Notes

Cutaneous Numerous Myeloma.
The 3D-printed large-scale multi-drive 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 reporter-labeled 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 genetically-encoded 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 over-expressed by lentiviral or adeno-associated 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 protein-DNA 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 AAV-HBV 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. Spliceosome associated factor 1 (SART1) has been described to inhibit hepatitis C virus infection through direct regulation of interferon stimulated genes. Selleck MLN0128 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 immune-related 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 anti-CTLA4 and/or the IDO1 inhibitor, epacadostat, or a 4-1BB agonist antibody.

ICI combination-induced hepatitis and the 4-1BB agonist mediated hepatitis share similar features yet maintain distinct immune signatures. Both were characterized by an expansion of peri-portal infiltrates and pan-zonal 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 ICI-mediated 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.
24-NorUrsodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid for immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC) where dysregulated T-cells including CD8
T-cells contribute to hepatobiliary immunopathology. We hypothesized that NorUDCA may directly modulate CD8
T-cell function thus contributing to its therapeutic efficacy.

NorUDCA's immunomodulatory effects were first studied in Mdr2
mice as cholestatic model of PSC. To dissect NorUDCA's immunomodulatory effects on CD8
T-cell function from its anti-cholestatic actions, the mechanisms were also explored in a non-cholestatic model of hepatic injury induced by excessive CD8
T-cell immune response upon acute non-cytolytic lymphocytic-choriomeningitis-virus (LCMV) infection. Studies included molecular and biochemical approaches, flow cytometry and metabolic assays in murine CD8
T-cells in vitro. Mass spectrometry (MS) was used to identify potential CD8
T-cell targets modulated by NorUDCA. NorUDCA' vivo and in vitro experimental approaches, we uncovered a yet-unrecognized immunometabolic regulatory property of NorUDCA adding to its previously established anti-cholestatic effects, explaining mechanistic aspects of its therapeutic potential in immune-mediated liver diseases including PSC.Proteases 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.
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