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Abstract The geochemical discriminate diagrams help to distinguish the volcanics recovered from different tectonic settings but these diagrams tend to group the ocean floor basalts (OFB) under one class i.e., as mid-oceanic ridge basalts (MORB). Hence, a method is specifically needed to identify the OFB as normal (N-MORB), enriched (E-MORB) and ocean island basalts (OIB). We have applied Artificial Neural Network (ANN) technique as a supervised Learning Vector Quantisation (LVQ) to identify the inherent geochemical signatures present in the Central Indian Ocean Basin (CIOB) basalts. A range of N-MORB, E-MORB and OIB dataset was used for training and testing of the network. Although the identification of the characters as N-MORB, E-MORB and OIB is completely dependent upon the training data set for the LVQ, but to a significant extent this method is found to be successful in identifying the characters within the CIOB basalts. The study helped to geochemically delineate the CIOB basalts as N-MORB with perceptible imprints of E-MORB and OIB characteristics in the form of moderately enriched rare earth and incompatible elements. Apart from the fact that the magmatic processes are difficult to be deciphered, the architecture performs satisfactorily.

Hypertension and metabolic syndrome frequently coexist to increase the risk for adverse cardiometabolic outcomes. To date, no drug has been proven to be effective in treating hypertension with metabolic syndrome. M-atrial natriuretic peptide is a novel atrial natriuretic peptide analog that activates the particulate guanylyl cyclase A receptor. This study conducted a double-blind, placebo-controlled trial in 22 patients and demonstrated that a single subcutaneous injection of M-atrial natriuretic peptide was safe, well-tolerated, and exerted pleiotropic properties including blood pressure-lowering, lipolytic, and insulin resistance-improving effects. (MANP in Hypertension and Metabolic Syndrome [MANP-HTN-MS]; NCT03781739)., Competing Interests: This work was supported by the Marie Ingalls Cardiovascular Research Career Development Award in honor of Dr Alexander Schirger, Mayo Clinic CV Circulatory Program Award, and Scientist Development Award of the American Heart Association 16SDG29930003 (to Dr Cannone); Postdoctoral Fellowship 903661 (to Dr Ma) from the American Heart Association; and grants R01HL134668 and R01HL136340 (to Dr Burnett) from the National Heart, Lung and Blood Institute. Dr Burnett is the inventor of MANP, which the Mayo Clinic has licensed to E-STAR BIO TECH. All other authors have reported that they have no relationships relevant to the contents in this paper to disclose., (© 2024 The Authors.)

Spinel (magnesium aluminate MgAl 2 O 4 ) ceramic-based polyphasic composite scaffold has been recently reported for craniofacial bone tissue engineering. Improving the osteogenic effects of such composite scaffolds with bone morphogenetic proteins (BMP2) is an intensely researched area. This study investigated the gene interactions of this scaffold with BMP2 and mesenchymal stem cells (MSCs). Human bone marrow MSCs were cultured in 3 groups: Group 1-Control (BMSCs), Group 2-BMSC with BMP2, and Group 3-BMSC with scaffold and BMP2. After RNA isolation, gene expression analysis was done by microarray. Differentially expressed genes (DEGs) (-1.0 > fold changes>1 and p value <.05) were studied for their function and gene ontologies using Database for Annotation, Visualization and Integrated Discovery (DAVID). They were further studied by protein-protein interaction network analysis using STRING and MCODE Cytoscape plugin database. Group 3 showed up regulation of 3222 genes against 2158 of Group 2. Group 3 had five annotation clusters with enrichment scores from 2.08 to 3.93. Group 2 had only one cluster. Group 3 showed activation of all major osteogenic pathways: TGF, BMP2, WNT, SMAD, and Notch gene signaling with effects of calcium and magnesium released from the scaffold. Downstream effect of all these caused significant activation of RUNX2, the key transcriptional regulator of osteogenesis in Group 3. STRING and MCODE Cytoscape plugin demonstrated the interactions. The enhanced MSC differentiation for osteogenesis with the addition of BMP2 to the polyphasic composite scaffold proposed promising clinical applications for bone tissue engineering., (© 2022 Wiley Periodicals LLC.)

ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI-sensitive oncogenic variants in cell-based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA-approved ROS1-TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA-approved ROS1-TKIs., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are produced in the heart and secreted into the circulation. As hormones, both peptides activate the guanylyl cyclase receptor A (GC-A), playing a role in blood pressure (BP) regulation. A significant role for ANP and BNP includes favorable actions in metabolic homeostasis. Sex-based high prevalence of risk factors for cardiovascular disease in males compared with females is well established, but sex-based differences on cardiometabolic protection have not been investigated in relation to ANP ( NPPA ) and BNP ( NPPB ) gene variants. We included 1,146 subjects in the general population from Olmsted County, Minnesota. Subjects were genotyped for the ANP gene variant rs5068 and BNP gene variant rs198389. Cardiometabolic parameters and medical records were reviewed. In the presence of the minor allele of rs5068, diastolic BP, creatinine, body mass index (BMI), waist measurement, insulin, and prevalence of obesity and metabolic syndrome were lower, whereas HDL was higher in males with only trends observed in females. We observed no associations of the minor allele with echocardiographic parameters in either males or females. Regarding rs198389 genotype, the minor allele was not associated with any BP, metabolic, renal, or echocardiographic parameters in either sex. In the general community, the minor allele of the ANP gene variant rs5068 is associated with a favorable metabolic phenotype in males. No associations were observed with the BNP gene variant rs198389. These studies support a protective role of the ANP pathway on metabolic function and underscore the importance of sex in relationship to natriuretic peptide responses. NEW & NOTEWORTHY Males are characterized by lower ANP and BNP with greater prevalence of cardiometabolic disease. The ANP genetic variant rs5068 was associated with less metabolic dysfunction in males, whereas no metabolic profile was related to the BNP genetic variant rs198389 in the general population. ANP may play a more biological role in metabolic homeostasis compared with BNP in the general population with greater physiological metabolic actions in males compared with females.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT 1 ) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT 1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT 1 /PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

