Genome-wide association studies (GWASs) identify single nucleotide polymorphisms (SNPs) that are enriched in individuals suffering from a given disease. Most disease-associated SNPs fall into non-coding regions, so that it is not straightforward to infer phenotype or function; moreover, many SNPs are in tight genetic linkage, so that a SNP identified as associated with a particular disease may not itself be causal, but rather signify the presence of a linked SNP that is functionally relevant to disease pathogenesis. Here, we present an analysis method that takes advantage of the recent rapid accumulation of epigenomics data to address these problems for some SNPs. Using asthma as a prototypic example; we show that non-coding disease-associated SNPs are enriched in genomic regions that function as regulators of transcription, such as enhancers and promoters. Identifying enhancers based on the presence of the histone modification marks such as H3K4me1 in different cell types, we show that the location of enhancers is highly cell-type specific. We use these findings to predict which SNPs are likely to be directly contributing to disease based on their presence in regulatory regions, and in which cell types their effect is expected to be detectable. Moreover, we can also predict which cell types contribute to a disease based on overlap of the disease-associated SNPs with the locations of enhancers present in a given cell type. Finally, we suggest that it will be possible to re-analyze GWAS studies with much higher power by limiting the SNPs considered to those in coding or regulatory regions of cell types relevant to a given disease. Asthma is a chronic inflammatory disease, characterized by reversible airway obstruction and increased bronchial hyperresponsiveness. This complex disorder is influenced by the interdependencies between various factors - genetic and environmental being the most important ones. The estimate that 35–80% of the variation in the risk of asthma can be attributed to genetic variation has spurred a number of genome-wide association studies (GWASs) of asthma Recently, there has been a vast acceleration in the identification of non-coding genomic elements that regulate gene transcription Notably, the location of enhancers is highly cell-type specific Based on these considerations, we tested the hypothesis that SNPs associated with a specific disease are more frequently found in enhancers specific to cells that are relevant for the disease. Using asthma as an example, we document a significant enrichment of asthma-associated SNPs in genomic regions marked by H3K4me1 in CD4+ T cells, which are known to contribute to asthma pathogenesis. In contrast, cells from brain, breast and skeletal muscle tissues unrelated to asthma, are depleted of asthma-associated SNPs in their enhancer regions. Thus, the methodology we report here can be utilized to make an unbiased prediction of which cell types contribute to disease pathogenesis, and which disease-associated SNPs are likely to be functionally important.2 = 0.8) based on the most recent release of HaploReg We retrieved all known asthma-associated SNPs from the GWAS integrator database In an elegant study by the Kellis group In contrast, regions with heterochromatin state and repetitive elements  showed high depletion for disease-associated SNPs, as did some states associated with transcription .Notably, we also found significant enrichment in States 9, and 34, which are associated with promoter and enhancer regions, suggesting that SNPs present in non-coding regions (enhancers and promoters) contribute to disease pathogenesis by perturbing the transcriptional regulation of an associated gene.Enhancers have previously been reported to be tissue and cell-type specific Next, we compared the enhancers identified in different datasets by calculating pair-wise Matthews correlation coefficients (MCC), which quantify the overlap of enhancers on a per nucleotide basis . To determine if there was differential enrichment of asthma-associated SNPs in the enhancers of different cell types, we merged the enhancers identified in each of the eight cell/tissue type specific clusters displayed in Next, we performed a similar analysis using a second enhancer marker, H3K27ac . H3K27acWe asked whether the enrichment for asthma-associated SNPs could be increased by excluding ubiquitous enhancers found in many cell types/tissues. The highest enrichment was determined by considering either the SNPs unique to CD4+ T cells, or SNPs found in up to two other cell types. By contrast, enhancers in skeletal muscle cells were clearly depleted of asthma-associated SNPs, and such depletion became increasingly pronounced when excluding enhancers also found in other cell types. A similar, but less pronounced, trend was found for cells from breast and brain tissues (Table S5 in File S1), whose enhancers are depleted of asthma-associated SNPs and become more depleted when eliminating enhancers shared with other cell types. For the remaining cell types (liver and adipose tissues), which showed an enrichment of asthma SNPs in their enhancers, the enrichment increased further when considering subsets of enhancers unique to these cells.To examine the putative functional mechanism of asthma SNPs in enhancers, we were interested if might modulate in Transcription Factor Binding Sites (TFBSs). In summary, our analysis demonstrates that focusing on enhancers present in a few cell types further enriched asthma-associated SNPs compared to background SNPs. Since by far the greatest enrichment was observed at enhancers of CD4+ T cells which are known to contribute to asthma, our approach is capable of simultaneously prioritizing those SNPs that are more likely to contribute to a given disease, and identifying the cell types involved.cis-regulatory elements (enhancers) present in cell types that drive disease pathogenesis such as CD4+ T cells, we predict that SNPs present in such cell-type-specific enhancer regions  project after our initial results were submitted for publication Our present analysis is meant more as a proof of concept than an optimized, definitive study; there are multiple ways in which it can be significantly improved. First, the amount of published asthma-associated SNPs is continuously increasing, with a gain of 65% from January to October 2012, and additional SNPs will continue to be discovered. Moreover, rather than relying on SNPs that reach statistical significance in whole genome studies, our approach would be even more powerful when GWAS data is re-analyzed from scratch, limiting the SNPs considered to those in active genomic regions of the cells of interest.cis-regulatory elements such as suppressors or isolators may well have similar importance to enhancers, and because even for enhancers, H3K4me1 in combination with other markers may provide a more accurate identification. We expect classifications such as those by the Kellis group to become available for multiple cell types in the near future. Also, the chromatin state classifications could be further tailored to our type of analysis by focusing on those states that show the highest correlation with disease-associated SNPs, and identifying the optimal set of chromatin marks that identifies these regions. Finally, we want to reiterate that asthma-associated SNPs are significantly enriched not only in enhancers and promoters, but also in coding and untranslated regions. The transcription of these regions could further depend on both genetic and epigenetic factors.Second, the peak-calling algorithm used (MACS) is not optimal for identifying histone modifications, but was designed for identifying much better-defined transcription factor binding sites. Preliminary analysis showed that we obtained a higher enrichment with other algorithms, such as SICER In conclusion, we have demonstrated a novel approach to GWAS data analysis that integrates epigenomic information to identify SNPs and cell types contributing to disease. We expect our approach to be broadly applicable, and to further enhance the value of the accumulating information from GWAS of disease. Future work needs to experimentally confirm the functional role of the identified SNPs.To create a collection of asthma related SNPs, we had all asthma-associated variants (SNPs) downloaded from GWAS Integrator http://genome.ucsc.edu/cgi-bin/hgLiftOver]. Some non-asthma SNPs where un-mappable by this tool, and discarded from that calculation. For a given chromatin state, we determined the fraction of asthma-associated SNPs located in the corresponding genomic region, and compared it to the fraction of background SNPs to calculate enrichments. Significance was assessed using the chi-square test.To estimate distribution of asthma SNPs in different chromatin states we used chromatin state prediction for CD4+ T-cells by Ernst and Kellis http://www.genboree.org/epigenomeatlas/edaccGridViewerPublic.rhtml].ChIP-seq H3K4me1 data together with the input data for 37 datasets were obtained from Human Epigenome Atlas, release 5. [The samples analyzed came from the following cell types. CD4+ T cells: three samples of CD4+ memory T cells, two samples of CD4+ naive T cells, one sample of CD4+ CD25- IL17- T cells, CD4+ CD25- T cells, CD4+ CD25- IL17+ Th17 cells, CD4+ CD25+ CD127- Treg cells; brain: brain germinal cells, brain mid frontal and inferior temporal lobe, two samples of brain hippocampus middle, two samples of brain anterior caudate, two samples of brain substantia nigra and brain cingulate gyrus; three samples of adipose derived mesenchymal (ADM) stem cells; three samples of liver cells; two samples of kidney cells; five samples of adipose nuclei; two samples of breast myoepithelial cells and three samples of skeletal muscle cells. Total number of all analyzed samples is equal to 37; the number of analyzed cell types is equal to 19 because for some cell types data for more than one donor was available. These 19 cell types belonged to eight different tissues.For each sample we calculated H3K4me1 enriched regions by enrichment of treatment reads under a local background model estimated from control reads by employing the statistical software MACS  All available ChIP-seq H3K27ac data for the samples that contained H3K4me1 datasets were obtained from Human Epigenome Atlas, release 5. Thus, we have analyzed H3K27ac marks for the same CD4+ T cells (9 samples), one sample of brain anterior caudate, one sample of adipose nuclei and one sample of skeletal muscle cells. The twelve cell types mentioned above belong to four different tissues and for each sample we have calculated H3K27ac peaks using exactly the same parameters as we used for H3K4me1 peak calling.In order to compare the enhancers in different datasets we calculated pair-wise Matthews correlation coefficients (MCC), which quantify the overlap of enhancers on a per nucleotide basis. For each pair of samples (datasets) the MCC was calculated using the following formula:To calculate distribution of asthma SNPs within and without Transcription Factor Binding Sites (TFBSs) we have used TFBSs by ChIP-seq data from the ENCODE (Release 2) Figure S1 in File S1Asthma-associated SNPs and H3K4me1 (enhancer) enriched regions in the human IKZF3 locus of different cell/tissue types. From top to bottom, using the UCSC genome browser, are displayed: the gene track (genes), all the SNPs not associated with asthma, the SNPs associated with asthma , H3K4me1 ChIP-seq track (green) for different cell/tissue types (named on the left) underlined by the corresponding peak-calling track (black boxes). For the blood CD4+ T cells, peak calling tracks from seven samples/cell-types are displayed. The red box shows an H3K4me1 peak that is present only in CD4+ T cells.(PDF)Click here for additional data file.


