Conclusion: In this review we summarize how platelets drive such pleiotropic actions and attempt to reconcile the paradox of platelets being both deleterious and beneficial to liver function; with increasingly novel methods of manipulating platelet function at our disposal, we highlight avenues for future therapeutic intervention in liver diseaseGrowing lines of evidence recognize that platelets play a central role in liver homeostasis and pathobiology. Platelets have important roles at every stage during the continuum of liver injury and healing. These cells contribute to the initiation of liver inflammation by promoting leukocyte recruitment through sinusoidal endothelium. They can activate effector cells, thus amplifying liver damage, and by modifying the hepatic cellular and cytokine milieu drive both hepatoprotective and hepatotoxic processes. .  CCLchemokine (C‐C motif) ligandCDcluster of differentiationCTLcytotoxic T lymphocyteCXCLchemokine (C‐X‐C motif) ligandHGFhepatocyte growth factorHSChepatic stellate cellHSEChepatic sinusoidal endothelial cell5HT5‐hydroxytryptamineIL‐6interleukin‐6NAFLDnonalcoholic fatty liver diseasePDGFplatelet‐derived growth factorTGF‐βtransforming growth factor‐βTNF‐αtumor necrosis factor‐αVEGFvascular endothelial growth factorTraditional paradigms of platelet function focus on the role of platelets in mediating hemostasis at points of endothelial disruption or vascular damage. The archetypal platelet role, starting from adhesion to the damaged vessel wall followed by activation and aggregation triggering the clotting cascade, has been well described; and antiplatelet therapy blocking one or multiple points of the above sequence is the cornerstone of pharmacological therapy aimed at preventing arteriothrombotic vasculo‐occlusive events. However, antiplatelet therapy is often halted or suspended in patients with chronic liver disease due to the association with a coagulopathy. The relatively common occurrence of portal venous thrombosis in patients with cirrhosis argues against such a strategy, and it is likely that the bleeding diathesis associated with chronic liver disease is overstated.in vitro studies highlights a role for platelets far beyond the confines of hemostasis as active players in liver inflammation  demonstrate that platelet adhesion is partly integrin (GPIIb/IIIa and αVβ3)‐mediated,Hepatic sinusoids are lined by unique fenestrated endothelial cells, which are exposed to only minimal shear stressPlatelet interaction with myeloid cells, particularly macrophages and neutrophils, has been extensively investigated in the context of cardiovascular disease, thrombosis, and atherosclerosis.In both humans and mice, neutrophils are recruited and activated during the acute phase of liver inflammation in response to a variety of injuries.Although the accepted paradigm is that, regardless of initial insult, neutrophils recruited to the site of liver damage exacerbate liver damage,In vitro studies using human cells demonstrated the ability of platelet‐derived CXCL4 to induce differentiation of blood monocytes to tissue macrophages.Macrophages are involved in both driving and helping resolve liver disease, being capable of mediating liver injury, fibrosis progression, fibrosis resolution,1 did not restore T cell‐mediated liver damage ‐mediated liver damage.in vitro by almost 50%,In vivo murine experiments examining the mechanisms of liver immunosurveillance by T cells reveal that platelets adhere to sinusoidal hyaluronan by CD44 promoting attachment of flowing effector CD8 T cells to the vessel wall .Depletion of platelets protects against cholestasis‐induced hepatic inflammation and injury, suggesting that platelet interactions within the microvasculature may be important in cholestasis‐induced liver damage.However, as with other injury models, the role of platelets in cholestatic liver injury evolves with the development of chronicity. Although perpetuating acute inflammation early on, in the late stages of cholestasis platelet activation is hepatoprotective. Inducing a thrombocytopenia in mice during the latter stages of chronic cholestasis therefore worsens liver function by causing hepatic fibrosis Table .61 StudiPlatelets are critical regulators of liver regeneration. After hepatic resection, platelets sequester at the resection margins and orchestrate the complex ontogenic processes necessary for functional hepatic architecture to develop.Platelet‐mediated hepatic regeneration is dependent on their ability to bind to sinusoidal endothelium, interact with Kupffer cells, and traverse the space of Disse to interact with the hepatocytes,In vitro studies with cultured HSECs reveal that platelets promote endothelial production of interleukin‐6 (IL‐6) and vascular endothelial growth factor (VEGF) through sphingosine 1‐phosphate. These two proteins simultaneously inhibit apoptosis and stimulate hepatocyte proliferation  2A and 2B messenger RNA is observed in the liver. Antagonism of these serotonin receptors (5HT 2A and 2B) inhibits liver regeneration.In the late stages of liver injury, however, HSCs participate in termination of the regeneration process and actually promote fibrosis. At this time point, platelet‐derived serotonin interacts with HSC 5‐HT 2b receptors stimulating HSC TGF‐β expression.Overall the contribution of platelets in general and platelet‐derived serotonin specifically favors regeneration or liver repair, but it is important to note that regeneration and fibrosis are both part of the repair response to injury. The ability of platelets to influence both processes demonstrates how pleiotropic these cells are. Platelets are crucial for regeneration to occur, and after acute liver injury, this is their dominant contribution. However, as liver damage approaches chronicity, platelets participate in fibrogenesis and actively block regeneration, in part through serotonin‐driven TGF‐β expression. Whether platelets provide predominantly promitogenic or profibrotic signals is determined by the cellular and cytokine microenvironment specific to the stage and type of liver injury.Platelets play a central role both in liver homeostasis and in the response of the liver to injury. The complexity of this role is becoming increasingly apparent as advances in genomic, proteomic, and transcriptomic analyses enable researchers to define roles for platelets beyond hemostasis Table .Recent work suggests that aspirin reduces the risk of liver fibrosis in patients who have been transplanted for hepatitis C.Given the complex and dualistic roles played by platelets outlined above, the evidence for recommending when to use antiplatelet therapy in liver disease is far from complete. It is important that future preclinical studies delineate the precise molecular basis of platelet activation and the functional consequences in the context of liver injury. Only by understanding the disease and disease stage‐specific platelet contribution will we be able to design logical and safe interventions with antiplatelet therapies. Evolving understanding of how platelets mediate inflammatory damage, modulate vascular integrity, and interact with leukocytesWith the repertoire of antiplatelet therapy already available, multiple potential avenues of novel treatment are possible. As Friedman et al. aptly state, “Still, it is heartening that platelets, which have always been present in the scene of hepatic injury during disease, are now also considered as actors in the scene.”Author names in bold denote shared co‐first authorship.


PLA) research approaches show promise, but there has been no detailed analysis of stakeholders’ and researchers’ experiences of PLA techniques for data generation and co‐analysis.The material practices which researchers use in research partnerships may enable or constrain the nature of engagement with stakeholder groups. Participatory learning and action  to enhance communication in cross‐cultural primary care consultations. We developed a purposeful sample of 78 stakeholders  from primary care settings in Austria, England, Greece, Ireland and The Netherlands. We used speed evaluations and participatory evaluations to explore their experiences of two PLA techniques—Commentary Charts and Direct Ranking—which were intended to generate data for co‐analysis by stakeholders about the GTIs under analysis. We evaluated 16 RESTORE researchers’ experiences using interviews. We conducted thematic and content analysis of all evaluation data.The PLA Commentary Charts and Direct Ranking techniques, with their visual, verbal and tangible nature and inherent analytical capabilities, were found to be powerful tools for involving stakeholders in a collaborative analysis of GTIs. Stakeholders had few negative experiences and numerous multifaceted positive experiences of meaningful engagement, which resonated with researchers’ accounts.PLA techniques and approaches are valuable as material practices in health research partnerships. Meaningful engagement reduces asymmetries of power, encourages participants’ ownership of the project and enables participants’ authentic perspectives to emerge clearly in research outcomes.“Material practices,” such as the types of methods and techniques that researchers use to involve stakeholders, can enable or constrain participation in research.Research to identify the best methods to achieve meaningful engagement is currently lacking.Tierney et al.'sParticipatory health research is an overarching term that refers to “bottom‐up” research approaches specifically designed for stakeholder involvement in research partnerships. These include, among others, participatory research (PR),PLA mode of engagement and PLA techniques. A PLA mode of engagement aims to create a trusting relational environment, a “safe space” where stakeholders are encouraged to respect a diversity of views and experiences, and to learn from each other's perspectives.Participatory approaches face challenges, such as the need to see community participation as a long‐term process of implementation and support for improved health outcomesPLA techniques evolved originally from PRA and are based on a shared stock of ideas and experiences from participatory trainers and stakeholders around the globe. They continue to be adapted to specific contexts as required.structured, integrated process. Stakeholders’ priorities and perspectives are meant to guide the generation and co‐analysis of data about the issue being explored, with researchers acting as catalysts rather than directors or top‐down decision‐makers.There are some recent positive examples of PLA applied to primary care health research. These studies describe meaningful involvement of migrants and other stakeholders in the development of a guideline to improve communication in cross‐cultural consultations;In this paper, we describe the use of two PLA techniques (Commentary Charts and Direct Ranking) used for data generation and co‐analysis, and the perceived utility of these by various stakeholders and researchers involved in a recent European primary health‐care implementation project.22.1RESTORE (Research into Implementation Strategies to Support Patients of Different Origins and Language Background in a Variety of European Primary Care Settings) was an EU‐funded primary health‐care research