Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

In vivo CAR T-cell generation in non-human primates using lentiviral vectors displaying a multi-domain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high cost and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVecTM platform, a lentiviral vector capable of generating CAR T-cells in vivo. Here we describe the incorporation of T cell activation and costimulatory signals onto the surface of VivoVecTM particles (VVPs) in the form of a multi-domain fusion protein and show enhanced in vivo transduction and improved CAR-T cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into non-human primates resulted in the robust generation of anti-CD20 CAR T-cells and the complete depletion of B cells for more than 10 weeks. These data validate the VivoVecTM platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Reduced sister chromatid cohesion acts as a tumor penetrance modifier.

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.

Dissecting the regulatory activity and sequence content of loci with exceptional numbers of transcription factor associations.

DNA-associated proteins (DAPs) classically regulate gene expression by binding to regulatory loci such as enhancers or promoters. As expanding catalogs of genome-wide DAP binding maps reveal thousands of loci that, unlike the majority of conventional enhancers and promoters, associate with dozens of different DAPs with apparently little regard for motif preference, an understanding of DAP association and coordination at such regulatory loci is essential to deciphering how these regions contribute to normal development and disease. In this study, we aggregated publicly available ChIP-seq data from 469 human DAPs assayed in three cell lines and integrated these data with an orthogonal data set of 352 nonredundant, in vitro-derived motifs mapped to the genome within DNase I hypersensitivity footprints to characterize regions with high numbers of DAP associations. We establish a generalizable definition for high occupancy target (HOT) loci and identify putative driver DAP motifs in HepG2 cells, including HNF4A, SP1, SP5, and ETV4, that are highly prevalent and show sequence conservation at HOT loci. The number of different DAPs associated with an element is positively associated with evidence of regulatory activity, and by systematically mutating 245 HOT loci with a massively parallel mutagenesis assay, we localized regulatory activity to a central core region that depends on the motif sequences of our previously nominated driver DAPs. In sum, this work leverages the increasingly large number of DAP motif and ChIP-seq data publicly available to explore how DAP associations contribute to genome-wide transcriptional regulation.

Genome-wide DNA methylation analysis implicates enrichment of interferon pathway in African American patients with Systemic Lupus Erythematosus and European Americans with lupus nephritis.

Systemic Lupus Erythematosus (SLE) is a chronic, multisystem, inflammatory autoimmune disease that disproportionately affects women. Trends in SLE prevalence and clinical course differ by ancestry, with those of African American ancestry presenting with more active, severe and rapidly progressive disease than European Americans. Previous research established altered epigenetic signatures in SLE patients compared to controls. However, the contribution of aberrant DNA methylation (DNAm) to the risk of SLE by ancestry and differences among patients with SLE-associated Lupus Nephritis (LN) has not been well described. We evaluated the DNA methylomes of 87 individuals including 41 SLE patients, with and without LN, and 46 controls enrolled in an ancestry diverse, well-characterized cohort study of established SLE (41 SLE patients [20 SLE-LN+, 21 SLE-LN-] and 46 sex-, race- and age-matched controls; 55% African American, 45% European American). Participants were genotyped using the Infinium Global Diversity Array (GDA), and genetic ancestry was estimated using principal components. Genome-wide DNA methylation was initially measured using the Illumina MethylationEPIC 850K Beadchip array followed by methylation-specific qPCR to validate the methylation status at putative loci. Differentially Methylated Positions (DMP) were identified using a case-control approach adjusted for ancestry. We identified a total of 51 DMPs in CpGs among SLE patients compared to controls. Genes proximal to these CpGs were highly enriched for involvement in type I interferon signaling. DMPs among European American SLE patients with LN were similar to African American SLE patients with and without LN. Our findings were validated using an orthogonal, methyl-specific PCR for three SLE-associated DMPs near or proximal to MX1, USP18, and IFITM1. Our study confirms previous reports that DMPs in CpGs associated with SLE are enriched in type I interferon genes. However, we show that European American SLE patients with LN have similar DNAm patterns to African American SLE patients irrespective of LN, suggesting that aberrant DNAm alters activity of type I interferon pathway leading to more severe disease independent of ancestry.

Genomic analysis reveals HDAC1 regulates clinically relevant transcriptional programs in Pancreatic cancer.