Increasingly, health workforces are undergoing high-level ‘re-engineering’ to help them better meet the needs of the population, workforce and service delivery. Queensland Health implemented a large scale 5-year workforce redesign program across more than 13 health-care disciplines. This study synthesized the findings from this program to identify and codify mechanisms associated with successful workforce redesign to help inform other large workforce projects.This study used Inductive Logic Reasoning (ILR), a process that uses logic models as the primary functional tool to develop theories of change, which are subsequently validated through proposition testing. Initial theories of change were developed from a systematic review of the literature and synthesized using a logic model. These theories of change were then developed into propositions and subsequently tested empirically against documentary, interview, and survey data from 55 projects in the workforce redesign program.Three overarching principles were identified that optimized successful workforce redesign: (1) drivers for change need to be close to practice; (2) contexts need to be supportive both at the local levels and legislatively; and (3) mechanisms should include appropriate engagement, resources to facilitate change management, governance, and support structures. Attendance to these factors was uniformly associated with success of individual projects.ILR is a transparent and reproducible method for developing and testing theories of workforce change. Despite the heterogeneity of projects, professions, and approaches used, a consistent set of overarching principles underpinned success of workforce change interventions. These concepts have been operationalized into a workforce change checklist. The Australian healthcare system faces a number of challenges that test its ability to deliver effective, efficient, and responsive services to the population. These challenges are well documented and include: an increasing demand for services ,2, a groThe Australian Allied Health Workforce - An Overview of Workforce Planning Issues; allied health staff felt pressured by [name] to attempt unachievable deliverables.’ 1.3 Better success of the new models of care is associated with top-down supportet al. [Best et al.  found thThis proposition was supported at several levels of our study:–A change of government towards the end of the MoC project resulted in a change of strategic priorities, uncertainty, and reallocation or cessation of funding. This change and uncertainty prevented the completion of some projects and prevented the sustainability of other successful projects due to realignment of priorities at policy and decision-making levels.–Leadership in the form of change management training for staff, and support for AHA staff to undertake certificate training contributed to the success of projects.–Strong executive sponsorship was associated with project success, whereas projects without executive support were discontinued or did not receive recurrent funding.1.4 Better success of the new models of care is associated with legislative scaffoldingAustralia’s Health Workforce Productivity Commission Research Report[Support for the development and introduction of the new roles came from national and state initiatives. The recommendations in the ch Report served a1.5 Better sustainability of the new models of care is associated with codification of the processes, practices, and training used to implement the roleOne of the major outputs from the QH MoC projects was the codification of processes necessary to implement new roles; specifically, this involved role definitions, service definitions, new competencies and competency frameworks, and new tools and pathways to support implementation of the new MoC. The codification of processes was important to help both the sustainability of the project in times of change, as well as the transfer of the project into a new environment/service/context. This was exemplified in one project, which specifically identified that having poorly defined role descriptions, objectives, and goals at the commencement of the project substantially slowed progress of the project; role descriptions were negotiated late in the project, delaying staff and patient recruitment.Five projects used the Calderdale Framework  in the d1.6 Better success of the models of care is associated with having powerful allies to drive the role forwardAs previously noted, the engagement of key and powerful allies  within an organization was paramount to the success of new roles and MoC. Support from these individuals can enable the necessary legislative changes required for acceptance of the new role and scope of practice by the regulatory body. They can also foster acceptance of the new MoC by others whose practice may be affected by the role changes.1.7 Better success is associated with implementing new models of care that are appropriate for the contextThe context into which the new MoC is implemented needs to be receptive to and supportive of the need for change . The characteristics of contexts that were identified as associated with successful implementation were:–Willingness to discuss the potential to implement change. This required being flexible, open minded, and openly engaging with new ideas.–Having a mindset that supports change.–Willingness of team members to work with the change leader throughout the entire process.–Ability to focus changes around those that will positively impact on patient outcomes.–Having the relevant participants located in close proximity to each other.A number of unsuccessful projects were initially started in contexts that were not receptive or supportive of the proposed changes and were discontinued; when moved to more supportive contexts that demonstrated the above characteristics, they were expected to be successful.2.1 More efficient use of the role is associated with clearly defined roles within the model of careThere was evidence from 15 projects that having a clearly defined worker role within the MoC was associated with increased efficiency and sustainability of the role. In particular, it was important to have clearly defined role descriptions and identified training needs. In the absence of role clarity, protection of role boundaries was more likely to arise, as well as inefficient delegation of tasks to new practitioners. An important area requiring further clarity was the differentiation between advanced roles and full scope of practice.2.2 More efficient use of the role is associated with clearly defined, understood, and unambiguous delegatory or allocatory models of careThe need for clearly defined delegatory or allocatory MoC was an important indicator of project success (8 projects). In particular, it was important to clearly identify tasks that could be delegated. Professional readiness for taking on extended practice varied between practitioners.2.3 More efficient use of the role is associated with delegating practitioners having confidence in delegation and trust in the practitionerTrust between practitioners was an important component of efficient delegation. This was supported by having a clear understanding of the roles, training, and competencies of the practitioners to whom they are delegating. Lack of trust resulted in a lack of confidence in the delegating practitioner, and consequently, in inefficient delegation.2.4 More efficient use of the role is associated with allowing practitioners to work to their full scope of practiceEnabling staff to work to their full scope of practice, and providing structures to reinforce this was associated with increased efficiency (six projects). One of the strongest examples of this was the orthopedic podiatry triage service, which enabled podiatrists to work to their full scope of practice and led to substantial reductions in orthopedic surgery waiting lists and more efficient use of the orthopedic surgeon’s time.Conversely, the inability or unwillingness of AHPs to delegate parts of their role to others, predominantly AHAs, resulted in inefficient use of the newly created roles. These challenges were related to:–Lack of clarity with overlapping roles.–Lack of understanding of how to delegate, and the need for education, supervision, and frameworks to support delegation. These structures needed to be in place from the beginning of the project.–Lack of trust in assistants and engagement with the training provided to assistants by AHPs.–Turf protection and reluctance to let go of some tasks to a workforce perceived to be less skilled.The use of the Calderdale Framework assisted in resolving these challenges as it allowed AHPs to understand how they could improve their own patient care by delegating specific tasks and functions to AHAs.3.1 Greater staff satisfaction is associated with models of care that provide for better career development opportunitiesThe implementation of new MoC improved career opportunities and led to greater staff satisfaction. Surveys to measure staff satisfaction with the new MoC were undertaken in some projects as part of their evaluation processes. The following outcomes were identified:–Development of more sustainable roles within the organization.–Perceived ability by allied health team members to complete more quality activities, therapy interventions and patient education, thus improving their job satisfaction.–More appropriate task to skill matching.–Increased sense of achievement through the learning of new skills and acquiring a broader knowledge base, particularly for AHA.–A perception of inclusion into a multidisciplinary team.–Improved staff morale.However, in projects where turbulent leadership, poor recruitment to leadership roles, and an influx of junior/inexperienced staff were reported, the staff satisfaction with the new MoC was poor. These barriers overshadowed the positive impacts the new MoC may have had on team members.3.2 Greater staff satisfaction is associated with role clarityThe data did not demonstrate a direct link between increased staff satisfaction and role clarity. However, there was evidence that improved understanding and acceptance of the new roles arose, in part, through appropriate consultative processes that were reinforced through processes of role clarity. This is consistent with findings from other studies that examined role clarity issues associated with role boundaries for workplaces with multi-disciplinary teams . Issues Better understanding and acceptance of new roles was associated with: 1) AHPs having the increased knowledge and ability to identify appropriate tasks for the proposed assistant role, 2) building trust in delegation models and a multi-professional assistant workforce, and 3) changes that resulted in more advanced AHP and advanced AHA positions being incorporated into services to improve efficiencies and reduce costs. Staff acceptance of these new roles within the teams was demonstrated in six projects with the majority of these projects being continued.This proposition is, therefore not upheld in this context.3.3 Greater staff satisfaction is associated with seeing value in/impact of the new models of careAppreciating the value and impact of a new role is largely associated with the new MoC being driven by a locally identified need or being driven from the bottom-up. The other main factor impacting on this is engagement. Both of these factors are described under Proposition 1.3.4 Greater staff satisfaction is associated with appropriate support for the development and implementation of the new models of careThis factor is also closely related to the importance of engagement of key stakeholders from across a multitude of levels within the organization, and therefore, is closely related to Proposition 1.4.1 Better patient outcomes are associated with greater engagement of patients in the decision making associated with their care deliveryFew of the projects reported the patient perspective; therefore, there is limited data to assess this proposition. Failure to facilitate patient engagement was highlighted in one unsuccessful project; the patients using the service did not feel that there was a need for additional services and therefore did not use them.4.2 Better patient outcomes are associated with putting the patient at the center of the new models of care, rather than the practitionerThis study found evidence that if patients were not involved in driving the new services then patient outcomes were less likely to be the specific focus of the project, or were not improved. In particular, one project, driven from the top-down, with the goal of reducing unplanned hospitalizations in a pediatric population was not successful because the patients (parents) did not see a need for more services, and therefore did not make use of the new services. Another project used a multidisciplinary team to shift the approach from professionally centred care to patient centred care. Some projects were focused on decreasing wait times for patient and improving patients safety; however, the outcome evaluations were generally