project that ran from 2011 to 2015. The objective of RESTORE was to investigate and support the implementation of guidelines and training initiatives (GTIs) intended to enhance communication in cross‐cultural primary care consultations. This qualitative, comparative case studyStage 1. Stakeholders were informed about RESTORE and invited to participate. Researchers mapped GTIs that were available in each RESTORE project country.Stage 2. Stakeholders examined a set of these GTIs that showed potential for implementation in their country and selected one that they deemed most suitable or relevant for their local primary care setting.Stage 3. Stakeholders successfully adapted their selected GTI at a local level and worked on its implementation, with evidence of some impact on daily practice.A detailed description of the study protocol is available elsewhere.RESTORE was the overall setting in which we explored and evaluated stakeholders’ and researchers’ experiences of two PLA techniques. These techniques were employed during Stage 2 and were intended to enable stakeholders  to work collaboratively and with RESTORE researchers to select a GTI for their local primary care setting.First, we describe sampling and recruitment for RESTORE—this is the sample for the evaluation reported in this paper. We then describe the two specific PLA techniques employed in RESTORE and the methods used to evaluate stakeholders’ and researchers’ experiences of these techniques. Finally, we present our analysis of the evaluation data.2.2In Stage 2, we used a combination of purposeful and network sampling to identify and recruit 78 stakeholder representatives across five research sites. A geographically defined area (district) was selected in each partner country. Selection was pragmatic, based on researchers’ knowledge of groups working in the district and proximity to the research teams, to facilitate data collection. Eligible organizations/agencies were those involved in primary health‐care planning and delivery  as well as those addressing migrant health issues . The aim was to identify individuals who were decision‐makers , service providers , primary care staff, community interpreters) or service users .In line with standard ethical procedures, stakeholders in all countries were provided with information leaflets and signed consent forms prior to participating in fieldwork sessions.2.3Two members of the RESTORE consortium in the Irish team PLA mode of engagement and PLA techniques in a PLA‐brokered dialogue between stakeholders. One of the striking features of PLA in general is the highly visual nature of the techniques used. Stakeholders work together to generate maps, charts and diagrams which function as powerful reference points (data displays) as they engage in verbal interaction, discussing, questioning, and learning from each other's perspectives, adding new data to maps and charts.inherent analytical capabilities of PLA techniques aim to enable stakeholders to assess, correlate, categorize and/or prioritize data they are co‐generating. PLA techniques, therefore, have the capacity to facilitate meaningful engagement that automatically incorporates co‐generation and co‐analysis of data “by” and “with” stakeholders. In a very practical way, then, stakeholders using PLA techniques engage in a structured, integrated, visual‐verbal‐tangible process of co‐generating and co‐analysing data which produces visual‐tangible results.The PLA process for RESTORE was based on a The two PLA techniques we used in RESTORE in Stage 2 were Commentary Charts and Direct Ranking. A Commentary Chart is an interactive knowledge‐exchange, knowledge‐enhancement technique which allows stakeholders to exchange differential knowledge, expertise and perspectives. Box Box 1Stakeholders record key data on Post‐It notes about the issue of interest. This provides a visual representation of their co‐generated data.‐analysis process.The Post‐Its are assigned to the relevant category on the chart. These categories may be determined before or during the sessions. This is the start of the coResearchers and stakeholders consider and discuss the emerging and completed Commentary Chart. This process automatically incorporates co‐generation and co‐analysis of data “by” and “with” stakeholders.Does the Commentary Chart make sense? Are stakeholders comfortable with their data display? Is there anything striking/odd about the data display? Are diverse views sufficiently and accurately represented? Does anything need to be added as we reflect on the Chart? Are stakeholders willing and content to “sign off” on the Chart? Can it now be presented to another stakeholder group (as needs be) for discussion and development?Researchers and stakeholders continue the co‐analysis with a visual‐verbal‐tangible process of “interviewing” the chart. The emphasis is on looking at the Commentary Chart and encouraging stakeholders to share their unique knowledge and insights, to exchange differential knowledge by asking: Physical objects and/or images are selected to represent the issues/entities being ranked. This provides a visual focus for the co‐analysis process.Stakeholders place the selected images randomly on a large flipchart sheet, to give each image equal visual “weight” and importance.Stakeholders engage in co‐analysis and clarify what the ranking criterion will be.Stakeholders discuss each object/image in relation to the agreed ranking criterion, listening, learning, questioning, reflecting and assessing, thus continuing the integrated processes of data generation and analysis.When discussion is complete, each stakeholder is provided with an equal number of “votes” .Stakeholders distribute their votes across the images.Stakeholders count the number of votes assigned to each image.Results are double‐checked, recorded in numerical form on “Post‐It” notes and attached to the relevant images.Stakeholders draw a line down the centre of the flipchart sheet and place the images on the line: highest scoring image at the top, lowest at the bottom, others in between in positions dictated by number of votes accrued.Do stakeholders feel comfortable with the outcome? Is there anything striking/odd about the result? Having been decided by democratic vote, is the result definitely acceptable? Are stakeholders willing and content to “sign up” to the result?Researchers and stakeholders continue the co‐analysis with a visual‐verbal‐tangible process of “interviewing” the results of the Direct Ranking process. The emphasis is on looking at the Direct Ranking chart and encouraging stakeholders to share their unique knowledge and insights, to exchange differential knowledge by asking: does it make sense? In RESTORE, PLA Commentary Charts were used to facilitate dialogue among stakeholders about the GTIs mapped in Stage 1. Full‐text copies, summaries and/or PowerPoint presentations of the identified GTIs were made available to stakeholders. The Commentary Charts comprised three analytical categories—”positive aspects of the GTI,” “negative aspects of the GTI” and “questions to be checked out.” Stakeholders recorded their key deliberations, perspectives and insights about each GTI on the Commentary Chart  to document their experiences of involvement in their PLA sessions. A “speed evaluation” is a brief verbal  or written evaluation, which provides an opportunity for stakeholders to describe experiences in their own words and suggest areas for improvement. It allows researchers to “take the temperature” of the group, to build on positives, and, where possible, to plan suggested improvements for forthcoming PLA sessions. Speed evaluations usually occur at the close of a PLA process or session. Participants respond to an open‐ended question in a rapid, interactive and spontaneous way.yourexperience of this.”For example, on completion of Commentary Charts, the question was as follows: “During this session, we worked together in a participatory way: listening, learning, creating our Commentary Charts. In a word or phrase, please comment on Stakeholders at the Irish site also completed a participatory evaluation (PE) because the facilitators there had extensive experience of this technique. This form of more in‐depth collaborative evaluation is judgement‐based and may be formative or summative. Stakeholders responded to questions focused on Direct Ranking and their experiences of engagement in the PLA process. An “open category” question also invited stakeholders to comment on any aspect of their experience, especially if there were any improvements and/or changes that could be made.Speed and participatory evaluations were either noted or audio‐taped and transcribed at each site, and collated as reports by the researchers. We had ethical approval to include data from all of these reports in the analysis for this paper, apart from the data from the English site.All academic researchers involved in PLA fieldwork participated in in‐depth reflection interviews (R Int), conducted by the PLA trainers, at the close of Stage 2 fieldwork (n=16). Interviews were conducted face‐to‐face or by Skype; interview length ranged from 50 minutes to 1 hour 45 minutes, depending mainly on the number of researchers involved . The interview schedule was circulated to all teams in advance. They were asked to reflect on the use of Commentary Charts and Direct Ranking with reference to their experiences of the techniques and their observations of stakeholders’ experiences of them , we achieved a measure of triangulation, or cross‐validation. As per Lincoln and Guba,prolonged involvement of researchers in fieldwork for “trust‐building” and “knowing the culture,” coupled with persistent observation of stakeholders’ reactions to PLA methods and peer debriefing by research teams when producing regular field reports, contributed to study depth. This, in conjunction with the triangulation or cross‐validation mentioned above, contributed to and enhanced the trustworthiness, credibility and dependability of the study.Using different enquiry techniques  to explore researchers’ 33.1There was appropriate representation of stakeholders at each site by gender, age group, country of origin and type of stakeholder group  during the fieldwork period (September 2012 to May 2013).There was strong representation of the academic research team, as 16 of 18 individuals participated in PLA fieldwork and its evaluation (see Table 3.2We identified five interrelated themes of stakeholders’ experiences that elucidate their positive perspective on the PLA techniques (see Table (1) meaningful engagement in a “safe space”: active inclusion, collegiality, collaboration. They reported that group dynamics were positive. The working environment was considered safe, allowing stakeholders to readily and safely express diverse views in a relaxed and enjoyable manner. This was also noted and commented on by researchers.Stakeholders described their overall involvement in the PLA process of co‐generation and co‐analysis in ways that speak powerfully of (2a) enhanced learning throughout the process of using Commentary Charts and Direct Ranking. The Commentary Charts facilitated exposure to each other's perspectives about the GTIs and contributed to positive experiences of enhanced learning. Researchers considered