Novel strategies are needed to combat multidrug resistance in pancreatic ductal adenocarcinoma (PDAC). We applied genomic approaches to understand mechanisms of resistance in order to better inform treatment and precision medicine. Altered function of chromatin remodeling complexes contribute to chemoresistance. Our study generates and analyzes genomic and biochemical data from PDAC cells overexpressing HDAC1, a histone deacetylase involved in several chromatin remodeling complexes. We characterized the impact of overexpression on drug response, gene expression, HDAC1 binding, and chromatin structure using RNA-sequencing and ChIP-sequencing for HDAC1 and H3K27 acetylation. Integrative genomic analysis shows that HDAC1 overexpression promotes activation of key resistance pathways including epithelial to mesenchymal transition, cell cycle, and apoptosis through global chromatin remodeling. Target genes are similarly altered in patient tissues and show correlation with patient survival. We also demonstrate that direct targets of HDAC1 that also show altered chromatin are enriched near genes associated with altered GTPase activity. HDAC1 target genes identified using in vitro methods and observed in patient tissues were used to develop a clinically relevant nine-transcript signature associated with patient prognosis. Integration of multiple genomic and biochemical data types enables understanding of multidrug resistance and tumorigenesis in PDAC, a disease in desperate need of novel treatment strategies.

Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers based on five-year survival rates. Genes contributing to chemoresistance represent novel therapeutic targets that can improve treatment response. Increased expression of ANGPTL4 in tumors correlates with poor outcomes in pancreatic cancer. METHODS: We used statistical analysis of publicly available gene expression data (TCGA-PAAD) to test whether expression of ANGPTL4 and its downstream targets, ITGB4 and APOL1, were correlated with patient survival. We measured the impact of ANGPTL4 overexpression in a common pancreatic cancer cell line, MIA PaCa-2 cells, using CRISPRa for overexpression and DsiRNA for knockdown. We characterized global gene expression changes associated with high levels of ANGPTL4 and response to gemcitabine treatment using RNA-sequencing. Gemcitabine dose response curves were calculated on modified cell lines by measuring cell viability with CellTiter-Glo (Promega). Impacts on cell migration were measured using a time course scratch assay. RESULTS: We show that ANGPTL4 overexpression leads to in vitro resistance to gemcitabine and reduced survival times in patients. Overexpression of ANGPTL4 induces transcriptional signatures of tumor invasion and metastasis, proliferation and differentiation, and inhibition of apoptosis. Analyses revealed an overlapping signature of genes associated with both ANGPTL4 activation and gemcitabine response. Increased expression of the genes in this signature in patient PDAC tissues was significantly associated with shorter patient survival. We identified 42 genes that were both co-regulated with ANGPTL4 and were responsive to gemcitabine treatment. ITGB4 and APOL1 were among these genes. Knockdown of either of these genes in cell lines overexpressing ANGPTL4 reversed the observed gemcitabine resistance and inhibited cellular migration associated with epithelial to mesenchymal transition (EMT) and ANGPTL4 overexpression. CONCLUSIONS: These data suggest that ANGPTL4 promotes EMT and regulates the genes APOL1 and ITGB4. Importantly, we show that inhibition of both targets reverses chemoresistance and decreases migratory potential. Our findings have revealed a novel pathway regulating tumor response to treatment and suggest relevant therapeutic targets in pancreatic cancer.

Healthcare workers' perceptions and experiences of primary healthcare integration: a scoping review of qualitative evidence.