based on the staff’s perceptions that patient outcomes improved.any care or service where the alternative is no service, or a long waiting list4.3 Better patient outcomes are associated with providing The data from this study predominantly related to providers and identified outcomes from the provider perspective; however, there was some empirical evidence to support this proposition. Eleven projects provided care or services where previously, there was limited or no service provided due to long waiting lists or geographic inequities in service distribution. However, because of the lack of data collected to describe outcomes from the patient’s perspective, we recommend that in future workforce change projects, data are captured that focus on the patient’s perspective of new MoC.This project identified and tested empirically a series of propositions associated with successful workforce change. The propositions that were supported by the data are listed below:1. Better sustainability of a new MoC is associated with:•full engagement of all key stakeholders first•bottom- up drivers (rather than top-down)•top-down support to drive, underpin, and sustain the new MoC•legislative scaffolding to reinforce the new MoC, including award and pay structures supported in industrial agreements, and ratified at the highest possible levels of government, to avoid undermining by professional boundary arguments•codification of the processes, practices, and training used to implement the role•powerful allies to drive the role forward•implementing new MoC that are appropriate for the context 2. More efficient use of health practitioner roles is associated with:•clearly defined roles within the MoC•clearly defined, understood, and unambiguous delegatory or allocatory MoC•delegating practitioners having confidence in their delegation, which comes from understanding the roles, training, and competencies of the practitioners to whom they are delegating•trust, derived from time and exposure to the new MoC , is important for establishing appropriate delegation/collaboration/referring practices•allowing practitioners to work to their full scope of practice and having structures that reinforce this.3. Greater staff satisfaction is associated with:•better career development opportunities•appreciating value / impact of the role•appropriate support for the development and implementation of the MoC.4. Better patient outcomes are associated with:•greater engagement of patients in the decision making associated with their care delivery•putting the patient at the centre of the MoC, rather than the practitioner•providing any care or service where the alternative is no service, or a long waiting listThese propositions have been further synthesized into three broad principles of workforce change:(1) Drivers for change need to be closely linked to clinical practice and patient care. Workforce change needs to be driven by perceived or potential benefits to patients, staff and /or services at the local level.(2) The context for workforce change must be supportive at all levels. This includes a supportive legislative and industrial environment, professional environment, and leadership and champions.(3) Mechanisms for workforce change should include engagement of key stakeholders, access to resources to support the implementation and performance of the role, a facilitated change management process, and appropriate governance and support structures.The first two propositions had the greatest volume of data to support them. While there was evidence to support the propositions within statements 3 and 4, they were not mutually exclusive to these domains, and tended to reinforce the statements within Propositions 1 and 2.The strength of the evidence largely reflects the nature and volume of data collected relating to each factor. In other words, ‘weak’ evidence was likely to be due to a lack of available data rather than findings that refuted the propositions. We were surprised by (a) the lack of patient engagement and (b) the lack of patient focus in the projects. Consistent with the literature in this field, the majority of the projects focus on professions and changing role boundaries or developing new MoC. This focus, by definition, leads to interdisciplinary challenges and rivalry because the emphasis of change becomes the renegotiation of roles, rather than the best way of allocating care to meet the needs of the patient. The lack of a clear patient focus meant that goals were often process-based, rather than outcome-focused.This project developed and tested a novel method, Inductive Logic Reasoning, to create and empirically test theories of workforce change. This method has the advantage that it enabled us to develop and develop theories from the existing literature to create a series of propositions that were then tested empirically and transparently against a large dataset. This method aimed to address some of the limitations of the existing theory-based evaluation approaches by using logic models in a transparent way to develop theories of change which can be transparently tested. The dataset incorporated a large range and volume of data sources of varying structure, content, and quality, and we were able to transparently extract data against pre-defined themes to create the initial logic model.This study involved the analysis and synthesis of a large volume of data from a range of sources, in varying formats, and over a short period of time. The combination of the logic model and proposition development and testing appears to have been an effective and transparent way to arrive at a high-level synthesis of the data, which was the goal of this study. However, in achieving that goal, we have lost a great deal of depth and detail of the raw data.One of the challenges of the evaluation was identifying measures of success due to the heterogeneity of the projects and their large range of potential impacts. We were unable to draw firm conclusions about the outcomes of the workforce projects for reasons identified above; however, there were clear process indicators associated with project success. The nature of the evidence of change makes it difficult to draw causal relationships.As with all research, there is the risk of researcher bias. Workforce research is highly contextually dependent, and we have framed this research within the Australian health workforce context. This approach automatically biased the researchers towards certain norms. We attempted to ensure objectivity by embedding our propositions within the literature first, and then testing them empirically against the data arising from the projects; however, the research still has the Australian health-care context as its normative setting.The project reports used as data were not written with the expectation that they would form part of a large-scale evaluation; hence, their findings were presented in different ways. We have triangulated a range of primary and secondary data sources from a variety of participants to ensure the validity of our findings.We acknowledge that methods to empirically develop and test change theories require further development and refinement. For instance, it may be possible to draw tighter conclusions if we were to re-assess each project against the success criteria. This would also help to validate the success criteria.The implementation of new MoC is a complex process and broad principles of change management apply. Based on the findings of this study, we developed a comprehensive Workforce Change Checklist: An Evidence Based Practice Guide for Implementing Successful Workforce Change , which iAHAs: Allied health assistants; AHPs: Allied health professionals; CASP: Critical Appraisal Skills Programme; EPOCH: The Cochrane Collaboration’s Effective Practice and Organisation of Care; ILR: Inductive logic reasoning; MoC: Models of care; QH MoC: Queensland Health Models of Care.Kerry Vanniekerk-Lyons was employed as a project officer by Queensland Health and was involved in the project management of the overall Models of Care projects. Her role in this research has been to help broker access to the data, provide support for the multiple site specific research governance requirements, and provide clarification of facts relating to the data. To maintain objectivity, the remaining members of the research team, who were external to the Queensland Health MoC processes, undertook the majority of interpretation and analysis of the data and have no competing interests.SN conceived the original project, obtained the funding, developed the methodology and was involved in the data analysis and synthesis and drafted the paper. AR was overall project manager and was responsible for much of the data management, synthesis and analysis. AM and SG were involved in the data analysis and synthesis. KV provided ethics and governance support. All authors provided input into the conceptual development and ongoing drafts of the paper. All authors read and approved the final manuscript.Susan Nancarrow, Director of Research at the School of Health and Human Sciences, Southern Cross University, Australia.Alison Roots and Anna Moran are research fellows at the School of Health and Human Sciences, Southern Cross University, Australia.Sandra Grace is a senior lecturer at the School of Health and Human Sciences, Southern Cross University, Australia.Kerry Vanniekerk-Lyons is a full-time PhD student examining sustainable workforce change at the School of Health and Human Sciences, Southern Cross University, Australia.Data extraction template for Queensland Health documents.Click here for fileSingle case study of the introduction of a social work assistant illustrating data output from the Google form[ogle form.Click here for fileQueensland Health models of care project summaries.Click here for fileSummary of project outcomes.Click here for fileProposition summaries and supporting evidence.Click here for file


Effective collaboration between the Ministry of Health, local government, and implementing partnersSocial mobilization to sensitize the community about the importance of net useDevelopment of a mobile application to facilitate data collection and analysisA school-based net distribution program, piloted in the Southern Zone of Tanzania to sustain ≥80% universal net coverage previously attained through mass campaigns, successfully issued nets to nearly all eligible students and teachers. Keys to success included: Effective collaboration between the Ministry of Health, local government, and implementing partnersSocial mobilization to sensitize the community about the importance of net useDevelopment of a mobile application to facilitate data collection and analysisA school-based net distribution program, piloted in the Southern Zone of Tanzania to sustain ≥80% universal net coverage previously attained through mass campaigns, successfully issued nets to nearly all eligible students and teachers. Keys to success included: Tanzania successfully scaled up coverage of long-lasting insecticidal nets (LLINs) through mass campaigns. To sustain these gains, a school-based approach was piloted in the country’s Southern Zone starting in 2013, called the School Net Program 1 (SNP1). We report on the design, implementation, monitoring, and outputs of the second round (SNP2) undertaken in 2014. SNP2 was conducted in all schools in Lindi, Mtwara, and Ruvuma regions, targeting students in primary  and secondary (Forms 2 and 4) schools and all teachers. In Lindi region, 2 additional classes (Standards 2 and 4) were targeted. LLIN distribution data were managed using an Android software application called SchoolNet. SNP2 included 2,337 schools, 473,700 students, and 25,269 teachers. A total of 5,070 people were trained in LLIN distribution , and 4,392  community change agents undertook sensitization and mobilization. A total of 507,775 LLINs were distributed to schools, with 464,510 (97.9% of those registered) students and 24,206 (95.8% of those registered) school teachers receiving LLINs. LLIN ownership and use is expected to have increased, potentially further reducing the burden of malaria in the Southern Zone of Tanzania. Keep-up strategies focus on long-term, continuous distribution through channels such as schools, health facilities, community initiatives, and the private sector; catch-up strategies are periodic mass distribution campaigns. NATNETS assessed all possible keep-up options and determined that a combined approach including the TNVS and school-based distribution, involving both push and pull systems, was the most cost-effective and efficient keep-up strategy for Tanzania.The Ministry developed a keep-up strategy in schools to complement the voucher scheme.In 2013, the NMCP and partners piloted the first round of the School Net Program (SNP1) to distribute LLINs in the Southern Zone of Tanzania . Each student in Standards 1, 3, 5, and 7 of primary schools, and Forms 2 and 4 of secondary schools, received an LLIN to take home. A total of 421,285 students (83% of those eligible) received LLINs.We describe here the