that this placed stakeholders in a more informed position from which to prioritize GTIs during Direct Ranking. For example, in Ireland, details about time demands in general practice surgeries were described by the general practice stakeholders. This enhanced other stakeholders’ understanding of the clinical setting and impacted on their assessment and ranking of the GTIs that they were examining (see File Stakeholders described how there was (2b) enhanced learning led to important new understandings and, on occasion, resulted in shifts away from long‐held positions and towards new possibilities. This suggests that the sharing of diverse perspectives during co‐analysis enabled stakeholders to broaden their horizons. Researchers’ comments reflected stakeholders’ sense of the “flow” between the two techniques. Researchers noted the interactive energy of stakeholders’ involvement in a PLA process that generated a “win‐win” outcome.It was clear that (3) democracy‐in‐action. Stakeholders enjoyed positioning GTIs and Post‐It notes (stickies) and distributing their 20 “paper clip” votes on the chart. Asymmetrical power relations were balanced in part by the fact that 20 votes per stakeholder meant equal voting power and equal opportunity to influence the outcome. Finally, by arranging the GTI images according to the vote count, the democratic outcome was immediately accessible to all and the chart showed the important result stakeholders had achieved.Furthermore, the visual nature of Direct Ranking allowed stakeholders to engage in a nuanced decision‐making process which was demonstrably democratic. This indicated Researchers noted the equalizing power of PLA, “levelling the playing field,” and that stakeholders with a lower level of language skills were not disenfranchised—they could “see” the result; the presence (density) compared to absence (paucity) of paper clips visually expressed what a numerical count confirmed.(4) power, ownership. Researchers commented on the genuineness/authenticity of stakeholders’ interactions—the willingness of some stakeholders to put others before themselves. This was even the case where the vested interests of typically powerful stakeholders might be expected to win the day, but where choices inclusive of all stakeholders actually won out.Stakeholders recognized that their choice represented a fundamental input into the progress and trajectory of the research. Exercising power in this way contributed to a genuine sense of “ownership” of the research project: (5) sustained engagement. Direct Ranking provided opportunities for stakeholders to experience “positive power,” and researchers noted that they often linked this to the “light” side of PLA: energy, enjoyment, fun and achievement. This “lightness” seemed to offset research fatigue and contributed to sustained meaningful engagement of stakeholders in Stage 2 research.An important outcome was Negative comments were also identified. Speed evaluation data from The Netherlands comprised a total of 21 stakeholder comments. Of these, there were five negative responses. Speed and participatory evaluation data from Ireland, where evaluations were more extensive, generated 80 stakeholder comments. There were two negative responses. No negative comments were recorded in data from Greece or Austria. However, our content analysis showed that five of 15 stakeholders in Austria chose not to offer evaluation comments of any nature.“Slow, too labour‐intensive.” Netherlands (NL) Nurse SE“Useful meeting, the presentation can be done faster.” NL General Practitioner SE“Learning‐full, but I felt a little like an outsider not involved in daily practice.” NL Practice Manager SE“Nice! Maybe without migrant participants there might be more space for interaction.” NL SH#76 SEAnalysis of the negative comments about the PLA techniques shows that the process was considered overly time‐consuming by some Dutch clinical stakeholders. There was also a reduced sense of inclusion for some stakeholders in The Netherlands due to the interstakeholder representation in the PLA sessions.“I am not sure of the potential benefits of assessing GTIs.” Ireland (IRL) Community Interpreter SE“Not clear on the outcome of the session/what we want to achieve by assessing GTIs?” IRL Community Interpreter SEThe other negative comments were RESTORE‐specific (rather than about the PLA processes per se) and came from The Netherlands and Ireland, early in Stage 2. They related to the view that the summaries of the GTI were too brief and needed to be longer to make better judgements, and a degree of uncertainty about what the objective of the process was.4In this paper, we have explored the perceived utility of two PLA techniques—Commentary Charts and Direct Ranking—for data generation and analysis with a diverse sample comprising migrants, general practice staff, community interpreters, service providers, service planners and academic researchers.4.1negative experiences, and numerous multifaceted positive experiences of meaningful engagement: PLA created a “safe space” and a trusting environment in which they learned from each other's perspectives, gained enhanced knowledge via the co‐generation of Commentary Charts and used these data to inform their co‐analysis during Direct Ranking. Using these two PLA techniques involved stakeholders in an experience of “democracy‐in‐action” which was empowering and energizing, promoting a sense of ownership and sustained engagement in the research project.Our findings show that Commentary Charts and Direct Ranking techniques, with their visual nature and inherent analytical capabilities, were experienced by stakeholders from both community and health‐care settings as powerful tools for collaborative decision making. There was consensus among stakeholders and researchers that there were few 4.2There is limited knowledge about suitable methods for involving stakeholders in a meaningful (rather than tokenistic) way in health research partnerships.how PLA techniques are capable of delivering this—their combined visual, verbal and tangible nature and inherently analytical capabilities involved stakeholders in structured, integrated co‐generation and co‐analysis of research data, which delivered practical democratic results at all five sites. This empirical evidence about the capacity of PLA to “deliver” meaningful engagement in data generation and co‐analysis is a significant addition to a literature that calls for methodological innovation in this sphere. These findings provide important empirical data about experiences of participation that are lacking in the literature.Furthermore, we provide new evidence about common ground upon which democratic decision making took place. This is an important function of PLA: to “level the playing field” where asymmetric power relations between stakeholders/stakeholder groups may exist.In keeping with Tierney et al.,Our results show that this levelling also occurred between stakeholders and researchers: throughout the Commentary Charts and Direct Ranking, it was stakeholders who exercised power and took on the key responsibility of selecting a GTI for implementation, thereby “setting the agenda” for the final stage of RESTORE fieldwork. Researchers, in their capacity as catalysts, facilitated but did not control this process. This shared ownership and agenda‐setting takes us firmly beyond tokenism and towards a “shared power” approach, enhancing the research partnership.The rare, negative experiences reported by stakeholders in this study warrant attention: the time and pace of research, the need for comprehensive information to facilitate equitable participation, and the possibility that bringing different types of stakeholders together may, on occasion, actually reduce a sense of active inclusion for a minority. While concerns about time demands and tokenistic participation have been reported previously,4.3We were unable, for site‐specific ethical reasons, to include the use of stakeholder evaluation comments from the English site in our thematic and content analyses. However, we were able to ameliorate this by including comments from researchers’ reflection interviews.We cannot claim representativeness of findings for the qualitative study data presented here. However, we emphasize that in this comparative case study, spanning five European countries with very different primary care systems, the same PLA techniques were used and successfully involved diverse stakeholders in data generation and co‐analysis.Regarding the PLA techniques employed, there was variation in our use of PLA Commentary Charts: stakeholders reviewed the Commentary Charts together at the same time in some settings, while some were physically removed from one another and reviewing the Commentary Charts after others had done so. This may have created variation across the sites and this possibility would be worth exploring in future work., the brevity of speed evaluations does not encourage rich, lengthy, in‐depth stakeholder responses. The additional participatory evaluation conducted did provide richer data, while researchers’ reflections also added to the quality and depth of evaluation data. Therefore, we suggest using an array of PLA evaluation techniques in future projects to explore all stakeholders’ experiences of involvement in greater depth.Regarding evaluation methods employed: speed evaluations are efficient and the key messages recorded were similar to those in the additional participatory evaluation conducted at the Irish site. HoweverRESTORE ended before our thematic analysis of stakeholders’ evaluation data took place, and we did not, therefore, benefit from their contribution to the development of codes and categories for thematic analysis, nor from their potential insights about the relevance and veracity of evaluation results. While we are confident that data saturation was achieved as the analysis reached a point where the codes and themes were comprehensive, we acknowledge the lack of member checking. In future projects, it would be apposite to invite stakeholders to co‐generate evaluation criteria and to co‐analyse the results of evaluation data, thus closing the circle of “involvement.”To add to the evidence base, we need further research and evaluation to explore whether and how PLA techniques might work when applied in projects with very different research foci and stakeholder groups to those in RESTORE. The specifics of involving community and health sector partners in analysis of other qualitative methods, such as interviews and focus groups, would also be valuable.5PLA Commentary Charts and Direct Ranking techniques, with their distinctive visual, verbal and tangible nature and inherently co‐analytical capabilities, are rated very positively by stakeholders and researchers. The positive benefits gained from the PLA process in this study  outweighed the negatives. The significant additional investment of resources was impactful and was worth the time and effort. Therefore, we recommend the use of these two PLA techniques as material practices to enable collaborative decision making and meaningful engagement in health research partnerships.None to declare. Click here for additional data file. Click here for additional data file. Click here for additional data file.


HIV‐1 management of persons on antiretroviral therapy (ART) has been recommended by the World Health Organization (WHO) to identify treatment failure. However, viral load testing represents a substantial cost in resource constrained health care systems. The central challenge is whether and how viral load monitoring may be delivered such that it maximizes health gains across the population for the costs incurred. We hypothesized that key features of program design and delivery costs drive the cost‐effectiveness of viral load monitoring within programs.Routine viral load monitoring for LMICs). We followed the Cochrane Collaboration guidelines and the PRISMA reporting guidelines.We conducted a systematic review of studies on the cost‐effectiveness of viral load monitoring in low‐ and middle‐income countries  Use of effective, lower cost approaches to viral load monitoring (e.g. use of dried blood spots); 2) Ensuring the pathway to health improvement is established and that viral load results are acted upon; and 3) Viral load results are used to simplify HIV care in patients with viral suppression . Within the context of differentiated care, viral load monitoring has the potential to double the health gains and be cost saving compared to the current standard (CD4 monitoring).We identified 18 studies that evaluated the cost‐effectiveness of viral load monitoring in HIV care is likely to be cost‐effective. Viral load monitoring in differentiated care programs provides evidence that reduced clinical engagement, where appropriate, is not impacting health outcomes. Introducing viral load monitoring without differentiated care is unlikely to be cost‐effective in most settings and results in lost opportunity for health gains through alternative uses of limited resources. As countries scale up differentiated care programs, data on viral suppression outcomes and costs should be collected to evaluate the on‐going cost‐effectiveness of viral load monitoring as utilized in practice.The cost‐effectiveness of viral load monitoring critically depends on how it is delivered and the program context. Viral load monitoring as part of differentiated  An emerging stream of research has shown that, for general health care, values are much lower than previous acknowledged – for example ~$60 to 100 in Malawi – due to high levels of unmet health needs We conducted a review of recent studies on the cost‐effectiveness of viral load monitoring in LMICs. Our hypothesis was that key assumptions about program design and costs of monitoring drive the cost‐effectiveness of programs that incorporate viral load monitoring. The primary review objective was to identify characteristics of programs that make viral load testing more or less likely to be cost‐effective and contribute to improvements in population health, recognizing there are other calls on limited HIV program resources.2We reviewed cost and cost‐effectiveness studies of viral load testing in HIV treatment programs in LMICs and summarized the key factors that determine cost‐effectiveness. We followed Cochrane Collaboration guidelines in conducting our review 2.1We conducted an electronic search of the PubMed database on 14 February 2017 for studies published from 1 January 2000, onwards, using the following MeSH terms:  OR “viral”(All Fields) AND “load”(All Fields) OR “viral load”(All Fields)) AND (“economics”(Subheading) OR “economics”(All Fields) OR “cost”(All Fields) OR “costs and cost analysis”(MeSH Terms) OR (“costs”(All Fields) AND “cost”(All Fields) AND “analysis”(All Fields)) OR “costs and cost analysis”(All Fields)). The EMBASE data base was searched using the equivalent search terms. Reference lists of papers meeting criteria were hand searched for additional articles. To ensure that we included unpublished data, abstracts were reviewed from the past meetings of the Conference on Retroviruses and Opportunistic Infections (CROI).Abstracts and full‐text articles of potentially relevant studies were reviewed independently by two authors (N.T. and R.V.B.) against pre‐defined criteria. Papers were eligible for inclusion if the analysis was conducted in a LMIC setting and a cost‐effectiveness result for viral load monitoring was included. Data were extracted using a standardized data extraction form. Discrepancies regarding eligibility of papers were discussed and consensus reached. The methodological quality of included studies was reviewed by N.T. and R.V.B. Discrepancies in quality rating were discussed and consensus reached. Studies were rated as low, moderate, or high risk of bias, dependent on whether they met standard guidelines for health economic evaluation reporting 3The electronic search yielded 1248 results of which 1165 were unique abstracts. We identified 23 manuscripts and four conference abstracts for review of which 18 met the search criteria and addressed the cost‐effectiveness of viral load monitoring . Two health economic analyses were based on clinical trials conducted in Uganda and Cameron Cost‐effectiveness analyses were conducted from 2004 4We found three main factors that make it more likely for viral load monitoring to be cost‐effective Table : 1) Use 4.1Several factors can ensure the cost of viral load testing and the fully loaded cost  is as low as possible: choosing an efficient specimen for measuring viral load such as DBS, using an assay and threshold that strikes the right balance between the risks of missing detectable viral load and switching unnecessarily, and limiting the frequency of viral load monitoring In addition to the costs of viral load testing itself, analyses have shown that downstream costs (in particular second line ART) notably affect the cost‐effectiveness of viral load monitoring. Although costs of protease inhibitor based regimens have fallen markedly in recent years . Protocols that delineate the next steps for a client on ART with a detectable viral load for example adherence counseling, viral load retesting three months after adherence counseling, resistance testing (where available) if viral load is still detectable despite adherence, and switching to second line ART in a timely manner, increase clinical effectiveness and decrease the emergence and transmission of resistance mutations. Some clients may require more regular visits and monitoring for complex disease or comorbidities. Notably, even with viral load monitoring, switching to second line ART generally does not occur within the timeframe assumed by most mathematical models of HIV and the proportion of people who are on second line regimens is generally below 5% 4.3Differentiated care for HIV allows simplifying of protocols for persons well controlled on ART, with client responsive viral load testing  providing feedback to inform on‐going individual and program effectiveness. This also supports viral load monitoring replacing CD4 count monitoring, spacing appointments, providing longer prescription refills, task shifting, community‐based ART, testing using DBS specimens to simplify specimen transport, and using clinical care resources for complex clinical cases and clients who are not suppressed on ART. In this differentiated care context, viral load monitoring enables greater comfort with less clinical engagement. The cost of viral load testing can be offset by savings in the clinical visit costs. Thus, differentiated care with viral load monitoring can save costs with greater health gains compared to standard of care clinic management  and the incremental health benefits (e.g. QALYs‐gained or disability adjusted life years (DALYs)‐averted); then assessing whether the cost‐per‐unit of health improvement represents sufficient value, compared to other claims on limited resources. These can all change depending upon how and where viral load monitoring is delivered, so any universal claims to cost‐effectiveness are misguided and it is vital to understand the place of viral load monitoring within HIV programs and how it may facilitate design of programs to improve population health from within the resources available.Cost‐effectiveness studies aim to inform the allocation of limited resources. This requires estimating the The results table Table  illustra4.4Lastly, notable programmatic changes are likely to impact cost‐effectiveness of viral load testing, specifically changing to ART regimens with a high barrier to resistance 5The cost‐effectiveness of viral load monitoring is critically dependent on context. Viral load monitoring in differentiated care programs provides evidence that reduced clinical engagement, where appropriate, is not impacting health outcomes The author have no competing interests.RVB, PR, and AP oversaw the review. RVB wrote the first draft of the paper, which was revised by all authors. NT did the electronic searches and reviewed the abstracts, with guidance from RVB. All authors contributed to design and execution of the review, as well as to the interpretation of findings. All the authors approved the final version of the paper for submission.


Reduced funding to public health care systems during economic downturns is a common phenomenon around the world. The effect of health care cost on family members of the patients has not been established. This paper aims to explore the relationship between affordability of health care and vulnerability of family members to distress levels.Data of a total of 262,843 participants were obtained from 17 waves (1997–2013) of the United States National Health Interview Survey. Multinomial logistic regression was used to investigate psychological distress level as a result of having family members who experienced unmet medical needs due to cost.Among participants without family members who experienced unmet needs for medical care due to cost, risks of having ‘moderate’ (score of 5–12) or ‘serious’ (score of 13 or above) level of psychological distress were 1.0% and 11.5%, respectively. Risks of having ‘moderate’ or ‘serious’ level of psychological distress were 3.1% and 23.4%, respectively among participants with family members who experienced unmet needs. The adjusted relative risk ratio of ‘moderate’ and ‘serious’, as compared to ‘normal’ level of psychological distress, were 1.58  and 2.09  if one’s family members experienced unmet medical needs.Unmet medical needs due to cost increases risk of distress levels experienced by family members. Careful planning and adequate funding to public health care system could be implemented to prevent any unnecessary detrimental effect on mental health among family members of the unwell and any further increment of the prevalence of mental illnesses. This recommendation aligns with the World Health Organization Mental Health Action Plan 2013–2020. Health care systems in many developed countries are funded from public sources to provide equitable access to all based on needs . DespiteWhile poor access to health care can lead to negative impact on physical health of patients, the cost to access health care may also add to one’s family financial burden . The finA search through the PubMed, Science Direct and Web of Science databases using the following keywords: “unmet medical needs” AND “mental distress” AND “family member” AND “cost” from 1st January 2010 to 1st January 2017 yielded 426 articles. However, after a closer examination, there is no publication on this topic. It is therefore paramount to investigate the impact of unmet medical care and the vulnerability of family members to psychological distress. The aim of this study was to investigate the effect of having a family member with unaffordable medical cost on one’s psychological distress level.http://www.ihis.us). The NHIS was conducted by the National Center for Health Statistics (NCHS), Centers for Disease Control and Prevention to provide estimations of health indictors at national level [Data from 17 waves (1997–2013) of National Health Interview Survey (NHIS) were collated from Integrated Health Interview Series of United States National Health Interview Survey: Minnesota Population Center and State Health Access Data Assistance Center, Integrated Health Interview Series: Version 5.0. Minneapolis: University of Minnesota, 2012 .Multistage probability sampling technique was applied to select representative sample of 100,000 people of the wider population  every year and weighted for key demographic variables to assure representativeness of the sample  moderate or serious (K6 score ≥ 5) psychological distress level and ii) serious (K6 score 13–24) psychological distress level in the past 30 days were first assessed in two multivariate Poisson regression models. Psychological distress level was further classified into three categories based on their K6 score: 0–4 , 5–12 (moderate) and 13–24 (serious). Multinomial logistic regression was then used to investigate the effect of having family members who had unmet needs for medical care on their psychological distress level. In order to control for the potential confounding effect of participant’s health on his/her distress level, analyses were restricted to participants who: i) were living with two or more members in a single family household; ii) rated their health status as good, very good or excellent; and iii) were free from any limitation due to physical or mental problems, without unmet needs of medical care. Potential confounding factors such as age, gender, race, marital status, highest educational attainment and health status, whether having any family members with activity limitations, family income comparing to poverty threshold and year of survey were controlled for in the multivariate models. Participants with missing information on these potential confounding factors were excluded in the multivariate analysis. Data from a total of 262,843 participants were included. Sampling weight was applied in all analyses . SignifiThe risks of having moderate and serious (K6 score ≥ 5) or serious (K6 score ≥ 13) level of psychological distress were 23.4% and 3.1%, respectively among participants with family members who had unmet needs for medical care. The risks among participants without family members had unmet needs were significantly lower at 11.5% and 1.0%, respectively. In other words, having family members with unmet needs for medical care were associated with about 100% and 200% increase in the risk of having at least moderate and serious level of psychological distress, respectively.Overall the multivariate analyses showed that the risk of moderate or serious and serious psychological distress level were significantly higher  among participants who had family members with unmet medical needs Tables  and 2. EIn the past, reduction or removal of health care funding in some countries contributed to poorer patients’ health outcomes as they could not afford medical care due to cost. This study provides a new insight by directly measuring the relationship between unmet medical care, specifically due to cost, and the vulnerability of family members (not just the main care-giver) to psychological distress. In this study, it was found that health care cost, when linked to unmet medical needs, affected psychological distress level of family members. Specifically, family members of those with unmet medical needs due to cost are at a significantly higher risk of suffering from ‘moderate’ and ‘serious’ level of psychological distress, as assessed by the K6 scale. The risk is comparable to those with a history of epilepsy .A person may experience high psychological distress when they have to make decision when there are conflicts in the choice, and has to ‘trade-off between attributes’ . Family Adequate resources can maintain if not prevent ill health outcome . EffectiThe detrimental impact on the mental health of these family members is not exclusive to those living in the United States of America (USA) but everyone who has to undergo difficult decision making process, particularly when the demand of illness is not static  and therThis study used a large representative sample of the USA population. The conclusion is thus representative of the wider USA population because it provided a more accurate estimate of the risk of having psychological distress due to unmet medical needs of family members. Due to the nature of cross-sectional design, long term impact on the family members’ distress level is not assessable in this study, particularly when the K6 scale only measures the psychological distress level over a 30 day period. Nevertheless, it is possible that the family members would continue to suffer from psychological distress as their loved ones have unmet medical needs [It is highlighted from this study that unmet medical needs can affect psychological distress of family members, not just the patients themselves. Specifically, family members of those with unmet medical needs due to cost are at a significantly higher risk of ‘moderate’ and ‘serious’ level of psychological distress. These important findings need to be taken into consideration while shaping the funding for our health care systems in order to avoid detrimental effect on mental health of the otherwise healthy family members and to prevent further rise in the prevalence of mental illnesses. This recommendation aligns with the WHO Mental Health Action Plan 2013–2020.


In this paper a fluid‐structure interaction (FSI) experiment is presented. The aim of this experiment is to provide a challenging yet easy‐to‐setup FSI test case that addresses the need for rigorous testing of FSI algorithms and modeling frameworks. Steady‐state and periodic steady‐state test cases with constant and periodic inflow were established. Focus of the experiment is on biomedical engineering applications with flow being in the laminar regime with Reynolds numbers 1283 and 651. Flow and solid domains were defined using computer‐aided design (CAD) tools. The experimental design aimed at providing a straightforward boundary condition definition. Material parameters and mechanical response of a moderately viscous Newtonian fluid and a nonlinear incompressible solid were experimentally determined. A comprehensive data set was acquired by using magnetic resonance imaging to record the interaction between the fluid and the solid, quantifying flow and solid motion. Numerical methods for simulating the interaction between fluids and solids are diverse with many variants proposed to address important factors such as large deformations, computational efficiency, ease of implementation naming a few.Determining the efficacy of FSI modeling approaches, both in numerical methods and modeling approaches, requires verification and validation. In the context of verification, a series of standard benchmark problems have been developed and proposed (see Table Re) number flows. These conditions are in contrast to those observed in many biomedical FSI applications, with flows ranging from 100 to 3000 for a typical medium‐/large‐sized artery.While verification and validation testing remains critical, extension to biomedically relevant problems in three dimension remains limited. To date, most benchmarks favor using water or highly viscous fluids. This leads to either high (15 400‐32 000) or low (20‐333) Reynolds , as has become common in patient‐specific biomedical applications. A precise definition of the geometry was obtained through the use of computer‐aided design (CAD) generated 3D printed geometry. Straightforward boundary conditions can be derived from measurement of inlet conditions along with devising the experiment to have simple‐to‐define outflow and fluid‐solid conditions. Laminar flow was measured for both steady‐state and transient test cases with Re numbers of 651 and 1283, respectively (on the basis of maximum inflow velocity and inlet diameter); reflecting Re numbers found in the cardiovascular system . A CAD‐defined geometryμm). The surface was smoothed and polished to remove staircase effects. Inlet and outlet pipes were selected with compatible inner diameters to avoid disturbance of flow. The distance between inlets and outlet of the flow phantom was 119 mm. A cylindrical insert held a solid body and slid into a prepared hole in the flow phantom, such that it was level with the flow phantom wall. The insert was designed using CADDesign of the model aimed to avoid sharp edges, kinks or gaps and further allow straightforward implementation of boundary conditions in the numerical setting ; however, recorded fluctuations for each of the test cases were negligible. Fluid density and viscosity were determined  aimed at minimizing stiffness of the material. Thus, forces required to drive solid motion and deformation are minimized. Density of the silicone material was measured using Archimedes' methodThe silicone material used can be approximated as incompressibleNote, experiments for phases I and II were performed at different time instances. Because the used silicone material undergoes a continuous curing process, stiffness properties may change. Therefore, additional measurements were performed to enable calibration of material models employed, see Section The flow evolving over one cycle was recorded at ten slices along the z‐axis of the flow phantom (spacing 10 mm) for 25 time frames, eg,Under zero flow conditions, the silicone filament exhibits a deflection in positive y‐direction because of a difference in fluid/solid density. Because phases I and II of the FSI experiment were performed at different points in time and considering the aforementioned potential change in silicone stiffness, deflection of the silicone filament was recorded under zero flow conditions to enable calibration of solid material models. This was done as part of the acquisition of geometry data in an image stack to obtain the orientation of the model.During phase I of the experiment, the pump was set to a constant pump rate of approximately 14.07 l/min. Selection of the pump rate aimed at increasing the observed deflection from the zero flow state while maintaining a low Reynolds number. Preliminary flow experiments and numerical investigations showed no solid motion, such that data could be acquired assuming steady‐state flow. In fact, violation of the steady‐state assumption would cause image blur in the magnitude images because of time averaging of fluctuating velocities causing artifacts (in particular near the solid). This, however, was not observed. Further high frequency (approximately 30 Hz) low‐quality datasets were acquired confirming no solid motion and supporting validity of the steady‐state assumption.% of the selected VENC if the selected VENC value is three times as large as the expected velocity. Thus, the VENC value was tuned for each velocity component for improved image accuracy, see Table Firstly, the position of the deformed silicone filament was recorded under flow conditions using a T1 TFE flow sequence. Secondly, flow images were acquired using individual image stacks for each velocity component. Here, care was taken to select an appropriate velocity encoding sensitivity (VENC) value for each component scan. This is to avoid velocity aliasing when real flow velocities exceed the VENC value and an incorrect phase is assigned. On the other hand, setting a much larger VENC was avoided, because the work of Lotz et alDuring phase II of the experiment, the pump was set to an oscillating pump rate with approximate maximum pump rate of 7.71 l/min. The period of the experiment was controlled by the scanner, with flow sequences modified to trigger low frequency current pulses from the scanner to the pump using a custom‐built current amplifier. Thus, any delays in the image acquisition process could not accumulate and lead to out‐of‐sync data acquisition. Pulsatile inflow and a repeatable deflection pattern of the silicone filament were observed in preliminary survey scans, such that a periodic steady‐state was assumed for final data acquisition. Similarly to phase I, violation of this assumption would result in blurring in the magnitude image, which was not observed. Again, further high frequency (approximately 30 Hz) low‐quality data were acquired confirming periodic solid motion.vx and vy and 44 time frames for vz using phase‐contrast MRI sequences. The reduced VENC for non‐axial velocity components yielded a longer acquisition time per time frameVelocity components were recorded individually at a single plane crossing both inlets of the flow phantom to provide boundary condition data for the numerical setting. Inflow scans were acquired at 28 time frames for 2.3MRI techniques were employed to acquire both geometry and flow data. A clinical 3T MRI scanner was used for data collection . For imaging of flow, 3T MRI scanners are generally superior to 1.5T MRI scanners because of a higher signal‐to‐noise ratio (SNR).Once acquired, scans were first registered to determine the mapping between the MRI coordinate frame and the model coordinate frame . Five and three image slices were processed for phases I and II, respectively. Image slices were cropped to focus on the region containing the silicone filament and the origin identified. For each slice, intensity minima were detected row‐ and column‐wise and averaged. Finally, these results were averaged and a polynomial of order 4 fit to the data  can be related to the SNR and VENC, σ=2VENC/(πSNR), a myriad of additional artifacts can also impact measurement accuracy.% when comparing velocity measures between PC MRI and stereoscopic particle image velocimetry (PIV).Measurement error in the velocity data extracted from PC MRI can be challenging and is often not determinable from the images directly. While noise in the velocity measurement  on top of the first phase.4.2Postprocessed data were presented in this paper and are available in the online supplement. This supplement contains surface geometries (CAD), tensile load‐displacement test data, and velocity boundary condition data in tabulated format. Further, data files containing extracted fluid/solid velocity and solid position for a set of equispaced data points on multiple slices through the FSI section.4.3On the basis of the experiment presented in this work, a 3D FSI Benchmark Challenge was initiated.4.4The experimental design was optimized, and additional data acquired to minimize measurement errors and assist with correct modeling of the FSI problem. Of course, measurement errors could not be fully avoided. For example, tolerances during manufacture  and measurement  impact precision as outlined in Table L2‐norm of the radial profile with the multifrequency Womersley solution to determine the unknown driving coefficients.State‐of‐the‐art modeling frameworks that are used for patient‐specific models increasingly integrate data obtained from clinical imaging techniques,The use of MRI also constrained the experimental design. For example, all parts exposed to the magnetic field needed to be MRI compatible, and the requirement for a periodic steady‐state was essential for data acquisition. The temporal resolution of MRI sequences used was also limited to avoid prolonged data acquisition. In this regard, PIV may provide better performance and would be less prone to averaging effects than MRI because of the underlying technology of tracing particles to obtain an instantaneous velocity map. Nevertheless, in our case temporal resolution of the employed MRI scanner was sufficient, and the employed sequences allowed us to record all three spatial velocity components whereas standard 2D PIV is limited to imaging in‐plane velocity components. An additional advantage of MRI over PIV is the lack for optical requirements, for example, accounting for the refraction index of the flow phantom material, as well as the less intrusive approach (no tracer particles required). Further, MRI tends to provide the input data commonly used in biomedical engineering research geared toward translation. Hence, the definition of input data and comparison of end results are considered a valuable step for translational FSI applications. For further details on PIV, see previous works.Gravitational forces make an important contribution to the mechanism that is employed to obtain a repeatable deflection pattern (see Section In the current study, a uniaxial tensile load‐displacement test was conducted manually. Although a device‐based test would be preferable, the data derived represent an adequate sampling to observe the material behavior. Moreover, due to the continuous curing process, calibration to the zero flow deflection data is recommended for estimating solid model material parameters in phases I and II. We note that the solid material exhibits viscoelastic behavior. However, viscous effects are minimal at low frequency and were thus deemed negligible in this study.5In this paper, an FSI experiment for validation of FSI modeling frameworks was presented, complementing and extending well‐known standard numerical and experimental FSI tests. Focus of the experiment was on aspects encountered in typical biomedical engineering applications. For example, flow was in the laminar regime with biologically relevant Reynolds numbers. A moderately viscous incompressible Newtonian fluid interacts with a nonlinear incompressible isotropic solid in a fully 3D setting under the influence of gravitational forces. Like in many biomedical engineering applications, MRI was used for acquisition. A comprehensive data set comprising of geometry and flow images quantifying fluid and solid motion in space and time was obtained and is made available in the online supplement.supporting info itemClick here for additional data file.


ATP production via oxidative phosphorylation, apoptosis, iron‐sulfur cluster biogenesis, heme and steroid synthesis, calcium homeostasis, and regulation of cellular redox state. One of the unique features of these organelles is the presence of an extrachromosomal mitochondrial genome (mtDNA), together with all the machinery required to replicate and transcribe mtDNA. The accurate maintenance of mitochondrial gene expression is essential for correct organellar metabolism, and is in part dependent on the levels of mtDNA and mtRNA, which are regulated by balancing synthesis against degradation. It is clear that although a number of mitochondrial nucleases have been identified, not all those responsible for the degradation of DNA or RNA have been characterized. Recent investigations, however, have revealed the contribution that mutations in the genes coding for these enzymes has made to causing pathogenic mitochondrial diseases.Mitochondria are cytosolic organelles that have many essential roles including  BERbase excision repairDNasedeoxyribonucleasemtDNAmitochondrial genomePNPasepolynucleotide phosphorylaseRNaseribonucleaseMitochondria are cytosolic organelles that have many essential roles including ATP production via oxidative phosphorylation, apoptosis, iron‐sulfur cluster biogenesis, heme and steroid synthesis, calcium homeostasis, and regulation of cellular redox state. One of the unique features of these organelles is the presence of an extrachromosomal mitochondrial genome (mtDNA), together with all the machinery required to replicate and transcribe mtDNA. The accurate maintenance of mitochondrial gene expression is essential for correct organellar metabolism, and is in part dependent on the levels of mtDNA and mtRNA, which are regulated by balancing synthesis against degradation. It is clear that although a number of mitochondrial nucleases have been identified, not all those responsible for the degradation of DNA or RNA have been characterized. Recent investigations, however, have revealed the contribution that mutations in the genes coding for these enzymes has made to causing pathogenic mitochondrial diseases Characterizing mitochondrial nucleases is particularly demanding as these may be present in more than one cellular compartment, for example, REXO2 is an RNase that is present in both mitochondria and cytosol. As a result, the biochemical classification of nucleases as truly mitochondrial rather than as a contaminant during purification can be challenging. Despite these difficulties, several human mitochondrial nucleases have been characterized, although the existence or the activity of some of these enzymes is still debated.Most of the identified DNases are involved in mtDNA maintenance or repair, and almost all have both endo‐ and exonucleolytic activity, although some such as EndoG also recognize RNA as a substrate Tailoring and targeting of specific nuclease activities to mitochondria, however, is gaining increasing attention as a therapeutic tool to remove mutated mitochondrial DNA and thereby ameliorate disease This review summarizes the recent advances in characterizing mitochondrial nucleases Fig. , and theThe number of mtDNA molecules that are found in human cells can vary from a few hundred to more than a thousand. This mtDNA is organized into complexes known as nucleoids. These structures not only include TFAM  to package the mtDNA One of the first DNases to be studied was AP endonuclease 1 (APE1), which is localized to both mitochondria and the nucleus in vitro 3′–5′ DNA exonuclease activity and is localized to mitochondria.Another AP endonuclease shown to localize to both the mitochondria and nucleus is APE2 in vitro have demonstrated a specificity for DNA intermediates that contain 5′‐flaps in vitro DNA2 and FEN1 are able to increase the efficiency of flap fragment maturation Long‐patch base excision repair (LP‐BER) occurs in human mitochondria and specifically requires a 5′‐exo/endonuclease activity It is believed that more DNases are required to complete LP‐BER. One of these, EXOG (endonuclease G‐like‐1), is implicated in the repair of single‐strand breaks in the mitochondrial genome Enzymes involved in the metabolism of mitochondrial nucleic acids are expected to localize to the matrix. Surprisingly, however, several mitochondrial nucleases have been found in the IMS and the mechanism by which these nucleases act on the mtDNA or mtRNA located in the matrix remains obscure. Endonuclease G (Endo G) is a paralogue of EXOG, which also localizes to the mitochondrial IMS in vitro and using this system have demonstrated that MGME1 is able to process 5′‐flap intermediates, crucial for the RNA primer removal during mtDNA replication, into ligatable nicks in combination with DNA polymerase One of the more recently characterized human mitochondrial nucleases is MGME1, also known as Ddk1 The human mitochondrial genome is transcribed as polycistronic units. This process gives rise to one short and two long transcripts. The former encompasses a ribosomal RNA transcription unit while the remaining two essentially transcribe the whole mtDNA molecule In vitro assays with purified recombinant PNPase show that it forms a heteromeric complex with the helicase SUV3 and efficiently degrades structured single‐stranded RNAs in vitro experiments have shown that PNPase and SUV3 can form a transient complex with the poly(A) polymerase (mtPAP) in order to modulate mt‐mRNA poly(A) tail lengths in response to energy changes in vivo function of PNPase One of the mitochondrial RNases that has been found in RNA granules is polynucleotide phosphorylase (PNPase) in vitro ability to degrade small single‐stranded RNAs, and also DNA fragments, in a 3′–5′ direction de novo mitochondrial protein synthesis The end product of such mitochondrial mRNA degradation is short oligoribonucleotides. PNPase is not able to degrade RNA fragments < 4 nucleotides in length in vitro RNase MRP can cleave R‐loop substrates at the physiological priming sites, which would promote the mtDNA leading‐strand replication Almost 30 years ago, the Clayton laboratory identified and partially purified from mouse mitochondria an endonucleolytic RNase activity The recent hypotheses about the primer processing and removal, as part of the mtDNA replication process, invoke the formation of flap intermediates and the involvement of different mitochondrial nucleases (reviewed in RNASEH1 gene RNase H1 possesses a hybrid binding domain (HBD) that allows it to specifically digest the RNA strand of RNA/DNA heteroduplexes RNA14 from MTCO3One of the features of mammalian mtDNA is the distribution of the tRNA genes, such that they are interspersed between the rRNA and mRNA genes. In most cases, the resulting polycistronic transcripts are processed according to the ‘tRNA punctuation model’ to release individual RNA species Mitochondrial RNase P was identified as the endonuclease responsible for the removal of 5′ tRNA extensions and is composed by three protein subunits, encoded by different genes that are MRPP1, MRPP2, and MRPP3. The subunit accountable for the catalytic activity on mitochondrial tRNA 5′‐ends is MRPP3 and shows a unique feature, considering the ribozymal origin of this enzyme: in contrast with nuclear RNase P, it has no RNA component First identified as a candidate prostate cancer susceptibility gene in vitro and in vivo 3′–5′ exonuclease activity, demonstrating preferential cleavage of A stretches. In addition to its function on mt‐mRNA, PDE12 has been also shown in vitro to trim oligo(A) tails to a single A at the 3′‐termini of mitochondrial tRNATyr, thereby completing tRNATyr, as the mtDNA sequence lacks the terminal 3′‐adenosine Mitochondrial gene expression is controlled by the balance of synthesis and degradation of transcripts, which in turn modulated by other factors such as polyadenylation. Other than completion of termination codons on seven open reading frames, the function of mitochondrial poly(A) tails in mtRNA turnover and/or translation, has not been determined. The length of this modification is potentially regulated by the actions of poly(A) polymerase (mtPAP) with the LRP SLIRP complex, and the nuclease, PDE12 in vitro endoribonucleolytic activity assays. In cultured cells, depletion of this protein causes an accumulation of a subset of mitochondrial transcripts and overexpression of LACTB2 has the opposite effect. However, these effects are potentially too modest for an enzyme that should be critical for mtRNA degradation in human mitochondria. Further investigations are required to confirm the in vivo role of LACTB2 in mtRNA processing, and to answer several remaining questions concerning missing ribonucleases including: is mt‐mRNA degradation initiated by an endonucleolytic cleavage? If so, which enzyme(s) is responsible for this cleavage? Are 5′–3′ exonucleases actually required for the complete degradation of mt‐mRNA? Since there is no tRNA gene placed between ATP8 and COX3 genes, is the maturation of ATP6/8 and COX3 mRNAs dependent upon an as yet uncharacterized nuclease?In this complex panorama of mitochondrial ribonucleases that act on mtRNA at different stages, neither an 5′–3′ exoRNase activity nor an endoRNase active on mitochondrial transcripts had been detected in the matrix until the report on LACTB2 (β‐lactamase‐like‐protein 2) Since each cell contains multiple copies of the mitochondrial genome, it is possible that different mtDNA molecules, carrying mutations or polymorphisms, can coexist in the same cell. The balance between wild‐type and mutant copies, a state known as heteroplasmy, is an important factor in determining whether or not there are pathological consequences that result in mitochondrial diseases Many approaches have been designed to try and redress the balance in favor of the wild‐type mtDNA Fig.  4. One oTo date, mitochondrial REs have been successfully expressed in several cellular systems, and in mice, by use of adenoviral transduction. Constructs that have been injected in specific mouse tissues in this manner have shown convincing switches in the levels of heteroplasmy ex vivo on cultured cells or in vivo on mice. The expression of these nucleases in patients' tissues, together with emergent genome editing technologies, has a strong therapeutic potential and may constitute a powerful tool to improve the health of people affected by mitochondrial disease.All the experimental works using mitochondria‐targeted endonucleases have been performed The authors have no conflicts of interest to be disclosed.FB, RNL and ZCL were all involved in writing the review.


Over the past centuries, humans have transformed large parts of the biosphere, and there is a growing need to understand and predict the distribution of biodiversity hotspots influenced by the presence of humans. Our basic hypothesis is that human influence in the Anthropocene is ubiquitous, and we predict that biodiversity hot spot modeling can be improved by addressing three challenges raised by the increasing ecological influence of humans: (i) anthropogenically modified responses to individual ecological factors, (ii) fundamentally different processes and predictors in landscape types shaped by different land use histories and (iii) a multitude and complexity of natural and anthropogenic processes that may require many predictors and even multiple models in different landscape types. We modeled the occurrence of veteran oaks in Norway, and found, in accordance with our basic hypothesis and predictions, that humans influence the distribution of veteran oaks throughout its range, but in different ways in forests and open landscapes. In forests, geographical and topographic variables related to the oak niche are still important, but the occurrence of veteran oaks is shifted toward steeper slopes, where logging is difficult. In open landscapes, land cover variables are more important, and veteran oaks are more common toward the north than expected from the fundamental oak niche. In both landscape types, multiple predictor variables representing ecological and human‐influenced processes were needed to build a good model, and several models performed almost equally well. Models accounting for the different anthropogenic influences on landscape structure and processes consistently performed better than models based exclusively on natural biogeographical and ecological predictors. Thus, our results for veteran oaks clearly illustrate the challenges to distribution modeling raised by the ubiquitous influence of humans, even in a moderately populated region, but also show that predictions can be improved by explicitly addressing these anthropogenic complexities. Specifically, for veteran oaks, we expect shifts in the realized niche toward inaccessible and low‐productive areas, and different responses to geographical and land cover variables in forests and open landscapes because of differences in past and present human land use. Moreover, because of the multiple ecological and anthropogenic drivers, we expect that the distribution of veteran oaks is better predicted with multiple predictors and models, combined in coherence with the structure of the landscape.22.1Quercus petrea and pedunculate oak Quercus robur. The two species frequently hybridize and may be difficult to separate in the field. Ecologically they are fairly similar, and we treat them together as oaks (Quercus spp.) in the analysis. The northern limit of oaks in Europe seems to be related to climatic factors that vary with latitude. On a coarse scale, Q. petrea follows the −4°C isotherm for the coldest month, whereas Q. robur, which reaches a little further north in Scandinavia, seems to be more limited by respiration sums  we sorted the plots into “oak present”‐plots and “oak presence unclear”‐plots. All “oak present”‐plots and 25% of the “oak presence unclear”‐plots were visited in the field, and the position of all large and hollow oaks in the plot was determined with a hand‐held GPS. To prepare for analysis, each plot was gridded to obtain information on the presence and absence of veteran oaks in 10 × 10 m cells matching the geographical resolution of the predictors.We used data on veteran oak presences and absences from the pilot study behind the recently established national monitoring of veteran oaks  plus forest patches with a size of 1 ha or less (see 2.32) was necessary to account for a nonlinear response.We developed prediction models for the full data set across all landscape types and for each landscape type separately by means of logistic regression  and different effects in forests and open landscapes (prediction 2). To evaluate the relative importance of these and other predictor variables we developed multiple regression models with several predictors. To evaluate model uncertainty and assess the need for multiple models (prediction 3) we also calculated AICc‐weighted average parameter estimates across multiple alternative models  areas, but this effect was much weaker and even opposite in some models for open landscapes Tables  & 4.Several local topographic aspects were also important. The multiple regression models suggested a unimodal pattern for slope in forest  in different landscapes depending on the kind and degree of human influence (prediction 1). Examples include differences in magnitude and steepness of linear responses, direction of linear responses, and position of realized optima along ecological gradients. This is clearly seen in the different responses of veteran oaks to forest edge, latitude, and slope, respectively Figure . Such shThis brings us to the second general challenge for spatial prediction modeling: different predictors may be relevant only in specific areas (prediction 2). Humans frequently modify ecosystems to the extent that original natural processes are no longer the most relevant and important processes structuring ecosystems . Ecological processes are complex, anthropogenic processes even more so. This challenge goes far beyond the time‐ and space‐dependent relevance and importance of single‐predictor variables discussed above. It is clear from our study of veteran oaks that no predictor can be singled out as the one and only or clearly most important one. Multiple predictors are needed, representing both ecological and anthropogenic processes. This is in accordance with the “multiple ecosystem drivers hypothesis” of oak forest dynamics  are important predictors of veteran oak presence, in accordance with the fundamental oak niche Dahl, , but witTo summarize and conclude, we find that veteran oaks are predictable despite the complexity of processes in human‐influenced landscapes and that considering how different human‐related processes operate in different landscapes helps both understanding veteran oak responses to environmental variables and prediction of distribution patterns. We expect predictions to be further improved with the extensive monitoring data set under establishment, especially after repeated visits. This will document recruitment and mortality patterns in veteran oaks and can be used to develop increasingly refined process‐oriented models. Regardless of the modeling approach, finding ways to account for human influence on ecological systems and address the challenges illustrated by the veteran oak case are likely to become increasingly important in the Anthropocene.None declared. Click here for additional data file.