BACKGROUND: Primary healthcare (PHC) integration has been promoted globally as a tool for health sector reform and universal health coverage (UHC), especially in low-resource settings. However, for a range of reasons, implementation and impact remain variable. PHC integration, at its simplest, can be considered a way of delivering PHC services together that sometimes have been delivered as a series of separate or 'vertical' health programmes. Healthcare workers are known to shape the success of implementing reform interventions. Understanding healthcare worker perceptions and experiences of PHC integration can therefore provide insights into the role healthcare workers play in shaping implementation efforts and the impact of PHC integration. However, the heterogeneity of the evidence base complicates our understanding of their role in shaping the implementation, delivery, and impact of PHC integration, and the role of contextual factors influencing their responses. OBJECTIVES: To map the qualitative literature on healthcare workers' perceptions and experiences of PHC integration to characterise the evidence base, with a view to better inform future syntheses on the topic. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 28 July 2020. We did not search for grey literature due to the many published records identified. SELECTION CRITERIA: We included studies with qualitative and mixed methods designs that reported on healthcare worker perceptions and experiences of PHC integration from any country. We excluded settings other than PHC and community-based health care, participants other than healthcare workers, and interventions broader than healthcare services. We used translation support from colleagues and Google Translate software to screen non-English records. Where translation was not feasible we categorised these records as studies awaiting classification. DATA COLLECTION AND ANALYSIS: For data extraction, we used a customised data extraction form containing items developed using inductive and deductive approaches. We performed independent extraction in duplicate for a sample on 10% of studies allowed for sufficient agreement to be reached between review authors. We analysed extracted data quantitatively by counting the number of studies per indicator and converting these into proportions with additional qualitative descriptive information. Indicators included descriptions of study methods, country setting, intervention type, scope and strategies, implementing healthcare workers, and client target population. MAIN RESULTS: The review included 184 studies for analysis based on 191 included papers. Most studies were published in the last 12 years, with a sharp increase in the last five years. Studies mostly employed methods with cross-sectional qualitative design (mainly interviews and focus group discussions), and few used longitudinal or ethnographic (or both) designs. Studies covered 37 countries, with close to an even split in the proportions of high-income countries (HICs) and low- and middle-income countries (LMICs). There were gaps in the geographical spread for both HICs and LMICs and some countries were more dominant, such as the USA for HICs, South Africa for middle-income countries, and Uganda for low-income countries. Methods were mainly cross-sectional observational studies with few longitudinal studies. A minority of studies used an analytical conceptual model to guide the design, implementation, and evaluation of the integration study. The main finding was the various levels of diversity found in the evidence base on PHC integration studies that examined healthcare workers' perceptions and experiences. The review identified six different configurations of health service streams that were being integrated and these were categorised as: mental and behavioural health; HIV, tuberculosis (TB) and sexual reproductive health; maternal, women, and child health; non-communicable diseases; and two broader categories, namely general PHC services, and allied and specialised services. Within the health streams, the review mapped the scope of the interventions as full or partial integration. The review mapped the use of three different integration strategies and categorised these as horizontal integration, service expansion, and service linkage strategies. The wide range of healthcare workers who participated in the implementation of integration interventions was mapped and these included policymakers, senior managers, middle and frontline managers, clinicians, allied healthcare professionals, lay healthcare workers, and health system support staff. We mapped the range of client target populations. AUTHORS' CONCLUSIONS: This scoping review provides a systematic, descriptive overview of the heterogeneity in qualitative literature on healthcare workers' perceptions and experience of PHC integration, pointing to diversity with regard to country settings; study types; client populations; healthcare worker populations; and intervention focus, scope, and strategies. It would be important for researchers and decision-makers to understand how the diversity in PHC integration intervention design, implementation, and context may influence how healthcare workers shape PHC integration impact. The classification of studies on the various dimensions (e.g. integration focus, scope, strategy, and type of healthcare workers and client populations) can help researchers to navigate the way the literature varies and for specifying potential questions for future qualitative evidence syntheses.

