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Biology subjects

Russo, R.

Publications and source records attributed to Russo, R..

13 recordsLinked to original sources

TNFR2 Agonism as a Sex-Specific Therapy for Novel Osteoarthritis-Induced Cardiac Dysfunction

Osteoarthritis (OA), a degenerative joint disease, is associated with increased systemic inflammation, chronic pain, and cardiovascular dysfunction. Epidemiological evidence establishes that OA increases the risk of cardiovascular disease (CVD) threefold, yet the causal role of OAs contributions remains underexamined. We assessed cardiac function longitudinally following destabilization of the medial meniscus (DMM) surgery to induce osteoarthritis in mice. DMM-mice exhibited significant, sexually dimorphic alterations in echocardiographic parameters. Female DMM mice developed impaired relaxation with altered E/A ratios, increased E/e ratios, and prolonged intraventricular relaxation time with no change in ejection fraction, while male DMM mice showed progressive systolic dysfunction with decreasing ejection fraction, increased E/e ratio, and prolonged intraventricular contraction time. Transcriptomic profiles and biochemical analyses demonstrated divergent cellular responses involving fibrosis and oxidative stress in female mice, whereas autophagic and apoptotic responses were observed in male mice. Using a tumor necrosis factor 2 (TNFR2) agonist shown to reduce systemic inflammation, we investigated its potential therapeutic role in the context of OA-induced cardiovascular dysfunction. TNFR2 agonism proved to be effective both prophylactically and therapeutically for female diastolic dysfunction. While prophylactic and therapeutic administration delayed male systolic dysfunction, the efficacy declined over time. Our findings demonstrate evidence of a novel sexually dimorphic model of OA-induced CVD that recapitulates the sexually dimorphic pattern of patient phenotypes and a promising new therapeutic approach to CVD. Translational RelevanceOsteoarthritis patients have higher, often unrecognized, cardiovascular risk, yet preclinical models linking joint disease to cardiac dysfunction remain unexplored. Using a murine preclinical model of OA reveals the key findings. First, OA alone drives sex-specific cardiac phenotypes - females develop diastolic dysfunction, whereas males develop progressive systolic impairment. Second, selective TNFR2 agonism prevents and reverses OA-induced diastolic dysfunction in female mice and delays systolic decline in males. These findings suggest sex-dependent cardiac monitoring in OA patients and indicate that TNFR2-targeted therapy will likely be a sex-informed intervention to provide cardioprotective benefit. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/736778v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@819a80org.highwire.dtl.DTLVardef@c6646eorg.highwire.dtl.DTLVardef@eb86aborg.highwire.dtl.DTLVardef@d68292_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

Chiral shift toward D-serine reflects intrathecal inflammation in multiple sclerosis and counteracts motor impairment in a murine model

Multiple sclerosis (MS) is characterized by chronic inflammatory demyelination involving complex interplay between the central nervous and immune systems. Neuroinflammation triggers cellular reorganization requiring O_SCPLOWLC_SCPLOW-serine for sustained syntheses of membrane lipids and nucleic acids, whereas it causes aberrant glutamatergic neurotransmission involving O_SCPLOWDC_SCPLOW-serine. However, significance of serine metabolism in MS pathology remains unexplored. Here we show that serine chiral homeostasis is disrupted in MS and endogenous O_SCPLOWDC_SCPLOW-serine prevents motor deficits caused by inflammatory demyelination. We found in a large cohort study that patients with MS exhibit elevated O_SCPLOWDC_SCPLOW-serine levels and the O_SCPLOWDC_SCPLOW-/total serine ratio in the cerebrospinal fluid at diagnosis. Steric deviation toward O_SCPLOWDC_SCPLOW-serine accords with emergence of the intrathecal inflammatory marker oligoclonal bands, and correlates negatively with proinflammatory cytokines. An in vivo animal model of MS, genetically engineered to exhibit distinct metabolic states of O_SCPLOWDC_SCPLOW-serine, revealed that endogenous O_SCPLOWDC_SCPLOW-serine synthesis mitigates the progression of motor deficits and suppresses proinflammatory and vascular endothelial pathogenic signaling. Moreover, pre-symptomatic oral supplementation with O_SCPLOWDC_SCPLOW-serine, but not O_SCPLOWLC_SCPLOW-serine, enhances production of extracellular matrices, preserves integrity of the blood brain barrier, attenuates demyelination, and improves motor function. Contrary to the previously recognized neurotoxic nature of O_SCPLOWDC_SCPLOW-serine, our findings reveal an unrecognized significance of O_SCPLOWDC_SCPLOW-serine metabolism in MS and a protective function of O_SCPLOWDC_SCPLOW-serine against neuroinflammation involving disruption of the blood brain barrier, which may present an untapped therapeutic target in MS. One Sentence SummarySerine chiral homeostasis is disturbed in multiple sclerosis and O_SCPLOWDC_SCPLOW-serine mitigates inflammatory demyelination.

