lost their job. of those who were still working, only . % were able to go to work "normally". the rest ( %) were working from home, either with the same schedule or with a more flexible schedule. of those still working, % considered it likely that they would lose their job. of those who were already or became unemployed, % knew that they were entitled to unemployment benefits, while the other % either knew that they were not entitled or did not know. our survey included questions regarding how much time children invested in schoolwork every day since the beginning of the school lockdown, how frequent the contact was with their school and teacher/s, how often they received online teaching lessons, whether they had to complete specific homework tasks, and how often these tasks were reviewed and returned to children. taking into account the intensity of all these tasks, we composed an index of opportunities to learn (otl). to compose the index, which ranged from to points, we normalised indicators and aggregated (combined) frequencies. we only used this index for a subsample of the older children aged between and , since assigned school tasks or online teaching were more unlikely for younger age groups. in terms of the index, . % of students had an otl equal to , meaning that they dedicated less than one hour a day to school tasks, had almost no communication with teachers and did not have homework to do or to be reviewed. at the other end of the index, . % of students had a maximum otl of , meaning that they dedicated more than four hours per day to schoolwork, had frequent contact with their teachers and received regular feedback for school assignments. the majority ( . %) of all surveyed students in this subsample had an otl score below points. interestingly, we found the otl score to be positively related to a number of variables. having greater access to digital devices, being enrolled in more advanced courses (older students had a higher otl), being native to spain or living in a higher-income household were all factors associated with higher otl scores. students enrolled in private schools, both independent and private subsidised ones, had significantly higher otl scores than those enrolled in public schools. there are explanations for this difference. for example, the catalan department of education announced that the first two weeks after the approval of the state of alarm would be a non-school period. therefore, a significant number figure opportunities to learn (otl) index, by school sector and educational level note: baccalaureate refers to two years of optional upper secondary education preparing students for tertiary level of public schools did not develop school tasks during these two weeks, waiting for new instructions from the department. despite this announcement, private subsidised and independent schools did not stop their teaching activity. one of the plausible explanations for this difference lies in the economic dependency of private schools on fees. they needed to keep providing a service to users despite the exceptional circumstances. figure shows the distribution of the otl index across school sectors for different educational levels. the bar chart reveals strong differences by school sector for students enrolled in the last years of primary education and for those in lower secondary education. the chart also shows how students in vocational education and training (vet) had the lowest otl of post-compulsory education. we also found the otl index to be clearly related to the level of parental education attainment and to family income. for instance, % of children in families from the richest quintile (q ) had an otl score of points or higher. this reduced to % in the case of the poorest quintile (q ). the absence of schooling increases the importance of families as teacher substitutes in the learning process. our survey included questions regarding whether adults in the family helped students in their school tasks during the relevant lockdown period. an initial remarkable result appeared in terms of gender: while % of female adults stated that they supported their children to do schoolwork, only % of male adults did. as expected, gender differences were clearly observed, and family support was higher in the case of younger children. figure shows that for children undertaking primary education, the support of mothers who had completed compulsory figure adult support for school tasks, by gender and highest parental education attainment education was comparable to that of mothers with higher educational (in bourdieu's terms) cultural capital. however, for students enrolled in lower secondary education, the differences increased dramatically: only % of mothers who had completed compulsory education helped with homework, while % of the most educated mothers did. providing support for school tasks to students in post-compulsory education declined to close to % for all groups. there are several reasons why support for schoolwork may not have been provided by adults in a student's household. these reasons are remarkably different depending on the level of parental education attainment. in those households in our survey with children enrolled in lower secondary education, % of the families with an adult who had a university degree and who did not provide support for schoolwork argued that the child did not need it. this reason was only argued by % of respondents from those households with adults who had completed only compulsory education. in this case, a lack of knowledge was proffered as a significant reason for not giving support (by % of respondents), which reduced to % for those parents with a university degree. differences in cultural capital are therefore reflected in the capacity and possibilities of families to help children with their school tasks. in addition, (and not just in times of school closure) families' cultural capital and everyday informal practices have effects on children's learning experiences and opportunities. interestingly, confinement, which maximises the interactive time between family members, provides ideal research conditions for assessing informal learning activities. figure reveals that in those households with children aged between and , there were three activities in which social differences were especially acute. first, accompanying children in reading was more frequent by far in families with high cultural capital: % of families with an adult who had completed university studies responded that their children aged between and read with an adult every day. this reduced to only % in the case of families with an adult who had completed compulsory education. second, differences were also visible in foreign language informal practices: % of families with an adult who had completed university studies responded that foreign language practice was done daily or several days a week, while this practice was carried out by only % of families with an adult who had completed compulsory education. third, sporting activities were practised several times a week by % of families with university degrees and only by % of families with compulsory education. by contrast, other activities were more frequent among families with lower cultural capital. the most relevant was playing video games: % of families with an adult who had completed compulsory education reported that their children of preschool age played video games every day or several times a week. this applied to only . % of families with parents who had completed university studies. the differences were also significant in the frequency of watching educational television programmes ( % of households with parents who had completed compulsory education and who had children enrolled in preschool education reported that their children did so every day, versus % of parents with a university degree), and in following and carrying out educational activities available via social media. in such an extreme situation of school absence, it appears that families with less cultural capital made more use of external resources to support their children's learning activities, while families with more cultural capital were more confident in their own abilities to respond to their children's learning needs. in the case of older children, the same differences were observed, although the gap increased in musical and other artistic activities and was less pronounced in playing video games. participation in after-school activities is a source of differential learning opportunities for children from different socioeconomic backgrounds (bradley and conway ; gonzález motos ; lauer et al. ; potter and morris ) . our survey compared whether children participated in one or more after-school activities before and after the beginning of the lockdown. we found that the effects of covid- on these kinds of activities have also increased the gap between socially advantaged and disadvantaged children. before lockdown, after-school activities were more frequent for students enrolled in primary education. this was particularly the case for children whose parent/s had completed a university degree ( %), compared to children from households with parent/s who had completed compulsory education ( %). this polarisation increased particularly for children enrolled in lower secondary education with at least one unversity-educated parent ( %) versus those whose parent/s had completed compulsory education ( %). we also found families' economic, social and cultural capital to be associated with the number of after-school activities in which children participated. for example, for families with two children in lower secondary education, the average number of after-school activities for highly educated households was . , compared to . for households with lower educational levels. sports, foreign languages and artistic practices were the most frequently reported after-school activities. by income level, students in families in the lowest quintile (q ) were more represented in sports and compensatory education activities, while children from the richest quintiles were over-represented in foreign language and musical after-school activities. as expected, we found that most after-school activities ( %) were interrupted during lockdown. however, economic and cultural inequalities were also visible in the probability of maintaining after-school activities despite the confinement. figure shows that children whose parent/s had lower educational levels already had lower participation in after-school activities before lockdown and were more likely to be unable to continue them after schools closed than children from families with higher educational levels. there are two main reasons for this difference in after-school activities after the closure of schools. first, families with higher economic, social and cultural capital participated in activities that were more likely to continue online (e.g artistic activities, foreign languages), compared to the activities more commonly practised by families with lower capital (e.g. sports). second, voluntary interruption of afterschool activities was also higher among families with lower ecoomic, social and cultural capital, due to difficulties in coping with the costs during times of crisis. for instance, compensatory education activities -which could be undertaken online -were voluntarily interrupted by % of families who had previously engaged in figure after-school activities before and after lockdown, by parental education attainment compensatory education, in this context, refers to those after-school classes that some students attend to reinforce their learning competencies. these classes are usually offered to students with marked learning difficulties. them. this voluntary interruption was particularly high among children whose parent/s attained compulsory education ( %), compared to those whose parent/s had a university degree ( %). despite schools' efforts to maintain learning activities during lockdown, our analysis reflects significant inequalities in exposure to school learning depending on family characteristics (income, level of educational attainment) and school characteristics (educational level, school sector). the absence of schooling neutralises the benefits of socialisation provided by early childhood education for the most vulnerable children. it also disrupts those processes of guidance and accompaniment which are especially important for adolescent students in their study, work and life transitions. in addition, the digital divide and visible differences in access to technological devices among students have left some children and young people without options to connect to learning for at least three months or, more probably, for six months. inequalities in being able to maintain school-based learning are not the only existing ones. our analysis shows that families with a lower level of parental education attainment have fewer resources and less knowledge to help their children with school tasks. these limitations become more acute when support from the school has been reduced and demands for autonomous work by students increase. likewise, family economic, social and cultural capital also influences the adoption of everyday informal learning practices that are more or less aligned with school logic, which readily increases differences in learning opportunities and familiarity with what is considered valid knowledge. after-school activities are also more likely to be maintained among children from wealthier families, which also increases the existing inequalities in this area. in sum, if the pre-covid- school system already had significant limitations in its ability to eliminate existing social inequalities, the closure of schools during this period of lockdown poses enormous challenges for developing effective policies to compensate for learning losses and learning inequalities. school lockdown has not affected all children in the same way, and significant material and human resources will be necessary to ensure that the most vulnerable children can catch up. an educational emergency plan with social and educational objectives will be needed to restore children's socio-emotional and cognitive skills. the individual and social costs of not intervening are just too high, unfair and unequal. it is the responsibility of states and countries to ensure the right to education in these difficult times. instructional time loss in developing countries: concepts, 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famílies sense ordinador o sense connexió per poder seguir les classes a distància covid- , school closures, and child poverty: a social crisis in the making is summer learning loss real? education next publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations he has been a member of the eu network of experts in social sciences and education (nesse) and is an editorial board member of several international journals on education policies and educational development. xavier has widely published in national and international journals and is the author of several books on the sociology of education, education policy and globalisation, and education and development. he has worked as a consultant for international organisations such as unesco, unicef, the european commission, and the council of europe she is a member of the institute of government and public policy (igop) and the globalisation, education and social policies (geps) research centre. her research interests include public policy analysis and social inequalities, with a special focus on education and migration. sheila has conducted several research projects in this field, and she has experience in policy analysis and policy evaluation the data that support the findings of this study are available from the corresponding author, upon reasonable request. authors' contributions: all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by xavier bonal and sheila gonzález. all authors read and approved the final manuscript. key: cord- -esc vwj authors: qiu, jingwen; wilkens, casper; barrett, kristian; meyer, anne s. title: microbial enzymes catalyzing keratin degradation: classification, structure, function date: - - journal: biotechnol adv doi: . /j.biotechadv. . sha: doc_id: cord_uid: esc vwj keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. it is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. keratin is packed by disulfide bonds and hydrogen bonds. based on the secondary structure, keratin can be classified into α-keratin and β-keratin. keratinases (ec . .-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. currently, the known keratinolytic enzymes belong to at least different protease families: s , s , s , s , s , m , m , m , m , m , m , m , m , m (merops database). the various keratinolytic enzymes act via endo-attack (proteases in families s , s , s , m , m , m ), exo-attack (proteases in families s , s , m , m , m , m ) or by action only on oligopeptides (proteases in families m , m ), respectively. other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. this review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. we also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications. livestock production is increasing rapidly throughout the world as a result of population growth, increasing incomes, changes in lifestyles and dietary habits. according to the un food and agriculture organization (fao), annual global poultry production is projected to reach more than . billion animals in and . billion in ( . kg carcass weight/animal), and bovine and pork meat production also continues to increase (alexandratos and bruinsma, ) . although the global coronavirus pandemic in has caused the united states department of agriculture to decrease their global animal protein prognosis in the spring of , the forecast for for global chicken meat production is still . million tons, and the global pork production is projected to be . million tons (united states department of agriculture, ). the waste or co-products from such animal meat production consists of keratinous materials such as chicken feathers, pig bristles, wool and horns and millions of tons of these co-products are produced each year. for instance, it has been estimated that in alone more than . million tons of chicken feathers were produced from poultry processing (li et al., ) . chicken feathers and other keratinous co-products from poultry processing plants and abattoirs are classified in category animal byproducts which entails that they are low-risk materials for animals, the public, and the environment (verma et al., ) . they can therefore be considered as an abundant protein or amino acid source for new upcycling processes targeting potential use in e.g. feed, fertilizers, cosmetics, and other applications (callegaro et al., ) . keratinous materials are unique because they are rich in certain amino acids, including in particular the sulfur-containing amino acid, cysteine, other amino acids like glycine, proline, arginine, and the essential amino acids valine, leucine, and threonine. the compact conformation and high stability of keratin is indeed mainly due to disulfide bridges formed among cysteine residues within and between keratin polypeptides (callegaro et al., ) . since keratin is an insoluble protein, its reutilization will have to include partial or complete degradation, without destroying the amino acids, to provide useful biorefinery options for the constituent protein and amino acids. keratinases belong to the serine-and metalloprotease families. the major keratinase family is the s family, and the subtilisin subfamily members in particular have keratinolytic activity. recently, an increasing number of other protease families are also reported to harbor enzymes having keratinolytic activity. for example, family m and m proteases secreted from the non-pathogenic fungus onygena corvina have been reported to catalyze degradation of pig bristle keratin, and moreover to act synergistically with enzymes from the s family . in addition, and as discussed later, several other fungal proteases from the m , s and s families have been reported to exhibit keratin degrading action (mercer and stewart, ) . thus, the efficiency of enzymatic keratin degradation may be improved by designing specific blends of keratinases originating from different protease families. additionally, other proteases, such as disulfide reductase (e.g. cysteine dioxygenase (kasperova et al., ) , thioredoxin-disulfide reductase), and other types of enzymes, e.g. lytic polysaccharide monooxygenases (lpmos) (lange et al., ) and certain enzymes involved in lipoprotein signaling or fatty acid degradation, have also been suggested as possible candidates for contributing to biocatalytic keratin degradation (lee et al., b) . although microbial and enzymatic keratin degradation has been a subject of research interest for some time, currently available overviews summarize keratinase data, but rarely discuss the enzymatic action details in relation to substrate structure and assessment method. also, an updated analysis of the protease family classification of keratin-degrading enzymes, possible activity synergies, and a discussion of other enzymes that may play a role in keratin degradation is not available. the present review aims to provide an overview of the current knowledge of keratin as a substrate for enzymatic attack in relation to enzyme-assisted upcycling of keratin. in addition to summarizing the essential knowledge of keratin, the review provides a systematic investigation of the currently known (sequenced) keratinolytic enzymes and their classification, an analysis of the available keratinase structures, and insight into the structural-functional traits of these enzymes. also included is a critical discussion of keratinase enzyme assays and a summary of other enzymes that have been suggested to contribute to keratin degradation. our goal is to provide a better understanding of the roles and functions of keratinases in nature, and help generate a better foundation for using keratin-degrading enzymes in keratin recycling processes. classification, especially regarding classification of skin. some authors classify skin as hard keratinous material (irwin mclean and moore, ; korniłłowicz-kowalska and bohacz, ) , while others classify skin as soft keratin based on that skin keratin only contains - % cysteine (safranek and goos, ; shavandi et al., ) . however, there is no doubt that feathers, claws, beaks, wool, and hair are all considered as hard keratinous biomass. keratin is also classified into α-keratin and β-keratin based on the secondary protein structure. in αkeratin, the α-helical-coils type i (acidic) and type ii (basic/neutral) protein chains are coiled together to form elongated α-helix filaments that form fibrils by interchain bonding (fig. , fig. ) , while β-keratin is mainly built of β-sheets (fig. ) . as indicated in table , α-keratin mainly occurs in mammals, while βkeratin is the major component of avian and reptilian tissues. in fact, β-keratin is found only in avian and reptilian epidermis (though one mammal, the pangolin, a unique scaly anteater, has been reported to have both α-and β-keratin in the keratin scales that cover its skin ), whereas α-keratin is found in mammalian as well as in avian and reptilian keratin (alibardi, ; greenwold et al., ; ng and li, ) . the α-keratin found in the epidermis located between the scales in reptiles and in feathers is responsible for the mechanical strength of the epithelial cells, their adhesiveness, and the changes in shape when stretched (alibardi, ; skieresz-szewczyk et al., ) . by contrast, the β-keratin in reptile scales exhibits limited extensibility yet significant microbiological resistance and hydrophobicity, and serves a protective function in nature (calvaresi et al., ) . in addition, some believe that α-and βkeratins are completely unrelated evolutionarily, and it has even been proposed recently that β-keratins should be renamed as corneous -proteins (ng and li, ) . (frenkel and gillespie, ) . α-keratin as regards α-keratin, the monomer molecular structure contains three partsa head domain (nterminal domain), a rod domain and a tail domain (c-terminal domain)presented in fig. (a) . although the central rod domain is the main driver that sustains self-assembly into filaments, the variable globular nonhelical head and tail domain at the end of intermediate filament proteins exert key roles in the assembly, organization and regulation of intermediate filaments (lee et al., ; rafik et al., ) . the rod domain consists of four right-handed α-helical subdomains, a, b, a and b, which are separated from one another by non-helical β-turns called ‗linker' regions (l , l and l ) (bragulla and homberger, ). as displayed in fig. (a) , the α-helical subdomains a, b, a and b are composed of repeats of seven amino acids in positions labeled a-g (a heptad pattern) (smith et al., ) . specific hydrophobic apolar amino acids in positions a and d, and charged amino acid residues in positions e and g, stabilize the α-helix (steinmetz et al., ) (fig. a) . two monomers (type i and type ii monomers) form a left-handed coiled-coil, a so-called heterodimer, as shown in fig. (b) (bragulla and homberger, ) . two heterodimers can aggregate to form a tetramer in four ways (fig. c ) -a , a , a and a cnvia disulfide bonds and hydrogen bonds (chou and buehler, ) . then, as illustrated in fig. (d) , two such tetramers associate into a protofibril while four protofibrils combine into socalled α-keratin intermediate filaments (ifs). an if is thus a structure comprised of these tetrameric α-helix structures in a packed assembly. the micro-fibrils contain several of these ifs that bind to each other and cross-link via disulfide bonds in a matrix of sulfide-rich keratin-associated proteins (kaps) (marshall et al., ) . the kaps include high-glycine tyrosine, high-sulfur, and ultrahigh-sulfur proteins (rogers, ) . in hard keratin such as hair, claws and horns, sulfur-rich and ‗ultra'sulfur-rich kaps are believed to determine the hardness of these corneous tissues (alibardi and segalla, ). the macro-fibrils thus appear to consist of larger assemblies of micro-fibril filaments with the kaps matrix surrounding them (fig. ) . on the basis of the structural analysis presented above, keratin is clearly a recalcitrant biomass mainly due to the bonding in the structure and the assembly of keratin monomers, dimers, tetramers, and ifs in the kap matrix. many intrachain and interchain disulfide bonds, hydrogen bonds and ionic bonds occur to reinforce the structural stability ( fig. a, b) . in particular, filaments are cross-linked with each other by disulfide bonds in a sulfur-rich kap matrix (fig. a, b) to form a highly resistant macromolecular assembly (gong et al., ) . beyond the primary structure post-translational modifications, such as phosphorylation or glycosylation also occur on keratins (bragulla and homberger, ). phosphorylation may enable or prevent the interaction of keratins with other molecules, such as signaling molecules, receptor molecules, etc. (kirfel et al., ) . specifically, the phosphorylation of amino acids in the head domain changes the overall net charge of this region to block any interactions with the rod domains of adjacent keratins, and thus prevents the assembly of keratin filaments (wöll et al., ) . glycosylation is another posttranslational modification of proteins, which results in an alteration of the binding or signaling functions of keratin (chou and omary, ) . the significance of glycosylation and phosphorylation in relation to enzymatic degradation of keratin appears unexplored. barbules (-hairs‖) fraction has slightly more α-helix than β-sheet structure, whereas the rachis (-stalk‖) has more β-sheet than α-helix structure (ng et al., ) . the available data for the other -keratin rich sources, e.g. for turtle scute, confirm that -keratin has higher glycine content and therefore more nonpolar amino acids than α-keratin (table ) . some interesting and curious details of the amino acid composition should be noted: penguin feather rachis appears for example to have more non-polar amino acids, especially glycine, than penguin feather barbs (table ) . this fact may explain why the feather rachis are more hydrophobic and more difficult to degrade than the feather barbs. the keratin amino acid sequences of rachis, barbules, barbules/barbs in chicken feathers have been recently reported (jin et al., ) . such data allow comparison of conserved keratin sequences of feather rachis (accession number: np_ . ), barbules (np_ . ) and barbules/barbs (np_ . ) (fig. a, b) . as many publications show, these three parts of feather keratin contain a -residue segment corresponding to the filament framework parry, , ) . based on the amino acid composition of the filament framework segments in such keratin (fraser and parry, ) , each β-sheet includes four β-strands as illustrated in fig. (c) for amino acid residues assigned a βsheet structure. these four β-strands can be parallel or antiparallel, and the chains are held together by hydrophobic interactions . from fig (c) , the apolar residues (mainly valine and some isoleucine) are also seen to be on the inner face of the sheet fold, while the charged and cysteine residues are on the sides or turns. the occurrence of a charged residue (arginine) in the outer β-strand in all three keratin structures (fig. c ) prevents the exposed edge of one sheet inappropriately bonding to the exposed edge of another sheet (fraser and parry, ). proline is found in the turn area in chicken feather keratin (see fig. c ) and also in other avian keratin (fraser and parry, ) ; proline plays the role of breaking the β-sheet which induces the formation of turns in the conserved β-rich central domain (calvaresi et al., ) . this four-strand β-sheet is the socalled monomeric structure of β-keratin (fig. d ). two such monomeric structures pack together via reverse stacking to form a dimer by hydrophobic face-to-face interactions ( fig. e ) (calvaresi et al., ) . in the dimeric structure, salt bridges, hydrogen bonds and disulfide bonds are formed between specific amino acids within the two monomers (calvaresi et al., ) . in addition, a head-to-tail assembly of four dimers forms a helix (fig. f ). this helix is created through hydrogen bonding and disulfide bonding to produce a left-handed helical ruled surface with four repeating units per turn (where the progressive rotation of the piled dimers is approx. °); this is the βkeratin filament (calvaresi et al., ; wang et al., ) (fig. f ). keratinases are principally type ec . .-.-peptidases (or peptide hydrolases) because their function is to catalyze the hydrolytic cleavage of peptide bonds. -keratinase‖ was formerly classified as ec . . . , but this particular classification was deleted in , and there is currently neither a or -digit ec number for keratinases. the lack of a detailed ec classification is a reflection of the peptidase function not being considered specific because the keratinases in fact merely catalyze keratin degradation by catalyzing the hydrolysis of peptide bonds in keratinous materials. however, a classification is obviously required in order to understand the enzymatic function on this unique substrate. we suggest classifying the keratinolytic enzymes according to their merops protease family relationship, which to some degree corresponds to their substrate attack preference. to our knowledge, all reported keratinolytic enzymes are serine proteases or metallo proteases, but they belong to different protease families (brandelli et al., ) . in addition to categorizing the keratinases according to their protease family type, it is highly useful to distinguish their mode of action according to whether they work via endo-attack, exo-attack or exert enzymatic action on peptide-oligomers (oligopeptide-acting). this attack preference differentiation will be discussed in section . the presence of multiple disulfide bonds in keratin is a key reason for the difficulty of degrading keratin enzymatically. thus, most keratinases can catalyze keratin degradation only after the disulfide bonds have been broken (reductively cleaved) (gupta and ramnani, ; wang et al., ) . this means that the keratinolytic process may involve two steps: sulfitolysis (s-s bond breakage) and proteolysis (lange et al., ; peng et al., ) . hence in practice, many reports of -keratinase‖ activity, in fact refer to keratinases that act synergistically with disulfide reductases or reducing agents (e.g. dtt) to break down the complex structure of keratin (lange et al., ) . however, what defines a true keratinase (or a superior keratinase) is that it does not rely on the presence of reducing agents or any accompanying disulfide reductase activity (navone and speight, ) . so far, only a few known proteases fit this criterion: for instance, an alkaline protease from bacillus sp. ah- , and an s serine protease from o. corvina, have been reported to exert high proteolytic activity on keratin biomass in the absence of any added reducing agents takami et al., ) . in any case, it is necessary to accommodate the fact that keratinases can actually catalyze hydrolysis of peptide bonds in a broad spectrum of protein substrates, including many soluble and insoluble proteins, such as casein, albumin, heme protein, collagen, gelatin, bovine serum albumin, globulin in addition to keratin biomass substrates such as wool, feathers, hair, and porcine bristles (brandelli et al., ) . hence, a ratio of keratinolytic activity to caseinolytic activity of more than . has been suggested as specifying the keratinase (gupta et al., b) . this ratio gives a relative reference of protease specificity, and works fine as a screening measurement to spot keratinolytic ability e.g. in a crude protease mixture, but the assay may not suffice to compare the rate or the specific keratinolytic activity between different keratinases. for purified proteases, this ratio mainly identifies the endo-keratinolytic proteases but not exo-nor oligo-attacking keratinolytic proteases. to date, by using different assays (see section . ), keratinolytic enzymes have been shown to be produced by both bacteria and fungi. some of the prominent producers are bacterial species, such as b. licheniformis, b. subtilis, and b. pumilus (fellahi et al., ; gupta and singh, ; ramnani and gupta, ) , as well as actinobacteria, such as streptomyces fradiae, nocardiopsis sp. (li et al., ; mitsuiki et al., ) . as regards fungi, most keratinolytic enzymes have been described in dermatophytes like t. rubrum and m. canis (descamps et al., ; zaugg et al., ) . furthermore, onygena spp. such as the non-pathogenic o. equina and o. corvina have also been found to possess keratinolytic ability because they grow on putrefying horns or hooves in nature and proteases from o. corvina have been reported to degrade pig bristles . the currently reported keratinolytic enzymes with sequences deposited in the ncbi database are listed in table . the keratinases were classified into different families according to their amino acid sequence similarities (homology), and validating the categorizations by submitting their sequences to pfam. this categorization therefore distinguishes the enzyme families according to a) their functional active sites, and b) their conserved domains in the merops database. this family classification distributes the keratinolytic enzymes into at least protein families across the serine and metallo proteases: the serine proteases include members of families s , s , s , s , and s , and the metallo proteases include members of families m , m , m , m , m , m , m , m and m (table ) . it should be noted that proteases in family m have been reported to have the capacity to degrade keratin , but no available sequences have been confirmed to have keratinolytic activity. the keratinolytic enzymes in the different families may play different roles in the keratin degrading process, due to differences in their active sites, the preferred substrate cleavage sites and recognition of amino acid length. as is evident from table , the source organisms of these keratinolytic enzymes come from a broad range of natural environments, such as keratin-rich environmental soil, compost, fungusinfected horns, human and animal dermatophytes, and other environments such as bathroom tile-joints, hot springs and even sugarcane molasses ( table ). the number of amino acids in each enzyme (including n-, c-and available signal peptides) range from - which gives the enzymes molecular weight range of between and kda (table ) . strikingly, the two largest enzymes (with molecular weights of and kda, respectively) are both from fervidobacterium species and both were isolated from hot springs. this wide range of habitats indicates the versatility of keratin-degrading organisms, and suggests that microbial keratinolytic enzymes may be quite diverse with regard to their mode of action, biochemical and biophysical properties. in order to qualify as being keratin degrading, the ability of keratinolytic proteases to degrade keratin has been validated on substrates such as keratin azure (a commercial, colored wool-based keratin), feathers, pig bristles, cat keratin, keratinized tissues and wool cuticles (table ) (assays are discussed in detail in section . ). the available data on the optimum reaction conditions of keratinolytic enzymes show that most proteases work at neutral to high alkaline ph of . to . and at temperatures of - °c (table ). this wide range of reaction conditions qualifies the keratinolytic enzymes for use in different industrial-scale waste processing facilities. however, in designing keratinolytic enzyme blends, the similarity of optimum conditions of the candidate enzymes also needs to be considered to ensure that all the proteases work efficiently together. j o u r n a l p r e -p r o o f (jarai, gabor, daniel kirchherr, many of the keratinolytic enzyme activities have been verified using natural keratin-rich substrates such as feather, pig bristles, wool cuticles etc. (table ) , where detailed enzyme kinetics studies are difficult. some more detailed keratinolytic enzyme activity studies using purified or recombinantly produced enzymes have employed colored, modified keratin-derivatives as substrates, e.g. azo-keratin (gonzalo et al., ) and keratin azure, but natural, insoluble keratin substrates such as feather (in fact ball-milled feather powder) (fang et al., ) , pig bristles, human hair (mukhopadhyay and chandra, ), cow's horn (dozie et al., ) have also been used (table ) . azo-keratin is a deep red-orange compound with an absorption maximum at - nm (riffel et al., ) . azo-keratin is not commercially available, but it is possible to prepare this keratinase assay substrate by coupling keratin (derived from various keratin sources) with a diazotized aryl amine to j o u r n a l p r e -p r o o f produce a chromophoric derivative, sulfanilic acid-azokeratin (riffel et al., ) . another synthetic colored keratin substrate is keratin azure (habbeche et al., ; nahar et al., ) . this substrate is available commercially from sigma-aldrich (st. louis, mo, usa) and is presumably prepared from sheep wool (scott and untereiner, ) . hence, the keratin azure from sheep wool is particularly useful for α-keratin-degrading enzymes, but β-keratinases are also detected. for deep kinetics analysis and comparisons of keratinases, it seems useful to combine measurements using the keratin azure substrate with parallel assays using azo-keratin prepared from -rich keratin such as feather. such comparisons allow assessment of any differentiation in substrate type preference among keratinases, an area that appears very little investigated. an additional point however, is that azo-keratin used as keratinase assay substrate appears to be more sensitive and robust than keratin azure ( -fold) (gonzalo et al., ) . the assessment of enzyme action on -genuine‖ keratin substrates, e.g. pulverized feather, will obviously be a better reflection of the degradation function of a specific keratinase. however, when real keratin substrates are used, more operations and various analytical methodologies need to be employed. hence, azo dying of genuine keratin biomass appears to be a good choice to develop more selective keratinase assays, and we envisage that more assay combinations may gradually emerge with more focus on keratinase enzyme discovery for keratin biomass refining. recently, a particularly elegant type of assay has been developed, involving the use of soluble recombinant chicken feather keratin substrates (jin et al., ) . with this assay, it is possible to obtain information about the substrate-enzyme interaction, such as which amino acid residues in the substrate are preferentially bound in the p /p ' sites of the enzyme (jin et al., ) . despite these developments, no standard keratinase enzyme assay currently exists and the enzyme unit definitions for keratinase activity vary (table ) , which makes comparisons of enzyme activities and kinetics across different studies challenging. the increase in absorbance at nm of . after min in the test reaction compared with the control reaction. (tork et al., ) the amount of enzyme causing an increase of . in absorbance at nm in min under the assay conditions. (habbeche et al., ) the amount of enzyme required to increase absorbance at nm by . in h. (nahar et al., ) human hair keratinase unit= . corrected absorbance at nm. (mukhopadhyay and chandra, ) cow horn an increase of . absorbance unit at nm ml - h - . (dozie et al., ) feather powder mmol tyrosine liberated per min. (fang et al., ) the amount of enzyme which liberates μmol tyrosine/min under the assay conditions. (iglesias et al., ) the amount of enzyme that produced nmole of free amino groups (equivalent to arginine) as products per min at the assay conditions. (jin et al., ) the protease families involved in keratin degradation (table ) may be further grouped into endoprotease, exo-protease and oligopeptidase families. this additional sub-division of the enzymes is valuable for understanding their roles and potential function in keratin degradation. the complete degradation of keratin to single amino acids requires (at least) three kinds of protease, namely endo-, exoand oligo-protease, as well as an s-s-bond breaking strategy, which may be done either chemically (such as dtt) or enzymatically, e.g. by disulfide reductase (lange et al., ; mercer and stewart, ) . endoproteases catalyze the cleavage of peptide bonds internally within a polypeptide, and keratinolytic endoproteases are classified in the s , s , s , m , m , m families (table ). in contrast, the keratinolytic proteases in the s , s , m , m , m and m families are exoproteases, which means that they attack the polypeptide chain at the terminal end (lange et al., ; mercer and stewart, ) . the exo-acting keratinolytic enzyme members in the different families attack from either the n-terminal (s , m and m family members) or the c-terminal (s , m and m family members); furthermore, synergistic action may take place as exo-acting proteases act on the free peptides released by the action of the keratinolytic endo-proteases. the keratinases acting on the peptide bonds in peptide-oligomers (oligopeptide-acting or oligopeptidases) are categorized in m and m families. these enzymes can act on shorter peptides and catalyze hydrolysis of small peptides to result in dimeric or trimeric peptides or even individual amino acids. some other enzymes may have a positive facilitation effect on keratin digestion. for example, lpmos have been suggested to potentially contribute to keratin degradation in nature (lange et al., ) . all hitherto identified endo-keratinolytic serine proteases belong to family s , s or s , whereas the members of family m , m and m are metallo proteases. family s is the largest protease family in terms of the number of proteins available in the merops database. all the peptidases in family s are endopeptidases and work via a -classic‖ catalytic triad involving a nucleophilic attack followed by an acid hydrolysis mechanism provided by his, asp and ser (presented according to their order in the amino acid sequence) (rawlings and barrett, a) . the four described keratinolytic s family endoproteases (ncbi id: bam , aao , aqx , cah , table ) were isolated from very different habitats and organisms. they were tested for their keratinolytic function using different types of keratin assay substrates, yet all four enzymes work optimally at alkaline ph of - . ( table ). the keratinolytic activity of the enzyme ncbi id: bam , also called -anisep‖, derived from paenarthrobacter nicotinovorans was about -fold higher than that of trypsin (sone et al., ) . protein aao from nocardiopsis sp. toa- had very high keratinolytic activity, especially at a high alkaline ph of . , where the activity was to -fold higher than that of proteinase k and subtilisin (mitsuiki et al., ) . the substrate specificity of protein aao was investigated using synthetic peptides. the k m results show preference for leu, ala and phe residues at the p position (mitsuiki et al., ) . the keratinolytic enzyme (cah ) from streptomyces fradiae var. k gave a keratin/casein ratio of . (li et al., ) . this results indicates that cah is indeed -keratinolytic‖ because this high ratio is similar to that of proteinase k, one of the most well described keratinases, considered by some as a benchmark keratinase, based on the same keratinase and caseinase assay. moreover, the keratinase activity of cah was much higher than that of proteinase k (li et al., ) . from the activity data, the s keratinases have comparable ability with s keratinases in catalyzing keratin biomass degradation. on this basis, the family s proteases deserve further investigation. there are plenty of keratinolytic proteases classified in family s . after sorting out doublets/identical sequences (using cd-hit protein sequence clustering with a % sequence identity threshold), we identified a total of unique keratinolytic proteases in the s protease family (table ) . like the s proteases, the s protease family members work via a catalytic triad mechanism, but the active sites with respect to the order of asp, his, ser in the sequence (laskar et al., ) . among the s -family members, the akr enzyme (also known as kerk) derived from b. amyloliquefaciens j o u r n a l p r e -p r o o f has been reported to have the highest keratin specificity with a keratinolytic:caseinolytic activity ratio of . in the presence of mm dtt and a purified recombinant kerk dosage of . u/ml . other bacterial s keratinases are produced by species of fervidobacterium, meiothermus, and stenotrophomonas. notably the enzyme from fervidobacterium pennivorans has been reported to be highly thermostable and to act at high ph levels (table ) ; the structure of this enzyme has already been determined (kim et al., ) , although it is not clear which structural features are responsible for the alkalinity and thermostability of the enzyme. dermatophyte fungi of the class eurotiomycetes, such as t. rubrum, t. benhamiae and m. canis also produce keratinolytic s endoproteases. for example, m. canis produce three different keratinolytic subtilisin-like proteases, sub (cad ), sub (cad ) and sub (cad ). the etiology of the pathogenesis strongly suggests that these proteases are produced by m. canis during invasion of keratinized structures (descamps et al., ) . similarly, sub (aar ) and sub (aar ) from t. rubrum show appreciable activity on keratin azure is comparable to that of subtilisin carlsberg and proteinase k (jousson et al., ) . in comparison to subtilisin carlsberg and proteinase k, sub and sub were less active on other protein sources, especially elastin, indicative of the specificity of sub and sub towards keratinous substrates (jousson et al., ) . sub (aar ) from t. rubrum has . % sequence identity with sub (cad ) from m. canis (therefore aar is not listed in table ). the non-pathogenic o. corvina is known to grow on hooves and horns. huang et al. reported that a recombinantly produced s protease from o. corvina appears to be a true keratinase because it is able to catalyze degradation of pig bristles and hooves without addition of reducing agent to break the cysteine bridges in the keratin . more detailed examination of s protease sequences has shown that the active site ser in the s family members is found within a gly-thr-ser-xaa-xaa-xbb-pro motif (where xaa is an aliphatic amino acid and xbb is a small amino acid) (rawlings and barrett, a) . in order to reveal the specific amino acid motif in s keratinolytic proteases (listed in table ), meme suite (bailey et al., ) was used for motif finding. the results (given in fig. ) indicates that the residues around active sites are highly conserved, with asp in the asp-thr/ser-gly motif, his in the his-gly-thr-his-val/thr-ala/ser-gly/ser-thr/ile motif and ser in the gly-thr-ser-met/ala-ala-ala/ser/thr-pro-his-val motif. these specific motifs may attribute specificity to keratinases compared with other proteases. unlike the peptidases in the s and s family, the active site of members in family s is a ser-lys catalytic dyad (rawlings and barrett, a) . one protease in the s family (derived from fervidobacterium islandicum, accession number: amw ) has recently been confirmed to be a keratinolytic protease (kang et al., ) . the enzymatic rates free amino acids release from chicken feather with addition of purified s protease is around . -fold higher than those of a crude f. islandicum extract. the comparison information available for s keratinolytic proteases is unclear. there are only two identified and sequenced keratinolytic endoproteases in the m family. one of them, the ajd enzyme is referred to as geoker, originates from the thermophilic bacterium geobacillus stearothermophilus ad- (table ) . a recombinant version of this enzyme, recgeoker (expressed in e. coli), has been found to have a consensus zinc-binding helth motif which indicates that the enzyme belongs to the zinc metalloproteinases of family m . based on studies of the recombinant enzyme, there are strong indications that the his , glu and his catalytic triad is essential for the geoker enzymatic reaction and the enzyme activity on different protein substrates was ranked as follows: wool keratin > collagen > sodium caseinate > gelatin > bsa (gegeckas et al., ) . the enzyme from pseudomonas aeruginosa (adp ) exerts keratin degrading ability (sharma and gupta, ) , however, the specific function has not been investigated. in addition to an s keratinolytic protease (see section . . ), the f. islandicum produce one m protease (accession number: amw ). addition of this enzyme on top of a f. islandicum extract also enhanced the feather degradation rate in vitro (more than . fold) compared with the crude f. islandicum extract only (kang et al., ) but the keratinolytic activity of this protease alone is unknown. furthermore, it is worth noting that not all family m proteases act by endo-attack, for instance, subfamily m a contains oligopeptidases denoted as insulysin and nardilysin (rawlings and barrett, b) . the detailed attack mode of family m keratinolytic proteases warrants further investigation. members of the m (fungalysins) metalloendopeptidase family have been shown to be involved in degrading different keratinous substrates, and this enzyme family includes proteases secreted by o. corvina , m. canis (brouta et al., ) and f. oxysporum (chaya et al., ) (table ) . family m is a metalloprotease family containing a his-glu-xaa-xaa-his (hexxh) motif of metallopeptidases from clan ma (rawlings and barrett, b) . the motif serve to coordinate the zinc ion (xu et al., ) . mep (accession number: ajd ) is induced when o. corvina grows on chicken feathers, pig bristle, or dog wool . the fusarium-derived enzyme krtc (accession number: bam ) can hydrolyze keratin and has been shown to have a preference for cleaving the amino side of hydrophobic residues with bulky side-chains (chaya et al., ) . mep (accession number: cad ) was found to be a gene encoding an isolated m. canis . -kda keratinolytic metalloprotease, and was successfully expressed in pichia pastoris (brouta et al., ) . it is noteworthy that these enzymes can overcome the limited proteolysis on the surface of insoluble keratin particles, which restricts enzyme-substrate interaction . current exo-keratinolytic enzymes are classified in the serine protease families s and s and in the metallo protease families m , m , m and m . proteases belonging to the s family exerts their catalytic function via a catalytic triad in the order ser, asp, his. the representative keratinolytic proteases in the s family are aan (dipeptidyl peptidase v, dppv) and aas (dipeptidyl peptidase iv, dppiv) ( table ) . both enzymes are from t. rubrum (monod et al., ) . recombinant rudppiv is able to catalyze the hydrolysis of synthetic dipeptides gly-pro-amc and lys-ala-amc, while recombinant rudppv hydrolyze only lys-ala-amc but not gly-pro-amc (monod et al., ) . another source of dppiv is the fungus t. tonsurans (preuett et al., ) . in addition, secretion of dppv peptidase activity has been detected from the fungal pathogen t. mentagrophytes (kaufman et al., ) in skin infections. although the in vitro activity of dppv and dppiv in degrading other keratin substrates than skin keratin is unknown, the trir allergen (aad ) from t. rubrum (woodfolk et al., ) , which has the same amino acid sequence as dppv (aan ), has shown keratinolytic activity on keratin azure. these results demonstrate that trir exerts weak activity against keratin azure ( units/mg). this keratinolytic activity is times lower than the activity of the more well-known endo-protease proteinase k ( units/mg) (woodfolk et al., ) . the family s exo-keratinases may play a key role in attacking products from endo-protease hydrolyzed keratin protein (lange et al., ; mercer and stewart, ) , but is not highly efficient in directly degrading keratin. endo-and exo-keratinolytic proteases may degrade keratin synergistically. similarly, the available research concerning synergism between endo-and exopeptidases is likely to be an essential prerequisite for potent dermatophyte virulence, e.g. when dermatophytes infect keratinized tissues (monod et al., ) . family s contains only carboxypeptidases (rawlings and barrett, a) which hydrolyze the peptides from c-terminal. the proteases aas (truscpa) and aas (truscpb) are secreted by t. rubrum when degrading compact keratinized tissues (zaugg et al., ) . the catalytic triad of truscpa (ser , asp , and his ) and truscpb (ser , asp , and his ) are in the order of ser, asp, his, and belong to the s family. in addition, truscpa and truscpb enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (gpi) anchor. during infection, gpi-anchored carboxypeptidases secreted by t. rubrum may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides (zaugg et al., ) . therefore, like the s keratinolytic proteases, the currently known s keratinolytic proteases also promote keratin tissue degradation in concert with keratinolytic endoproteases. in a medium containing keratin-soy as sole nitrogen and carbon source, t. rubrum secretes the protease abg (mcpa), which is classified in the m protease family (zaugg et al., (zaugg et al., , . all members of family m contain the motif his-xaa-xaa-glu. most of the peptidases in the m family are carboxypeptidases that catalyze the hydrolytic removal of single c-terminal amino acids from polypeptide chains. these enzymes all have a recognition site for the free c-terminal carboxyl group, which is a key determinant of specificity (riffel et al., ) . in the mcpa enzyme from t. rubrum, the residues his , glu and his are ligands for the catalytic zinc, while other amino acids, e.g. arg , arg , tyr , tyr and glu , are important for substrate binding and catalysis in the m a family (zaugg et al., ) . the mcpa from t. benhamiae is also known to be highly expressed during in vitro growth on keratin (tran et al., ) . thus mcpa in the m family may assist in degrading keratin. another purified m keratinolytic metalloprotease, q (no available protein sequence), originating from chryseobacterium sp. kr has also been examined, and reported to have specific keratinolytic activity of u/mg protein with keratin azure as substrate (riffel et al., ) . the m family is the most researched exo-keratinolytic protein family. as listed in table , five proteases in the m family have been tested for activity on pig bristle, feathers and keratin. the aminopeptidase cah in family m , produced by the keratin-degrading bacterium streptomyces fradiae var. k , is known to play a role in the processing of proenzymes (wu et al., ) . the conserved motifs h , d , e , d , and h , which are all implicated in the coordination of two zinc atoms, are found in this protease (cah ). two t. rubrum derived aminopeptidases aas (rulap ) and aas (rulap ) have also been reported (monod et al., ) . substrate specificity analysis using different fluorogenic aminoacyl- -methylcoumaryl- -amide derivatives has shown that rulap is the most selective for leu-amc, while ser-amc and pro-amc are more efficiently cleaved by rulap (monod et al., ) . the tested laps were not capable of cleaving the gly-pro-amc substrate, which indicates that the presence of a pro residue in position p ' affects the action of these enzymes (monod et al., ) . thus, the aminopeptidases lap and lap in family m family hydrolyze peptides from the n-terminus until they reach x-pro or x-ala motifs, which act as a stop point (mercer and stewart, ) . two exopeptidases (m with accession numbers ajd and ajd ) together with two endopeptidases (s ) from the non-pathogenic fungus, o. corvina, displayed a higher j o u r n a l p r e -p r o o f degree of pig bristle degradation compared with degradation by s proteases only . to our knowledge, this is the first record of in vitro synergies of s and m keratinolytic proteases in degrading keratin, and these data support the view that a combination of exo-and endo-acting proteases can efficiently degrade keratin. the m and m keratinolytic proteases, with accession number amw and amw , respectively, are also derived from f. islandicum. both of the purified m and m proteases improved the free amino acid release rate by comparison of the rate of f. islandicum extract only (kang et al., ) . during keratin degradation by f. islandicum, the m protease is presumably responsible for regulating proteolysis, while m protease might be activated during starvation conditions and involved in controlling the cell's flow of peptides as degradation products (kang et al., ) . an oligopeptidase is an enzyme that catalyze cleavage of peptides but not proteins. this property is due to its structure, namely that the active site of this enzyme is located at the end of a narrow cavity that can be reached only by peptides. in the keratin degradation process, oligopeptidase plays an important role in degrading the peptides generated by endo-and exo-protease digestion of compact keratinized tissues. among the keratinolytic enzymes listed in table , proteins in family m and m function as oligopeptidases. the m and m family are in the ma (e) clan which contains zinc-dependent metallopeptidases. the two zinc ligands are the histidines in the motif his-glu-xaa-xaa-his (rawlings and barrett, b) . protease family m includes intracellular oligopeptidases from mammals, fungi and bacteria, which only degrade peptides of certain lengths (between and residues, in the case of thimet oligopeptidase) (rawlings and barrett, b) . thus the m keratinolytic enzymes can only catalyze hydrolysis of small keratin peptides, i.e. those that are produced from endo-and exo-keratinolytic action on keratin. the first and currently the only described m keratinolytic protease, ajd , is from the non-pathogenic fungus o. corvina. previous research has revealed a synergistic function in keratin degradation between three o. corvina proteases, a family m keratinase (oligopeptidase), an s protease (endo-keratinase), and an m protease (exo-keratinolytic enzyme) . however, the substrate specificity and the oligopeptide size preferences of this m protein are not known at present. metallo-carboxypeptidases (mcp) of the m family are found in many organisms. these enzymes catalyze hydrolysis of the peptide bond at the c-terminus of peptides and have been widely studied (lee et al., ) . the protease amw (fiscp) plays a pivotal role in the decomposition of keratin when added to reaction mixtures containing whole cell extractions derived from f. islandicum aw- cells (lee et al., a) . the results show that amino acid production was -fold higher in fiscp-supplemented whole cell extractions than in whole cell extractions alone. this feature supports the notion that fiscp together with numerous other highly up-regulated proteases on keratin are mainly involved in the degradation of native chicken feathers (lee et al., a) . as mentioned earlier, the first step in enzymatic keratin degradation is the reductive breakage of the disulfide bonds in keratin (peng et al., ) . the enzymes that have been suggested as being able to catalyze the breaking of disulfide bonds include cysteine dioxygenase (ec . . . ) , glutathione reductase (ec . . . ), alkyl hydroperoxide reductase (ec . . . ), thioredoxin reductase (ec . . . ), dihydrolipoyl dehydrogenase (ec . . . ), peptide methionine sulfoxide reductase (ec . . . ), j o u r n a l p r e -p r o o f phospho-adenosine phosphosulfate reductase (ec . . . ) , ribonucleoside-diphosphate reductase (ec . . . ) (peng et al., ) . among these, disulfide reductase, cysteine dioxygenase and glutathione reductase have indeed been shown to play an important role in keratinolysis (grumbt et al., ; Łaba et al., ) . however, research data is scant in this area, so the efficient action of other types of reductases in keratin breakdown cannot be ruled out. cysteine dioxygenase is a key enzyme in homeostatic regulation of the cysteine level in eukaryotic cells. because this enzyme is involved in the production of important oxidized metabolites of cysteine, such as pyruvate, sulfite, sulfate, hypotaurine, and taurine in all eukaryotic cells (kasperova et al., ) . the relevant research has indicated that when dermatophytes grow in keratin tissues, sulfite formation from cysteine relies on cysteine dioxygenase cdo and sulfite secretion is supported by the sulfite efflux pump ssu (grumbt et al., ) .the presence of the reducing agent sulfite may break the s-s bonds in keratin biomass and in this way facilitate keratin degradation. furthermore, γ-glutamyl transpeptidase (ggt) is a periplasmic enzyme that catalyzes the hydrolysis of glutathione (gsh) to produce cysteinyl-glycine, which is a strong reductant that further reduces disulfide bonds. it has been hypothesized that a ggt-gsh mediated redox might be one of the pathways of sulfitolysis for feather degradation and that cysteinyl glycine might be the active redox moiety (sharma and gupta, ) . the lyase cystathionine gamma-synthase (ec . . . ) secreted by b. subtilis , which catalyzes the breakdown of carbon-sulfur bonds, has also been confirmend to be involved in and able to promote decomposition of feather keratin (he et al., ) . many disulfide reductases derived from keratinolytic organisms have not yet been identified to a specific type of reductase, but their disulfide reduction activity has been identified using dtnb ( , '-dithiobis-( -nitrobenzoic acid)) as substrate. some researchers have reported efficient synergies between keratinolytic enzymes and disulfide reductases. for instance, bacillus sp. mts produces an extracellular alkaline keratinolytic protease and a disulfide reductase while growing in media containing feathers (rahayu et al., ) . when these two enzymes are combined, the keratinolytic activity of the mixture on feather substrates ( . u/mg) is greatly increased compared to the activity of purified alkaline protease alone ( . u/mg) or purified alkaline protease in the presence of reducing agents dtt ( . u/mg) (rahayu et al., ) . stenotrophomonas sp. also produce a serine protease and a disulfide bond-reducing protein and is thus capable of degrading native keratin (human hair) (yamamura et al., ) . interestingly, the keratinolytic activity of the serine protease alone was only ku/mg, however, when the serine protease was mixed with the disulfide reductase, the keratinolytic activity was ku/mg, which was more than -fold higher than that of the disulfide reductase combined with proteinase k ( ku/mg) (yamamura et al., ) . in addition, the disulfide reductase mixed with the serine protease resulted in higher keratinolytic activity compared with the serine protease in presence of the reducing agent dtt (rahayu et al., ; yamamura et al., ) . this illustrates that some disulfide reductases are better choices for use in catalyzing the breakage of s-s bonds in keratin. an interesting research focus is the investigation of the keratinolytic efficiency of different disulfide reductases in combination with keratinolytic proteases. other enzymes such as lpmos may exert a possible auxiliary function in degrading keratin (lange et al., ) . lpmos are copper-dependent and utilize molecular oxygen and an electron donor (e.g. ascorbic acid) to catalyze cleavage of glycosidic bonds as has been demonstrated by the action of lpmos on cellulose, hemicellulose, chitin, and starch. interestingly, lpmo genes occur consistently in dermatophytic and keratin-degrading fungi (busk and lange, ) as well as in keratinolytic non-pathogenic fungi, e.g. o. corvina . the putative function of (aa ) lpmos in keratin-degrading fungi is presumed to be the catalytic breakage of glycosyl-bonds between n-acetyl-glucosamine and serine and threonine in the non-coiled head structure of the keratin filaments. such changes have been shown to loosen the keratin structure or even to de-assemble the keratin filaments (lange et al., ) . besides, enzymes involved in fatty acid degradation may contribute to keratin digestion (lee et al., j o u r n a l p r e -p r o o f b). the surface of wool fiber is rich in lipids, with an outer lipid layer consisting of methyleicosanoic acid along with other fatty acids, which are bound largely through thioester linkages to the cysteine-rich underlying proteins (ghosh et al., ) . enzymes relevant for degradation of fatty acids may be envisaged to assist in breaking the outer lipid layer of keratin so that keratinolytic endoproteases can gain easier access to the internal keratin structure. however, in vitro testing of lpmos and fatty acid degradation enzymes in hydrolyzing keratin substrates still needs further research. although the detailed mechanism of keratin degradation by enzymes is not fully understood, several hypotheses have been suggested to explain the enzymatic degradation events. as outlined above, keratin is packed with disulfide bonds and hydrogen bonds, and both inter-and intra-chain disulfide bonds exist (fig. ) . to obtain amino acids from keratin-rich materials using keratinolytic enzymes, a preliminary degradation of disulfide bonds is necessary to loosen the keratin structure and make the amino acid chains available for keratinase attack (gupta et al., b; peng et al., ) . the more enzyme attack sites are exposed, the better for enzyme hydrolysis. based on present research on biodegradation of keratin, an efficient biocatalytic process invariably involves two main processes: reduction of disulfide bonds and hydrolysis of the keratin-polypeptide chain by keratinolytic proteases (fig. ). if the disulfide bonds are not broken, most keratinases cannot degrade native keratin effectively (okoroma et al., ) .for instance, unless reducing agents are present, the wf -protease from bacillus sp. cannot catalyze the degradation of feathers (liang et al., ) . analogously, the enzyme cibenza dp did not produce detectable levels of soluble peptides during degradation of hair unless a reducing agent was added to the reaction (navone and speight, ) . as regards the keratin biomass degradation by purified keratinases, the disulfide bonds can be broken either by adding chemical reducing agents or, as mentioned above, by means of enzymes such as cysteine dioxygenase or glutathione reductase (the latter requires the presence of glutathione to accomplish the reaction i.e. the redox half reaction) (burmester et al., ; descamps et al., ) . regarding the process of keratin degradation by keratinolytic organisms, the reducing agent sulfite or disulfide reductase may be secreted from the organism and may be involved in cleavage of disulfide bonds in the keratinous biomass (grumbt et al., ; kang et al., ) . some organisms cannot secrete disulfide reductase or sulfite (e.g. streptomyces pactum), in this case, the reduction of disulfide bonds must depend on the presence of metabolically active cells and the membrane potential may play a key role (böckle and müller, ). once the disulfide bonds in keratin