Engelmeldgaard5450

As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Formins direct the elongation of unbranched actin filaments by binding their barbed ends and processively stepping onto incoming actin monomers to incorporate them into the filament. Binding of profilin to actin monomers creates profilin-actin complexes, which then bind polyproline tracts located in formin homology 1 (FH1) domains. Diffusion of these natively disordered domains enables direct delivery of profilin-actin to the barbed end, speeding the rate of filament elongation. In this study, we investigated the mechanism of coordinated actin delivery from the multiple polyproline tracts in formin FH1 domains. We found that each polyproline tract can efficiently mediate polymerization, but that all tracts do not generate the same rate of elongation. In wild-type FH1 domains, the multiple polyproline tracts compete to deliver profilin-actin to the barbed end. This competition ultimately limits the rate of formin-mediated elongation. We propose that intrinsic properties of the filament-binding FH2 domain tune the efficiency of FH1-mediated elongation by directly regulating the rate of monomer incorporation at the barbed end. A strong correlation between competitive FH1-mediated profilin-actin delivery and FH2-regulated gating of the barbed end effectively limits the elongation rate, thereby obviating the need for evolutionary optimization of FH1 domain sequences. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Myostatin (or growth/differentiation factor 8 [GDF8]) is a member of the transforming growth factor β (TGF-β) superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage monoclonal antibody that prevents extracellular proteolytic activation of pro- and latent myostatin. Here, we used integrated structural and biochemical approaches to elucidate the molecular mechanism of an antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. read more This epitope is highly sequence divergent, sharing only limited similarity to other closely related members of the TGF-β superfamily. Hydrogen/deuterium exchange-MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen-antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 11 (antibodymyostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Non-alcoholic fatty liver disease (NAFLD) is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of NAFLD and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease, and these may represent targetable pathways to treat liver disorders.  Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH.  Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants (BAS) are orally administered polymers that bind bile acids in the intestine forming nonabsorbable complexes. BAS interrupts intestinal reabsorption of bile acids, decreasing in their circulating levels. We determined that treatment with the bile acid sequestrant sevelamer reversed the liver injury and prevented the progression of NASH, including steatosis, inflammation, and fibrosis in a western diet-induced NASH mouse model. Metabolomics and microbiome analysis revealed that this beneficial effect is associated with changes in the microbiota population and bile acid composition, including reversing microbiota complexity in cecum by increasing Lactobacillus and decreased Desulfovibrio. The net effect of these changes was improvement in liver function and markers of liver injury, and the positive effects of reversal of insulin resistance. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Background Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex® Investigational Use Only Blood Culture Identification Fungal Pathogen Panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram stain. We aimed to determine the performance of the BCID-FP in a multi-center clinical study.Materials and Methods Blood culture samples collected at 10 US sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, PNA-FISH and MALDI-TOF MS. Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples.Results A total of 866 clinical samples, 120 retrospectively- and 21 prospectively- collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of the Candida species (C. albicans, C. auris, C. dubliniensis, C.