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Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.

Pathologically altered stromas are a common contributing factor to cancer progression and fibrogenesis. This report uncovers the role of LOXL2 in the creation and maintenance of the pathological microenvironment of human cancers and fibrotic diseases and presents the development of a LOXL2-specific antibody that shows therapeutic activity in tumor as well as lung and liver fibrosis models. We have identified a new role for the matrix enzyme lysyl oxidase–like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-β (TGF-β) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor β-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.

Serum transferrin (sTf) plays a pivotal role in regulating iron biodistribution and homeostasis within the body. The molecular details of sTf Fe(III) binding blood transport, and cellular delivery through transferrin receptor-mediated endocytosis are generally well-understood. Emerging interest exists in exploring sTf complexation of nonferric metals as it facilitates the therapeutic potential and toxicity of several of them. This review explores recent X-ray structural and physiologically relevant metal speciation studies to understand how sTf partakes in the bioactivity of key non-redox active hard Lewis acidic metals. It challenges preconceived notions of sTf structure function correlations that were based exclusively on the Fe(III) model by revealing distinct coordination modalities that nonferric metal ions can adopt and different modes of binding to metal-free and Fe(III)-bound sTf that can directly influence how they enter into cells and, ultimately, how they may impact human health. This knowledge informs on biomedical strategies to engineer sTf as a delivery vehicle for metal-based diagnostic and therapeutic agents in the cancer field. It is the intention of this work to open new avenues for characterizing the functionality and medical utility of nonferric-bound sTf and to expand the significance of this protein in the context of bioinorganic chemistry.

Background: The use of antioxidants of natural origin is an alternative to improve the cryopreservation of sheep semen. Objective: To evaluate the effect of Erythrina americana diet supplementation in the seminal characteristics of rams. Methods: Ten rams were randomly distributed in two treatments: l) group with addition of E. americana (20% dry matter basis) in the diet (AEA, n=5), and 2) the control group without addition of E. americana (SEA, n=5). Both diets were balanced iso-proteinically and iso-energetically. The feeding period of the rams was eleven weeks. The ejaculates were obtained through an artificial vagina and were evaluated fresh (37 °C) and refrigerated (5 °C). Volume (VOL) was evaluated with a tube graduated in milliliters, sperm concentration (CON x106) with a photometer and mass motility (MM, 1-5), individual motility (IM), normality (N) and live sperm (L) by microscopic observation. Results: The fresh semen showed that the inclusion of E. americana in the diet of rams did not modify (p>0.05) the parameters of VOL, CON, MM, and N, but decreased (p<0.05) the IM and L. In refrigerated semen the variables L and N were not different (p>0.05) among the treatment groups; however, IM was improved by the AEA group (p<0.05) compared to the SEA group. Conclusion: The supplementation with E. americana in the diet of rams reduces the sperm quality in fresh semen but is beneficial in the cooling process for refrigerated semen.

The goal of this study was to evaluate the significance of biliary dilatation proximal to the level of obstruction in liver transplant recipients with anastomotic strictures. A retrospective review of all liver transplants with duct-to-duct biliary reconstruction performed at our institution was conducted to identify patients with anastomotic stricture. Maximum diameter of the donor bile duct proximal to the stricture was measured from cholangiographic images taken at diagnosis and most recent follow-up. The change in duct size during this time was compared between those who responded to treatment (responder group) and those who did not (nonresponder group). Mean maximum duct diameter at diagnosis in the responder group was 10.1 mm at diagnosis and 12.1 mm at follow-up. This compared to a mean of 10.6 mm at diagnosis and 12.4 mm at follow-up in the nonresponder group. This corresponded to an average change in duct size between diagnosis and follow-up of +1.9 mm in the responders and +1.8 mm in the nonresponders. In posttransplant patients with obstructive cholestasis, duct dilatation does not correlate with clinically significant obstruction. Further, the change in duct size after treatment is not indicative of cholangiographic improvement.

Exosomes, small membrane vesicles （30-100 nm） of endocytic origin secreted by most cell types, contain functional biomolecules, which can be horizontally transferred to recipient cells [1]. Exosomes bear a specific protein and lipid composition, and carry a select set of functional mRNAs and microRNAs [2]. Recently, our group has shown that c-Met shed in exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-derived progenitor cells during melanoma progression [3]. In previous research, retrotransposon RNA transcripts, single-stranded DNA （ssDNA）,