have been broken, the keratinolytic endoproteases (members of families s , s , m , and m ) act to accomplish the keratinolytic substrate degradation. keratinolytic exo-proteases (members of the families s , s , m , and m ) cleave peptide chains from both ends, while oligopeptides (members of family m and m ) work on oligo-peptides to release individual amino acids or shorter peptides. this sequential degradation, outlined in fig. , has been suggested to take place during for example infectious keratin tissue degradation (mercer and stewart, ) and during fungal keratin decomposition (lange et al., ) . apparently, efficient and complete decomposition of keratin to free amino acids cannot be achieved by one keratinolytic protease alone, but seem to require a combination of enzymes, plus presence of a reducing agent or a reducing enzyme to help cleave the s-s bonds. in dermatophytes infecting keratinized tissues, such as aspergillus spp. and lactobacillus spp., the laps (in family m ) and dppiv (in family s ) were shown to synergistically digest the large peptides generated by the endoproteases to give free amino acids and x-pro dipeptides (byun et al., ) . laps degrade peptides from their n-terminus, while x-pro acts as a stop sequence. in a complementary manner, these x-pro sequences can be removed j o u r n a l p r e -p r o o f by dppiv and thus allow the laps access to the next residues (giddey et al., ) . investigations of the keratinases of the keratinolytic non-pathogenic fungus o. corvina suggest that a blend of fungal keratinasesnamely an endoprotease (s ), exoprotease (m ), and an oligopeptidase (m )act synergistically to break down pig bristle keratin . moreover, the enzymology of feather degradation by f. islandicum aw- , which has recently been reported in detail (kang et al., ) , points out that synergies among different types of proteases help the degradation. hence, after sulfitolysis of feathers, the cpbp family (m protease family) intramembrane metalloprotease and five additional membrane proteases (of family s , s , s , m , a , respectively) act as key keratinases to catalyze the feather degradation. subsequently, the released amino acid and peptides are transported into f. islandicum aw- for further degradation (involved proteases include proteases belonging to family m , m , m , s and t ) (kang et al., ) . in addition, the high degradation rates achieved on recalcitrant keratinous material by bacterial consortia also indicates synergies between keratinolytic and other enzymes (nasipuri et al., ) . however, counteracting effects between these different enzymes may also happen during enzyme catalyzed degradation of keratin biomass, mainly because the proteases may attack the other enzymes and/or self-digest. for instance, disulfide bonds in keratinolytic enzymes function to stabilize the enzyme structure. the disulfide reductases or reducing agent added in the hydrolysis reaction may in fact break both keratin and keratinase disulfide bonds; the latter will cause lower enzyme activity and reduce the enzyme stability. moreover, keratinolytic enzymes themselves can exhibit -autolysis‖, especially in the presence of reducing agents (khan and ahmad, ; liang et al., ) . in order to prevent autolysis, the introduction of prolines at the autolytic sites was demonstrated to increase the autolysis resistance of the enzyme under reducing conditions, though sometimes the mutation also changed enzyme specificities (liang et al., ) . synergies and counter-acting effects between the enzymes should be considered when designing keratinase blends for keratin refining. the interactions between the enzymes are complicated and partly unknown, however, and further research is required to identify optimum enzyme combinations for efficient keratinolytic enzyme blends. the structures of five keratinolytic enzymes have been x-ray structure analyzed. in addition, the structures of some keratinases in the s protease family have been homology modeled based on the crystal structures of subtilisin-like proteases (e.g. pdb identifier of mee, scj, scn, whi, lpa, lpc, lpd, ti , ti , dbi, thm, afg) (fang et al., ) . the five keratinases for which a crystal structure is known are anisep from p. nicotinovorans (sone et al., ) , proteinase k from p. album (betzel et al., ; ebeling et al., ; jany et al., ) , fervidolysin from f. pennivorans (friedrich and antranikian, ; kim et al., ) , mtaker from m. taiwanenisis wr- (wu et al., ) , and fiscp from f. islandicum (lee et al., a) (table ). according to the active sites, anisep belongs to family s . proteinase k, fervidolysin, mtaker and subtilisin all belong to family s , while fiscp is in the metallo-protease family m . his -glu -his -glu (co + ) (lee et al., a) *the active site his of fervidolysin is mutated to ala and also show as ala in the structure (pdb id aak ). journal pre-proof . keratinolytic protease structure in family s anisep (fig. a) from p. nicotinovorans is significantly more active on keratin azure than on trypsin (sone et al., ) . the conserved catalytic triad of anisep consists of his , asp , and ser (fig. a) , which indicates that anisep belongs to clan pa(s) of the serine protease family (rawlings and barrett, a; sone et al., ) . the catalytic theory is discussed below (section . ). the active site ser is located in a ggssg motif (residues - ), corresponding to the conserved motif of serine proteases gxsyg (x and y for any amino acids) (sone et al., ) . in clan pa(s) the tertiary structure consists mainly of β-sheets that form a double β-barrel at the core of the enzymes (rawlings and barrett, a) , which is also the case with anisep (fig. a) . the active site is located in a shallow cleft that spans the entire enzyme situated between the two β-barrels (fig. a) (sone et al., ) . more than half of all characterized keratinolytic enzymes belong to the s family (table ), but the crystal d structure has been determined for only three of them, namely proteinase k (pdb id ic ), fervidolysin (pdb id r v) and mtaker (pdb id wsl) ( table ). the catalytic domain (cd) of each of these structures consists of seven parallel β-sheets flanked by six (proteinase k, mtaker) or nine (fervidolysin) α-helices (fig. b , violet: proteinase k, green: fervidolysin, salmon: mtaker). two nearly parallel α-helices are also found in all three structures; these parallel α-helices form the main sites of interaction with the propeptide domain (pd) (kim et al., ) . though the overall folding of various s keratinolytic proteases may differ, they all follow the same mechanism of action through an identical stereochemistry of the catalytic triad (fig. ) (betzel et al., ) . the catalytic triad of the s keratinolytic enzymes constitutes asp, his and ser in that order in the wild type enzymes (table and fig. b ) (betzel et al., ; kim et al., ; wu et al., ) . the his plays a dual role as proton acceptor and donor at different steps in the reaction, while the asp is thought to bring the his residue into the correct orientation to facilitate nucleophilic attack by ser (fig. ) (betzel et al., ) . the whole reaction theory (hedstrom, ) (fig. ) is as follows: the keratin substrate binds to the surface of the keratinolytic protease. the nucleophilic ser acts first as the nucleophile to attack the carbonyl carbon of keratin protein. a tetrahedral intermediate is generated because a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen (fig. ) . next, the electrons move back from the negative oxygen to recreate the bond, generating an acyl-enzyme intermediate and releasing the n-terminus of the peptide. thus, a larger space is formed for water coming into the reaction to facilitate a nucleophilic attack on the carbonyl carbon of the substrate. the bond between the oxygen of water and the carbon in the substrate is formed to generate another tetrahedral intermediate. in the final stage of the reaction, the carbonyl carbon reforms the double bond with the oxygen. as a result, the cterminus of the peptide is released. in addition to the catalytic triad, another characteristic of the reaction mechanism is the presence of the oxyanion hole (fig. ) . the oxyanion hole plays a key role in stabilizing the catalytic tetrahedral intermediate anion and protecting the substrate's negatively charged oxygen from water molecules. in proteinase k, the negatively charged oxygen ion is stabilized by ser and asn to form an oxyanion hole. as regards fervidolysin, two peptide nitrogen atoms of thr and ser form the oxyanion hole, where a water molecule mimics the carbonyl oxygen of the scissile peptide bond (kim et al., ) . the crystal structure of mtaker shows that the tetrahedral acyl enzyme intermediate can be stabilized by the oxyanion hole of asn and thr (wu et al., ) . one of the major determinants of the substrate specificity of the s enzymes is hydrophobic surface pockets (de kreij et al., ; wu et al., ) . according to the nomenclature (schechter and berger, ) , the active site residues in the protease are composed of contiguous pockets termed subsites. amino acid residues in the substrate sequence are consecutively numbered outward from the cleavage sites as j o u r n a l p r e -p r o o f pn…-p -p -p '-p '-…pn', where the scissile bond is located between the p and p ' positions. the subsites in the enzyme are correspondingly labelled as sn…-s -s -s '-s '-…sn'. the s subsites in proteinase k are ser -leu -gly -gly and residues ala -ala -gly -asn at the bottom . the s ' subsites are formed by the residues ser , his , tyr and glu in fervidolysin defined enzyme specificity (kim et al., ) . in mtaker, the s binding subsites consists of ser -leu -gly and ala -ala -gly (wu et al., ) . overall, the s keratinolytic enzyme contains a substrate-binding pocket that is relatively abundant in the nonpolar and smaller-side-chain amino acid residues ala and gly. the s subsites structure may be associated with specificity for keratin. s keratinolytic enzymes prefer to cleave the phe, tyr, and arg at the p site of a synthetic pna substrate (brandelli et al., ) . many proteases in the s family contain one or more calcium ion-binding sites. two ca + ions are found in proteinase k (fig. b , yellow ca + ): the first ca site contributes to the stabilization of surrounding regions, especially the long connecting loops. the second ca site stabilizes to some extent the n-and c-terminal regions of the molecular structure . in mtaker, two ca + ions are also observed in the structure (fig. b , ca + ion colored in olive). the first ca + (ca + ) is conserved with the first ca + (ca + ) of proteinase k (fig. b , yellow ca + ion), and, similarly, with the first ca + of mtaker involved in stabilizing the surface loop. the second ca + is not conserved and this ca + comes into contact with the main-chain carbonyl oxygen atoms of the surrounding amino acids residues and two water molecules (wu et al., ) . by comparison, only one ca + ion is found in fervidolysin (fig. b , orange ca + ) (kim et al., ) . the second calcium is not conserved. fiscp from f. islandicum is an m keratinolytic protease. unlike anisep (s family), which consists primarily of β-sheets and s keratinolytic enzymes whose structures are a mix of α-helices and βsheets, fiscp primarily consists of α-helices with a three-stranded β-sheet near the active site (fig. a) . the active site of fiscp is located in the hexxh motif (table ) -two his (his and his ) and a solvent water molecule coordinated with co + bound to the third active site with glu ( fig. a and b ) (lee et al., a) . coordination of the substrate through the scissile carbonyl group is assisted by the presence of the positively charged co + (fig. c) . after deprotonation of the water molecule, the resulting hydroxide nucleophilically attacks the scissile carbonyl carbon; this results in protonation of the scissile amide group and cleavage of the peptide bonds to form the peptide products ( fig. c) (szeto et al., ) . most m proteins are oligopeptidases and the length of their substrate is governed by the length of the active site groove, which closes upon substrate binding (sharma et al., ) . the arg (arg in pfucp), located at the back of the substrate groove, is % conserved in over sequences of m cps (lee et al., ) . the region surrounding this conserved arginine is re-adjusted during gate closure. the -gate‖ was found in m carboxypeptidases subfamily i (e.g. thermus aquaticus (taqcp), pyrococcus furiosus (pfucp), leishmania major (lmacp)). hence, m subfamily i should be able to cleave only those substrates that are small enough to fit into the narrow substrate groove and allow the active site gate to be closed (sharma et al., ) . furthermore, taqcp, pfucp and lmacp in subfamily i have broad substrate specificity with a c-terminal amino acid preference in the order basic > aliphatic > aromatic >> acidic and the substrate is limited to - residues (lee et al., ). however, not all m proteins have length restriction. for example b. subtilis (bsucp) and thermus thermophilus (tthcp) lack conserved arg, which is why they have no substrate length limitations, although the rate of catalysis may vary depending on the substrate length (lee et al., ) . additionally, unlike the broad substrate specificity in subfamily i, bsucp does not cleave c-terminal aliphatic and polar amino acids when tested with a series of benzyloxycarbonyl-ala-x (zax) substrates (where x is various amino acids) (lee et al., ) . alignment of fiscp with other m proteins demonstrates that fiscp lacks a key arg residue of substrate j o u r n a l p r e -p r o o f length restriction, whereas it has a lysine residue, as also found in bsucp, which indicates that the substrate length restriction of fiscp differs from cps in the m subfamily i (lee et al., a) . the substrate specificity of fiscp may also be narrower than that of proteases in subfamily i. this substrate discrimination is attributed to the keratin structure. nevertheless, the exact substrate restriction length and specificity of fiscp still need to be clarified. keratinolytic enzymes are already applied industrially, for example in detergents, in production of leather, and in bioremediation. keratinases are attractive detergent agents because they have broad specificity for both soluble and insoluble proteins as substrates. a bio-detergent formulated with alkaline keratinase (from paenibacillus woosongensis tkb ) can remove blood and egg yolk stains efficiently (paul et al., ) . keratinolytic enzymes produced by paecilomyces lilacinus (cavello et al., ) , gibberella intermedia (zhang et al., ) and b. pumilus (gong et al., ) have also shown potential in detergent applications. additionally, keratinolytic enzymes may find use in detergents for cleaning drains and clogged pipes caused for example by hair (gupta et al., a) . in the leather industry, dehairing by (keratinolytic) enzymes is also considered environmentally friendly (fang et al., ) . keratinolytic enzymes are ideal for this purpose because they exhibit high dehairing activity but no or only very weak collagenolytic activity and elastinolytic activity. for example, a keratinolyic enzyme from brevibacillus sp. as-s -ii with no collagen-degrading activity was demonstrated to work as a dehairing agent when tested on goat skin (rai and mukherjee, ) . the enzymes may also reduce the toxicity of wastewater effluents from the leather industry. in addition, industries involved in bio-remediation of soil and wastewater also offer some important applications of keratinolytic enzymes (brandelli et al., ; gupta et al., a; sharma and devi, ; tesfaye et al., a; verma et al., ) . in the agroindustry, one of the most important and potentially significant high-volume applications of keratinases is in keratin co-product management. keratin harbors at least amino acids (see table ). these amino acids may be recycled for use as fertilizer or in animal and aquaculture feed in processes offering high-impact applications of enzymatically degraded keratin such as feathers and pig bristles. keratinolytic enzymes derived from bacillus sp. slii- have been shown to be able to degrade chicken feathers and the hydrolysate could in turn substitute about % of soybean meal protein in broiler feed. the performance of broiler chickens on this feed was better than with conventional soybean meal protein (larasati et al., ) . similarly, addition of -versazyme‖ (a keratinase feed additive produced by b. licheniformis pwd- ) or other keratinases to feed mixtures may improve growth performance, breast meat yield, and gut villus structure of broilers fed diets based on corn and soybean meal (huang et al., ; wang et al., wang et al., , . chicken feather hydrolysates could also serve as a cheap source of liquid organic fertilizer. b. licheniformis as-s -i keratinase coupled to iron-oxide magnetic nanoparticles hydrolyzed chicken feathers, and the filtered sterile hydrolysate produced a significant increase in the length and growth of bengal gram seedlings and increased the soil microbial population (rai and mukherjee, ) . furthermore, keratinolytic enzymes have been used to suppress nematodes and action of entomopathogenic microorganisms (brandelli et al., ; gupta et al., a; verma et al., ) . a keratinase from bacillus sp. - is able to kill meloidogyne incognita (a root-knot nematode) may be used as biological pesticide (yue et al., ) . overall, keratinolytic enzymes have potential in the agroindustry, and the most relevant enzymes are obtained from bacillus sp. mostly belonging to the protease s family. however, the present review shows that other efficient and specific keratinolytic enzymes may be worth further investigation as well for agroindustrial applications. in biomedicine, keratinolytic enzymes are applied in treatment of nails, calluses, acne, scars, prions, and skin, and as a cosmetics supplement. for instance, pure keratinase is marketed for treating nail j o u r n a l p r e -p r o o f disorders, calluses and for prion decontamination (gupta et al., a) . keratinolytic enzymes can even contribute to improving drug delivery through the skin (epidermis) (gupta et al., a) . indeed, keratinolytic enzymes have been added as supplements to cosmetics for use in skin whitening and in exfoliation and for assisting drug permeation (anandharaj et al., ; gupta et al., a) . keratinolytic enzymes formulated in dehairing cream have also been reported to be particularly effective in removing hair compared to conventional marketed formulations (sanghvi et al., ) . use in prion decontamination is another function of keratinolytic enzymes because the enzymes cleave β-plated protein that is particularly prevalent in prion proteins. several keratinolytic enzymes showing ability to digest prion protein are produced by b. licheniformis pwd- (langeveld et al., ) , streptomyces sp. (tsiroulnikov et al., ) , nocardiopsis sp. toa- (mitsuiki et al., ) , thermoanaerobacter, thermosipho, and thermococcus sp. (suzuki et al., ) . furthermore, microbial keratin-rich material hydrolysis can generate bioactive peptides, and this constitutes a point of utmost interest for development of functional ingredients with elevated value (callegaro et al., ) , for example, for use in pharmaceuticals and cosmetics yeo et al., ) . keratinolytic enzymes may also be used in bioenergy applications by assisting the production of biofuel and biogas. this function is mainly related to keratin hydrolysates serving as nitrogen rich sources for microbial production of methane, fuel pellets and bio-hydrogen. the contribution of keratinolytic organisms during biohydrogen production has been investigated. keratinolytic bacillus sp. degrades keratin first, and then the hyperthermophilic archaeon produces h highly efficiently from keratin-derived breakdown products (bálint et al., ) . chicken feathers hydrolyzed by keratinolytic enzymes during pretreatment with bacillus sp. yielded % more ch during biogas fermentation than non-pretreated feather (patinvoh et al., ) . however, research on keratin-based biogas production is still limited, and further investigations are needed into such fermentation processes. in conclusion, certain microbially derived proteolytic enzymes exhibit significant keratinolytic activity, and thus readily catalyze the degradation of keratin. keratinolytic enzymes are therefore highly useful for various applications in biomedicine, industry, agroindustry, and bioenergy. compared with chemical treatment of keratin, enzymes act more gently and do not denature the amino acids. applications of keratinolytic enzymes are therefore being increasingly explored, and some keratinolytic enzyme-based products have already been commercialized (gupta et al., a) . despite such developments, the current knowledge on how keratinolytic enzymes function is limited, especially with regard to the structure-function aspects, substrate specificity, kinetic traits (rate, substrate affinity), biological diversity of the enzymes and their reaction robustness. hence, further investigations are warranted. in particular, the mechanisms, kinetics, and free energy of catalytic conversion and the significance of supplying reductases for keratinases deserve investigation. such research will help understand both natural bioconversion of keratin (and its significance in larger natural c-and n-cycles), and the practical use and selection of efficient keratin-converting biocatalysts for new technical applications. a standardized set of assays with proper keratin substrates, i.e. able to distinguish any differences in kinetic rates on αand -keratin, and more stable and accurate measurement methods are recommended. besides, wider use of bioinformatics tools may help further discovery and understanding of keratinases, in particular when it comes to rationally identifying efficient keratinases for use in new upcycling processes of keratinous biomass. in this review, keratinases are classified into different protease families that may relate to different functional theories and products. therefore, for application purposes, it is important to study the design and optimization of efficient enzyme blends towards different keratin-rich substrate or special products. (c-terminal domain) . in addition, the repeating heptad pattern in α-helix is labeled from -a-g‖ shown in different colors; (b) heterodimer structure formed from two monomers (one type i keratin monomer and one type ii keratin monomer) in parallel alignment; (c) two heterodimers assembled into a tetramer in four different ways: a , a , a , acn (chou and buehler, ) ; (d) the composition of hair: two tetramers associate into a protofibril, four protofibrils combine into intermediate filament (if) . the ifs are surrounded by sulfiderich keratin-associated proteins (kaps). the formation of disulfide bonds (cysteinecysteine), hydrogen bonds (e.g. glutamine-serine) and ionic bonds (e.g. aspartic acid-lysine) in protein. fig. . feather structure and secondary β-keratin structure. (a) feather structure is shown as barbules/barbs, barbules, rachis; (b) conserved amino acids of the barbules/barbs (ncbi accession number: np_ . ), barbules (np_ . ), feather rachis (np_ . ) (jin et al., ) with the β-strands in each sequence indicated in red; (c) the monomeric central structure of βkeratin in feathers. the structure is based on the amino compositions of the filament framework segments in avian feather keratin (fraser and parry, ) ; (d) the central monomeric structure of β-keratin, where the four strands are usually antiparallel; (e) dimeric structure (β-sandwich) of β-keratin; (f) the basic repeating structure of the β-filaments, with the four dimers rotated by approx. ° to form a helix structure, with the fifth dimer at the bottom assuming an inverted sandwich position compared to the first dimer (calvaresi et al., ) . . catalytic triad and catalytic mechanism of keratinolytic serine protease enzymes. the nucleophilic ser first acts as a nucleophile to attack the carbonyl carbon of the keratin protein. a tetrahedral intermediate is generated because a pair of electrons from the double bond of the carbonyl oxygen moves to the oxygen. next, the electrons move back from the negative oxygen to recreate the bond, generating an acyl-enzyme intermediate and releasing the n-terminus of the peptide. thus, a larger space is formed to allow water coming into the reaction to facilitate a nucleophilic attack on the carbonyl carbon of the substrate. a bond between the oxygen of water and the carbon in the substrate is formed generating another tetrahedral intermediate. in the final stage of the reaction, the carbonyl carbon reforms the double j o u r n a l p r e -p r o o f bond with the oxygen. as a result, the c-terminus of the peptide is released. the active sites of fiscp (his -glu -his -glu (co + )) are displayed as sticks; (c) the mechanism of catalyzing the hydrolysis of protein by fiscp: deprotonation of the nucleophilic water molecule; nucleophilic attack of the resulting hydroxide on the scissile carbonyl carbon; protonation of the scissile amide group, followed by cleavage of the peptide bond (szeto et al., ). j o u r n a l p r e -p r o o f  overview of keratin as an enzyme substrate for circular economy biorefining  details of α-keratin and -keratin  examination of microbial keratinases: protein family, origin, reaction optima  critical assessment of keratinase enzyme assays  structure-function analysis of all known endo-keratinolytic proteases j o u r n a l p r e -p r o o f journal pre-proof beyond plucking: feathers bioprocessing into valuable protein hydrolysates the molecular organization of the beta-sheet region in corneous beta-proteins (beta-keratins) of sauropsids explains its stability and polymerization into filaments bioprocessing of -hair waste‖ by paecilomyces lilacinus as a source of a bleach-stable, alkaline, and thermostable keratinase with potential application as a laundry detergent additive: characterization and wash performance analysis sequence analysis and heterologous expression of the wool cuticle-degrading enzyme encoding genes in fusarium oxysporum - prokaryotic expression and characterization of a keratinolytic protease from aspergillus niger structure and mechanical properties of human trichocyte keratin intermediate filament protein mitotic arrest-associated enhancement of o-linked glycosylation and phosphorylation of human keratins and the effect of changing the hydrophobic s ' subsite of thermolysin-like proteases on substrate specificity isolation of a microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo a thermostable, alkaline-active, keratinolytic proteinase from chrysosporium keratinophilum production of keratinase by bacillus subtilis s proteinase k from tritirachium album limber ft-raman spectroscopic study of keratotic materials: horn, hoof and tortoiseshell an investigation on keratin extraction from wool and feather 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packing in the feather keratin filament the proteins of the keratin component of bird's beaks. aust keratin degradation by fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order thermotogales keratinous waste decomposition and peptide production by keratinase from geobacillus stearothermophilus ad- cloning of feather-degrading minor extracellular protease from bacillus cereus dcuw: dissection of the structural domains thermal effects of ionic liquid dissolution on the structures and properties of regenerated wool keratin comprehensive analysis of proteins secreted by trichophyton rubrum and trichophyton violaceum under in vitro conditions a comparison of lizard claw keratin proteins with those of avian beak and claw an updated nomenclature for keratin-associated proteins (kaps) a surfactantstable bacillus pumilus k α-keratinase and its potential application in detergent industry azo dying of α-keratin material improves microbial keratinase screening and standardization dynamic evolution of the alpha (α) and beta (β) keratins has accompanied integument diversification and the adaptation of birds into novel lifestyles keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump biotechnological applications and prospective market of microbial keratinases microbial keratinases and their prospective applications: an overview revisiting microbial keratinases: next generation proteases for sustainable biotechnology statistical modeling and optimization of keratinase production from newly isolated bacillus subtilis rse purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete actinomadura keratinilytica strain cpt isolated from poultry compost soluble derivatives of feather keratin. . isolation, fractionation and amino acid composition biodegradation of feather waste keratin by the keratin-degrading strain bacillus subtilis some properties of keratin biomaterials: kerateines effect of keratinase on ileal amino acid digestibility in five feedstuffs fed to growing pigs genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus onygena corvina newly isolated bacillus sp. g from patagonian wool produces an enzyme combination suitable for felt-resist treatments of organic wool a review: biodegradation and applications of keratin degrading microorganisms and keratinolytic enzymes, focusing on thermophiles and thermostable serine proteases keratin disorders: from gene to therapy production, characterization, gene cloning, and nematocidal activity of the extracellular protease from stenotrophomonas maltophilia n amino acid sequence of proteinase k from the mold tritirachium album limber: proteinase k-a subtilisin-related enzyme with disulfide bonds cloning and characterization of the pepd gene of aspergillus niger which codes for a subtilisin-like protease development of a keratinase activity assay using recombinant chicken feather keratin substrates identification of matrix metalloproteinase- -suppressive peptides in feather keratin hydrolysate secreted subtilisin gene family in trichophyton rubrum the possible role of dermatophyte cysteine dioxygenase in keratin degradation markers for host-induced gene expression in trichophyton dermatophytosis identification of keratinases from fervidobacterium islandicum aw- using dynamic gene expression profiling in vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of cinnamomum-, syzygium-and cymbopogon-species against aspergillus fumigatus and trichophyton rubrum crystal structure of fervidolysin from fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin keratins: a structural scaffold with emerging functions biodegradation of keratin waste: theory and practical aspects the release of sulfur compounds during degradation of feather keratin by two bacillus strains microbial decomposition of keratin in nature-a new hypothesis of industrial relevance enzymatic degradation of prion protein in brain stem from infected cattle and sheep keratinase from newly isolated strain of thermophilic bacillus for chicken feed modification modeling and structural analysis of pa clan serine proteases structural basis for heteromeric assembly and perinuclear organization of keratin filaments insight into the substrate length restriction of m carboxypeptidases: characterization of two distinct subfamilies carboxypeptidase taq, a thermostable zinc enzyme, from thermus aquaticus yt- : molecular cloning, sequencing, and expression of the encoding gene in escherichia coli biochemical and structural characterization of a keratin-degrading m carboxypeptidase from fervidobacterium islandicum aw- genome sequence of a native-feather degrading extremely thermophilic eubacterium functional expression of the keratinolytic serine protease gene sfp from streptomyces fradiae var. k in pichia pastoris independent expansion of zincin metalloproteinases in onygenales fungi may be associated with their pathogenicity characterization of chicken feather biocarbon for use in sustainable biocomposites enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions structural and dynamic basis of serine proteases from nematophagous fungi for cuticle degradation pure keratin membrane and fibers from chicken feather expression and characterization of a keratinase encoding gene gm in streptomyces pactum act strain. sheng wu gong cheng xue bao structure and biochemistry of mammalian hard keratin challenges and opportunities in identifying and characterising keratinases for value-added peptide production keratin hydrolysis by dermatophytes degradation of prp sc by keratinolytic protease from nocardiopsis sp. toa- molecular characterization of a keratinolytic enzyme from an alkaliphilic nocardiopsis sp. toa- aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte trichophyton rubrum keratinase of a streptomycete amino acid composition of feather barbs and rachises in three species of pygoscelid penguins: nutritional implications mozammel hoq, m, . cloning, expression and structure simulation of keratinase from bacillus licheniformis strain mzk . malays community-intrinsic properties enhance keratin degradation from bacterial consortia understanding the dynamics of keratin weakening and hydrolysis by proteases genetic and molecular basis of feather diversity in birds the chicken frizzle feather is due to an α-keratin (krt ) mutation that causes a defective rachis identification and characterisation of a bacillus licheniformis strain with profound keratinase activity for degradation of melanised feather biological pretreatment of chicken feather and biogas production from total broth an efficient cloth cleaning properties of a crude keratinase combined with detergent: towards industrial viewpoint keratin waste recycling based on microbial degradation: mechanisms and prospects comparative analysis of secreted enzymes between the anthropophilic-zoophilic sister species trichophyton tonsurans and trichophyton equinum the intermediate filament architecture as determined by x-ray diffraction modeling of hard α-keratin degradation of keratin by keratinase and disulfide reductase from bacillus sp. mts of indonesian origin optimization for production of liquid nitrogen fertilizer from the degradation of chicken feather by iron-oxide (fe o ) magnetic nanoparticles coupled β-keratinase optimization of production of an oxidant and detergent-stable alkaline β-keratinase from brevibacillus sp. strain as-s -ii: application of enzyme in laundry detergent formulations and in leather industry optimization of medium composition for keratinase production on feather by bacillus licheniformis rg using statistical methods involving response surface methodology introduction: serine peptidases and their clans introduction: metallopeptidases and their clans structure and properties of chicken feather barbs as natural protein fibers purification and characterization of a keratinolytic metalloprotease from chryseobacterium sp. kr characterization of a new keratinolytic bacterium that completely degrades native feather keratin hair follicle differentiation and regulation degradation of wool by saprotrohic fungi a novel alkaline keratinase from bacillus subtilis dp with potential utility in cosmetic formulation on the size of the acitive site in proteases determination of keratin degradation by fungi using keratin azure active site gate of m carboxypeptidases illuminated by crystal structure and molecular dynamics simulations versatility and commercial status of microbial keratinases: a review coupled action of c-glutamyl transpeptidase-glutathione and keratinase effectively degrades feather keratin and surrogate prion protein, sup nm extracellular expression of keratinase ker p from pseudomonas aeruginosa in e. coli sustainable management of keratin waste biomass: applications and future perspectives keratin: dissolution, extraction and biomedical application characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic bacillus halodurans jb exhibiting dehairing activity localization of alpha-keratin and beta-keratin (corneous beta protein) in the epithelium on the ventral surface of the lingual apex and its lingual nail in the domestic goose (anser anser f. domestica) by using immunohistochemistry and raman microspectros modeling effects of mutations in coiled-coil structures: case study using epidermolysis bullosa simplex mutations in segment a of k /k intermediate filaments structural characterization reveals the keratinolytic activity of an arthrobacter nicotinovorans protease a distinct residue site triggers coiled-coil formation in cortexillin i decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria qm/mm study on the mechanism of peptide hydrolysis by carboxypeptidase a characterization of an alkaline protease from bacillus sp. no. ah- valorisation of chicken feathers: a review on recycling j o u r n a l p r e -p r o o f and recovery route-current status and future prospects valorisation of chicken feathers: characterisation of physical properties and morphological structure purification and partial characterization of serine-metallokeratinase from a newly isolated bacillus pumilus nrc rna sequencing-based genome reannotation of the dermatophyte arthroderma benhamiae and characterization of its secretome and whole gene expression profile during infection hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies livestock and poultry: world markets and trade microbial keratinases: industrial enzymes with waste management potential keratin: structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration effects of dietary supplementation of keratinase on growth performance, nitrogen retention and intestinal morphology of broiler chickens fed diets with soybean and cottonseed meals beneficial effects of versazyme, a keratinase feed additive, on body weight, feed conversion, and breast yield of broiler chickens degradation of intact chicken feathers by thermoactinomyces sp. cdf and characterization of its keratinolytic protease production and characterization of keratinolytic proteases by a chicken feather-degrading thermophilic strain, thermoactinomyces sp. yt p mapk-dependent shaping of the keratin cytoskeleton in cultured cells trichophyton antigens associated with ige antibodies and delayed type hypersensitivity a new aminopeptidase from the keratin-degrading strain streptomyces fradiae var. k the discovery of novel heat-stable keratinases from meiothermus taiwanensis wr- and other extremophiles serine proteases and metalloproteases fungus zoophthora radicans keratin degradation: a cooperative action of two enzymes from stenotrophomonas sp construction of a rapid feather-degrading bacterium by overexpression of a highly efficient alkaline keratinase in its parent strain bacillus amyloliquefaciens k low-molecular weight keratins with anti-skin aging activity produced by anaerobic digestion of poultry feathers with fervidobacterium islandicum aw- flippase (flp) recombinase-mediated marker recycling in the dermatophyte arthroderma vanbreuseghemii separation and purification of a keratinase as pesticide against root-knot nematodes trichophyton rubrum secreted and membrane-associated carboxypeptidases gene expression profiling in the human pathogenic dermatophyte trichophyton rubrum during growth on proteins production and characterization of surfactant-stable fungal keratinase from gibberella intermedia ca - with application potential in detergent industry characterisation of keratin biomass from butchery and wool industry wastes this work is supported by the technical university of denmark and the china scholarship council (csc) grant # . key: cord- -e pmyvdq authors: kaiper-marquez, anna; wolfe, emily; clymer, carol; lee, jungeun; mclean, elisabeth grinder; prins, esther; stickel, tabitha title: on the fly: adapting quickly to emergency remote instruction in a family literacy programme date: - - journal: int rev educ doi: . /s - - -y sha: doc_id: cord_uid: e pmyvdq the covid- pandemic has led to extraordinary changes in family literacy instruction, forcing face-to-face programmes to shift rapidly (or “on the fly”) to online, remote instruction. this study is one of the few on online teaching and learning in family literacy and, to the knowledge of the authors, the first on emergency remote instruction in a family literacy programme during the covid- pandemic. this article examines how the goodling institute for research in family literacy at the pennsylvania state university in the united states has responded to the pandemic by converting its face-to-face family literacy classes into emergency remote instruction using online platforms. serving eight immigrant families in – who live in the state college area in central pennsylvania, the family pathways programme includes adult education, parent education and interactive parent–child literacy activities. the article discusses how teachers created online learning opportunities for parents and children to learn together, the strategies and resources instructors used to teach remotely, how challenges such as discomfort with technology were addressed, and what has been learned from the experience. although covid- presents unprecedented challenges for educators and learners in family literacy programmes more broadly, it has also compelled instructors in this particular programme to use remote instruction creatively and has revealed the critical importance of family literacy programmes as an educational support system for low-income and immigrant families. distance education (de) -especially the kind delivered through radio and television -has been used for decades to provide adult literacy education, particularly in lower-income countries (aderinoye ) . today, de is increasingly offered online via computers, mobile (cell) phones, tablets and other digital devices. de classes are typically planned and designed months beforehand, whereas emergency remote teaching is "a temporary shift of instructional delivery to an alternate delivery mode due to crisis circumstances" (hodges et al. , n.p.) . the covid- pandemic offers a distinctive opportunity to explore how adult basic education (abe) and family literacy programmes have moved to emergency remote teaching. adult basic education (abe) refers to an array of educational services offering adults the opportunity to acquire basic literacy and numeracy skills, as well as language skills, thus enabling them to become fully participating members of their community. family literacy emerged in the late s in the united states (us). family literacy or learning includes programmes, services or activities that provide education for both adults and children, encourage reading in families and/or help parents support their children's education. from to , many family literacy programmes were funded by federal even start legislation. even start integrated "early childhood education, adult literacy (adult basic and secondary-level education and instruction for english language learners), parenting education, and interactive parent and child literacy activities for low-this article examines how instructors at the goodling institute for research in family literacy (gi) at the pennsylvania state university have responded to the pandemic by converting face-to-face family literacy classes into online, remote instruction. as researchers at gi, we used a case study approach to respond to four research questions: ( ) how were de strategies, digital tools and online resources in the family pathways programme used to provide remote instruction to families? ( ) how did the family literacy instructors and programme help parents and children continue learning during the covid- pandemic? ( ) what instructional challenges did instructors encounter, and how were they addressed? ( ) what was learned from this experience that can inform others using remote instruction and de modalities to support families? to address these questions, the instructors (anna kaiper-marquez and emily wolfe; the first and second authors of this article) analysed and reflected on their emergency remote teaching practices. the author team then collectively identified wider implications for family literacy. we argue that although the pandemic has presented unprecedented challenges for adult educators and learners in the united states (us) and around the world, it has also compelled instructors to use remote instruction creatively and has revealed the critical importance of family literacy programmes as an educational support system for families. this section begins with a summary of insights from very recent literature on a new teaching mode termed "emergency remote instruction". there is no published research on this type of teaching in our specific field of interest, because modern-day abe and family literacy programmes have never experienced a global pandemic. since remote instruction and de share some similar instructional strategies, technologies and challenges, we also review pertinent literature on de in abe and family literacy, including parent education and parent-child interactive literacy activities (ilas). income families" (us department of education , para ). although this four-component model is still widespread, many other family literacy models exist. see gadsden ( ) on the development of family literacy in the us, and hanemann ( ) for examples of international family literacy. footnote (continued) the term emergency remote teaching (or instruction) was coined in during the covid- pandemic. online distance education is an established, planned approach to designing educational content and using a suite of instructional strategies for online learners, whereas "emergency remote teaching should be considered a temporary solution to an immediate problem" (bozkurt and sharma , p. ii) -in this case, pandemic-induced educational disruptions. thus, emergency remote teaching and online distance education are not synonymous. although focused on formal education in k- schools and higher education, the emerging literature on emergency remote instruction includes teaching and planning recommendations pertinent to adult education. these include engaging in "creative problem solving" (hodges et al. , n.p.) , providing active learning opportunities (quality matters ), providing scaffolding and cognitive aids (pohan ) , fostering community and collaboration among learners (pohan ; quality matters ), creating "a climate of empathy and care" through "emotional presence" (bozkurt and sharma , p. iii) , and providing adequate training and support for teachers (karalis ) . a webinar sponsored by the unesco institute for lifelong learning (uil ) highlighted how the pandemic has affected family and community learning in poland, ireland, germany and pakistan. salient insights included the expectation for parents and caregivers -particularly mothers and older girls -to assume a more active role in young children's education at home and revealed challenges related to low parental and child literacy rates. programmatic and policy responses included supporting social and emotional learning, providing self-directed distance learning, organising online meetings for families and experts (e.g. psychologists, educators), and providing creative family learning opportunities (e.g. online stories series with actors reading fairy tales, online science centres, family music projects). these practices have emerged in various unesco learning cities, for whom the webinar was organised. sharing their experiences with these new forms of family learning, programme representatives highlighted the importance of cultivating a learning environment at home. in us-based abe programmes, de is nearly always online and asynchronous, meaning that teachers and students do not meet at the same time. there are four de models: ( ) pure (no in-class instruction); ( ) blended (de and in-class instruction are integrated); ( ) hybrid (online curriculum is not necessarily aligned with in-class instruction); and ( ) supplemental (online work is optional and outside of regular class time) (murphy et al. ; vanek et al. ) . online instruction typically involves approved (commercial) products rather than teacher-created content. instructors also use various online tools and resources to support and communicate with learners. the blended model is considered most effective and prevalent in abe; structured programmes with in-person instruction combined with de tend to be more beneficial than the de model using digital technologies alone, particularly for beginner-level learners and those who need more guidance and support (murphy et al. ) . empirical studies indicate several benefits of de. with flexible delivery, de can serve geographically remote learners (prins et al. ) and allow students to work at their own pace (murphy et al. ; prins et al. ) . online de products can help teachers "identify struggling students", "provide immediate feedback" and "differentiate instruction" based on students' needs, thus helping to close educational gaps (murphy et al. , p. ; see also newman et al. ) . as learners' comfort with technology and their confidence in using it grows, their de experiences can motivate them to continue learning (porter and sturm ) and apply their knowledge to real-life tasks (rosen and vanek ) . many of the hallmarks of high-quality de in abe overlap with recommendations for remote instruction (as described above). developing a consistent, personal teacher-learner relationship with regular communication is critical for enrolment, retention and learning in de (gungor and prins ; inverso et al. ; porter and sturm ) . for example, a meta-analysis showed that instructors' involvement and active online presence predicted de programme effectiveness and learning outcomes (zhao et al. ) . de teachers also need to build community among learners, for instance, by using communication applications (apps) such as whatsapp (rosen and stewart ) . in addition, de instructors should provide meaningful, relevant, high-quality learning material (inverso et al. ; vanek et al. ) . digital access is a pervasive challenge in online de in abe and online remote instruction. to participate in online platforms, learners need to access the internet and computers or digital devices, however, this is inequitably distributed by income, formal education, ethnicity and geographic location (ryan ) . access is not only material; it also includes other domains: mental (e.g. anxiety stemming from limited basic computer experience), skills (e.g. limited knowledge of how to use digital devices) and usage (e.g. limited opportunities to use technologies) ( van dijk and hacker ) , as well as the ability to use technology to solve problems (vanek et al. ). since smartphones have become widespread and mobile apps for learning are increasing, using mobile devices is one way to lessen the digital divide (inverso et al. ; rosen and vanek ) . because some learners have limited experience with digital technologies, teachers should assess their readiness for de (gungor and prins ; vanek et al. ) . they should also provide well-planned orientations and continuous technical support (porter and sturm ) ; however, these may not be feasible in emergency remote teaching. lastly, since de depends on instructors who can use technology effectively, high-quality professional development is needed (gungor and prins ; newman et al. ) . next, we turn to de in family literacy, parent education and ilas. we identified few studies on this topic, which underscores the need for more research. research on distance learning for family literacy programmes is scarce. the available literature concentrates on integrating technology into the family, the educational possibilities of technology (at home or in a programme), and/or the need for programmes to foster families' digital literacy skills (e.g. lynch and prins forthcoming; marsh et al. ; rideout ; stephen et al. ). similar to marsh et al. ( ) , we "use the term 'digital' in relation to literacy to reflect the way in which reading and writing practices are increasingly mediated by new technologies in the new media age" (ibid., p. ). this research shows that in wealthier countries, digital technologies are already integrated into most families' lives, making online de or remote instruction a viable delivery method for family literacy. typically, parent education and ilas are delivered face-to-face (debruin-parecki ); however, covid- has reinforced the need for different delivery methods. in one of the only studies examining de in family literacy, beschorner and hutchison ( ) compared the experiences and outcomes of adult learners completing a parent education class through face-to-face classes with those who had received online instruction. the differences in parental experiences included participants' characteristics (various family members attended in-person classes, but only mothers attended online), attendance (higher for online classes) and social networking opportunities (face-to-face participants valued these opportunities more). however, the children of participants in both groups performed similarly, indicating that both methods were equally effective for teaching dialogic reading (i.e. shared reading in which the adult and child engage in an active conversation about the story) (whitehurst et al. ) . a key focus of family literacy programmes is the opportunity for parents and children to interact to enhance children's language and print literacy skills, while also allowing parents to practise what they have learned in adult education. coupled with parent education, ilas are opportunities for parents to discover how to positively influence their children's learning and development. research has consistently demonstrated that parent involvement enhances children's language and print literacy development (fan and chen ; sénéchal and lefevre ) , and that shared reading is related to language, literacy and other academic skills development (bus et al. ; shalaeian et al. ) . recognising that families already incorporate digital literacies in their everyday lives (marsh et al. ; rideout ) , the digital parenting workgroup at the university of wisconsin (clarkson ) produced a parent education programme (eparenting®) that combined online and face-to-face learning opportunities, focusing on "meaningful, positive uses of digital media with young children" (clarkson , p. ) . both the face-to-face and online classes helped parents learn new parenting ideas and techniques for using digital media. this programme, along with beschorner and hutchinson's ( ) study, demonstrates the need for further research on providing parent education online because this mode of teaching appears effective and there are myriad online teaching tools and learning platforms available. drawing on behavioural science insights regarding what researchers in that field term "nudges" (hummel and maedche ), family literacy programmes have also used text messaging and/or e-mailing to reach parents with tips and ideas for parenting and ilas, and reminders about classes, resources and activities. for instance, parents in head start (a us early childhood education programme for low-income families) who received text messages about child activities as well as parent encouragement, engaged in more learning activities with their children compared to parents who did not receive messages (hurwitz et al. ) . similarly, an eight-month textmessaging intervention showed that parents' home literacy engagement and school involvement increased (york et al. ) . finally, in a randomised experiment to prevent summer (holiday) reading loss, parents received text messages with ideas for athome literacy activities (kraft and monti-nussbaum ) . consequently, attendance at parent-teacher conferences (interviews) increased, as did reading comprehension for third and fourth graders (but not first and second graders). these studies emphasise the importance of supplementing de and remote instruction parent education with targeted text messages about family literacy resources and activities. launched in july in williamsport, pennsylvania, in the us, gi's family pathways programme expanded to the central pennsylvanian town of state college in january with funding from the pennsylvania department of education (pde), division of adult education. normally, the programme components included faceto-face instruction in adult and parent education, face-to-face and take-home ilas for parents and children to learn together, and early childhood education (children are required to enrol in an early childhood education programme or in school -kindergarten to third grade). a university town, state college is home to many international families. in - , the family pathways programme served eight immigrant families from china, egypt, iran, japan, panama and turkey. the parents had second to fifth grade reading levels in english, and their educational attainment ranged from high school to post-secondary. children (aged months to eight years) were enrolled in different early childhood facilities or schools throughout the state college area. classes were taught by two instructors (the first and second authors of this article: anna kaiper-marquez [adult education/english language development] and emily wolfe [parent education/ilas]). before the outbreak of the worldwide covid- pandemic and the issue of the statewide stay-at-home order in pennsylvania (mid-march ), classes were held twice per week; each session included . hours of adult education and hour of parent education. face-to-face ilas (with parents and children) were held on evenings, weekends or when schools were closed. the adult education, parent education and ila components often had integrated themes such as environmental and social justice issues, culturally relevant topics and school engagement. powerpoint slides were used to organise adult education classes, which typically included an introduction and check-in, warm-up activity, homework review, and a mini lesson with accompanying exercises and readings. online videos and website activities were also frequently used in classes, and instructors organised a whatsapp chat group to send parents information and updates about classes and invite learners to share pictures, recipes, questions and resources. this case study describes the instructors' experiences transitioning from faceto-face to remote instruction from the beginning of the statewide shutdown (march ) to june . five parents and children were participating in the programme during this time. three of the families were classified as low-income, according to us federal poverty criteria. as of this writing (august ), classes are still meeting remotely. in the next section, we provide findings related to our research questions. in pennsylvania, the covid- quarantine was implemented swiftly with little time to prepare and shift from face-to-face teaching to remote instruction. nonetheless, the transition to remote instruction in the family pathways programme was fairly smooth, since instructors already used a consistent structure in the face-to-face classes. before beginning remote classes, instructors quickly conducted an extensive online resource review and identified helpful resources and websites (e.g. usa learns, raising a reader and epic!) that matched families' interests and needs. many of the resources and websites were then woven into the remote lesson plans developed for parent education and ilas. similar to the face-to-face parent education class and ila take-home assignments, online tools and resources were presented and reviewed before parents implemented the activities at home with their children; parents then reported their experiences at the next class. instructors also provided a weekly remote ila class that parents could attend virtually with their children. as instructors recast the classes for remote instruction, they maintained the importance of integrating the programme components. at the start of the pandemic, covid- was on everyone's mind; parents were quarantined with their children and stressed about having to oversee both their children's and their own education. instructors responded by organising lessons related to these topics, especially during the first month of the transition. as the pandemic experience became less of a shock, instructors shifted to topics that helped families experience some normalcy and fun while continuing to emphasise learning (e.g. exploring recommended family movies on common sense media). the types of activities were also adjusted as instructors learned what worked and what did not. for example, as the pandemic progressed, families' needs and schedules changed, prompting wolfe to videotape the ila class so it could be viewed asynchronously. she added individual mentoring sessions to review how the ila went and provide other assistance. instructors also realised early on that revising face-to-face lessons for remote classes took much more time, requiring the use of creative problem-solving to adapt activities for online instruction. the remote instruction format included adult education with a focus on english language development (introduction to the lesson, warm-up activity, homework review, mini lesson, and exercises and practice); parent education (check-in and ila debrief, online resource lesson); and ila (assignment and remote ila). a synchronous (real-time) class was offered on mondays and wednesdays for . hours per session (approximately . hours of adult education and minutes of parent education). instructors used powerpoint slides to organise instruction and zoom videoconferencing to interact with parents and their children. initially, the synchronous ila class met on tuesdays for to minutes, depending on attendance and the topic; however, as mentioned earlier, the instructor soon took to videotaping the class because parents were having trouble attending the ila class at the scheduled time. adult education classes started with a parent reading the date to remind everyone of the pronunciation of ordinal and cardinal numbers. another parent read the "schedule for today" for more pronunciation practice and to preview class content; then all parents participated in a -to -minute warm-up with a vocabulary review and a fun learning activity. homework was a normal part of the face-to-face class and continued to be an important activity during remote instruction because it gave parents something to work on in addition to the supplemental distance learning activities (described below). homework included worksheets on concepts, grammar or punctuation discussed in class; readings to complete before class; and writing prompts that connected to parents' lives. with writing prompts, parents edited a screenshot of one learner's writing together, looking for concepts, grammar or punctuation that needed review or would be covered in the mini lesson. writing prompts helped the instructor informally assess parents' progress, develop future lessons and identify concepts that required more instruction. the mini lesson involved a writing, reading and/or speaking activity. lessons often took more than one class to complete and new lessons were introduced using pow-erpoint presentatios and videos. a typical lesson included a pre-reading vocabulary exercise followed by parents' taking turns reading an article to practise pronunciation and comprehension. after reading the article, comprehension questions were presented using powerpoint for parents to write down their answers at home. kaiper-marquez then called on parents to read their answers aloud and used power-point's animation feature to reveal the correct answer. lesson content was informed by parents' needs and requests as well as their progress in writing, reading and speaking, and assessments. many lessons focused on information, vocabulary and ideas parents heard daily related to the pandemic. for example, opportunities to build basic skills and develop language were provided when parents responded to writing prompts such as: think about a time in the past when you were sick. how did you get sick? how did it make you feel? what did you do to feel better? what have you done since that time to help prevent getting sick again? vocabulary was connected to covid- , since many parents had questions about what they were learning from the news. other activities included covid- -related reading passages, learning verb tenses and new vocabulary words from the readings, and discussing the pandemic. initially, lesson content focused on being healthy and safe, learning at home and establishing family routines. since the end of may, content included other important current events such as the death of george floyd and other victims of police brutality. supplemental distance learning programmes (such as khan academy and learning chocolate) were used more frequently after transitioning to remote instruction. when the quarantine began, instructors set up individual learner accounts in khan academy. this free programme tracks the time participants spend using it, which was required for reporting programme performance. it also has a plethora of videos, exercises and quizzes on english grammar that helped offset the reduction in grammar instruction due to fewer hours in class. instructors connected content from khan academy to lessons offered through zoom classes and homework. george floyd was a -year-old african american man who was killed by police during an arrest in minneapolis, minnesota, on may . khan academy is a non-profit educational organisation that offers video tutorials for children and adults on a wide range of subjects in about languages. visit https ://www.khana cadem y.org [accessed august ]. learning chocolate is a vocabulary learning platform designed for child and adult learners preparing for proficiency tests such as the international english language testing system (ielts). the platform offers language learning materials such as songs, films, themed illustrated interactive worksheets, etc. for free download in english, japanese, chinese (mandarin), german and spanish. visit https ://www.learn ingch ocola te.com [accessed august ]. wolfe began parent education classes with a review of the agenda and a check-in which helped parents practise their verbal skills, chat about how their families were doing and discuss any challenges they were facing (e.g. children perceiving their time at home as a holiday). similar to face-to-face parent education classes, parents debriefed about the previous week's ila assignment. this allowed wolfe to learn about what was happening at home and to help parents understand the skills their child gained during the ila. many parents needed assistance communicating with schools and teachers to help their children continue learning when instruction went online. at first, much of the parent education class time focused on a selected online resource such as when school is closed: resources to keep kids learning at home (reading rockets ). wolfe also reviewed the online resource "wonder of the day" on wonderopolis with parents, talked about the activities available, discussed how to adapt the activities for different age groups and children's needs, and outlined what children would learn. for example, reading rockets resources were often used as they were tailored to children's ages and included a variety of interesting topics (e.g. flight, insects, planets and dinosaurs can be explored in start with a book) that parents could select with children, and they came with recommendations for books and activities. class discussions usually occurred over two sessions: the resource would be introduced in the first session and the topic more thoroughly discussed in the second session to prepare parents for the weekly ila. while reviewing the online resource, parents had opportunities for additional adult education instruction as they took turns reading sections, explored new vocabulary and concepts, and navigated the online resource. created by the national center for families learning (ncfl), wonderopolis is a learning platform that offers information about a wide range of everyday subjects. posting an interesting new question every day, it inspires children (and adults) to think about things, do research and find answers. visit https ://wonde ropol is.org [accessed august ]. reading rockets is a multimedia resource for parents, teachers, librarians, school psychologists, etc. addressing reading needs and challenges (e.g. dyslexia), and providing strategies and insights from current research. visit https ://www.readi ngroc kets.org [accessed august ]. focusing on books, start with a book is a companion website to the multimedia platform of reading rockets. visit https ://www.start witha book.org/ [accessed august ]. after reviewing and discussing the online resource together during the parent education class, parents selected activities that were age-appropriate for their child and could be done interactively with materials already in the home (e.g. making pizza related to the book pete the cat and the perfect pizza party (dean and dean ); or reading and discussing the seventh national geographic kids volume of weird but true!: outrageous facts (national geographic kids ). then completing fun national geographic kids online quizzes). once parents