Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate ...the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.
Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate ...the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues.
The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and ...its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.
Summary
Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism and renewable energy technologies. While considerable effort has been put into ...understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up‐regulated during growth with xylan containing media. In‐frame deletion analysis of these genes found that only gly43F is critical for utilization of xylo‐oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo‐oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are (i) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and (ii) a single cytoplasmic β‐xylosidase is essential for the utilization of xylo‐oligosaccharides.
The saprophytic bacterium Cellvibrio japonicus is proficient at degrading the polysaccharides found in plant biomass. Using a combination of transcriptomic and mutational analyses this report updates the model of how the bacterium depolymerizes and transports the hemicellulose xylan into the cell. An essential Carbohydrate Active Enzyme (CAZyme) and a critical oligosaccharide transporter for xylan metabolism in C. japonicus are also characterized in this study.
Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known ...cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.
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•NS1 partitions into the nucleus upon RSV infection•NS1 coimmunoprecipitates with chromatin-associated proteins•NS1 binds regulatory elements of differentially expressed genes in RSV infection•NS1 modulates host gene transcription
Pei et al. show that NS1 is present in the nucleus in primary airway epithelial, MDDCs, and A549 cells upon RSV infection. This study finds that NS1 functions as an immune antagonist by binding chromatin at the promoters and enhancers of immune response genes and modulating host gene transcription.
Cholangiocarcinoma is an epithelial malignancy originating in the biliary tracts and frequently recurs even with surgical resection. Unresectable disease has a 5-year overall survival of less than ...10%. Given this poor prognosis, additional therapies are urgently needed. Chemotherapy has been the mainstay of treatment for many years. However, with the incorporation of immunotherapy into the treatment of other malignancies, there has been a great deal of interest in immunotherapy for biliary cancers. Recently, durvalumab was approved in combination with gemcitabine and cisplatin for the treatment of unresectable cholangiocarcinoma in the first-line setting. However, predicting which patients may respond to immunotherapy remains a challenge due to the lack of a reliable biomarker.
Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of ...bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.
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•Measuring bacterial growth with insoluble substrates is challenging.•3-D printed biomass containment devices (BCDs) hold insoluble substrates.•BCDs facilitate optical density measurements with recalcitrant biomass in real time.
Background
Genetic testing is recommended for all pancreatic ductal adenocarcinoma (PDAC) patients. Prior research demonstrates that multidisciplinary pancreatic cancer clinics (MDPCs) improve ...treatment‐ and survival‐related outcomes for PDAC patients. However, limited information exists regarding the utility of integrated genetics in the MDPC setting. We hypothesized that incorporating genetics in an MDPC serving both PDAC patients and high‐risk individuals (HRI) could: (1) improve compliance with guideline‐based genetic testing for PDAC patients, and (2) optimize HRI identification and PDAC surveillance participation to improve early detection and survival.
Methods
Demographics, genetic testing results, and pedigrees were reviewed for PDAC patients and HRI at one institution over 45 months. Genetic testing analyzed 16 PDAC‐associated genes at minimum.
Results
Overall, 969 MDPC subjects were evaluated during the study period; another 56 PDAC patients were seen outside the MDPC. Among 425 MDPC PDAC patients, 333 (78.4%) completed genetic testing; 29 (8.7%) carried a PDAC‐related pathogenic germline variant (PGV). Additionally, 32 (9.6%) met familial pancreatic cancer (FPC) criteria. These PDAC patients had 191 relatives eligible for surveillance or genetic testing. Only 2/56 (3.6%) non‐MDPC PDAC patients completed genetic testing (p < 0.01). Among 544 HRI, 253 (46.5%) had a known PGV or a designation of FPC, and were eligible for surveillance at baseline; of the remainder, 15/291 (5.2%) were eligible following genetic testing and PGV identification.
Conclusion
Integrating genetics into the multidisciplinary setting significantly improved genetic testing compliance by reducing logistical barriers for PDAC patients, and clarified cancer risks for their relatives while conserving clinical resources. Overall, we identified 206 individuals newly eligible for surveillance or genetic testing (191 relatives of MDPC PDAC patients, and 15 HRI from this cohort), enabling continuity of care for PDAC patients and at‐risk relatives in one clinic.
Genetic testing is recommended for all pancreatic ductal adenocarcinoma (PDAC) patients. Limited information exists regarding the utility of integrated genetics in the multidisciplinary pancreatic cancer clinic setting. We found that integrating genetics into the multidisciplinary setting significantly improved genetic testing compliance by reducing logistical barriers for PDAC patients, and clarified cancer risks for their relatives while conserving clinical resources.
Epstein-Barr virus (EBV) causes multiple human cancers, including B-cell lymphomas. In cell culture, EBV converts healthy human B-cells into immortalized ones that grow continuously, which model ...post-transplant lymphomas. Constitutive signaling from two cytoplasmic tail domains of the EBV oncogene latent membrane protein 1 (LMP1) is required for this transformation, yet there has not been systematic analysis of their host gene targets. We identified that only signaling from the membrane proximal domain is required for survival of these EBV-immortalized cells and that its loss triggers apoptosis. We identified key LMP1 target genes, whose abundance changed significantly with loss of LMP1 signals, or that were instead upregulated in response to switching on signaling by one or both LMP1 domains in an EBV-uninfected human B-cell model. These included major anti-apoptotic factors necessary for EBV-infected B-cell survival. Bioinformatics analyses identified clusters of B-cell genes that respond differently to signaling by either or both domains.
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