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Evidence-based practice in educational development includes leveraging data to iteratively refine center for teaching and learning (CTL) services. However, CTL data collection is often limited to counts and satisfaction surveys rather than direct measures of outcomes. To directly assess impacts of consultations on course and syllabus design, we analyzed 94 clients' syllabi (32 faculty, 62 graduate students and postdocs) before and after consultations. Faculty and non-faculty clients demonstrated significant change following consultations (6% and 10% gains in syllabus rubric scores, representing 50% and 31% of possible gains and effect sizes of 0.73 and 1.04 standard deviations, respectively). We compared faculty clients to quasi-experimental control groups that did not receive consultations. Syllabi from non-clients scored lower and did not demonstrate similar changes across semesters. Attendance at a CTL seminar on course and syllabus design did not explain variation in clients' syllabi. We discuss implications for assessment of CTL services and how we leveraged formative assessments to inform and iteratively refine our educational development practices.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50–100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection. Author summary: Dengue, caused by dengue virus (DENV), is the most prevalent mosquito-transmitted disease in humans and a significant public health issue worldwide. DENV nonstructural protein 1 (NS1) has been shown to contribute to dengue pathogenesis through the induction of endothelial barrier dysfunction, resulting in hyperpermeability and vascular leak–a key feature of severe dengue disease. DENV NS1 has also been shown to activate innate immune cells; however, the exact mechanisms and consequences of this interaction are unclear. Here we show that DENV NS1 triggers activation of the inflammasome pathway–a family of cytosolic innate immune sensors–in mouse and human macrophages. We find that DENV NS1-induced inflammasome activation is independent of the NLRP3 inflammasome but dependent on caspase-1 and CD14. We also find that, in a mouse model of DENV disease, caspase-1/11-deficient mice succumb to infection at higher rates compared to caspase-1/11-functional mice, whereas NLRP3 deficiency had no effect on survival. Altogether, our study demonstrates that the inflammasome pathway acts as a sensor of DENV NS1 and contributes to protective responses during DENV infection, potentially identifying the inflammasome pathway as a therapeutic target. [ABSTRACT FROM AUTHOR]

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins β-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of β-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of β-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3β (GSK-3β). Supporting this model, we found that a chemical inhibitor of GSK-3β reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3β as a potential therapeutic target for treatment of vascular leakage during severe dengue disease. [ABSTRACT FROM AUTHOR]

How can educational developers best formatively assess impacts of their services? Standard practices tend to rely on indirect measures, such as counts of participants and feedback surveys. This paper responds to recent calls for more robust approaches directly measuring outcomes. We describe how to implement a new, transferable, evidence-based approach for directly measuring instructors' learning gains within and across educational development workshops: the Thin-Slice Embedded Direct Assessment (T-SEDA) Process. Although this approach does not measure longterm retention of learning or effects on future teaching behaviors, it significantly enhanced our toolkit for formatively assessing educational development workshops. Through case studies, we illustrate principles underlying our approach, impacts on instructors' learning, and how we iteratively refine our programs and practices using the T-SEDA process. We also discuss lessons learned for fostering an inclusive, collaborative culture of formative assessment among educational developers. [ABSTRACT FROM AUTHOR]

Abstract Background Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized in vitro and in vivo the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells. Results We show that Ens-1 LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the Ens-1 gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of Ens-1. Conclusion Our results show that Ens-1 LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, Ens-1 LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.

The protein of retroviral origin ENS-1/ERNI plays a major role during neural plate development in chick embryos by controlling the activity of the epigenetic regulator HP1γ, but its function in the earlier developmental stages is still unknown. ENS-1/ERNI promoter activity is down-regulated upon differentiation but the resulting protein expression has never been examined. In this study, we present the results obtained with custom-made antibodies to gain further insights into ENS-1 protein expression in Chicken embryonic stem cells (CES) and during their differentiation. First, we show that ENS-1 controls the activity of HP1γ in CES and we examined the context of its interaction with HP1γ. By combining immunofluorescence and western blot analysis we show that ENS-1 is localized in the cytoplasm and in the nucleus, in agreement with its role on gene's promoter activity. During differentiation, ENS-1 decreases in the cytoplasm but not in the nucleus. More precisely, three distinct forms of the ENS-1 protein co-exist in the nucleus and are differently regulated during differentiation, revealing a new level of control of the protein ENS-1. In silico analysis of the Ens-1 gene copies and the sequence of their corresponding proteins indicate that this pattern is compatible with at least three potential regulation mechanisms, each accounting only partially. The results obtained with the anti-ENS-1 antibodies presented here reveal that the regulation of ENS-1 expression in CES is more complex than expected, providing new tracks to explore the integration of ENS-1 in CES cells regulatory networks. [ABSTRACT FROM AUTHOR]

The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. “Helfensteiner” (cross of cv. “Pinot noir” and “Schiava grossa”) instead of a single “Pinot noir”. These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome. [ABSTRACT FROM AUTHOR]

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 2023 1 ., Competing Interests: DECLARATION OF INTERESTS The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has a financially compensated consulting agreement with Maze Therapeutics. N.J.K. is the President and is on the Board of Directors of Rezo Therapeutics, and he is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. M.B. is a scientific advisor for Gen1e LifeSciences.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1β in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1β. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection., Competing Interests: Declaration of Interests R.E.V. consults for Ventus Therapeutics and X-biotix Therapeutics.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.