design, implementation, monitoring, and outputs of the second round of the School Net Program (SNP2), conducted in 2014.SNP2 aimed to increase access to LLINs and maintain at least 80% coverage in the 3 regions by distributing LLINs free of charge to primary and secondary school students and teachers. Teachers were included as beneficiaries in SNP2 because there was an excess supply of LLINs from SNP1. In Lindi, an additional 2 grades (Standards 2 and 4) were targeted for LLIN distribution to reduce the expected remaining stock. As in SNP1, each student from Standards 1, 3, 5, and 7 and Forms 2 and 4 in Mtwara and Ruvuma received 1 LLIN.An SNP task force, chaired by the NMCP, was created to oversee the implementation of SNP2 . The tasNational Malaria Control ProgrammeMinistry of Health and Social Welfare School Health ProgramMinistry of Education and Vocational TrainingPrime Minister’s Office Regional Authority and Local GovernmentSwiss Tropical and Public Health Institute’s NETCELL projectU.S. President’s Malaria InitiativeWorld Health OrganizationJohns Hopkins Center for Communication ProgramsU.S. Peace CorpsMennonite Economic Development AssociatesRTI InternationalTanzania Red Cross SocietyPopulation Services InternationalJohn Snow, Inc.Members of the SNP2 task force included representatives from the:www.vector-works.org/resources/netcalc-planning-tool) to estimate the quantity of nets required to maintain at least 80% universal coverage in the Southern Zone of Tanzania.Before the SNP1 pilot, we used the NetCALC Planning Tool  at the district level, who then trained implementers (teachers) at the ward level. This was a shift away from a more centralized model used in SNP1, which required a substantial period of time, as one team of trainers moved from one district and region to another to conduct trainings. The training period for SNP2 was also reduced from a 2-day session to a half-day session at both the district and ward levels. The TOT targeted district malaria focal persons, district school health coordinators for health and education, and the ward education coordinators. The training of implementers was at the ward level, whereby head teachers and school health teachers from all schools within a ward attended the training. The ward education coordinators were the principal trainers who were supervised by national, regional, and district-based staff. Training focused on the use of LLINs, continuous distribution of LLINs through schools, monitoring, and reporting. Participants learned a systematic approach to managing the LLIN distribution process, including handling data collection forms, enumerating students, recording LLIN issuance data, summarizing data, and submitting data.The IEC/BCC subcommittee of the SNP task force planned advocacy and sensitization meetings; reviewed information, education, and communication (IEC) and behavior change communication (BCC) materials; and trained community change agents and volunteers. The task force distributed IEC materials, and aired radio spots and programs on local radio stations in Ruvuma, Lindi, and Mtwara regions to sensitize the community on the importance of LLIN ownership and use. IEC materials and radio spots focused on the community aspect of LLIN distribution. Spots provided details on the importance of using LLINs, emphasizing that students who received LLINs served as conduits for delivering them to the community.The M&E subcommittee of the SNP task force was set up to revise data collection tools and training manuals. The M&E team developed registration and issuance booklets as well as summary booklets to capture data at the school level during LLIN issuance. They used the summary booklets, filled in by the school principal, to compare the number of LLINs issued against the number of students in the registration and issuance booklets, filled in by class teachers.We developed a database application called SchoolNet to facilitate the collection and management of data from SNP2 schools. The SchoolNet app runs on Android tablets. We trained data clerks to use the SchoolNet app and developed an operative manual for guidance. Data clerks were responsible for entering data from the respective schools onto the tablets. Data were transmitted in real time through the local mobile phone network to a central database where the M&E team cleaned and analyzed the data in a timely manner for programmatic decision making. This approach to data management reduced the time-lag between LLIN issuance and data reporting, which enabled prompt analysis while maintaining high standards of data quality.A tablet-based application called SchoolNet enabled real-time data collection from schools on LLIN issuance.The logistics subcommittee of the SNP task force was responsible for reviewing the transport and logistics protocols, streamlining distribution procedures, and developing a program and timeline to distribute LLINs from Dar es Salaam to all SNP2 schools. The bulk transportation and distribution of LLINs to schools was led by the Tanzania Red Cross Society (TRCS), a humanitarian organization with expertise in logistics and supply chain management. Moving the LLINs from Dar es Salaam to schools involved geographical reconnaissance, transportation planning, rebundling, storage, and transport to the schools through the districts. TRCS transported LLINs from the central-level medical store department warehouse to the district level, rebundled them according to the quantification data, and stored them in warehouses until distribution to respective SNP2 schools.Logistics teams were dispatched to all districts to collect data regarding storage facilities, availability of council and private transports, and maps to design distribution routes. The geographic reconnaissance data were critical to developing a comprehensive logistics and transportation schedule, as it took into account geographical opportunities and challenges, such as poor road infrastructure in hard-to-reach areas.Rebundling LLINs involved grouping them into bales according to the needs of each school in the district, based on quantification data gathered during the SNP2 planning phase. TRCS packed LLINs in smaller volumes and labeled each with the quantity of LLINs and the respective school name. Labeled bundles were loaded onto smaller vehicles for ease of access on rural roads to ensure that they reached the targeted schools. To properly account for the LLIN inventory, SNP2 used delivery notes, warehouse journals, and bin cards to document and track net movement at every point of delivery. We conducted an independent procedural audit to track the LLINs from the central warehouse in Dar es Salaam, to a sample of 3 districts warehouses and to a sample of 27 schools. The audit reported that the logistics chain was effective in delivering LLINs and that there were no losses.LLIN delivery and issuance to all primary and secondary schools took place over a 3-day period on regular school days. Class teachers organized preregistered students by their respective classes. Each student and teacher receiving an LLIN were required to sign against his or her name in the registration and issuance booklets as proof of having received an LLIN. Supervision and documentation focused on the integrity of school data; adherence to procedures for distribution; and feedback on successes, challenges, and areas for improvement related to the LLIN distribution and issuing. We defined LLIN coverage as the proportion of eligible students and teachers who received LLINs.With technical assistance and supervision from the M&E team, data clerks entered data from hard copy training reports, inventory booklets, attendance registers, and school registers in central databases via the SchoolNet app. All information was subjected to periodic data quality checks through regular supervision and review of documents. The head teacher or school principal filled in summary booklets for each school, which the M&E team used to compare the number of LLINs issued against the number of students registered in each class. The issuance forms also captured the number of LLINs that schools received from the district warehouses, thus enabling the schools to keep track of remaining stocks.Issuance forms captured the number of LLINs that schools received, enabling them to track remaining stock.Data from each school were submitted to ward education coordinators for verification and subsequently sent to the district education officers. The M&E team then verified and cleaned the data. The M&E team visited all district offices to supervise the data collection process and to verify the quality, tools, and methodology used. To verify the data entered onto the tablets by the data clerks, the M&E team cross-checked them with hard copies of the issuance forms and summary booklets. All hard copy records were reviewed for accuracy and consistency, and filed in a central repository. Naming conventions were assigned for easy storage and retrieval of the records on hand. Relevant data were extracted from the SchoolNet app database and exported to a Microsoft Excel spreadsheet for analysis.The M&E team used Stata 12.0 to conduct data analysis. The team used descriptive statistics to investigate frequency distributions and proportions, and box plots to investigate the distributions of number of students eligible for SNP by class and region.SNP2 was undertaken as part of public health programming, and therefore ethical clearance was not required a priori. The National Health Research Ethics Subcommittee granted written permission to publish these data.PataPata children’s radio programs were broadcast through local radio stations, including Jogoo FM, Pride FM, Newala FM, and the Tanzania Broadcasting Corporation. A total of 4,392 community change agents  delivered 1,500 sensitization and mobilization sessions, reaching 42,626 people.Comprehensive social mobilization activities complemented LLIN distribution, including IEC and BCC materials, radio spots, and sensitization sessions.All 2,337 schools participated in SNP2-targeted districts, the majority of which were primary schools . The numSchools received a total of 507,775 LLINs, of which 91.5% and 4.8% were issued to students and teachers, respectively . After dOverall, 98% of eligible students and 96% of eligible teachers received LLINs in the 3 regions.,,The school-based net distribution program, SNP2, successfully issued LLINs to 98% of eligible students and 96% of eligible teachers in the 3 targeted regions of Tanzania. Our findings are consistent with a similar pilot study in Ghana where LLINs were distributed to students in grades 2 and 6 of primary schools, teachers, and district office team members, and achieved nearly 100% LLIN coverage among eligible participants.We recommend implementing continuous LLIN distribution through schools within 9 to 12 months of any mass distribution campaign.,,Compared with SNP1, SNP2 achieved higher LLIN coverage of students (98% versus 83%), distributed 16% more nets, and had fewer LLINs remaining after distribution (4% versus 17%) due to a change in the program design to include teachers in all regions and additional grades in Lindi region. SNP2 was built on effective collaboration and teamwork between the Ministry of Health and Social Welfare, local government, and implementing partners. Specific roles and responsibilities were assigned to working groups to implement elements of the SNP framework—a crucial ingredient for the success of SNP2 activities, as demonstrated in previous campaigns conducted in Tanzania.Slight differences between school enrollment data provided by districts (used for quantification) and student registration at schools  may have resulted in a deficit or oversupply of LLINs in a small proportion of schools. Such deficits were resolved through redistribution of excess LLINs from neighboring schools. We therefore recommend that head teachers should provide school enrollment data for net quantification. Also, based on feedback from participants, the half-day training for trainers and implementers was not adequate to cover all necessary SNP2 details; trainings should be extended to 1 day in future rounds of the SNP. We also recommend that the training of key implementers (teachers) should be coordinated by the Ministry of Education and Vocational Training, who should be more engaged in SNP. Finally, models have shown that 35% of households are potentially not reached by either the TNVS or SNP.Future school-based LLIN distribution should consider targeting primary school students only.SNP2 was successful in reaching 98% of eligible students and 96% of eligible teachers in 3 regions in Tanzania. Effective partnerships, coordination, and teamwork facilitated the successful implementation of SNP2. LLIN ownership and use in the community is expected to increase and therefore reduce the burden of malaria in the 3 regions. This program can serve as a model for other countries who wish to implement a school-based approach as a keep-up strategy to maintain universal coverage of LLINs for malaria prevention and control.