ANTECEDENTES: La integración de la atención primaria de salud (APS) se ha promovido en todo el mundo como herramienta para la mejora del sector sanitario y la cobertura sanitaria universal (CSU), especialmente en contextos con pocos recursos. Sin embargo, por diversas razones, la aplicación y el impacto todavía son variables. La integración de la APS, en su forma más simple, se puede considerar una forma de prestar conjuntamente servicios de APS que en ocasiones se han prestado como una serie de programas sanitarios separados o "verticales". Se sabe que el personal sanitario determina el éxito de la aplicación de las intervenciones de mejora. Por lo tanto, conocer las percepciones y experiencias de los trabajadores sanitarios sobre la integración de la APS puede ayudar a comprender la función que desempeñan en la configuración de los esfuerzos para la aplicación y el impacto de la integración de la APS. Sin embargo, la heterogeneidad de la base de evidencia complica la comprensión de su función en la configuración de la aplicación, la prestación y el impacto de la integración de la APS, así como el papel de los factores contextuales que influyen en sus respuestas. OBJETIVOS: Identificar la literatura cualitativa sobre las percepciones y experiencias del personal sanitario en relación con la integración de la APS para caracterizar la base de evidencia, con vistas a informar mejor las futuras síntesis sobre el tema. MÉTODOS DE BÚSQUEDA: Se utilizaron los métodos exhaustivos estándar de búsqueda de Cochrane. La última fecha de búsqueda fue el 28 de julio de 2020. No se buscó literatura gris debido a los numerosos registros publicados identificados. CRITERIOS DE SELECCIÓN: Se incluyeron estudios con diseños cualitativos y de métodos mixtos que informaran sobre las percepciones y experiencias de los profesionales sanitarios sobre la integración de la APS de cualquier país. Se excluyeron los contextos distintos de la APS y la atención sanitaria comunitaria, los participantes que no fueran profesionales sanitarios y las intervenciones que abarcaran más que los servicios sanitarios. Para revisar los registros que no estaban en inglés se contó con la traducción realizada con la ayuda de colegas y el programa Google Translate. En los casos en que la traducción no fue posible, estos registros se clasificaron como estudios pendientes de clasificación. OBTENCIÓN Y ANÁLISIS DE LOS DATOS: Para la extracción de los datos, se utilizó un formulario de extracción de datos personalizado que contenía ítems elaborados mediante enfoques inductivos y deductivos. La extracción independiente por duplicado de una muestra del 10% de los estudios permitió alcanzar un acuerdo suficiente entre los autores de la revisión. Los datos extraídos se analizaron cuantitativamente contando el número de estudios por indicador y convirtiéndolos en proporciones con información descriptiva cualitativa adicional. Los indicadores incluían descripciones de los métodos de estudio, el contexto del país, el tipo de intervención, el alcance y las estrategias, el personal sanitario encargado de aplicarla y la población destinataria. RESULTADOS PRINCIPALES: La revisión incluyó 184 estudios para el análisis sobre la base de 191 documentos incluidos. La mayoría de los estudios se publicaron en los últimos 12 años, con un fuerte aumento en los últimos cinco. La mayoría de los estudios emplearon métodos con un diseño cualitativo transversal (principalmente entrevistas y debates en grupos de discusión), y pocos utilizaron diseños longitudinales o etnográficos (o ambos). Los estudios abarcaron 37 países, con una proporción casi equitativa de países de ingresos altos (PIA) y países de ingresos bajos y medios (PIBM). Tanto en los PIA como en los PIBM, la distribución geográfica presentaba carencias y algunos países eran más dominantes, como EE. UU. en los países de ingresos altos, Sudáfrica en los de ingresos medios y Uganda en los de ingresos bajos. Los métodos fueron principalmente estudios observacionales transversales con pocos estudios longitudinales. Una minoría de estudios utilizó un modelo conceptual analítico para orientar el diseño, la aplicación y la evaluación del estudio de integración. El principal hallazgo fue los distintos niveles de diversidad encontrados en la base de evidencia sobre estudios de integración de la APS que examinaron las percepciones y experiencias de los trabajadores sanitarios. La revisión identificó seis configuraciones diferentes de flujos de servicios sanitarios que se estaban integrando y que se clasificaron como: salud mental y del comportamiento; VIH, tuberculosis (TB) y salud sexual y reproductiva; salud materna, de la mujer y del niño; enfermedades no transmisibles; y dos categorías más amplias, a saber, servicios generales de APS y servicios afines y especializados. Dentro de los flujos sanitarios, la revisión clasificó el alcance de las intervenciones como integración total o parcial. La revisión identificó el uso de tres estrategias de integración diferentes y las clasificó como estrategias de integración horizontal, ampliación de los servicios y vinculación de los servicios. Se identificó el amplio abanico de profesionales sanitarios que participaron en la aplicación de las intervenciones de integración: responsables de políticas sanitarias, altos directivos, directivos intermedios y de primera línea, médicos, profesionales sanitarios asociados, trabajadores sanitarios no técnicos y personal de apoyo de los sistemas sanitarios. Se identificó la variedad de poblaciones destinatarias. CONCLUSIONES DE LOS AUTORES: Esta revisión sistemática exploratoria proporciona una revisión global sistemática y descriptiva de la heterogeneidad de la bibliografía cualitativa sobre las percepciones y experiencias de los profesionales sanitarios con respecto a la integración de la APS, señalando la diversidad con respecto a los contextos nacionales, los tipos de estudio, las poblaciones de clientes, las poblaciones de profesionales sanitarios y el enfoque, el alcance y las estrategias de la intervención. Sería importante que los investigadores y los responsables de la toma de decisiones comprendieran cómo la diversidad en el diseño, la aplicación y el contexto de la intervención de integración de la APS podría influir en la forma en que los trabajadores sanitarios conciben el impacto de la integración de la APS. La clasificación de los estudios en función de las distintas dimensiones (p. ej., enfoque de la integración, alcance, estrategia y tipo de trabajadores sanitarios y poblaciones de clientes) puede ayudar a los investigadores a orientarse en la forma en que varía la bibliografía y especificar posibles preguntas para futuras síntesis de evidencia cualitativa.