had chosen their preferred activity, they reviewed other educational app ideas, child-friendly websites and writing ideas available on the resource to extend learning at home. in addition to the weekly ila assignment selected from the website, parents were encouraged to access books related to ilas and use the online resource unite for literacy, a collection of free books that can be narrated and translated into over languages. parents were asked to review the unite for literacy website for homework, find at least one book related to their chosen topic and read the book with their child as part of their weekly ila assignment. at the beginning of the quarantine, the parent education instructor maintained a traditional "take-home" model for ilas by dropping off packets with ila materials at families' homes. however, after settling into a routine, wolfe added a weekly ila experience via zoom, in which parents and children listened to her read a story, engaged in conversations about the story and reviewed book extension activities for the family to do at home. this remote ila was first offered synchronously, but eventually became an asynchronous activity to accommodate parents' changing schedules. instructors moved relatively quickly from face-to-face to remote instruction, primarily because they maintained a consistent class structure and continued using some of the same technologies. in addition, parents adjusted to new methods of teaching and learning fairly easily because they had access to mobile phones and/or computers with internet. however, some challenges did arise and had to be addressed creatively. during the first week of remote instruction, local schools closed, and parents and children of all ages were at home together. this made some programme components easier to implement (e.g. at-home ilas) and others more difficult (e.g. children popping in during adult or parent education zoom classes). common national geographic kids is a print magazine for children aged - . it is complemented by an online component that offers educational games and videos about the natural world. visit https ://kids. natio nalge ograp hic.com/ [accessed august ]. regional websites are maintained in the united kingdom, ireland, south africa, australia and new zealand. unite for literacy, a free online library, offers digital books in english and spanish for reading -and in languages, including a number of indigenous ones, for listening. visit https ://www.unite forli terac y.com [accessed august ]. challenges included dealing with digital inequities and discomfort with technology, managing instruction remotely, and addressing attendance and participation inconsistencies. parents experienced several digital challenges. first, they had difficulty signing into zoom, which was a new platform for all learners. some parents could not remember their app store log-in and password, which complicated setting up their account. using zoom features (e.g. logging in, turning the camera and microphone on and off, adjusting settings, using the chat feature) was troublesome at first because parents had to learn how to navigate the technology, but they caught on quickly as spouses and children helped them to log on and instructors used screenshots of directions to guide them. parents' comfort with using digital devices and online resources also differed. pre-covid- , parents were confident using their smartphones. they used google translate to understand new vocabulary words, social media websites (e.g. facebook, instagram, whatsapp) to connect with friends and family, and youtube videos for entertainment and information. although digital literacy skills development was part of face-to-face instruction, many parents were not totally comfortable using a tablet or computer (e.g. starting up and shutting down, using a mouse or keyboard), or using the other online resources for supplemental learning such as khan academy. this discomfort was exacerbated during remote instruction. finally, to respond to parents' preferences for receiving information, instructors sent information through both whatsapp and e-mail. although parents had various levels of comfort with technology, they also had support systems that other learners both in the us and globally might not have. for example, the state college parents received more support from spouses and children to help them access remote classes than did the parents in our williamsport family pathways classes. moreover, williamsport parents had substantial difficulty transitioning to online learning due to unstable or no access to the internet. further, the ongoing technological support that instructors provided before the pandemic in face-to-face classes, and during the pandemic in remote instruction, was essential to increasing parents' overall digital comfort; instructors spent considerable time helping parents troubleshoot technological issues whenever necessary. many organisations might not have access to two instructors and a tutor, but for the state college family pathways programme's successful transition to online learning, having more than one instructor was vital. for example, effective use of the we have insufficient documentation from williamsport to be able to make additional comparisons between the sites. zoom features took some time for instructors to learn, and they found it easier to deliver both adult education and parent education classes jointly, with one instructor teaching and the other managing technical difficulties, such as muting a learner with too much background noise. in addition, it was helpful to have the tutor engage in conversations with the parents if the instructor was having a technical difficulty or had to quickly adapt the lesson if, for example, an online link in the powerpoint did not work. drawing not only on parents' feedback, but also on her reflection on individual families' needs, wolfe decided to divide the lesson into two sessions because parents were overwhelmed with the amount of information they had to process. on mondays, she provided a mini overview of everything about the ila, including the resource, possible activities and a corresponding book. on wednesdays, wolfe provided more detail about the activity, such as how to tailor it to children of different ages. this curricular change helped keep families engaged and stemmed from wolfe's continual desire to understand families' academic and emotional needs to determine what would work in this uncharted instructional environment. parents were preoccupied with the changes in their lives, so instructors sent a text or e-mail before class to remind them about ila times or adult education homework assignments. wolfe also reminded parents about the remote ila at the beginning of each parent education class with a powerpoint slide about the book and activity; she then sent a reminder with information about the next parent education class and links for completing the ila assignment. for instructors and learners with limited data allowance or no internet access, these types of reminders might be more difficult to implement; however, the instructors also telephoned parents and dropped off instructional materials at their homes to maintain communication about upcoming classes and ilas. about a month after the quarantine began, remote ila attendance became sporadic due to families' scheduling conflicts or other issues. wolfe began recording a story time on zoom so parents could access it asynchronously. the recorded, interactive story time followed children's tv presenter fred rogers' philosophy of emphasising human relationships in teaching children by interjecting individualised comments to children. rogers (also known as "mister rogers") believed it was important to model listening to others and he did so by showing how to empathise with children (johnson and howard ) . first, wolfe greeted each child and, drawing on previous interactions with the family, shared an anecdote to get children's attention and personalise their experience -for example, "yasser [pseudonym], your mom says you are working on compound words! that's great." next, she read the book, made comments about it, asked questions and paused to acknowledge an imagined response, as if the children were present. after the story, wolfe referred to the ila discussed during parent education class or showed prompts for extension activities. she then sent links of the ila recording and other activities related to the book to the families. another challenge was that parents did not complete their homework as often during remote instruction as they did when they attended face-to-face classes. this was likely due to more demands on their time, in particular their needing to supervise and support their children's education throughout the day. since homework review was a critical part of the lesson, the instructors created sample homework assignments to use as examples in case those attending did not complete the assignment. the instructors understood that without internet access, activities such as these might be hard to administer. nonetheless, these activities were successful primarily because the instructors emphasised building human relationships, which helped them to continuously address the families' needs. the experience of adapting face-to-face classes to remote instruction demonstrated the critical importance of family literacy for low-income and immigrant families in the us. for example, the immigrant families were unfamiliar with school policies and requirements before the pandemic, and their limited understanding and ability to read english compounded their stress as they tried to navigate school websites, understand online school learning sites, read teachers' e-mails and help their children learn at home during school closures. the lessons that instructors learned can help other educators provide family literacy programming at a distance, be it by choice or by necessity. first, instructors' existing relationships with families were critical to the transition to remote instruction. families were eager to continue classes because of the trust they had in their instructors. before covid- , the instructors had made a concerted effort to develop a relationship with each family and to be aware of their individual circumstances. by having this personal connection, families did not feel like they were "on a screen" but were instead having a conversation with their teachers and friends. the instructors also continued to develop these relationships by scheduling individual ilas on zoom with parents and their children if needed. this finding supports literature showing that building caring relationships with instructors is crucial both in remote teaching (bozkurt and sharma ) and de (inverso et al. ; porter and sturm ) . in family literacy programmes that have few or no in-person meetings -whether because they use a pure de model or because of unexpected disruption -relationship building may be more challenging and instructors would have to add activities that help families get to know each other virtually. second, shifting to remote instruction significantly increased instructors' class preparation time, which may not be feasible for part-time instructors or those with heavy class loads. to keep lessons interactive and interesting to parents and children, instructors had to develop new lesson plans, look for or create videos, research useful multimedia resources and find creative ways to construct virtual interactive activities. simple actions in a face-to-face class such as distributing handouts, creating pairs or small groups, or walking around the room to monitor learners' progress are more complex and time-consuming in remote instruction. after planning the objectives and content of the lesson, instructors varied the presentation modalities by using short youtube clips, finding or creating online quizzes, and adding animation, website links and screenshots of the powerpoint slides. instructors also asked students to have certain materials available during class time such as a reading selection that was sent beforehand. these actions illustrate the principle that "involved instruction" is critical for successful de (vanek et al. ) and remote instruction. in this approach, teachers actively engage in students' learning, particularly by mediating between learner and online content rather than relying solely on pre-established online curricula (ibid., p. ). through their supportive, personalised teaching and creative use of online materials and digital tools, instructors modelled this teaching approach. involved instruction is inevitably more difficult for instructors with large numbers of learners who have multiple academic and emotional needs, but for this programme it was crucial for maintaining families' interest and persistence. the covid- pandemic was challenging for instructors, but also engendered innovation and a positive outlook that are likely to remain when face-to-face classes resume. there were two unanticipated outcomes. first, the instructors are now planning to continue incorporating some aspects of remote instruction. specifically, they will use zoom when face-to-face ilas are not possible (e.g. school snow days or parents needing to be home with sick children), and parents will continue to be enrolled in supplemental online programmes such as khan academy. another outcome is that parents increased their digital literacy skills (e.g. their ability to use computers, smartphones, apps and digital tools) because they wanted to continue participating in the class. however, the instructors realised the need to make digital literacy skills development a more intentional part of their lesson planning. these plans illustrate how a supplemental de model can enhance family literacy programming and build digital literacy skills. finally, instructors found that parents were able to model lifelong learning for their children, an essential component in family literacy programmes. for example, children often sat on their mother's lap while she participated in her adult education class. children's presence in adult education classrooms is often viewed as disruptive to learning, but during the quarantine children watched their parents be students too. moreover, seeing their parents as students helped some children be less unruly because this was their "new normal". modelling is a foundational teaching practice within families, as children observe parents and other caregivers engage in language, reading, print (burgess et al. ) and digital literacy practices. in this case, parents also modelled a positive orientation towards learning and adapting to unexpected circumstances. the covid- pandemic has led to extraordinary changes in family literacy instruction, forcing face-to-face programmes to shift rapidly to online, remote instruction. although our findings are derived from a small number of families who participated in a specific type of family learning intervention in one location in central pennsylvania, this study has global implications for remote instruction. the strategies used for teaching the family literacy components remotely are applicable to any programme that aims to support emotional and self-directed learning and enhance parents' and children's education. moreover, although the family pathways programme instructors used us-based online resources, any programmes providing creative family learning opportunities will foreseeably find these or similar resources useful. this study is one of the few on online teaching and learning in family literacy and, to our knowledge, the first on emergency remote instruction in a family literacy programme during the covid- pandemic. we found that the consistent structure used in face-to face classes helped both instructors and parents transition smoothly to an online format. moreover, the shift to online, remote instruction encouraged instructors to find new ways to promote learning (e.g. supplemental distance learning programmes, online resources, recorded instructional videos, expanded use of social media messaging apps) that can be continued when face-to-face classes reconvene. instructors also exhibited many of the features of high-quality de and remote instruction cited in the literature, particularly in terms of maintaining caring, supportive relationships with learners, thus creating an education support system for low-income and immigrant families. converting face-to-face programming to remote instruction was not without its difficulties, including parents' digital challenges, instructors' need to manage multiple distractions during class, and sporadic attendance due to parents' responsibility for overseeing children's schoolwork. such challenges, particularly for programmes in areas with high poverty and limited internet access, must be further examined and mitigated to ensure effective and equitable programming for all families, both in de and emergency remote instruction. however, even with these obstacles, this experience revealed that moving to remote instruction incited some benefits such as improvement in parents' digital skills, greater participation between parents and children engaging in ilas at home, and continued family learning that might otherwise have ceased due to the covid- quarantine. ultimately, this case study supports a conclusion drawn by the unesco webinar on family learning (uil ), namely that the covid- pandemic has led to greater acknowledgement, in policy and practice, of parents' and families' pivotal roles in children's learning and of parents' and adults' own learning needs. emphasis was previously placed on formal education, but now we are more clearly seeing the important role played by informal, intergenerational and family learning (ibid., n.p.). our study helps elucidate how instructors can nurture family learning through remote instruction. anna kaiper-marquez, phd, is the associate director and assistant teaching professor of the institute for the study of adult literacy (isal) and the goodling institute for research in family literacy at the pennsylvania state university. she has published several journal articles, book chapters and book reviews on adult basic education (abe), family literacy, english language learning, and qualitative methodologies in national and international contexts. dr kaiper has been an abe and english language instructor in the united states, thailand, argentina and south africa, and currently teaches online courses on adult and family literacy topics through penn state. emily wolfe is the adult education and family literacy services coordinator and family literacy instructor at the goodling institute and the family pathways programme. she has over years of experience in family literacy programming providing direct service to families throughout pennsylvania, pa. her work focuses on creating and delivering technical assistance to family literacy programmes, developing curricula, and designing and implementing professional development opportunities on the topics of early childhood education, parent education, and interactive literacy activities. carol clymer, phd, is the co-director for isal and the goodling institute at penn state university. dr clymer has + years of experience implementing and evaluating programmes to improve education and employment prospects of low-income individuals. as a senior program director at public/private ventures, she participated in the design and implementation of professional development projects and largescale evaluations and outcomes studies related to performance benchmarking, employment retention, sectoral employment, out-of-school youth, and college access. as the director of literacy and workforce development at el paso community college, her work focused on adult education, workplace and family literacy. jungeun lee, phd, is the assistant teaching professor/associate co-director at isal and the goodling institute at penn state. she completed her phd in adult education at the university of georgia. her research interests include adult literacy and adult learning policy and practices, education policy analysis, educational inequality from comparative and international perspectives, and quantitative and mixed methodologies. elisabeth grinder mclean, phd, is an assistant teaching professor at the goodling institute at penn state. dr mclean has over years of experience implementing, evaluating and providing professional development to early childhood and family literacy programmes that serve low-income families. her work focuses on early literacy development, parent engagement, and parent-child interactive literacy activities that enhance children's school success. dr mclean is also the lead faculty member for the online family literacy certificate at penn state and teaches several online courses about interactive literacy activities, parent engagement and early childhood education. esther prins, phd, is a professor in the lifelong learning and adult education program at penn state, where she also serves as the co-director of isal and the goodling institute. her research interests include critical and participatory approaches to adult education (particularly adult and family literacy), community development, and research. she has conducted research in the usa, el salvador, belize, ireland, ghana and tanzania. her research has been published in journals such as adult education quarterly, international journal of lifelong education, studies in the education of adults, american educational research journal, and teachers college record. tabitha stickel is a phd candidate at penn state university's lifelong learning and adult education programme; her research focuses on the dynamics of classroom belonging in an adult vocational education programme on native american tribal land. tabitha works as research assistant at the goodling institute. she received her edm in adult learning and leadership from teachers college, columbia university, in may . her master's thesis examined native american student experiences in an adult basic education programme. prior to graduate school, tabitha taught adult basic and developmental 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cord- - ll hzfr authors: giordano, chiara title: freedom or money? the dilemma of migrant live‐in elderly carers in times of covid‐ date: - - journal: gend work organ doi: . /gwao. sha: doc_id: cord_uid: ll hzfr nan dana is a -year-old romanian woman, who works as a live-in elderly carer in brussels. after ten years as a caregiver first in italy and then in france, she moved to belgium years ago, when a friend proposed her a better working opportunity. for the last year and a half, she has been working for the same employer, where she looks after an old lady, seriously impaired due her mobility problems and a cognitive degeneration linked to the alzheimer's disease. thanks to her job as a caregiver, she managed to provide her two daughters with secondary schooling and to help them buy the apartments where they currently leave with their own families. with the money that she sends home she helps her husband, unemployed, and contributes to the bank mortgages of her daughters. when the covid- stroke belgium and the lockdown measures were announced by the belgian government on the th of march, she used to live with the older woman six days per week, and she used to spend her day off in the small apartment that she rents with other romanian caregivers. during her day off, she was used to go to the church and enjoy the apartment, talking in romanian and cooking romanian food together with her friends. she was planning to welcome her husband in june and eventually move to a day-time hourly job. since the th of marcha few days before the official lockdown startedshe lives permanently with the older woman. at the request of the employer family, concerned about the health of the elderly woman, dana has not left the elderly person's residence: she has lost her day off, which represented her only time of leisure and social activities, and the freedom to go out for a small walk or for grocery shopping during the day, as she was used to do before. the only people she meets in person are the frail elderly lady and the lady's daughter, who comes by once a week to bring supplies. with her family, confined to romania, and with her friends all over europe, she remains connected through the internet. dana is one of the many invisibles of our era: invisible in normal times and even more invisible in covid- times. to preserve and protect the safety of another invisible (the older lady confined to her private domicile), dana had to choose between keeping her jobat the detriment of her personal freedom and her psychological, physical and mental healthand preserving her freedom but losing her jobjeopardising her survival in a country other than her own, and the survival of her family in her country of origin. as it is the case of other livein elderly caregivers, she had to choose between freedom and money. this paper explores the dilemma faced by migrant live-in elderly carers in times of covid- , by focusing on five interlocking systems of oppressionor domains of powerwhich are likely to determine both their experiences (namely, their living and working conditions during the pandemic) and their choices (namely, their solutions to the dilemma). drawing from the intersectionality theory (hill collins, ; hill collins and bilge, ) , which highlights the centrality of interlocking systems of oppression in forging the identities and experiences of women, i use the term 'identity locations' to refer to the positioning of migrant live-in elderly carers within these systems of oppression (or domains of power). specifically, i look at their 'identity locations' as women, as migrants, as elderly care workers, as family breadwinners, and as 'quasi-family members' in their employing families, which are in turn related to larger domains of power (namely gender, ethnicity/citizenship, labour market, care economy and interpersonal power relations, respectively). i argue that the position of migrant live-in elderly carers at the intersection of these five interlocking systems of oppression transposes the dilemma to a purely symbolic level. their economic survival and that of their families, as well as the ethical dimension associated to their work as caregivers, among other factors, resolves the dilemma by turning it into a nondilemma, where the decision to keep the job and to accept the newly defined working conditions and the consequent loss of freedom becomes unavoidable. what are the consequences of the unprecedented crisis generated by the covid- on our lives and on our society at large? this question, so vast and so simple, has become in the past few months the new mantra in politics, in the media, as well as in the academia. while the attention of governments is currently focused on analysing the economic consequences of this crisis, in order to contain its effects and to 'relaunch the economy' after the covid- , many voices have raised to emphasise other aspects of this phenomenon, such as the social, psychological and emotional consequences that this pandemic has had, has and will have on our lives and livelihoods. as an article appeared in the belgian press highlights (le soir, / / ), visions diverge greatly when it comes to identifying the predominant social reactions triggered by the covid- crisis. on the one hand, there are those who, mainly referring to the health dimension of the crisis, highlight the 'social justice' of the virus. the fact that everyoneincluding doctors and politicians, who symbolise the true powerful in our societyhave been indiscriminately affected by the virus would demonstrate the equalising power of the pandemic. on the other hand, other analyses, mainly referring to the economic, social, family and psychological dimensions, highlight the intensification of existing inequalities and of social hierarchies, and the resurgence of class divisions, in a marxist structuralist interpretation. from a purely health perspective, although certain categories at the top rungs of the socioeconomic ladder, such as medical doctors, are among the highest-risk categories as a result of their position at the front line of the emergency, the first available data show that vulnerable populations have suffered higher rates of infections, proving that even the health equality in front the pandemic is an illusion (fassin, ) . leaving aside the inevitable preponderance of the health dimension over any other, what strikes the most in this chaotic and unprecedented situation is the dramatic intensification of existing inequalities and the resurgence of structural inequalities sank into oblivion. in a broader perspective and including other dimensions of our livelihoods, the crisis has proved to disproportionately affect two segments of the population: the most vulnerable, in terms of economic, social and cultural capitalthat is, lower classes (major and machin, ; adams-prassl, )and women, regardless of their socioeconomic position (alon et al., ; etuce, ; eige, ; iweps, ) . concerning the latter, as it has repeatedly been stressed, the covid- crisis has and will have an impact on various aspects of gender equality, including the economic hardship falling on women whose economic position is precarious, which is the case of many single-parent households; the sharpening of domestic violence; the hazardous situation of workers in the frontline, the majority of whom are women; and the increased burden of unpaid care and housework responsibilities during the lockdown period . trapped in a sort of reverting vortex, some women saw the resurgence of those inequalities that were supposedly become obsoleteor at least hiddenfor many. as emphasised by feminist scholars, despite the progress made by european women in terms of equal opportunities and gender equality, their equality remains intrinsically precarious (evans, ) . this risk of relapse affects not only women's performances in the labour market, but alsoand especiallythe division of labour within households. in the context of this crisis, the closure of schools and childcare facilities and the imposition of a system of teleworking have disproportionately affected working women, who 'reassumed' the greatest part of housework and caring responsibilities, regardless of their ability to telework and/or to reduce their working hours. rather than representing for working women a real backlash in the labour market, the crisis had the effect of bringing back to the fore the traditional dichotomy that assigns men the economic function and women the private sphere (anderson, ; gerhard et al., ; lutz, ) . in other words, the crisis reopened the debate, well-known to the feminist literature, on the division between productive and reproductive work and the essentialisation of gender roles around these criteria, which visibly still represents a crucial node of gender relations. however, although this crisis had consequences in terms of unequal gender relations even for the most privileged segments of the population, it has not affected all women in the same way. as the literature based on intersectionality has long stressed, the identities of women are heterogeneous and distinct, and depend on the ways in which interlocking systems of oppression intersect and act upon them in a matrix of domination (carastathis, ; hill collins, ) . women's experiences are profoundly shaped by these systems of oppression and depend on their position as actors at their intersection. their 'identity locations'that is, their position within these systems of oppressionare not fixed and static, nor mutually exclusive, but they represent the place at the intersection of which women's experiences and choices are distinctively negotiated. looking specifically at the situation of migrant live-in elderly carers, at least five interlocked systems of oppression can be recognized which act simultaneously in shaping their work and livelihood, as well as their agency and their responses in times of covid- . their position as women, as migrants, as care workers, as family breadwinners, and as 'quasi-family members' in the employer's family, represent specific 'identity locations' which are likely to affect their experiences and their choices. specifically, it is their position at the intersection of without neglecting the importance of the debate on the misappropriation of intersectionality by western scholars and the displacement from the gender/class/race triad to a variety of other categories, adapted to the european context (davis, ) , in this paper i position my analysis in this stream. therefore, i use an intersectional approach for the analysis of the 'identity locations' which are specifically relevant to migrant elderly carers in the context of belgium. these systems of oppression, which correspond to different 'identity locations', which make their situation unique and determine their work and living conditions in times of covid- . this paper digs into these five 'identity locations' and in the way in which they interact in determining their response, when faced with the dilemma between freedom and money. the material used for this paper draws from two main sources. first, it draws from the material collected in the framework of an ongoing research on the undeclared work in the home elderly care sector in brussels. in the framework of this research, whose fieldwork had to be interrupted as a result of the covid- lockdown measures, i had collected qualitative data in the form of in-depth interviews, one focus group and fieldwork notes on migrant women working as elderly carers in belgium, without a regular contract. the second source derives from the exchanges i had during the lockdown period with the migrant elderly carers i had previously interviewed. thanks to the strong relationship that i had built with some of them, and in preparation for a longitudinal study that will be pursued in the following years, i had the chance to regularly exchange with them, mainly by text messages (whatsapp or sms), during the lockdown period. the direct quotations, as well as the empirical situations that i describe in this paper and the resulting reflections are issued from these exchanges and are based on their experiences. as long stressed by the scholarship, domestic and care work are different from any other employment relationship, for a variety of reasons (anderson, ; gerhard et al., ; cox, ; lutz, lutz, , devetter et al., ). this has important consequences on the working conditions and lives of elderly carers. on the one hand, the private nature of the workthat is, the fact that it is performed in the private home of the care receiverhas direct consequences for caregivers, including the lack of control over their working conditions (rollins, ; devetter et al., ) . because the work is not subject to inspections or control by third parties, not only care workers are potentially at risk of abuse, be that physical, verbal, or psychological, but they are also subject to poor working conditions, such as low salaries, physical and emotional hardship and long working hours, among others. the latter element is especially problematic for live-in caregivers: even when working hours are contractually defined, live-in elderly carers are practically required to work on a system of uninterrupted availability, resulting in a lack of separation between leisure and working time. linked to this element is the fact that for live-in caregivers the work itself implies a lack of separation between 'home' and 'work', with a dangerously skewed predominance of the latter. because their living environment is a type of home with diminished characteristicsa 'quasi-home', which is never completely felt as home the result is that the caregiver always feels 'on duty', without having the opportunity to completely disconnect from work. on the other hand, the work itself is endowed with specificities which make it essentially different from other employment relationships and which have enormous consequences for caregivers. first, even when remunerated, care work involves tasks which are traditionally part to the realm of the reproductive labour. the demarcation line between productive and reproductive labour remains blurred and contributes to the difficulties in recognising it as 'real work' (anderson, ; cox, ; devetter et al., ) . furthermore, because of its association with the reproductive function of the family, it is traditionally associated to feminine attributes and expected to be assumed by female members of the family (anderson, ; lutz, ) . the main consequences of the naturalisation and essentialisation of care work as feminine are the lack of valorisation of care work, the persistence of marked gender stereotypes and the persistence of employers' expectations in the form of love and abnegation from the caregiver, which correspond to what a female family member would supposedly do. other elements that are found to be problematic for caregivers include the ambiguous employment relationship at the basis of care work, which continuously oscillates between a real employment relationship and a family relationship (rollins, ; cox, ) and the emotional labour required from caregivers (hochschild and ehrenreich, ; anderson, ) the two elements being closely linked. given the emotional bonds that are established between the caregiver and the care receiver and the ambiguity of the employment relationship, the worker is no longer considered as a worker, but rather as 'a member of the family', as employers tend to define her/him (rollins, ; cox, ; lutz, , parreñas, . the fact that the main qualities (or 'soft skills') which are usually requested and appreciated in elderly carers are the ability to establish emotional ties, empathy, humanity, as well as a sort of vocation, testifies of the importance of the emotional dimension in care work (anderson, ) . these features of care work assume a crucial importance for live-in elderly carers, who are neither treated as workers, nor as members of the family. their status as 'quasi-family members' is such that they are required hard physical and mental labour (very long hours, h availability, nurse-like competencies, among others), but also self-denial, sacrifice and love, just as one would expect from a family member. the fact that the employer may coincide with the person who receives care further complicates the picture and makes the relationship between the different parties highly ambiguous. both elementsthe emotional ties between caregiver and care receiver and the position of live-in caregivers as 'quasi-family members'have crucial consequences on the lives and choices of live-in elderly carers in times of covid- as mentioned in the previous section, the very nature of care work, including the gendered construction of the ideals and values associated to it and the emotional labour that shapes their this article is protected by copyright. all rights reserved. position as 'quasi-family members', are all crucial locations at the core of the identity formation and the experiences of live-in elderly carers. looking at the situation of live-in elderly carers under the prism of migration and family relationship allows to identify other important elements that are likely to affect their livelihoods and their specific responses to the covid- crisis. as the literature on domestic and care work and on global care chains has long emphasised, the position of domestic and care workers cannot be reduced to their employment situation, but is defined and continuously reshaped by other factors, such as their administrative status and work permits, which are in turn determined by the migration regime specific to each country; their transnational lives and that of their families; as well as their family situation and caring responsibilities in the host country and in the sending country (hochschild and ehrenreich, ; parreñas, parreñas, , yeates, ; lutz, ; anderson and shutes, ) . concerning family and caring responsibilities, the existence of a family in the country of origin and the intensity of the caring responsibilities 'left behind' (the presence, for instance, of young children and/or frail elderly parents), as well as the economic responsibilities linked to the maintenance of the family, are all elements that affect the livelihood and the choices of elderly carers (hondagneu-sotelo and avila ; fresnoza-flot, ; parreñas, parreñas, , kabeer, ) . the fact that domestic and care workers are often the main family breadwinners has different consequences. on the one hand, it may have an emancipatory power and contribute to a redefinition of gender roles within their families. on the other hand, it means that various members of the family (husbands, younger or older children, parents, but also other kinship relationships) will depend of the income of the elderly carer and of her ability to adapt to adverse circumstances. these economic and caring responsibilities have gendered meanings for elderly carers. because they have to assume the economic function within their family at the same time as maintaining their caring responsibilities, they bear a double burden. to use the traditional distinctions between productive/reproductive labour, public/private sphere and the gendered constructions associated to it, not only they incarnate both masculine and feminine attributes, but they do so in a complex matrix. in their migration journey they first undergo a process of emancipation from the caring responsibilities linked to their own families, and then they reassume these same responsibilities, in a double form: they financially (and emotionally) support their families left behind, while taking care of the members of another family in the host country, as part of their job. as the scholarship has stressed, this has important social, psychological and emotional consequences, for both elderly carers themselves and their families abroad (hochschild and ehrenreich, ; parreñas, ; graham et al., ; fresnoza-flot, ) . a last element to be taken into account with regard to their position as migrants is the impact of their migration status, which include the conditions of entry, stay and work permits enforced in the country of destination (shutes, ; anderson, ; anderson and shutes, ) . the migration status is likely to influence not only their professional and migratory career, but also their living conditions, including their access to health, leisure, loans and educational opportunities, among others. as scholars highlighted, among all the possible migration statuses, it is undocumented workers who are more susceptible to be found in live-in jobs, for a series of reasons (ambrosini, ) . first, by reason of the poor reputation and the low value accorded to care work, and especially to live-in types of arrangements, live-in care work is often the first and only entry point of the most vulnerable segments of the migrant population into the economy (lutz, ) . because undocumented migrants are less likely to find better paid jobs and/or to negotiate better working conditions, they are more likely to accept live-in jobs, which are discarded by locals and other migrants (ambrosini, ; schutes, ) . second, live-in care work may be considered an advantage for migrants, because it allows to save money on food and accommodation, and because at the same time it represents a sort of protection. this latter point may be a crucial element of the migratory strategy of undocumented workers, as the house of the employer becomes a sort of shelter, which protects against the risk of being caught by the authorities (ambrosini, ) . in order to understand the impact of covid- on the lives and work of migrant live-in caregivers in brussels and their choices during the lockdown period, i explore their position as women, as migrants, as care workers, as breadwinners of their families, and as 'quasi-family members' in the host families for which they work. by looking at the intersection of these 'identity locations', i focus on how their situation affected their choices and how they resolve the dilemma between stayingat the detriment of their freedom and healthand leavingat the detriment of their survival and that of their families. as mentioned in the introduction, the exacerbation of the inequalities between men and women during the lockdown period of the covid- crisis has been especially visible in the domestic sphere. not only women have suffered the harshest consequences in terms of domestic violence, but they also had to assume a disproportionate increase of domestic and care responsibilities compared to their male counterparts, with a parallel reduction in the number of hours allocated to paid work and an increase in the physical and mental chargewhich in turn will have repercussions on their professional career (eige, ; alon et al., ). one of the greatest difficulties encountered by (tele)working women is related to the lack of separation between 'work' and 'home', which corresponds to the lack of a clear demarcation this article is protected by copyright. all rights reserved. between professional and domestic spheres. as already pointed out by the scholarship, although it allows to combine work and caring responsibilities, the system of teleworking may have clear disadvantages for women, who may end up trapped in their home and be disproportionally located in the peripheral labour market (haddon and lewis, ) . the imposition of a teleworking system upon adults with caring responsibilities had the result of blurring the boundaries between all life domains: when all activities (work, leisure, family, etc.) are realised in the same environment the result is a mental charge, experienced as a feeling of being always 'on duty'. ironically, what working mothers (and fathers, to a certain extent) experienced during the lockdown is part of the 'usual business' for live-in caregivers. similarly to other women, also live-in caregivers experienced an intensification of caring responsibilities, which in their case were distributed between their own families and the families who employ them. on one side, some of them had to intensify the financial support to their families, because of health and/or financial problems, which in some cases were engendered by the pandemic in their countries. also, to counteract the fears and anxieties linked to the pandemic, and in order to provide an emotional and symbolic support to their families at distance, they intensified the contacts with the different members of the family, through the use of technology (emails, chats, video). on the other side, their care duties and responsibilities increased dramatically in the framework of their jobs as caregivers. starting from the announcement of the lockdown measures by the government, and to compensate for the potential health risk of having multiple external actors intervening in the care of the older person, live-in caregivers were required to cover the whole care needs of the older person. when asked whether she had received any support from the employing family or from external people, maria replies: this article is protected by copyright. all rights reserved. maria takes care of an older woman who does not have any relatives. because the other caregiver who was taking over during maria's day off was dismissed, since the beginning of the lockdown the responsibility for the care of the elderly woman falls entirely on maria, who is now alone to cover her duties as a caregiver while at the same time replacing the support of the family. according to maria, live-in caregivers are left with the full responsibility for the elderly people, regardless of the existence or not of a family behind. indeed, she reports that also the other elderly caregivers with whom she remained in contact are facing the same situation, despite the presence of a family who could potentially take over the responsibilities linked to the care of the older persons. the growth of both productive and reproductive labour, which in the case of live-in caregivers accumulate within the same employment relationship, as well as at their family level, results in a significant increase of the care burden. while other working women had to learn to combine their external professional responsibilities with the care of their own families, live-in elderly carers had to take over the whole caring responsibility of another family, while ensuring the care of their own families at distance (hochschild and ehrenreich, ) . the migrant status of live-in elderly carers affected their life and their response to the covid- crisis with many respects. first of all, the situation of lockdown, including the closure of borders and the reduction on the freedom of movement inside and outside the country, meant that migrants had to live the covid- crisis within the host country, without any possibility to travel or to draw near their own families. this equally affected all migrants, irrespectively of their country of origin. moreover, the interruption of the economic activities not considered 'essential' (that is, a certain number of activities, with the exception of mainly health and medical jobs and scientific research, food processing industry and grocery shops, but also chemical and building industry, among others) disproportionately hit the migrant population, whose presence in the labour market is more precarious. this had dramatic consequences for many working migrants, who lost their jobs and incomes without a system of safety net behind them (social protection, social networks, etc.). the loss of income had tremendous consequences for their survival in the host country, but also in terms of sending of remittances and for repaying the indebtedness engendered by migration. the situation of migrant caregivers followed two separate paths. on one side, the majority of domestic and care workers working on an hourly basis lost their jobs and were faced with the above-mentioned obstacles, without means of support and without the possibility to move closer to their families. in some cases, the possibility of survival entirely depended on the good will of the employers, as it is the case for kristina, a young filipino domestic and care worker: kristina, who had lost her primary job as a live-in elderly carer because of the death of the older woman she was looking after, had maintained her secondary job as a domestic worker in another family, where she used to clean once per week to complete her weekly salary. when the covid- emergency stroke the country, she had not found another full-time employment. during the lockdown period, her only means of living depended on the generosity of her employer, thanks to whom she managed to go through her living expenses. this article is protected by copyright. all rights reserved. on the other side, live-in elderly carers experienced other difficulties: while they were given the possibility to save their jobsin return for the intensification of their efforts (working extra hours, performing health tasks which were not allowed in normal times, renouncing their time off, etc.)they were locked down with a family other than their own. therefore, live-in elderly carers had to bear both the practical disadvantages of the lockdown (not being able to go out, being always 'on duty', among others) and the emotional and psychological burden of being confined in the private home of strangers, rather than their own families. being confined to other people's intimate space, without being able to take care of your own confined family abroad, certainly contributed to amplify the distress of migrant elderly carers. the experience of elena, a spanish elderly carer, exemplifies the inner torment of the separation from her loved ones in a country other than her own. immediately after the outbreak of the pandemic she was ready to leave belgium to join her family in spain: in addition to the increased amount of work and of care responsibilities that they have experienced as women and as care workers, migrant live-in elderly carers have suffered from a sense of displacement. this sense of displacement is not only the result of the geographical and physical distance from their countries and their families, but also to the unprecedented situation of experiencing the covid- lockdownand the enormous emotional charge associated to itbeing lonely and isolated (from their loved ones) and at the same time nearby and wanted (by non-family members). if the isolation and the lack of social activities imposed by the lockdown measures represent a burden that hit the entire population, regardless of their socioeconomic level or their origin, it had harshest consequences for migrants. first, contrary to the majority of the population, they were physically isolated with respect to their families and their countries, which, as mentioned above, constitutes a source of emotional distress. second, they suffered from the sudden isolation from the social networks they have built in belgium, which represent for many migrants a crucial element for the construction of community ties and of their sense of belonging. if some of them managed to maintain some of the activities with the community, mainly through the use of technology, other live-in caregivers entirely lost the connexion with their community. as it emerges from the words of dana, this double isolation is likely to have important psychological and mental health consequences and to contribute to serious forms of anxiety and distress. despite the lack of a global and long-term vision on the effects of the covid- , the data on the infection rates registered in all countries affected by the pandemic suggest that elderly people, as well as the professionals who take care of them (home carers, domiciliary nurses, health and care personnel in rest homes, etc.) are among the most affected by the covid- , in every respect. if the elderly population has been heavily impacted because of its health vulnerability to the virus, health and care workers have been greatly affected, because of the high risk of contamination given by their front-line position at close contact with the most vulnerable population. thus, elderly caregivers have suffered specific consequences, including from a health point of view. as attested by numerous articles appeared in the belgian press during the first two months of the lockdown, the lack of adequate equipment (masks, hand sanitiser and screening tests, among others) for home carers and the personnel working in rest homes has been a crucial factor in the spread of the virus and the deterioration of their working conditions. however, while the working conditions of 'official' care workers in times of covid- have been relatively visible in the media, the situation of the most invisible among care workersthat is migrant live-in carers, sometimes with an irregular administrative statushave been completely hidden from the public opinion. the story of carmen highlights the situation that some of the live-in caregivers had to face in terms of their own health conditions. Ça c'est bien passé, je étais contaminée après le carnaval, mais tout était bien passé sans complications. après je suis restée avec une certaine distance avec la dame, son fils a pris en charge jusque j'étais bien. je suis bien, et la famille que je travaille aussi. [it went well, i was infected after the carnival period, but everything went well, without complications. afterwards, i kept a certain distance from the lady and his son took over until i felt better. now i feel fine and the family for which i work is fine too] -carmen carmen, a brazilian undocumented migrant who works as a live-in caregiver, did not have access to public health services. after being hit by the covid- virus, she spent the quarantine and the convalescence period in the home with the older person, where her work was partially replaced by the son of the older lady. the experience of carmen not only confirms the crucial role that these women play within the family and the essentiality of their work in times of covid- , but it also says a lot about the particular situation of live-in elderly carers with regards to their own access to health services and with regards to their working conditions. in addition to the health consequences, elderly carers suffered from a deterioration in their working conditions in other respects. contrary to most sectors of the economy, forced to stop or to drastically reduce their activities, care workers had to guarantee the continuity of their services and even intensify their efforts, because of the vital nature of their work during the pandemic. however, the intensity of the activities of care during the lockdown period depended on multiple factors, including the wish of the care receivers and their families (whether or not the family was ready to take over the caring responsibilities, thus dismissing the worker) and the professional status of care workers (whether they worked on hourly basis or as live-in carers, for instance). this means that while some workersmainly elderly carers working on an hourly basis for more than one employerlost their job or drastically reduced their work and their income, othersmainly live-in elderly carershad to consistently increase their work commitment. the situation of live-in elderly carers is unique with this respect. as mentioned in the introduction, they had to choose between losing their jobs or losing their freedom. those who have chosen the loss, albeit temporary, of their personal freedoms have been locked up in the home of the care receiver for several months so far. maria, talking about her own situation and that of her friends, who are also live-in elderly carers, describes the situation by saying that they are all 'stuck' in the house of the older person. oui, nous sommes bloquée en permanence dans la maison avec les personnes âgées qui nous soignons. [yes, we are stuck permanently in the house with the elderly people who we take care] -maria far from being a simple redistribution of time and tasks, this new working arrangement heavily affects their livelihoods. not only have they had to deal with the profound stress and anxiety generated by this health emergency situationand have had to do so in a situation of isolation and distance from their loved onesbut they have also suffered a considerable deterioration of their working conditions. longer hours, heavy tasks without any external support, especially in the case of heavy physical and mental dependencies on the side of older people, no time off, permanent availability, isolation, no social contact, no access to basic services, are just a few examples of what they recall when they describe their work during the lockdown. as the literatures on global care chains and on transnational motherhood have emphasised, many of the women who emigrate for economic reasons and go to fulfil the shortages of reproductive labour in western societies, are also mothers with caring responsibilities in their countries of origin (hochschild and ehrenreich, ; fresnoza-flot, ; parreñas, parreñas, , yeates, ; lutz, ; anderson and shutes, ) . whether or not their migration contributes to changing gender roles within their families and how roles are renegotiated within couples are not without contradictions. however, because they assume the economic function within their family, thus becoming the primary family breadwinners, while at the same time maintaining their caring responsibilities, they bear a double burden, as both economic and care providers. the situation of live-in elderly carers as family breadwinners places them in a special position, which is likely to have a crucial impact on their professional and life choices during the crisis. not only they had to bear the costs to cover for the indebtedness relative to their migration, in addition to their living costs in the host countries, but they had to ensure the continuity of the remittances. therefore, the existence of family and care obligations abroad, as well as their role as main family breadwinners, generated an additional pressure on them. i can't go out because i taking care of old people. my family is doing well, it's hard but they are fine, but i heard that some of the girls is jobless, it's hard for their families. - marilise, from the philippines, used to work as a live-out caregiver for an older man affected by alzheimer, where she used to spend hours per day, while at the same time working as a cleaner and a babysitter in two other families. while marilise feels safe, because she managed to maintain at least her primary job with the older man, by transforming it into a live-in job during the lockdown period, she is worried about the economic hardship of her friends. the words of marilise point to the link between the situation of migrant elderly carers and that of their families. the economic hardshipnamely, the loss of the job that some of them experiencedis not only an issue for the survival of elderly carers themselves, but it has dramatic repercussions on their families. when she talks about her friends losing their job, she immediately recalls the economic distress that might fall on their families. because their role as family breadwinners is so anchored in their migration process and family obligations guide their migration-related choices, the situation they live during the covid- lockdown is assessed through the lens of this role and the survival of their families becomes the prevailing issue over their own survival (fresnoza-flot, ). if live-in elderly carers could maintain their role as family breadwinners by accepting to be entirely confined with the older person they take care of, some of them were nevertheless financially hit by the covid- crisis. on the one hand, some had to bear extra costs, linked to their health condition or that of their families, this reducing the possibility to maintain their remittances at previous levels. on the other hand, some of them had to renounce to other economic activities, such as other part-time jobs. this is the case of many among the filipino caregivers that i have interviewed, who chose to maintain the live-inmore remunerativejob and to renounce to their other part-time jobs in other families, with a subsequent loss of income. the ambiguous position of caregivers as 'quasi-family members', which is particularly visible in the case of live-in care arrangements, has direct consequences on caregivers. this 'identity location', which is embedded in a larger system of domination that refers to interpersonal power relations, shapes the identity and the experiences of elderly carers, with many respects. first, the ambiguity of the employment relationship, oscillating from work to family relations, as well as the emotional labour implied in care work, are built on a series of expectations, ideals and stereotypes which associate care work to the 'natural' responsibility of female family members. care workers, and especially live-in caregivers, are expected to take care of the older person in a family-like way, that is 'with love'. not only they have to show qualities such as empathy, positive and joyful attitude, and patience, but they are expected to develop close emotional ties with the person they take care of. although the dilemma over the possibility to 'commodify' love cannot be resolved (anderson, ) , the expectations linked to care work and the status of workers in an intermediate position between work and family represent a source of pressure that acts upon care worker. second, the ambiguous status of 'quasi-family members' contributes to construct and reinforce dependencies and power relations (rollins, ) . their intermediate position in the intimate space of the family transforms the dynamics of power and the role of the elderly caregiver within the family. while still obliged to ensure good quality and professional work, the live-in caregiver becomes a member of the family without really being one: she acquires all the family obligations linked to it, but without being fully entitled to family rights. the combination of the emotional tie between the caregiver and the care receiver, as well as the ambiguous position of caregivers as 'someone of the family' creates a logic of dependency of the caregiver from the employer (anderson, ; cox, ; parreñas, ) . these dependencies and power relations are found to be strong drivers with respect to the choices of elderly caregivers during the covid- crisis. because of the emotional ties they have established with the care receiver, they feel responsible for her/his well-being, as if the person was actually someone of their own family. this article is protected by copyright. all rights reserved. despite the exceptional harshness of her job since the lockdown measures were implemented, according to maria caring for the elderly person is not a burden, but rather a 'pleasure'. although some defining elements of the employment relationship, such as the salary, are maintained, the work as such goes beyond the employment relationship, and results in a form of self-denial and love. furthermore, the rhetoric of the employers, who repeatedly emphasise the membership of the caregiver as part of the family (in terms of obligations, rather than rights), generates a strong dependency on the side of the elderly carer, who feels grateful and obliged to compensate by offering extra services. i argue that this ambiguity of the employment relationship, and the dependencies that it generates, are important drivers in shaping their choices in times of covid- and that their choice to stay with the older person is not only economically-driven, but becomes a sort of moral responsibility. the position of migrant live-in elderly carers is at the intersection of multiple systems of oppression that make for the distinctiveness of their experiences. in this paper, i have analysed five 'identity locations' that define their experiences as women, as migrants, as live-in elderly carers, as family breadwinners and as 'quasi-family members'. looking at these five 'identity locations' simultaneously helps understanding their individual experience and responses to this specific pandemic crisis. the solution to the dilemma to which live-in elderly carers were faced at the beginning of the lockdown periodwhether they should keep their job and temporarily renounce to their freedom, or whether they should renounce to the employmentis to be understood in the light of these five intersecting 'identity locations'. inequality in the impact of the coronavirus shock: new survey evidence for the uk. briefing report the impact of covid- on gender equality parenting from a distance and processes of family reunification: a research on the italian case doing the dirty work? the global politics of domestic labour 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the new economy i'm here but i'm there': the meanings of latina transnational motherhood institut wallon de l'évaluation, de la prospective et de la statistique (iweps) ( ) covid- , travail et genre en wallonie the rise of the female breadwinner. reconfigurations of marriage, motherhood and masculinities in the global economy the new maids. transnational women and the care economy covid- is increasing the divide in life chances between rich and poor servants of globalization. women, migration and domestic work long distance intimacy: class, gender and intergenerational relations between mothers and children in filipino transnational families migrant domestic workers as 'one of the family covid- pandemic shines a light on a new kind of class divide and its inequalities. the guardian between women. domestics and their employers global care chains key: cord- -tbmtg k authors: sharma, shreela v.; haidar, amier; noyola, jacqueline; tien, jacqueline; rushing, melinda; naylor, brittni m.; chuang, ru-jye; markham, christine title: using a rapid assessment methodology to identify and address immediate needs among low-income households with children during covid- date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tbmtg k objective: brighter bites is a school-based health promotion program that delivers fresh produce and nutrition education to low-income children and families. due to covid- -related school closures, states were under “shelter in place” orders, and brighter bites administered a rapid assessment survey to identify social needs among their families. the purpose of this study is to demonstrate the methodology used to identify those with greatest social needs during this time (“high risk”), and to describe the response of brighter bites to these “high risk” families. methods: the rapid assessment survey was collected in april across houston, dallas, washington dc, and southwest florida. the survey consisted of items on disruption of employment status, financial hardship, food insecurity, perceived health status and sociodemographics. the open-ended question “please share your greatest concern at this time, or any other thoughts you would like to share with us.” was asked at the end of