Biology subjects

Hinton, A.

Publications and source records attributed to Hinton, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Optimisation of Solid-Phase Antimicrobial Susceptibility Testing for Hydrophobic Antibiotics Using an Agarose-Based MIC Assay

The minimum inhibitory concentration (MIC) assay is the gold standard for evaluating antimicrobial activity1. However, conventional agar-based MIC methods often underestimate the potency of physicochemically complex compounds[1, 2]. Hydrophobic and adhesive molecules, such as lipoglycopeptide antibiotics, exhibit poor diffusion and non-specific binding to agar, leading to artificially elevated MIC values compared to broth-based methods[3]. This issue complicates accurate potency assessment and is particularly an issue when attempting resistance frequency (FOR) studies, which must be conducted on solid media. Here, we developed a modified miniaturised agar MIC assay using 1% agarose, 0.002% Tween 80-supplemented tryptic soy broth (TSB), and a 24-well plate format[4]. These modifications improved compound dispersion, reduced matrix interactions, and lowered compound requirements. The optimised assay was validated with vancomycin, oritavancin, and dalbavancin against Staphylococcus aureus ATCC 43300 (MRSA) and Streptococcus pneumoniae ATCC 700677. This efficient, cost-effective, high-throughput platform overcomes the limitations of traditional agar methods, enhancing reliability in evaluating challenging antimicrobials and supporting next-generation antibiotic development.

microbiology↗

bioRxiv2026

Senescence and DNA Damage-Induced Inflammation Drive Heart Failure with Preserved Ejection Fraction in Cardiovascular Kidney Metabolic Syndrome

IntroductionHeart failure with preserved ejection fraction (HFpEF) is strongly associated with cardiometabolic comorbidities, including obesity, diabetes, hypertension, chronic kidney disease and aging, yet the mechanistic contribution of cellular senescence to HFpEF pathogenesis remains poorly defined. Methods and ResultsTo model clinically relevant HFpEF, we subjected p16-3MR mice to a novel chronic "four-hit" cardiovascular-kidney-metabolic stress regimen (10 months of a high-fat diet, low-dose streptozotocin, L-NAME, and aging). These mice developed a robust HFpEF phenotype characterized by left ventricular hypertrophy, impaired diastolic function (reduced E'/A' and elevated E/E'), preserved ejection fraction, reduced -dP/dt, exercise intolerance, pulmonary congestion, and increased cardiac CD68 macrophage infiltration. Cardiac proteomics identified 821 proteins significantly altered by four-hit stress. Selective genetic ablation of p16 senescent cells using ganciclovir ameliorated HFpEF phenotypes, reduced cardiac p16 expression and inflammation, and normalized proteomic remodeling, without affecting body weight or glycemic status. Comparative network analysis of mouse and human HFpEF cardiac proteomes revealed highly concordant upstream regulatory networks, prominently involving cell-cycle control, DNA damage responses, and inflammatory signaling. Immunohistochemical analysis of human HFpEF cardiac biopsies confirmed increased p16, {gamma}H2AX, STING, IRF3, NF-{kappa}B p65, and CD68 macrophages, mirroring the murine findings. The 4-Hit mice also developed chronic diabetic kidney disease with increased kidney inflammation, both of which were attenuated by Senolytic therapy. Mechanistically, the cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) is activated in response to damaged DNA, which in turn activates the downstream immune responses, including NF-{kappa}B and interferons. Cross-species validation further demonstrated that combined metabolic stress impaired cardiac function and nephrocyte function in Drosophila. Cardiac and nephrocyte dysfunctions were independently rescued by cardiomyocyte-specific and nephrocyte-specific inhibition of the cGAS-STING pathway, respectively. In human iPSC-derived cardiomyocytes, irradiation and palmitate induced senescence, DNA damage sensing via ZBP1, and activation of the cGAS-STING-IRF3 signaling axis; ZBP1 knockdown or senolytic treatment suppressed this inflammatory axis. ConclusionsAcross mouse, human, fly, and human iPSC models, our findings identify DNA damage-driven senescence and ZBP1-cGAS-STING signaling as conserved, causal mechanisms linking cardiovascular-kidney-metabolic comorbidities to HFpEF, highlighting senescence and innate immune pathways as promising therapeutic targets.

pathology↗

bioRxiv2026

Predicting Antibody Self-Association with Sequence Structure Fusion Models: The Central Role of CSI-BLI in Early Developability Screening