NSP4) is a critical protein for rotavirus (RV) replication and assembly. This protein has multiple domains and motifs that predispose its function and activity. NSP4 has a sequence divergence in human and animal RVs. Recently, 14 genotypes (E1-E14) of NSP4 have been identified, and E1 and E2 have been shown to be the most common genotypes in human.Non-structural protein 4 (NSP4 in RV-positive samples were inspected with the aim of NSP4 genotyping and variation analysis in viroporin and other domains. P and G typings of RV samples were carried out by WHO primers using a semi-multiplex PCR method. Non-typeable RV samples were amplified by conserved primers and sequenced.The gene and protein sequence of In viroporin and enterotoxin, conserved sequence was detected, and amino acids substitution with the same biochemical properties was found.NSP4 genotype with P or G genotyping G1/G9 correlates with E1 genogroups. In electrophoretyping of RV, E2 genotype had a short pattern when compared to E1.Association of  Reoviridae family with 100 nm diameter and contains icosahedral nucleocapsid symmetry, 1T-1 P[8], and 3T-1 P[6] forward and reverse primers, respectively. The cDNA was constructed with RevertAid First Strand cDNA Synthesis Kit  and gene specific consensus End 9 and con3 primers . Typically, the first-round PCR was performed in a 50-µl volume containing 1 × PCR buffer  for 1 to 2 min until the appearance of dark-stained bands on the yellow background in 17 (62.9%) samples, P[4] in 8 (29%), and P[6] in 1 (3%). Amplicon sizes of P[4], P[8], P[6], and P[9] genotypes were 484, 346, 260, and 392 bp, respectively; P[9] genotype was not identified . In all NSP4 genotyping assay  and E2  were obtained. In general, E1 and E2 genotypes are dominant E genotypes in human RV samples/E1, and G2P[4]/E2 were detected, and no inter-genogroup reassorting containing VP7, VP4, and Based on previous epidemiological studies from Iran-42, ther


Objective: The purpose of this study was to validate our previous genetic association findings related to the endoglin (ENG) pathway from an American Caucasian preeclampsia cohort in independent preeclampsia cohorts. We also sought to explore the ENG pathway for new genetic associations in these independent cohorts. Study design: We used a tagging single nucleotide (tSNP) approach to assess genetic variability across five ENG pathway genes  in a Caucasian cohort from Norway (n = 77 preeclampsia cases & n = 63 normotensive controls) and a White Hispanic cohort from Southern California (n = 69 preeclampsia cases & n = 106 normotensive controls). Main outcome measures: Univariate analyses  and multivariate logistic regression were conducted to evaluate the association between tSNP genotype distributions and pregnancy outcome in each cohort. Logistic regression models were adjusted for maternal age at delivery, infant sex, parity, smoking during pregnancy, and pre-pregnancy BMI. Results: Although we were unable to replicate our previous SNP-specific findings , we found that genetic variation in TGFβR1[ALK5] (rs6478974) and TGFβR2  was significantly associated with preeclampsia in the Norwegian cohort and genetic variation in ALK1 (rs706819) and TGFβR2 (rs9843942) was significantly associated with preeclampsia in the Latina cohort. Conclusion: Overall, our results provide further support for the involvement and investigation of the endoglin pathway in preeclampsia. Characterized as one of the “great obstetrical syndromes,” ENG mRNA expression is elevated in the placenta and the maternal blood (cellular and cell-free components) in women who develop preeclampsia ENG mRNA expression is significantly elevated in microvascular endothelial cells isolated from adipose tissue obtained at cesarean section in women with preeclampsia trans-membrane ENG receptor, is postulated to enter the maternal circulation from the placenta where it sequesters transforming growth factor beta (TGFβ) and inhibits TGFβ-mediated cell signaling and endothelial function Endoglin  is a membrane-bound co-receptor of transforming growth factor beta (TGFβ) that regulates vascular tone ENG and other members of the TGFβ1 signaling system may play in the development of preeclampsia. Using a case-control genetic association study design and a tagging single nucleotide polymorphism (tSNP) approach, we previously found that genetic variation in ENG  and TGFβR2 (rs6550005) was significantly associated with the development of preeclampsia in American Caucasian women, while genetic variation in TGFβ1 , TGFβR1 (rs10739778), and TGFβR2  was significantly associated with the development of preeclampsia in African American women These elevations in endoglin mRNA expression and circulating endoglin protein levels in women with preeclampsia, assumed to be due to the increased contribution from a dysfunctional placenta, prompted us to investigate the role that maternal genetic variation in ENG pathway for new associations in these two cohorts.The purpose of this current study was to 22.1De-identified clinical data and DNA samples were obtained from the Oslo Pregnancy Biobank and the “Pilot Study of Novel Candidate Genes for Preeclampsia” research study conducted at the University of Southern California, for Norwegian and Latina cohort participants, respectively.Participants of the Oslo Pregnancy Biobank were women with singleton pregnancies who delivered via elective cesarean section between the years 2001 and 2008. Women with a history of chronic hypertension, renal disease, diabetes, or other chronic diseases were excluded. Preeclampsia was defined as new-onset hypertension and proteinuria after gestational week 20, in accordance with the American College of Obstetricians and Gynecologists 2002 criteria that were in place when the study was conducted Participants of the “Pilot Study of Novel Candidate Genes for Preeclampsia” case-control research study  were retrospectively recruited from delivery logs at the Los Angeles County and University of Southern California Women’s and Children’s Hospital or during their postpartum stay at the Women’s and Children’s Hospital between the years of 1999 and 2008. Women with lupus, chronic renal disease, multiple gestation, or sickle cell disease/trait were excluded from participation. Preeclampsia was also defined in alignment with the blood pressure and proteinuria criteria outlined by the American College of Obstetricians and Gynecologists in 2002 (please see criteria above) 2.2TGFβR2 AND rs11129420) to learn about the potential function of the tSNPs that were significantly associated with pregnancy outcome in the multivariate models.DNA aliquots obtained from the Norwegian cohort participants were extracted from EDTA peripheral blood samples using the MagNA Pure LC DNA Isolation Kit . DNA aliquots obtained from the Latina cohort participants were extracted from either peripheral blood samples or buccal swabs using the QIAamp DNA mini kit , saliva samples using the Oragene kit , or mouthwash samples using a phenyl-chloroform protocol previously described 2.3t-test, Fisher’s exact test). For the main analyses, we utilized a tiered analytic approach. For the first analytic tier, we used Chi-square or Fisher’s exact tests  and multivariate regression models to evaluate three tSNPs that were found to be significantly associated with pregnancy outcome in our previous study . In the second analytic tier, we evaluated the remaining tSNPs that had been genotyped. Univariate analysis with either Chi-square or Fisher’s exact tests was conducted to initially explore the relationship between pregnancy outcome and each tSNP. A p-value of p ≤ 0.15 was considered as potentially important. Multiple logistic regression models were then used to assess the relationship between potentially important tSNPs and pregnancy outcome while controlling for maternal age at delivery, infant sex, parity, self-reported smoking status during the index pregnancy, and pre-pregnancy body mass index (BMI). The adjusted odds ratio, 95% confidence interval, and the p-value of the tSNPs were reported. The overall goodness-of-fit of the logistic models were assessed by the Hosmer-Lemeshow test. We did not correct for multiple comparisons. Power analysis revealed that a sample size of 140 achieves 80% power to detect moderate effect size (W) of 0.2368 using a 1 degree of freedom Chi-square Test with a significance level  of 0.05.Demographic and clinical characteristics were compared between cases and controls using parametric and non-parametric tests . We used gestational age at delivery as a surrogate marker for preeclampsia onset  and compared. tSNP genotype distributions for each preeclampsia subgroup to the healthy control group using Fisher’s exact tests.33.1TGFβR2 tSNP (rs6550005) was associated with preeclampsia in both an American Caucasian and an African American cohort. We found additional tSNPs that were associated with preeclampsia in the African American cohort that were not found to be associated with preeclampsia in the American Caucasian cohort, but here we focused on tSNPs that were associated with preeclampsia in the American Caucasian cohort given that our replication cohorts were not of African ancestry.The demographic and clinical characteristics for the Norwegian and Latina cohorts are presented in ENG: rs11792480, rs10121110; TGFβR2: rs6550005) and preeclampsia status were not replicated in both the unadjusted and adjusted analyses (p’s > 0.05)  and TFFβR2 (Six intronic tSNPs in two 3773663)   and TGFBR2 (Our exploratory subgroup findings suggest that certain tSNPs in two ntronic) . Because4ENG pathway are associated with an increased susceptibility to preeclampsia in both American Caucasian and African American women ENG mRNA expression and circulating sENG protein levels is well documented across multiple studies, few studies have explored the role of ENG pathway genetic variation in preeclampsia development.Preeclampsia is a heterogeneous disorder of pregnancy whose etiology may stem from a variety of underlying mechanisms. Women who develop early-onset and late-onset preeclampsia differ on average on several aspects, including their risk of future cardiovascular disease ENG pathway and susceptibility to/protection from preeclampsia in independent, well-characterized preeclampsia cohorts. We first sought to validate our genetic association findings from an American Caucasian preeclampsia cohort  in Norwegian and Latina cohorts. We further explored the other gene candidates from the ENG pathway for new genetic associations with preeclampsia in these cohorts. We were unable to validate/replicate