each survey to triage “high risk” families. responses were then used to articulate a response to meet the needs of these high risk families. results: a total of families completed the covid- rapid response survey, of which families were triaged and classified as “high risk” ( . % of survey respondents). during this time, % of the “high risk” participants reported being food insecure, % were concerned about their financial stability, % concerned about the availability of food, and % concerned about the affordability of food. a qualitative analysis of the high-risk group revealed four major themes: fear of contracting covid , disruption of employment status, financial hardship, and exacerbated food insecurity. in response, brighter bites pivoted, created, and deployed a framework to immediately address a variety of social needs among those in the “high risk” category. administering a rapid response survey to identify the immediate needs of their families can help social service providers tailor their services to meet the needs of the most vulnerable. a a a a a on march , , the world health organization declared the sars cov- (covid- ) a pandemic [ ] . on march , the united states (u.s.) declared a national emergency concerning the covid- outbreak, leading to numerous social distancing measures including, school closures, cancellation of public gatherings, and remote working [ ] . starting the week of march th , through the week of march rd , states began ordering schools to close for the academic year and issuing statewide stay-at-home orders, with many companies implementing work from home policies [ ] . these social distancing measures had the detrimental consequences of disrupting food systems, businesses, and economies throughout the u.s. [ ] [ ] [ ] . rapid epidemiological assessment (rea) refers to post-disaster assessment methods that attempt to accurately assess a population by using the fewest resources in the shortest time [ ] . these measures have included surveys, door-to-door assessments, surveillance methods, and screening and individual risk assessment using qualitative and quantitative methods [ ] . typically, during an outbreak investigation, these methods may be used to assess symptoms of disease to contain and prevent further outbreaks [ ] . however, the application of a rapid assessment could extend to identifying social determinants of health needs during a disaster such as food insecurity, financial instability, unemployment, housing insecurity, and access to healthcare among vulnerable populations. this is particularly important to covid- , given that reducing risk of covid- complications is related to maintaining optimal immune function and health, all of which are linked to these social determinants of health [ ] [ ] [ ] . brighter bites is a non-profit, school-based health promotion program implemented across six cities (houston, dallas, austin, new york city, washington, d.c., and southwest florida areas) in the u.s., with the goal of delivering fresh produce and nutrition education to lowincome children and families in underserved communities to mitigate food insecurity and improve dietary habits. brighter bites currently has an ongoing partnership with university of texas health science center at houston (uthealth) school of public health for research and evaluation. during the month of april , amid the covid- pandemic, while states were under "shelter-in-place" orders, brighter bites conducted a rapid assessment survey of families across four of the six cities (houston, dallas, washington dc, and florida). the survey screened families for food security, housing security, financial stability, employment status, transportation needs, access to childcare, and access to healthcare, to better understand the immediate needs of families, identify those with the greatest need, and provide them with critical resources during this time of crisis. the purpose of this study is to demonstrate the methodology used to conduct the rapid assessment of needs using qualitative and quantitative measures among low-income households with children during the covid- pandemic to identify those in greatest need, to present our findings, and describe the strategies taken to meet the participants' needs. the rapid assessment survey was collected in april across houston, dallas, washington dc, and southwest florida. the self-report survey was administered electronically using formsite (vroman systems inc., illinois, usa) in english and spanish to , bb families who were enrolled in the program in the - school year and had provided their contact information to the program. the participants in our study were predominately hispanic ( %), while % were african american, and % were white/other. this is significantly higher than the sampled cities where the hispanic population makes up at most % of the population in houston, % in dallas, % in florida, and % in washington d.c. completion of the survey was voluntary and informed consent was obtained from all participants prior to the start of the study. data is collected by brighter bites non-profit organization, and then is de-identified and shared with the university of texas health science center (uthealth) for analysis as part of a data sharing agreement and approved by the uthealth committee for protection of human subjects. data obtained in the first wave of survey responses, while cities were under "shelter-in-place" orders, were used for this paper. the self-reported rapid assessment survey consisted of a -item questionnaire on covid- related concerns, social determinants of health, and sociodemographics. the - minute in length survey was administered in english and spanish. sociodemographic variables included child gender, respondents' relationship to child, race/ ethnicity of both parent and child, parent education level, parent employment status, and government assistance program enrollment. program options included the special supplemental nutrition program for women, infants, and children (wic), supplemental nutrition assistance program (snap), double dollars, medicaid, medicare, national school lunch and/or breakfast programs, and children's health insurance program. household food security status was self-reported by the participants using the two-item hunger vital sign™ screening questionnaire developed and validated by hager et al. [ ] . if the participant responded "often true" or "sometimes true" to either of the two questions, then the household was considered food insecure. if a participant answered "never true" to both questions, then the household was considered food secure. participants were asked about their concerns regarding various social determinants of health including financial stability, employment status, access to food, affordability of food, availability and affordability of housing, access to reliable transportation, access to childcare, access to clinic/doctor, or other concerns, which was open-ended for participants to fill in. participants could check all that applied. participants were asked to rate their health status. responses included poor, fair, good, very good, and excellent. stata . (statacorp college station, tx) was used to perform all data analysis. means, standard deviations, and frequency distributions were computed. significance was set at p< . . the open-ended question "please share your greatest concern at this time, or any other thoughts you would like to share with us." was asked at the end of each survey. the responses to this question were collated and analyzed for themes. two trained uthealth project staff conducted a thematic analysis to analyze the responses using an inductive approach, where codes and themes are derived based on the content from the survey data [ , ] . the coding of the data was done initially through microsoft word using an open coding method by each coder. to ensure reliability of codes, coders collectively re-coded the data in microsoft excel. a codebook of codes and definitions was created, and codes were used to search for patterns and to identify possible themes. triaging for "high risk" families: "high risk" families were defined as the following: ) if indicated in the open-ended question "please share your greatest concern at this time, or any other thoughts you would like to share with us "the following response": a) running out of food, b) diagnosed with covid- and/or living with someone who has been diagnosed with covid- and experiencing challenges, c) is ill and needs assistance, d) is about to lose their place of living, e) is about to lose their utilities, or f) no one at home is making an income; or ) if indicated on the survey as "poor" on the question when they were asked to rate their current health status. if they met any of these categories, they were classified as "high risk". subsequently, these data were used to articulate a response to meet the needs of these high risk families. the sociodemographic characteristics of the high-risk families are presented in table . a total of families completed the covid- rapid response survey, of which families were triaged and classified as high risk ( . % of survey respondents). overall, the mean age of high- during this time frame, % of the high risk participants reported being food insecure, % were concerned about their financial stability, % concerned about the availability of food, and % concerned about the affordability of food. they were also concerned about the availability/affordability of housing ( %), while % were concerned their employment status would change in the near future. other concerns were access to reliable transportation ( %), access to childcare ( %), and access to a clinic/doctor ( %). almost half the participants ( %) reported being of fair or poor health status during this time. while overall results of the qualitative thematic analysis for the n = participants are presented elsewhere (sharma et al., under review) , a qualitative analysis of participant responses in the high-risk group revealed four major themes presented in table : fear of contracting covid- , disruption of employment status, financial hardship, and exacerbated food insecurity. fear of contracting covid- was an immediate threat for the families as is witnessed in one of the comments "my daughter was diagnosed with coronavirus and i am very scared for all the members of my family". moreover, loss of employment was of major concern leaving parents with little or no resources to meet their family's needs. one parent said, "i don't have enough work, i'm a single mother, i have two children and i no longer have enough for food for my children and i'm behind with the rent." table "my daughter was diagnosed with coronavirus and i am very scared for all the members of my family." {{e.g. bb produce vouchers provided, grocery drop off}} • provided language-appropriate information and resources on safety measures-hand washing/sanitizer, wearing masks (talked about various items that could be a mask if they did not have access to any) social distancing and staying home. • encouraged to seek testing if there was a concern of potential covid- infection. • provided information on community resources for clinics, doctors and testing sites by way of family resource links "we are worried that my husband does not have a job and we do not know how we will be able to buy food, pay the mortgage, electricity, water, the internet, what will happen to our future, we are very afraid, regarding the virus, of taking our children to the doctor, to the dentist, etc." "i don't want to go to the store and i no longer have food, afraid of going out." "i was left without work, and i don't have for supplies, or bills, or food." • provided information on community resources for employment and unemployment benefit services. • provided electronic $ gift card to a local retail store. if they did not have access to an email, the $ gift card was printed and handdelivered to them. "i'm very worried because i haven't had work for three weeks and i don't have money to pay rent, electricity, or water. my work has been reduced by %, i'm an assistant housecleaner." "i don't have enough work, i'm a single mother, i have two children and i no longer have enough for food for my children and i'm behind with the rent." "there's no work, and you still have to pay rent, and there's not enough money for food." financial hardship "my biggest worry is not being able to pay next month's rent and not knowing where to go." • provided information on community resources for assistance programs, housing, transportation, child care and other services such as free or reduced internet access by way of family resource links • cross-checked that they were receiving the produce vouchers to local grocery retail store and updated contact information accordingly. • sent electronic $ gift card to local retail store. multiple electronic $ gift cards were sent to families that communicated continued hardship. if they did not have access to an email, the $ gift card was printed and hand-delivered to them. "i am worried about not being able to pay my rent next month (may_ ) because we are out of work." "i don't have enough food for my children." • provided dates and times for community food distributions and brighter bites food distributions. • sent electronic $ gift card to local retail store • if they did not have access to an email, the $ gift card was printed and hand-delivered to them. • if they could not go shopping due to barriers such as lack of transportation, groceries were bought and delivered to them. (family was asked to share food preferences) • families received a $ produce voucher to the local grocery retail store. "i am worried about how to feed the children because there is no work right now, i am worried about paying my debts" "my biggest worry at the moment is food for my family. loss of employment further led to financial instability leaving parents unable to pay their bills or buy food. one parent commented, "my biggest worry is not being able to pay next month's rent and not knowing where to go." this financial uncertainty also left parents concerned about their food security, and about how they were going to access healthy foods with the limited produce available and the increasing prices in the grocery stores. one parent described their concerns by saying, "i feel worried because i'm not giving enough vegetables and fruit to my children since my husband only works days, and i'm not working because my baby was just born, so there are children and adults and i'm short of food and diapers for my baby, but what i need is food for the family." brighter bites response to high risk families: in response to these aforementioned needs, brighter bites pivoted and created an infrastructure and a standardized framework to meet the needs of these high-risk families (see fig ) . a core, centralized group of brighter bites staff was assigned specifically to address these families' immediate concerns. following the triage, trained brighter bites staff made follow up phone calls to each high-risk family and obtain more details regarding their concerns and assistance needed. a tracking database was created, in which all phone calls and family concerns were tracked for each family. this information was then relayed back to the brighter bites leadership where a tailored response was generated for each family. brighter bites partnered with public health and medical schools' faculty and students to develop educational materials and informational resources to provide to the families (haidar et al., under review). responses ranged from a) providing immediate financial relief in the form of gift cards to local retail stores, b) grocery drop off to families unable to leave home, and c) providing area-specific resources via text, email and phone regarding food distributions, financial assistance, safety from eviction due to inability to pay rent, covid- testing sites near their homes, participation in government assistance programs etc. all responses were documented in the tracking database for each family. a % of the high-risk families were reached through this process. the covid- pandemic has had a major impact on the u.s. economy leading to a worldwide economic crisis [ ] . the most vulnerable populations in our communities have been disproportionality affected by the direct and indirect health, social, and financial hardships of covid- [ , ] . low-income, vulnerable populations contribute the most to healthcare costs, are more likely to suffer from health disparities, have increased risk factors, and have fewer resources for promoting health and treating illness [ ] . it is therefore imperative that pertinent health, financial, and critical needs resources are provided in a timely matter to families and communities in need. this study provided insight into how organizations could potentially identify and respond to the needs of communities in times of crisis and demonstrates how to effectively reach families that are struggling and need support. social factors play a critical role in determining health outcomes [ , ] . addressing the social determinants of health of underserved communities through rapid response is important to reduce health disparities and improve health outcomes associated with covid- and other comorbidities [ ] . minority populations, specifically african-american and hispanic have been disproportionately affected by covid- , with increased risk of complications and mortality [ ] [ ] [ ] . these same populations also suffer the most from chronic diseases such as obesity, diabetes, respiratory disorders, and other pre-disposing conditions, accounting for the majority of healthcare costs [ ] . often times, these diseases are also rooted in unequitable social and structural needs surrounding employment, transportation, housing, poverty, food, education, and health literacy [ , ] . conducting a covid- rapid response survey was a purposeful decision on part of brighter bites to identify those with highest need during this time of crisis and develop a framework to immediately address a variety of social needs among those in the "high risk" category. due to the rapid transmission of coronavirus, many cities including the four in our study were rapidly brought to a halt with many states issuing statewide stay-at-home-orders, closing schools, restaurants, and businesses [ ] . for low-income families this resulted in loss of wages and increased financial instability adding further strain to previous economic hardships. administering a rapid response survey during such times is important to help identify the immediate needs of these families can help social service providers re-direct their services to meet the needs of this vulnerable population. in response to the covid- pandemic, brighter bites pivoted rapidly to invest its network into addressing urgent needs of their participating families. brighter bites used the expertise of public health faculty and students of their partner institutions to create and rapidly provide resources for their families. for example, if a parent indicated that they were fearful of contracting covid- , the team discussed safety measures such as handwashing, social distancing, and various items that could serve as a mask. if a parent was concerned about paying for rent or food, then bright bites team provided information on community resources for food, bill relief, and ensured they were receiving vouchers, and gift cards to local grocery stores. details regarding these strategies and their dissemination is provided elsewhere (haidar et al., under review). by having a triaging and tracking system, along with a centralized response team and strategic partners, in place, brighter bites was able to have a targeted response to meet the needs of % of families with the highest needs during this time. social service organizations can use similar strategies to identify and address the needs of their populations rapidly during times of disaster. this study has some limitations and strengths. the study sample is a sub-sample of families who participated in brighter bites in the - school year. the sample size varied across each city as family enrollment in the brighter bites program is not proportional to size of the city, and likely those families who needed the most help responded to the survey. finally, survey was conducted to families electronically which introduces a selection bias as a limitation due to the fact that only families that had access electronically could participate. however, in this phase of the pandemic, given closures of schools and businesses, this was the most effective way to reach people. finally, the qualitative themes observed in our study cannot be sorely attributed to the pandemic, and could be a result of other multitude of factors which remain to be seen. the purpose of this study was to identify the families with the highest need for social services during a time when all cities surveyed were in "shelter-in-place" and deploy an immediate response, which was successfully achieved. finally, the timeline from the pandemic occurring to our response to these families is one of the major strengths of this paper. our study provides the methodology and framework to screen at-risk low-income families for social needs during the time of the covid- pandemic, and to provide a timely response to these critical needs. in the future, this framework could be used for other pandemics and times when an immediate response to screen at-risk families will be needed. world health organization. who director-general's opening remarks at the media briefing on covid- - the covid- pandemic in the usa: what might we expect? map: coronavirus and school closures the early food insecurity impacts of covid- feeding low-income children during the covid- pandemic the socio-economic implications of the coronavirus and covid- pandemic: a review rapid epidemiological assessment of health status in displaced popula-tionsâ€"an evolution toward standardized minimum essential data sets development of rapid epidemiologic assessment methods to evaluate health status and delivery of health service development and validity of a -item screen to identify families at risk for food insecurity using thematic analysis in psychology transforming qualitative information: thematic analysis and code development.: sage centers for disease control and prevention. covid- in racial and ethnic minority groups impacts of covid- on vulnerable children in temporary accommodation in the uk vulnerability and vulnerable populations. foundations for population health in community/public health nursing-e-book addressing social determinants of health and health inequalities the social determinants of health: it's time to consider the causes of the causes racial health disparities and covid- â €"caution and context ná poles am, pé rez-stable ej. covid- and racial/ethnic disparities covid- and african americans multiple chronic conditions in the united states prevalence of single and multiple leading causes of death by race/ethnicity among us adults aged to years social determinants of health the authors would also like to acknowledge the families who participated in this study. conceptualization: shreela v. sharma, christine markham. key: cord- -hi u w q authors: xu, xinglong; zhou, lulin; asante-antwi, henry; boafo-arthur, ama; mustafa, tehzeeb title: reconstructing family doctors’ psychological well-being and motivation for effective performance in china: the intervening role of psychological capital date: - - journal: bmc fam pract doi: . /s - - - sha: doc_id: cord_uid: hi u w q background: family practice and family doctors are critical part of china’s primary healthcare delivery in a constantly evolving society. as the first point of contact with the medical system, family practices require physically and psychologically sound and a well-motivated family doctors at all times. this is because an error can lead to loss of lives as gatekeepers of the medical system. our study explored the extent to which positive psychological capital promotes higher performance among family doctors. methods: a questionnaire was used to collect data from family doctors in shanghai, nanjing, and beijing. we applied a structural equation analysis to analyze the causal relationship among the variables. results: we found out that psychological well-being and job involvement significantly influences the performance of family doctors in china. the study also noted that psychological capital moderates the relationship between psychological well-being attainment, job involvement, and performance. conclusions: studies have shown that these pressures affect their well-being considerably. for this reason, a healthcare professional who experiences positive emotions affects the total behavior which culminates into performance. healthcare services to both individuals and families within the community as the first line of contact with the overall national health system [ ] . this role requires fundamental requires continuous fundamental reform to strengthen its capacity to provide a vast network of primary care, especially to the majority of its population that lives in rural areas [ ] . the current primary healthcare system in china is organized into three tiers and the intensity of care increases with a higher tier. it starts with primary care facilities in the villages and towns. then second tier comprises of the county hospitals while the third tier includes the tertiary hospitals located in the major cities but each tier has its own challenges [ ] . the first challenge relates to the number of patients. two decades ago, the outpatients' medical doctor in a normal hospital treated around - patients in a single day. this number has doubled in and nearly patients visit a single medical doctor's consulting room in urumqi, zhenjiang, xian, hefei, nanning, kunming, nanchang, etc. every day. the situation is worse in major cities such as shanghai, beijing, nanjing, guangzhou, shenzhen, hangzhou, suzhou, etc. and these can have a significant effect on the mental and psychological wellbeing of the primary care doctor. there are other challenges that face primary health doctors in china [ ] . generally, the healthcare industry has little room for errors but the constraints of working as a family practitioner makes doctors susceptible to errors. if these results in the loss of lives and other fatal injuries, they can significant impact on the family doctors mental and psychological well-being [ ] . gallagher [ ] , recommends that each primary health system must put in place appropriate coping strategies to help medical professionals to deal with the emotional, mental and psychological impacts on of their work on their wellbeing. thus the psychological and mental well-being of the primary health doctor and nurse is the catalyst for their happiness, satisfaction, creative thinking, pro-social behaviour, good physical health, sense of purpose and meaning to life needed to thrive professionally despite the ups and downs of primary healthcare in china [ ] . another source of vulnerabilities of mental and psychological health faced by family care practitioners in china is that they have a very low social status, receives low salary. compared with specialist clinicians in hospitals, family practitioners receive limited recognition for their work and this occurs in both urban and rural areas in china [ ] . these conditions do not befit the status of family doctors as a special gateway to health services for families before referral to advanced medical facilities. this duty entails managing risk and uncertainty, caring for individuals in the context of their families and communities, and addressing the totality of the patient. yuan [ ] explains that a vibrant family practice system potentially lessens the burden of work provided by specialist physicians, nurses, and other public health workers in the community health clinics in the urban areas. through effective planning and provision of comprehensive primary health care, regardless of age or sex, continuingly, family doctors support public health services by offering health management, disease prevention, disease control, health assessment to patients. china's initial attempt to revamp the family doctor system after the sars virus did epidemic in did not achieve much success hence the effort to resuscitate the system in . four years on, the hope that family doctor contract services will mature and become an integral part of the primary healthcare system in china is still far from being attained. a study conducted by xu et al. [ ] suggests that an average proportion of . % of china's family doctors intend to quit direct patient contact in the next years if the current system continues. in most instances, work-related mental and psychological health issues underscore the decision to opt for other related practices. selvaggio [ ] appropriately captures thus when they allude to the fact that a more pathogenic work environment appears to be developing for a population already known to be at risk of mental and psychological ill-health including burnout, depression, and addiction. prior studies that support this situation among china's family doctors argue that this category of workers must abide by several rules and regulations that affect their rate of pay. most family doctors consider the strict official guidelines given to them to follow as unpleasant interference and manipulation by people of inferior social status. in addition, family doctors in china believe that a lot of the paperwork they fill is unnecessary and tortuous. they are subject to strict, unfriendly monitoring and evaluation requirements that are deemed unsuitable to the healthcare industry. for this reason family, doctor practice has become an unattractive field of specialization relative to medical specializations. the persistence of these challenges significantly increases the potential for mental and psychological decay among family doctors which can affect their performance. ryff, [ ] explains that psychological well-being is affected by several factors which consist of motivation, job satisfaction, self-efficacy, achievement, de-individuation, physical and psychological fatigue, environment and organization identification psychological fatigue, environment, and organization identification [ , ] . doctors with negative mental and psychological wellbeing will exhibit poor mental and emotional health. this can potentially create psychological, mental, and emotional disorders that can interfere with their ability to enjoy life as primary care providers and participate as active members of the society in general. thus psychological and mental wellbeing is the precursor to a desirable or positive work outcome which includes the sustainable performance of family doctors in china. however, robertson & cooper [ ] assert that psychological wellbeing alone is not enough to achieve the sustainable performance of family doctors. a strand of contemporary literature in human resources for health is revisiting the concept of psychological capital and its influence on family doctor motivation particularly in the case of china. according to robertson & cooper [ ] , positive psychology builds the human resources to be dedicated and engaged to create and guarantee the competitiveness that organizations so much desire. this position is attested in the extant literature in other. for example, pan, mao, zhang, wang, and su [ ] investigated the mediation role of psychological capital on the association between nurses' practice environment and work engagement among chinese male nurses and noted that creating a supportive nursing practice environment could increase male nurses' work engagement by developing their psychological capital. they suggested that nurse managers can then provide reasonable workload and pathways for male nurses to achieve goals, thereby fostering their hope. this research on "psychological capital-work engagement-job performance" mostly focused on employees' individual dimensions leaving the organizational dimensions. the work of tien-ming cheng, hong, and yang [ ] examined the moderating effects of service climate on psychological capital, work engagement, and service behavior. the study noted that attendants who showed high psychological capital tended to show more work engagement and better service behavior. the study further indicated that work engagement is the mediator between psychological capital and service behavior. comparatively, studies have shown that there is much difference in the impact of psychological capital on government and private employees. comparing the impact of psychological capital dimensions on the level of happiness of private and government employees, singh and khan [ ] found that private-sector employees had higher happiness based on higher forms of the various dimensions. the government workers had a lower level of happiness as a result of lower forms of psychological capital development; an indication that an enhanced psychological capital significantly influences employees' level of happiness which subsequently impact their well-being. the current crisis in family doctor motivation in china offers an opportunity to explore the intermediating role of psychological capital in the relationship between psychological wellbeing and job involvement on one hand and sustainable performance of family doctors. this is because, in the traditional human resource strategy literature, psychological well-being is conceptualized to mean the experience of positive emotions that is global than negative emotions. although most studies continue to concentrate on the negative emotions, lazarus [ ] new findings emanating from the proponents of positive psychology point to the need to advance argument on how positive emotions influence the total functioning of the individual, [ ] . in this paper, we apply a novel structural equation model to explore effect of interplay between psychological wellbeing, psychological capital and job involvement on family doctor motivation in shanghai, nanjing and beijing in china. first we explain the methods used in the study and present our analytical procedure and results. next we discuss the findings and make our conclusions. figure shows the final conceptual model for the current study. a quantitative research study was designed to explore the direct influence of the psychological wellbeing of family doctors and job involvement (independent variables) on family doctor's performance (dependent variable). secondly the study also explored the moderating effect of psychological capital (hope, the self-efficacy, the optimism, the resilience) on the two direct relationships. this crosssectional study was conducted among practicing family doctors, potential family doctors and past family doctors selected in shanghai, beijing, and nanjing cities between july and december . these cities were chosen because family doctor contract services are relatively developed in chinese megacities than in other parts of china. an initial number of questionnaires were designed and sent to family doctors who were randomly selected by cluster sampling to accommodate the three different groups of targeted doctors. the number of years of work experience of the doctors was a major factor in deciding a qualified participant. a target least years post-qualification experience was deemed sufficient. a similar effort was put into recruiting both male and female participants to compare responses were necessary. a selfadministered questionnaire was directly administered to them after obtaining their written consent. a questionnaire was given to each worker that contained a declaration of anonymity, information secrecy, voluntary participation, and voluntary withdrawal. the co-investigators directly distributed, retrieved, codded, and managed the questionnaires, and where possible respondents were provided with an explanation without inducement for any unclear questionnaire items. items in the questionnaire were based on appropriate items in pre-existing measurement scales and respondents needed between and min to complete a full questionnaire. at the end of the research period, questionnaires were not returned and questionnaires with missing data were regarded invalid. thus effective response was made up of respondents which represent a response rate of approximately %. the study was approved by the ethics committee of centre for health and public policy research and validated by the human experimentation of jiangsu university in china. psychological wellbeing was measured using the shortened version of the psychological wellbeing (pwb) scale developed by ryff [ ] . this model measures six aspects of psychological wellbeing and happiness namely; autonomy, environmental mastery, personal growth, positive relations with others, purpose in life, and self-acceptance (ryff et al., [ ] ; adapted from [ ] ). the item version is configured with a point likert scale where ( = strongly agree; = strongly disagree). response to the individual questions is composed of six subscales and a composite value of the subscales is used as a proxy score of psychological wellbeing of each doctor. the psychological capital questionnaire (pcq) was used in measuring psychological capital (psycap). it is the most widely used standard scale and has been validated in a different research context. this questionnaire was developed by [ ] and consists of items composed into four subscales namely hope, optimism, resilience, and self-efficacy. the scale is configured on a sixpoint likert scale measure ranging from ('strongly disagree') to ('strongly agree'). kanungo's [ ] job involvement scale was used to measure the concept of job involvement. the model is a item job involvement questionnaire. this questionnaire was adopted due to its high internal consistency and wide applicability in a different research context. the response scale to this questionnaire is on a -point likert scale ranging between "do not agree/not applicable to me" to "fully agree/fully applicable. performance sustenance, on the other hand, was measured based on four constructs which include frequency of patients' visits [ ] , frequency of patients complain [ ] , intention to stay or re-enrol in family practice [ ] , doctors days worked [ ] . the response scale for items measured was ( = strongly agree to = strongly disagree). the composite value was determined and used as a proxy for performance sustenance. firstly descriptive statistics were computed using spss and inferential statistics was conducted using amos version . the pearson moment correlation coefficient was conducted to determine the significance and possible multicolinearity in the relationship between the variables. subsequently, a mixed structural equation model was modelled to establish the direct effect of family doctors' psychological wellbeing and involvement on performance sustenance. similar model was done to determine the moderating effect of psychological capital on the direct relationships. a structural equation model is a better analytical model for this research because it produces more robust inferences compared to the traditional regression model. a structural equation helps to analyse the influence of predictor variables on different dependent variables simultaneously. the specific model used in our study is expressed in a matrix form as follows: where for each of the causal parameters (psychological wellbeing and job involvement), the γ's and the β's, the subscripts follow the same pattern. in the matrix, the p by p b matrix holds the coefficient of regression of the y variables whereas the other y variable with 's on the diagonal indicates that a variable cannot cause itself. the model also contains the p by q Γmatrix which contains the coefficients of the y's on the x's. the error vector (errors-in-equations or specification errors) ζ, is p by . for example, we assumed that e(y) = and e(x) = and these have absolutely no impact on both the variance and the covariances of the variables if we assume the independence of x and ζ. finally, we adopted a secondorder factor model that assumes that the factors can form higher-order factors. we ensured that the correlation among the factors has the correct structure as a result of the latent variables. this leads to the following path diagram indicated below; (adopted from bediako et al., [ ] ) in this model each η has a unique factor and a known variable while ξ represents the higher-order factor. generally, we write the second-order factor analysis of the mixed structural equation as follows; this special case of an sem with latent variables can be rewritten more compactly as in this model, it is assumed that cov(ε, ζ) = , cov(ζ, ζ) = , v(ε) = Θε, v(ζ) = Ψ and v(ζ) = Φ. in this model also, the variance of y, Σ, assumes a unique aesthetic form mathematically expressed as follows; where [ΓΦΓ ' + Ψ] represents v(η). ( ) next, we used summed indicators to "specify" the latent variables (psychological wellbeing and job involvement) and the regression model for the structural equation used the "no origin" option. consistent with prior works of rabenu et al. [ ] , we set a strict level of significance for the regression coefficients ( % confidence interval) for each latent variable. we accordingly evaluated the reliability, validity and internal consistency of each latent variable using appropriate respected techniques and each latent variable met the criteria based on pallant et al's [ ] minimum threshold. next, the single-construct measurement model (mm) for each latent variable was evaluated to ensure it fitted the data. the reliability, validity, and internal consistency of the latent variables were evaluated and subsequently averaged to obtain the error attenuated covariance matrix (cn). using the procedure set out in ping [ ] , the resultant matrix was adjusted to obtain the measurement errors. the resulting error-adjusted (err-adj) cm was used to estimate eqs. and without omitting the variables. this approach is much superior to existing approaches because a measurement error is determined by measuring model loadings and measurement error variances from the "no dummies". in this instance using the measurement model loadings and measurement error variances from the "no dummies" for eq. was applied. related studies that have used this approach include; anderson et al., [ ] ; asante-antwi, zhangxiao tian et al. [ ] , kong, akomeah et al. [ ] . next, the err-adj cm was used as inputs into the least square structural regression model. again ping [ ] as cited in kong, akomeah et al. [ ] judges this approach to be a much superior method of validating the integrity of a data relative to other traditional options since it produces fairly accurate and consistent structural coefficients in a model. finally, we inputted the parameter estimates obtained from the no dummies mm into the latent variable regression excel spreadsheet to produce the err-adj cm matrix based on the following computations; cov where var(ξ x ) represents the expected error-adjusted variance of the regression inputs x, var(x) represents the error attenuated variance of x. Λ x represents the average λ x + λ x + λ x … λ xn ), where avg (θ x ) = var(ε x ) + var(ε x ) + var(ε x ) … + var(ε xn ), in which case the ε x 's and the λ's represent the measurement error and the measurement model respectively. the cov(ξ x ξ z ) represents the desirable level of erroradjusted covariance of x and z, and cov (x, z) represents the error attenuated covariance of x and z, finally, we inputted the err-adj cn into the regression function based on the "regression-through-the-origin" option. as recommended in warren, white et al. as cited in bediako et al. [ ] , we corrected the coefficient standard error (ses) i.e. the ses of β , β , … ... in eq. ( ) produced by the err-adj for measurement error by adjusting the se from regression using the root mean square error (rmse) as expressed as follows; where y i represents the observed andŷ i represents the predicted values based on the err-adj cm [ , ] . this implies that the correct standard errors for the err-adj cm structural coefficients involve the standard error from the regression based on the err-unadj cm. this leads to the formulation of the ratio of the standard error (se) from err-unadj cm regression and the standard error (se) from err-adj cm regression which is formulated as; in which case se a a is the err-adj cm regression standard error, se u is the standard error obtained from the err-unadj cm regression, rmse u is the standard error obtained from the err-unadj cm regression, and rmse a is the standard error obtained from the err-unadj cm regression. descriptive statistics table shows the inter-correlation matrix between the latent variables as well as the relationship between the latent variables and the moderator and dependent variables. the results indicates that psychological well-being was positively and significantly correlated with psychological capital (r = . ) and performance (r = . ) but not job involvement. this means that an increase in the psychological wellbeing of a family doctor is positively associated with their performance and their psychological capital. this also supports the absence of multicolinearity among the independent variables (psychological wellbeing and job involvement). similarly, a higher job involvement is positively associated with psychological capital (r = . ) and performance (r = . ). finally, the analysis also shows that psychological capital is positively associated with performance (r = . ) and this is statistically significant at a % confidence interval. confirmatory factor analysis (cfa) was adopted to assess the adequacy of the measurement components. reliability and validity of the constructs was also assessed and showed that the item-to total correlations were stronger. some scholars have argued that factor loadings from . should be an acceptable loading. for this reason, the constructs that reported standardized factor loadings above the . criteria were accepted as recommended by hair et al. [ ] . based on this threshold, all, the measures of the study were considered as showing satisfactory reliability. again all the constructs showed high average variance extracted (ave) and that further strengthens the evidence of strong evidence of convergent validity. the results of cfa, reliability, and convergent validity are shown in table confirmatory factor analysis confirmatory factor analysis (cfa) is an important statistical tool for probing the nature of relationships among latent constructs. it is important because it helps to measure the construct validity, identify method effects, and helps in evaluating the factor invariance through time and groups. the use of confirmatory factor analysis (cfa) continues to gain ground in the psychological literature as a result of the belief researchers have in the structural equation model as a robust model specifically. given the key impact cfa makes in the measure development and due to the understanding that having a tool that manages the measurement of variables effectively, it can be presumed to be paramount quantitatively simply because its role is crucial to the results a researcher reports we sought to find out the relationship between the latent variables using amos version . the model consisted of the latent variables psychological wellbeing, job involvement, the moderating variable (psychological capital and its constructs i.e. optimism, self-efficacy, resilience, hope) and the dependent variable (performance sustenance) among family doctors in china. the items in the observed variables with low factor loadings were eliminated leaving the strong ones to be included. for instance, on hope, h , h were eliminated, psychological well-being pwb , pwb , pwb pwb were also eliminated. performance sustenance jp and jp which showed low loadings were also eliminated. figure shows the second-order confirmatory factor analysis of psychological capital and well-being, involvement, and performance. the fit indices for the model were, (χ = . , df = , χ /df = . tli = . , cfi = . , nfi = . , ifi = . , rfi = . rmsea = . . we tested the significance of psychological capital as a moderator between psychological well-being and job involvement. the model showed adequacy as an indication of a good fit for the data. the fit indices of the final measurement model were, χ = . , df = χ /df = . , cfi = . , tli = . , rfi = . , ifi = . , and rmsea = . . the model confirmed h a: that psychological wellbeing and job involvement directly influence performance sustenance of family doctors with pwb . (p > . ), job involvement . (p > . ) and h b: was confirmed . (p > . ) that psychological capital moderates significantly between job satisfaction and performance sustenance. psychological capital interacts with both psychological well-being and job involvement to predict the performance sustenance of family doctors. figure shows the final path analysis of the moderated model of psychological wellbeing, job involvement, psychological capital and performance sustenance. we did a bootstrap of the sample to find out the indirect effect of the interaction, details are presented in table table shows the results of the path coefficient analysis of the structural equation model. a unit change in a family doctors' psychological wellbeing influences performance positively by . and is statistically significant at a % confidence interval. similarly, a change in job involvement causes a positive change in the performance of family doctors in the selected cities. the coefficient value of . and a p-value of . statistically justify this claim. the moderating effect of psychological capital on the relationship between family doctor's psychological wellbeing and their performance is also positive. the coefficient is statistically significant at a % confidence interval. this is also the case with the moderating effect of psychological capital on the relationship between job involvement and performance. these cases support the assumption that psychological capital is a significant factor in stimulating positive mental, psychological and emotional behavioural outcomes among family doctors in china china's healthy china initiative has changed the view of the family practice profession from a mere gatekeeping practice to the pivot of overall primary healthcare delivery in china. family doctors have assumed extended responsibility to manage risks and uncertainties, care for individuals in the context of their families and communities, and address the totality of patients. this underscores the ambition of china to train , more gps in the next decade to consolidate its primary health gains. however, the results of this study suggest that endemic psychological and mental wellbeing concerns among family doctors may erode the gains already achieved. traditionally, family doctors in china have considered themselves "people's doctors" yet, a stressful workday, endless rules and regulations, poor wages and salaries are perceived dehumanizing routines attenuating the growth of family doctors and the primary healthcare system in china [ , ] . these and many other hindrances have taken away a strong sense of the mutually beneficial long-term therapeutic relationship between family doctors and patients which is the central psychological factor for doctors' willingness to accept family doctor jobs. the result of these challenges is that young doctors, in particular, see family doctor services as less prestigious and an unfulfilling arena to build a noble career [ ] . the results of this study show that when psychological capital can interact with other job-related attitudes to generates positive and significant results thereby sustaining the performance of family doctors. consequently, enhancing the well-being of health professionals such as the family doctor system, for instance, is a sure way of bettering the lives of the larger society. similarly, a health employee who feels satisfied with the level of responsibilities and a positive work environment identifies with the organization by way of job involvement. in countries such as uk (where the family doctor system is well established), fig. second order confirmatory factor analysis of psychological capital and well-being, involvement, and performance it contributes to solving nearly % of health-related problems and only rare or complex conditions are referred to specialists in secondary or tertiary hospitals. to ensure china reaps the benefits of the family doctor system and guarantee its sustainability, a strong and well-motivated family doctor workforce must be pursued. the results of this study support the need to build and develop family doctors that are mentally, psychologically and emotionally healthy. to achieve the target of training and retaining , more gps human resource managers in china's health sector must focus on helping doctors overcome the challenges that negatively affect psychological and mental well-being. some of these include high workload, avoidable bureaucratic requirements, and problems of low salary and limited recognition of their competency by the public, compared with hospital specialists. being able to satisfy family doctors' psychological needs can offer them the belief that they are masters of their own decisions, which when enabled, reflects in various jobrelated attitudes of satisfaction, peak performance, and increase in commitment levels, job involvement, and ultimately psychological well-being. theoretically, this study confirms the self-determination theory and further contributes to the theoretical outcomes of the development of self-determination theory and its application to supporting human resources for healthcare from an emerging country perspective. precisely because this study extends the theory in practice by providing an additional outcome of sustained positive performance to the self-determination theory (sdt) aside from what the theorist propounded. this is particularly relevant to the expansion of the self-determination theory discourse because the essence of developing individuals to be self-reliant and selfmotivated in an organization or field of specialization is to ensure their commitment to the said organization based on strong mental, emotional and psychological wellbeing. in this regard, when employees are emotionally glued to an organization, it guarantees high-level performance because they begin to appreciate the fact that they can balance their personal goals and that of the organization. a condition that ensures a win-win situation enables employees to give off their best in the execution of tasks assigned to them. most importantly, when employees such as family doctors are emotionally committed to an organization, they become intrinsically motivated. intrinsic motivation is the foundation of the self-determination theory. an intrinsically motivated family doctor who has developed a more emotional attachment to the job will place the clients or patients ahead of any challenges faced in the line of duty. this provides additional validity of the confirmation of the self-determination theory especially among the healthcare professionals in china. additionally, family doctors and other health employees who develop affective commitment dedicate and consign themselves to making the work environment a better place. an effectively committed employee takes on additional responsibilities to enhance individual and organizational performance and becomes autonomous in motivating himself and others. importantly, when a healthcare employee develops affective commitment, it makes an individual a team player in the task execution of the institution. in terms of the setting, our extension of the theory is relevant because we conducted our study in a different environment outside the areas the theory was conducted. this study intended to examine the relationship between job involvement, psychological well-being, and performance among family doctors in china. it again considered the influence of the interaction effect of psychological capital on psychological well-being, job involvement leading to performance sustenance on the family doctor system. the study confirmed all the hypotheses that job involvement correlates positively and significantly with performance sustenance while psychological well-being significantly correlates with performance sustenance,. secondly, the interaction effect of psychological capital significantly affects the performance sustenance of healthcare employees positively. health professionals such as family doctors experience a lot of pressure that weighs heavily on their psychological and emotional wellbeing. for this reason, a healthcare professional who experiences positive emotions affects the total behaviour which culminates in a performance. the results lay credence to preceding studies that observed that healthcare professionals who demonstrated subjective happy feelings brought on by the pleasure in the profession and work environment they appreciate shows in their performance. additionally, health professionals who experience sentiments or emotions that give the impression of having purpose and meaning in the life they live translate it the task performance family doctors who experience a sense of drive, happiness, and satisfaction identifies with the organization in terms of task execution, relationship with co-workers, and forming an identity with the organization they serve.. again the higherorder construct of psychological capital relate significantly with job involvement to sustained performance. psychological capital which consists of constructs such as hope, resilience, optimism, and self-efficacy reflects significantly on performance. this suggests that an upsurge in psychological capital equally leads to an improvement in psychological well-being. similarly, an increase in the level of psychological capital leads to the job involvement of employees. the data for this research is held by the authors and will be made available upon reasonable request. ethics approval and consent to participate ethical approval was granted by the ethics committee of the center for health and public policy research of jiangsu university china and confirmed by the jiangsu university. this was approved by the zhenjiang administration bureau for scientific and industrial research. written consent to participate was obtained from the study participants. the questionnaire included an opening statement explaining the ethical responsibilities of confidentiality of data, the anonymity of respondents, and participants responsibility to opt-out of the study at any time. respondent's decision to fill the questionnaire constituted their consent to participate in the research-informed. consent for publication "not applicable" the author declares that there is no conflict of interest amongst them. author details job displacement and the duration of joblessness: the role of spatial mismatch china's "left behind children" and the consequences of distributive injustice: a systematic review protection of migrant workers under new chinese labour law: a critical review immigrant generational status and developmental problems among prematurely born children job satisfaction in health-care organizations the emotional impact of medical error involvement on physicians: a call for leadership and organizational accountability sarcopenia in asia: consensus report of the asian working group for sarcopenia examining the moderating effects of service climate on psychological 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satisfaction in the medical profession: a cross-sectional survey in zhejiang province, eastern china healthcare data analysis system for regional medical union in smart city healthcare data analytics: using a metadata annotation approach for integrating electronic hospital records socialized healthcare service recommendation using deep learning publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the support of colleagues and staff at the department of public management of the school of management and the center for health and public policy research and the overseas education college of jiangsu university is deeply appreciated. we are also grateful to the zhenjiang administration bureau for scientific and industrial research for their support. received: january accepted: june authors' contributions xx: conceived the idea, collected the data, and revised the manuscript in line with the objectives. lz: is the supervisor of the project and sequentially aligned the parts of the research paper, haa: conducted the analysis of the data and drafted manuscript. ab: collected data and analyzed the data. tm: collected the data, conducted the analysis. all authors read and approved the final manuscript. the national natural science foundation of china ( ), the social science application research project of jiangsu province ( syb- ) and the universities' philosophy and social science researches in jiangsu province ( sja ) provided assistance for pre-study, data collection and other administrative expenses incurred for the study as part of a larger project to key: cord- - gtsqyfj authors: rawlings, neil d.; barrett, alan j. title: introduction: the clans and families of cysteine peptidases date: - - journal: handbook of proteolytic enzymes doi: . /b - - - - . -x sha: doc_id: cord_uid: gtsqyfj the third edition of the handbook of proteolytic enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over chapters. each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. the subject of chapter is clans and families of cysteine peptidases. peptidases in which the nucleophile that attacks the scissile peptide bond is the sulfhydryl group of a cysteine residue are known as cysteine-type peptidases. the catalytic mechanism is similar to that of the serine-type peptidases in that a nucleophile and a proton donor/general base are required, and the proton donor in all cysteine peptidases in which it has been identified is a histidine residue, as in the majority of serine-type peptidases. in some families only the dyad of cys and his seems to be essential for catalysis, whereas in others there is evidence that a third residue is required to orientate the imidazolium ring of the his (a role analogous to that of the essential aspartate in some serine peptidases). the catalytic mechanisms of cysteine peptidases are described fully in chapter . the clans and families of cysteine peptidases are listed in table . . the first clearly recognized cysteine peptidase was papain (chapter ), and this forms the foundation of clan ca. the crystal structure (figure . ) shows two structural domains separated by the active site cleft. the n-terminal domain consists largely of a bundle of α-helices whereas the c-terminal domain contains a β-barrel. a long helix runs through the middle of the molecule, and the catalytic cysteine is at the start of this. clan ca contains over families (see table . ). almost all of these have been brought into the clan on the basis of structures that are known from crystallographic data to be similar. the other families are assigned to clan ca because they contain similar sequence motifs around the catalytic residues (figures . À . ) . besides cys and his of the catalytic dyad, two other residues are important for catalysis in papain and its relatives (numbering as in merops database). these are a gln that helps in the formation of the 'oxyanion hole', an electrophilic center that stabilizes the tetrahedral intermediate, and asn (sometimes asp in families c , c and c ), which is thought to orientate the imidazolium ring of the catalytic his (chapter ). the order of these residues in the sequence is gln, cys, his, asn/asp, and in mature papain, the numbering is gln , cys , his and asn . in family c , asparagine serves the same function as gln . no equivalent of gln has been identified in the foot-and-mouth disease virus l-endopeptidase. the catalytic cysteine is usually followed by an aromatic, hydrophobic amino acid, but glycine occupies this position in some peptidases of family c and all those of family c . figure . shows secondary structure diagrams of example peptidases from the clan. all the examples shown are α/β proteins, and all have the nucleophilic cys at the start of a helix and the catalytic his at the start of a β-sheet. inhibitors are of some use in identifying peptidases of clan ca. many members of the clan are irreversibly inhibited by compound e- , but reversible inhibition by the arginine-like side chain of e- is not significant and should be ignored. proteins of the cystatin family also inhibit many peptidases in clan ca, but it should be noted that some cystatins have a second reactive site that can inhibit legumain, a clan cd peptidase [ ] , and some cystatins can inhibit metallopeptidases [ ] . family c . the evolutionary tree (see the merops database) shows that the family is divided into two subfamilies, c a (or papain subfamily) and c b (or bleomycin hydrolase subfamily). it may well be that a peptidase of family c was present in the universal ancestor of all organisms, and that this evolved into a subfamily c a-type protein in early archaea and a subfamily c b-type protein in early bacteria. further divergence would have occurred in the separate groups of organisms, and horizontal transfers can explain the presence of a c a homolog in clostridium and of bleomycin hydrolase in eukaryotes. subfamily c a is the larger of these subunits, and consists of secreted and lysosomal enzymes. subfamily c a contains mainly endopeptidases from dna viruses, protozoa, plants and animals, and exopeptidases from gram-positive bacteria, fungi and animals. members but the majority of completed archaean genomes lack c a homologs. homologs are patchily distributed in bacteria, with most genomes lacking a homolog. most bacterial homologs are from members of the phyla bacteroidetes and chloroflexi. even within a genus, some species will possess a homolog and some will not; for example, half of the mycoplasma species with completed genomes have homologs. the subfamily includes the plant enzymes papain, chymopapain and actinidain, and the animal lysosomal enzymes cathepsins b, h and l. figure . shows the three-dimensional structure of papain. there is a clear division within the subfamily between the cathepsin b-like enzymes and the papain-like enzymes. among the cathepsin b-like enzymes are dipeptidyl-peptidase i, and the endopeptidases from giardia. included among the papain-like enzymes are cathepsins o, h, l, k and s. cathepsin o is very divergent, whereas the others are more closely related. the close relationship between mammalian cathepsin h and plant aleurain is apparent. the distinction between exopeptidases and endopeptidases is blurred for some members of subfamily c a. dipeptidyl-peptidase i (chapter ) acts principally as an exopeptidase, removing n-terminal dipeptides, but may have limited endopeptidase activity. cathepsins b and h (chapters and ) both possess endopeptidase activity, but arguably are more important for their exopeptidase activities. cathepsin b acts as a peptidyldipeptidase, releasing c-terminal dipeptides, and this activity is attributable to the existence of an extended loop that forms a cap to the active-site cleft, and carries a pair of his residues that are thought to bind the c-terminal carboxylate of the substrate (figure . ) . cathepsin x is a rather specific carboxypeptidase (chapter ). the enzymes that enter the secretory pathway, destined either for secretion or for the lysosomal system, are synthesized as precursors, with n-terminal propeptides as well as the signal peptides. most members of family c have propeptides homologous to that of papain, but the propeptide of cathepsin b is much shorter and very different in sequence. however, the propeptides of both cathepsin b and papain are thought to act in the same way in the proenzymes, blocking the active site by being bound in the reverse orientation to a substrate, as can be seen from the structure of procathepsin b (figure . ). papain-like propeptides may be identified from the presence of the erfnin motif, in which some of the following residues (numbered according to the papain propeptide) are conserved: glu , arg , phe , asn , asn , phe , asp and glu . the papain propeptide is homologous to proteins ctla- α and ctla- β of activated t cells, which have been shown to be cysteine endopeptidase inhibitors [ ] . it is notable that residue pro is often conserved in the mature peptidases of family c (see the alignment in merops), and this may prevent attack by aminopeptidases, since the xaa-pro bond is resistant to many such enzymes. most members of subfamily c a are monomeric, but dipeptidyl-peptidase i is a homotetramer in which each figure . richardson diagram of foot-and-mouth disease virus l-peptidase. the image was prepared from the protein data bank entry ( qmy) as described in the introduction (p. li). one molecule of