Healthcare workers' perceptions and experiences of primary healthcare integration: a scoping review of qualitative evidence.

BACKGROUND: Primary healthcare (PHC) integration has been promoted globally as a tool for health sector reform and universal health coverage (UHC), especially in low-resource settings. However, for a range of reasons, implementation and impact remain variable. PHC integration, at its simplest, can be considered a way of delivering PHC services together that sometimes have been delivered as a series of separate or 'vertical' health programmes. Healthcare workers are known to shape the success of implementing reform interventions. Understanding healthcare worker perceptions and experiences of PHC integration can therefore provide insights into the role healthcare workers play in shaping implementation efforts and the impact of PHC integration. However, the heterogeneity of the evidence base complicates our understanding of their role in shaping the implementation, delivery, and impact of PHC integration, and the role of contextual factors influencing their responses. OBJECTIVES: To map the qualitative literature on healthcare workers' perceptions and experiences of PHC integration to characterise the evidence base, with a view to better inform future syntheses on the topic. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 28 July 2020. We did not search for grey literature due to the many published records identified. SELECTION CRITERIA: We included studies with qualitative and mixed methods designs that reported on healthcare worker perceptions and experiences of PHC integration from any country. We excluded settings other than PHC and community-based health care, participants other than healthcare workers, and interventions broader than healthcare services. We used translation support from colleagues and Google Translate software to screen non-English records. Where translation was not feasible we categorised these records as studies awaiting classification. DATA COLLECTION AND ANALYSIS: For data extraction, we used a customised data extraction form containing items developed using inductive and deductive approaches. We performed independent extraction in duplicate for a sample on 10% of studies allowed for sufficient agreement to be reached between review authors. We analysed extracted data quantitatively by counting the number of studies per indicator and converting these into proportions with additional qualitative descriptive information. Indicators included descriptions of study methods, country setting, intervention type, scope and strategies, implementing healthcare workers, and client target population. MAIN RESULTS: The review included 184 studies for analysis based on 191 included papers. Most studies were published in the last 12 years, with a sharp increase in the last five years. Studies mostly employed methods with cross-sectional qualitative design (mainly interviews and focus group discussions), and few used longitudinal or ethnographic (or both) designs. Studies covered 37 countries, with close to an even split in the proportions of high-income countries (HICs) and low- and middle-income countries (LMICs). There were gaps in the geographical spread for both HICs and LMICs and some countries were more dominant, such as the USA for HICs, South Africa for middle-income countries, and Uganda for low-income countries. Methods were mainly cross-sectional observational studies with few longitudinal studies. A minority of studies used an analytical conceptual model to guide the design, implementation, and evaluation of the integration study. The main finding was the various levels of diversity found in the evidence base on PHC integration studies that examined healthcare workers' perceptions and experiences. The review identified six different configurations of health service streams that were being integrated and these were categorised as: mental and behavioural health; HIV, tuberculosis (TB) and sexual reproductive health; maternal, women, and child health; non-communicable diseases; and two broader categories, namely general PHC services, and allied and specialised services. Within the health streams, the review mapped the scope of the interventions as full or partial integration. The review mapped the use of three different integration strategies and categorised these as horizontal integration, service expansion, and service linkage strategies. The wide range of healthcare workers who participated in the implementation of integration interventions was mapped and these included policymakers, senior managers, middle and frontline managers, clinicians, allied healthcare professionals, lay healthcare workers, and health system support staff. We mapped the range of client target populations. AUTHORS' CONCLUSIONS: This scoping review provides a systematic, descriptive overview of the heterogeneity in qualitative literature on healthcare workers' perceptions and experience of PHC integration, pointing to diversity with regard to country settings; study types; client populations; healthcare worker populations; and intervention focus, scope, and strategies. It would be important for researchers and decision-makers to understand how the diversity in PHC integration intervention design, implementation, and context may influence how healthcare workers shape PHC integration impact. The classification of studies on the various dimensions (e.g. integration focus, scope, strategy, and type of healthcare workers and client populations) can help researchers to navigate the way the literature varies and for specifying potential questions for future qualitative evidence syntheses.