neuroscience↗

Tandem Mass Tag-Based High-Resolution LC-MS/MS identifies free D-aspartate-induced expression of proteins linked to schizophrenia and autism spectrum disorder

D-aspartate is an endogenous agonist of NMDA and mGlu5 receptors, with a distinctive spatiotemporal expression profile that peaks in the prenatal and early postnatal brain. This suggests a critical role for D-aspartate metabolism in modulating neurodevelopmental processes linked to glutamatergic neurotransmission. However, the precise mechanisms through which D-aspartate exerts its effects remain unclear. To elucidate the molecular pathways orchestrated by early D-aspartate signalling, we employed a knockin mouse model characterized by constitutive D-aspartate depletion due to the prenatal expression of its degradative enzyme, D-aspartate oxidase. Using an advanced quantitative proteomic approach based on Tandem Mass Tag isobaric labelling and nano-liquid chromatography coupled with high-resolution tandem mass spectrometry, we investigated the proteomic variations induced by D-aspartate depletion during postnatal brain development comparing Ddo knockin mice with their wild-type littermates. Our findings reveal that D-aspartate modulates the neonatal expression of proteins involved in glutamatergic neurotransmission, nervous system development, and cytoskeleton organization. Moreover, proteomic analysis identified a subset of D-aspartate-regulated proteins mapping molecular pathways associated with autism spectrum disorder and schizophrenia. These findings offer new perspectives on the complex protein networks influenced by D-aspartate metabolism in the developing brain and highlight its potential impact on cerebral function in health and psychiatric disorders.

biochemistry↗

Structural bases for Nuclear Factor 1-X activation and DNA recognition. Prototypic insight into the NFI transcription factor family

Nuclear Factor I (NFI) proteins were first identified in adenovirus DNA replication and later as regulators of gene transcription, stem cell proliferation, and differentiation. They play key roles in development, cancer and congenital disorders. Within the NFI family, NFI-X is critical for neural stem cell biology, hematopoiesis, muscle development, muscular dystrophies and oncogenesis. Here, we present the first structural characterization of the NFI transcription factor, NFI-X, both alone and bound to its consensus palindromic DNA site. Our analyses reveal a novel, MH1-like fold within NFI-X DNA-binding domain (DBD) and identify crucial structural determinants for activity, such as a Zn{superscript 2} binding site, dimeric assembly, activation mechanism and DNA-binding specificity. Given the >95% sequence identity within the NFI DBDs, our structural data are prototypic for the entire family; a NFI Rosetta Stone that allows decoding a wealth of biochemical and functional data and provides a precise target for drug design in a wider disease context.

biochemistry↗

A conformational fingerprint for amyloidogenic light chains.

Immunoglobulin light chain amyloidosis (AL) and multiple myeloma (MM) both share the overproduction of a clonal light chain (LC). However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics (MD) simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

biophysics↗

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu

The estrogen receptor- (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations --receptor conformational heterodimers--mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. SignificanceThe estrogen receptor- (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

biophysics↗

High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653nm vs. 510nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, Ksv, were observed during the initial 40 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4 to 8, with a small ratio reduction for pH>10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6 to 0.8s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels < 5% within 3 -10 hours for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics in vitro for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.

bioengineering↗

Irreversible inhibition of TRF2TRFH recruiting functions: a strategy to induce telomeric replication stress in cancer cells.

The shelterin component telomeric repeat-binding factor 2 (TRF2) is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, resulting in a telomeric DNA damage response. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response in the absence of fusions, increased telomeric replication stress, and impaired recruitment of regulator of telomere elongation helicase 1 (RTEL1) and structure-specific endonuclease subunit (SLX4) to telomeres. We demonstrate that APOD53 impairs cell growth in both a telomerase-positive and an ALT cell line, while sparing the viability of non-cancerous cells. Finally, we find that co-treatment with APOD53 and the G4 stabilizer RHPS4 further exacerbates telomere replication stress.

cancer biology↗

Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

Rapid emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is one of the most significant healthcare challenges of our time. The cause of drug resistance is multifactorial, with the long course anti-tubercular therapy required to treat tuberculosis (TB) constituting a major contributing factor. Introduction of pyrazinamide (PZA) resulted in shortening of TB treatment from twelve to six months and consequently played a critical role in curbing drug resistance that developed over long course therapy. Nevertheless, because PZA is a prodrug activated by a nonessential amidase, PncA, resistance to PZA develops and frequently results in treatment failure. Here, we leveraged a whole cell drug screening approach to identify anti-tuberculars with unconventional mechanisms of action or activation that could be further developed into compounds effective at killing Mtb resistant to PZA. We discovered an amide containing prodrug, DG160, that was activated by the amidase, Rv2888c (AmiC). This amidase was capable of metabolizing a variety of amide containing compounds including a novel pyrazinoic acid-isoquinolin-1-amine prodrug, JSF-4302, which we developed as a potential PncA-independent replacement for PZA. As predicted, AmiC activation of JSF-4302 led to the generation of POA in Mtb including in a PZA resistant clinical isolate, thereby successfully delivering the active component of PZA while bypassing the need for activation by PncA. This work provides a framework for a new approach to drug development and prodrug activation in Mtb. SIGNIFICANCEPyrazinamide (PZA) is a vital component of Mycobacterium tuberculosis (Mtb) treatment since its inclusion shortened tuberculosis therapy by six months. However, PZA is a prodrug and resistance develops at a high frequency due to mutations in its activator PncA. Here, we present the discovery of amide-containing anti-tubercular prodrugs that are activated intracellularly by the Mtb amidase, AmiC. Taking advantage of this finding, we successfully designed and synthesized pyrazinoic acid (POA) prodrugs that were activated by AmiC and found that these compounds delivered intracellular POA to PZA- resistant Mtb isolates that contained a nonfunctional PncA. This new approach to prodrug development provides a method for delivering conjugated drugs into Mtb with the potential to overcome clinical drug resistance.