the SNP-specific previous findings in these independent cohorts. However, we identified other associations between ENG pathway candidate genes and preeclampsia in the Norwegian  and Latina  cohorts in our adjusted models. We have previously demonstrated that variation in TGFβR1[ALK5] was associated with preeclampsia in an African American cohort TGFβR2 is associated with susceptibility to/protection from preeclampsia in all of the populations we have studied. Although the TGFβR2 tSNPs that were associated with a particular cohort may differ, these findings, along with the other associations in the Norwegian and Latina cohorts, provide additional evidence to support the ENG pathway’s role/involvement in the development of preeclampsia.In this study we investigated the association between maternal genetic variation in the 4.1Several potential explanations could account for our inability to validate our previous results in independent cohorts. First, these cohorts may not have been sufficiently powered to detect statistically significant associations when they truly existed. Second, tSNPs that were selected based on Caucasian ancestry (CEU population-European descendants living in Utah) may not be as informative in the Norwegian and Latina cohorts if the haploblocks tagged by the tSNPs differ by ancestry. Finally, differences in the definition of preeclampsia phenotype may underlie our failure to validate our previous results. In the original cohort, a diagnosis of preeclampsia was based on hypertension, proteinuria, and hyperuricemia. Hyperuricemia was included in the original cohort’s research definition as a way to identify a more homogeneous and potentially more severe form of preeclampsia. In both the Norwegian and Latina cohorts, hyperuricemia was not included as a criterion for the preeclampsia phenotype. Given the heterogeneous nature of preeclampsia, these cohorts may represent different preeclampsia subtypes with differing etiologies.4.2TGFβR1[ALK5] and TGFβR2 was associated with susceptibility to/protection from preeclampsia in the Norwegian cohort, while genetic variation in ALK1 and TGFβR2 was associated with susceptibility to/protection from preeclampsia in the Latina cohort. Seven of the eight significantly associated tSNPs .In addition to small sample size and differences in ancestry that were previously discussed, there were several other limitations associated with this study. First, the cohorts’ sample sizes were further reduced when we carried out our exploratory preeclampsia subgroup analyses, which limited our ability to adequately evaluate preeclampsia subgroups. Second, we did not adjust for multiple comparisons and therefore we cannot rule out the potential for type 1 error. As such, follow-up studies with larger sample sizes, including larger preeclampsia subgroups, will be needed to validate these results. Third, we were unable to successfully genotype three SNPs (5Overall, our results provide further support for the involvement and investigation of the endoglin pathway in preeclampsia, yet additional research with larger sample sizes, including larger samples of different preeclampsia subgroups, is needed. Due to the relatively low frequency of preeclampsia, there is a need for investigators across the globe to collaborate and share data/biological samples with each other so that we can continue to put preeclampsia’s puzzle pieces together.


In vitro studies assessed the role of caspase-2 in adipose tissue proliferative properties and susceptibility for lipoapoptosis. Caspase-2-deficient mice fed a Western diet were protected from abdominal fat deposition, diabetes mellitus, dyslipidemia and hepatic steatosis. Adipose tissue in caspase-2-deficient mice was more proliferative, upregulated mitochondrial uncoupling proteins consistent with browning, and was resistant to cell hypertrophy and cell death. The liver was protected from steatohepatitis through a decrease in circulating fatty acids and more efficient hepatic fat metabolism, and from fibrosis as a consequence of reduced fibrogenic stimuli from fewer lipotoxic hepatocytes. Caspase-2 deficiency protected mice from diet-induced obesity, metabolic syndrome and nonalcoholic fatty liver disease. Further studies are necessary to assess caspase-2 as a therapeutic target for those conditions.Obesity and its resulting metabolic disturbances are major health threats. In response to energy surplus, overtaxed adipocytes release fatty acids and pro-inflammatory factors into the circulation, promoting organ fat accumulation , insulin resistance and the metabolic syndrome. Recently, caspase-2 was linked to lipoapoptosis, so we hypothesized that caspase-2 might be a critical determinant of metabolic syndrome pathogenesis. Caspase-2-deficient and wild-type mice were fed a Western diet  for 16 weeks. Metabolic and hepatic outcomes were evaluated.  Ectopic fat accumulation in the liver, dubbed nonalcoholic fatty liver disease (NAFLD), strongly associates with the metabolic syndrome.4 Whereas metabolic disturbances, including insulin resistance undoubtedly can induce hepatic steatosis, whether or not hepatic steatosis can induce insulin resistance is still a matter of debate. On one hand, epidemiological studies suggest that patients with NAFLD are at increased risk for developing T2DM.5 On the other, several mouse models of NAFLD do not develop insulin resistance, and even exhibit increased sensitivity to insulin.8Western societies exist in an era of caloric excess that is at odds with evolutionary adaptations to the ancestral low-calorie lifestyle. As Western dietary habits have spread, obesity prevalence has nearly doubled worldwide. In 2008, 1.4 billion adults were overweight and half a billion obese, comprising 35 and 11% of the world population.9 Fat-swollen adipocytes become insulin resistant, decrease expression of adipokines such as the anti-inflammatory insulin-sensitizer adiponectin, and increase expression of the satiety factor leptin.10 Stressed adipocytes undergo cell death in mice with genetic- or diet-induced obesity, and in morbidly obese humans.11 Adipocyte cell size and cell death correlate with the presence of metabolic syndrome, insulin resistance and NAFLD.12Adipocytes respond maladaptively to chronic energy surplus, resulting in adipocyte hypertrophy.13 The primary function of caspase-2 remains to be determined, however, because it has also been implicated in several other vital processes including regulation of cell cycle checkpoints, oxidative stress responses, autophagy and senescence.17 Intriguingly, caspase-2 mRNA expression is elevated in adipose tissue of rats fed high-fat diet.18 Recently, caspase-2-initiated apoptosis was associated with lipotoxicity caused by accumulation of saturated fatty acids in frog oocytes,19 cultured mouse hepatocytes, and in mouse and human NAFLD.20 Caspase-2 expression strongly correlated with liver disease severity in patients with NAFLD.20 Further, caspase-2 depletion protected mice with methionine/choline-deficient (MCD) diet-induced steatohepatitis from hepatocyte apoptosis and fibrosis progression.20Caspase-2 is an initiator caspase for cellular apoptosis.We hypothesized that caspase-2 may be a key link between energy surplus, adipocyte death and the development of the metabolic syndrome and NAFLD. Our data support this concept because caspase-2-deficient mice fed a Western diet were protected from many aspects of the metabolic syndrome, namely increased adiposity, insulin resistance, dyslipidemia and NAFLD development and progression. These results suggest caspase-2 as a target for managing the metabolic syndrome, obesity, T2DM and NAFLD.versus subcutaneous distribution of body fat is deleterious.21Caspase-2-deficient mice and wild-type (WT) controls were fed standard chow diet or high-fat, high-sugar Western diet for 16 weeks. At the end of treatment, WT mice fed Western diet weighed 10% more than WT mice-fed chow diet. However, Western diet induced minimal additional weight gain in caspase-2-deficient mice . SimilarDifferences in adiposity could not be explained by decreased food intake, as caspase-2-deficient mice ate 8% more solid food and drank 40% more sugar-supplemented water than WT mice. Indeed, WT mice had higher food efficiency, gaining more weight per gram of food ingested .23 Western diet induced hyperplasia of pancreatic Langerhans islands in WT mice, but this expansion was significantly blunted in caspase-2-deficient mice . Caspase-2-deficient mice were protected from dyslipidemia . The fai10 Consistent with the increased number of smaller cells in caspase-2 knockout mice, these cells also had increased expression of cyclins, suggesting increased proliferative activity in the adipose cells of caspase-2-deficient mice , suggesting browning of the adipose tissue . UCPs deogenesis .28 Relative to WT mice, expression of crucial genes that regulate adipocyte metabolism and differentiation were increased in the adipose tissue of capase-2 deficient mice, including lipoprotein lipase, peroxisome proliferator activator receptor-γ (PPAR-γ); sterol regulatory element-binding protein-1 (SREBP-1), CCAAT-enhancer-binding protein-α, (CEBP-α) and liver X receptor-α (LXR-α) (Caspase-2 expression decreases as 3T3-L1 pre-adipocytes become adipocytes during culture-induced differentiation. (LXR-α) . Further (LXR-α) . Accumul29 Western diet induced massive hepatomegaly in WT mice, with an almost twofold increase in liver mass, and 50% increase in liver-to-body weight ratio. However, caspase-2-deficient mice maintained normal liver weight and liver-to-body weight ratio on Western diet , correlating with fibrosis severity in patients. of NASH . Western of NASH . We also of NASH , TUNEL s of NASH  and immu of NASH .30 Western diet induced more liver fibrosis in WT mice than in caspase-2-deficient mice, as assessed by Sirius Red staining, hydroxyproline content, qRT-PCR for collagen-1α1, and immunohistochemistry for the myofibroblast markers, α-smooth muscle actin (α-SMA) and desmin (20 Fibrosis progression in NASH directly correlates with accumulation of reactive appearing bile ductules and inflammatory cells (the ductular reaction).31 Compared with WT mice fed Western diet, caspase-2-deficient mice were also protected from diet-induced ductular reaction  that induces cell death and is expressed in most tissues, and caspase-2S (short isoform) that antagonizes cell death, and is expressed mainly in brain, heart and skeletal muscle.32 This suggests an ancient role for caspase-2 in preserving the most essential organs by sacrificing less critical tissues in order to