Fibroadipogenic progenitor (FAP) cells are implicated as a major source of fatty infiltration (FI) in murine rotator cuff (RC) injury, but FAP cell response after RC tear in a rabbit model is unknown. This study determined whether changes in FAP cell count after an RC tear predate muscle degeneration in a clinically relevant rabbit model. We hypothesized increases in FAP cell count correlate temporally with RC degeneration. New Zealand white rabbits (n = 26) were evaluated at 1, 2, 4, and 6 weeks after unilateral full-thickness tenotomy of supraspinatus and infraspinatus tendons. FI area and adipocyte size were histologically analyzed, muscle density was measured by computerized tomography, and quantification of FAP cells was measured by flow cytometry and immunohistochemistry. The percentage of intrafascicular adipocyte area increased over time in supraspinatus muscle samples (p = 0.03), significantly between 1- and 6-week samples (p = 0.04). There were no differences in perifascicular adipocyte area percentages between time points. Peak increase in FAP cell count occurred at 1-week (p = 0.03), with a decrease in the following weeks. There was a negative correlation between supraspinatus adipocyte area and FAP cell count (p < 0.05). On computed tomography (CT) scan, maximal decrease in muscle density was observed in the 4th to 6th weeks. In summary, FAP cell response occurred early after tenotomy and did not correlate temporally with increases in FI. This suggests that FAP cell response may predate degenerative changes, and early targeting of FAPs before adipocyte maturation could blunt FI after RC tear., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear., Methods: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice., Results: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased., Conclusions: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.

This article presents a literature review of the 81 articles accepted in the Finance Research Letters Special Issue titled "COVID-19 and the Economy". The articles are classified into five broad areas (investments and asset pricing, the macroeconomy and banking, commodities, corporate finance, and other topics). We summarize the key findings of the articles by area and highlight the influence of the articles as measured by Google Scholar citations. We conclude by presenting directions for future research., (© 2022 Elsevier Inc. All rights reserved.)

Targeted therapy of ROS1-fusion-driven non-small cell lung cancer (NSCLC) has achieved notable clinical success. Despite this, resistance to therapy inevitably poses a significant challenge. MYC amplification was present in ∼19% of lorlatinib-resistant ROS1-driven NSCLC. We hypothesized that MYC overexpression drives ROS1-TKI resistance. Using complementary approaches in multiple models, including a MYC-amplified patient-derived cell line and xenograft (LUAD-0006), we established that MYC overexpression induces broad ROS1-TKI resistance. Pharmacologic inhibition of ROS1 combined with MYC knockdown were essential to completely suppress LUAD-0006 cell proliferation compared with either treatment alone. We interrogated cellular signaling in ROS1-TKI-resistant LUAD-0006 and discovered significant differential regulation of targets associated with cell cycle, apoptosis, and mitochondrial function. Combinatorial treatment of mitochondrial inhibitors with crizotinib revealed inhibitory synergism, suggesting increased reliance on glutamine metabolism and fatty-acid synthesis in chronic ROS1-TKI treated LUAD-0006 cells. In vitro experiments further revealed that CDK4/6 and BET bromodomain inhibitors effectively mitigate ROS1-TKI resistance in MYC-overexpressing cells. Notably, in vivo studies demonstrate that tumor control may be regained by combining ROS1-TKI and CDK4/6 inhibition. Our results contribute to the broader understanding of ROS1-TKI resistance in NSCLC., Implications: This study functionally characterizes MYC overexpression as a novel form of therapeutic resistance to ROS1 tyrosine kinase inhibitors in non-small cell lung cancer and proposes rational combination treatment strategies., (©2022 American Association for Cancer Research.)

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following in vivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders., Competing Interests: Declaration of interests C.S., P.A., K.R.W., and G.L. are scientific founders and shareholders and P.A. and G.L. serve as scientific advisors of Vita Therapeutics. K.R.W. is a current employee of F. Hoffmann La-Roche. C.S. is a current employee of Vita Therapeutics. C.S., I.Y.C., H.L., P.A., K.R.W., and G.L. are inventors of a patent related to this study. The Lee lab receives a funding from Vita Therapeutics. R.M.L. is a current employee at National Institutes of Health, but this work was prepared while R.M.L. was employed at University of Maryland School of Medicine. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: cGMP MANP (M-atrial natriuretic peptide) is a best-in-class activator of the pGC-A (particulate guanylyl cyclase A) receptor. Furosemide increases the effectiveness of antihypertensive agents, but activates renin-angiotensin-aldosterone system. We aimed to investigate for the first time cardiorenal and neurohumoral actions of MANP in a genetic model of hypertension in spontaneously hypertensive rats. We also assessed how MANP would potentiate the blood pressure (BP)-lowering actions of furosemide while reducing the production of aldosterone., Methods: Spontaneously hypertensive rats (N=60) were randomized in vehicle, MANP, furosemide, or MANP+furosemide groups. Furosemide (1, 5, 10 mg/kg) was given as a single bolus which in MANP+furosemide groups was followed by a 60-minute infusion of MANP., Results: BP was reduced in MANP300 (300 pmol/[kg·min]) and MANP600 (600 pmol/[kg·min]) groups ( P <0.05) and was accompanied by significant increase in plasma cyclic guanosine monophosphate. Furosemide alone reduced BP but less compared with MANP with no change in plasma cyclic guanosine monophosphate. MANP+furosemide resulted in the greatest BP reduction and significant increase in plasma cyclic guanosine monophosphate in Fs5+MANP300, Fs10+MANP300, and Fs10+MANP600. Plasma aldosterone increased in furosemide groups, which was significantly attenuated in MANP+furosemide groups. Natriuresis and diuresis increased in all treated groups ( P <0.05) with no significant differences between furosemide and furosemide+MANP. In vitro, MANP increased cyclic guanosine monophosphate level in human vascular cells., Conclusions: We provide novel evidence that MANP potentiates the BP-lowering actions of furosemide, suppresses the activation of renin-angiotensin-aldosterone system, and preserves renal function. These data are highly relevant to clinical needs in the treatment of hypertension and heart failure.

