tyler jones
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The 3Dprinted largescale multidrive platform we described here may serve as a powerful new tool for future studies of brain circuitry functions Protein expansion microscopy proExM is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples The resulting increased resolution 70nm and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues such as the brain However the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles mitochondria and the Golgi apparatus within reporterlabeled neurons in fixed mouse brain tissue We found that immunostaining before proExM and using a proteinase K based digestion for 8h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and geneticallyencoded reporters With these methods we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines reducing effort time and resources on the optimization process This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines reducing effort time and resources on the optimization process Our understanding about the interaction of the Hepatitis B virus HBV with its host cells is still quite limited Spliceosome associated factor 1 SART1 has recently been found to restrict Hepatitis C Virus We aim to dissect its role in HBV infection that remains a big threat to global health SART1 was knocked down by RNA interference and overexpressed by lentiviral or adenoassociated virus AAV vectors in HBV infected cell cultures and in vivo in HBV replicating mice evaluating HBV replication markers Luciferase reporter assays were used to determine viral or host factor promoter activities and chromatin immunoprecipitation ChIP to investigate proteinDNA interactions In HBV infected cell cultures downregulation of SART1 did not affect HBV cccDNA but resulted in markedly enhancing HBV RNA antigen expression and progeny virus production On the other hand HBV transcription and replication were significantly inhibited by overexpression of SART1 Similar results were observed in AAVHBV infough direct regulation of interferon stimulated genes In this study by using various HBV models we demonstrate that SART1 restrains HBV cccDNA transcription through suppression of HBV key transcription factor HNF4α Hepatitis B virus HBV infects hepatocytes and establishes a covalently closed circular DNA cccDNA which remains a major obstacle for antiviral treatments Akt inhibitor Spliceosome associated factor 1 SART1 has been described to inhibit hepatitis C virus infection through direct regulation of interferon stimulated genes In this study by using various HBV models we demonstrate that SART1 restrains HBV cccDNA transcription through suppression of HBV key transcription factor HNF4α Immune checkpoint inhibitors ICIs are associated with immunerelated adverse events irAEs which are more severe when ICIs are used in combination We aim to use a mouse model to elucidate the immune related molecular mechanisms of hepatitis one of the common ICI irAEs Immune phenotyping and molecular profiling were performed on PD1 mice treated with antiCTLA4 andor the IDO1 inhibitor epacadostat or a 41BB agonist antibody ICI combinationinduced hepatitis and the 41BB agonist mediated hepatitis share similar features yet maintain distinct immune signatures Both were characterized by an expansion of periportal infiltrates and panzonal inflammation albeit with different morphologic characteristics In both cases infiltrates were predominantly CD4 and CD8 T cells with upregulated T cell activation markers ICOS and CD44 Depletion of CD8 T cells abolished the ICImediated hepatitis Single cell transcriptomics revealed that the hepatitis induced by combination of ICIs is associated wi we identify key molecular mechanisms mediating immune intracellular crosstalk between liver T cells and macrophages in response to ICI in a mouse model 24NorUrsodeoxycholic acid NorUDCA is a novel therapeutic bile acid for immunemediated cholestatic liver diseases such as primary sclerosing cholangitis PSC where dysregulated Tcells including CD8 Tcells contribute to hepatobiliary immunopathology We hypothesized that NorUDCA may directly modulate CD8 Tcell function thus contributing to its therapeutic efficacy NorUDCAs immunomodulatory effects were first studied in Mdr2 mice as cholestatic model of PSC To dissect NorUDCAs immunomodulatory effects on CD8 Tcell function from its anticholestatic actions the mechanisms were also explored in a noncholestatic model of hepatic injury induced by excessive CD8 Tcell immune response upon acute noncytolytic lymphocyticchoriomeningitisvirus LCMV infection Studies included molecular and biochemical approaches flow cytometry and metabolic assays in murine CD8 Tcells in vitro Mass spectrometry MS was used to identify potential CD8 Tcell targets modulated by NorUDCA NorUDCA vivo and in vitro experimental approaches we uncovered a yetunrecognized immunometabolic regulatory property of NorUDCA adding to its previously established anticholestatic effects explaining mechanistic aspects of its therapeutic potential in immunemediated liver diseases including PSCProteases are the most abundant enzyme gene family in vertebrates and execute essential functions in all living organisms Their main role is to hydrolase the peptide bond within proteins a process also called proteolysis Contrary to the conventional paradigm proteases are not only random catalytic devices but can perform highly selective and targeted cleavage of specific substrates finely modulating multiple essential cellular processes Lysosomal protease cathepsins comprise three families of proteases with preferential activity within acidic cellular compartments but can also be found in other cellular locations They can operate alone or as part of signalling cascades and regulatory circuits playing important roles in apoptosis extracellular matrix remodelling HSC activation autophagy and metastasis contributing to the initiation development and progression of liver disease This review comprehensively summarizes the current knowledge to date on the role and contribution of lysosomal cathepsins to liver disease with a particular emphasis in liver fibrosis NAFLD and HCC