the trimer is shown. catalytic residues are shown in ball-and-stick representation: cys (engineered to be ala), his and asp (numbering as in pdb entry). the image was prepared from the protein data bank entry ( cv ) as described in the introduction (p. li). catalytic residues are shown in balland-stick representation: cys , his and asn (numbering as in pdb entry). e- is shown in gray in ball-and-stick representation. monomer consists of three chains as a result of proteolytic processing. some peptidases of subfamily c a have c-terminal extensions relative to papain. among the endopeptidases from plants, the extensions at the c-terminus of oryzains α and β, and cysteine endopeptidases from tomato (uniprot p ), arabidopsis thaliana (uniprot: p ), douglas fir (uniprot: q ), carnation (uniprot: q ) and pea (uniprot: q ) are homologous. the c-terminal extensions found in endopeptidases from trypanosoma and leishmania are unique in the family. inserts relative to papain occur within the catalytic domain in other members of the family. in cathepsin b, the 'occluding loop' that carries the histidine residues important for peptidyl-dipeptidase activity is inserted between the catalytic cys and his residues. this loop is not present in enzymes from giardia [ ] that are the most divergent of the cathepsin b-like proteins and probably represent the primitive state. some endopeptidases from dictyostelium possess glycine-and serine-rich inserts between the active site his and asn. the specificity subsite that is dominant in most peptidases of subfamily c a is s , which commonly displays a preference for occupation by a bulky hydrophobic side chain, and not a charged one. unusually, the s subsite of cathepsin b readily accepts arg, however, so that z-arg-argknhmec is a good selective substrate for the enzyme. this distinctive specificity of cathepsin b can be explained by the residue lying at the bottom of the s pocket, which in papain is ser , but in cathepsin b is glu (numbering as in the merops c alignment). glu also occurs in cysteine endopeptidases from figure . ) contains several soluble, intracellular peptidases. these are aminopeptidases that commonly show selectivity for release of n-terminal arg residues, and include aminopeptidase c (pepc) from bacteria (chapter ), and bleomycin hydrolase (chapter ) from eukaryotes. note that no bleomycin hydrolase homolog is the aminopeptidase c-like enzymes are oligomeric. the yeast bleomycin hydrolase is probably representative, and is a homohexamer, with the active sites arranged on the inner face of the central channel, in an arrangement reminiscent of that in the proteasome. this arrangement evidently allows only small molecules to interact with the catalytic site. unlike papain and cathepsin b, the aminopeptidases of family c b do not contain disulfide bonds and are synthesized without propeptides. as is discussed in chapter , the mature bleomycin hydrolase subunit consists of three domains, the peptidase domain, an oligomerization (or 'hook') domain, and a helical domain. half of the hook domain is n-terminal to the catalytic domain, but the other half is an insert (relative to papain) preceding the catalytic his. the helical domain corresponds to two inserts in the catalytic domain with respect to papain. figure . shows the tertiary structure of the yeast bleomycin hydrolase subunit. there are a number of proteins homologous to papain that lack peptidase activity because catalytic residues have been replaced. these include an oil-bodyassociated protein from soybean (uniprot: p ) in which the catalytic cys is replaced by gly, a surface protective protein from plasmodium (uniprot: p ) and a protein from schistosoma japonicum (genbank: x ) in which the catalytic cys has been replaced by ser. in mammals, non-peptidase homologs include figure . richardson diagram of the human cathepsin b/ca complex. the image was prepared from the protein data bank entry ( csb) as described in the introduction (p. li). catalytic residues are shown in ball-and-stick representation: cys , his and asn (numbering as in pdb entry). one of the pair of histidines (his ) on the occluding loop is also shown in ball-and-stick representation. figure . richardson diagram of rat procathepsin b. the image was prepared from the protein data bank entry ( mir) as described in the introduction (p. li). catalytic residues are shown in ball-and-stick representation: cys (engineered to be serine), his and asn (numbering as in pdb entry). one of the pair of histidines (his ) on the occluding loop is also shown in ball-and-stick representation. the propeptide is shown in gray. testin, which is secreted by sertoli cells of the testis but has no known function, and tubulointerstitial nephritis antigen, an extracellular matrix basement protein that is a target for antibody-mediated interstitial nephritis [ ] . both mammalian proteins have the catalytic cys replaced by ser. several pseudogenes are known in mammals, including a human cathepsin l-like pseudogene (genbank: l ). family c contains the calpains. the molecule of a 'typical' calpain is a heterodimer of a heavy chain and a light chain (chapters , ). the heavy chain is a mosaic protein containing the peptidase domain (domain ii) (figure . ) and also a c-terminal domain (domain iv) with four calcium-binding ef-hand structures. the calcium-binding domain is homologous to calciumbinding domains in other proteins. the functions of heavy chain domains i and iii are unknown. crystal structures of calcium-free human calpain have been described [ , ] , and they show that in the absence of calcium the peptidase unit adopts a conformation that disrupts the catalytic site. in most mammalian cells there are two calpains with different calcium requirements for activity: calpain- (chapter ) and calpain- (chapter ). calpain (chapter ) has a residue insert in the catalytic domain between the catalytic cys and his. a number of atypical calpains are known, in which the domains n-terminal and c-terminal to the protease domain are different from those in the typical calpains, and a small subunit may not be required for activity. a drosophila homolog of calpain possesses an insert in the c-terminal domain, and there is evidence that differential splicing can give rise to a form lacking the calcium-binding domain [ ] . another calpain homolog exists in drosophila and is involved in the development of the small optic lobe. this putative endopeptidase is the product of the sol gene, and the protein is predicted to be multidomain, including six zinc fingers as well as a peptidase domain, but no calcium-binding domain [ ] (figure . ). again, an alternatively spliced form exists, this time lacking the peptidase domain. the calpain family is not restricted to animals: there are sequences from fungi, including aspergillus and saccharomyces, the protozoa trypanosoma and leishmania, the plant arabidopsis, and the cyanobacterium nostoc (but not synechocystis). homologs are also known from members of a wide variety of bacterial phyla, but homologs are absent from archaea. the only bacterial homolog that has been characterized is the tpr peptidase from porphyromonas gingivalis, which is so dissimilar in sequence to other members of the family that it had been considered a member of a separate family, and is now the only representative in subfamily c b. the absence of homologs from archaea and many bacteria with completely sequenced genomes, implies that horizontal gene transfer has occurred, probably from a protist or fungus to a bacterium (see the evolutionary tree in the merops database). aspergillus palb protein and a hypothetical protein from caenorhabditis are unusual among peptidases in clan ca in that the catalytic cys is followed by ser rather than a hydrophobic residue (see the alignment in merops). both the aspergillus and caenorhabditis proteins are multidomain, but show only limited sequence similarity to domains i and iii of human calpain. neither protein possesses the calcium-binding domain iv. a non-peptidase homolog is known from trypanosoma brucei in which the catalytic cys has been replaced by ser. family c contains streptopain (chapter ) and a few similar enzymes, all of which are from gram-positive bacteria. streptopain is inactivated by e- much more slowly than papain, but has a similar specificity, with a preference for a hydrophobic residue at p [ , ] . in some members of the family, asn (numbering as in figure . ) is replaced by asp. the prtt peptidase from porphyromonas is a mosaic protein with a c-terminal hemagglutinin domain unrelated to those in gingipains r and k in family c but % identical to the c-terminal domain of hemin-regulated protein from porphyromonas (uniprot: p ). surprisingly, streptopain homologs are found in some bacteroides species but not others. there are several families in clan ca in which the peptidases release ubiquitin from conjugated proteins. ubiquitin is a amino acid protein that is attached to other proteins as a signal for intracellular translocation or degradation (usually mediated by the proteasome). ubiquitin is attached through the carboxyl group of its c-terminal glycine residue either to the n-terminus of another polypeptide or to the α-amino group of a lysine residue, which forms an isopeptide bond (chapters À ). ubiquitin may also be attached to another molecule of ubiquitin, and proteins targeted for degradation may be polyubiquitinated. in order for ubiquitin to be recycled there is a need for peptidases to liberate the free ubiquitin by hydrolysis of the c-terminal glycyl bond. peptidases that release ubiquitin from conjugates are known as 'ubiquitin c-terminal hydrolases' or 'deubiquitinylating enzymes'. because release of ubiquitin may involve cleavage of an isopeptide bond, ubiquitin c-terminal hydrolases are not classified as peptidases in enzyme nomenclature but as thiolester hydrolases. there are at least eight families of deubiquitinylating enzymes in clan ca, and there are enzymes in other, unrelated, families, including the metallopeptidase amsh (family m ) which is a component of the s proteasome (chapter ). there are over different deubiquitinylating enzymes in humans, which implies a sophisticated network controlling translocation and degradation of proteins which is only just being understood. family c includes peptidases that can hydrolyze the ubiquitin-conjugate glycyl bond whether it is an α-peptide bond or an isopeptide bond, with various specificities. the tertiary structure of the human ubiquitin c-terminal hydrolase uch-l has been solved [ ] , and is shown in figure . . there are many structural similarities to papain. the molecule is bilobed, one lobe consisting mainly of helices, the other containing a β-barrel surrounded by helices. the catalytic cys is at the start of one of the helices, and the catalytic his is the start of a β strand. a difference from papain is that the first strand of the β-barrel precedes the helix bearing the catalytic cysteine in the sequence, whereas in papain the helix precedes all the strands of the β-barrel. the spacing between gln and cys (numbering as in figure . ) is identical to that in families c , c and c . peptidases in family c are synthesized without propeptides, and are intracellular; they are known only from animals, plants and fungi. family c contains the second group of ubiquitin c-terminal hydrolases. these are also intracellular peptidases, but are able to release ubiquitin from much larger peptides that have been polyubiquitinated (chapters À ). because ubiquitination is also essential for the assembly of multimeric proteins, there is a need to remove the ubiquitin molecules from polyubiquitinated subunits, which is presumably one of the functions of peptidases of family c . peptidases in family c have more complicated structures than those in c , and many are multidomain proteins. there is also a much greater variety, with saccharomyces possessing at least homologs, and even greater numbers in mammals. the tertiary structure of ubiquitin-specific peptidase has been solved and a catalytic triad consisting of cys, his and asp was identified (figure . ; also see later figure . ). the asp is equivalent to the asn of papain. the side chain of an asparagine stabilizes the oxyanion hole and is equivalent of gln in papain. the fold has been likened to that of an open hand, consisting of three subdomains, with the active site cys on the 'thumb' subdomain, the active site his on the 'palm' and the active site cleft between the 'thumb' and the 'palm.' the 'fingers' interact with ubiquitin [ ] . members of family c are present in all the eukaryotic genomes so far completely sequenced, including encephalitozoon cuniculi. family c sequences are known from several protozoan species that do not contain family c . c homologs are known from bacteria such as the pathogen burkholderia pseudomallei, but the sequences are shorter (c. residues) and lack inserts between the catalytic cys and his. the bacterial homologs are derived from the most divergent branch in the evolutionary tree. the tssm protein from burkholderia mallei has been shown to be a deubiquitinating enzyme [ ] . other families of deubiquitinating enzymes were initially postulated by makarova et al [ ] , which have now been experimentally verified. the human cezanne protein, from family c , was shown to release ubiquitin from linear or branched synthetic ubiquitin chains and from ubiquitinated proteins [ ] , and from the solved structure of tumor necrosis factor α-induced protein , the fold was shown to be similar to that of papain [ ] . family c includes the otubains (see chapter ). the tertiary structure of otubain- has been determined and shows that the fold is similar to that of papain [ ] . otubain- has been shown to cleave lys -linked polyubiquitin [ ] . the cyld protein from family c (see chapter ) has been shown to have de-ubiquitinating activity that is directed towards non-lys -linked polyubiquitin chains [ ] . the tertiary structure has been solved [ ] . family c includes the duba deubiquitinylating peptidase, which has been shown to cleave the lysine- -linked polyubiquitin chains on tumor necrosis factor receptor-associated factor [ ] . however, no tertiary structure has been solved for any member of the family, and the active site residues are predictions only [ ] . ataxin- (chapter ) is yet another deubiquitinating enzyme and a member of family c . the nuclear magnetic resonance solution structure of the domain containing the active site of ataxin- , known as the josephin domain, has been determined [ ] , and shows the characteristics of a cysteine peptidase in clan ca. family c includes otu peptidase from saccharomyces cerevisiae (chapter ) which preferentially cleaves longer polyubiquitin chains with lys linkages. the tertiary structure has been solved for the catalytic domain, and the structure is similar to that of other clan ca members [ ] . surprisingly, enzymes that releases ubiquitin from proteins have been discovered in viruses. the ul protein from herpes simplex virus releases ubiquitin from proteins ubiquitinylated via lys and is the founder of family c (chapter ). the tertiary structure has been solved, and shows a papain-like fold [ ] . the crimean-congo hemorrhagic fever virus (a nairovirus) also possesses a deubiquitinating enzyme which can downregulate antiviral responses of the host by release of ubiquitin and interferon-stimulated gene product from proteins of the antiviral and inflammatory pathways [ ] . the nairovirus enzyme is a member of family c , and the order of active site residues, identified by mutagenesis [ ] , led us to include the family in clan ca. other proteins act like ubiquitin and can be tagged to proteins, these include sumo and nedd- . ubiquitin-fold modifier- (ufm ) is another ubiquitinlike protein, and like ubiquitin attaches to proteins via the c-terminal glycine. the physiological relevance for tagging proteins with ufm is unknown, but tagged proteins are not degraded. peptidases in family c , which includes ufsp peptidase (chapter ), release ufm from conjugates and process the ufm precursor by removing a c-terminal dipeptide [ ] . once again, resolution of the tertiary structure has confirmed the relationship to papain [ ] and the family c is included in clan ca. the aspartic acid that is the third member of the catalytic triad unusually is n-terminal of the histidine, whereas in most papain-like proteins follows the histidine. the residue stabilizing the oxyanion hole is a tyrosine (whereas it is glutamine in papain). in both these respects, members of family c resemble members of the family c . family c includes the yeast peptidase aut that is responsible for the removal of a c-terminal arginine residue from the apg (or aut ) protein. the apg fg protein, as the processed substrate is known, is then able to form a conjugate with an unidentified protein via the newly exposed c-terminal gly and the conjugate binds tightly to membranes permitting the formation of the double-membrane vesicles required for the transport of material from the cytoplasm to an autophagic vesicle. homologs are known from human, mouse, drosophila melanogaster, caenorhabditis elegans and arabidopsis thaliana. inhibition by n-ethylmaleimide and site-directed mutagenesis of cys (see alignment c in merops) has shown that yeast peptidase aut is a cysteine peptidase (chapter ), but other catalytic residues have not been determined. the tertiary structure of human autophagin- has been solved [ , ] , and shows an catalytic triad consisting of cys , asp and his that is unusual amongst papain-like proteins, in which the third member of the catalytic triad follows the histidine in the sequence (see figure . ). families c and c are unique in having the third member (in both families an aspartic acid) preceding the histidine. members of both families are also unique in having a tyrosine helping to stabilize the oxyanion hole. family c includes staphopain (see chapters and ), a cysteine peptidase from staphylococcus epidermidis. the tertiary structure has been determined and shows a papain-like fold [ ] (see figure . ). staphopain is inhibited by e- and cystatin a. family c includes endopeptidases that are involved in the processing and export of bacteriocins. bacteriocins are antibiotic proteins secreted by some species of bacteria that have the effect of inhibiting the growth of other bacterial species. the bacteriocin is synthesized as a precursor with an n-terminal leader peptide, and processing involves removal of the leader peptide at a gly-glyk bond, followed by translocation of the mature bacteriocin across the cytoplasmic membrane. the endopeptidase serves both functions, and is also known as an atpbinding cassette transporter or abc-transporter. the endopeptidases are integral membrane proteins containing an n-terminal peptidase domain followed by six or more transmembrane domains and a c-terminal atpbinding domain. the structure of the peptidase domain has been solved, and shows a papain-like fold (see figure . ) [ ] . the non-peptidase-domains are homologous to a variety of other abc-transport proteins. peptidase specificity is not unlike that of the ubiquitin peptidases. the complex structure of bacterial cell walls means that there are a large number of peptidases involved in cell wall processing and lysis. newly synthesized bacteriophage virions escape from the host cell by lysing it, and the bacteriophage genomes encode a cell wall lytic enzyme that acts either as a peptidase to degrade the cell wall crosslinks, or as an amidase to disrupt the acetylmuramyl bonds. staphylococcus aureus has a multi-domain enzyme known as autolysin the c-terminal domain of which is related to lysostaphin (peptidase family m ). the autolysin gene has been acquired by a number of staphylococcal phages, and in bacteriophage ϕ , autolysin has been shown to cleave the cell wall cross-linking peptide at d-alakgly. the peptidase activity is expressed by the nterminal domain [ ] . an alignment shows that residues gln , cys , his and asn are conserved, suggesting that the n-terminal domain is a cysteine peptidase with a papain-like fold, which we include in family c . pseudomonas syringae is a plant pathogen which secretes disease-causing factors into the host cell via its type iii secretion pathway [ ] . the avrrpt protein (chapter ) is one of these virulence factors which activates itself and probably cleaves the rin protein of the host plant cell. by comparison with staphopain, avrrpt protein has been predicted to be a cysteine peptidase with a cys, his and asp, papain-like, catalytic triad, and mutation of any of these residues prevented autoactivation or rin digestion [ ] . the avrrpt protein is included in family c . family c includes the yopt effector protein from the plague organism yersinia pestis. the yop effectors are proteins secreted into host cells that disrupt the immune response; two of these effectors are now known to be peptidases, yopt and yopj (see chapter ). the yopt effector has been shown to be a cysteine peptidase, and is known to remove a c-terminal prenylated cysteine (or a short peptide containing the c-terminal cysteine) from gtpases. the effect is to release the gtpase from the membrane. the tertiary structure has been solved for a homolog from the plant pathogen pseudomonas syringae, which is the avirulence protein avrpphb (see figure . ) . the only proteolytic activity that could be demonstrated was that for autocatalytic activation of the precursor protein. a papain-like catalytic triad (cys/ his/asp) has been identified in both the yopt effector and the avrpphb protein by site-directed mutagenesis. however, there does not appear to be a conserved residue in the family that would act like gln in papain [ ] . another related peptidase from pseudomonas syringae, hopn is a virulence factor and is injected into host plant cells by a type iii secretion system where is suppresses cell death [ ] . the hopn peptidase is only distantly related and consequently, family c is divided into two subfamilies with yopt and avrpphb members of subfamily c a and hopn in c b. the ides peptidase (chapter ) from streptococcus pyogenes is included in family c . this peptidase cleaves the γ-chains of human igg. from the crystal structure [ ] , a papain-like fold is apparent and a catalytic triad consisting of cys , his and asp (see figure . ). family c includes phytochelatin synthases (chapter ), which are γ-glutamylcysteine dipeptidyltranspeptidases, from cyanobacteria and plants. these enzymes remove the c-terminal gly from glutathione (γ-glu-cys-gly). this is in contrast to the action of the γ-glutamyltransferases (chapter ) that remove the n-terminal residue. removal of the c-terminal gly is an important step towards formation of phytochelatin from linear polymers of the γ-glu-cys, and phytochelatin is important because ions of heavy metal ions such as mercury and cadmium are rendered harmless by forming tight complexes with phytochelatin. the tertiary structure of γ-glutamylcysteine dipeptidyltranspeptidase from the cyanobacterium nostoc sp. pcc has been solved, and shows a papain-like fold and an active site also similar to that of papain, containing gln, cys, his and asp [ ] (see figure . ). the 'papain-like' endopeptidases of rna viruses form a large group of cysteine peptidases that contain the catalytic dyad residues in the order cys, his. all of the families had been included in clan ca, even though for many no tertiary structure for any member had been solved. when the crystal structure of the nsp (nonstructural protein ) from sindbis virus (a member of family c ) was determined, the fold was quite unlike that of papain, and the family was assigned to its own clan (cn, see below). consequently, many families of peptidases from rna viruses that had been included in clan ca have been removed, because until a tertiary structure has been solved it will not be possible from sequence alone to determine if any of these families belongs to clan ca or clan cn. the families of rna viruses retained in clan ca, and for which tertiary structures have been solved, are c , c , c and c . each family contains peptidases from only one family of viruses. thus, endopeptidases from aphthoviruses are in family c ; arteriviruses in c and c , and nairoviruses in c . the cleavages mediated by the clan ca viral endopeptidases usually show gly in p (contrasting with gln in p for many cleavages by clan pa(c) endopeptidases). in most of these viruses there is a single polyprotein containing a single peptidase, but there may be up to three peptidases in a single polyprotein. in the foot-andmouth disease virus, potyviruses and togaviruses, the other cysteine peptidases are not related to papain (having the catalytic dyad in the order his, cys). in the coronaviruses and arteriviruses, all the endopeptidases have a cys, his catalytic dyad, suggesting papain-like tertiary folds, and are probably derived from one or two sequence duplications. such duplications appear to have been as ancient as the speciation events that gave rise to the different viruses, because there is no significant sequence similarity, and hence the peptidases are in different families. the crystal structure of the l-peptidase from the footand-mouth disease virus (in family c ) shows a clear relationship to papain. although aphthoviruses such as the foot-and-mouth disease virus possess picornain c (chapter ) as a general polyprotein-processing enzyme, they lack a picornain a, and the functions of that endopeptidase are performed by the l-peptidase or an autolytic activity (see chapter ). the l-peptidase is sited at the n-terminus of the polyprotein, from which it releases itself. the l-peptidase also cleaves the eukaryotic initiation factor eif- g (formerly known as p or eif- γ), thus preventing cap-dependent synthesis of hostcell proteins. family c contains the mouse hepatitis virus p endopeptidase, which releases itself from the n-terminus of the polyprotein encoded by gene a. the catalytic dyad has been identified by mutagenesis [ ] . the polyprotein encoded by gene a includes a picornain-like endopeptidase and a putative second papain-like endopeptidase, formerly assigned to a separate family, c , but now considered to be a member of family c . the compound e- c inhibits polyprotein processing in the mouse hepatitis virus [ ] , presumably by inhibiting the action of one or both of the two papain-like endopeptidases. the tertiary structure of the papain-like endopeptidase from sars virus has been solved (see figure . ) . families c , c and c include polyprotein-processing endopeptidases from arteriviruses. in the lelystad (porcine reproductive and respiratory syndrome) virus, the three papain-like endopeptidases are consecutive in the pol polyprotein, with pcpα at the n-terminus, followed by pcpβ and then nsp , which are so different in sequence that they are assigned to the separate families c , c and c , respectively. pcpα and pcpβ together constitute the nsp protein. the catalytic dyads have been determined by mutagenesis for all three endopeptidases [ , ] . besides the three papain-like cysteine endopeptidases, the arteriviruses possess a fourth, serinetype, endopeptidase (see chapter ) in the pol polyprotein. not all arteriviruses require all four endopeptidases, because in the equine arteritis virus, pcpα is inactive, the catalytic cys being replaced by lys. the pcpα endopeptidase releases itself from the n-terminus of the nsp protein [ ] . the pcpα endopeptidase cleaves between the nsp and nsp proteins in the pol polyprotein [ ] . the nsp endopeptidase cleaves between the nsp and nsp proteins, and, unusually, the catalytic cys is followed by gly. cys-gly is generally regarded as diagnostic for the subclan pa(c) viral endopeptidases, but it has also been seen in some calpain homologs [ ] and in family c . in the archaean methanothermobacter, the cell wall is analogous to that of bacteria, but the crosslinking peptide contains amino acids only in the normal, laevo, orientation. the crosslink is made between the ε-amino group of l-lys and the α-carboxyl group of l-ala. it is this bond that is broken by the pseudomurein endoisopeptidase (family c ) from methanobacterium phage psim . the enzyme can be inhibited by edta, suggesting that metal ions are important for activity. however, a cysteine-type catalytic triad (cys, his, asp), similar to that of papain, has also been proposed [ ] , and family c is included in clan ca. a second clan of cysteine peptidases contains processing endopeptidases of rna viruses. formerly termed clan cb, this has the catalytic dyad in the order his, cys in the sequence. a comparison of sequence motifs led bazan and fletterick [ ] to suggest that the polyprotein processing cysteine endopeptidases of several single, positive-stranded rna viruses were structurally related to chymotrypsin, and this was confirmed when the crystal structures of picornains c from human hepatitis a virus (chapter ) (figure . ) and human rhinovirus type (chapter ) were found to share the two β-barrel fold of chymotrypsin. there now seems no doubt that these peptidases have evolved from a common ancestor with chymotrypsin. the catalytic his of picornain c (in family c ) is equivalent in position to his of chymotrypsin, and the cys replaces ser (see the representation of two-dimensional structures for clan pa, see figure . ). in rhinovirus picornain a a his, asp, cys catalytic triad is seen [ ] , but the asp is replaced by glu in some other members of family c . the viral endopeptidases and chymotrypsin clearly belong in the same clan, and because the clan contains both cysteine-type and serine-type peptidases it has been named clan pa. the clan is divided into subclan pa(s), for the serine peptidases, and subclan pa(c) for the cysteine peptidases. families c , c , c , c and c also contain endopeptidases from rna viruses that are predicted to have the chymotrypsin fold, and are placed in subclan pa(c) together with family c . the viral peptidases in subclan pa(c) contrast in several respects with those in clan ca. in subclan pa(c) the active site cys is followed by gly (like the ser in chymotrypsin) whereas in clan ca the catalytic cys is generally followed by a large hydrophobic residue. as regards function, the endopeptidases of subclan pa(c) tend to be general processing enzymes, cleaving several bonds in the polyprotein, whereas those in clan ca generally perform only a single cleavage. there is also a difference in p substrate specificity: in subclan pa(c) cleavage is commonly at a glnkgly bond, whereas in clan ca cleavage usually occurs at a glykgly bond. the subclan pa (c) endopeptidase is usually positioned between the helicase and the rna polymerase in the polyprotein, whereas the clan ca enzyme usually precedes both the helicase and the rna polymerase. taken together with other indications that the positive-stranded rna viruses are evolutionarily related, the similar locations of the subclan pa (c) endopeptidases in the genomes is entirely consistent with a common evolutionary origin of all of the peptidases in the subclan. the high rate of mutation of rna viruses, lacking proofreading in the replication of the genome, could rapidly have obscured sequence relationships between the sequences. family c contains the processing endopeptidases of picornaviruses, aphthoviruses, nepoviruses and comoviruses. the first group includes animal viruses such as those that cause polio and encephalomyocarditis. picornaviruses encode one polyprotein that contains coat and core proteins, an rna polymerase and two endopeptidases that are homologous to each other and responsible for excising the individual proteins. the larger of the endopeptidases, picornain c, is responsible for most of the cleavages, mainly at glnkgly bonds, whereas the smaller endopeptidase, picornain a, releases itself by cleavage of a tyrkgly bond. picornain a has the further function of cleaving the eukaryote initiation factor g of the host cell [ ] . picornaviruses are unusual in possessing two homologous endopeptidases; in aphthoviruses, for example, cleavage of the eukaryote initiation factor g is performed by an endopeptidase unrelated to the picornains, the l-peptidase of family c , clan ca (chapter ). although the picornains c and a are homologous, the sequences are so different that they are assigned to the separate subfamilies c a and c b, respectively. the polyprotein processing enzyme from aphthoviruses, which include foot-and-mouth disease, is so different in sequence from the picornains that it too is assigned to a separate subfamily, c c. the processing endopeptidase for the hepatitis a virus is a member of a fourth subfamily, c e, and a fifth subfamily, c f, includes the protease from parechovirus . not all members of family c are animal pathogens, and subfamily c d includes processing peptidases for the polyprotein of cowpea mosaic virus and other plant pathogens. a seventh subfamily, c g, includes a polyprotein processing endopeptidase from rice tungro spherical virus. catalytic activity has been demonstrated and the cleavage sites determined by site directed mutagenesis to be gln kasp and gln kala [ ] ; the catalytic residues are predicted to be his , asp and cys (see alignment c g in merops). a homolog is also known from maize chlorotic dwarf waikavirus. family c contains one of the three polyprotein processing endopeptidases from potyviruses, which are plant pathogens. the nia endopeptidase has been shown by site-directed mutagenesis to have at least a catalytic dyad that is in the order his, cys [ ] . another similarity with family c is the preference for cleavage of glnkgly bonds. the endopeptidase is a multifunctional molecule, acting also as the vpg protein, which is attached covalently to the viral rna [ ] . family c includes the processing endopeptidase from caliciviruses. catalytic residues have been identified in the rabbit hemorrhagic disease virus, and a his, cys dyad is the most likely order in the sequence, but a histidine c-terminal to the cysteine is also important, probably for substrate binding [ ] . the endopeptidase cleaves glukgly bonds in the polyprotein [ ] . family c includes the processing endopeptidase from coronaviruses. again, there is a his, cys catalytic dyad, and cleavage is preferentially at glnkgly bonds [ ] . a representation of the three-dimensional structure of the processing peptidase from porcine transmissible gastroenteritis virus main protease is shown in figure . . family c contains the processing endopeptidases of southampton and norwalk caliciviruses. the catalytic cysteine has been identified in southampton virus, and there is a preference for cleavage at glnkgly bonds [ ] . family c comprises a putative endopeptidase from parsnip yellow fleck virus identified by comparison with the picornavirus polyprotein [ ] . family c includes the ns protein, one of the three polyprotein processing peptidases from pestiviruses. the catalytic triad has been established by mutagenesis as his, glu, cys, consistent with other families in subclan pa(c) [ ] . clan cd contains endopeptidases in which only a catalytic dyad appears to exist, in the order his, cys in the sequence. chen et al. [ ] recognized that sequence motifs around the catalytic residues in mammalian legumain were similar to those in the caspases (family c ). moreover, they saw similar motifs in the cysteine peptidases of families c (clostripain) and c (gingipain r). they proposed the existence of a clan cd of cysteine peptidases with a protein fold similar to that already known for the caspases (family c ). the proposal soon received support from the crystal structure of gingipain r . the structure of caspase (see figure . , chapter ) shows a distinctive α/β fold quite unlike those of clans ca and pa. in the sequence motif described by chen et al. [ ] , the catalytic histidine is preceded by a block of hydrophobic residues and a glycine, and the catalytic cysteine is preceded by a second block of hydrophobic residues, a glutamine and an alanine (see figures . and . . a similar motif has been recognized in separase (family c ), rtx selfcleaving toxin(family c ) and prth peptidase (family c ), and all three families have been added to clan cd. similar motifs are present in proteins not yet known to be peptidases [ ] so the clan may expand still further. the peptidases in clan cd have some biochemical similarities. they are all endopeptidases with restricted specificity dominated by selectivity for the p residue of the substrate. thus, in family c cleavage occurs predominately after asp, in c after asn, in c and c after arg, and in c after arg or lys depending on the peptidase. this p -dominated type of specificity resembles that of the unrelated chymotrypsin-like serine peptidases in subclanclan pa(s), and is quite unlike clan ca. the endopeptidases in the clan are not irreversibly inhibited by e- , in contrast to most enzymes in clan ca. clostripain and legumain react more rapidly with iodoacetamide than with iodoacetate, whereas iodoacetate is the faster inhibitor for papain and other members of family c (chapter ). crma, a serpin from cowpox virus, is an inhibitor of the caspases [ ] that also inhibits the serine endopeptidase granzyme b (chapter ) [ ] . rather than reflecting any structural relationship, however, this is undoubtedly the result of a similar specificity of the enzymes for aspartyl bonds. family c comprises a number of cytosolic endopeptidases that have strict specificity for hydrolysis of aspartyl bonds, the best known of which are caspase- (chapter ) and caspase- (chapter ). as can be seen in the alignment in merops, there is a catalytic dyad in the order his, cys in the sequence, as expected in clan cd. caspase- is synthesized as a single-chain precursor, and the activation by cleavage of four aspartyl bonds is presumably autocatalytic. the mature endopeptidase is a heterodimer of a kda heavy chain and a kda light chain [ ] . caspase- also mediates the processing of interleukin β at aspartyl bonds [ ] , and was formerly known as interleukin β-converting enzyme (ice). the discovery that the product of the ced gene in caenorhabditis elegans, which when defective increases the lifespan of the nematode [ ] is a homolog of caspase- led to the discovery of a proteolytic cascade that leads to apoptosis in mammalian cells. apoptosis is receptor mediated, and once the system is activated, proteins associate with one another by means of 'death domains' and 'deatheffector domains'. death-effector domains occur in a number of intracellular proteins and in caspases- and - [ ] . flip, a newly reported inhibitor of caspase- , occurs in two alternatively spliced forms, the longer of which, flip l , contains two death-effector domains, and a caspase-like domain in which the catalytic cys is replaced by tyr, and the his by arg (human) or leu (mouse) [ ] (see the alignment for family c in merops). the evolutionary tree shows a pronounced divergence within the family, which is therefore divided into two subfamilies. subfamily c a includes caspases, whereas subfamily c b includes metacaspases (see chapter ) and paracaspases (see chapter ). paracaspase is known from a variety of animal species, including human, and provides an important link to a number of bacterial species, including homologs in the completed genomes of nostoc, mesorhizobium loti and xylella fastidiosa [ ] . the family is generally absent from archaea, but homologs have been detected in the proteomes of methanosarcina barkeri, methanothermus fervidus and thermococcus onnurineus, which are presumably derived from horizontal gene transfers. there are also homologs in two insect ascoviruses, which are double-stranded dna viruses. family c includes a number of endopeptidases that specifically cleave asparaginyl bonds. an asparaginyl endopeptidase was first identified in seeds of leguminous plants and was named 'legumain' [ ] . the legumain of plant seeds has now been found in a wide variety of dicotyledonous plants and is proposed to be responsible for the post-translational processing of seed proteins prior to storage [ ] . the processing of a few of these proteins, such as concanavalin a, also involves protein splicing at asparaginyl bonds, and this transamidase reaction also can be mediated by legumain [ ] . homologous asparaginyl endopeptidases have been characterized from the blood flukes schistosoma mansoni and s. japonicum; they were initially thought to be responsible for hemoglobin breakdown and were termed 'hemoglobinases' but this name is no longer appropriate. mammalian legumain has been characterized and shown to be a lysosomal enzyme [ ] . legumain is not inhibited by compound e- and like clostripain it reacts more rapidly with iodoacetamide than with iodoacetate. the enzyme is inhibited by egg-white cystatin, which is also an inhibitor of peptidases in family c (clan ca), but it has been found that cystatin possesses two reactive sites, one for legumain and one for the papain-like enzymes, and is able to inhibit both simultaneously and independently [ ] (chapter ). not a conventional endopeptidase, but also in the legumain family, is glycosylphosphatidylinositol:protein transamidase. this has been identified in saccharomyces and humans, and is responsible for attaching glycosylphosphatidylinositol (gpi) anchors to the c-termini of newly synthesized proteins in the endoplasmic reticulum. a c-terminal peptide is removed before the preformed gpi anchor is attached, apparently in a single reaction. the transamidase differs considerably in sequence from the legumains, and the cys residue that had been predicted to be catalytic in schistosoma legumain [ ] is replaced by leu. the evolutionary tree demonstrates the marked difference between the forms of legumain and the gpi transamidase (chapter ). members of family c are known principally from animals, plants and fungi: the family is absent from the genome of the microsporidian encephalitozoon cuniculi and not known from any other protozoa. however, despite generally being absent from prokaryotes, homologs are present in the gram-negative bacteria pseudomonas aeruginosa and caulobacter crescentus. the pseudomonas homolog is the most divergent member of the family appears to be derived from a divergence that predates that of legumain and glycosylphosphatidylinositol:protein transamidase. family c contains only clostripain (chapter ). this is an endopeptidase discovered in clostridium histolyticum that has a preference for hydrolysis of arginyl bonds. clostripain is synthesized as a proprotein, and activation requires loss of a residue propeptide and autolytic removal of an internal nonapeptide to produce a heterodimer consisting of a -residue light chain and a -residue heavy chain that contains the residues of the catalytic dyad. clostripain differs from enzymes of the papain family in a number of respects: it is calcium dependent, it is more rapidly inactivated by iodoacetamide than by iodoacetate, and e- gives only reversible inhibition attributable to the presence of an arginine-like sidechain in the compound. forms of clostripain are known from clostridium perfringens and thermotoga maritima. family c is represented by gingipains r and k (chapters and ), secreted endopeptidases from the pathogenic bacterium porphyromonas gingivalis. gingipain is a multidomain protein, containing not only a peptidase unit but also a c-terminal hemagglutinin domain (figure . ) . gingipain r has specificity for arginyl bonds, whereas gingipain k cleaves after lysine residues. both enzymes are thought to contribute to the disease processes in gingivitis. family c contains separase (chapter ), an endopeptidase that is involved in the separation of sister chromatids during the anaphase periods of mitosis or meiosis. in mitosis, the chromatids are bound together by the multi-subunit protein cohesin, and cleavage of the scc subunit of cohesion by separase promotes chromatid separation. cleavage occurs at the arg karg and arg karg bonds of the scc subunit. separase also cleaves the kinetochore-associated protein slk at the start of anaphase, which is necessary for the development of a stable spindle. in meiosis, the rec protein performs the same role as cohesin and is also susceptible to cleavage by separase. a simple consensus for cleavage is an acidic residue at p , gly in p , arg in p and arg, lys or ser in p . members of family c are known only from eukaryotes, and are found in every eukaryote genome that has so far been completely sequenced. family c contains self-cleaving proteins that are precursors of bacterial toxins, including vibrio cholera rtx toxin (chapter ), and clostridium difficile toxins a and b. however, peptidase activity is not restricted to self-cleavage and the mature rtx toxin has been shown to degrade the leucine-rich protein yopm [ ] . the tertiary structure of the cholera rtx toxin has been solved [ ] and shows a structure similar to that of caspase- (see figure . ). family c is therefore included in clan cd. the prth protein, a virulence factor from tannerella (formerly bacteroides) forsythensis, is the only characterized peptidase in family c . however, very little is known about prth, except that it hydrolyzes bz-val-gly-arg-p-nitroanilide, which can be inhibited by standard cysteine peptidase inhibitors and the metal chelator edta [ ] . the prth gene is associated with the transition from a commensal to pathogenic organism, and increased levels are associated with release of cells from the substratum [ ] . although there is no information about structure or active site residues, a caspases-like structure has been predicted along with a his, cys catalytic dyad. the his and cys residues occur in motifs similar to those around the active site residues of caspases and legumains [ ] . clan ce contains four families: c , c , c and c . three-dimensional structures have been solved for adenain (chapter ) in family c and the ulp endopeptidase (chapter ) in family c . the families c , containing the yopj protease from yersinia, and c , containing the vaccinia virus i protein, are included in clan ce because the order of putative catalytic residues (his, glu or asp, gln, cys) is the same in each. several of the peptidases in clan ce show specificity for cleavage in the gly-glykxaa motif. an alignment showing conservation of sequence around the catalytic residues is shown in figure . , and secondary structures are compared in figure . . family c contains adenain, an endopeptidase from adenoviruses. adenoviruses are double-stranded dna viruses that do not encode polyproteins but have a separate gene for each protein. activation of the proteins involves processing at the n-terminus to remove a propeptide, and it is the adenovirus endopeptidase that is responsible for this [ ] . the tertiary structure of adenain from human adenovirus type (figure . ) shows an α/β fold with some remarkable similarities to that of papain but with the catalytic residues in the order his, cys in the sequence. the catalytic dyad is complemented by two other residues: glu (most commonly asp, in the clan as a whole; numbering as in figure . ), which has the same role of orientating the imidazolium ring of the catalytic his as does asn of papain, and gln , which helps form the oxyanion hole, similarly to gln of papain. the catalytic cys is also positioned at the start of a long helix, as in papain [ ] . however, the structures are so different that we conclude that they have resulted from convergent evolution. the adenovirus endopeptidase is synthesized as an active enzyme without a propeptide. the endopeptidase is highly selective for xaa-xbb-gly-xbbkxbb bonds, in which xaa is either met, leu or ile, and xbb is any amino acid. a virally encoded, eleven residue cofactor is also required for activity [ ] , and the enzyme is apparently further activated by interaction with dna. in family c the tertiary structure of the yeast ulp endopeptidase (chapter ) shows a similar protein fold to adenain (figure . ). the ulp endopeptidase is responsible for the release of the smt proteins. smt and the animal equivalent sumo- (also known as sentrin) are proteins that target other proteins for export from the nucleus via the nuclear pore complex protein ranbp . smt and sumo- are distantly related to ubiquitin, and like ubiquitin possess c-terminal glycine residues that can be linked in isopeptide bonds. unlike ubiquitin, smt and sumo- do not target proteins for degradation, and do not form homopolymers, because the lysine essential to that process is not present. the ulp endopeptidase releases smt from its precursor by removing three c-terminal residues to expose the -gly-gly c-terminus; the mammalian equivalent, senp -endopeptidase, removes four c-terminal residues. ulp endopeptidase can also release smt from targeted proteins. the enzyme is normally localized to the periphery of the nucleus, but is found within the nucleus during mitotic anaphase. family c is that of the yopj endopeptidase from yersinia (chapter ). the yopj protein contributes to the disease process by causing host macrophages to undergo apoptosis and suppressing the action of cytokines such as tnfα and nf-κb. yopj is thought to act by cleaving the bid protein, which then induces apoptosis by translocating to the mitochondrion, causing the release of cytochrome c and the activation of caspases , and [ ] . yopj is an outer membrane protein that is encoded on a plasmid. there are a number of homologs in this family that are not known to be peptidases (even though all the catalytic residues are conserved) including avirulence protein avrbst from xanthomonas. the family is known only from proteobacteria. although no catalytic residues have been identified for any member of the family, they are predicted to occur in the order his, glu, gln, cys. family c contains the i protein from the vaccinia virus (chapter ), which is believed to be a polyprotein processing endopeptidase. although no catalytic residues have been identified for any member of the family, they are predicted to occur in the order his, asp/asn, gln, cys. the polyprotein processing endopeptidase present in the african swine fever virus (chapter ) is included in family c . although no tertiary structure has been solved, the order of active site residues (his, asn, gln, cys) is consistent with membership of clan ce, as is the restricted specificity for gly-glykxaa bonds [ ] . bacterial homologs are known from chlamydia, xanthomonas, pseudomonas and mesorhizobium but have low similarity to the eukaryotic sequences. the pseudomonas and mesorhizobium homologs are multidomain, mosaic proteins. no peptidase activity has yet been demonstrated for any of the bacterial homologs, but the gene has been shown to be encoded on a plasmid and essential for virulence in pseudomonas [ ] . no homologs are known from archaea. not all deubiquitinating enzymes are proteins with a papain-like fold. the elad peptidase from escherichia coli is able to release ubiquitin from synthetic constructs with a preference for lys -linked ubiquitin chains, but also cleaving at lys- [ , ] . this peptidase is included in family c . this family is included in clan ce because we propose a his, cys catalytic dyad (see the alignment in merops). clan cf, family c . the crystal structure of pyroglutamyl-peptidase i from thermococcus (figure . ) shows a catalytic triad with the residues in the order glu, cys, his in the sequence, which is different to any other clan of cysteine peptidases. the enzyme is an intracellular omega peptidase that removes an n-terminal pyroglutamyl residue from a polypeptide. cys and his have been identified as catalytic residues [ ] . human pyroglutamyl-peptidase i has been cloned and expressed [ ] ; (chapter ), and appears to be structurally very similar to the archaean and bacterial forms of the enzyme. family c contains the hedgehog proteins. the hedgehog proteins are formed of two structural domains, and the only peptidase activity is an autolytic reaction that separates the two domains. the reaction is cleavage of a conserved gly kcys bond, and a cholesterol moiety is simultaneously attached to the new c-terminal glycyl residue of the n-terminal domain. the -kda nterminal domain is structurally related to a zinc d-ala-d-ala carboxypeptidase in clan md; it is not known to have any peptidase activity but has a function in the embryological development of dorsal-ventral patterning. mutational studies have shown that residues cys and his in the -kda c-terminal domain are essential for the autolytic cleavage [ ] . the reaction is not inhibited by any standard peptidase inhibitors, but requires dtt in vitro. the cleavage mechanism is believed to be similar to that of the activation of prohistidine decarboxylase [ ] , involving activation of cys by his for a nucleophilic attack on the carbonyl of gly . there is a motif around the cleavage site that is similar to that found at the n-terminal splice junction of self-splicing proteins (chapter ), and splicing may involve a thioester intermediate [ ] . the crystal structure of the sonic hedgehog c-terminal domain (figure . ) shows that it consists of two tandem intein-like repeats [ ] , and family c is now included in a clan of mixed catalytic types, pd, along with the three intein families (see chapter ) [ ] . peptidases within clan cl have active site residues in the order his, cys in the sequence, and a tertiary fold consisting of a closed β-barrel surrounded by helices (see figure . ). the active site is at the end of the barrel. the fold is similar to that of the ykud transpeptidase from bacillus subtilis [ ] . members of the clan are involved in hydrolysis of bacterial cell wall peptides. family c includes peptidases known as sortases (chapter ) from gram-positive bacteria, and sortase a from staphylococcus aureus cleaves proteins at a leu-pro-xaa-thrkgly motif, and then catalyzes the formation of an amide bond between the newly exposed c-terminal thr and the cross-linking pentapeptide of the bacterial cell wall [ ] . the proteins that are attached in this way to the exterior of the cell wall can be virulence factors, and inhibition of sortases has been shown to decrease virulence. surprisingly, given a lack of structural relationship to members of clan ca, sortases are inhibited by e- . the evolutionary tree shows a deep divergence within the family, which is divided into two subfamilies. subfamily c a includes sortase a, whereas c b includes sortase b. sortase b, also from staphylococcus aureus, recognizes a different cleavage motif to that of sortase a [ ] . the only other family in clan cl is c . this also includes peptidases involved in bacterial cell wall metabolism. the l,d-transpeptidase from enterococcus faecium (chapter ) cleaves the l-lyskd-ala bonds from the crosslinking tetra-and pentapeptides, and also possesses cell-wall crosslinking activity [ ] . the structure of a fragment of the peptidase has been solved and shows a fold similar to that of sortases [ ] . the hepatitis c polyprotein is processed by virally encoded endopeptidases as well as by host endopeptidases. most of the cleavages are performed by hepacivirin (chapter ), which is a serine-type endopeptidase with a trypsin-like fold. cleavage of the ns / site at a leukala bond is mediated by the second virally encoded endopeptidase, endopeptidase , often termed the 'ns - endopeptidase', not because of the cleavage site but because the endopeptidase consists of the ns protein and one-third of the ns protein (chapter ). endopeptidase is included in family c . site-directed mutagenesis had identified his and cys as the catalytic dyad [ ] . however, evidence that the ns - endopeptidase is zinc dependent, including inhibition by edta and increased activity with the addition of zinc [ ] , had cast doubt on the identification of the catalytic type. the tertiary structure has been determined, and shows a fold unrelated to that of any other peptidase, hence family c is the only family in clan cm (see figure . ). the active peptidase is a homodimer, and each monomer comprises an n-terminal, helical subdomain, a c-terminal subdomain with a β-sheet and an extended linker region. the active site his and glu are situated in the n-terminal domain and the cys in the cterminal domain. an active site is only formed in the dimer, when the his and glu from one monomer oppose the cys from the other, thus the ns dimer has two active sites [ ] . the formation of an active site between different subunits in a dimer is unusual amongst peptidases, but does happen with retropepsins (see chapter ) and has been proposed for the pyrococcus furiosus protease (see chapter ). the tertiary structure of the ns dimer also revealed the presence of a structural zinc ion, which explains the metal-dependence. family c includes the non-structural polyproteinprocessing endopeptidase from togaviruses, the nsp endopeptidase. the catalytic dyad has been identified by site-specific mutagenesis [ ] , and the nsp endopeptidase is a bifunctional molecule with the peptidase unit at the c-terminus and an n-terminal unit that is probably involved in rna-binding during replication. the n-terminal domain is homologous to a similarly located domain in the cucumber green mottle mosaic tobamovirus. the crystal structure of the nsp endopeptidase from venezuelan equine encephalitis virus has been solved, and shows a fold unrelated to that of any other peptidase [ ] (see figure . ). hence c is the only family in clan cn. until the structure was solved, it had been expected that members of family c would have a fold similar to that of papain. clan pc is another clan containing peptidases of different catalytic types, and subclan pc(c) contains the cysteinetype peptidases in the clan. members of the clan have a the image was prepared from the protein data bank entry ( hd ) as described in the introduction (p. li). one homodimer is shown. catalytic residues are shown in ball-and-stick representation for each monomer: his in purple, glu in blue and cys in yellow (numbering as in pdb entry). fold similar to that of class i glutamine amidotransferase, which consists of an α/β/α sandwich. the nucleophile can be either a cysteine or a serine, and these are in equivalent positions. family c , contains γ-glutamyl hydrolase (chapter ). the tertiary structure of human γ-glutamyl hydrolase (figure . ) shows a homotetramer. a cys, his catalytic dyad is evident, and the histidine is hydrogenbonded to a glutamate and a tyrosine, either of which could be a third member of a catalytic triad [ ] . family c contains only protease i from pyrococcus (see chapter ). although the crystal structure has been solved ( figure . ) , the catalytic mechanism is not known with certainty. the endopeptidase is a homotrimer, and cys and his have been implicated in the catalytic mechanism, with the suggestion that a catalytic triad is formed between residues from opposing monomers, with glu completing the active site. this would be a unique catalytic mechanism amongst the peptidases. his is poorly conserved, however, which may mean that very few members of the protein family are peptidases. his is also not in an equivalent position to his from γ-glutamyl hydrolase. the image was prepared from the protein data bank entry ( l x) as described in the introduction (p. li). one monomer of the tetramer is shown. catalytic residues are shown in ball-and-stick representation: cys and his (numbering as in pdb entry). figure . richardson diagram of pyrococcus horikoshii pfpi endopeptidase. the image was prepared from the protein data bank entry ( g i) as described in the introduction (p. li). one monomer of the homotrimer is shown. catalytic residues are shown in ball-and-stick representation: cys and his (numbering as in pdb entry). family c contains dipeptidyl-peptidase vi from bacillus sphaericus. this is an enzyme that is expressed during sporulation, and is responsible for degradation of bacterial cell wall components. because the enzyme is cytoplasmic and is synthesized without signal and propeptides, it is presumably acting at a late stage in cell wall component turnover. there are a number of homologs of the bacillus endopeptidase, but similarity is restricted to a c-terminal domain of residues. these include nlpc lipoprotein from escherichia coli, invasion-associated protein p from listeria species, a starch-degrading enzyme from clostridium acetobutylicum, and a phosphatase-associated protein from bacillus subtilis. there are single conserved cys and his residues). the crystal structures of several members of the family have been solved, and the fold has been described as 'papain-like' but primitive [ ] . however, unlike papain where the catalytic cys is at the n-terminus of a helix, in the structures from family c the active site cys is at the c-terminus of a helix, which means the sequence must run in the opposite direction to that in papain (see figure . ) . no similarity between the structures can be detected using the dali algorithm [ ] . the structural resemblances are therefore assumed to be derived from convergent rather than divergent evolution, and proteins in c are assigned to the clan co. all members of this family have either a signal peptide or a transmembrane region, indicating a cell-surface location. clan pb contains n-terminal nucleophile hydrolases, amongst which the most important peptidase is the proteasome. in the proteasome the n-terminal nucleophile is threonine, but there are members of the clan in which the nucleophile is cysteine. these are exclusively self-processing proenzymes that carry out a single peptide bond cleavage to form the active enzyme, but in contrast to the proteasome, the enzyme that is formed is not a peptidase. the cysteinetype enzymes of clan pb form a subclan pb(c), and are discussed more fully in chapter . there are a few other families of cysteine peptidases for which no tertiary structures are available and too little is known about the catalytic machinery to permit any conclusions about their relationships. family c contains one of the two cysteine endopeptidases found in potyviruses, the helper-component endopeptidase. (the second is the nia peptidase, chapter ) the only cleavage performed by the helper-component endopeptidase is its own release from the polyprotein by cleavage of a glykgly bond; further processing is performed by the serine-type p endopeptidase (chapter ). the helper-component endopeptidase is a two-part molecule with the peptidase unit at the c-terminus, and the helper component, required for virus transmission from plant to plant by aphids, at the n-terminus. the catalytic residues have been identified by site-directed mutagenesis in tobacco etch virus [ ] . families c and c contain the chestnut blight hypovirus endopeptidases p and p . chestnut blight is caused by the fungus cryphonectria parasitica, but the symptoms are reduced if the fungus is infected by the double-stranded rna hypovirus. the virus encodes two polyproteins, and the enzymes responsible for their processing constitute the smaller polyprotein. the p endopeptidase cleaves a single glykgly bond in the polyprotein to release itself and the p endopeptidase [ ] . the p endopeptidase cleaves a single glykala bond in the larger polyprotein [ ] . catalytic residues have been identified by mutagenesis for both endopeptidases [ , ] . the endopeptidases show no significant sequence similarity to any other peptidase, and each is considered a representative of a distinct family, although there is conservation of a gly-tyr-cys-tyr motif containing the catalytic cys between the p endopeptidase (family c ) and family c . family c contains the polyprotein-processing endopeptidase from tymoviruses. the kda polyprotein is cleaved at an alakthr bond [ ] to release the kda polymerase from the c-terminus and a kda protein figure . richardson diagram of the nlp/p -like putative peptidase from nostoc punctiforme. the image was prepared from the protein data bank entry ( evr) as described in the introduction (p. li). catalytic residues are shown in ball-and-stick representation: his and his in purple and cys in yellow (numbering as in pdb entry). that includes the endopeptidase and a helicase from the n-terminus. the catalytic residues have been identified by mutagenesis [ ] . family c includes the p polyprotein processing endopeptidase from the blueberry scorch virus. cleavage is at a single glykala bond, and site-directed mutagenesis has been used to identify the potential catalytic residues [ ] . family c includes the non-structural polyproteinprocessing endopeptidase from rubella virus. processing occurs at a single glykgly bond, and the cys, his catalytic dyad has been identified [ ] . family c is represented by a polyprotein processing endopeptidase beet necrotic yellow vein virus [ ] . family c includes a polyprotein-processing endopeptidase from the beet yellows closterovirus. processing occurs at a single glykgly bond, and the catalytic dyad has been identified by site-directed mutagenesis [ ] . family c contains the npro endopeptidase of pestiviruses (chapter ). pestiviruses possess two polyprotein-processing endopeptidases: the serine-type ns - endopeptidase (chapter ), which is the more general processing activity; and the npro endopeptidase, which is the n-terminal protein and releases itself from the polyprotein by autolytic cleavage of a cyskser bond. the residue cys has now been identified as a catalytic residue [ ] . family c includes the agrb protein from staphylococcus aureus. the agr operon includes four genes whose products are involved in quorum sensing. the arga protein is a response regulator, argc is a sensor kinase and agrd is a polypeptide that is integrated into the cytoplasmic membrane via its n-terminal region, and is the precursor for an autoinducing peptide (aip) that is the ligand for agrc. the agrb protein is the peptidase that releases aip from agrd [ ] , following the removal of an n-terminal peptide by type i signal peptidase [ ] . the active site residues his and cys were identified in staphylococcus aureus agrb by [ ] . agrb is an integral membrane protein, with the active site close to the cytoplasm [ ] . inhibition of mammalian legumain by some cystatins is due to a novel second reactive site bj a, a snake venom metalloproteinase inhibitor. isolation, characterization, cloning and insights into its mechanism of action inhibition of cathepsin l-like cysteine proteases by cytotoxic t-lymphocyte antigen- beta a primitive enzyme for a primitive cell: the protease required for excystation of giardia molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen crystal structure of calpain reveals the structural basis for ca -dependent protease activity and a novel mode of enzyme activation the crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium calpa, a drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells molecular cloning and analysis of small optic lobes, a structural brain gene of drosophila melanogaster on the mechanism of action of streptococcal proteinase. ii. comparison of the kinetics of proteinase-and papain-catalyzed hydrolysis of n-acylamino acid esters l-trans-epoxysuccinyl-leucylamido( -guanidino)butane (e- ) and its analogues as inhibitors of cysteine proteinases including cathepsins b, h and l crystal structure of a deubiquitinating enzyme (human uch-l ) at . Å resolution crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde burkholderia mallei tssm encodes a putative deubiquitinase that is secreted and expressed inside infected raw . murine macrophages a novel superfamily of predicted cysteine proteases from eukaryotes, viruses and chlamydia pneumoniae a novel type of deubiquitinating enzyme structure of the a otu domain and mechanistic insights into deubiquitination crystal structure of human otubain evidence for bidentate substrate binding as the basis for the k linkage specificity of otubain the tumour suppressor cyld negatively regulates nf-kappab signalling by deubiquitination the structure of the cyld usp domain explains its specificity for lys -linked polyubiquitin and reveals a b box module duba: a deubiquitinase that regulates type i interferon production the solution structure of the josephin domain of ataxin- : structural determinants for molecular recognition structural basis for ubiquitin recognition by the otu ovarian tumor domain protein structure of a herpesvirusencoded cysteine protease reveals a unique class of deubiquitinating enzymes ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses two novel ubiquitin-fold modifier (ufm )-specific proteases, ufsp and ufsp structural basis for ufm processing by ufsp the crystal structure of human atg b, a processing and de-conjugating enzyme for autophagosome-forming modifiers structural basis for the specificity and catalysis of human atg b responsible for mammalian autophagy crystal structure of a thiol proteinase from staphylococcus aureus v in the e- inhibitor complex crystal structure of the peptidase domain of streptococcus coma, a bifunctional atp-binding cassette transporter involved