Background: Laser etching addresses the disadvantages of conventional acid etching technique, such as enamel decalcification and formation of white spot lesions. The aim of the present study was to evaluate and compare the shear bond strength (SBS), adhesive remnant index (ARI), and the surface characteristics of the samples treated with conventional acid etching and Er, Cr: YSGG laser etching with variable output power and time durations., Methodology: The study sample included 78 extracted teeth divided into six groups of 13 teeth each, and 3 samples from each group were utilized for analyzing etch patterns, and the remaining 10 teeth from each group were used for evaluating the shear bond strength. In Group I phosphoric acid etching was done, whereas in Group II- VI Laser etching 1.5 W/10 s, 1.5 W/15 s, 3 W/5 s, 3 W/10 s, 3 W/15 s. Statistical analysis for shear bond strength testing was performed using one-way ANOVA followed by Post HOC tests., Results: The mean shear bond strength of Group I was 7.16 Mpa and Group III of 5.43 Mpa. Group II, IV, V, and VI had mean shear bond strength of 4.93 Mpa, 3.88 Mpa, 4.05 Mpa, and 4.88 Mpa, respectively. The ARI scores Group I had a significant number of samples with scores 2 other groups showed increased Score 0. The etch pattern of groups I, II, III showed the combined dissolution of both prism cores, and peripheries were seen. In group IV, the etching pattern was irregular with the pitted type of surface. In groups V and VI, relatively flat and smooth enamel surface was seen., Conclusion: The bond strength attained by laser etching (1.5 W/10 s and 1.5 W/15 s) was comparable to that obtained by the acid etching technique., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Orthodontic Science.)

The use of air quality monitoring networks to inform urban policies is critical especially where urban populations are exposed to unprecedented levels of air pollution. High costs, however, limit city governments' ability to deploy reference grade air quality monitors at scale; for instance, only 33 reference grade monitors are available for the entire territory of Delhi, India, spanning 1500 sq km with 15 million residents. In this paper, we describe a high-precision spatio-temporal prediction model that can be used to derive fine-grained pollution maps. We utilize two years of data from a low-cost monitoring network of 28 custom-designed low-cost portable air quality sensors covering a dense region of Delhi. The model uses a combination of message-passing recurrent neural networks combined with conventional spatio-temporal geostatistics models to achieve high predictive accuracy in the face of high data variability and intermittent data availability from low-cost sensors (due to sensor faults, network, and power issues). Using data from reference grade monitors for validation, our spatio-temporal pollution model can make predictions within 1-hour time-windows at 9.4, 10.5, and 9.6% Mean Absolute Percentage Error (MAPE) over our low-cost monitors, reference grade monitors, and the combined monitoring network respectively. These accurate fine-grained pollution sensing maps provide a way forward to build citizen-driven low-cost monitoring systems that detect hazardous urban air quality at fine-grained granularities., Competing Interests: Competing interestsProf. Subramanian declares no competing non-financial interests but the following competing financial interests: Prof. Subramanian is a co-founder of Entrupy Inc, Velai Inc, and Gaius Networks Inc and has served as a consultant for the World Bank and the Governance Lab. Dr. Subramanian reports that Velai Inc broadly works in the area of socio-economic predictive models. All other authors declare no competing interests., (© The Author(s) 2022.)

Background. The Flip-Lock Herbst (TP Orthodontics Inc.) is a fixed functional appliance, a variant of the Herbst appliance, introduced by Miller. It is claimed to have better patient tolerance due to its increased freedom for the mandible's lateral movements. There have been no studies on the flip lock Herbst till date. This study was undertaken to assess the efficiency of the Flip-Lock Herbst appliance in correcting Angle's class II division 1 malocclusion. Methods. Eight subjects in their active growth period with class II division 1 malocclusion due to a retrognathic mandible were included in the study. Standardized lateral cephalometric radiographs were used to evaluate skeletal and dental changes with the SO analysis. Paired samples t-test was used to assess statistical significance. Results. Statistically significant increases in mandibular length (pg/OLp) and effective mandibular length (ar/OLp + pg/OLp) were observed. There was a significant maxillary restraining effect. Dental effects were significant and exhibited class II correction features except for the position of lower incisors within the mandible (ii/OLp - pg/OLp). Skeletal changes accounted for 61% and dental changes for 39% of the total treatment for molar correction. For overjet correction, skeletal changes contributed to 63% and dental changes to 37% of the total treatment. Conclusion. The Flip-Lock Herbst appliance was efficient in correcting Angle's class II division 1 malocclusion due to a retrognathic mandible. Both skeletal and dental changes were evident, with the former predominating (60:40)., (© 2021 The Author(s).)