Antibody-based biologics are expanding rapidly, yet challenges in development from self-association, high viscosity, aggregation, and unfavorable clearance underscore the need for accurate in silico screening. Clone self-interaction biolayer interferometry (CSI-BLI) is a plate-based, low-material assay of weak, reversible self-association that serves as an early proxy for high-concentration viscosity and a complementary predictor of in vivo clearance. In a 246-mAb panel, CSI-BLI moderately correlates with viscosity; further, in hFcRn Tg32 mice (41 antibodies), CSI-BLI strongly associates with clearance. Here, we present an end-to-end framework that distinguishes high versus low self-interacting clones (CSI-BLI class) by coupling a fine-tuned protein language model (ESM-2) with residue-aligned 3D context from AlphaFold-predicted structures encoded as residue graphs. Disentangled multi-stream attention fuses sequence content, chain-aware positional information, and structural signals to capture spatially proximate interactions that are distant in sequence. Edit-distance-controlled splits across 1499 IgGs and 988 VHHs assess generalization. The structure-aware model achieves the highest hold-out performance (VHH-Fc F1 = 0.76; IgG F1 = 0.57), while a sequence-only disentangled variant outperforms a standard PLM baseline without structural inputs. Complementary biophysical feature-based models, built from AlphaFold structures and sequence/structure-derived physicochemical descriptors with cluster-aware selection, deliver robust, interpretable performance (VHH F1 = 0.72; IgG F1 = 0.57), with SHAP analyses highlighting charge/dipole, hydrophobicity, and aggregation-propensity drivers across CDRs and frameworks. This interaction-aware sequence-structure framework, supported by interpretable feature models, is extensible to other developability endpoints and broader protein classification tasks where joint modeling of language-derived representations and residue-level geometry is advantageous.

bioinformatics↗

bioRxiv2026

ATF4 Coordinates Transcriptomic and Structural Adaptations in Aging Muscle

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

cell biology↗

bioRxiv2026

Stress-Encoded Mitochondrial Plasticity: ATF4 Control of Mega-Mitochondria and Nanotunnel Communication

Mitochondrial structural plasticity is a critical adaptive response to cellular stress, yet the transcriptional networks governing the formation of specialized mitochondrial architectures remain poorly defined. Here, we identified and demonstrated that activating transcription factor 4 (ATF4), the master regulator of the integrated stress response, directly regulates mitochondrial morphological remodeling through a novel ATF4-NRF1/Nrf2-MFN2 signaling axis. Using serial block-face scanning electron microscopy and three-dimensional reconstruction in Drosophila flight muscle, primary myotubes, and human skeletal muscle, we show that overexpression of ATF4 promotes significant mitochondrial elongation, increased cristae concentration, enhanced mitochondrial-endoplasmic reticulum contact site (MERC) formation, and the initiation of Mitochondrial Nanotunnels. In contrast, loss of ATF4 results in mitochondrial fragmentation and impaired aerobic capacity. Chromatin immunoprecipitation sequencing reveals direct ATF4 binding at the promoters of the genes encoding NRF1 and Nrf2, which in turn regulate MFN2 expression. Small-molecule inhibition studies further establish that activation of this hierarchical pathway is both necessary and sufficient for stress-induced mitochondrial structural adaptation. Together, these findings position ATF4 as a master regulator of mitochondrial architectural plasticity, providing a direct mechanistic link between cellular stress signaling and organelle remodeling.

cell biology↗

bioRxiv2026

Derlin-mediated ERAD of lipid regulator ORMDL3 safeguards mitochondrial function

Mammalian Derlin proteins (Derlin-1, Derlin-2, and Derlin-3) are conserved components of the endoplasmic reticulum-associated degradation (ERAD) machinery that mediate the retrotranslocation and proteasomal degradation of misfolded ER-resident proteins. However, their paralog-specific contributions to cellular homeostasis remain poorly understood. Here, we show that Derlin deficiency disrupts mitochondrial architecture and results in mitochondrial fragmentation and tightening of ER-mitochondria contact sites (MERCs) in HEK293 cells. Mechanistically, we identify ORMDL proteins, evolutionarily conserved negative regulators of sphingolipid biosynthesis, as substrates of Derlin-2- and Derlin-3-dependent ERAD. Derlin deficiency leads to selective accumulation of ORMDL3 and its dose-dependent enrichment at MERCs, where it drives mitochondrial dysfunction in respiration and calcium handling. Reducing ORMDL3 levels restores mitochondrial function, establishing ORMDL3 as a key effector downstream of Derlin loss. Our work establishes ERAD as a critical mechanism of protein quantity control that safeguards organelle homeostasis by preventing aberrant accumulation and mislocalization of ER clients at inter-organelle contact sites. Given that ORMDL family members are central regulators of sphingolipid metabolism and are genetically linked to inflammation, cancer, asthma, inflammatory bowel disease, type 1 and type 2 diabetes, multiple sclerosis, obesity, and nonalcoholic fatty liver disease, these findings connect ERAD-dependent spatial control to sphingolipid homeostasis and a broad spectrum of human pathologies.

