Matriglycan maintains t-tubule structural integrity in cardiac muscle.

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites.

Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine deacylases regulating metabolism and stress responses; however, characterization of the removed acyl groups and their downstream metabolic fates remains incomplete. Here we employed untargeted comparative metabolomics to reinvestigate mitochondrial sirtuin biochemistry. First, we identified N-glutarylspermidines as metabolites downstream of the mitochondrial sirtuin SIR-2.3 in Caenorhabditis elegans and demonstrated that SIR-2.3 functions as a lysine deglutarylase and that N-glutarylspermidines can be derived from O-glutaryl-ADP-ribose. Subsequent targeted analysis of C. elegans, mouse and human metabolomes revealed a chemically diverse range of N-acylspermidines, and formation of N-succinylspermidines and/or N-glutarylspermidines was observed downstream of mammalian mitochondrial sirtuin SIRT5 in two cell lines, consistent with annotated functions of SIRT5. Finally, N-glutarylspermidines were found to adversely affect C. elegans lifespan and mammalian cell proliferation. Our results indicate that N-acylspermidines are conserved metabolites downstream of mitochondrial sirtuins that facilitate annotation of sirtuin enzymatic activities in vivo and may contribute to sirtuin-dependent phenotypes.

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

Long-chain fatty acyl coenzyme A inhibits NME1/2 and regulates cancer metastasis.

SignificanceThe study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.

IGF2BP2 promotes cancer progression by degrading the RNA transcript encoding a v-ATPase subunit.

IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

Simultaneous creatine and phosphocreatine mapping of skeletal muscle by CEST MRI at 3T.

PURPOSE: To confirm that CrCEST in muscle exhibits a slow-exchanging process, and to obtain high-resolution amide, creatine (Cr), and phosphocreatine (PCr) maps of skeletal muscle using a POlynomial and Lorentzian Line-shape Fitting (PLOF) CEST at 3T. METHODS: We used dynamic changes in PCr/CrCEST of mouse hindlimb before and after euthanasia to assign the Cr and PCr CEST peaks in the Z-spectrum at 3T and to obtain the optimum saturation parameters. Segmented 3D EPI was employed to obtain multi-slice amide, PCr, and Cr CEST maps of human skeletal muscle. Subsequently, the PCrCEST maps were calibrated using the PCr concentrations determined by 31 P MRS. RESULTS: A comparison of the Z-spectra in mouse hindlimb before and after euthanasia indicated that CrCEST is a slow-exchanging process in muscle (<150.7 s-1 ). This allowed us to simultaneously extract PCr/CrCEST signals at 3T using the PLOF method. We determined optimal B1 values ranging from 0.3 to 0.6 µT for CrCEST in muscle and 0.3-1.2 µT for PCrCEST. For the study on human calf muscle, we determined an optimum saturation time of 2 s for both PCr/CrCEST (B1 = 0.6 µT). The PCr/CrCEST using 3D EPI were found to be comparable to those obtained using turbo spin echo (TSE). (3D EPI/TSE PCr: (2.6 ± 0.3) %/(2.3 ± 0.1) %; Cr: (1.3 ± 0.1) %/(1.4 ± 0.07) %). CONCLUSIONS: Our study showed that in vivo CrCEST is a slow-exchanging process. Hence, amide, Cr, and PCr CEST in the skeletal muscle can be mapped simultaneously at 3T by PLOF CEST.

Creatine mapping of the brain at 3T by CEST MRI.