[Figure: see text].

Intermediate filaments (IFs) are a primary structural component of the cytoskeleton extending throughout the muscle cell (myofiber). Mechanotransduction, the process by which mechanical force is translated into a biochemical signal to activate downstream cellular responses, is crucial to myofiber function. Mechanical forces also act on the nuclear cytoskeleton, which is integrated with the myofiber cytoskeleton by the linker of the nucleoskeleton and cytoskeleton (LINC) complexes. Thus, the nucleus serves as the endpoint for the transmission of force through the cell. The nuclear lamina, a dense meshwork of lamin IFs between the nuclear envelope and underlying chromatin, plays a crucial role in responding to mechanical input; myofibers constantly respond to mechanical perturbation via signaling pathways by activation of specific genes. The nucleus is the largest organelle in cells and a master regulator of cell homeostasis, thus an understanding of how it responds to its mechanical environment is of great interest. The importance of the cell nucleus is magnified in skeletal muscle cells due to their syncytial nature and the extreme mechanical environment that muscle contraction creates. In this review, we summarize the bidirectional link between the organization of the nucleoskeleton and the contractile features of skeletal muscle as they relate to muscle function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iyer, Folker and Lovering.)

Objectives: This study sought to characterize urinary and plasma C-type natriuretic peptide (CNP) in acute decompensated heart failure (ADHF) to define their relationship with clinical variables and to determine whether urinary and plasma CNP together add prognostic value., Background: CNP is a protective hormone that is synthesized in the kidney and endothelium and possesses antiremodeling properties. Urinary and plasma CNP levels are elevated in pathophysiological conditions; however, their regulation and prognostic value in heart failure (HF) is unclear., Methods: Urinary and plasma CNP were measured in 109 healthy subjects and 208 patients with ADHF; the 95th percentile of CNP values from healthy subjects established the normal contemporary cutoffs. Patients with ADHF were stratified based on urinary and plasma CNP levels for clinical characterization and the assessment of risk for adverse outcomes., Results: There was no significant correlation between urinary and plasma CNP in both cohorts. Urinary and plasma CNP were significantly elevated in patients with ADHF, and both increased with disease severity and were positively correlated with plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP). Of the patients with ADHF, 23% had elevations in both urinary and plasma CNP, whereas 24% had normal CNP levels. During a median follow-up of 3 years, patients with elevated urinary and plasma CNP had a significantly higher risk of rehospitalization and/or death (HR: 1.79; P = 0.03) and rehospitalization (HR: 2.16; P = 0.01) after adjusting for age, sex, left ventricular ejection fraction, renal function, and plasma NT-proBNP. The C-statistic and integrated discrimination analyses further supported that the addition of urinary and plasma CNP to established risk models improved the prediction of adverse outcomes in patients with ADHF., Conclusions: Urinary and plasma CNP are differentially regulated in ADHF, and elevations in both provided independent prognostic value for predicting adverse outcomes., Competing Interests: Funding Support and Funding Disclosures This work was supported by National Heart, Lung and Blood Institute grants R01 HL36634 (to Dr Burnett) and R01 HL132854 (to Dr Sangaralingham). This work was also supported by the Division of Circulatory Failure, Department of Cardiovascular Medicine at Mayo Clinic and the Mayo Foundation. Drs Ma, Y. Chen, H. Chen, Burnett, and Sangaralingham, and the Mayo Foundation have issued or filed patents for assessing the use of CNP as well as for natriuretic peptide-based or particulate guanylyl cyclase enhancing therapeutics. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

The neuromuscular junction (NMJ) is a specialized synapse that bridges the motor neuron and the skeletal muscle fiber and is crucial for conversion of electrical impulses originating in the motor neuron to action potentials in the muscle fiber. The consideration of contributing factors to skeletal muscle injury, muscular dystrophy and sarcopenia cannot be restricted only to processes intrinsic to the muscle, as data show that these conditions incur denervation-like findings, such as fragmented NMJ morphology and corresponding functional changes in neuromuscular transmission. Primary defects in the NMJ also influence functional loss in motor neuron disease, congenital myasthenic syndromes and myasthenia gravis, resulting in skeletal muscle weakness and heightened fatigue. Such findings underscore the role that the NMJ plays in neuromuscular performance. Regardless of cause or effect, functional denervation is now an accepted consequence of sarcopenia and muscle disease. In this short review, we provide an overview of the pathologic etiology, symptoms, and therapeutic strategies related to the NMJ. In particular, we examine the role of the NMJ as a disease modifier and a potential therapeutic target in neuromuscular injury and disease.