microbiology↗

A novel oral GyrB/ParE dual binding inhibitor effective against multidrug resistant Neisseria gonorrhoeae and other high-threat pathogens

Drug resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance, as well as limit development of new resistance. We describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6- fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2- oxa-8-azaspiro[4.5]decan-3-yl)methanol], which simultaneously binds to ATP binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae including drug-resistant strains. A phosphate prodrug JSF-2659 was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram positive organisms, and JSF-2659 was shown in a deep tissue model of VRSA and a model of C. difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

microbiology↗

TRPV1 in arteries enables a rapid myogenic tone

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly, reaching a plateau in minutes. In the heart and skeletal muscle, however, myogenic tone is rapid; activation occurs in tens of seconds and arterial constrictions or raised extravascular pressure as brief as 100 ms remove tone. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and the adipose. Here we reveal a critical role for TRPV1 in the myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these effects of TRPV1 antagonists were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells, or raised intravascular pressure in arteries (with or without endothelium), triggered Ca2+ signaling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the rapid vasodilation following brief constriction of arterioles was also dependent on TRPV1, consistent with a rapid deactivation or inactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates a phospholipase C/protein kinase C signaling pathway to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle.

physiology↗

Genetic analysis of the novel SARS-CoV-2 host receptor TMPRSS2 in different populations.

The infection coronavirus disease 2019 (COVID-19) is caused by a virus classified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At cellular level, virus infection initiates with binding of viral particles to the host surface cellular receptor angiotensin converting enzyme 2 (ACE2). SARS-CoV-2 engages ACE2 as the entry receptor and employs the cellular serine protease 2 (TMPRSS2) for S protein priming. TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host. Understanding how TMPRSS2 protein expression in the lung varies in the population could reveal important insights into differential susceptibility to influenza and coronavirus infections. Here, we systematically analyzed coding-region variants in TMPRSS2 and the eQTL variants, which may affect the gene expression, to compare the genomic characteristics of TMPRSS2 among different populations. Our findings suggest that the lung-specific eQTL variants may confer different susceptibility or response to SARS-CoV-2 infection from different populations under the similar conditions. In particular, we found that the eQTL variant rs35074065 is associated with high expression of TMPRSS2 but with a low expression of the interferon (IFN)-/{beta}-inducible gene, MX1, splicing isoform. Thus, these subjects could account for a more susceptibility either to viral infection or to a decrease in cellular antiviral response.

genetics↗

TRPV1 expressed throughout the arterial circulation regulates vasoconstriction

The capsaicin receptor, TRPV1, is a key ion channel involved in inflammatory pain signaling. Although mainly studied in sensory nerves, there are reports of TRPV1 expression in isolated segments of the vasculature, but whether the channel localizes to vascular endothelium or smooth muscle is controversial and the distribution and functional roles of TRPV1 in arteries remain unknown. We mapped functional TRPV1 expression throughout the mouse arterial circulation. Analysis of reporter mouse lines TRPV1PLAP-nlacZ and TRPV1-Cre:tdTomato combined with Ca2+ imaging revealed specific localization of TRPV1 to smooth muscle of terminal arterioles in the heart, fat and skeletal muscle. Capsaicin evoked inward currents and raised intracellular Ca2+ levels in arterial smooth muscle cells, constricted arterioles ex vivo and in vivo and increased systemic blood pressure in mice and rats. Further, capsaicin markedly and dose-dependently reduced coronary flow. Pharmacologic and/or genetic disruption of TRPV1 abolished all these effects of capsaicin as well as vasoconstriction triggered by lysophosphatidic acid, a bioactive lipid generated by platelets and atherogenic plaques. Notably, ablation of sensory nerves did not affect the responses to capsaicin revealing a vascular smooth muscle-restricted signaling mechanism. Moreover, unlike in sensory nerves, TRPV1 function in arteries was resistant to activity-induced desensitization. Thus, TRPV1 activation in vascular myocytes of resistance arterioles enables a persistent depolarizing current, leading to constriction of coronary, skeletal muscle, and adipose arterioles and a sustained increase in systemic blood pressure.

physiology↗