survive prolonged fasting. However, in modern times, energy surplus can also lead to accumulation of toxic lipids, leading to maladaptive caspase-2 activation.The involvement of a protein known to regulate cell death and the cell cycle in regulating whole-body metabolism is non-intuitive. Natural selection optimized mechanisms to cope with food deprivation. When presented with energy surplus, these adaptive mechanisms become deleterious and lead to the development of the metabolic syndrome. Caspase-2, the most evolutionarily conserved caspase,18 and complement an earlier report showing that caspase-2-deficient mice resist age-induced glucose intolerance.16 Our findings are in concordance with previous work from other labs showing an increase in adipocyte apoptosis both in mouse models of diet-induced obesity and obese humans.33 Adipocyte apoptosis correlates with insulin resistance and metabolic disturbances in obesity.34 Moreover, adipocyte apoptosis likely plays a causal role in both processes because deletion of the pro-apoptotic factor, Bid, prevented adipose tissue inflammation, systemic insulin resistance, and hepatic steatosis in mice with diet-induced obesity.33 The mechanisms involved are being uncovered. For example, there is evidence that apoptosis-effector caspases cleave PPAR-γ and GLUT-4, leading to decreased lipogenic gene transcription and impaired glucose uptake, effects that promote insulin resistance.35 In aggregate, these data suggest a unifying hypothesis whereby adipocyte apoptosis both leads to a massive decrease in adipose tissue capacity for fat storage by limiting the expansibility of the adipose tissue and induces an insulin resistant and pro-inflammatory state that promotes the metabolic syndrome. As a key initiator of adipocyte apoptosis, caspase-2 activation is a critical driver for this state of ‘relative lipodystrophy' that limits the adaptive response to energy surplus.We propose the following paradigm: Western diet leads to accumulation of toxic lipids in adipocytes. Lipotoxic adipocytes activate caspase-2, inducing cell stress and death, leading to the release of toxic mediators , decreased uptake of NEFAs from blood, and release of NEFAs from dying cells. The net result is an increase in circulating NEFAs and perturbed adipokine secretion, promoting the metabolic syndrome. Supporting this hypothesis, caspase-2-deficient mice fed a Western diet showed some evidence of reduced activation of apoptosis in abdominal adipose tissue, normal adipokine secretion profile, decreased levels of circulating NEFAs, and protection from the development of insulin resistance/T2DM and the metabolic syndrome. These new findings reveal the relevance of increased caspase-2 in adipose tissue of rats fed a high-fat diet,36 Indeed, in twin pairs discordant for obesity, the prevalence of insulin resistance, metabolic syndrome and NAFLD in obese twins with hyperplastic adipose tissue is similar to that of their lean siblings, whereas obese twins with hypoplastic adipose tissue have higher rates of these conditions than their lean counterparts.37 In other studies, adipocyte size also had a strong positive correlation with risk for developing T2DM, cardiovascular disease and NAFLD.39 Several putative mechanisms for insulin resistance have been demonstrated in enlarged adipocytes including impaired insulin signaling due to decreased concentration of cholesterol in the cellular membrane,40 cytoskeleton disassembly,41 and excessive local accumulation of NEFAs.42 Fat depots with hypertrophic adipocytes also exhibit oxidative stress,43 ER stress,44 hypoxia,45 inflammation46 and fibrogenesis.47 These cellular stresses can culminate in adipocyte death, decreasing adipose tissue storage capacity during energy surplus, with the resultant relative lipodystrophy promoting an inflammatory state that characterizes human obesity.We identified several mechanisms in caspase-2-deficient mice that promote adipocyte homeostasis in response to energy surplus. Abdominal adipose tissue from caspase-2-deficient mice is fundamentally different from that of WT mice: adipose stem cells are more proliferative and hence, caspase-2-deficient mice have a larger number of small adipocytes, even when fed chow diet. Fat mass expansion by hyperplasia (increased cell number) is considered a healthier mechanism to compensate for energy surplus than expanding fat mass via adipocyte hypertrophy (increased cell size).48 In addition, female caspase-2-deficient-mice are known to have more primordial follicles than WT mice, although this results from reduced apoptosis, rather than increased cell division.49 Here we show not only that untransformed adipose stem cells are more proliferative in vitro, but also that chow-fed caspase-2-deficient mice have more adipocytes in abdominal adipose tissue in vivo than WT mice, and this difference persists after challenge with Western diet. The mechanism by which caspase-2 depletion confers a proliferative advantage is not yet fully understood. Caspase-2 is likely to inhibit replication because its activity is typically repressed during mitosis by a mechanism involving cyclin B1-mediated phosphorylation of caspase-2.50 Caspase-2 has been proposed to regulate cell cycle checkpoints through regulation of p53;51 p53 also regulates caspase-2 by controlling transcription of p53-induced protein with a death domain (PIDD), a scaffold protein required for caspase-2 activation. On the other hand, caspase-2 negatively regulates the NFkB survival and proliferative pathways through cleavage and degradation of receptor-interacting serine/threonine protein kinase 1 (RIPK1)52 and competition for assembly with PIDD.13Our study is the first to report increased proliferative capacity in untransformed somatic cells of adult caspase-2-deficient mice. Embryonic fibroblasts from caspase-2-deficient mice were reported to have a higher growth rate after oncogenic transformation than comparable cells from WT mice.53 Metabolic reprogramming of adipose tissue can also help explain the phenotype of caspase-2-deficient mice. We found increased expression of adipocyte differentiation markers, PPAR-γ, CEBP-α and SREBP-1c in caspase-2-deficient mice. These genes control adipocyte triglyceride storage capacity, and are expressed at high levels in adipose tissues of metabolically healthy obese individuals.54 In addition, PPAR-γ is a crucial regulator of gene networks involved in glucose homeostasis, controlling adiponectin expression.55 LXR-α expression was also increased in caspase-2-deficient adipose tissue. LXR-α promotes lipolysis and fatty acid stimulated oxygen consumption, and associates with decreased adipocyte size.56 The net outcome of these various responses to caspase-2 deletion is adipose depots with a larger number of adipocytes that are able to consume more energy. Hence, caspase-2-deficient mice do not develop stressful enlargement of adipocytes when challenged with an energy-dense Western diet. This is important as increased size/volume of adipocytes correlates with the development of T2DM.10 These beneficial effects of caspase-2 deletion of adipose tissue metabolism are likely to contribute to the profound protection from hepatic steatosis in caspase-2-deficient mice fed Western diet. Indeed, caspase-2-deficient mice were not protected from hepatic steatosis when fed MCD diet,20 a model in which hepatic steatosis is a consequence of reduced hepatic lipid export, rather than increased import of adipose-derived NEFAs. Additionally, the livers from caspase-2-deficient mice coped better with Western diet-related energy surplus by reprogramming hepatic metabolism, decreasing de novo hepatic lipogenesis, increasing hepatic fatty acid oxidation, and increasing fat export from the liver as lipoproteins. As in our prior study of MCD diet-fed mice,29 caspase-2 depletion protected Western diet-fed mice from liver injury, inflammation, and fibrosis. Thus, modulating caspase-2 activity is an appealing therapeutic approach for obesity and related metabolic syndrome pathology, including NAFLD.Our data also show, for the first time, that white adipocytes from caspase-2-deficient mice have evidence of browning, as indicated by UCP induction to more effectively cope with energy excess. Caspase-2 can also regulate metabolism through other mechanisms that are not fully understood. For instance, it has been suggested that caspase-2 regulates expression of FoxO1 and FoxO3a, important players in insulin signaling.57 Our studies, on the other hand, demonstrated a decrease in the level of liver oxidative stress caused by Western diet. This result is consistent with evidence that caspase-2 deficiency reduced diet-induced lipid accumulation, because ectopic fat accumulation is a major driver of oxidative stress.Capase-2 inhibition appears to be safe in mice. Caspase-2-deficient mice have a near normal phenotype and do not develop excessive tumors with aging, despite the fact that caspase-2 is an inhibitor of apoptosis and as such, might have some tumor suppressing functions. Specific inhibition of caspase-2 is also predicted to be safer than compromising the function of multiple caspases by treatment with pan-caspase inhibitors. Nevertheless, some reports have suggested that caspase-2 deficiency might lead to premature aging by reducing the clearance of cells damaged by oxidative stress.Our work raises several questions that must be further addressed. The biochemical mechanism by which caspase-2 deficiency protects from metabolic dysregulation remains unidentified. This must be clarified in order to design an appropriately targeted intervention that abrogates the deleterious effects of caspase-2 during energy excess. Current understanding of caspase-2 biochemistry suggests that caspase-2 might drive metabolic stress either via its proteolytic-catalytic activity (which is pro-apoptotic) or its scaffolding functions (which mediate the molecule's actions on proliferation and gene expression). Further, because no specific inhibitor of caspase-2 has been developed yet, it has not been possible to test the ability of such an agent to recapitulate the effects of caspase-2 gene deletion. Nevertheless, the current study provides compelling novel evidence that caspase-2 is a potential target to correct obesity and its associated comorbidities: metabolic syndrome, insulin resistance/T2DM and NAFLD. These diseases are the major killers of the Western world, and approaches to treat or prevent them will have tremendous implications for overall human health.et al. inactivated the caspase-2 gene by homologous recombination in 129S4/SvJae-derived J1 embryonic stem cells, and the founder mice were backcrossed to C57BL/6J inbred mice for 10 generations by Bergeron et al. before being donated to the Jax Lab, where they were C57Bl/6J-backcrossed for at least one more generation, resulting in genetic uniformity. Mice were fed either chow diet  or Western diet  for 16 weeks, beginning at 4 weeks of age. Diet specifications are in Male caspase-2-deficient and congenic WT C57Bl/6J mice were obtained from Jackson Laboratory . Bergeron During the last week of diet, glucose and insulin tolerance tests were performed. Glucose tolerance test was performed after 12 h of fasting, by IP injection of glucose (2 g/kg). Insulin tolerance test was performed after 5 h of fasting, with IP injection of 0.6 units/kg of human regular insulin. Glucose levels were measured at sequential time points by tail vein sampling using a glucometer.2, as previously described.58In mice, body mass index was calculated as the body weight (g)/(crown-rump length (mm))Animal care and procedures were approved by the Duke University Institutional Animal Care and fulfilled National Institutes for Health and Duke University IACUC requirements for humane animal care.