Mechanical forces are conducted through myofibers and into nuclei to regulate muscle development, hypertrophy, and homeostasis. We hypothesized that nuclei in aged muscle have changes in the nuclear envelope and associated proteins, resulting in altered markers of mechano-signaling., Methods: YAP/TAZ protein expression and gene expression of downstream targets, Ankrd1 and Cyr61, were evaluated as mechanotransduction indicators. Expression of proteins in the nuclear lamina and the nuclear pore complex (NPC) were assessed, and nuclear morphology was characterized by electron microscopy. Nuclear envelope permeability was assessed by uptake of 70 kDa fluorescent dextran., Results: Nuclear changes with aging included a relative decrease of lamin β1 and Nup107, and a relative increase in Nup93, which could underlie the aberrant nuclear morphology, increased nuclear leakiness, and elevated YAP/TAZ signaling., Conclusion: Aged muscles have hyperactive nuclear-cytoplasmic signaling, indicative of altered nuclear mechanotransduction. These data highlight a possible role for the nucleus in aging-related aberrant mechano-sensing., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the Fe II active site to form a highly reactive Fe IV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-Fe IV = O intermediate and characterized its structure as a Fe II -peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary Fe II active site before the O 2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of Fe IV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the Fe II site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the Fe II site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes., Competing Interests: The authors declare no competing interest.

The importance of canonical versus noncanonical mechanisms for the generation of angiotensins remains a major challenge that, in part, is heavily swayed by the relative efficacy of therapies designed to inhibit renin, ACE (angiotensin-converting enzyme), or the Ang II (Angiotensin II) receptor. Ang (1-12) (angiotensin [1-12]) is an Ang II forming substrate serving as a source for Ang II-mediated tissue actions. This study identifies for the first time the presence of Ang (1-12) in the blood of 52 normal (22 women) and 19 (13 women) patients with hypertension not receiving antihypertensive medication at the time of the study. Normal subjects of comparable ages and body habitus had similar circulating plasma Ang (1-12) concentrations (women: 2.02±0.62 [SD] ng/mL; men 2.05±0.55 [SD] ng/mL, P >0.05). The higher values of plasma Ang (1-12) concentrations in hypertensive men (2.51±0.49 ng/mL, n=6) and women (2.33±0.63 [SD] ng/mL, n=13) were statistically significant ( P <0.02) and correlated with elevated plasma renin activity, systolic and pulse pressure, and plasma concentrations of NT-proBNP (N-terminal prohormone BNP). The increased plasma Ang (1-12) in patients with hypertension was not mirrored by similar changes in plasma angiotensinogen and Ang II concentrations. The first identification of an age-independent presence of Ang (1-12) in the blood of normotensive subjects and patients with hypertension, irrespective of sex, implicates this non-renin dependent substrate as a source for Ang II production in the blood and its potential contribution to the hypertensive process.

Mitochondrial derangement is an important contributor to the pathophysiology of muscular dystrophies and may be among the earliest cellular deficits. We have previously shown that disruption of Mss51, a mammalian skeletal muscle protein that localizes to the mitochondria, results in enhanced muscle oxygen consumption rate, increased endurance capacity, and improved limb muscle strength in mice with wildtype background. Here, we investigate whether Mss51 deletion in the mdx murine model of Duchenne muscular dystrophy (mdx-Mss51 KO) counteracts the muscle pathology and mitochondrial irregularities observed in mdx mice. We found that mdx-Mss51 KO mice had increased myofiber oxygen consumption rates and an amelioration of muscle histopathology compared to mdx counterparts. This corresponded with greater treadmill endurance and less percent fatigue in muscle physiology, but no improvement in forelimb grip strength or limb muscle force production. These findings suggest that although Mss51 deletion ameliorates the skeletal muscle mitochondrial respiration defects in mdx and improves fatigue resistance in vivo, the lack of improvement in force production suggests that this target alone may be insufficient for a therapeutic effect., (© 2021 Federation of American Societies for Experimental Biology.)