Pooled CRISPR screening in pancreatic cancer cells implicates co-repressor complexes as a cause of multiple drug resistance via regulation of epithelial-to-mesenchymal transition.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients suffer poor outcomes, including a five-year survival of below 10%. Poor outcomes result in part from therapeutic resistance that limits the impact of cytotoxic first-line therapy. Novel therapeutic approaches are needed, but currently no targeted therapies exist to treat PDAC. METHODS: To assess cellular resistance mechanisms common to four cytotoxic chemotherapies (gemcitabine, 5-fluorouracil, irinotecan, and oxaliplatin) used to treat PDAC patients, we performed four genome-wide CRISPR activation (CRISPRact) and CRISPR knock-out (CRISPRko) screens in two common PDAC cell lines (Panc-1 and BxPC3). We used pathway analysis to identify gene sets enriched among our hits and conducted RNA-sequencing and chromatin immunoprecipitation-sequencing (ChIP-seq) to characterize top hits from our screen. We used scratch assays to assess changes in cellular migration with HDAC1 overexpression. RESULTS: Our data revealed activation of ABCG2, a well-described efflux pump, as the most consistent mediator of resistance in each of our screens. CRISPR-mediated activation of genes involved in transcriptional co-repressor complexes also conferred resistance to multiple drugs. Expression of many of these genes, including HDAC1, is associated with reduced survival in PDAC patients. Up-regulation of HDAC1 in vitro increased promoter occupancy and expression of several genes involved in the epithelial-to-mesenchymal transition (EMT). These cells also displayed phenotypic changes in cellular migration consistent with activation of the EMT pathway. The expression changes resulting from HDAC1 activation were also observed with activation of several other co-repressor complex members. Finally, we developed a publicly available analysis tool, PancDS, which integrates gene expression profiles with our screen results to predict drug sensitivity in resected PDAC tumors and cell lines. CONCLUSION: Our results provide a comprehensive resource for identifying cellular mechanisms of drug resistance in PDAC, mechanistically implicate HDAC1, and co-repressor complex members broadly, in multi-drug resistance, and provide an analytical tool for predicting treatment response in PDAC tumors and cell lines.

Factors that influence parents' and informal caregivers' views and practices regarding routine childhood vaccination: a qualitative evidence synthesis.