PURPOSE: To assess the feasibility of CEST-based creatine (Cr) mapping in brain at 3T using the guanidino (Guan) proton resonance. METHODS: Wild type and knockout mice with guanidinoacetate N-methyltransferase deficiency and low Cr and phosphocreatine (PCr) concentrations in the brain were used to assign the Cr and protein-based arginine contributions to the GuanCEST signal at 2.0 ppm. To quantify the Cr proton exchange rate, two-step Bloch-McConnell fitting was used to fit the extracted CrCEST line-shape and multi-B1 Z-spectral data. The pH response of GuanCEST was simulated to demonstrate its potential for pH mapping. RESULTS: Brain Z-spectra of wild type and guanidinoacetate N-methyltransferase deficiency mice show a clear Guan proton peak at 2.0 ppm at 3T. The CrCEST signal contributes ∼23% to the GuanCEST signal at B1 = 0.8 µT, where a maximum CrCEST effect of 0.007 was detected. An exchange rate range of 200-300 s-1 was estimated for the Cr Guan protons. As revealed by the simulation, an elevated GuanCEST in the brain is observed when B1 is less than 0.4 µT at 3T, when intracellular pH reduces by 0.2. Conversely, the GuanCEST decreases when B1 is greater than 0.4 µT with the same pH drop. CONCLUSIONS: CrCEST mapping is possible at 3T, which has potential for detecting intracellular pH and Cr concentration in brain.

Pre-clinical evaluation of biomarkers for the early detection of nephrotoxicity following alpha-particle radioligand therapy.

PURPOSE: Cancer treatment with alpha-emitter-based radioligand therapies (α-RLTs) demonstrates promising tumor responses. Radiolabeled peptides are filtered through glomeruli, followed by potential reabsorption of a fraction by proximal tubules, which may cause acute kidney injury (AKI) and chronic kidney disease (CKD). Because tubular cells are considered the primary site of radiopeptides' renal reabsorption and potential injury, the current use of kidney biomarkers of glomerular functional loss limits the evaluation of possible nephrotoxicity and its early detection. This study aimed to investigate whether urinary secretion of tubular injury biomarkers could be used as an additional non-invasive sensitive diagnostic tool to identify unrecognizable tubular damage and risk of long-term α-RLT nephrotoxicity. METHODS: A bifunctional cyclic peptide, melanocortin 1 ligand (MC1L), labeled with [203Pb]Pb-MC1L, was used for [212Pb]Pb-MC1L biodistribution and absorbed dose measurements in CD-1 Elite mice. Mice were treated with [212Pb]Pb-MC1L in a dose-escalation study up to levels of radioactivity intended to induce kidney injury. The approach enabled prospective kidney functional and injury biomarker evaluation and late kidney histological analysis to validate these biomarkers. RESULTS: Biodistribution analysis identified [212Pb]Pb-MC1L reabsorption in kidneys with a dose deposition of 2.8, 8.9, and 20 Gy for 0.9, 3.0, and 6.7 MBq injected [212Pb]Pb-MC1L doses, respectively. As expected, mice receiving 6.7 MBq had significant weight loss and CKD evidence based on serum creatinine, cystatin C, and kidney histological alterations 28 weeks after treatment. A dose-dependent urinary neutrophil gelatinase-associated lipocalin (NGAL, tubular injury biomarker) urinary excretion the day after [212Pb]Pb-MC1L treatment highly correlated with the severity of late tubulointerstitial injury and histological findings. CONCLUSION: Urine NGAL secretion could be a potential early diagnostic tool to identify unrecognized tubular damage and predict long-term α-RLT-related nephrotoxicity.

Gene Therapy With the N-Terminus of Junctophilin-2 Improves Heart Failure in Mice.

BACKGROUND: Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. METHODS: We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NTΔbNLS/ARR) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. RESULTS: Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. CONCLUSIONS: Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.

Mitochondrial Creatine Kinase Attenuates Pathologic Remodeling in Heart Failure.

BACKGROUND: Abnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF. METHODS: First, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice. RESULTS: In people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling. CONCLUSIONS: In the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

Validating the Reliability and Clinical Relevance of a Nasolabial Fold Photonumeric Scale.