Objectives: This study sought to assess associations between longitudinal change in atrial natriuretic peptide (ANP) and reverse cardiac remodeling following initiation of sacubitril/valsartan in patients with heart failure with reduced ejection fraction (HFrEF)., Background: Neprilysin inhibition results in an increase of several vasoactive peptides that may mediate the beneficial effects of sacubitril/valsartan, including ANP., Methods: In a prospective study of initiation and titration of sacubitril/valsartan in patients with HFrEF, blood was collected at scheduled time points into tubes containing protease inhibitors. This pre-specified exploratory analysis included patients in whom ANP was measured at baseline and serially through 12 months of treatment., Results: Among 144 participants (mean age: 64.5 years; left ventricular ejection fraction: 30.8%), following initiation of sacubitril/valsartan, there was an early and significant increase in ANP, with the majority of rise from 99 pg/ml at baseline to 156 pg/ml at day 14 (p < 0.001). There was a further trend toward a second increase from day 30 to day 45 (p = 0.07). At maximal rise, ANP had doubled. In longitudinal analyses, early rise in ANP was followed by a subsequent increase in urinary cycle guanosine monophosphate. Larger early increase in ANP was associated with larger later improvements in left ventricular ejection fraction and left atrial volume index (p < 0.001 for both)., Conclusions: Concentrations of ANP doubled after initiation of sacubitril/valsartan in patients with HFrEF. Larger early increases in ANP were associated with a greater magnitude of subsequent reverse cardiac remodeling. (Effects of Sacubitril/Valsartan Therapy on Biomarkers, Myocardial Remodeling and Outcomes [PROVE-HF]; NCT02887183)., Competing Interests: Funding Support and Author disclosures This work was supported by Novartis Pharmaceuticals, Inc. Dr. Januzzi is supported in part by the Hutter Family Professorship. Dr. Prescott is an employee of Novartis Pharmaceuticals, Inc. Dr. Maisel has received consulting income from Abbott Vascular, Ortho Clinical Diagnostics, and Novartis. Dr. Felker has received research grants from the National Heart, Lung, and Blood Institute, American Heart Association, Amgen, Merck, Cytokinetics, and Roche Diagnostics and has acted as a consultant to Novartis, Amgen, Bristol Myers Squibb, Medtronic, Cardionomic, Relypsa, V-Wave, Myokardia, Innolife, EBR Systems, Arena, Abbott, Sphingotec, Roche Diagnostics, Alnylam, LivaNova, and SC Pharma. Dr. Butler has received research support from the National Institutes of Health, Patient-Centered Outcomes Research Institute, and the European Union; and serves as a consultant for Abbott, Adrenomed, Amgen, Array, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, CVRx, G3 Pharmaceutical, Innolife, Janssen, LinaNova, Luitpold, Medtronic, Merck, Novartis, Novo Nordisk, Relypsa, Roche, Sanofi, V-Wave Limited, and Vifor. Dr. Piña has received consulting income from Relypsa and Novartis. Dr. Ibrahim has received honoraria from Novartis Pharmaceuticals and Roche Diagnostics. Dr. Abbas is an employee of Novartis Pharmaceuticals, Inc. Dr. Solomon has received research grants from Alnylam, Amgen, AstraZeneca, Bellerophon, Bayer, Bristol Myers Squibb, Celladon, Cytokinetics, Eidos, Gilead, GlaxoSmithKline, Ionis, Lone Star Heart, Mesoblast, MyoKardia, National Institute of Health/National Heart, Lung, and Blood Institute, Novartis, Sanofi Pasteur, and Theracos; and has consulted for Akros, Alnylam, Amgen, AstraZeneca, Bayer, Bristol Myers Squibb, Cardior, Corvia, Cytokinetics, Gilead, GlaxoSmithKline, Ironwood, Merck, Myokardia, Novartis, Roche, Takeda, Theracos, Quantum Genetics, Cardurion, AoBiome, Janssen, Cardiac Dimensions, and Tenaya. Dr. Januzzi is a Trustee of the American College of Cardiology; has received grant support from Novartis Pharmaceuticals and Abbott Diagnostics and consulting income from Abbott Diagnostics, Janssen, Novartis, and Roche Diagnostics; and participates in clinical endpoint committees/Data Safety Monitoring Boards for Abbott, AbbVie, Amgen, Bayer, and Takeda. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Our aim was to assess key muscle imaging and contractility parameters in the Duchenne muscular dystrophy (DMD) rat model (Dmd-KO rat), which have not yet been characterized sufficiently., Methods: We performed in-vivo magnetic resonance imaging (MRI) for thigh and leg muscles, and performed hematoxylin and eosin (H&E) staining and in-vivo muscle contractility testing in specific hindlimb muscles., Results: MRI prior to testing muscle contractility revealed multiple, unevenly distributed focal hyperintensities in the Dmd-KO rat quadriceps and tibialis anterior muscles. H&E staining showed corresponding areas of inflammation and ongoing regeneration. In-vivo contractile testing showed maximal force generated by Dmd-KO muscles was significantly lower, and susceptibility to injury was ~ two-fold greater in the Dmd-KO rats compared to wild-type (WT) rats., Discussion: Together, the MRI findings, histological findings, and the low strength and high susceptibility to injury in muscles support use of the Dmd-KO rat as an animal model of DMD., (© 2020 Wiley Periodicals LLC.)

The α-ketoglutarate (αKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin Fe IV ═O intermediate that, in most reactions, abstract a hydrogen atom from the substrate. Previously, the Fe IV ═O intermediate in the αKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the Fe IV ═O complex in taurine dioxygenase (TauD), the αKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate Fe IV ═O complexes in two different substrate orientations (one more along [σ channel] and another more perpendicular [π channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the π channel by the significantly larger oxyl character present in the pπ orbital oriented toward the substrate, which leads to an earlier transition state along the C-H coordinate.

The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the weakness, susceptibility to injury, and progressive wasting associated with DMD., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 μm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: This study sought to identify the role of annexin A1 (AnxA1) as a congestion marker in acute heart failure (AHF) and to identify its putative role in predicting clinical outcomes., Background: AnxA1 is a protein that inhibits inflammation following ischemia-reperfusion injury in cardiorenal tissues. Because AHF is a state of tissue hypoperfusion, we hypothesized that plasma AnxA1 levels are altered in AHF., Methods: In the Renal Optimization Strategies Evaluation (ROSE) trial, patients hospitalized for AHF with kidney injury were randomized to receive dopamine, nesiritide, or placebo for 72 hours in addition to diuresis. In a subanalysis, plasma AnxA1 levels were measured at baseline and at 72 hours in 275 patients. Participants were divided into 3 tertiles based on their baseline AnxA1 levels., Results: The prevalence of peripheral edema 2+ increased with increasing AnxA1 levels (P < .007). Cystatin C, blood urea nitrogen, and kidney injury molecule-1 plasma levels were higher among participants in tertile 3 vs tertiles 1 or 2 (P< .05). Patients with a congestion score of 4 had a mean baseline AnxA1 level 8.63 units higher than those with a congestion score of 0 (P = .03). Patients in tertiles 2 and 3 were twice as likely to experience creatinine elevation as patients in tertile 1 (P = .03). Patients in tertiles 2 and 3 were at a higher risk of 60-day all-cause mortality or heart failure hospitalization and 180-day all-cause mortality (P < .05)., Conclusions: Among patients hospitalized for AHF with impaired kidney function, elevated AnxA1 levels are associated with worse congestion, higher risk for further creatinine elevation, and higher rates of 60-day morbidity or all-cause mortality and 180-day all-cause mortality., Clinical Trial Registration: clinicaltrials.gov Identifier: NCT01132846., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of dystrophin with progressive degeneration of skeletal muscles. Most studies regarding DMD understandably focus on muscle, but dystrophin is also expressed in the central nervous system, potentially resulting in cognitive and behavioral changes. Animal models are being used for developing more comprehensive neuromonitoring protocols and clinical image acquisition procedures. The recently developed DMD rat is an animal model that parallels the progressive muscle wasting seen in DMD. Here, we studied the brain and temporalis muscle structure and neurochemistry of wild type (WT) and dystrophic (DMD) rats using magnetic resonance imaging and spectroscopy. Both structural and neurochemistry alterations were observed in the DMD rat brain and the temporalis muscle. There was a decrease in absolute brain volume ( WT = 1579 mm 3 ; DMD = 1501 mm 3 ; p = 0.039, Cohen's d = 1.867), but not normalized ( WT = 4.27; DMD = 4.02; p = 0.306) brain volume. Diffusion tensor imaging (DTI) revealed structural alterations in the DMD temporalis muscle, with increased mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD). In the DMD rat thalamus, DTI revealed an increase in fractional anisotropy (FA) and a decrease in RD. Smaller normalized brain volume correlated to severity of muscle dystrophy ( r = -0.975). Neurochemical changes in the DMD rat brain included increased GABA and NAA in the prefrontal cortex, and GABA in the hippocampus. Such findings could indicate disturbed motor and sensory signaling, resulting in a dysfunctional GABAergic neurotransmission, and an unstable osmoregulation in the dystrophin-null brain., (Copyright © 2020 Xu, Tang, Li, Iyer and Lovering.)