BACKGROUND: Childhood vaccination is one of the most effective ways to prevent serious illnesses and deaths in children. However, worldwide, many children do not receive all recommended vaccinations, for several potential reasons. Vaccines might be unavailable, or parents may experience difficulties in accessing vaccination services; for instance, because of poor quality health services, distance from a health facility, or lack of money. Some parents may not accept available vaccines and vaccination services. Our understanding of what influences parents' views and practices around childhood vaccination, and why some parents may not accept vaccines for their children, is still limited. This synthesis links to Cochrane Reviews of the effectiveness of interventions to improve coverage or uptake of childhood vaccination. OBJECTIVES: - Explore parents' and informal caregivers' views and practices regarding routine childhood vaccination, and the factors influencing acceptance, hesitancy, or nonacceptance of routine childhood vaccination. - Develop a conceptual understanding of what and how different factors reduce parental acceptance of routine childhood vaccination. - Explore how the findings of this review can enhance our understanding of the related Cochrane Reviews of intervention effectiveness. SEARCH METHODS: We searched MEDLINE, Embase, CINAHL, and three other databases for eligible studies from 1974 to June 2020. SELECTION CRITERIA: We included studies that: utilised qualitative methods for data collection and analysis; focused on parents' or caregivers' views, practices, acceptance, hesitancy, or refusal of routine vaccination for children aged up to six years; and were from any setting globally where childhood vaccination is provided. DATA COLLECTION AND ANALYSIS: We used a pre-specified sampling frame to sample from eligible studies, aiming to capture studies that were conceptually rich, relevant to the review's phenomenon of interest, from diverse geographical settings, and from a range of income-level settings. We extracted contextual and methodological data from each sampled study. We used a meta-ethnographic approach to analyse and synthesise the evidence. We assessed methodological limitations using a list of criteria used in previous Cochrane Reviews and originally based on the Critical Appraisal Skills Programme quality assessment tool for qualitative studies. We used the GRADE-CERQual (Confidence in the Evidence from Reviews of Qualitative research) approach to assess our confidence in each finding. We integrated the findings of this review with those from relevant Cochrane Reviews of intervention effectiveness. We did this by mapping whether the underlying theories or components of trial interventions included in those reviews related to or targeted the overarching factors influencing parental views and practices regarding routine childhood vaccination identified by this review. MAIN RESULTS: We included 145 studies in the review and sampled 27 of these for our analysis. Six studies were conducted in Africa, seven in the Americas, four in South-East Asia, nine in Europe, and one in the Western Pacific. Studies included urban and rural settings, and high-, middle-, and low-income settings. Many complex factors were found to influence parents' vaccination views and practices, which we divided into four themes. Firstly, parents' vaccination ideas and practices may be influenced by their broader ideas and practices surrounding health and illness generally, and specifically with regards to their children, and their perceptions of the role of vaccination within this context. Secondly, many parents' vaccination ideas and practices were influenced by the vaccination ideas and practices of the people they mix with socially. At the same time, shared vaccination ideas and practices helped some parents establish social relationships, which in turn strengthened their views and practices around vaccination. Thirdly, parents' vaccination ideas and practices may be influenced by wider political issues and concerns, and particularly their trust (or distrust) in those associated with vaccination programmes. Finally, parents' vaccination ideas and practices may be influenced by their access to and experiences of vaccination services and their frontline healthcare workers. We developed two concepts for understanding possible pathways to reduced acceptance of childhood vaccination. The first concept, 'neoliberal logic', suggests that many parents, particularly from high-income countries, understood health and healthcare decisions as matters of individual risk, choice, and responsibility. Some parents experienced this understanding as in conflict with vaccination programmes, which emphasise generalised risk and population health. This perceived conflict led some parents to be less accepting of vaccination for their children. The second concept, 'social exclusion', suggests that some parents, particularly from low- and middle-income countries, were less accepting of childhood vaccination due to their experiences of social exclusion. Social exclusion may damage trustful relationships between government and the public, generate feelings of isolation and resentment, and give rise to demotivation in the face of public services that are poor quality and difficult to access. These factors in turn led some parents who were socially excluded to distrust vaccination, to refuse vaccination as a form of resistance or a way to bring about change, or to avoid vaccination due to the time, costs, and distress it creates. Many of the overarching factors our review identified as influencing parents' vaccination views and practices were underrepresented in the interventions tested in the four related Cochrane Reviews of intervention effectiveness. AUTHORS' CONCLUSIONS: Our review has revealed that parents' views and practices regarding childhood vaccination are complex and dynamic social processes that reflect multiple webs of influence, meaning, and logic. We have provided a theorised understanding of the social processes contributing to vaccination acceptance (or not), thereby complementing but also extending more individualistic models of vaccination acceptance. Successful development of interventions to promote acceptance and uptake of childhood vaccination will require an understanding of, and then tailoring to, the specific factors influencing vaccination views and practices of the group(s) in the target setting. The themes and concepts developed through our review could serve as a basis for gaining this understanding, and subsequent development of interventions that are potentially more aligned with the norms, expectations, and concerns of target users.

Intervention development of a brief messaging intervention for a randomised controlled trial to improve diabetes treatment adherence in sub-Saharan Africa.

BACKGROUND: Brief messaging interventions, including Short Message Service (SMS) text-messages, delivered via mobile device platforms, show promise to support and improve treatment adherence. To understand how these interventions work, and to facilitate transparency, we need clear descriptions of the intervention development process. METHOD: We describe and reflect on the process of designing and pretesting an evidence- and theory-informed brief messaging intervention, to improve diabetes treatment adherence in sub-Saharan Africa. We followed the stepwise approach recommended by the Medical Research Council, United Kingdom (MRC UK) Framework for Development and Evaluation of Complex Health Interventions and guidance for mobile health intervention development. RESULTS: We used a four-phase, iterative approach that first generated primary and secondary evidence on the lived experience of diabetes, diabetes treatment services and mobile-phone use. Second, we designed a type 2 diabetes-specific, brief text-message library, building on our previous hypertension text-message library, as well as drawing on the primary and secondary data from phase one, and on expert opinion. We then mapped the brief text-messages onto behaviour change (COM-B) theoretical constructs. Third, we refined and finalised the newly developed brief text-message library through stakeholder consultation and translated it into three local languages. Finally, we piloted the intervention by pre-testing the automated delivery of the brief text-messages in the trial sites in Malawi and South Africa. The final SMS text Adherence suppoRt for people with type 2 diabetes (StAR2D) intervention was tested in a randomised controlled trial in Malawi and South Africa (trial registration: ISRCTN70768808 ). CONCLUSION: The complexity of public health interventions requires that we give more attention to intervention development work. Our documentation and reflection on the StAR2D intervention development process promotes transparency, replicability, assessment of intervention quality, and comparison with other studies.