BACKGROUND: The use of tissue fillers to treat age-related deepening of the nasolabial fold (NLF) has increased and become the standard clinical approach, creating a need for evidence-based, objective evaluation for pre- and post-procedure assessment of the NLF. METHODS: A 5-point rating scale was developed to assess the NLF, specifically the presence of depression and shadowing. Live validation of the scale was performed with a total of 73 participants representing the full range of NLF severities. Physicians board-certified in a core aesthetic specialty (3 trained raters) performed the scale validation over 2 rounds, 2 weeks apart. Training was carried out, and test-retest reliability was quantitated through the determination of intra- and inter-rater reliability by percentage of agreement, weighted kappa statistic with 95% confidence interval (CI), and intraclass correlation coefficient with 95% CI. To evaluate the clinical relevance of a 1-grade difference, rater assessments of 90 photo pairs were compared with previous designations of clinically different or not clinically different. RESULTS: The NLF scale achieved near-perfect intra- and inter-rater reliability when utilized by trained raters to assess a diverse group of live participants. Furthermore, clinically relevant differences between grades were established, and a 1-point difference was detectable by trained evaluators using the NLF scale. CONCLUSION: The clinically relevant and highly reliable validated NLF scale provides a standardized grading system with a user-friendly design for objectively assessing NLF in clinical practice and as a research tool for clinical approval studies of new aesthetic products and technologies. J Drugs Dermatol. 2024;23(1):1284-1291.   doi:10.36849/JDD.7316.

Incipient genome erosion and metabolic streamlining for antibiotic production in a defensive symbiont.

Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but "Streptomyces philanthi" also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that-compared to in vitro symbiont cultures-"S. philanthi" in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host-symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.

dATP elevation induces myocardial metabolic remodeling to support improved cardiac function.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Core transcriptional networks in Williams syndrome: IGF1-PI3K-AKT-mTOR, MAPK and actin signaling at the synapse echo autism.

Gene networks for disorders of social behavior provide the mechanisms critical for identifying therapeutic targets and biomarkers. Large behavioral phenotypic effects of small human deletions make the positive sociality of Williams syndrome (WS) ideal for determining transcriptional networks for social dysfunction currently based on DNA variations for disorders such as autistic spectrum disorder (ASD) and schizophrenia (SCHZ). Consensus on WS networks has been elusive due to the need for larger cohort size, sensitive genome-wide detection and analytic tools. We report a core set of WS network perturbations in a cohort of 58 individuals (34 with typical, 6 atypical deletions and 18 controls). Genome-wide exon-level expression arrays robustly detected changes in differentially expressed gene (DEG) transcripts from WS deleted genes that ranked in the top 11 of 12 122 transcripts, validated by quantitative reverse transcription PCR, RNASeq and western blots. WS DEG's were strictly dosed in the full but not the atypical deletions that revealed a breakpoint position effect on non-deleted CLIP2, a caveat for current phenotypic mapping based on copy number variants. Network analyses tested the top WS DEG's role in the dendritic spine, employing GeneMANIA to harmonize WS DEGs with comparable query gene-sets. The results indicate perturbed actin cytoskeletal signaling analogous to the excitatory dendritic spines. Independent protein-protein interaction analyses of top WS DEGs generated a 100-node graph annotated topologically revealing three interacting pathways, MAPK, IGF1-PI3K-AKT-mTOR/insulin and actin signaling at the synapse. The results indicate striking similarity of WS transcriptional networks to genome-wide association study-based ASD and SCHZ risk suggesting common network dysfunction for these disorders of divergent sociality.

Contribution of skeletal muscle-specific microRNA-133b to insulin resistance in heart failure.

Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15, the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF.NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.

Coronary artery endothelial function and aging in people with HIV and HIV-negative individuals.

Coronary artery disease (CAD) is a common comorbidity in people with human immunodeficiency virus (HIV) (PWH) and impaired coronary endothelial function (CEF) plays a central role in the pathogenesis of CAD. Age-related impaired CEF among PWH, however, is not well characterized. We investigated the association between CEF and age in males and females with and without HIV using 3-T magnetic resonance imaging (MRI). We measured the changes in coronary cross-sectional area (CSA) and coronary blood flow during isometric handgrip exercise (IHE), an established endothelial-dependent stressor with smaller increases in CSA and coronary blood flow indicative of impaired CEF. We included 106 PWH and 82 individuals without HIV. Differences in demographic and clinical characteristics between PWH and individuals without HIV were explored using Pearson's χ2 test for categorical variables and Welch's t test for continuous variables. Linear regression models were used to examine the association between CEF and age. CEF was significantly lower in PWH as compared with individuals without HIV. Coronary endothelial dysfunction was also present at younger ages in PWH than in the individuals without HIV and there were significant differences in CEF between the PWH and individuals without HIV across age groups. Among the individuals without HIV, the percent changes in CSA were inversely related to age in unadjusted and adjusted models. There was no significant association between CEF and age in PWH. To the best of our knowledge, this is the first study to examine the relationship between age and CEF in PWH, and our results suggest that factors other than age significantly impair CEF in PWH across the life span.NEW & NOTEWORTHY This is the first study to examine the relationship between age and coronary endothelial function (CEF) in people with human immunodeficiency virus (HIV) (PWH). CEF was assessed using magnetic resonance imaging (MRI) in people with and without HIV. Although age and CEF were significantly inversely related in individuals without HIV, there was no association between age and CEF in PWH.

The exchange rate of creatine CEST in mouse brain.

PURPOSE: To estimate the exchange rate of creatine (Cr) CEST and to evaluate the pH sensitivity of guanidinium (Guan) CEST in the mouse brain. METHODS: Polynomial and Lorentzian line-shape fitting (PLOF) were implemented to extract the amine, amide, and Guan CEST signals from the brain Z-spectrum at 11.7T. Wild-type (WT) and knockout mice with the guanidinoacetate N-methyltransferase deficiency (GAMT-/- ) that have low Cr and phosphocreatine (PCr) concentrations in the brain were used to extract the CrCEST signal. To quantify the CrCEST exchange rate, a two-step Bloch-McConnell (BM) fitting was used to fit the CrCEST line-shape, B1 -dependent CrCEST, and the pH response with different B1 values. The pH in the brain cells was altered by hypercapnia to measure the pH sensitivity of GuanCEST. RESULTS: Comparison between the Z-spectra of WT and GAMT-/- mice suggest that the CrCEST is between 20% and 25% of the GuanCEST in the Z-spectrum at 1.95 ppm between B1 = 0.8 and 2 µT. The CrCEST exchange rate was found to be around 240-480 s-1 in the mouse brain, which is significantly lower than that in solutions (∼1000 s-1 ). The hypercapnia study on the mouse brain revealed that CrCEST at B1 = 2 µT and amineCEST at B1 = 0.8 µT are highly sensitive to pH change in the WT mouse brain. CONCLUSIONS: The in vivo CrCEST exchange rate is slow, and the acquisition parameters for the CrCEST should be adjusted accordingly. CrCEST is the major contribution to the opposite pH-dependence of GuanCEST signal under different conditions of B1 in the brain.

A Potent Auto-Umpolung Ligand for Conjugative Radical Stabilization.

Carbenes with conjugatively connected redox system act as "auto-umpolung" ligands. Due to their electronic flexibility, they should also be particularly suitable to stabilize open-shell species. Herein, the first neutral radical of such sort is described in form of a dialkylamino-substituted bis(dicyanomethylene)cyclopropanide. Despite the absence of steric shielding, the radical is stable for an extended amount of time and was consequently characterized in solution via EPR measurements. These data and accompanying X-ray structural analyses indicate that the radical species is in equilibrium with aggregates (formed via π-stacking) and dimers (obtained via σ-bond formation between methylene carbons).