μm serial sections for H&E and Oil red staining. Immunohistochemistry was performed as previously described,20 with the primary antibodies specified in Formalin-fixed, paraffin-embedded liver, pancreas and epididymal adipose tissue biopsies were cut into 5 Insulin was measured with Ultrasensitive Mouse Insulin ELISA kit , after 12 h fasting. Lipids were measured with Triglyceride Colorimetric Assay kit , Free Fatty Acid Quantification Kit,  and Cholesterol Quantification kit ; serum leptin and adiponectin were determined with Abcam mouse ELISA kits, ab100718 and ab108785, respectively.59 For primers, see Total RNA was extracted from tissue using TRIzol . RNA was reverse transcribed into cDNA templates using random primer and SuperScript RNAse HReverse Transcriptase (Invitrogen). Semiquantitative qRT-PCR was performed using iQ-SYBR Green Supermix  on a StepOne Plus Real-Time PCR Platform , as previously described.Total proteins were extracted from liver and adipose tissue using RIPA buffer . Equal amounts of protein were separated by electrophoresis on 4–20% Criterion gels (Bio-Rad), transferred onto polyvinylidene difluoride membranes, and incubated with the primary antibodies specified in 60Mouse adipose tissue stem cells were isolated from caspase-2-deficient and WT mice as previously described.61 and differentiation confirmed with Oil red staining. Differentiated adipocytes were treated with 1 mM palmitate without or with 20 μM caspase-2 inhibitor Z-VDVAD-FMK  for 48 h.3T3-L1 cells were differentiated into adipocytes,Apoptosis was measured using ApoTox-Glo Triplex Assay  and proliferation using BrdU assay .P<0.05 considered significant.Results are expressed as mean±S.E.M. Significance was established using two-way ANOVA, with


The measurement of Pavlovian forms of fear extinction offers a relatively simple behavioral preparation that is nonetheless tractable, from a translational perspective, as an approach to study mechanisms of exposure therapy and biological underpinnings of anxiety and trauma-related disorders such as post-traumatic stress disorder (PTSD). Deficient fear extinction is considered a robust clinical endophenotype for these disorders and, as such, has particular significance in the current “age of RDoC (research domain criteria).” Various rodent models of impaired extinction have thus been generated with the objective of approximating this clinical, relapse prone aberrant extinction learning. These models have helped to reveal neurobiological correlates of extinction circuitry failure, gene variants, and other mechanisms underlying deficient fear extinction. In addition, they are increasingly serving as tools to investigate ways to therapeutically overcome poor extinction to support long-term retention of extinction memory and thus protection against various forms of fear relapse; modeled in the laboratory by measuring spontaneous recovery, reinstatement and renewal of fear. In the current article, we review models of impaired extinction built around (1) experimentally induced brain region and neural circuit disruptions (2) spontaneously-arising and laboratory-induced genetic modifications, or (3) exposure to environmental insults, including stress, drugs of abuse, and unhealthy diet. Collectively, these models have been instrumental in advancing in our understanding of extinction failure and underlying susceptibilities at the neural, genetic, molecular, and neurochemical levels; generating renewed interest in developing novel, targeted and effective therapeutic treatments for anxiety and trauma-related disorders. Canst thou not minister to a mind diseased,Pluck from the memory a rooted sorrow,Raze out the written troubles of the brain…Shakespeare, Macbethfear extinction, the CS is typically a previously neutral stimulus that, through conditioning, has come to be associated with an aversive outcome, such that its occurrence alone is capable of eliciting some form(s) of fear/anxiety/defensive CR. The CR, and the degree of its diminution in amplitude and frequency under extinction, can be objectively measured and quantified in the laboratory in human  and rodent  subjects and has even been documented in invertebrates  described extinction as a phenomenon whereby repeated non-reinforced presentation of a conditioned stimulus (CS) led to the reduction in the magnitude of the conditioned response(s) (CR) Pavlov . In the In addition to being relatively easy to compare readouts of fear extinction between humans and rodents, given the similarity of laboratory testing procedures, there are a number of other factors that give extinction translational appeal. First, as summarized below, many of the brain regions that have been connected in some way to effective or deficient fear extinction, respectively, are common across humans and rodents—lending credence to the idea that findings at the neural level in one species are informative to the other  of these factors, and is distinguished from the use of fear extinction as a test or assay  inactivation, have been supplemented by newer optogenetic and chemogenetic approaches that allow for access over specific neural populations and pathways with better spatial and/or temporal precision. From these studies, it is clear that the learning of and the memory for extinction is distributed in a network fashion across brain areas including the prefrontal cortex (PFC), amygdala and hippocampus, but also a range of additional structures such as periaqueductal gray (PAG), bed nucleus of the stria terminalis (BNST), VTA, striatum, and others.The broad strokes of the principal findings to date are as follows: Using a range of techniques, including temporary inactivations, immediate-early gene analyses, single-unit recordings, and optogenetics, activity in the dorsomedial PFC (dmPFC) positively associates with high fear/poor extinction, via reciprocal connections to pro-fear subpopulations of projections neurons in the basal amygdala (BA) .The well-studied vHPC-mPFC-BA system subserving extinction is itself supported by other structures that are being revealed by recent work. These include the ventrolateral periaqueductal gray (vlPAG), which when inhibited impairs extinction (and fear learning)   encoded within this genomic region , suggesting this gene affects extinction by modulating a key stress-regulating system  (C57BL/6 J) mice could be split into extinction-resilient and susceptible subgroups, that were in turn associated with specific patterns of corticoamygdala activity Table  and geneThere are interesting overlaps between the effects of exposure to stress and drugs of abuse, another risk factor for anxiety disorders and PTSD, on extinction. For example, chronic cannabis use is associated with impaired extinction in humans (Papini et al. Unlike most other abused drugs, alcohol is consumed like a food and is a source of calorific intake. The degree to which this contribute to the deleterious physiological effects of chronic drinking is debated but may be relevant here given recent evidence that abnormal diet can affect fear extinction. Rats fed a high-fat/high-sugar diet over the course of 6 weeks developed poor extinction Table  and alteWe wish to highlight these latter findings not to overstate the potential importance of poor diet as a risk factor for trauma-related conditions, but rather to underscore the expanding range of environmental insults found to disrupt fear extinction. A challenge for future work will be to model the real-world combination of dietary factors, exposure to drugs and life stressors faced by most at-risk individuals, and to use the rodent models to decipher how these interact and potentially synergize to affect clinical outcomes.The value of rodent models of impaired extinction is ultimately gauged by their utility as a platform for the identification of novel mechanisms for therapeutically normalizing extinction. While there are examples of targeting circuit abnormalities via deep brain stimulation in these models (Rodriguez-Romaguera and Quirk Once a promising pharmacological target is identified, a critical question is how it should be clinically administered to maximize its therapeutic potential and mitigate risk of failure in clinical trials. In this regard, the administration of a single drug concomitant to fear extinction in extinction-deficient individuals does often not suffice to support the extinction memory-augmenting mechanism to an extent that prevents temporal, spatial, or stress-dependent fear relapse (Singewald et al. Supporting the utility of this dual pharmacotherapeutic concept, it was demonstrated that fear relapses in extinction-deficient mice can also be reduced by combined administration of L-DOPA and the HDAC-inhibitor MS-275, concomitant to extinction training (Whittle et al. Beyond pharmacological approaches, less conventional approaches have to date been less studied in extinction-deficient models, but are certainly worthy of investigation. One interesting modification to changing the way extinction memories are formed simply involves training in multiple contexts (de Jong et al. A final point to underscore is that eventual success of novel treatments will be bolstered by grounding them in a solid understanding of how they act at the neural level. The field can draw upon the great advances that have been made in delineating the neural circuitry of fear extinction, as discussed above (Hariri and Holmes