A genome-wide interactome of DNA-associated proteins in the human liver.

Large-scale efforts like the ENCODE Project have made tremendous progress in cataloging the genomic binding patterns of DNA-associated proteins (DAPs), such as transcription factors (TFs). However, most chromatin immunoprecipitation-sequencing (ChIP-seq) analyses have focused on a few immortalized cell lines whose activities and physiology differ in important ways from endogenous cells and tissues. Consequently, binding data from primary human tissue are essential to improving our understanding of in vivo gene regulation. Here, we identify and analyze more than 440,000 binding sites using ChIP-seq data for 20 DAPs in two human liver tissue samples. We integrated binding data with transcriptome and phased WGS data to investigate allelic DAP interactions and the impact of heterozygous sequence variation on the expression of neighboring genes. Our tissue-based data set exhibits binding patterns more consistent with liver biology than cell lines, and we describe uses of these data to better prioritize impactful noncoding variation. Collectively, our rich data set offers novel insights into genome function in human liver tissue and provides a valuable resource for assessing disease-related disruptions.

Chromodomain Helicase DNA-Binding Protein 7 Is Suppressed in the Perinecrotic/Ischemic Microenvironment and Is a Novel Regulator of Glioblastoma Angiogenesis.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453-462.

Classroom sound can be used to classify teaching practices in college science courses.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Pex35 is a regulator of peroxisome abundance.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1.

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

The expression of MHC class II molecules on murine breast tumors delays T-cell exhaustion, expands the T-cell repertoire, and slows tumor growth.

The expression of MHC class II molecules (MHCII) on tumor cells correlates with survival and responsiveness to immunotherapy. However, the mechanisms underlying these observations are poorly defined. Using a murine breast tumor line, we showed that MHCII-expressing tumors grew more slowly than controls and recruited more functional CD4+ and CD8+ T cells. In addition, MHCII-expressing tumors contained more TCR clonotypes expanded to a larger degree than control tumors. Functional CD8+ T cells in tumors depended on CD4+ T cells. However, both CD4+ and CD8+ T cells eventually became exhausted, even in MHCII-expressing tumors. Treatment with anti-CTLA4, but not anti-PD-1 or anti-TIM-3, promoted complete eradication of MHCII-expressing tumors. These results suggest tumor cell expression of MHCII facilitates the local activation of CD4+ T cells, indirectly helps the activation and expansion of CD8+ T cells, and, in combination with the appropriate checkpoint inhibitor, promotes tumor regression.

R2DGC: threshold-free peak alignment and identification for 2D gas chromatography-mass spectrometry in R.

Summary: Comprehensive 2D gas chromatography-mass spectrometry is a powerful method for analyzing complex mixtures of volatile compounds, but produces a large amount of raw data that requires downstream processing to align signals of interest (peaks) across multiple samples and match peak characteristics to reference standard libraries prior to downstream statistical analysis. Very few existing tools address this aspect of analysis and those that do have shortfalls in usability or performance. We have developed an R package that implements retention time and mass spectra similarity threshold-free alignments, seamlessly integrates retention time standards for universally reproducible alignments, performs common ion filtering and provides compatibility with multiple peak quantification methods. We demonstrate that our package's performance compares favorably to existing tools on a controlled mix of metabolite standards separated under variable chromatography conditions and data generated from cell lines. Availability and implementation: R2DGC can be downloaded at https://github.com/rramaker/R2DGC or installed via the Comprehensive R Archive Network (CRAN). Contact: sjcooper@hudsonalpha.org. Supplementary information: Supplementary data are available at Bioinformatics online.

powerTCR: A model-based approach to comparative analysis of the clone size distribution of the T cell receptor repertoire.

Sequencing of the T cell receptor (TCR) repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR.