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Abstract Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We developed a novel “two-hit” model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57Bl6/NJ mice fed a high-fat diet (HFD) for > 10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. HFD-Renin (aka “HFpEF”) mice demonstrated obesity and insulin resistance, moderate left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by echocardiographic measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to human HFpEF. Treatment with empagliflozin, an effective but incompletely understood HFpEF therapy, improved multiple endpoints. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Additional preclinical HFpEF models allow for orthogonal studies to increase validity in assessment of interventions.

Background Heart failure with preserved ejection fraction (HFpEF) currently accounts for more than half of patients with HF, with limited approved evidence‐based therapies. HFpEF is a complex multifactorial disease associated with hypertension, obesity, diabetes, and renal dysfunction. In addition to our limited understanding of HFpEF pathophysiology, the development of new therapies is partially hindered by the existing translationally relevant preclinical HFpEF models. Methods and Results Here, we report the development of a novel 2‐hit HFpEF male mouse model through adding hypertensive stress from the activation of the renin‐angiotensin‐aldosterone system with adeno‐associated viral‐renin (AAV‐renin), to the metabolic syndrome nonalcoholic steatohepatitis (MSNASH, formerly FATZO) mouse (MSNASH+AAV‐renin). To further demonstrate model translatability, MSNASH+AAV‐renin mice were then treated with approved HFpEF therapies sacubitril/valsartan or a combination of sacubitril/valsartan and empagliflozin. We found that the MSNASH+AAV‐renin mouse model demonstrates clinically relevant features of human HFpEF, including preserved ejection fraction, cardiac hypertrophy, left atrial enlargement, diastolic dysfunction, elevated natriuretic peptides, inflammation, and low exercise capacity. We also demonstrate that treatment with approved HFpEF therapies sacubitril/valsartan and the combination of sacubitril/valsartan with empagliflozin improves the cardiac phenotypes of HFpEF in this model. Conclusions The MSNASH+AAV‐renin model faithfully represents a severe cardiometabolic HFpEF phenogroup that can be used as a new tool for HFpEF preclinical research and drug discovery.

Abstract Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

Abstract Background Inflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms. Methods Female C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study. Results Chronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P

Summary: Cancer cachexia is a disorder characterized by involuntary weight loss and impaired physical performance. Decline in physical performance of patients with cachexia is associated with poor quality of life, and currently there are no effective pharmacological interventions that restore physical performance. Here we examine the effect of GDF15 neutralization in a mouse model of cancer-induced cachexia (TOV21G) that manifests weight loss and muscle function impairments. With comprehensive assessments, our results demonstrate that cachectic mice treated with the anti-GDF15 antibody mAB2 exhibit body weight gain with near-complete restoration of muscle mass and markedly improved muscle function and physical performance. Mechanistically, the improvements induced by GDF15 neutralization are primarily attributed to increased caloric intake, while altered gene expression in cachectic muscles is restored in caloric-intake-dependent and -independent manners. The findings indicate potential of GDF15 neutralization as an effective therapy to enhance physical performance of patients with cachexia.

Muscle wasting is one of the main characteristics of cachexia associated with cancer and other chronic diseases and is often exacerbated by antineoplastic agents. Increased oxidative stress is associated with muscle wasting, along with depletion of glutathione, the most abundant endogenous antioxidant. Therefore, boosting endogenous glutathione has been proposed as a therapeutic strategy to prevent muscle wasting. Here, we tested this hypothesis by inactivating CHAC1, an intracellular glutathione degradation enzyme. We found CHAC1 expression is increased under multiple muscle wasting conditions in animal models, including fasting, cancer cachexia, and chemotherapy. The elevation of muscle Chac1 expression is associated with reduced glutathione level. CHAC1 inhibition via CRSPR/Cas9 mediated knock-in of an enzyme inactivating mutation demonstrates a novel strategy to preserve muscle glutathione levels under wasting conditions but fails to prevent muscle wasting in mice. These results suggest that preserving intracellular glutathione level alone may not be sufficient to prevent cancer or chemotherapy induced muscle wasting.

Objective: Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. Methods: The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr−/−). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. Results: A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr−/− mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice. Conclusions: These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a “fed signal” to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a “fasting signal” to prolong the catabolic state.

The precious rare edible fungus Morchella conica is popular worldwide for its rich nutrition, savory flavor, and varieties of bioactive components. Due to its high commercial, nutritional, and medicinal value, it has always been a hot spot. However, the molecular mechanism and endophytic bacterial communities in M. conica were poorly understood. In this study, we sequenced, assembled, and analyzed the genome of M. conica SH. Transcriptome analysis reveals significant differences between the mycelia and fruiting body. As shown in this study, 1,329 and 2,796 genes were specifically expressed in the mycelia and fruiting body, respectively. The Gene Ontology (GO) enrichment showed that RNA polymerase II transcription activity-related genes were enriched in the mycelium-specific gene cluster, and nucleotide binding-related genes were enriched in the fruiting body-specific gene cluster. Further analysis of differentially expressed genes in different development stages resulted in finding two groups with distinct expression patterns. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment displays that glycan degradation and ABC transporters were enriched in the group 1 with low expressed level in the mycelia, while taurine and hypotaurine metabolismand tyrosine metabolism-related genes were significantly enriched in the group 2 with high expressed level in mycelia. Moreover, a dynamic shift of bacterial communities in the developing fruiting body was detected by 16S rRNA sequencing, and co-expression analysis suggested that bacterial communities might play an important role in regulating gene expression. Taken together, our study provided a better understanding of the molecular biology of M. conica SH and direction for future research on artificial cultivation.

Summary: Growth differentiation factor 15 (GDF15) causes anorexia and weight loss in animal models, and higher circulating levels are associated with cachexia and reduced survival in cancer and other chronic diseases such as sepsis. To investigate the role of sepsis-induced GDF15, we examined whether GDF15 neutralization via a validated and highly potent monoclonal antibody, mAB2, modulates lipopolysaccharide (LPS)-induced anorexia, weight loss, and mortality in rodents. LPS injection transiently increased circulating GDF15 in wild-type mice, decreased food intake and body weight, and increased illness behavior and mortality at a high dose. GDF15 neutralization with mAB2 did not prevent or exacerbate any of the effects of LPS. Similarly, in GDF15 knockout mice, the LPS effect on appetite and survival was comparable with that observed in wild-type controls. Therefore, effective inhibition of circulating active GDF15 via an antibody or via gene knockout demonstrated that survival in the LPS acute inflammation model was independent of GDF15.

Growth and differentiation factor 15 (GDF15) is a cytokine reported to cause anorexia and weight loss in animal models. Neutralization of GDF15 was efficacious in mitigating cachexia and improving survival in cachectic tumor models. Interestingly, elevated circulating GDF15 was reported in patients with pulmonary arterial hypertension and heart failure, but it is unclear whether GDF15 contributes to cachexia in these disease conditions. In this study, rats treated with monocrotaline (MCT) manifested a progressive decrease in body weight, food intake, and lean and fat mass concomitant with elevated circulating GDF15, as well as development of right-ventricular dysfunction. Cotreatment of GDF15 antibody mAb2 with MCT prevented MCT-induced anorexia and weight loss, as well as preserved lean and fat mass. These results indicate that elevated GDF15 by MCT is causal to anorexia and weight loss. GDF15 mAb2 is efficacious in mitigating MCT-induced cachexia in vivo. Furthermore, the results suggest GDF15 inhibition is a potential therapeutic approach to alleviate cardiac cachexia in patients.

Fiona C Baker,1,2 Massimiliano de Zambotti,1 Ian M Colrain,1,3 Bei Bei4,5 1Center for Health Sciences, SRI International, Menlo Park, CA, USA; 2Brain Function Research Group, University of the Witwatersrand, Johannesburg, South Africa; 3Melbourne School of Psychological Sciences, University of Melbourne, 4Monash Institute of Cognitive and Clinical Neurosciences, School of Psychological Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, 5Centre for Women’s Mental Health, Department of Psychiatry, University of Melbourne, Royal Women’s Hospital, Melbourne, VIC, Australia Abstract: A substantial number of women experience sleep difficulties in the approach to menopause and beyond, with 26% experiencing severe symptoms that impact daytime functioning, qualifying them for a diagnosis of insomnia. Here, we review both self-report and polysomnographic evidence for sleep difficulties in the context of the menopausal transition, considering severity of sleep complaints and links between hot flashes (HFs) and depression with poor sleep. Longitudinal population-based studies show that sleep difficulties are uniquely linked with menopausal stage and changes in follicle-stimulating hormone and estradiol, over and above the effects of age. A major contributor to sleep complaints in the context of the menopausal transition is HFs, and many, although not all, HFs are linked with polysomnographic-defined awakenings, with HF-associated wake time contributing significantly to overall wakefulness after sleep onset. Some sleep complaints may be comorbid with depressive disorders or attributed to sleep-related breathing or movement disorders, which increase in prevalence especially after menopause, and for some women, menopause, age, and environmental/behavioral factors may interact to disrupt sleep. Considering the unique and multifactorial basis for sleep difficulties in women transitioning menopause, we describe clinical assessment approaches and management options, including combination treatments, ranging from cognitive behavioral therapy for insomnia to hormonal and nonhormonal pharmacological options. Emerging studies suggest that the impact of severe insomnia symptoms could extend beyond immediate health care usage and quality of life issues to long-term mental and physical health, if left untreated in midlife women. Appropriate treatment, therefore, has immediate benefit as well as advantages for maintaining optimal health in the postmenopausal years. Keywords: insomnia, midlife women, hormone therapy, estradiol, hot flashes, vasomotor symptoms

In order to improve the conductivity and anticorrosive resistance of bipolar plates in fuel cells, the characterizations and corrosion behaviors of the DLC-coated SUS316L steel deposited with different gas ratios CH4/H2 and deposition times were investigated and evaluated. The chemical bonding structure and composition of the DLC coatings were confirmed by Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The micromorphology and surface roughness of the DLC were observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The element compositions of cross section were determined by electron probe microanalysis (EPMA). The interfacial contact resistance (ICR) was measured. Furthermore, the DLC-coated SUS316L steel was corroded by potentiostatic polarizations in a 0.5 M H2SO4 solution at 0.8 V, 90°C for 168 h, and the corrosion behaviors were investigated in the solution using electrochemical techniques. In addition, the metal ions in sulfuric acid corrosion solution were detected by inductively coupled plasma-atomic emission spectroscopy. The results indicate that the DLC coatings deposited at a lower gas ratio CH4/H2 of 1 : 1 could result in a decreasing ID/IG ratio of 1.03, low Ra roughness of 3.77 nm, low ICR of 12.9 mΩ cm2, and low metal ion concentration of 16.60 ppm in the corrosion solution. The significant improvement in the anticorrosion resistance of the DLC film was mainly due to the increased sp3 element and formation of the passive film. As a result, the DLC coating deposited on the SUS316L steel at CH4/H2 = 1 : 1 has better anticorrosion properties. However, the DLC film-coated SUS316L steel still cannot meet the corrosive resistance of the bipolar plate.

BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in our cell-based druggable genome siRNA screen hold the potential to serve as novel pharmacological targets for the treatment of T2D.

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.

Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We developed a novel "two-hit" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57Bl6/NJ mice fed a high-fat diet (HFD) for > 10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d. HFD-Renin (aka "HFpEF") mice demonstrated obesity and insulin resistance, moderate left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by echocardiographic measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to human HFpEF. Treatment with empagliflozin, an effective but incompletely understood HFpEF therapy, improved multiple endpoints. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Additional preclinical HFpEF models allow for orthogonal studies to increase validity in assessment of interventions. [ABSTRACT FROM AUTHOR]

Abstract Purpose Severe combined immunodeficiency (SCID) is a set of rare monogenic inherited diseases that together represent the most severe form of the primary immunodeficiency disease phenotype. Preimplantation genetic testing for monogenic defects (PGT-M) is an effective reproductive technology strategy to prevent disease-causing gene mutations from being transmitted to offspring. The aim of this study was to report the use of PGT-M strategy based on karyomapping in four families to avoid the birth of SCID children. Methods Four couples underwent the PGT-M strategy due to SCID. The strategy of PGT-M started with a biopsy of the trophectoderm cells of embryos, and the whole genome was amplified by multiple replacement amplification (MDA). Then, the single nucleotide polymorphisms (SNPs) in the region upstream and downstream of the mutation site were subsequently identified via karyomapping, and the results were analyzed via SNPs linkage analysis. The aneuploids of the embryos were identified simultaneously. Finally, prenatal amniocentesis was used to verify the validity of the PGT-M results. Results We identified three novel variants (case1: IL2RG c.720_726delGAGCCAC; case 3: RAG2 c.770 C > T; and case 4: LIG4 c.1347 A > T). All four couples with SCID pathogenic gene mutations were subjected to karyomapping linkage analysis, and embryos with the pathogenic gene mutation were successfully identified. Euploid blastocysts without pathogenic alleles were transplanted, and healthy offspring were ultimately born. Prenatal diagnosis also confirmed the validity of our results. Conclusion This study revealed that karyomapping is an efficient approach for identifying SCID. Through PGT-M with karyomapping linkage analysis, healthy babies were born to families carrying mutations in the SCID pathogenic gene.

Author(s): Paul J Emmerson [1]; Feng Wang [2]; Yong Du [2]; Qian Liu [2]; Richard T Pickard [1]; Malgorzata D Gonciarz [1]; Tamer Coskun [1]; Matthew J Hamang [1]; Dana [...]