in the quorum-sensing pathway multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi . identification of a d-alanyl-glycine endopeptidase activity cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity initiation of rps -specified disease resistance in arabidopsis is coupled to the avrrpt -directed elimination of rin a yersinia effector and a pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis hoppton is a pseudomonas syringae hrp (type iii secretion system) cysteine protease effector that suppresses pathogeninduced necrosis associated with both compatible and incompatible plant interactions structure of the streptococcal endopeptidase ides, a cysteine proteinase with strict specificity for igg a papain-like enzyme at work: native and acyl-enzyme intermediate structures in phytochelatin synthesis identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d processing and evolution of the n-terminal region of the arterivirus replicase orf a protein: identification of two papain-like cysteine proteases the arterivirus nsp protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases pseudomurein endoisopeptidases peiw and peip, two moderately related members of a novel family of proteases produced in methanothermobacter strains viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications the structure of the a proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis a single amino acid change in protein synthesis initiation factor g renders cap-dependent translation resistant to picornaviral a proteases rice tungro spherical virus polyprotein processing: identification of a virus-encoded protease and mutational analysis of putative cleavage sites characterization of the catalytic residues of the tobacco etch virus -kda proteinase autocatalytic activity of the tobacco etch virus nia proteinase in viral and foreign protein sequences identification and characterization of a c-like protease from rabbit hemorrhagic disease virus c-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the orf polyprotein and analysis of cleavage specificity characterisation and mutational analysis of an orf a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus a/ b polyprotein polyprotein processing in southampton virus: identification of c-like protease cleavage sites by in vitro mutagenesis sequence analysis of the parsnip yellow fleck virus polyprotein: evidence of affinities with picornaviruses temporal modulation of an autoprotease is crucial for replication and pathogenicity of an rna virus identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases classification of the caspasehemoglobinase fold: detection of new families and implications for the origin of the eukaryotic separins viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin- beta converting enzyme granzyme b is inhibited by the cowpox virus serpin cytokine response modifier a activation of the native -kda precursor form of interleukin- -converting enzyme interleukin- β converting enzyme: a novel cysteine protease required for il- β production and implicated in programmed cell death the caenorhabditis elegans cell-death protein ced- is a cysteine protease with substrate specificities similar to those of the human cpp protease apoptosis. placing death under control inhibition of death receptor signals by cellular flip the two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays vacuolar processing enzyme responsible for maturation of seed proteins in vitro splicing of concanavalin a is catalyzed by asparaginyl endopeptidase cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase expression and partial characterization of a cathepsin b-like enzyme (sm ) and a proposed 'haemoglobinase' (sm ) from schistosoma mansoni structural and molecular mechanism for autoprocessing of martx toxin of vibrio cholerae at multiple sites small molecule-induced allosteric activation of the vibrio cholerae rtx cysteine protease domain cloning, expression, and sequencing of a protease gene from bacteroides forsythus atcc in escherichia coli a -year longitudinal study of tannerella forsythia prth genotype: association with loss of attachment prediction of a caspase-like fold in tannerella forsythia virulence factor prth adenovirus endopeptidases crystal structure of the human adenovirus proteinase with its amino acid cofactor activation of adenovirus-coded protease and processing of preterminal protein yersinia enterocolitica yopp-induced apoptosis of macrophages involves the apoptotic signaling cascade upstream of bid gly-gly-x, a novel consensus sequence for the proteolytic processing of viral and cellular proteins isolation and characterization of virulence gene psva on a plasmid of pseudomonas syringae pv. eriobotryae elad, a deubiquitinating protease expressed by e. coli ssel, a salmonella deubiquitinase required for macrophage killing and virulence mutational analysis of the active site of pseudomonas fluorescens pyrrolidone carboxyl peptidase pyroglutamyl-peptidase i: cloning, sequencing, and characterisation of the recombinant human enzyme autoproteolysis in hedgehog protein biogenesis sitedirected alteration of serine causes nonproductive chain cleavage in prohistidine decarboxylase the product of hedgehog autoproteolytic cleavage active in local and long-range signalling crystal structure of a hedgehog autoprocessing domain: homology between hedgehog and self-splicing proteins asparagine peptide lyases: a seventh catalytic type of proteolytic enzymes crystal structures of staphylococcus aureus sortase a and its substrate complex an iron-regulated sortase anchors a class of surface protein during staphylococcus aureus pathogenesis specificity of l,d-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes crystal structure of a novel β-lactam-insensitive peptidoglycan transpeptidase a second hepatitis c virus-encoded proteinase two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis c virus structure of the catalytic domain of the hepatitis c virus ns - protease identification of the active site residues in the nsp proteinase of sindbis virus the crystal structure of the venezuelan equine encephalitis alphavirus nsp protease threedimensional structure of human γ-glutamyl hydrolase. a class i glutamine amidotransferase adapted for a complex substrate dali: a network tool for protein structure comparison identification of essential residues in potyvirus proteinase hc-pro by site-directed mutagenesis cotranslational autoproteolysis involved in gene expression from a double-stranded rna genetic element associated with hypovirulence of the chestnut blight fungus gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. essential residues and cleavage site requirements for p autoproteolysis the autocatalytic protease p encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease hc-pro identification of the cleavage site recognized by the turnip yellow mosaic virus protease identification of the essential cysteine and histidine residues of the turnip yellow mosaic virus protease autocatalytic processing of the -kda protein of blueberry scorch carlavirus by a papain-like proteinase characterization of the rubella virus nonstructural protease domain and its cleavage site evidence for in vitro and in vivo autocatalytic processing of the primary translation product of beet necrotic yellow vein virus rna by a papain-like proteinase beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease nterminal protease of pestiviruses: identification of putative catalytic residues by site-directed mutagenesis identification of the putative staphylococcal agrb catalytic residues involving the proteolytic cleavage of agrd to generate autoinducing peptide a role for type i signal peptidase in staphylococcus aureus quorum sensing identification of a staphylococcal agrb segment(s) responsible for group-specific processing of agrd by gene swapping rawlings the wellcome trust sanger institute email: ab @sanger.ac.uk handbook of proteolytic enzymes, rd edn © key: cord- -jib fo authors: guidotti, m.; gateau, a.; malvy, j.; bonnet-brilhault, f. title: does autism protect against covid quarantine effects? date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: jib fo introduction: covid- outbreak has imposed an eight-week confinement in france. during this period, children and their families were exposed to a full-time home life. the aim of this study was to assess the emotional experience and tolerance of children with autism spectrum disorder (asd) in this particular context. method: a clinical survey was proposed to parents and rated by professionals once a week during the quarantine period in france. autistic children followed by the child and adolescent psychiatry department of tours university hospital were assessed from the th of march to the th of may. the following clinical points were investigated: child anxiety, family anxiety, behavior problems, impact on sleep, impact on appetite, impact on school work, family tension, confinement intolerance, difficulties to follow a schedule, isolation behavior. results: despite minor changes in family anxiety and school work, no difference was highlighted between clinical scores collected at the beginning and at the end of this period. asd children with or without intellectual disability had non-significant clinical changes during quarantine. this evolution was also independent of the accommodation type (house or apartment) and the parental status (relationship, separated or isolated). conclusion: the sameness dimension in autism and parents adaptation may be involved in this clinical stability during covid confinement. moreover, specialized tools and support provided by professionals could have participated to these outcomes and must be regularly promoted in order to help families in this still difficult period. covid- outbreak began in china at the end of and quickly spread to europe. in france, the confinement of the population began on the th of march and schools closed the day before. this new situation, which lasted officially until the th of may, exposed children and parents to a full-time home life. we know that this lifestyle can be difficult for families, especially for children suffering from mental diseases [ ] . the psychological impact of quarantine is heterogeneous and can be long lasting, including anxiety, depression symptoms and feelings of anger and fear. to limit these consequences, it is recommended to reduce boredom and improve communication [ ] . in order to help parents at home, some advice was published to manage children suffering from autism spectrum disorder (asd) [ ] . furthermore, health professionals regularly contacted families to get news and to provide some personal advice. considering these clinical resources, we investigated how well the covid- quarantine was tolerated by autistic children who benefit from regular rehab programs in the child and adolescent psychiatry department of tours university hospital. a telephone survey was proposed to families, once a week, during french lockdown. the following clinical points were investigated by health professionals (doctors, nurses and psychologists): child anxiety, family anxiety, behavior problems, impact on sleep, impact on appetite, impact on school work, family tension, confinement intolerance, difficulties to follow a schedule, isolation behavior. each of these ten clinical items was rated on a -point scale in reference to the basal clinical state of the child: /not at all, /a little, /moderately, /a lot or /enormously. the mean of the completed clinical items, labelled emotional score, was computed. two time points were considered: the first weeks (t ) and the last weeks is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint of the recruitment period (t ). furthermore, the accommodation type (apartment or house) and the parental status were also collected. additionally, parents were regularly contacted by professionals and many supporting materials were sent to families (sensorimotor activities, timetables, simplified covid explanations) during this period. sphinxonline v . was used for data collection. student t-test and linear mixed-effect models with repeated measures (patients as random effect; time and id, accommodation type or parental status as fixed effects) were carried out to study the effect of the quarantine between t and t time points. approval of tours hospital ethical committee has been granted to conduct this project. results children with asd, aged from to years old, were recruited from the th of march to the th of may in the child and adolescent psychiatry department of tours university hospital (table ) . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint eight clinical items (child anxiety, behavior problems, impact on sleep, impact on appetite, family tension, confinement intolerance, difficulties to follow a schedule, isolation behavior) had an average score of less than and two items (family anxiety and impact on school work) were greater than at both t and t time points (fig. ) . the emotional score (n= ) was . (sd: . ) at t and . (sd: . ) at t , no statistical difference was observed (t( )= . , p= . )). moreover there was no di effect (di effect: p= . , interaction di * time: p= . ), no accommodation type effect (accommodation type effect: p= . , interaction accommodation type * time: p= . ), and no parental status effect (parental status effect: p= . , interaction parental status * time: p= . ). no difference was highlighted between clinical scores collected at the beginning and at the end of french covid confinement in our sample. despite minor changes in family anxiety and impact on school work, the emotional score was unchanged. this evolution was not found by colizzi et al. ( ) , who highlight more frequent and intense behavior problems during confinement [ ] . however, unlike colizzi's study, our investigation was a prospective survey and included children followed by professionals. the latter regularly contacted families and offered adapted and personalized tools during quarantine. indeed, we argue that the materials sent and regular support provided by professionals prevented an increase of anxiety and behavioral disturbances. our outcomes may also be linked to autism's sameness dimension [ ] , associated to the tendency to seek immutability and to be afraid of changes [ ] . furthermore, the decrease of social interactions and sensory exterior inputs during the confinement could have helped to maintain environmental stability. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint another result of our survey was the minor changes in family anxiety and school work. during quarantine, parents had to ensure their children's schooling in addition to their work. this double task was complicated, especially since the education of autistic children presents several particularities and needs specific materials [ ] . moreover, lei et al. ( ) described an increased prevalence of anxiety and depression in adults affected by quarantine [ ] , which could have participated to our parents' distress. furthermore, we found that the accommodation type and parental status did not influence the emotional experience and tolerance of children to quarantine. these independent factors can highlight the significance of routine and environmental stability. on the other hand, autistic children were allowed to go out longer (> hour per day) than other children from the nd of april (from the mid-time of quarantine); this political decision could have impacted the effect of these variables. finally, our survey has some limits. firstly, clinical points were evaluated by parents and rated by professionals who know the children: this method does not ensure the uniformity of measurements. secondly, our study was based on children followed by a single center with specific procedures which limits generalization to other situations. this prospective study underlines the unchanged emotional experience and behavior in asd children during quarantine in france. asd particularities and parents' adjustments to the situation probably contributed to this clinical stability; moreover, professionals' support and specialized tools likely participated to these results as well and must be promoted in order to help families in this still difficult period. finally, going back to school and the resumption of is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october , . . https://doi.org/ . / . . . doi: medrxiv preprint activities with covid restrictions will be fragile moments in asd children's lives, and would deserve a specific assessment. how is covid- affecting the mental health of children with special educational needs and disabilities and their families? the psychological impact of quarantine and how to reduce it: rapid review of the evidence. the lancet parenting in a time of covid- psychosocial and behavioral impact of covid- in autism spectrum disorder: an online parent survey investigating the structure of the restricted, repetitive behaviours and interests domain of autism diagnostic and statistical manual of mental disorders: dsm- tm transition to school for children with autism spectrum disorder: a systematic review comparison of prevalence and associated factors of anxiety and depression among people affected by versus people unaffected by quarantine during the covid- epidemic in southwestern china the authors declare that they have no conflict of interest. key: cord- -q trgj authors: robert, rené; kentish-barnes, nancy; boyer, alexandre; laurent, alexandra; azoulay, elie; reignier, jean title: ethical dilemmas due to the covid- pandemic date: - - journal: ann intensive care doi: . /s - - - sha: doc_id: cord_uid: q trgj the devastating pandemic that has stricken the worldwide population induced an unprecedented influx of patients in icus, raising ethical concerns not only surrounding triage and withdrawal of life support decisions, but also regarding family visits and quality of end-of-life support. these ingredients are liable to shake up our ethical principles, sharpen our ethical dilemmas, and lead to situations of major caregiver sufferings. proposals have been made to rationalize triage policies in conjunction with ethical justifications. however, whatever the angle of approach, imbalance between utilitarian and individual ethics leads to unsolvable discomforts that caregivers will need to overcome. with this in mind, we aimed to point out some critical ethical choices with which icu caregivers have been confronted during the covid- pandemic and to underline their limits. the formalized strategies integrating the relevant tools of ethical reflection were disseminated without deviating from usual practices, leaving to intensivists the ultimate choice of decision. in their daily practice, intensivists are used to facing to ethical concerns related to admission or non-admission to icu, to withholding or withdrawing life support and to communication with families. the devastating pandemic that has stricken the worldwide population induced an unprecedented influx of severe ards patients dramatically exceeding icu bed capacities in several areas of many countries. as a result, four new options never applied to date were considered with the common aim of saving a maximum number of lives: to prioritize icu beds for patients with the best prognosis; to increase at all costs the number of icu beds, thereby creating stepdown icus; to organize transfer to distant icus with more beds available, or to accelerate withdrawal of life support in icus. additionally, to protect the patients' relatives, visits for families were prohibited or strongly limited and adequate communication between caregivers and families was disrupted, counteracting more than years of research aimed at improving interaction with families and quality of care during eol [ ] . moreover, since most health care facilities were being used for covid- patients, the situation also raised concerns inside the icu for patients without covid- requiring icu admission. in such a crisis, there are ingredients liable to shake up our ethical principles, sharpen our ethical dilemmas, and lead to situations of suffering for caregivers [ ] . faced with these profound changes in patient management, intensivists were caught off guard, forced by the density of work, the lack of immediately available beds and the possibilities of transferring patients to make painfully experienced choices that were contrary to their basic ethical principles and source of immediate burden [ ] [ ] [ ] . the aim of this paper is to focus on and to discuss the main ethical concerns raised during the pandemic, especially with regard to icus. since there are differences between health organizations in different countries around the world, ethical perception may vary according to legal or societal specificities. however, even though our thinking was based on french management of the crisis, similar approaches were assessed in other countries, especially in europe and ethical questioning is commonly shared by intensivists throughout the world. the massive influx of patients raised questions on the eventual modification of our admission criteria to the detriment of the most vulnerable populations. the decision to refuse admission of a severely ill patient to an icu is a regular part of the intensivist's work. guidelines have been drawn up to guarantee fairness, avoid unreasonable obstinacy and ensure respect for the patient's wishes and transparency with families [ ] . theoretically, even during an epidemic icu patient admission decision-making should be identical to that of a routinely applied decision-making method. however, the number of requests for admission made at a time of extreme scarcity of icu beds dramatically increased. it has been shown that in case of shortage of icu beds, the criteria for patient selection are modified, patients being more frequently considered as necessitating mainly palliative comfort care [ , ] . it is also necessary to underline the increased risk of mortality for patients who cannot be admitted to icu due to lack of beds, whatever the secondary course adopted: delayed admission, transfer to another distant unit or admission to a less specialized unit [ ] . faced with a massive influx of patients and extreme scarcity of icu beds, the theoretical risk of "sacrificing the most vulnerable patients" shakes our ethical convictions. herein, a triage plan with ethical justifications (table ) has been proposed to maximize benefit for the greatest number of people [ , , , ] . were the plan to be applied, utilitarian ethics would take precedence over individual ethics and employ the means least restrictive to individual liberty in view of accomplishing the public health goal. in other words, an unprecedentedly dramatic experience has taken place in which, due to compressed temporality, exacerbated emotional factors and massive influx of patients, a choice in the sorting cursor is made to the detriment of a reasoned strategy. such a situation is likely to contradict our caregiving-based ethical values [ ] . indeed, in addition to the elements linked to the lack of available beds, several factors in the decisionmaking process were sources of concern: reduction of the minimum time necessary to make such occasionally "life-or-death" decisions, decrease due to containment measures in the essential time to be spent with relatives and pressure from the continuous flow of arriving icu patients. in parallel with war medicine or disaster situations, prioritization strategies have been proposed [ , ] . although such prioritization is not supposed to be opposed to the ethical issues of icu access, in a specific epidemic situation this approach is nevertheless in conflict with our principles insofar as it allows utilitarian ethics to take precedence over ethics based on personhood. in this strategy, doing the greatest good for the greatest number may be inadequate insofar as it ignores other ethically relevant considerations. among the ethical principles ( table ) , prediction of number of years to live is posited as the priority selection criterion, which means that the youngest individuals should receive priority, thereby applying the life-cycle principle in allocation decisions [ ] . however, this appears to be only the least bad of existing or proposed justifications. decision trees have been proposed and simplified specific criteria have been requested, so to shorten the previously implemented regulatory period; this is in contradiction with a recommended practice, which privileges clinical contact with the patient. a simple score integrating the sofa score and the estimate of a probability of death at or years has been used, leading to the creation of a three-grade priority standard [ , ] . although numerous studies have demonstrated the relevance of such scores on an overall population scale, their lack of sensitivity or specificity at the individual level has been repeatedly underlined [ ] [ ] [ ] . indeed, the crude auroc for sofa score predicting in-hospital mortality is only . , leaving one out every four patients with an inappropriate decision [ ] . similarly, the ability to predict a given patient's life expectancy or risk of mortality at or years is generally poor. when applied, such first come, first served lottery strategies must assume "mistake of prophecy" and the eventual sacrifice of wrongly predicted patients. similarly, age becomes a potentially easy operational cursor, which we do not know how to place rationally [ ] . however, whatever the angle of attack, we can only make our choices using ethically flawed approaches. thus, shared recommendations including an admission decision-making checklist incorporating frailty score, comorbidities and, quality of life evaluation (table ) , have been developed and published on covid-crisis websites [ , ] helping intensivists to make such decisions. to conclude, rather than promoting unrefined and imprecise outcome prediction, a pragmatic multimodal approach taking into account frailty score and, comorbidity indices while leaving room for physician judgment should be considered as the best possible [ ] . as another application of the societal concept, it has been proposed to prioritize for icu care the caregivers who have become critically ill, not due to their intrinsic quality or for so as to "reward" them, but rather for the possibility, once they are cured, of being returning to the operational caregiving circuit [ ] . this raises at least two issues: first, the illusion of a rapid return to the caregiver circuit after resuscitation care for a severe form of the disease [ ] , and second, the choice of target actors for such prioritization. this appears to be an insoluble brain teaser: why not prioritize other societal actors who may favor the fight against pandemic such as researchers or other professionals helping to maintain the balance of our society in times of acute crisis? and with respect to the ethical principle of distributive justice, how is one to say that one life is worth more than another? moreover, utilitarian theories of emergency icu bed allocation have been criticized in the theoretical literature, especially on the ground of inequity in application of criteria that may disadvantage existing vulnerable populations [ ] . one solution to overcome the shortage of icu beds during a pandemic is to quickly set up new icus. this requires available rooms in the hospital or the rapid construction of new units, as has been done in china. this option effectively increased the number of icu beds by almost % in several countries and facilitated on-the-spot admission of large number of patients requiring mechanical ventilation. it was rendered possible by the dedication of volunteer health care workers (hcws) having agreed to work in a new and singularly stressful environment. however, this option has been associated with a significant risk of reduced quality of care for several reasons associated with the difficulties in meeting nationwide standards for critical care facilities in this type of emergency context. first, rooms converted from intermediate care units or post-operative recovery rooms are not adequately designed for the all the equipment and organization required in critical care. second, volunteer hcws recruited to work in icus may not adequately be trained for specific and sophisticated icu work despite the hastily improvised teaching sessions or "crash courses" organized to help them learn. along with the risk of decreased skill level, insufficient training of these hcws increases the burden of work [ ] . third, in the context of a pandemic, highly sophisticated devices, especially ventilators, are frequently lacking. this leads to use of inappropriate devices for the complex care of severe ards patients. to sum up, while the possibility of quickly setting up "neo-icus" permits admission of a large number of very severe critically ill patients, it also entail a possible risk of downgraded quality level of care and subsequent impaired prognosis, as shown in other situations [ , ] . additionally, this type of organization may imply distributive inequality, with access to icu facilities of heterogeneous efficiency and with a selection criterion recording in the patient's medical file that would be close to first come-first served, which could become first come-best served. epidemic intensity and icu bed availability were reported to vary strongly across countries and also within regions in a single country. to mitigate these "geographic" inequalities, patient transfers from regions with dramatic shortages of icu beds to areas less affected by the outbreak and with a large amount of available icu beds along with including optimal material and icu staff, have been implemented. these transfers require aircrafts, helicopters or trains that have been sophistically adapted to the care of critically ill patients and necessitate the involvement of a large number of dedicated physicians and nurses to ensure adequate organization and optimal patient safety. notwithstanding its complexity, in order to be efficient this transfer strategy should be organized within a short period of time and should allow the transfer of a significant number of patients. it is associated with increased costs that should not be charged to the patient or his or her relatives. the first ethical issue surrounding such transfers is related to the benefit/risk balance. for the patient, the benefit of being in the hands of highly qualified teams is counterbalanced by the risk of clinical worsening during transfer. during patient selection, close attention should be paid to severity status: not too severe (transfer would be too risky), and not too well (to avoid unnecessary transfer). while informed patient consent should theoretically be part of the decision, most of the transferred patients were unconscious and unable to approve such a transfer, thereby ruling out the autonomy principle. informed consent was consequently obtained from their next of kin (patients whose next of kin refused were not transferred). a second ethical issue concerns the icu departments accepting patients from a distant region and possibly aggravating the risk of a suddenly increased epidemic wave in their own area. indeed, covid pandemic experience has shown that we did not have efficient predictive tools to precisely anticipate the kinetics of icu bed requirements. finally, such transfers may be associated with increased suffering and psychological trauma for the relatives. indeed, long distance and limitation of travels for epidemic control will strongly impede if not altogether rule out the presence of relatives at the patient's bedside and prevent adequate communication between them. this could exacerbate pain for the families, especially if specific communications are not developed (see below). it has been proposed to relieve the icu teams in charge of patient care of the responsibility of admission or nonadmission decisions and to entrust this work to a dedicated triage team headed by a triage officer [ , ] . the advantage of this approach is that it relieves the healthcare team of the emotional impact of a potentially painful ethical dilemma [ ] . however, the composition of these triage teams must be specified. mentions of volunteers, leaders recognized by their peers and by the medical community have been put forward [ ] . it should no doubt be specified that the triage leaders will be intensivists recognized for their ethical sensitivity, and an overly "military" strategy should be scrupulously avoided [ ] . if not, the potentially protective role of independent triage teams can be a source of additional injury for caregivers, disappointed with their patient's unfavorable outcome and even blamed for an unshared therapeutic cessation decision or dehumanization of care [ ] . it has been suggested that patient severity assessments be intensified during their progress in icu stay, so that the withdrawal of one patient's mechanical ventilation can benefit another patient [ ] . in this way, withdrawal of artificial ventilation might be decided when the improvement is not fast enough, while hopes of survival may persist. similarly to the triage team, it has been proposed to use triage committee to buffer clinician from potential harm [ , ] . again, the risk of ethical drift must be emphasized. despite an influx of patients and lack of beds, it does not seem ethically acceptable to lose a chance for patients for whom treatment does not seem to be unreasonable obstinacy. moreover, the appreciable time taken to make these decisions is an element that risks being called into question during an epidemic emergency. finally, under the pretext of risk of contamination and need for confinement, exchanges with relatives to share final decisions could be reduced if not eliminated, a factor entering once again into contradiction with basic ethical concepts. it must be admitted that in a crisis situation with an unprecedented influx of patients in icu, no single strategy fully corresponds to our ethical values. whatever the approach adopted, imbalance between societal and individual ethics leads to unsolvable discomforts that caregivers will have to overcome. in other words intensivists would have to consider their own tension between utilitarianism (making icu beds available rapidly, potentially sacrificing patients without rapid improvement for new admissions) and virtue (accept to prolong icu stay for an icu patient even if there is no bed available to admit another patient) ethics. fortunately, the formalized strategies of ethical reflection associated with decision-making for withdrawal of life support therapies have long since been part and parcel of routine practice, leaving the ultimate choice of decision up to the intensivist. the heterogeneity in eol-decision-making is probably huge across hospitals and icu. postponed decision-making or even paralysis at eol may have created excess in mortality due to shortage of icu beds. nevertheless, confidence should be given to icu teams to manage the eventual withdrawal of life support decision through a bedside decision-making process taking into account the exceptional difficulties linked to the epidemic situation. since discrepancies may exist between experts' ethical recommendations and public perception, general public opinion has been investigated based on the basis of deliberative democracy [ , ] . a -participant panel placed in a simulated context of a severe influenza pandemic favored ethical principles of saving the most lives (surviving current illness) and saving the most life-years (living longer) over a first come first serve scenario [ ] . however, a significant number of participants were opposed to the idea of ventilator reallocation [ ] . in this study, subgroup differences associated with age or ethnicity of the participants were pointed out [ ] . in another survey, the pragmatic constraints imposed by an assumption of extreme scarcity were not accepted by the canadian participants, who expressed difficulties in making priority-setting decisions because these were perceived as psychologically burdensome, no-win situations [ ] . transparent communication is also important during such a crisis so as to allow public opinion to be able to better understand place the decisions of icu teams. the covid- epidemic is a threat to family-centered care in icus. during the st weeks of the epidemic, visits were prohibited to ensure that relatives did not contaminate other family members, patients, or healthcare professionals. family members could no longer be at the patient's bedside and the icu team was unable to propose structured communication and support to family members. involvement in decision-making was compromised, and it was felt that this situation was harmful both for patients and family members. indeed, over the last decade, research has shown that post-icu syndrome (pics-f) [ ] in family members is a cause of major concern. the major risk factors for pics-f are poor communication with an icu team, being in a decision-making role, low educational level, and having a loved one who died or was close to death. indeed, many studies have shown that communication with caregivers is one of the most highly valued aspects of care and that impacts-on family members' experience during and after the patient's stay, including in the aftermath of the patient's death [ , ] . communication perceived as inconsistent, unsatisfactory or uncomforting is associated with higher risk of post-icu burden [ ] . risk of ptsd-related symptoms increases when relatives, both non-bereaved and bereaved, feel that the information given is incomplete [ ] . after death in the icu, bereaved family members are at high risk of presenting symptoms that negatively affect their quality of life, such as anxiety, depression, ptsd symptoms [ , ] and complicated grief [ ] . interestingly, family members who witness a relative of theirs suffering from dyspnea are at higher risk of developing ptsd-related symptoms and those who are not able to say goodbye to relative of theirs are at higher risk of developing complicated grief symptoms [ ] . in the context of the covid- pandemic, risk factors for developing post-icu burden are numerous, thereby increasing exposure to anxiety, depression, ptsd and complicated grief. as said in the new york times, "of all the ways the coronavirus pandemic has undermined the conventions of normal life, perhaps none is as cruel as the separation of seriously ill patients and their loved ones, now mandated at hospitals around the world" [ ] . faced with these various difficulties and risks, recommendations have been published regarding communication with family members in this specific context. first, patients and family members should receive clear explanations, both directly (over the phone or when present) and on institutional websites, concerning the imposed restrictive policies: it is important that they understand why they cannot visit their loved one [ ] . in other words, the restriction must have meaning. second, icu teams are encouraged to proactively schedule routine telephone calls with family members to maintain continuity of communication [ ] . the calls must follow a plan so that family members know when to expect contact. the phone calls will not only address the patient's health status, but also provide reassurance regarding comfort and dignity [ ] . conversations are important to help the icu team better understand the patient as a person (values, advance directives, etc.) and to help family members think about possible difficult decisions. in this context, goal-concordant care is particularly important and icu teams must strive to avoid intensive life-sustaining treatments that would be unwanted by patients [ ] . on a parallel track, strategies to reinforce communication between the patient and the family have been developed. icu teams should encourage patient and family to call, text, and videoconference with each other as often as wanted [ ] . they may also help the patient and family members record and send audios, videos, or written messages to one another. if the patient is unconscious, the icu team can print written messages or family photos and stick them in a diary that can then be given to the patient. staying in touch is vital, both for the patient and for the family members. moreover, many icu teams have made visitation policies more flexible. these units have adapted themselves to the influx of patients while respecting a predetermined protocol. the visitor must have a dedicated time appointment and wait in a room where he/she may not meet other visitors. instructions on hygiene are given by the nurses. psychological support for each visit and followup calls by the icu psychologist are also recommended. visiting a loved one in intensive care is very upsetting in the best of times, but when in addition one has been separated for days, perhaps weeks, there is also all the emotional pressure of a long-awaited reunion. in end-of-life (eol) situations, the icu team must avoid depriving family members of the opportunity to say goodbye to the patient [ ] . if visitation is usually forbidden in the icu, it should be made possible in an eol situation. if the family cannot or does not want to come to the icu, letting him/her speak to the patient one last time over the phone is important. family members need to prepare for bereavement, meaning they must understand what is happening: end-of-life family conferences should be organized, remotely if needed [ ] . honest conversations are important, as helping family members prepare for death is an important part of anticipatory grief [ ] . not being prepared is associated with increased risk of complicated grief. when possible, respecting the family's wishes is particularly important in a context where the grief process may be more complex as families are unable to see their loved one's body, to physically share their emotions with other relatives and, sometimes even to attend their loved one's burial. in the current pandemic, sources of psychological disorders for hcws are multiple. they are affected by distress similar to than the general population regarding the effects of lockdown and containment, the risk of personal or families' and friends' illnesses, the uncertainty about pandemic duration and, the lack of effective specific treatment. this dearth of knowledge has given rise to a great deal of contradictory information that has forced health care professionals to constantly readapt and to cope with the experience of powerlessness and personal ineffectiveness [ , ] , and they also experience "front line" specific factors [ ] . the factors include extended workloads, feelings of powerlessness when trying to contain the large number of patients, concerns about the suffering and potential poor outcomes of their patients, preoccupations about potential shortages of intensive care resources (including personal protective equipment), the fear of transmitting the disease to their loved ones, and apprehension about possible involvement in ethically difficult resource allocation decision-making. this situation has created a high level of uncertainty and insecurity that constitutes a risk to the mental health of caregivers [ , ] . to date a few studies have reported a quantification of symptoms amongst hcws. all of them have shown an increase in psychological disorders compared with different control groups providing no direct care to patients: [ , [ ] [ ] [ ] ] . fear was more frequent than anxiety and depression with incidences varying from , , % to , , %, respectively [ , ] . assessments by other scales confirmed two-thirds of mental health disorders, especially in young women [ ] . sleep disorders were also reported [ ] . in some countries, e.g., in italy or in france, healthcare workers are applauded by the population each evening at pm. societal reward and "glorification" [ ] of the caring function appears to be a protective factor in the short term [ ] and in his first address to the nation, the president of france, emmanuel macron, called healthcare workers "the heroes with a white coat". it may be dangerous for healthcare workers to fall into this trap. altruism has long since been recognized as a core value of this profession. moreover, a hero must keep silence about his feelings, a factor which is known to favor burnout [ ] . insecurity and uncertainty are reflected not only at an individual level, but also at a collective level. the covid- epidemic requires reinforcement of the icu teams with new staff members or even reorganization of the unit, weakening the reference points and trust within the team. this context creates a feeling of vulnerability and loss of control for professionals [ , ] . a lack of interaction between caregivers and families induces a feeling of exclusion and even a significant emotional burden when patients die, highlighted in certain cases by a feeling of guilt [ ] . psychological support has been set up for caregivers, as many hospitals have initiated telephone hotlines, psychologists within units, relaxation sessions, meditation, discussion groups, and optimization techniques. these responses should ideally vary according to the phase of the pandemic [ ] . at the early phase, the best way to prevent psychological disorders is to acknowledge staffers' work by providing adequate human resources and material supplies [ ] . both frequency and transparency in hospital communication likewise play a key role [ , ] . concrete measures to set up rest areas, to facilitate the logistics of meals, daily life, and the possibility of having leisure and relaxation time are optimally appropriate to the needs of the caregivers during the crisis. at this stage, this type of collective support could be more effective than individual support. however, individual assessment of mental health may later become relevant. in a study in wuhan, the most valued psychological resources consisted in social media ( %) and psychological guidance books ( %) [ ] . requests for therapist-driven video calls or consultations were less frequent ( %) and rose the question of their availability, given the large number of affected hcws [ ] . similarly, a form of reluctancy, or even an absence of solicitation of the listening units in times of health crisis has been reported [ , ] . to overcome the covid- pandemic, in many places throughout the world, new resources were developed in a short period of time, dramatically increasing the number of icu beds allowing admission of a huge number of critically ill patients. the massive patient influx highlighted numerous ethical concerns that icu caregivers are likely to face. some models have proposed ethical justifications to difficult decision-making, usually based on deontological (or societal) rather than individual ethics. we wished to draw attention to the risk of taking refuge behind ethical alibis notwithstanding the fact that the specific pandemic context there is no single satisfactory solution. in such a situation each option is associated with its own strengths and weaknesses, and intensivists should make their choices in full awareness of intractable ethical dilemmas. in many circumstances, caregivers have no choice but to adopt less than perfect solutions even though the price to be paid consists in undermining patients' , relatives' and caregivers' psychological wellbeing. lessons should be learnt from this experience and ethical reflections should be developed in order to anticipate a potential new pandemic in the close or more distant future. abbreviations eol: end-of-life; hcws: healthcare workers; icu: intensive care unit. guidelines for family-centered care in the neonatal, pediatric, and adult icu a coronavirus cautionary tale from italy: don't do what we did a framework for rationing ventilators and critical care beds during the covid- pandemic triage of scarce critical care resources in covid- an implementation guide for regional allocation factors associated with mental health outcomes among health care workers exposed to coronavirus disease recommendations for end-of-life care in the intensive care unit: a consensus statement by the american college [corrected] of critical care medicine intensive care unit bed availability and outcomes for hospitalized patients with sudden clinical deterioration influence of icu-bed availability on icu admission decisions refusal of intensive care unit admission due to a full unit: impact on mortality surge capacity logistics: care of the critically ill and injured during pandemics and disasters: chest consensus statement fair allocation of scarce medical resources in the time of covid- allocating medical resources in the time of covid- scarce resource allocation during disasters priorisation des traitements de réanimation pour les patients en état critique en situation d'épidémie de covid- avec capacités limitées. société française d' anesthésie réanimation, service de santé des armées too many patients…a framework to guide statewide allocation of scarce mechanical ventilation during disasters understanding articles describing clinical prediction tools. evidence based medicine in critical care group predicting death and readmission after intensive care discharge the proliferation of reports on clinical scoring systems: issues about uptake and clinical utility prognostic accuracy of the sofa score, sirs criteria, and qsofa score for in-hospital mortality among adults with suspected infection admitted to the intensive care unit effect of systematic intensive care unit triage on long-term mortality among critically ill elderly patients in france: a randomized clinical trial décision d'admission des patients en unités de réanimation et unités de soins critiques dans un contexte d'épidémie à covid- clinical ethics recommendations for the allocations of intensive care treatments, in exceptional, resource-limited circumstances long-term outcome after the acute respiratory distress syndrome: different from general critical illness? priority setting of icu resources in an influenza pandemic: a qualitative study of the canadian public's perspectives prepared to respond? exploring personal disaster preparedness and nursing staff response to disasters physician staffing patterns and clinical outcomes in critically ill patients: a systematic review esicm working group on quality improvement, valentin a, ferdinande p. recommendations on basic requirements for intensive care units: structural and organizational aspects the toughest triage-allocating ventilators in a pandemic ventilator triage policies during the covid- pandemic at u.s. hospitals associated with members of the association of bioethics program directors emotional impact of end-of-life decisions on professional relationships in the icu: an obstacle to collegiality? post-intensive care syndrome: an overview in their own words: patients and families define high-quality palliative care in the intensive care unit improving comfort and communication in the icu: a practical new tool for palliative care performance measurement and feedback. qual saf health care family response to critical illness: postintensive care syndrome-family risk of post-traumatic stress symptoms in family members of intensive care unit patients predictors of symptoms of posttraumatic stress and depression in family members after patient death in the icu complicated grief after death of a relative in the intensive care unit family-centered care during the covid- era a -point strategy for improved connection with relatives of critically ill patients with covid- covid-ready communication skills: a playbook of vitaltalk tips the importance of addressing advance care planning and decisions about do-not-resuscitate orders during novel coronavirus (covid- ) not dying alonemodern compassionate care in the covid- pandemic grief during the covid- pandemic: considerations for palliative care providers les professionnels de santé face à la pandémie de la maladie à coronavirus (covid- ): quels risques pour leur santé mentale? l'encéphale a study on the psychological needs of nurses caring for patients with coronavirus disease from the perspective of the existence, relatedness, and growth theory clinician wellness during the covid- pandemic: extraordinary times and unusual challenges for the allergist/immunologist mental health care for medical staff in china during the covid- outbreak psychological impact of the covid- pandemic on health care workers in singapore psychological status of medical workforce during the covid- pandemic: a cross-sectional study online mental health services in china during the covid- outbreak impact on mental health and perceptions of psychological care among medical and nursing staff in wuhan during the novel coronavirus disease outbreak: a crosssectional study social capital and sleep quality in individuals who self-isolated for days during the coronavirus disease (covid- ) outbreak in january in china on behalf of the entire editorial and publishing staff of jama and the jama network. health care heroes of the covid- pandemic an official critical care societies collaborative statement-burnout syndrome in critical care health-care professionals hospital preparedness and sars immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers timely mental health care for the novel coronavirus outbreak is urgently needed annals for hospitalists inpatient notes-preparing for battle: how hospitalists can manage the stress of covid- protecting healthcare workers during the coronavirus disease (covid- ) outbreak: lessons from taiwan's severe acute respiratory syndrome response publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank jeffrey arsham for reviewing and editing the original english-language manuscript. authors' contributions rr: conception and design of the work. rr, nkb, ab, al, jr, ea: drafted the manuscript and revised it. all authors read and approved the final manuscript. not applicable. not applicable. not applicable. not applicable. the authors declare they have no conflict of interest. key: cord- -dsnbh t authors: dew, jeffrey title: ten years of marriage and cohabitation research in the journal of family and economic issues date: - - journal: j fam econ issues doi: . /s - - - sha: doc_id: cord_uid: dsnbh t i reviewed the marriage and cohabitation studies from the journal of family and economic issues articles published between – . nearly all of the studies used quantitative methods, and two-thirds of them used publicly available nationally-representative data. the studies fell into roughly five, unevenly sized groups: family structure, relationship quality, division of labor/employment, money management, and an “other” category. suggestions for future research include applying some of the important questions within the articles to underrepresented groups, further examining the process of how finances and relationship quality interrelate and doing more applied and translational research. financial issues and adult romantic relationships interface in many important ways. whether in marriage or cohabitation, living with a romantic partner may modify how one approaches financial issues (e.g., kenney ) . this association may work in the other direction, too; financial issues may influence relationship quality (see dew for a review). although many scholars study marriage and cohabitation, few of them study these couples within the financial contexts that surround them or the financial aspects that may influence the relationship processes themselves. the journal of family and economic issues, therefore, is a key outlet where scholars can publish studies that explore the nexus of financial issues and adult romantic relationships. this review focuses on the studies of marriage and cohabitation from - in the journal of family and economic issues. the editor/editorial staff of jfei assigned these studies to me. in the first section, i provide a synopsis of the articles that i reviewed. in the second section, i discuss the future research directions that might further build this topic. for the purposes of this review, i define marriage as two adults whose union has been legally recognized by a state entity. cohabitation, by way of contrast, generally denotes two unmarried persons living together in a sexual union. social norms and behaviors regarding family structure have shifted over the past years. for example, % of all us households with children present were single-parent households in (united states census bureau ). in , the comparable statistic was %. furthermore, an analysis of us data from - suggested that around % of people aged - cohabited during that time (nugent and daugherty ) . comparable statistics for do not exist. governments and researchers did not ask individuals if they were cohabiting due to the social stigma attached to it at the time in the united states. additionally, in % of us adults were married; in , the percentage has dropped to only % (parker and stepler ) . i could cite similar statistics regarding changes in the average age at first marriage, the total fertility rate, and so forth. this is one of several papers published together in journal of family and economic issueson the "special issue on virtual decade in review". at the same time family structures were changing, national economies all over the world fluctuated as well. in the us, manufacturing jobs decreased, and service sector jobs increased. unionized jobs, which often provided living wages regardless of individuals' education level, declined. men's wages stagnated after accounting for inflation. many married women with young children in the home moved into the paid labor force. thus, although no one aspect, theme, or methodology links the studies i reviewed, many of them examined issues related to family structure and/or economic changes that have occurred over the past sixty years in the us and other nations. many researchers applied "older" questions regarding financial and family issues to newer and growing family forms. other researchers updated the fields' knowledge regarding previous findings. still others examined existing family and finance process models and added additional nuance. the methods and analyses that scholars use as they examine the association between family and financial issues can strongly influence the findings. consequently, as i reviewed the studies, i noted the analyses the authors' used to examine their data. i also studied the data, samples, and demographic characteristics of the participants. i offer an overview of the methodology here. as a body, the researchers used quantitative analyses more than any other type. that is, of the articles, used quantitative analyses. three studies used qualitative analyses, one study used a mixed methods design, one study was a theoretical piece, and one study was an erratum. of the studies that used quantitative analyses, used large data sets. i categorized any study as using a large data set if the sample size was at least participants/couples, etc. i used this cutoff because when a study size reaches or exceeds participants, single-item measures have psychometric properties similar to multi-item scales (johnson ) . all other things equal, larger sample sizes yield more precise estimates. most of these data sets were publicly available (e.g., the national longitudinal survey of youth, the general social survey), though a few were large proprietary data sets (e.g., the survey of marital generosity). another important consideration was whether researchers studied marriage and cohabitation among underrepresented populations. understanding the research coverage of these underrepresented groups is important and is one of the recommendations i make for future research (see below). studies using large representative samples facilitate understanding adult romantic relationships. they may, however, miss crucial relationship or financial processes that vary within and across subgroups. thus, i did not count these large data sets as focusing on underrepresented groups. for the purposes of this review, i categorized a study as examining an underrepresented group if the sample was largely composed of individuals from race/ethnic minority groups, interracial couples, sexual minorities, low-income families, or from countries outside the united states. although the studies that used large us national samples obviously included individuals from some of those groups, the studies did not focus on underrepresented groups. some of the other quantitative studies used convenience sampling techniques, but still did not explicitly sample any underrepresented groups. using these criteria, nine of the studies i reviewed focused on underrepresented populations. jones ( ) and jang and danes ( ) studied couples who were racially/ethnically intermarried. oshio et al. ( ) used data from the general social surveys in korea, japan, and china. evertsson and nyman ( ) had a swedish sample. further, % of the couples in their study were in same-sex relationships. the maclean et al. ( ) research took place in australia, while cantillon et al. ( ) took place in ireland. finally, addo ( ) , högnäs and williams ( ) , and jamison ( ) focused on low-income couples. as i reviewed the articles, i categorized them based on what i felt was the overarching theme of each piece. i have published many studies on relationship formation and dissolutionas well as studies examining the role of financial issues within adult romantic relationships. i have also edited two special issues in peer-reviewed journals on money and relationships and written several review articles and public scholarship pieces regarding the subject. consequently, i used my own expertise to assign the studies to different domains. from my previous experience, i knew that studies often focus on financial issues and family structure issues (e.g., the financial consequences of divorce). i also knew that many previous studies have focused on relationship quality or process issues as they relate to couples' finances (e.g., the association between consumer debt and relationship happiness). finally, i knew that employment and the division of household labor (e.g., the paid labor force participation of mothers) have been important research foci in many fields for at least five decades. i established these three domains prior to categorizing the studies. after putting studies that belonged in the domains of family structure, relationship quality, and labor/employment, i examined the remaining studies. i created a fourth domain, financial management, from some of those studies. the last five studies did not fit in any of these categories or with each other. as family forms and macro-economic characteristics have shifted, scholars have examined how these changes have influenced individuals, families, and societies. for example, one of the first studies linking changing family structure and child poverty was released in the early 's (eggebeen and lichter ) . given the enormity of the social changes, it is not surprising that studies of family structure, whether as a predictor or as an outcome, was the domain that had the most articles in my review. these articles used family structure as either a main independent variable or as the dependent variable. sub-themes in this area included the association between family structure and financial issues, the association between family structure and other outcomes, and marital stability. i assigned articles to the category of family structure. many of these studies focused on how changing/new family structures related to financial issues. for example, one study researched whether, and under what conditions, men enjoyed a cohabitation premium (i.e., higher wages) relative to both single, non-cohabiting men, and married men (mamun ) . men in cohabitations that led to marriage realized a wage premium relative to single men; men in other types of cohabitations did not. married men enjoyed the largest wage premium. painter and vespa ( ) also examined financial issues regarding newer family forms by comparing rates of networth gain between those who married without cohabiting first, and those who married after cohabitation. interestingly, the rate of net-worth gain was higher for those who cohabited prior to marriage. painter and vespa studied the financial changes closely and found that those who married following a cohabitation had more debt when they married, and so they could increase their net-worth more quickly by paying debt down. further, those who had cohabited increased their home-equity more quickly. as an alternative to studying old questions using newer family forms, some of the studies that researched the association between family structure and finances added nuance to previous findings. for example, tamborini et al. ( ) estimated the changes in women's labor force participation before and after divorce. although this question has been studied for decades, these scholars studied additional moderators that might influence the association among divorce, changes in women's labor force participation, and changes in earnings. they found that education was positively associated with earnings gains. having a child after the divorce was negatively associated. in a similar study, frech et al. ( ) investigated the association between divorce and women's net worth. in the initial models, divorce reduced women's overall net-worth as previous studies have demonstrated. however, after using advanced modeling techniques to account for selection into divorce and selection into remarriage, the difference between stably married wives and divorced wives who had remarried disappeared. the divorce difference was still present for divorced women who had not remarried and remarried women who went through another divorce. sharma ( ) researched wealth change for one of the fastest growing group of divorced persons-individuals who are years or older. this is an important population to study because the divorce rate has steadily decreased for the past years except for those who are years or older (allred ) . for example, for women aged or older, the divorce rate per married women has increased from . in to . in (allred ). sharma found that both older men and women lost money following a divorce; the average loss was between $ , and $ , . interestingly, the difference between men's and women's loss was not statistically significant, unlike other studies of couples at younger ages (e.g., zagorsky ) . other studies expanded the field by combining novel approaches with timely new questions. for example, using qualitative methods and a diverse sample, jamison ( ) examined participants' transitions into and out of residential cohabitation (i.e., living in the same domicile in an unmarried sexual union), as well as into and out of relationships (i.e., considering oneself in a couple). the innovative insight of this piece is that residential cohabitation and one's romantic relationship may or may not overlap, especially among low-income cohabiters. indeed, sometimes individuals would stop a residential cohabitation for various reasons, while still considering themselves a romantic couple. other times, individuals who had been a couple in the past, but who had broken up, would reunite as a couple and as residential cohabiters. jamison's ( ) qualitative study captured the fluidity of these relationships. the use of novel approaches extended to policy issues. maclean et al. ( ) used a series of hypothetical vignettes to assess australian participants' views of whether, and under what conditions, step-fathers should financially support their step-children. they found that marriage and the employment status of the step-children's mother raised people's expectations that a man would financially support his step-children. lerman et al. ( ) investigated variation in state-level economic indicators as a function of the proportion of married adults and/or the proportion of married parents. their results suggested that states that had higher proportions of married adults and/or married parents also had higher per capita gdp levels, equivalent-adult adjusted median household incomes, and median personal incomes. further, these states had lower child poverty levels. other studies examined family structure issues, without focusing on financial outcomes or predictors. for example, jones ( ) assessed the stability of interracial marriages and found that most stability differences between interracial marriages and racially homogenous marriages attenuated after controlling for demographic characteristics. kendall ( ) found no difference across state level divorce rates based on their level of broadband internet penetrations. using the general social survey (us), horner ( ) found that women's happiness declined when their state moved to a low-barrier-to-divorce regime. men, by way of contrast, increased their happiness. hussey et al. ( ) studied the effects of moving from a two-parent household to a oneparent household on adolescent outcomes. they used propensity