Despite international bodies calling for increased patient and family involvement, these concepts remain poorly defined within literature on critical and intensive care settings.This scoping review investigates the extent and range of literature on patient and family involvement in critical and intensive care settings. Methodological and empirical gaps are identified, and a future agenda for research into optimizing patient and family involvement is outlined.MEDLINE, CINAHL, Social Work Abstracts and PsycINFO were conducted. English‐language articles published between 2003 and 2014 were retrieved. Articles were included if the studies were undertaken in an intensive care or critical care setting, addressed the topic of patient and family involvement, included a sample of adult critical care patients, their families and/or critical care providers. Two reviewers extracted and charted data and analysed findings using qualitative content analysis.Searches of A total of 892 articles were screened, 124 were eligible for analysis, including 61 quantitative, 61 qualitative and 2 mixed‐methods studies. There was a significant gap in research on patient involvement in the intensive care unit. The analysis identified five different components of family and patient involvement: (i) presence, (ii) having needs met/being supported, (iii) communication, (iv) decision making and (v) contributing to care.Three research gaps were identified that require addressing: (i) the scope, extent and nature of patient involvement in intensive care settings; (ii) the broader socio‐cultural processes that shape patient and family involvement; and (iii) the bidirectional implications between patient/family involvement and interprofessional teamwork. Critical care research, policy and best practice increasingly recognize that patients admitted to acute care hospitals are members of a wider patient–family networkCrossing the Quality Chasm was a seminal document in acknowledging patient‐centred care as a key component of health‐care quality.The movement towards patient involvement is evident in a number of supranational policy statements and directives drafted since the late 1970sDespite international bodies calling for increased patient and family involvement, this concept remains unclear within the critical care literature. Little consensus exists on what involvement actually means to varying stakeholders,Recent literature reviews on family members' involvement in the ICU have primarily focused on family needsThe purpose of this scoping review is to map out the extent and range of literature on patient and family involvement in critical and intensive care settings, with attention to key concepts, topics and methodological approaches. More specifically, the scoping review aims to identify empirical and methodological gaps within the existing literature in order to inform an emerging research agenda in patient and family involvement and interprofessional collaboration.Scoping reviews are an exploratory review methodology used to rapidly map the literature on a well‐defined topic, reveal methodological and empirical gaps within a body of research and identify critical areas for investigation.The research questions that guided this review were developed in collaboration with researchers and the advisory board on a larger study examining interprofessional collaboration and patient and family involvement in intensive care settings.Studies were selected for this review through searches conducted on OVID MEDLINE, CINAHL, PyschINFO and Social Work abstracts. These databases were used to reach a broad range of English‐language literature published in the last decade 2003–2014) within peer‐reviewed health and social science journals. This publication range was selected to provide insight into the expansion of literature and interest in this topic during this particular period. Two reviewers developed the search strategies . The quantitative literature also includes 10 observational studies, 4 pre–post studies, 1 randomized controlled trial and 1 non‐randomized controlled trial. Amongst the 61 qualitative studies, most are described as interview‐based exploratory qualitative designs (n = 21). The qualitative literature also includes 17 interview‐based phenomenological studies, 9 ethnographic studies, 2 grounded theory studies and 2 action research studies.Of the 124 studies included, 61 are quantitative, 61 are qualitative and 2 are mixed‐methods studies. Findings on study characteristics are reported in Table n = 79), with most of the remaining studies published in critical care medicine journals (n = 32). In terms of geographical distribution, the United States is the leading site for research (n = 48), followed by Sweden (n = 22), Canada (n = 7), Australia (n = 6) and Norway (n = 6). The participants for these studies are most commonly family members of critically ill patients (n = 41) or nurses (n = 35)  patient experience and (ii) patient participation. The variety and volume of research conducted on family involvement far surpasses that on patient involvement. Notably, ‘patient involvement’ is a concept that has not been significantly explored in critical care research, with only six qualitative articles retrieved on the topic. Three of these studies focus broadly on ‘patient experience’, through interviews with patients, and only peripherally discuss any aspect of patient involvement in communication or decision making.Two other studies employ interviews and focus groups with nurses to explore their perspectives on communication with patients,The patient involvement studies reviewed tend to focus on the ability of patients to communicate with providers and family members. In investigating the experience of ICU patients, however, the findings from these studies shed light on contextual factors that limited patient involvement in intensive care units. These factors include technologically intensive ICU environments, clinical objectification of patients, voicelessness and breathlessness caused by intubation, and assumptions around cognitive ability and illness severity.We identified five main components of family involvement that have been investigated within the studies on intensive care units. We propose that these categories of involvement are not mutually exclusive, but rather represent aspects of involvement that range along a continuum from relatively passive to active involvement. Fig.  Dreyer &n = 16) measure provider perspectives, attitudes and preferences towards family presence during resuscitation.‘Family presence’ is a component of family involvement that has been extensively researched within the critical care literature, accounting for nearly one‐third of all included studies. The term ‘presence’ connotes a relatively passive role for families as visitors to the ICU,Another research topic that features prominently in the included literature is the identification of family needs in the ICU. Family members of critically ill patients are routinely represented within the literature as recipients of care with distinct psychological, social and physical needs.The third largest research body is the study of communication between patients, providers and families in the ICU. The communication and information literature relates closely to family needs literature in that many of these studies seek to explore how family members perceived and used informational support from health‐care providers.During the course of a patient's stay in the ICU, family members must often assume responsibility over health‐related decision making, including choices about diagnostics, treatment and therapeutic care. Family member involvement in decision making was the subject of 7 quantitativeAmongst the quantitative studies, two are longitudinal studies in which investigators examine factors associated with surrogate‐decision‐makers’ satisfaction.The fifth and least researched component of family involvement in the critical care research is family member contribution to patient care. In eleven qualitative studies and one quantitative study, researchers seek to identify and explore the contributions that family members made to patient care.families as recipients of care) than the latter (families as active participants in care). Where family involvement has been studied, the gaze tends to be oriented towards relatively passive forms of involvement, such as family presence during resuscitation. One implication of this trend is that the family involvement literature often views family members as vulnerable subjects that must be brought into the fold of care (i.e. as patients) or as resources for improving patient outcomes, but very rarely as individuals to be partnered with by health‐care professionals in the care of the patient. As such, the family involvement literature may not be interacting effectively with the hidden care work that family members do in critical care settings,The widespread shift towards patient and family‐centred care has been characterized by Garrouste‐Orgeas and colleagues as, ‘a global philosophical approach in which families are both recipients of care aimed at optimizing their well‐being and active participants in care provided to the patient’.This scoping review identified a distinct lack of research on the nature and extent of patient participation and involvement in their own treatment and care. Although patient participation has become a pillar of health services research and practice,The nature of patient participation in the ICU is particular to the ICU setting and may be less obvious to researchers and health‐care providers than it would be in other health‐care settings. The appointment of a surrogate‐decision‐maker and/or an advanced directive (written treatment plan) are other possible ways in which patients may participate in decision making.Another limitation of the current literature is the disproportionate focus on provider‐family relationships and provider perceptions as factors affecting patient and family involvement. In particular, the relationship between family members and nurses has received considerable attention, a trend that is common in the broader literature on patient participationOne such factor that likely shapes patient and family involvement, but which has not been investigated extensively, is the built environment of the ICU. Some studies have explored the effects of sound environment on patient experience,The literature on patient and family involvement could also be strengthened by attention to broader contextual factors shaping the setting under investigation. With the exception of a few studies examining cultural preferences of patientsJust as unit culture shapes the possibilities of patient and family participation, patients and family members bring their own diverse set of experiences, expectations and beliefs about what participation should entail. There is evidence from other health‐care settings to support that patients and their families perceive and conceptualize participation or involvement differently depending on their social position, cultural expectations and previous experiences with health‐care consultation.A significant gap within the literature is consideration of the ways in which interprofessional dynamics shape opportunities for family–patient involvement and in turn, the implications of patient and family involvement on interprofessional teamwork. As Table As a final observation, there were very few studies in the included literature that triangulated methodologically, with most studies using either quantitative surveys or qualitative interviews as their sole data collection method. This methodological gap is particularly pronounced in the family needs literature, where investigators relied almost exclusively on the CCFNI survey as a data collection method. The dominance of the CCFNI as a tool to understand family needs has led some researchers to conclude that its five dimensions  represent a ‘universal and predictable set of needs’ experienced by families in ICUs.say happens and what actually happens in practice.The lack of data triangulation was also a limiting feature within the qualitative literature, given the common use of provider interviews alone to investigate issues related to family presence, involvement and visitation. Although interviews offer a valuable way to access insider accounts on events within the ICU, there are often meaningful differences between what people There are three key limitations to this review. First, only English‐language articles were considered for inclusion in the study. As such, this review misses potentially relevant articles written in other languages and primarily covers research conducted in North America. Secondly, our review did not target studies on advance directives as an expression of patient involvement, which may partly explain the very small number of articles we retrieved on patient involvement in the ICU. Finally, because the reviewers limited their searches to academic research articles published in the last decade, the scoping review cannot speak to how involvement has been conceptualized within grey literature such as media‐sources, commentaries, policy documents and patient and family education materials. This restriction on grey literature was necessary to limit the volume of articles reviewed and maintain a focus on critical care research. It would be constructive to further investigate the grey literature, given its potential influence on critical care research priorities and clinical practice.Through this scoping review, we set out to map out the extent and range of research on patient and family involvement investigated in ICUs. This scoping review identified five main components of family involvement that have been investigated in critical care research: (i) presence and visitation, (ii) having needs met or being supported, (iii) communication/receiving information, (iv) decision making and (v) contribution to care. A key finding to emerge from this review is that patient involvement has not, in fact, received much attention within critical care literature. Where patient involvement has been explored, the focus has been primarily been on communication with families and providers around a limited set of medical decisions. While the movement for family‐centred care calls for families to be incorporated as partners in care,In light of the research gaps identified by this review, we suggest that a future research agenda should focus on the following: (i) the scope, extent and nature of patient involvement in intensive care settings; (ii) the broader socio‐cultural processes that shape patient and family involvement, including processual, organizational and contextual factors; and (iii) the relationship between patient and family involvement and interprofessional teamwork processes. In terms of methodology, future research could be strengthened through incorporation of ethnographic approaches that produce in‐depth, context‐specific accounts of patient and family involvement. We argue that this research agenda will at least move the critical care research literature towards evidence that can inform the creation of context‐sensitive and sustainable interventions to improve the involvement of families and patients in the treatment and care of ICU patients.This study was funded by The Betty and Gordon Moore Foundation, which took no part in the conception, design, conduct, interpretation or preparation of this manuscript. The Betty and Gordon Moore Foundation is located at 1161 Page Mill Road, Palo Alto, CA 94304.The authors have no conflicts of interest to declare.