Background: Clinical use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has gained momentum as treatment for muscle injuries. Exosomes, or small cell-derived vesicles, could be helpful if they could deliver the same or better physiological effect without cell transplantation into the muscle., Hypothesis: Local delivery of exosomes derived from PRP (PRP-exos) or MSCs (MSC-exos) to injured muscles hastens recovery of contractile function., Study Design: Controlled laboratory study., Methods: In a rat model, platelets were isolated from blood, and MSCs were isolated from bone marrow and expanded in culture; exosomes from both were isolated through ultracentrifugation. The tibialis anterior muscles were injured in vivo using maximal lengthening contractions. Muscles were injected with PRP-exos or MSC-exos (immediately after injury and 5 and 10 days after injury); controls received an equal volume of saline. Histological and biochemical analysis was performed on tissues for all groups., Results: Injury resulted in a significant loss of maximal isometric torque (66% ± 3%) that gradually recovered over 2 weeks. Both PRP-exos and MSC-exos accelerated recovery, with similar faster recovery of contractile function over the saline-treated group at 5, 10, and 15 days after injury ( P < .001). A significant increase in centrally nucleated fibers was seen with both types of exosome groups by day 15 ( P < .01). Genes involved in skeletal muscle regeneration were modulated by different exosomes. Muscles treated with PRP-exos had increased expression of Myogenin gene ( P < .05), whereas muscles treated with MSC-exos had reduced expression of TGF-β ( P < .05) at 10 days after muscle injury., Conclusion: Exosomes derived from PRP or MSCs can facilitate recovery after a muscle strain injury in a small-animal model likely because of factors that can modulate inflammation, fibrosis, and myogenesis., Clinical Relevance: Given their small size, low immunogenicity, and ease with which they can be obtained, exosomes could represent a novel therapy for many orthopaedic ailments.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. In the present study, when human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patient hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD-relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD Pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these 2 hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study revealed that fenofibrate could inhibit mitochondrion-induced apoptosis in DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified 2 promising small molecules with in vivo efficacy.

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed., (©2020 American Association for Cancer Research.)

Based on the cardiac hormone atrial natriuretic peptide (ANP) and its seminal role in blood pressure (BP) homeostasis, we investigated the chronic BP lowering actions of a novel ANP analog currently entering clinical trials for hypertension. Previous reports demonstrate that this analog MANP activates the guanylyl cyclase A receptor (GC-A) and results in more potent biological actions compared with ANP; thus, it may represent a new therapeutic drug for hypertension. A major goal of this study was to establish that chronic subcutaneous delivery of MANP is feasible and hypotensive together with cGMP effects. We investigated the BP-lowering and cGMP-activating actions of acute and chronic subcutaneous delivery in normal and hypertensive rats. Furthermore, we explored vascular mechanisms of MANP in human aortic smooth muscle cells (HASMC) and ex vivo in isolated arteries. In normal rats with a single subcutaneous injection, MANP promoted robust dose-dependent BP-lowering actions and natriuresis, together with cGMP activation. Most importantly in hypertensive rats, once-a-day subcutaneous injection of MANP for 7 days induced cGMP elevation and long-term BP reduction compared with vehicle. Mechanistically, in HASMC, MANP activated cGMP and attenuated angiotensin II-mediated increases in intracellular Ca 2+ levels while directly vasorelaxing arterial rings. Our study demonstrates for the first time the effectiveness of subcutaneous administration of MANP for 7 days and provides innovative, vascular mechanisms of BP regulation supporting its continued development as a novel therapeutic for hypertension.

Determining the requirements for efficient oxygen (O 2 ) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O 2 ) and a sequential mechanism (cofactor binding and reaction with O 2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the Fe II ), αKG binding to Fe II -DAOCS results in ∼45% five-coordinate sites that selectively react with O 2 relative to the remaining six-coordinate sites. However, this reaction produces an Fe III species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to Fe II -DAOCS produces five-coordinate sites that rapidly react with O 2 to form an Fe IV =O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes., Competing Interests: The authors declare no competing interest.

Background: In heart failure with reduced ejection fraction (HFrEF), elevated soluble neprilysin (sNEP) levels are associated with an increased risk of cardiovascular death, and its inhibition with sacubitril/valsartan has improved survival., Objectives: This study sought to determine the relevance of sNEP as a biomarker in heart failure with preserved ejection fraction (HFpEF) and to compare circulating sNEP levels in patients with HFpEF with normal controls., Methods: A case-control study was performed in 242 symptomatic patients with HFpEF previously enrolled in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure with Preserved Ejection Fraction (RELAX) and Nitrates's Effect on Activity Tolerance in Heart Failure With Preserved Ejection (NEAT-HFpEF) clinical trials and 891 asymptomatic subjects without HF or diastolic dysfunction (confirmed by NT-proBNP levels <200 pg/ml and echocardiography) who were enrolled in the Prevalence of Asymptomatic Left Ventricular Dysfunction study. sNEP was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) in all subjects., Results: Overall, sNEP levels were lower in HFpEF compared with controls (3.5 ng/ml; confidence interval [CI]: 2.5 to 4.8 vs. 8.5 ng/ml; CI: 7.2 to 10.0; p < 0.001). After adjusting for age, gender, body mass index (BMI), and smoking history, mean sNEP levels were also lower in HFpEF compared with controls (4.0 ng/ml [CI: 2.7 to 5.4] vs. 8.2 ng/ml [CI: 6.8 to 9.7]; p = 0.002). The cohorts were propensity matched based on age, BMI, diabetes, hypertension, smoking history, and renal function, and sNEP levels remained lower in HFpEF compared with controls (median 2.4 ng/ml [interquartile range: 0.6 to 27.7] vs. 4.9 ng/ml [interquartile range: 1.2 to 42.2]; p = 0.02)., Conclusions: Patients with HFpEF on average have significantly lower circulating sNEP levels compared with controls. These findings challenge our current understanding of the complex biology of circulating sNEP in HFpEF., (Copyright © 2020 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, β-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid β-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid β-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.

Atrial natriuretic peptide (ANP) is a peptide hormone that in response to atrial stretch is secreted from atrial myocytes into the circulation, where it stimulates vasodilatation and natriuresis. ANP is an important biomarker of heart failure where low plasma concentrations exclude cardiac dysfunction. ANP is a member of the natriuretic peptide (NP) family, which also includes the B-type natriuretic peptide (BNP) and the C-type natriuretic peptide. The proforms of these hormones undergo processing to mature peptides, and for proBNP, this process has previously been demonstrated to be regulated by O- glycosylation. It has been suggested that proANP also may undergo post-translational modifications. Here, we conducted a targeted O- glycoproteomics approach to characterize O- glycans on NPs and demonstrate that all NP members can carry O- glycans. We identified four O- glycosites in proANP in the porcine heart, and surprisingly, two of these were located on the mature bioactive ANP itself. We found that one of these glycans is located within a conserved sequence motif of the receptor-binding region, suggesting that O -glycans may serve a function beyond intracellular processing and maturation. We also identified an O -glycoform of proANP naturally occurring in human circulation. We demonstrated that site-specific O -glycosylation shields bioactive ANP from proteolytic degradation and modifies potency at its cognate receptor in vitro Furthermore, we showed that ANP O -glycosylation attenuates acute renal and cardiovascular ANP actions in vivo The discovery of novel glycosylated ANP proteoforms reported here significantly improves our understanding of cardiac endocrinology and provides important insight into the etiology of heart failure., (© 2019 Hansen et al.)

Background Neprilysin is a metalloprotease involved in proteolysis of numerous peptides, including natriuretic peptides, and is of prognostic and therapeutic importance in heart failure with reduced ejection fraction. No studies have investigated circulating neprilysin in the community, its clinical correlates, or its relationship to cardiovascular disease in the general population. Methods and Results Plasma neprilysin was measured in 1536 participants from Olmsted County, Minnesota, using a commercially available sandwich ELISA assay. Clinical and echocardiographic correlates and subsequent outcomes were determined. Soluble neprilysin is non-normally distributed in the community (median: 3.9 ng/mL; interquartile range: 1.0-43.0 ng/mL). There was no relationship between plasma neprilysin and age (Spearman correlation: -0.04, P=0.16); body mass index (Spearman correlation: -0.04, P=0.16); glomerular filtration rate (Spearman correlation: -0.007, P=0.8); or A-, B-, or C-type natriuretic peptides (Spearman correlation: 0.03, P=0.22; -0.001, P=0.96; 0.01, P=0.67, respectively). Among tertiles of neprilysin, the lowest tertile group had the highest prevalence of smokers (P<0.001), hypertension (P=0.04), dyslipidemia (P=0.03), and diastolic dysfunction (P=0.02). Soluble neprilysin was not prospectively associated with death or heart failure over a median of 10.7 years. Conclusions In a large community-based cohort, for the first time, we described the distribution of circulating neprilysin in the general community. We observed that neprilysin does not correlate with natriuretic peptide levels and is not independently associated with adverse outcomes. The novel associations observed between low soluble neprilysin levels and an adverse cardiometabolic and smoking profile requires further investigation.

Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin ( mdx ), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.

Rationale: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential., Objective: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI., Methods and Results: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca 2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels., Conclusions: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.