score matching to partly mitigate selection issues and found negative effects in the short term, medium term, and long term. these many studies demonstrate the utility of both examining "old" research questions in the context of growing family forms and of striving to add nuance to "old" findings. for example, finding a male cohabitation premium among only men who transitioned to marriage (mamun ) indicates that cohabiting unions are not monolithic relationships. this finding also further reinforces the link previous studies have found between marriage and upward economic mobility. finding that selection accounts for wealth differences between never-divorced and divorced-but-remarried women (frech et al. ) , generates a new avenue of research. specifically, this finding suggests that we should examine the characteristics that account for non-divorced women's higher net worth in a bivariate analyses, but that disappear upon controlling for selection. as family forms continue to change, scholars will likely conduct similar studies. the name of the journal suggests a natural fit for studies of the association between financial issues and adult romantic relationship quality. eight of the eleven articles i assigned to this domain focused on the interface between financial issues and relationship quality. three others focused on relationship quality and other issues (e.g., pornography). these studies highlight researchers' continued interest in the predictors of relationship quality. this interest in unsurprising, given how strongly relationship happiness and individual well-being are correlated (spuhler and dew ) . four studies examined the association between financial issues and relationship quality using either a unique population and/or a unique predictor. the first, schramm and william harris ( ) , used data from low-income couples to study the association between income, government assistance, and different aspects of marital quality. both receiving government assistance and having an income less than $ , was associated with lower marital satisfaction, commitment, and higher levels of divorce-proneness, negative marital interactions, and feeling trapped. an interaction did emerge, however. couples who had an income level between $ , -$ , and received government assistance reported higher levels of marital satisfaction and commitment than couples with the same income level, but who did not receive government assistance. using data from the married women in the national longitudinal study of youth, britt and huston ( ) studied the association between financial arguments and marital quality. not surprisingly, they found that the frequency of financial arguments was negatively associated with women's reported marital satisfaction. interestingly, by using the longitudinal aspect of the data, they also found that when women reported increased financial arguments over time, they reported lower marital satisfaction. finally, higher levels of financial conflict at the beginning of marriage was associated with greater likelihood of divorce. klein's ( ) study tested the association between financial issues and relationship quality and used a unique predictor-changes in home values. negative price shocks (i.e., declines in home values) were unrelated to the hazard of divorce. however, positive price shocks (i.e., increases in home values) did negatively predict the hazard of divorce. these positive price shocks needed to last at least four years to reduce the likelihood of divorce, though. lebaron et al. ( ) was likewise unique in that they examined how materialism was associated with marital satisfaction. materialism was negatively associated with marital satisfaction. one's feelings of importance about marriage partially mediated the association. that is, materialism was related to decreased feelings of marital importance; marital importance was positively related to marital satisfaction. many of the studies of the association between financial issues and relationship quality over the past three years have focused on the family stress model of economic pressure and marital distress (conger et al. ), or simply "family stress model." since its inception in , many scholars have used this model to research the association between negative financial events, feelings of economic pressure, and marital quality. the family stress model suggests that when individuals feel economic pressure, they respond affectively with greater levels of anxiety, depression, and hostility. this in turn increases marital distress (conger et al. ). ross et al. ( ) , tested the family stress model (conger et al. ) in the context of military couples. this research topic is important, given the unique pressures that military couples face (park ) . ross et al. ( ) study is the first of which i know to use the family stress model to examine military couples. their findings suggested that husbands' economic pressure was associated with receiving less warmth and greater hostility from their wives. wives' economic pressure was likewise related to reports of receiving less warmth from their husbands and increased hostility. further, wives' economic pressure was associated with their own reports of giving their husbands less warmth. dew and jackson ( ) ( ) found that relationship maintenance behaviors moderated the association between feelings of economic pressure and marital quality for wives. that is, husbands' performance of relationship maintenance behaviors, such as doing small favors for their spouses, protected wives' marital satisfaction from declining despite wives' feelings of economic pressure. modeled responses to a specific question that asked participants whether the recession had increased their marital commitment. factors that were positively associated with both wives and husbands stating that the recession had increased their commitment including religious marital sanctification, relationship maintenance behaviors, and financial support from families and friends. interestingly, the more economic pressure both wives and husbands felt, the more likely they were to say that the recession increased their marital commitment. wheeler et al. ( ) was the final study that used the family stress model. these researchers examined an additional mediator in the model using longitudinal data. relational aggression, such as social sabotage and love withdrawal, mediated the association between feelings of economic pressure and marital quality. wheeler et al. found these associations happening both within and across longitudinal waves. in other words, negative affect is not the only mechanism through which feelings of economic pressure incite marital distress. rather, worse relationship behaviors might arise because of economic pressure. these behaviors might then increase marital distress. these four studies contribute to the family stress model by adding specificity while, paradoxically, also broadening the potential relationship processes that may occur when couples experience negative financial events. ross et al. ( ) drew attention to a specific family context (i.e., military families). by doing so, they uncovered important sex differences as it relates to actor effects in the family stress model. broader studies of the family stress model have not often found these differences. the other four studies suggested additional mediators and moderators that researchers have previously not studied within the family stress model. for example, wheeler et al. ( ) studied a very specific relationship process, i.e., relationship aggression, as a potential mediator in the family stress model, and found that it was important. dew and jackson ( ) and found additional protective factors that helped couples weather the [ ] [ ] [ ] recession with their marital quality intact. the first study of relationship quality that did not deal with financial issues was doran and price ( ) . these researchers used the general social survey (us) to study the association between pornography use and marital quality. their data were drawn from the currently-married gss participants to test some of the hypotheses, and both the currently-married and ever-married participants for other hypotheses. their findings on the associations were too numerous to list specifically, but, in general, they found a negative association between pornography use and marital quality. for example, currently-married individuals were less happy in their marriages if they had watched an x-rated movie in the prior year. further, pornography use decreased the association between the frequency of sex and overall life happiness for men. the second study that investigated relationship quality without also including financial issues was a methodological piece. leppel ( a) illustrated a new technique "generalized ordered probit with selectivity" (gops) to estimate marital happiness. gops is useful when a dependent variable is discrete (i.e., not continuous), ordered, and incorporates information that may also be associated with selection into or out of a specific state. leppel made the argument that marital happiness ratings are an example of this type of dependent variable and that the gops is a superior estimation method relative to conventional ordered probit and generalized ordered probit without selectivity. the journal published an erratum (leppel b) , because some of the equations were misprinted in the original study. dew and tulane ( ) was the third study that did not examine the association between financial issues and relationship quality. instead, they studied how interactive media was associated with relationship quality in a national sample of married dyads. a negative linear association existed between husbands' social networking website use and wives' and husbands' marital quality. specifically, the more time husbands spent on social networking websites, the less maritally happy wives were, the more conflict both spouses reported, and the lower marital stability both spouses perceived. time spent playing video games was only problematic when differences in time use were considered. the greater the difference between the spouses in terms of video game usage, the lower they reported their marital quality, on average. synthesizing these studies was difficult. however, together they do suggest that relationship quality is a multifaceted construct that also has many predictors-from media use, to governmental aid, to personal attitudes. many of the predictors tested might seem somewhat pedestrian or prosaic. however, they are also the topics that daily concern families daily (daly ) . further, given that the studies that tested the association between financial issues and relationship quality averaged almost one per year may suggest that this area of relationship quality research continues to possess importance. like family structure, labor force participation and the division of household labor have changed over the past seventy years. married mothers participate in paid labor much more than in the past whereas men engage in household chores and childcare more. researchers have studied how these changes have influenced family life. four of the studies i reviewed related to labor and employment. one of the studies examined paid labor force participation. specifically, quinn and rubb ( ) researched the bidirectional association between being overeducated (i.e., having more education than one's employment merits), labor force participation, and moving house. both wives' and husbands' overeducation was associated with the likelihood of moving. interestingly, moving, in turn, was associated with an increased likelihood of wives leaving the paid labor force, but was associated with a decreased likelihood of a husband being overeducated. the other three studies researched the association between household division of labor and relationship happiness. oshio et al. ( ) studied this association in china, japan, and korea. they found no aspect in common across the three countries except that good health was positively associated with marital satisfaction. in china, dual-earning couples were happier. in korea, the more housework wives or husbands had to do, the less happy they were in their relationship. finally, income positively predicted marital satisfaction in japan and korea. britt and roy ( ) used the nlsy cohort to assess the relationship between the household division of labor and marital happiness. they found that perceived unfairness in the housework division was negatively associated with having high levels of marital satisfaction for wives, but not husbands. arguments about money and affection were negatively associated with marital quality for both wives and husbands. the final paper on division of labor and relationship quality was a theoretical and econometric piece. skåtun ( ) , outlined two types of marital bargaining. coasean bargaining behavior within marriage occurs if all marital/family goods (whether tangible or intangible) were shared between spouses and they could transfer utility to each other without cost. non-coasean bargaining behavior within marriage would occur if the marital/family goods were not all shared. skåtun asserted that the question of which of these two forms marital bargaining takes is unsettled in the literature, and that paid labor force participation behavior following divorce might help answer it. not many studies were in this category. it may be that scholars viewed other types of journals, such as economics journals and gender studies journals, as outlets more likely to publish their studies. it may also be because another review covered employment and wages. labor and employment studies will continue to be important, however, as macroeconomic conditions continue to change. the actual behavior that families use to manage their financial resources is an important topic because managing these resources is associated with families being able to meet their goals (national council on family relations ). further, financial products, instruments, and regulations have grown increasingly complex over time. this trend toward more financial complexity may influence how individuals and families manage their money. four studies examined family money management. the first study used qualitative methodology to discover how stable, happy couples engaged in money management (skogrand et al. ) . a phenomenological analysis revealed that couples typically had one spouse managing the day-to-day aspect of their finances, that they exercised financial trust and communication, that they had little-to-no debt, and that they stayed within their financial means. evertsson and nyman ( ) also used qualitative methods to examine family money management. they scrutinized how cohabiting and living-apart-together couples who claimed they manage their money independently actually manage their money. evertsson and nyman found that many couples had systems in place to handle joint expenses. however, sometimes the joint expenses made the distinctions between "my," "your," and "our" money less clear. furthermore, these couples would sometimes intentionally engage in joint consumption as a symbol of their union. in addition to the strong qualitative analysis, this study was unique in that it included many same-sex couples. cantillon et al. ( ) researched predictors of individual deprivation (e.g., doing without a substantial meal in the past two weeks, feeling unable to spend money on oneself) vis-à-vis family money management. they found that having children in the household was associated with being in the "female-only" deprivation group, while female-only employment/income was associated with being in the "male-only" deprivation group. many family characteristics were associated with being in the "both deprived" group, including income (negative), full income pooling (positive), and children in the home (positive). finally, addo ( ) examined an old family money management question using a newer population. family scholars have examined how married couples divided the money that came into their households (e.g., pahl ) . but addo studied the bidirectional association between the ways in which cohabiting couples integrated their finances and their plans for marriage. those cohabiting couples with definite plans to marry were much more likely to have joint bank accounts, credit card accounts, and mortgages. further, the more joint practices cohabiting couples engaged in, the more likely they would marry. three studies did not fit any categorization. hall and willoughby ( ) examined the importance that emerging adults felt for different roles (e.g., career, parenthood). the found that these attitudes were linked to both future expectations and behaviors. for example, those in the child/marriage centered group and marriage centered group had less sexual experience than young adults in other groups. jang and danes ( ) studied the quantity of social capital to which intermarried couples had access. social capital are resources, whether tangible or intangible, that individuals and couples can access based on their social networks. a methodological strength of this study was that the authors examined race, ethnicity, and national origin rather than just looking at one source of heterogeneity. jang and danes found that interracially married couples reported less access to social capital; this was not the case for interethnic or international couples. högnäs and williams ( ) assessed fatherhood identity among non-resident low-income men. a negative association existed between their partners' extended family involvement and the strength of men's fatherhood identity. that is, the more the women's extended family was involved in the raising and care of the child, the less the men reported feeling like fathers. finally, shamblen et al. ( ) evaluated a program meant to strengthen marriage and family life. they found the program had modest effects for the participants in some life domains, but no effects in other domains. they also estimated the return on investment (roi) by comparing the cost of implementing their curricula and counseling regime with the benefits. under most considerations, the roi for the program was positive. one of the ways researchers might grow the boundaries of this field is in continuing to apply important research questions we have already investigated to new relationship structures (i.e., beyond cohabitation). that is, by the editor's assignment, my review covered marriage and cohabitation research that appeared in the journal over the past ten years. all papers were strong representations of marriage and cohabitation research -at least for heterosexual individuals. gay and lesbian couples were not well represented in the literature i reviewed. only one study, evertsson and nyman ( ) , had a sample where at least % of the participants were in same sex relationships. of course, part of the reasons for this lack of research arises from the fact that same sex marriage was only legal in seven countries prior to , the beginning of my review period. as of april , countries have legalized same sex marriages. because many more countries legally recognize same sex cohabitations and marriages now than in the past, it would be important to study these relationships-particularly regarding financial issues. furthermore, it is the case that over the past years, other types of adult romantic relationships besides marriage and cohabitation have emerged and are slowly gaining cultural mainstream acceptance. for example, consensual non-monogamy (i.e., a romantic and/or sexual relationship with more than one partner in which all partners consent to the relationship), has become as a topic of mainstream conversation. inviting individuals and couples in these newer family forms to participate in research and studying them, generally, may be difficult. participants may be hard to find simply because there are not many in the population. for example, a recent national study revealed that only % of adults in the us reported ever having been in a consensually nonmonogamous relationship, and only % currently reside in such a relationship (hawkins and smith ) . furthermore, studying heterosexual marriage, researchers could take the number of spouses, gender configurations, and legal issues within the marriage for granted. this is simply no longer the case. having so much variance in family structure and smaller groups of newer family forms certainly complicates statistical models. in addition to studying underrepresented forms of adult romantic relationships, researchers who study marriage, cohabitation, and financial issues would serve the field and the public well by specifically studying groups that research has historically underrepresented. this includes studying different race and ethnic groups, and low-income families (beyond traditional "poverty outcomes" research). this also includes conducting more research with samples drawn from outside the united states. the suggestion to focus on underrepresented populations may be even more important given the financial difficulties caused by the covid- pandemic during . for example, dew and jackson ( ) found relationship attitudes and processes that helped protect couples' relationship quality during the - recession using a national sample. however, it is unknown whether these findings apply to underrepresented families during the current macro-financial problems because dew and jackson did not run any interactions by race or income. another way to grow this field is to more closely examine the process of how financial issues and relationship quality interrelate. in other words, while many studies have shown that financial issues and relationship outcomes relate, not as many have investigated how and why that is the case. our understanding of marriage, cohabitation, and other romantic relationship forms would expand if we understood the role of money within them. indeed, many of the studies i reviewed regarding relationship quality uncovered links between financial issues and relationship quality. for example, lebaron et al. ( ) tested whether attitudes about marriage mediated the negative association between materialism and marital quality. further, wheeler et al. ( ) tested some intriguing potential mediators (e.g., love withdrawal) of the association between economic pressure and marital quality within the family stress model. a number of new directions might help this area of study flourish. first, studies of the interface between financial issues and relationship quality would benefit by greater efforts in theory construction. the family stress model is an undeniably excellent model that has generated much research. however, studies in this area cannot grow without moving beyond the family stress model. the association between financial issues and relationship quality encompasses more than negative financial events and feelings of economic pressure. second, nearly all the studies in this area have the causal direction running from financial issues to relationship quality. but a few economic studies suggest that the opposite direction of causality is possible, even likely. that is, it may be that a strong marital or cohabiting relationship makes sound financial management behaviors more likely. individuals with a strong relationship are more likely to invest in it (becker ) -including by investing in their joint financial futures. studies have shown that couples spend down wealth or hold less of it as they approach divorce relative to couples who are stable (finke and pierce ; zagorsky ) . consequently, a relatively untapped area of research is to make great use of causal and longitudinal data to detangle issues of causal direction in the association between financial issues and relationship quality. the last aspect of process that i recommend for future study is to understand the attitudinal, relational, and behavioral aspects that protect romantic couples during financial difficulties. almost all couples will experience negative financial events and/or feelings of economic pressure. knowing what individual partners, spouses, and couples can do to maintain their relationships would benefit researchers, practitioners, and lay families. some of the studies i reviewed did exactly that (e.g., dew and jackson ) . however, much work remains to be done in this area. related to my last point, a final call for future marriage and cohabitation research is to generate more applied and translational research. only one of the studies i reviewed went beyond basic research (shamblen et al. ) . interestingly, many of the studies that i reviewed covered prosaic, that is every day or mundane, issues with which couples regularly struggle. i believe that is one of the strengths of the journal of family and economic issues. it might not be difficult to take some of the issues covered in this review -the division of household labor, money management, etc. -and begin working on applied and translational research. although the journal of family and economic issues is not a practice journal, applied and translational research would make the journal more widely relevant. funding jeffrey dew is a fellow of the wheatley institution. the wheatley institution did not directly contribute any funding toward this manuscript. conflict of interest the author declares that he has no conflicts of interest regarding this manuscript and its publication in the journal of financial and economic issues. ethical approval because this manuscript is a review of previously published studies, it does not meet the definition of human subjects research. therefore it needed neither irb approval nor informed consent. financial integration and relationship transitions of young adult cohabiters gray divorce rate in the us: geographic variation a treatise on the family the role of money arguments in marriage relationship quality among young couples from an economic and gender perspective family financial management and individual deprivation linking economic hardship to marital quality and stability family theory versus the theories families live by revisiting financial issues and marriage commitment and relationship maintenance behaviors as marital protective factors during economic pressure can stress build relationships? predictors of increased marital commitment resulting from the - recession the association between time spent using entertainment media and marital quality in a contemporary dyadic national sample pornography and marriage race, family structure, and changing poverty among american children perceptions and practices in independent management: blurring the boundaries between "mine precautionary savings behavior of maritally stressed couples marital biography and mothers' wealth relative work and family role centralities: beliefs and behaviors related to the transition to adulthood national survey reveals generational differences in consensual non-monagamy. institute for family studies blog maternal kinship involvement and father identity in fragile families continued pursuit of happily ever after: low barriers to divorce and happiness disruption in parental co-habitation and its effects on short-term, medium-term, and long-term outcomes of adolescents cohabitation transitions among low-income parents: a qualitative investigation of economic and relational motivations social capital accessibility of intermarrieds are single-item measures of marital quality valid? the case of marital happiness.paper presented at the annual conference of the national council on family relations stability of men's interracial first unions: a test of educational differentials and cohabitation history the relationship between internet access and divorce rate cohabiting couple, filing jointly? resource pooling and us poverty policies house price shocks and individual divorce risk in the united states money over marriage: marriage importance as a mediator between materialism and marital satisfaction the method of generalized ordered probit with selectivity: application to marital happiness erratum to: the method of generalized ordered probit with selectivity: application to marital happiness marriage and state-level economic outcomes perceptions of stepfathers' obligations to financially support stepchildren cohabitation premium in men's earnings: testing the joint human capital hypothesis family life education content areas: content and practice guidelines a demographic, attitudinal, and behavioral profile of cohabiting adults in the united states division of household labor and marital satisfaction in china his money, her money: recent research on financial organization in marriage the role of cohabitation in asset and debt accumulation during marriage military children and families: strengths and challenges during peace and war as us marriage rate hovers at %, education gap in marital status widens spouse overeducation and family migration: evidence from the us money matters in marriage: financial concerns, warmth, and hostility among military couples marital quality and income: an examination of the influence of government assistance the economic benefits of marriage and family strengthening programs divorce/separation in later-life: a fixed effects analysis of economic well-being by gender bargaining on your spouse: coasean and noncoasean behaviour within marriage financial management practices of couples with great marriages sound financial management and happiness: economic pressure and relationship happiness as mediators women's earnings before and after marital dissolution: evidence from longitudinal earnings records matched to survey data national single parent day economic hardship, financial distress, and marital quality: the role of relational aggression marriage and divorce's impact on wealth publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. jeffrey dew is an associate professor in the school of family life at brigham young university. he teaches family theory, family finance, and graduate statistics courses. dr. dew's research focuses on the "daily life" issue of the association between family resources (i.e., money and time) and relationship quality. examples of dr. dew's recent projects include examining factors that protect couples' marital quality during financial difficulties, and a qualitative strengths-based analysis of how african american couples talk about the role of finances in their marriage. dr. dew is a fellow of the wheatley institution. key: cord- - bx lsc authors: nan title: the covid- pandemic: a family affair date: - - journal: j fam nurs doi: . / sha: doc_id: cord_uid: bx lsc nan the assumption that disease and its prevention is a family affair is manifested in the full spectrum and scale of the current coronavirus pandemic. measures that have been taken in many countries to control the spread of the coronavirus are having a disruptive effect on relationships in general and family relationships specifically. families are reporting loss of community and freedom of movement in response to quarantine/lock down measures. other tangible losses include income, access to resources, and planned activities or celebrations. compelling and heartbreaking stories of the challenges and suffering that families are experiencing engulf us. individuals and families who are most vulnerable are particularly at risk. residents in long-term care facilities miss their partners and children who are no longer allowed to visit because of the covid- policies to contain the spread of the coronavirus. people with intellectual disabilities who live in institutions are upset because their father, mother, brothers, or sisters are no longer allowed to visit, and they often cannot understand why. distressing stories abound of patients who have to deal with the news of their covid- diagnosis all by themselves without a family member present and those patients who are admitted to an intensive care unit (icu) who have to say goodbye to their family in the emergency department not knowing whether they will see each other again. a nurse working at the front line of triage reported, "his family was not allowed to come to the hospital, because they may also be infected. he was alone and couldn't say goodbye." many of these patients, clients, and residents are members of families who miss their loved ones and who are worried. mothers, fathers, and other family members of children receiving psychiatric care report being unable visit their child for an extended period of time and are afraid their child will become ill from the coronavirus. families of very seriously ill and dying patients are not allowed to visit their loved one and may not be able to say a final goodbye. the lockdown/quarantine measures instituted in many countries have also invited vulnerability and risk within families. schools are being closed which leads to distress in many families not accustomed to being so closely confined for a long time period. moreover, as a result of the covid- crisis, much, if not all, of the support given to families who provide long-term care for an ill parent, partner, or child is lost. families with a child who requires specialized care and guidance now have to care for their child hr a day without the outside guidance provided by medical nursery, daycare, or special education services. families who care for a father, mother, or partner with dementia or other serious illness now have to manage without day care or daytime activity. school closures have created a family environment where children are rarely allowed to leave the house and are confronted by the vulnerabilities of a family member's addiction, aggression, and violence. children of divorced co-parents are suddenly being refused alternating parental care because one of the parents now works from home and cannot provide child care. all of these families and their interrelationships are often under great pressure as a result of the stresses created by coronavirus pandemic. health care professionals, including nurses and doctors, are also going through a very intensive and perhaps traumatic time. as a nurse working in the icu reported, "many people die without family present. the sorrow that comes with it hurts the nurses mentally." it is encouraging to see how innovative and creative many nurses are becoming during this pandemic as they find ways to involve families. despite being committed to the care of the ill person, they assure families that their family member is being cared for and will not die alone. they are sometimes able to connect family members to each other using new technology. mobile phone or video conference calls made by the nurse allow family members to "see" the patient in the icu or in the nursing home. our concerns also focus on the long-term implications for patients and their families; how will they cope once the coronavirus is under control? how will they be able to resume normal life again? individuals and families are often flexible and resilient (walsh, ) and many will likely be able to process the experiences of this pandemic and resume their lives. however, there will also be long-term mental and physical health consequences or even permanent damage. for example, how will patients and families recover after a long period of intensive care? research has documented that many patients experience many physical and psychological problems after such a long period of ventilation, even after discharge to their home environment (rawal et al., ) . we also know that family members of these patients also suffer greater levels of depression and anxiety (davidson et al., ) . we also hold our hearts out for families who have lost someone without being able to say goodbye and without being able to be present in the final dying phase of their loved one. how will we assist these families to cope with their loss and complex grief? our concerns also go out to the health care professionals, especially nurses and doctors, in the aftermath of this covid- crisis. they too will need support in recovering from their suffering and distress. the good news is that there is compelling evidence that our family nursing assessment and intervention skills can assist families to heal. this pandemic makes us more deeply aware of the important role of family in the lives of patients, clients, and residents. we anticipate that this increased awareness will help us advocate even more strongly for the importance of family nursing during and after this coronavirus crisis. rightfully, a great amount of money and resources are now being spent to fight the covid- virus. but lives saved must also be lives worth living afterwards. we believe that family nursing knowledge, developed over the last years, unequivocally offers the necessary skills to help families recover and heal from the expected and unexpected long-term consequences of this pandemic (international, family, nursing, & association, , . the aftermath of covid- calls for a substantial increase in the resources needed to (a) enable nurses to assess and intervene with families in need of support, (b) educate nurses to offer highly skilled family nursing care, and (c) conduct research which provides compelling evidence that family nursing assessment and intervention is effective in addressing illness suffering (wright, ; wright & bell, ) and optimizing family health. family nursing has never been more relevant or more urgently needed than now. this guest editorial has been written by members of the family health in europe-research in nursing (fame-rn) group: birte Østergaard, phd, associate professor, department of clinical research, university of southern denmark, denmark. email: boestergaard@health.sdu.dk orcid: https://orcid.org/ - - - family response to critical illness: postintensive care syndromefamily an interactive web-based dashboard to track covid- in real time. the lancet. advance online publication ifna-position-statement-on-generalist-competencies-forfamily-nursing-practice/ international family nursing association post-intensive care syndrome: an overview applying a family resilience framework in training, practice, and research: mastering the art of the possible suffering and spirituality: the path to illness healing beliefs and illness: a model for healing nurses and families: a guide to family assessment and intervention key: cord- -hf ndv f authors: cook, mackenzie; zonies, david; brasel, karen title: prioritizing communication in the provision of palliative care for the trauma patient date: - - journal: curr trauma rep doi: . /s - - -x sha: doc_id: cord_uid: hf ndv f purpose of review: communication skills in the icu are an essential part of the care of trauma patients. the goal of this review is to summarize key aspects of our understanding of communication with injured patients in the icu. recent findings: the need to communicate effectively and empathetically with patients and identify primary goals of care is an essential part of trauma care in the icu. the optimal design to support complex communication in the icu will be dependent on institutional experience and resources. the best/worst/most likely model provides a structural model for communication. summary: we have an imperative to improve the communication for all patients, not just those at the end of their life. a structured approach is important as is involving family at all stages of care. communication skills can and should be taught to trainees. in , the american college of surgeons called upon surgeons to provide high-quality palliative care to all patients with serious illness, not just those approaching the end of their life [ ••] . comprehensive care of the acutely injured and critically ill patient is the defining skill set of the acute care surgeon and an essential part of that skill set is the ability to communicate clearly, effectively, and efficiently with the patient and their family. communicating clearly as part of providing high-quality palliative care in the intensive care unit (icu) is about guiding difficult decisions, defining goals of care, explaining the impact of acute surgical problems on future quality of life, and facilitating transitions to end of life care [ , ] . it is critically important to understand that essential communication skills can be learned and taught [ , ] . the goal of this review is to summarize key aspects of our understanding of communication with injured patients in the icu. we will discuss communication through all phases of trauma care, review specific literature on guiding family meetings, and discuss strategies to teach these skills. the acuity of trauma patients admitted to icus in the usa is very high, with a diversity of pathologies [ ] . roughly one in five americans will die in hospital after receiving icu level care [ ] . when approaching the acutely injured and critically ill trauma patient, it is important to keep in mind the general observation that the seriously ill often value quality of life over extending quantity of their life [ , ] . while individual variation is wide, this point should guide initial approaches to communication with patients and their surrogates. there is a clear clinical need to improve our communication regarding goals and wishes; as even in patients with a stated desire to favor quality over quantity of life, preserving quality of life is not reliably honored [ ] . even in patients with established living wills or advanced directives, fulfillment of end of life wishes in the icu remains inconsistent [ ] . these data emphasize the imperative to improve the communication between patients, their families, and the care team through an earlier consensus on goals of care (goc) [ •] . attending to and this article is part of the topical collection on palliative care in trauma following a patient's goc, even if that includes comfort measures only, do not negatively impact mortality or length of stay [ , ] . this clinical need is matched by an urgent research need. as our ability to care for patients through their initial phases of injury improves, the need to focus on the key communication issues unique to trauma patients in the icu increases [ •, •, •] . this was recently emphasized in by the critical care committee of the american association for the surgery of trauma (aast). their statement on research priorities in adult surgical critical care highlighted addressing goc in the acute care setting as the top research priority [ • ]. there is only limited data specific to the trauma population available to review-with end of life/goc articles comprising just . % of the articles retrieved for the aast statement of research priorities [ •] . while frequently extrapolated, data generated on the optimal communication practices in elective surgical patients and non-surgical icu patients may have incomplete generalizability to trauma. much of the data available on the goc discussions pertained to older patients or those patients with a preexisting life-limiting illness. there is a wide span of injured patients and indeed the leading cause of death between and years old is injury [ ] . in addition to a wide span of ages, trauma patients may be socially marginalized, homeless, have pre-existing mental health and substance abuse disorders, and be estranged from their family and surrogates. the wide diversity of ages and associated medical and social challenges faced by trauma patients are unique. this raises the importance of an organized, consistent, and transparent approach to communication and palliative care in the trauma icu. communication with injured patients can be organized by phases of care and is an ethical imperative that must not be forgotten in the hustle of acute medical interventions [ •] . perhaps most easily organized by the physical location of the patient (prehospital setting, resuscitation bay, icu), communication challenges and priorities during these phases are distinct and build upon each other. a defining feature of trauma care is the unexpected nature of injury and the near instantaneous transition between normal life and high-intensity medical interventions. this temporal uniqueness of trauma care poses a challenge when communicating with families and surrogates. while everyone in medicine hopes that patients have had meaningful conversations with their surrogate decision makers prior to injury, the reality is that few have [ ] . we must help with the sudden news of a major injury while coaching them through a new and potentially uncomfortable role as a surrogate. the prehospital care of an acutely injured patient can be an extraordinarily challenging and time-pressured phase. as physiology is assessed and stabilized, in-depth de novo discussions are neither feasible nor appropriate. it is important, however, that any pre-established advanced directives are retrieved. this takes on critical importance when transporting patients from long-term care facilities, as trauma mortality in the frail elderly can be quite high. prehospital personnel can and do uphold advanced directives and established end of life wishes while managing family emotions near death [ , ] . the role of the hospital trauma service and state trauma system at this point is to support the medics as they seek to obtain and uphold established advanced directives. having access to an advanced directive on a patient's arrival, or shortly thereafter, opens a door to address goc in the resuscitation area and potentially avoid high-intensity interventions that a patient has previously decided are not consistent with their goals [ ] . it should come as no surprise that the presence of a pre-admission do not resuscitate (dnr) order is associated with a high risk of adverse in hospital events and death after injury, likely reflecting the high-risk nature of injury in patients who are already nearing the end of their life [ ] . it is far better to find out that a patient has an advanced directive limiting aggressive care before intubation, and prehospital personnel can help set the trauma team up for success. advanced care plans may be documented in a variety of different ways, including advanced directives, living wills, and durable power of attorney of health care documents. it is not feasible for prehospital personnel to read these documents in the acute setting, although it is helpful to bring them with the patient. what may be applicable in the acute prehospital, however, and can change acute management is a form similar to the physician orders of life-sustaining treatment (polst) form. designed to be portable and applicable across the spectrum of care, including prehospital care, polst forms are medical orders and valid in the states where programs have been established [ ] . they are designed to be easily and rapidly interpreted and honored, regardless of a patient's physical location [ ] . the state of oregon has a robust and easily searchable polst registry, and the presence of an active polst form at the time of contact is associated with a higher chance of dying not at a hospital (i.e., dying in a way concordant with wishes) [ ] [ ] [ ] [ ] . at our institution, trauma activation pages may include information on a pre-existing polst form, and it is not uncommon for a physical poslt form to be given to the attending surgeon by the responding prehospital providers, while the team is completing the primary survey. even in the absence of a physical polst form, a social worker in the emergency department immediately searches the statewide registry and, if a polst exists, it is available to the care team within min of patient's arrival. polst programs fill an important gap where other advanced directive documents fail, namely limiting unwanted resuscitative measures in the urgent/emergent setting if patients are unable to speak for themselves [ , ] . advanced trauma life support (atls) principles are an essential part of trauma care, and when a patient arrives at a trauma hospital, efforts should be focused on efficiently completing the primary and secondary survey. as much as possible, subject to available resources, communication with the patient, family, and surrogate decision makers should proceed in parallel with the trauma resuscitation. this is easier to support with the increasingly robust data on family presence during a trauma resuscitation. having families observe trauma evaluations can result in reduced family anxiety and stress in the short term [ ] . importantly, there were no long-term psychological effects from being at trauma resuscitations, and high-quality trauma care was able to be provided with families present; indeed, all family members present for a trauma resuscitation in one study reported they would choose to be present again [ , ] . having families at the bedside for trauma resuscitations can actually build trust in the health care team as well as fill information needs [ ] . these are concordant data with the findings on family presence during resuscitation from cardiac arrest and in pediatric trauma [ , ] . protocols to allow families to be present during trauma resuscitation do require an institutional commitment as well as support from the physician and nursing staff. a designated person, most frequently a social worker, should be present to provide context, guidance, and support. the trauma resuscitation is also a moment to review polst forms and assure that the care plan is concordant with pre-existing wishes. geriatric trauma patients with a polst specifying limited intervention that was available on admission spent fewer icu days with no change in in-hospital mortality, compared to age-matched controls without limitations on care [ •] . presumably this comes from limiting/focusing icu utilization and care based on a patient's pre-injury goals of care and reflects the sizeable mortality of patients admitted to the trauma system with pre-established limitations on medical interventions. in the absence of a polst, a well informed and empowered surrogate or otherwise well-established preinjury wishes, trauma bay communication with the family is often focused "just" on delivering bad news about the nature of the injury and further planning is left to the icu team. the paucity of data on the provision of high-quality palliative care in the icu is even more significant when the injured patient is considered [ , ] . three key points warrant close attention: clarifying goc, providing goal concordant care and transitioning goals to comfort measures only. given that the vast majority of severely injured trauma patients in the icu have limited abilities to function as their own decision makers, this discussion will primarily focus on the interactions with family and surrogate decision makers as this is the group most in need of high-quality communication [ , ] . the principles outlined below, however, would apply if a patient was competent to and able to make their own decisions. it is important to recognize that efforts to prioritize communication in the icu are difficult and multi-faceted with potentially disparate effects on patients and their families. as an example, among critically ill patients and their surrogates, a family-support intervention delivered by the icu team did not significantly affect the surrogates' burden of psychological symptoms, but their ratings of the quality of communication were higher and the length of stay in the icu was shorter with the intervention than with usual care [ •] . in a similar study, the use of palliative care-led informational and emotional support meetings compared with usual care did not reduce anxiety or depression symptoms and may have increased posttraumatic stress disorder symptoms [ ] . as we seek to advocate for the patient and aim for providing goal concordant care, it is essential to realize that the families and surrogates are under tremendous stress and a consistent, organized, and empathetic approach is likely the optimal approach to decision-making. one of the first questions that must be addressed as we think about communication in the icu setting is who will be primarily responsible for family facing communication, clarifying goc, guiding goal concordant care, and transitioning goals as warranted. this can be thought of both at the team level (trauma surgery, neurosurgery, orthopedics, etc.) and the individual level (attending, fellow, resident, advanced practice provider, etc.). the individual tasked as primary communication point for the family should be experienced enough to provide compassionate, meaningful, and nuanced updates. it is, furthermore, our belief that the primary trauma icu team should be in charge of overall communication with the family as they have the perspective to integrate multiple organ system problems and the recommendations of the whole care team. there are essentially three structural options: primary palliative care by the icu team, integrated subspecialty palliative care into the icu setting, and consult only subspecialty palliative care. there are strengths and weaknesses to each of these designs, and it is likely that the optimal design for any given icu will be dependent upon institutional level resources and culture. with that in mind, we attempt to outline potential benefits and challenges associated with each model. primary palliative care (ppc) is defined as palliative care provided by the primary treating service. this approach emphasizes the co-provision of palliative intensive care and may best integrate palliative communication principles into the icu [ ] . increasingly, this is being viewed as part of the routine job description of an acute care surgeon [ ] . primary palliative care has been shown to promote stronger clinician and patient relationships and reduce the fragmentation of care and can be integrated with other critical care interventions and therapy [ ] [ ] [ ] . the details of how ppc is integrated into routine icu care remain an area of active research, and defining therapeutic goals in a nuanced and sensitive way is essential [ , ] . the primary benefit to the ppc model is that, at least theoretically, all patients have their palliative needs, including communication, addressed on a daily basis. the primarily limitation to the ppc model is that at times of high icu acuity or with an intensive care team with less experience in complex communication, family communication may suffer. a particular challenge may be encountered when interacting with non-critical care surgical colleagues with different understandings of the patient's goals and the attendant conflict [ , ] . this highlights the need for additional subspecialty input which can be accomplished in one of two ways. the first model for providing subspecialty palliative care input in the icu is integrated subspecialty palliative care in the icu [ ] . this is most commonly framed as a subspecialty trained palliative care provider who routinely sees all patients within the icu, either as part of the rounding team or as a scheduled check in-ameliorating the lack of physician contact noted by family members of patients who died in hospital [ , ] . this emphasis on the routine integration of trained palliative care specialists helps to overcome a major barrier to involving palliative care to aid with communication, the initial call [ ] . the routine integration of trained palliative care subspecialists in the icu has been associated with improved quality of life, higher rates of formal advance directives, and greater utilization of hospice service as well as less frequent use of certain non-beneficial life-prolonging treatments for critically ill patients at the end of life [ ] . the challenges of this integrated subspecialty model are limited resources and the concurrent need for subspecialty palliative care across the inpatient and outpatient settings. it is likely that as the ppc skills of the rounding intensivist teams increase, the need for integrated subspecialty palliative care will decrease. the second model for integrating subspecialty palliative care is consultative care. this system works the same way as any other consultant service in the icu. the assumption in this model is that there is a baseline degree of comfort from the icu team with defining goc, providing goal concordant care and transitioning goals to comfort only. additionally, this model presumes a low barrier for the primary team to engage the palliative care consultant when appropriate, providing palliative and surgical care as an integrated whole [ ] . the primary failing of this structure comes if those presumptions are not true and the communication needs of the family and patient are not attended to. when this structure of care is studied, patients with a palliative consult are older, more seriously ill, and more likely to be at the end of life [ ] . palliative care consultations tend to be within the last h of life and are primarily symptom management at the end of life, suggesting an earlier opportunity to involve palliative care [ ] . the risk of late consultation can be mitigated through structured triggers for palliative consultation. demonstrated to increase palliative care involvement in critically ill non-surgical icu patients, triggers are established in only a minority of surgical icus, despite a sizeable identified need for palliative and communication support [ , , ] . as the poor to moderate performance of the "surprise" question in non-cancer diseases demonstrated, established screening systems designed in non-trauma patients have only limited ability to perform well in trauma patients [ ] . within the trauma population, triggers for consultation are poorly defined, beyond injury severity score and profound neurologic injury in older patients [ , ] . within the general geriatric trauma population, the geriatric trauma outcomes score (gtos) is a validated predictive tool that can provide a reliable estimate of in-hospital mortality risk using data available early in a patient's hospital course, allowing palliative consultations for the highest risk patients [ ] [ ] [ ] . predicting patients at risk for death in the year after injury, however, has proven much more challenging despite a clear need to identify patients who survive their hospital stay only to die in the coming year [ ] [ ] [ ] [ ] [ ] . it may be that a sustained focus on changing the culture of an icu unit towards one accepting and supportive of palliative care involvement is a key first step in routinely involving the consultative palliative care service [ ] . the optimal structure to prioritize communication in a given icu is likely dynamic and driven by local expertise and should be tailored to the individual institution's resources. the driving variable here is the quality of intensivist provided ppc as well as the culture of the institution and icu. at an institution where the attending physicians and the medical infrastructure are supportive of early approaches to goc discussions and supportive of a proactive approach to palliative principles, then consulting only subspecialty palliative care can be reserved for only the most difficult or complex cases. in the setting where the attending physicians or the hospital or patient cultures are less open to an early focus on understanding goc and considering less than maximally interventionist care plans, it may be necessary to integrate a palliative care subspecialist into the icu team. this will lower the barrier to integration of palliative care principles, help to prioritize communication, and, over time, will improve the ability of the treating team to provide high-quality primary palliative care in the icu. in the icu setting, communication often occurs with surrogate decision makers-particularly in the most complex patients at highest risk for a poor outcome. when organizing family meetings, we seek to use the broadest definition of "family" as is reasonable-essentially any person of importance in the patient's life who might attend a conference related to their care, as not all family is genetically related. as a general rule, most surrogates are not well prepared for their new, emotionally intense role and can suffer long-term psychological consequences as a result [ , ] . while we hope that all patients have had in-depth discussions about their wishes prior to injury, this is unfortunately not the case. as a result, when communicating with a surrogate in a complex and difficult clinical situation, we have a dual role of not only communicating the medical situation to the surrogates but also preparing them for the role of decision maker. involvement of surrogate decision makers and high-quality communication with them is essential, as physicians tend to systematically underestimate quality of life in favor of life-sustaining treatments, and this can often be mitigated by input from surrogate decision makers [ , ] . a structured communication tool or education program for family members may be helpful in supporting surrogates as they take on their new role. there are several predictable errors that are commonly made while communicating with critically ill patients and their families that can be mitigated by training and a standardized approach. the most common pitfalls are not budgeting appropriate time, entering into communication with family members without adequate information about the prognosis, and harboring a fear of inaction in face of a complex and highrisk scenario [ ] . this last pitfall is all too common among surgeons, who tend to have a "fix-it" mentality-focusing on the disease as an isolated anatomic or physiologic abnormality that can be corrected. this mental model fails in the complex critically ill and, when used to frame a high-risk communication, may lead to overly permissive decision-making that favors intervention even when the chance of the desired outcome is low [ ] . an additional error, more common in junior trainees, is terminating the discussion after a directive to "do everything." such a general statement is difficult to turn into actionable medical decisions. it is very important for the clinician, if asked to "do everything," to not prematurely truncate discussions but rather respond with empathy and explore what is meant, therefore understanding and capturing values that can be used to help guide treatment [ ] . a specific structured tool to help communication in the icu that is gathering a solid evidence base is the "best case/worst case" (bc/wc) model of communication. this model can help facilitate difficult decisions in high-risk scenarios with support from surgeons, patients, and family members [ ] . the bc/wc tool presents a range of possible outcomes ranging from the best case to the worst case, starting from where the patient is now, and using the spectrum to discuss what is possible. when using the tool to facilitate communication, a "most likely" case is marked and then used to make a recommendation that is informed by the patient's values and goc (fig. ) . in an adaptive tool that can be updated with changed in clinical condition, it has been shown to be effective with older patients considering invasive and acute medical treatments as well as high-stakes surgical decisions [ , ] . there are several routine communication scenarios that are frequently used in the icu and can be an opportunity, if correctly done, to build trust, support decision-making, and improve the psychological well-being of family members [ , , ] . communication with families must take the potential for long-term disability, and not just the potential for death, into account when discussing options. the astute surgeon needs to account for media portrayals of trauma care that deemphasize the impact and frequency of long-term physical and neurologic disability [ ] . . the first family meeting is a time to take a breath from the initial icu admission and acute stabilizing interventions. this is an opportunity for the treating icu team to do a comprehensive patient assessment, including not only their anatomic and physiologic status but also their goals, wishes, and values while providing a medical update to the family. this should occur within h of admission in order to optimize the quality of the communication, reduce icu length of stay, and understand patient's preinjury wishes, before proceeding with care that they would not want [ , , ] . early updates to the family and surrogate decision makers have been associated with earlier transition to comfort measured only as well as reduced length of stay, reduced ventilator days, and reduced chance of dying during a medical code (i.e., a dnr order placed for medical futility) [ ] . a proactive discussion regarding the patient's wishes early in the course of an icu admission is vastly preferable to an urgent discussion in the middle of the night following an acute deterioration [ , ] . while there are many ways to structure this first meeting, one of the most effective that we have found is as described by the vitaltalk group using the guide mnemonic [ ••] . get ready: pre-meet, get the right people, find quiet place, and sit down understand: what the patient knows inform: starting with a headline and stop for questions and emotions demonstrate: empathy and respond to emotion equip: the family to understand the next step in care (an opportunity to use the bc/wc tool) it is important to note that within this framework, there is no space reserved for decision-making. this is intentional as the first family meeting is framed as a meeting to deliver bad news and medical updates, gather information, and build relationships. the emotional toll of this meeting can overwhelm surrogates, limiting their ability to make complex decisions. ) the next common meeting type encountered in the surgical icu is the general family meeting, an update or planned check in following the initial meeting. the structure of this meeting can vary somewhat based upon the relationship with family and the urgency and scope of issues to be addressed. however, there are several essential components to the general family meeting. the first is a "pre-meeting"; this should include clinicians only and is an opportunity for multiple consulting teams to share their opinions and come to consensus regarding the medical situation, establish a shared understanding of prognosis, and agree on therapeutic options. it is not effective for clinician disagreements to be sorted out in front of the patient or the family, as discord in the care team often leads to family and patient distress and mistrust [ ] . as referenced earlier, since we are often coaching surrogate decision makers through a new and uncomfortable role, specifically saying things such as "what would they say" or "imagine that they are sitting here with us" to emphasize that we are not asking the surrogate for a decision but rather are seeking to hear the patient's voice. planning next steps together is an essential part of these meetings. the degree and nature of this plan, however, will depend significantly on the scope of the discussion [ ••] . ) clinician anxiety can be understandably high when heading into a meeting to talk about the third specific scenario, transitioning to end of life and comfort measures only. discussions regarding limiting life-sustaining interventions tend to be triggered by clinicians after an acute clinical deterioration and by family after a longer period where the patient fails to improve [ ] . in a study of new york state level trauma centers, advancing age, traumatic brain injury with an advanced directive, preexisting dementia, and pre-existing bleeding disorders are all associated with transition to comfort focused measures after injury [ ] . when discussing end of life issues, it is important to avoid an overly detailed review of clinical data, as this may obscure the overall life-limiting nature of the disease/injury, which is often multifactorial [ ] . similarly, it is essential to integrate patient preferences into medical recommendations, specifically patient preferences regarding quality of life, pain, chances of meaningful neurologic recovery, and anticipated degree of functional recovery [ ] . the bc/wc tool can be very helpful in this setting, particularly when the "best case" is profound disability or impairment. interestingly, fig. a graphical distribution of the use of the best case/worst case communication tool. within a theoretical case of a young man with a severe traumatic brain injury. the tool is used to integrate all available clinical information and estimate the range of future outcomes. the circle represents the absolute best case outcome, while the square represents the absolute worst case outcome and the star represents the clinicians' best judgment about what the future may bring. there is a clinical deterioration between day and day , as manifested by the "most likely" scenario moving closer to the "worst case." this is an original figure using the structure of taylor et al. [ ] while family satisfaction falls with increasing time in the icu, it actually rises when the process of transitioning to comfort only care takes greater than day-suggesting that additional time to adjust to the realities and see the ongoing medical care their loved one is receiving is helpful [ ] . family satisfaction, similarly, is actually higher when their loved one dies than when they survived their icu stay-a difference attributed to the improved patient and family-centered care that dying patients receive [ ] . as with the first family meeting, a structured approach to a "transition of goals" talk is essential, and we have found much success with the remap mnemonic, again supported by vitaltalk [ ••] . reframe: it is important to warn the family that a change in status update is coming and that the clinical scenario duration may have changed from previous updates [ •] . expect emotion and empathize: the nurse (name, understand, respect, support, explore) mnemonic of emotional response can be helpful, as responding to emotions from the family improves information retention [ , , ] . map the future: use an understanding of patient's goals to frame medical recommendations [ ] . the bc/ wc model can be particularly helpful in this setting as alternative approaches (recovery focused vs. comfort focused care) are considered [ ] . it is essential to have bidirectional flow of information when mapping the future as the care team brings medical information and options while the family brings patient values. it is not uncommon to have to make decisions in light of incomplete prognostic information [ ] [ ] [ ] [ ] . align with values: clinicians must align themselves with the family's and patient's values. using "i wish" statements (e.g., "i wish that were different too" or "i wish that was possible") to reflect family requests for clinically impossible options allows clinicians to maintain a therapeutic alliance while not offering false hope. these are difficult decisions for patients and their surrogates and conflict with the care team benefits nobody. plan treatments to match values: a clear statement of the plan moving forward is important, as well as a statement of hope. hope is an essential part of palliative care and even if we cannot hope for survival or recovery, we can still hope for time with family, an opportunity to say goodbye and an end to suffering. when considering the actual transition to comfort only care, specific clinical recommendations from the american college of critical care medicine are pertinent and informative [ ] . we strongly avoid using the words "withdrawal of care" as we never stop caring for patients, and we just shift our goal away from survival and recovery and towards comfort and acceptance of impending death. using "withdrawal of life-sustaining treatments" or "transition to comfort only goals" may be more acceptable. a final tip is to discuss cardiopulmonary resuscitation and code status last. once you have aligned goals and planned transitions of goals with the family, not doing cpr is a foregone conclusion. if, however, you open the meeting with the discussion about chest compressions before setting the ground work, there is a strong likelihood that the entire meeting will derail on this point. it should hopefully go without saying that, as patients transition to comfort measures only, it becomes increasingly important to care for the family [ ] . building and supporting family-clinician relationships and continuing to communicate well in the final hours of a patient's life help the family prepare for their upcoming death [ ] . the limited amount of research as to communication patterns and skills in the icu setting has focused, nearly exclusively, on formal "family meetings." there is, however, a significant amount of information delivery and care planning that occurs outside the structure of formal meetings. this usually happens at the bedside and is based on family and clinician availability-an interaction format that is very poorly studied [ ] . structured communication tools and family may help to improve physician and family relationships and reduce the risk of ptsd in family members [ , ] . difficult communication is a learned skill and must be taught to the next generation of surgical trainees. a cursory education about end of life care and high-stakes communication based on clinical exposure is no longer adequate, and there is very limited education in medical school on this topic [ , ] . as might be expected from the limited efficacy data, there are precious little data on the optimal approach to teaching residents and medical students key communication skills [ , ] . resident self-reported comfort with providing end-oflife care does not correlate well with experience providing end-of-life care, although a formal curriculum may [ ] . residents can effectively learn to use structured communication tools, such as the bc/wc tool, although do need skilled feedback in order to improve [ , ] . in the current medical training environment, it is likely that the clinical education of the vast majority of general surgery residents in high-stakes communication will occur in the surgical icu, where brief intervention can improve self-reported comfort with key communication skills [ ] . as we continue to improve our understanding of the optimal approach to high-stakes communication within injured patients, the educational process should proceed in parallel. families have traditionally and nearly universally been included at the bedside in surgical icus [ ] . as of the writing of this article, the covid- pandemic is raging across the world. as physical distancing becomes ubiquitous and hospitals severely restrict the number of visitors, high-stakes communication is becoming increasingly challenging. technological solutions have provided a bridge, but video conferencing will never be a full replacement for in-person communication and bedside discussions. while we suspect that many of the same principles and structures outlined in this article remain applicable in the current pandemic, adaptations to technological limitations will be required. the weight of the published work, to date, focuses on managing death and dying in the setting of a resource-limited pandemic. much work remains to be done and with families not at the bedside. conversations will need to become more intentional, more structured, and integrated more intentionally into the routine care of patients in the icu. the realities of physical distancing and limited family visitation emphasize the need for rapid and universal acceptance of surgical palliative care principles, particularly structured and empathetic communication in the icu [ •]. communication skills in the icu are an essential part of the care of trauma patients. we have an imperative to improve the communication for all patients, not just those at the end of their life [ ] . the need to communicate effectively and empathetically with patients and identify primary goc is an essential part of trauma care in the icu [ , ] . while the optimal design to support complex communication in the icu will be dependent on institutional experience and resources, a ppc model or integrated palliative care model will allow assessment of every patient's communication needs and may be superior to a purely consult-based format. a structured approach to family meetings may improve our ability to communicate succinctly and clearly in high stress situations. communication skills are teachable and can be improved. in an area of active and dynamic research, we look towards a coming explosion of research on surgical palliative care and complex communication challenges. this is an exciting time to be an acute care surgeon and an exciting time to build palliative and communication capabilities. conflict of interest the authors declare that they have no conflict of interest. human and animal 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end-of-life care palliative care and surgical training: are we being trained to be unprepared? training surgeons and anesthesiologists to facilitate end-of-life conversations with patients and families: a systematic review of existing educational models evaluation of palliative care training and skills retention by medical students resident education in end-of-life communication and management: assessing comfort level to enhance competence and confidence training surgical residents to use a framework to promote shared decision-making for patients with poor prognosis experiencing surgical emergencies surgical palliative care education a brief educational intervention to teach residents shared decision making in the intensive care unit to face coronovirus disease , surgeons must embrace palliative care publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - ocsf authors: amorin‐woods, deisy; fraenkel, peter; mosconi, andrea; nisse, martine; munoz, susana title: family therapy and covid‐ : international reflections during the pandemic from systemic therapists across the globe date: - - journal: aust n z j fam ther doi: . /anzf. sha: doc_id: cord_uid: ocsf the covid‐ pandemic has convulsed human communities across the globe like no previous event in history. family therapists, paradoxically, given the core of their work is with systems, are also experiencing upheaval in professional and personal lives, trying to work amidst a society in chaos. this paper offers a collection of reflections by systemic and family therapists from diverse cultures and contexts penned in the midst of the pandemic. the main intention in distilling these narratives is to preserve the ‘cultural diversity’ and ‘ecological position’ of the contributors, guided by phenomenology, cultural ecology, and systemic worldviews of ‘experiencing.’ the second intention is to ‘unite’ promoting solidarity in this isolating situation by bringing each story together, creating its own metaphor of a family: united, connected, stronger. as a cross‐cultural family practitioner, with a strong mission for collaboration, the lead author acknowledges the importance of context – the nation and location of the experience; culture – the manner in which culture impacts on experience; collaboration – enhancing partnership, enriching knowledge, and mapping the journey’s direction; and connectedness – combating isolation while enhancing unity. since the key transmission of culture is through language, raw reflections were sought initially in the practitioners’ own language, which were translated for an english‐speaking readership. these narratives are honest and rich descriptions of the authors’ lived experiences, diverse and distinctive. the contributors trust colleagues will find these reflections helpful, validating and acknowledging the challenges of this unique period in history. deisy amorin-woods this compilation of reflections, while not a research project per se, is written in an auto-ethnographic style (ellis & bochner, ; rhodes, ) by five systemic therapists from varied cultures and contexts about the covid- pandemic. their narratives, experiences, perceptions, ponderings, and feelings were composed as they were living the experience; unique in their meaning making and in the way they contribute to this collection. as the lead author, i live and practice in perth, australia. reflecting my international and collective soul, my curiosity has given me the impetus to facilitate global exchange and initiate cross-cultural conversations. peter fraenkel, in new york, united states, incorporates his buddhist insight and psycho-musical intervention to provide a message of connection and hope to help others preserve a capacity for joy. andrea mosconi, from padua, italy, provides students and colleagues with a gentle message of wisdom, and insights to stimulate critical thinking and opportunities for new ways forward. martine nisse, from paris, france, delivers a 'mood note' which introduces a sense of 'lightness' in the midst of her complex and challenging work with domestic violence and incest. susana munoz from santiago, chile, with a background as a midwife, uses the metaphor of labour and birth to demonstrate survival. this 'pandemic project' initially arose mid-march as i reached out to colleagues abroad to check how they were navigating through the pandemic chaos. through our 'cross-cultural' conversations, the sense of feeling understood through shared experience and empathic means led to strengthened connection. this gave me the impetus to ask myself: wouldn't it be helpful if we could write something together to find some healing, whilst sharing our stories with others, in order for them to also find healing? i realised this was a global crisis; a collective narrative unfolding daily which has left no one untouched, and we as family therapists had important and individual stories to tell. time was of the essence, the view from this window was unique and so i drafted a brief. given my strong interest in collaboration and collaborative practice, over the years i had fostered relationships and developed partnerships including with family therapists abroad (amorin-woods, ). thus, i reached out to this wide network of colleagues and invited them to join me in telling our stories. most i invited responded enthusiastically to this initiative almost immediately. regrettably, due to the tight deadline, a few were understandably overwhelmed with other priorities. as a cross-cultural family therapist, my mission is to be diverse and inclusive when meeting the varied cultural needs of my clients, developing programs, delivering services, and working with families. this project was no different. as i heard the international collective voices it was thus vital to me that contributions were garnered from a cross-section of nations and continents as i wanted to honour different cultural backgrounds and language representation. i believed that by tackling phase sharing our experiences, we could capture each of our cultural and ecological worldviews and thus unite in this process. then, we could contemplate stepping into phase regenerative process in order to rebuild our own lives post-pandemic. after we are given the space to process the raw and organic heaviness of our own experience, we are able to support our clients to come up with practical responses, but not before. it was critical to put the stories together before the 'immediacy' of the raw accounts was lost. we are in the midst of the experience, in 'situ,' in confinement, confused, hypervigilant. we live in a world where we seem to 'rush' to come up with labels, treatments, and solutions to human distress, often bypassing our lived experience of suffering, loss, and isolation. i felt as if these early collections merged into one, represent the 'holding, healing space' that we as therapists need, before we dare consider what life may look like on the 'other side.' without this, how could we support our clients in putting themselves, their families, and their lives back together, in even contemplating what a world of 'functionality' or 'normality' may look like? while family therapists are aware of the importance of 'unmasking' to remain authentic to our clients and to self-disclose to deepen connection and trust, therapists do not often have the chance to share their accounts as they too travel through challenging life experiences. these reflections provide that opportunity. finally, any conversation about culture must include the element of language. since the key transmission of culture is through language, organic reflections were sought in the practitioner's primary language. as in therapy, authenticity of feelings and vulnerability cannot be truly transmitted except in the primary language (amorin-woods, ). however, since the main demographic of this journal speak english as their primary language, these reflections are translated and offered in english. i am deeply appreciative to each contributor for responding as i reached out. from the bottom of my heart, thank you andrea, peter, martine, and susana. deisy amorin-woods, perth, australia this pandemic is a collective trauma event and its multi-faceted impact cannot be underestimated. this leads us to first acknowledge and respect the interconnectedness and relationship between systems, within our body, between us and our environment, and between one another. this also causes us to question the notion of permanence versus impermanence and certainty versus uncertainty. as a cross-cultural family and systemic therapist, i have developed a thirst for collaboration within programs, across organisations, within communities, and across the globe (lee, thompson, & amorin-woods, ). as a migrant to australia from peru, i have always felt drawn to the idea of collectivism (amorin-woods, ), a sense of 'being together' and of 'joining with the other,' and that notion has never left me. whether working in government, non-government organisations (ngos), in private practice, or in academia, i have ensured i bring my 'collective soul' into my profession in the way i practice and in my teaching. i try to connect with the issues that impact on the lives of our families regardless of where they are located or where they originate (amorin-woods, a , b . the issues are varied and the systems diverse, whether a couple facing grief, families with intergenerational trauma, or refugees who have fled persecution. when the news broke in early about a possible epidemic, i had not conceptualised either the extent or the utter devastation that was to follow. i was due to travel to basel, switzerland in early march to present at the world congress of family global family therapists reflect on covid- ª australian association of family therapy therapy under the international family therapy association (ifta). ironically, my presentation was titled, therapeutic conversations and trauma informed systemic practice: acknowledging meaning making in the backdrop of relentless fear and unpredictability. two days before my departure, the conference was cancelled due to the pandemic, which was rapidly spreading around the globe. in the weeks that followed, the whole world convulsed through a rapid period of change. the need to find refuge from this potent, yet invisible, force left us vulnerable, confused, distressed, fearful, and ambivalent. in my own space, due to the need to isolate, and the closure of state borders, i have not been able to see my daughters or embrace my little 'grandies.' this has left me profoundly sad and nostalgic. having family, friends, and colleagues around the world, many located in the countries worst hit, i was driven to connect, to check-in on how they were navigating through the crisis. i heard directly their accounts of anguish, dread, panic, powerlessness, loss, and grief. this virus was burning, just like the recent ignition of highly combustible dry eucalyptus trees in the australian bush, spreading rapidly like a wildfire, loud and forceful, the echoed thoughts keeping alive the nagging fear. people wondering, will i be next? will i be a carrier and infect my loved ones? families losing parents, grandparents . . . a whole generation seemingly vanishing as in a puff of smoke. listening to these raw and distressing stories became rather confronting. i found myself holding their collective pain in my arms and with a virtual embrace. i was attempting to ease their suffering. personally, these stories transported me back to a similar experience as a child living in peru. this was a time of 'sendero luminoso' (shining path) a guerrilla group, along with 'sinchis' an anti-subversive 'police sub-group,' both of whom were responsible for committing human rights violations, terrorising and decimating over , people, most of whom were indigenous. this led to cases of intergenerational trauma, the stories of fear, sorrow, and confusion passed down as through the 'mother's breast milk' for generations. these painful memories accumulate in one's body becoming a historical site which is transported through time (rueda, ) . i remember my mother distraught as the situation evolved given my sibling was studying at the university at the epicentre. while not directly impacted by the war and the trauma, i felt them indirectly, yet latently, through the suffering of my mother and the stories told by my sibling who witnessed young students being murdered. this brings me back to the present time and leaves me concerned wondering whether we will see stories of pain and sorrow passed down as a result of the covid- war? will this generation transmit trauma to those to follow? professionally, as the epidemic evolved, so did the need to transition my practice from face-to-face to purely tele-health. while australia has been fortunate not having been impacted as heavily as other countries, i have observed among my clients signs of anxiety and hypervigilance related to the fear of transmission, as well as new manifestations of suicidal ideation previously absent. these signs are connected to a number of issues: the stress related to job loss and financial uncertainty; the sadness and loneliness linked to the inability to see family and friends; grandparents' sorrow not seeing their grandchildren, an embrace appearing so distant. children unsettled, mothers feeling stressed and exhausted juggling extra roles, including home-schooling. while some people see isolation ('iso') as simply confinement, others see it as imprisonment, fearing the threat of imminent danger if leaving their safe and familiar bubble. for others the uncertainty, the not knowing, the unpredictable re-organisation of families and communities, is too much to bear and leads to heightened levels of suicidal ideation. people ask themselves: will there be an end to this? are we facing the end of the world? there is documented concern for heightened risk of suicide among communities continuing past the pandemic (gunnell et al., ) . further to this, given my extensive experience working with people with acute mental health issues and with complex trauma, i am aware that people who have experienced trauma are highly impacted by threats about the safety and stability of the world (brown, ; james & mackinnon, ) . i have also observed how vulnerable families (such as families with intergenerational trauma and child abuse survivors) are particularly at high risk, and consequently presenting with clear signs of re-traumatisation. throughout this pandemic transition, i have noticed various community stances rapidly forming. one that blames, with xenophobic undertones, pointing to race and culture as the original cause of the virus, discriminating against certain cultural groups. another that labels, describing people as 'paranoid' or 'hysterical' as people frantically accumulate food supplies or simply voluntarily decide to self-isolate. a third, with traces of denial and avoidance about the existence or magnitude of the situation, that judges and ridicules those taking a more conservative approach. within this stance, there is an underlying expectation and pressure from strangers or loved ones to 'move on' rapidly through the stages of grief (kubler-ross & kessler, ) from shock and denial to acceptance, without recognition that people need to be allowed to 'feel their feelings.' on the other hand, i have also seen demonstrations of kindness, generosity, and solidarity for fellow community members, much of which has been led by children. teddies perched in trees, messages of encouragement and hope written around neighbourhoods to lighten the tone and brighten people's days. boxes of fresh produce or non-perishables left to help others financially disadvantaged. i have observed some people viewing this pandemic as an opportunity to start over and live in a healthier, humble, more acknowledging and respectful way towards self, towards family and their environment. i have seen occasions when cut-off families have re-connected, and couples considering separation have learned to appreciate and not take each other for granted. i have been mindful of being available for my clients, to provide the holding space to acknowledge and validate their feelings and allow them to process their experiences. the need for self-care and self-compassion takes on a new meaning in its recognition of our shared humanity. i thus ponder: are we able to provide clients with a holding space to process things if we are not connecting with ourselves? . . . even though we too are living amidst the chaos. we do not have to abnegate ourselves or our experiences and feelings to show we are capable practitioners, because when we abnegate ourselves we tell our clients to let go of parts of self in order to function, in order to accept themselves, in order to relate. we need to 'be,' before we can 'do.' remaining authentic is key in therapy. i often say to my students and supervisees, authenticity is to a therapist what breath is to life. we need to be authentic with the families who come to us with hope and trust and this includes possessing heightened awareness of ourselves, hence the need to uncover the 'self'. i recall a similar description often during my fellowship at the accademia di psicoterapia della famiglia in rome. andolfi would often make reference to ridding self of the mask and being fully self, fully human (andolfi, ) . it is only through such a process that we arrive to a place of recognition of what is ours and what is theirs. it is crucial that we are able to name and process our experiences in order to support our global family therapists reflect on covid- ª australian association of family therapy clients to do the same, in order to provide the holding space they need, and joining-in with them (minuchin, ) . this then allows us to welcome the rich exchange between each other in order to develop an empathic connection and trusting relationship with them (aponte, ) , because how can we join in with families and trust be elicited without this important element? this also helps not to transfer or project our experiences onto our clients (lum, ) . as systemic therapists we are in a privileged position as collaborators and partners influencing the family system and the environment and context where families are placed. this gives us the opportunity to awaken deeper knowledge and understanding within a given system, within the family who puts their trust in us. we become responsible in co-creating and co-constructing the therapeutic reality and in eliciting change (von foerster, ) . we influence the environment whether we bring empathy, healing, anxiety, or fear. understanding the self and understanding each other becomes the impetus for healing. around the globe, we are all living the experience of the covid- pandemic as a collective trauma. the chaos is real to us all. we are dealing with challenges that are comparable yet distinct. while we are all impacted, the impact is different depending on where we are located, in our culture, our ecological system, and the politics of our day. a frequently used expression suggests we are all in the same boat; however, i would like to use the metaphor, while we may be sailing the same storm, we are in different boats. how robust our ship is, and how we manoeuvre it, may determine how we survive the storm. in writing this i am mindful of families as they navigate through this pandemic. i am interested in supporting them to acknowledge their being 'human,' while rejecting reductionist ideas of 'experiencing' that rush them to numb or bypass their experience. instead, it is time to pause and connect with the basics, our relationships. the lesson from gregory bateson was the importance of the interconnectedness of living things to their natural environment (bateson, ) . this is so relevant to our current situation. we hear loudly the desire of communities to go back to 'normal' . . . however, do we really want to go back to the normal that we knew? or do we want to look at this as an opportunity for social change? for a change in direction? in the way we do things, in the way we relate to self, to other people, to other cultures, to our environment. instead let's nurture families and preserve relationships. let's propose a paradigm shift about the way we think of ourselves not as passive closed-off beings, but as active authors of our life, insightful, creative, purposeful, and connecting. let us use this time to co-create a space for healing and holding the human spirit. coping with covid- : a time to focus on the simple gifts of life peter fraenkel , new york, united states as news of the covid- pandemic quickly grew in march of this year, i immediately found myself re-immersed in the traumatic disorientation of september, . terrorist planes crashing into the world trade center launched new york city and the united states more generally, into what i termed 'the new normal' (fraenkel, b) . yet this is different: an invisible virus with an unpredictable course, unseen yet life threatening. where to go? what to do? the answers to those questions quickly were determined in march by the governor of new york with guidance from federal and state health experts: go home, work at home. on march, i delivered the last in-person workshop at the ackerman institute for the family. attendees spread out across the room as a safety precaution to decrease the likelihood of communicating the virus among us. none of the usual handshakes between newlymet colleagues, none of the usual hugs between old friends, my former students, and professional acquaintances. we carried on, focusing on effective techniques in couple therapy. but we all had a nagging sense, mostly at the periphery of our consciousness, that the world outside was growing increasingly fearsome, and that we were better off in here, held by the warm embrace of what mary pipher has called the 'shelter of each other.' as i wrote in another article after / , we therapists were fortunate to be helped by the act of helping othersto belong to a community of care (fraenkel, ) . i've specialised for many years in work with families traumatised by incest, domestic violence, and homelessness. in my private practice, i've specialised in what i've called 'last chance couples'those who've often seen one or more couple therapists, without much improvement, and who are now on the brink of divorce or its nonmarried equivalent of relationship dissolution (fraenkel, ) . these two areas of my work now overlap: distressed, disengaged partners already strongly considering separating, or already living apart, now stuck together with kids and sometimes their elders and even other families. precipitous layoffs and loss of income, infection with the virus, and in some cases, the known death of relatives, friends, or colleagues, have led to the kind of traumatic effects of living through a tsunami, gathering force every day with no end in sight. at the same time, accurate information, guidance, and leadership from the federal government is sorely lacking, alarming in its own wayno comfort there. my clients are experiencing all the usual symptoms of psychological trauma: intrusive thoughts and nightmares, edginess and hypervigilance, sleeplessness, anxiety, and depression, as well as relational trauma and ruptures between couple partners, and among family members. the factor most clearly correlated with resilience in the face of trauma and other disruptive experiencessocial supportis in short supply, despite inventive use of social media and online meetings, including teletherapy. i have occasionally ventured into a nearby park with my percussion instruments to record myself playing along with inspiring songs about social justice and love, posting these videos on facebook and instagram. the response has been overwhelmingly one of gratitude and unexpected pleasure, and those responses have gratefully been received by me, giving me a slight sense that through my 'out-of-the-box' psycho-musical 'intervention,' i've helped others preserve their energy, hope, and capacity for joy. but i then return to my apartment, to be alone once again, as my kids are now attending college in europe. we're in close touch, as i am with friends and colleagues, but i surely feel isolated; and when i occasionally venture out and on to the streets to shop or bank, there's the uneasy, unreal sense of being on another planet, or at least, a familiar but altered landscape, with fearful masked neighbours strolling six feet apart. coincidentally, just a few weeks prior to news about the coronavirus, i had started writing a book for the general public about lessons to be learned from taoism about love and other relationships (fraenkel & akambe, ) . as a long-time buddhist and taoist, i've practiced noticing the little, familiar things and accustomed events of life that can bring unexpected joy once brought into the gentle gaze of the 'now.' for decades, i've tried to live my life as if inside a haiku poem, taking pleasure just from being sensate, breathing, and alive. musing on the nature of existence, the creator of taoism, th-century b.c. court librarian, philosopher, and social critic lao tzu, writes in the first passage of his classic text, the way of life ( th century bc): existence is beyond the power of words to define terms may be used but are none of them absolute . . . if name be needed, wonder names them both from wonder into wonder existence opens i've been advising all my clientsindividuals, couples, and familiesto use this disorienting time to reflect upon the following taoist suggestions, to look for opportunities to experience wonder, just as i have as a fellow traveller through this time. specifically, i've suggested they: look for pleasure and joy in small, everyday experiences. i took a break from writing this article, went to the kitchen window in my fifth-floor apartment, and watched the rain falling upon the black-painted fire escape. it was beautiful. a few hours later, i took another break, and saw my two kittens, whom i adopted from a shelter in october, cuddled together in my furry chair, sleeping contentedly. it was beautiful. it calmed me down. slow down. our usual life pace in nyc is frenetic, our lives overstuffed with aspiration, achievement, a never-ending quest for riches, possessions, and when we've accumulated enough of those, the relentless search for new experiences (which i've termed 'experience greed'), for unique travel destinations and novel experiences to empty our constantly refilling 'bucket list.' lao tzu ( th century bc) writes: gravity is the root of grace, the mainstay of all speed . . . what lord of countless chariots would ride them in vain, would make himself fool of the realm, with pace beyond rein, speed beyond helm? it's a time to stop, and look closely at what and who we already have in our lives. it's a time to breathe, walk, eat, and talk slowly, compassionately, and patiently with loved onesthose living with us or those whom the virus has separated from us. and while slowed down, take a good long look at your partner, your child, your parent, your friend, your neighbour, your colleague (online, most likely!)listen to their voices, and enjoy their unique aliveness. in the timeless words of songwriter cole porter and a song made famous by frank sinatra ('i get a kick out of you'), let yourselves 'get a kick' out of them (fraenkel, a) . look at them like there's no tomorrow for them, or for you. let go of control. on a related note, we cannot definitively avoid illness, and we cannot insure through our hand-washing and social distancing and mask wearing that we will survive this pandemic, or at least, leave it unscathed health-wise, financially, or emotionally. nor can we avoid the suffering of learning that a family member, a neighbour, a student, a mentor, or a colleague is sick, or has died. radical humility is called for in this troubling, unpredictable time. take precautions, yes, but recognise that this virus is nature, and nature has its way of overcoming even the best-laid plans of mice and men. learn from persons who inhabit oppressed social locations. lao tzu ( th century bc) advocated wisdom over knowledge: leave off find learning! end the nuisance of saying yes to this and perhaps to that, what slightest use are they! if one man leads, another must follow, how silly that is and how false! communities that have experienced generations of oppression due to race, ethnicity, geographic region/country, culture, and economic status have much wisdom to share about surviving and even thriving under hardship, and about how to make do with few resources. one hysterically funny video that circulated around the internet early in the emergence of the coronavirus came from the philippines, in response to privileged westerners' panic about running out of toilet paper. it was a lively music video, with dancing women behind the male lead singer, demonstrating how to use a little colourful plastic pot called the 'tabo' to wash up after defecating. it's high time we erase the terms 'first world' and 'third world' and the power and knowledge hierarchy between the countries and cultures belonging to each politically constructed group. the world must come together to defeat this virus, and to survive, as a unified, mutually respectful people in harmony with the earth and nature itself. my mother mimi bialostolsky fraenkel, who died of cancer in just before / , believed the world might eventually come together in a time she called 'creative chaos.' i think of her now, and whether this worldwide pandemic is enough to unite us in a common effort to live on. i'd like to hope so . . . but i'm not sure. what we are going through is not an ordinary crisis, this is an epochal crisis! it seems to me that it is the most obvious consideration which echoes with many. the question then may be the systemic one: what do we do in each situation for every person who presents to us with a problem? as i often say, a kind of systemic mantra: perfection, what does this allow me to learn? it is now clear that this virus has three aspects that makes it scary: ) it is new and we cannot be immunised; ) it also passes through healthy carriers who, even if they have a lower viral load, are still capable of contagion and therefore cannot be easily controlled; and ) it is not a flu virus like most flu viruses. this virus directly attacks the lungs and therefore gives viral pneumonia; it also attacks many other global family therapists reflect on covid- ª australian association of family therapy systems in our body. from this point of view, respect for the required government measures is essential as the only defence, while there is no effective drug or vaccine. the alternative is to build herd immunity, through a selection of the species. only those with resistance will survive. on the level of interaction between this virus and the social system, the question is: how long will it last? if we take into account the date of government measures, the fact that the need to tighten up has often been felt, and also the fact that it is always difficult both to impose and to accept strict rules and that the incubation time is days, it is not difficult to hypothesise that the so-called 'peak' of the infections is still to be reached. from there, it takes time for the sick to heal and for additional time to pass before the virus is eliminated by the carriers. by putting all this together, i believe that we must accept that the current situation will last a long time. epidemiologists seem to be moving increasingly in this direction, obviously, barring unforeseen events for the better and for the worse. let's have patience! however, let us look wider, at the macrosystem where all this happens. if we take into consideration the relationship between us, 'homo sapiens,' and the natural system into which we are inserted, this story changes some rules of the relationship. it allows us to realise that we are not omnipotent, but that we are part of a system that can destroy us in a short time if we do not take into account the feedback that comes from the interaction with the other elements of the system itself. its 'liberation' is part of this process of inappropriate invasion by us 'sapiens' of natural contexts not coordinated with the human system. from this point of view, this virus is a warning, it is almost a homeostatic mechanism that is produced in a system that is already at the limit of its range of possible interactions and is reaching a possible 'bifurcation point' (prigogine, p. ) . this is a test of a general catastrophe, a global crisis. basically there are other 'covid' around which we do not want to be aware of and which can equally undermine our survival in a global way: global warming, pollution, deforestation, desertification, water consumption, economic imbalances within societies and between different parts of the world. just like the case of 'covid,' we pretend not to see them yet they advance in a hidden and creeping way until they explode. just like covid- ! the 'good' thing we can say about the virus is that, unlike wars which can be taken out on someone who is considered guilty, the same cannot happen as this virus does not discriminate; it is a common enemy of the whole system. the virus applies equally to everyone, just like in the family systems with which we want to work. just as the members of a family system at the end of a session should not be able to say who is right or who is wrong (selvini palazzoli, boscolo, cecchin, & prata, ) , so as humans we are driven to look at each other, to confront each other, to even try to understand and help those, who until yesterday, rejected each other. yes, there are those who, not understanding, continue to wave the flag of personal interest, or hunt the culprit, or act in their own defence, but for now they are exposed and forced to admit or to try to make amends. for now, solidarity prevails, and there is admiration for the commitment and generosity of those on the front line, a sense of being a community. and we see the increasingly clear signs that, where man takes a step back, the natural system of which we are part takes a breath, pollution decreases, and everything seems to show us what we must consider for development and the future. a great opportunity, don't you think? but several times i have said: for now . . . the real question is: will we be able to take this into account? this brings another consideration regarding the social system. in recent years, perhaps blinded by the race for well-being or perhaps because well-being itself had allowed many to take advantage of it incorrectly even in public structures, the focus has been rebalancing national budgets by destroying the network of structures that had functioned as the skeleton, blood, and neurological system of society by supporting and bringing food throughout the social system. i am talking about healthcare, public education, welfare . . . let's consider what it means not to have continued to be aware of the value of these 'elements' of the social system. i say this both for those who have dishonestly taken advantage of it from within and for those who, from their own ideology, have fought them. here, this crisis can help us rearrange the scale of values regarding what is most important to preserve in a social system, counterbalancing the excessive importance given to the production system, to take into due consideration what is at the basis of a possible safe and civil coexistence. even regarding the enormous development of the computer network that is becoming more and more the neuronal network of the planet: on the one hand it allows us to accelerate the feedback between parts of the system facilitating the possibility of co-building solutions; on the other hand tomorrow it will put us in the face of problems such as the enormous possibility of controlling, even more than now, individual lives and determining who and how to manage the enormous power that all this enters . . . and in whose hands? of course, it is always the two polarities of the life of a system that must be balanced: the competitive system and the collaborative one. the same goes for natural selection which is the survival of the fittest and the other towards the idea that the average good of the most is better than the maximum good of the few. but here the question is: will we remember it later? and what does all this say about our systems of daily interaction, to our closest systems? of course, stopping puts us in a position to change our position in the systems of which we are a part and the relationship rules change. we feel distant from those who were close to us, communicating virtually with those we used to touch, look in the face, caress, shake hands, pat on the shoulder, take by the arm. in contrast, we find ourselves living with those who we did not have close to us for so many hours or who we were even in conflict with, seeing him/her every day and maybe having to talk to each other. we can no longer take advantage of dissipative structures, as they are called, which diluted and differentiated, distributed the tensions of the systems: work, travel, school, various activities. maybe if we choose to use the avoidance mechanism that is so useful to delude ourselves that there is a balance in relationships? no, we can't back off now, we're in touch! and then? this is an opportunity to stop and listen to ourselves and others, try to look at them with different eyes, re-appreciate the small gestures, let things arise from building together and patiently look for solutions, not problems. so, in the infinite space we can perhaps rediscover the value of everyday life, of silence, of simple things, of doing together, of allowing our imagination to bring to mind an idea. sometimes it's simpler than it seems! and with our patients . . . 'clients?' here too our position changes. from time to time we may find ourselves inventing different ways of making ourselves feel close, exploring new and different tools for staying in relationship and offering help, believing that, beyond the tool used, the relationship is what matters. cecchin's words echo on us: we all need to feel seen, and to feel seen we are willing to invent all the colors! (cecchin, p. ) . and then of course: hypothesis, circularity, and neutrality! (selvini palazzoli et al., ) . so, our patients will feel us present, even if in a different way, and they will appreciate our sincere and attentive commitment. as milton erickson (erickson & rossi, , p. ) said: there are no difficult or incurable patients, there are only therapists who are able or unable to find a way to communicate with them. another thing comes to mind. let's see how a small virus, which affects the system at a specific point, achieves great effects. this can be a teaching for our therapy. the 'saltology' mentioned by the milan team in the first pages of paradox and counterparadox comes to mind: the results confirmed that, when it is possible to discover and change a fundamental rule of a system, pathological behavior can quickly disappear. this leads us to accept rabkin's proposed idea: that in nature events of radical importance can happen suddenly when a fundamental rule of a system is changed. rabkin proposes the term "saltology", that is saltology (from the latin saltus) for the discipline that should study these phenomena. this finds its correspondence in the general theory of systems whose theorists speak of "p.s." as of that point of the system on which the maximum number of essential functions converges to a system, changing when obtaining the maximum change with a minimum of energy expenditure. (selvini palazzoli, boscolo, cecchin, & prata, , p. ) so, these are my reflections these days which i wanted to share. all this 'allows us to learn' and perhaps finally arrive. it is an opportunity to become aware what this covid virus . . . offers us as systemic therapists. let's go ahead, let's build better times together! small mood note from a french family therapist in times of pandemic (petit billet d'humeur d'une th erapeute familiale franc ßaise en temps de pand emie) the family therapy sessions i conduct are very rarely by videoconference. when they are, they involve my patients who, for one reason or another, have gone abroad to settle temporarily or permanently. but since the arrival of the pandemic in france, one of my patients, herself a psychotherapist and familiar with consultations by videoconference, has pushed me towards this mode of therapy. two days after the announcement of the confinement, i was equipped, and i launched the invitations to my patients. after researching what angle of view for my webcam would symbolically give an open message about the future, i decided to turn my back to my patio, so that the view behind me would lead them towards the sun and the plants. the weather is extraordinarily nice, the pollution in paris has dropped dramatically, the sun is shining without any halo of pollution. birds are heckling in the city. i half draw two curtains, in the colour of my therapy room, which has been abandoned since the confinement, and adjust lights towards my face to gently counterbalance the brightness of the outside. it is, finally, a kind of 'chiaroscuro' (light/dark) that makes my first affected and surprised patients say, keep going . . . it reminds us of vermeer . . . the surprises are mutual, i arrive directly in their living room, or their office, even if they have passed through my 'virtual waiting room' of zoom. i note that some have asked their spouse to use their one-hour right of exit during the consultation to be quiet, others are gathered on the living room sofa, a cup of tea in hand with their pet. sometimes a decorative detail jumps out at me, so 'connected' with the patient's problem that i cannot avoid referring to it. one of these surprised patients says to me, do you want me to show you where i live? i pose and respond, no thanks. or . . . maybe . . . yes, you could ask all your patients to show you their home, you could learn a lot from that? . . . don't you think so? . . . mmm, no, i don't think so. holding the structure is one of the signatures of family therapy. most of my patients have been exposed to violence that has broken into their psyche (nisse, ; nisse & sabourin, ) . re-establishing boundaries or maintaining distance from others requires an ongoing effort. it requires constant therapeutic adjustment. i find that they are grateful for the availability of their therapist. i find that after the first few sessions, i feel as if i am regenerated. despite the shock of this epidemic, i have not forgotten anything about my way of being a therapist with each of them, nor their family history. my abilities as a therapist seem to be naturally at my disposal. the new and artificial proximity of the screen requires me to be attentive to maintain a therapeutic atmosphere (nisse, ) , in conformity with the pre-existing one: that is to say, intimate and respectful at the same time. i also note that since the beginning of the pandemic their psychological work seems to be more productive between sessions. some patients have refused videoconference sessions. they cannot imagine hearing themselves or me talking at home about the violence, especially sexual violence, for which they have consulted; they don't have housing where it's possible to really isolate themselves, or they are single women raising their children. sometimes, a patient who has not responded to the offer checks in with her therapist with a fear of illness. some of them tested positive for covid- . it is nothing too serious, but a great deal of fatigue and fear for the impact on others. i don't have a pre-established bilateral agreement for the therapeutic meeting by videoconferencethis bothers me a little, but i know my patients, i trust them. it will be possible to establish it afterwards for the next sessions. the french or european family therapy societies provide support, stimulate reflection on this subject, offer platforms for exchange with family therapists, or platforms for helping families stressed by covid- or affected by it and also affected by sudden bereavement. a large part of our patient population is made up of children who have been placed in care by the juvenile judges. they come from all over france, and half of the country is currently in the 'red zone,' which means that children are not allowed to go to school or to travel more than kilometres to go to court. as for each of the people living in this 'red zone,' sometimes, as i know from the supervisions i give by videoconference, a certain number of them paradoxically relax knowing they will not receive any more parental visits, mediated at the same time . . . recently, the status of social workers has changed. they are now considered during this time of pandemic as health professionals, and as such they have the right to travel to meet with children. the idea emerges to organise family network therapy sessions by videoconference before the end of the confinement. the centre des buttes-chaumont is again in demand. the conventions established with each of the participants in the network session and the therapeutic tandem usefully frame the new disturbing context for the most vulnerable among them, by calming the fears of abandonment, or on the contrary of dictatorial control through this means of communication. a spontaneous energy flow appears as the homage 'at the windows' is paid every evening at pm to the nursing staff. i too hit something, a shell casing from world war i (empty!)it reminds me of the spanish flu pandemicand as a drumstick, a saharan jewel offeredwell, well, well!by one of my former patients who went to the sahel to offer solidarity to women. it makes a rather high-pitched bell sound, somewhat close to the bells of the tibetan monks . . . everything blends into this positive energy. a neighbour who is ill with cancer is the most alert to beat the call of the neighbours . . . her care continues during this time. the calmness is conducive of reflection. what do i want to change in my life as a therapist? . . . nothing, other than taking more time for myself. sports, baking, tidying up, painting, talking . . . i miss my friends, but the family exchange is nicely intensifying. confinement slogans tirelessly spread their message, one week, two weeks, three weeks, we don't count the weeks anymore, time has changed value, the pace has slowed down. look, why don't i take the time to check my pension rights? no, i'd rather watch a good series . . . humanity in times of pandemic (humanidad en pandemia) therapists and patients living through times of pandemic have evolved in a context of threat and uncertainty. due to the regulations to avoid contagion, family members have been forced to stay in the same space for indefinite times. the personal and group impact of this dynamic unfolds in multiple dimensions and has an unsuspected scope. the experience of physical space has evolved as hours, days, weeks have passed. although at first it could be perceived as a break from the whirlwind of everyday life, the 'forced' stay has turned into a kind of narrowing of the limits; of the physical space, of the psychic/corporal world, relational, bonding. no matter how many people inhabit a place, the members resent the isolation and helplessness on many occasions. in some way, previous forms of relationship, ties and family, group, and social functioning have pointed out the dominant styles of behaviour in this type of closure (muñoz, ) . without external compensatory systems, which operate as sedatives of the sense and deniers of finitude, we observe how the first attachment style appears in a dynamic that goes through different levels depending on the global context and the verbal and non-verbal information that emerges from authority (bowlby, ) . groups with primarily disorganised attachments generate contexts where friction, punishment, and violence emerge quickly as a way to relieve anxiety, fear, and tension. when the primary attachments are associated with anxiety and ambivalence, the group is submerged in fear that spreads through networks infiltrating the psychocorporal world, facilitating extreme care behaviours alternated with reckless risk behaviours that increase fear and anxiety. systems with predominantly anxious-avoidant attachment tend to focus on demand and performance by amplifying effective control systems at the expense of body and emotions. these are subject to the dominance of reason, dissociating body and its messages controlling fear, anxietiy, and uncertainty. on the contrary, groups with predominantly secure attachments creatively and adaptively go through this turbulence at the pole of action, creating and recreating new realities and ways of living. then, the isolation gradually infiltrates the family system, so fear and emptiness take over the bonds, as a melody that silences with consumer products. therefore, an impossible gap associated with the absence of meaning and a deep fear of damage and death is attempted to be filled. so, time becomes a waiting time. simultaneously, we witness an institutional and organisational crisis with the ensuing collapse of credibility and trust. a massive disconnection of people who in this paralysis have lost their jobs confirms those premises about the hope and credibility violated, and although cognitively it is explainable emotionally and affectively, the experience is overwhelming. uncertainty, fear, and the experience of injustice increase in a context that is impossible to decode. on the other hand, teleworking has been a way of maintaining continuity of work and staying on the move. however, in many organisations, this system has forced workers to be permanently available online. for executive women workers, the demand has increased exponentially due to the exercise of multiple roles that overlap and require time, effort, and dedication. uncertainty in an emotional, relational, social, global context reduces security for people; groups, institutions, when faced with the threat, exacerbate control and defence mechanisms. they bring solutions that only increase the problem, generating fear and pain expressed in different ways. they deny mourning in the face of loss at all levels: stability, power, status, the lives of loved ones, and their own lives (sluzki, ) . and the body? often forgotten and uninhabited due to the predominance of the image, it becomes the repository of emotions that, given the context, are impossible to symbolise and integrate. raising these emotions to consciousness reminds us that we hurt ourselves in bonds and heal in them, so that it is possible to agree to feel enough fear for self-care and care for others. holding the bond of intimacy that the therapeutic space provides in this transit that emerges as a new context from the face-to-face to virtual, implies an opening to newness. in turn, group networks that met in transit rituals establishing contact, providing support, direction, and meaning when assisting life and death, have also been impacted and injured. however, the voice and face have enhanced as a contact image and company (sluzki, ) . the question of my being a therapist . . . being also a midwife refers me to processes and learning; my history and its multiple resonances weaved into a systemic psycho-dramatic tapestry that includes myself and humanity. i feel that we cross a threshold similar to labour, being delivered and giving birth, simultaneously, in a channel whose timelessness is felt in our bodies. we are leaving a womb that could no longer contain us. today by force of contractions, we remain at times with fear, compressed by narrow walls that adhere to the personal, group, social and human body, with fears of harm and death. simultaneously and in another polarity, with an unknown force and with the survival instinct to the maximum, we open the virtual space in search of the exit. ª australian association of family therapy where are we going? just like before we were born, it is a mystery; however, from another perspective i know that we will look to another territory, with keys, codes, and ways of survival different from what is known. a place where we will need to put into operation new approaches that, probably without much awareness, we have developed in this previous gestation process. these are bodily, personal, group, social, and of humanity. i feel that, as in any process, we may reach the other side crying, it is also possible that we remain detained in a space 'in-between,' without being able to advance. so, as humanity we are at risk; it is the essential trust, the conscience and bonding that sustains us to arrive someday at that 'other side. ' nowadays, for me to be a therapist is to be a midwife, creating contexts that, in the intimacy of psychotherapy, allow me to accompany in uncertainty, in fear, in the pain of losses, in silence and respect for the expression of grief. i trust that the strength of the bonds will allow us to be born to other unimaginable dimensions from the prisms in which we contact today. i only miss hugging my son and daughter. i carry with me the nostalgia and smell of their bodies. the loneliness of the therapist . . . my being a therapist cuts across the many roles i carry out. with maturity and conscience, the person of the therapist talks and integrates with my entire person. on one level, i feel lonely, but, on another, it is a joy to feel the strength of the bonds of systemic therapists around the world to reach out. this unique practice collection offers readers a glimpse into the professional and personal experiences and reflections of an international group of family and systemic therapists across the globe as they experience the first phase of the covid- pandemic. the world is navigating through unpredictable times. therapists need to 'be,' before they can 'do.' systemic therapists are able to heal and in turn support the healing of the families, couples, and individuals they work with through the process of reaching out to fellow therapists in shared experience. it is only then they can contemplate stepping into a next phase; the 'regenerative process' where they can rebuild their lives post-pandemic. there needs to be time to pose and consider; whether the familiar pre-pandemic 'normal' is the 'normal' that is desperately sought . . . or in fact whether this calls for the creation of new opportunities for social change. the pandemic illustrates the reality that society remains at its essence 'collectivist.' we are all in this together, as a collective humanity. it is evident humans are inter-dependent on one another. there is an inescapable inter-connectedness and relationship between systems, within the body, between one another; humanity cannot separate from the environment, just as therapists cannot separate from their families or fellow therapists. this crisis may assist therapists to rearrange the scale of values regarding what is important. beyond the interventions used, preservation of the relationship is what really matters. it seems ironic that an enforced need to 'stay apart' from one another (in order to stay alive), has birthed an invitation to be more human (in order to stay emotionally and relationally alive), and be closer to each other than ever before. this collection illustrates now more than ever the importance of looking at the 'macro' issues presenting for people and society from a systemic perspective. the more complex the issues, the more important they be considered and addressed through a systemic lens. approaching these complexities from a sequestered, individual perspective is reductionist, invalidating, unrealistic, but also disrespectful to other cultures. this present challenge also causes a questioning of the notion of permanence and certainty to give room to impermanence and uncertainty; while distressing, and unsettling, this provides opportunities. this is an unpredictable and crucial time. if there has ever been a need for systemic therapists and the 'world of systems' to advocate for systems change, this is the time. humanity is part of a system that can destroy it in a short time if it does not listen to the feedback that comes from the interaction with the other elements of that system. as the pathway to the 'other side' is navigated, there is a need to value context, culture, collaboration, and connectedness in order to combat isolation and trauma while enhancing unity. together we can begin to think about some of the implications and recommendations for family therapy practice and research in relation to covid- . first, family and systemic therapists are in a key position to advise stakeholders such as governments and health departments in developing and implementing a response to the covid- pandemic. second, there are numerous advantages to understanding the effects of the pandemic through a systemic lens. it is unrealistic, illogical, and unscientific to imagine that complex issues like covid- can be considered simply by focusing on individuals. third, this suggests an integrated approach to the management of pandemic trauma and suicide prevention utilising 'systemic thinking' as a foundation. future collaborative research could focus on: the collective nature of trauma to consider the consequences of traumatic events shared by a 'social collective,' and how this may differ from 'interpersonal trauma'; mental health consequences that take into account the impact of pre-existing and co-existing mental health issues; the relational consequences of covid- in exploring whether collective traumas create greater resilience given the collective shared experience; further rigorous qualitative and phenomenological studies to capture the experiences of family therapists honouring different cultural backgrounds and languages. associate professor of psychology, subprogram in clinical psychology, department of psychology director of the paduan center of family therapy, academic of the milanese center of family therapy. email: mosconia @gmail.com member ipscan (international society for prevention of child abuse and neglect il multi-linguaggio e il multi-tempo dell'amore: il lavoro con le coppie interculturali (the multi-language and the multi-time of love: work with intercultural couples). paper presented at the convegno residenziale apf 'il processo terapeutico. tempi e fasi della terapia familiare my story, your story: the role of culture and language in emotion expression of cross-cultural couples. the mi culture model reflection on aaft family therapy conference getting together with like minded people': a conference edition habla mi idioma? an exploratory review of working systemically with people from diverse cultures: an australian perspective children in the margins training the person of the therapist in structural family therapy mind and nature: a necessary unity el apego (attachment). barcelona: paid os treating complex traumatic stress disorders: an evidence-based guide revisione dei concetti di ipotizzazione, circolarit a, neutralit a: un invito alla curiosit a (hypothesizing, circularity, and neutrality revisited: an invitation to curiosity) autoethnography, personal narrative, reflexivity: researcher as subject hypnotherapy: an exploratory casework getting a kick out of you: the jazz taoist key to love the new normal: living with a transformed reality the helpers and the helped: viewing the mental health profession through the lens of love in action: an integrative approach to last chance couple therapy the tao of love: life lessons learned from laotzu and the way of life suicide risk and prevention during the covid- pandemic integrating a trauma lens into a family therapy framework: ten principles for family therapists the five stages of grief the way of life one service, many voices: enhancing consumer participation in a primary health service for multicultural women the use of self of the therapist families and family therapy v ınculo, percepci on y conciencia en la coordinaci on grupal: la persona del coordinador (link, perception and consciousness in group coordination: the person of the coordinator) g enogramme et inceste: tempo th erapeutique et tempo judiciaire, in i les g enogrammes aujourd'hui la clinique syst emique en mouvement quand la famille marche sur la tête: inceste, p edophilie, maltraitance (when the family walks on your head: incest, pedophilia, abuse) la fin des certitudes. temps, chaos et lois de la nature (the end of certainties. time, cahos and the laws of nature) how family therapy stole my interiority and was then rescued by open dialogue memory, trauma, and phantasmagoria in claudia llosa's 'la teta asustada paradosso e controparadosso. un nuovo modello nella terapia della famiglia a transazione schizofrenica (paradox and counterparadox. a new model in schizophrenic transaction family therapy) ipotizzazione, circolarit a, neutralit a: tre direttive per la conduzione della seduta (hypothesizing, circularity, neutrality: three guidelines for the conductor of the session) la red social: frontera de la pr actica sist emica observing systems the authors look forward to further systemic themed papers on the family therapy response to covid- such as focusing on the regenerative phase of the pandemic and the reporting of practice and practical responses. lyndon amorin-woods for assistance in preparation of the draft manuscript.