cell biology↗

bioRxiv2026

Intercellular Communication via Mitotic Nanotubes is Influenced by Connexin-43 Trafficking and Actin Remodeling

Gap junction communication is reduced during mitosis as the junction protein connexin-43 (Cx43) is redistributed from gap junction plaques on the plasma membrane to cytoplasmic annular vesicles and actin-based mitotic nanotubes that transiently connect mitotic cells to neighboring cells. However, the dynamic details of Cx43 redistribution during cell entry into and exit from mitosis, and the roles of mitotic nanotubes and associated Cx43 in intercellular communication, remain poorly understood. Here, using confocal live-cell imaging, we show that as cells enter mitosis, plaque-derived Cx43 structures are transferred to mitotic nanotubes. Over time, these structures fragment and migrate along the length of the nanotubes, either being transferred to the cytoplasm of adjacent cells or being positioned at the nanotube ends where they could potentially enable communication. Functionally, mitotic nanotubes indeed facilitate gap junction-dependent intercellular communication, though at reduced rates compared interphase cells. Interestingly, knockdown of Cx43 resulted in impaired nanotube formation and intercellular communication while inhibition of Rho kinase (ROCK) with Y-27632 prevented mitotic cell rounding and nanotube elongation, and increased cell-cell communication during mitosis, suggesting that nanotube function is influenced by Cx43 expression and trafficking as well as actin remodeling via ROCK. Overall, these findings provide valuable insights into the mechanisms that regulate Cx43 and mitotic nanotube dynamics and reveal a novel role for mitotic nanotubes in facilitating cell-cell communication during cell division.

cell biology↗

bioRxiv2025

A zebrafish seizure model of cblX syndrome reveals a dose-dependent refractory response to mTor inhibition.

Mutations in the transcriptional cofactor HCFC1 cause methylmalonic aciduria and homocystinemia, cblX type (cblX) (MIM#309541), non-syndromic X-linked intellectual disability (XLID), and focal epilepsy. Zebrafish studies have revealed increased activation of the Akt/mTor signaling pathway after mutation of hcfc1a, one ortholog of HCFC1. mTOR hyperactivation is linked to seizures and its inhibition alleviates epilepsy in other preclinical models. We hypothesized that mTor overactivity in hcfc1a mutant zebrafish increases seizure susceptibility and/or severity. We employed a two-concentration model of the seizure inducing agent, pentylenetetrazol (PTZ), with or without pretreatment of the mTor inhibitor, torin1. Mutation of hcfc1a did not increase seizure susceptibility at sub-optimal concentrations of PTZ and the pharmaceutical inhibition of mTor reduced seizure severity when utilized at a dose of 250nM. Higher doses of torin1 treatment exacerbated seizure response in mutant larvae but not in wildtype siblings. These data suggest that in an hcfc1a deficient background, moderate inhibition of mTor signaling may partially alleviate seizure phenotypes, however, over-inhibition of the pathway causes a refractory response to PTZ. Collectively, we present a model that can be used to test dose response and for the development of combinatorial treatment approaches in a high throughput manner.

developmental biology↗

bioRxiv2024

Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

Pro-inflammatory cytokines are secreted via the classical pathway from secretory vesicles or the non-classical pathway via extracellular vesicles (EVs), that together, play critical roles in triple-negative breast cancer (TNBC) progression. Annexin A6 (AnxA6) is a Ca2+-dependent membrane-binding protein that in TNBC is implicated in cell growth and invasiveness. AnxA6 is associated with EVs, but whether it affects the secretion of proinflammatory cytokines and/or EVs remains to be fully elucidated. To assess if AnxA6 influences the secretion of cytokines and extracellular vesicles, we used cytokine arrays to analyze secreted factors in cleared culture supernatants from control AnxA6 expressing and AnxA6 downregulated MDA-MB-468 TNBC cells. This revealed the diminished secretion of monocyte chemoattractant protein 1 (MCP-1/CCL2), interleukin 8 (IL-8), dickkopf1 (DKK1), throbospondin-1 (TSP-1), and osteopontin (OPN) following AnxA6 downregulation. We also show that the secretion of small EVs is strongly reduced in AnxA6 downregulated cells and that upregulation of AnxA6 promoted the secretion of treatment was also associated with increased EVs associated Rab7, cholesterol, and MCP-1 levels. Moreover, cholesterol content in EVs was significantly higher in AnxA6-expressing cells than in AnxA6 downregulated cells and following chronic lapatinib induced upregulation of AnxA6. Mechanistically, we demonstrate that the secretion of MCP-1 and/or EVs is AnxA6 dependent and that this requires the translocation of AnxA6 to cellular membranes and its interaction with SNAP23. AnxA6 neutralizing antibodies strongly diminished the survival of AnxA6 low TNBC cells but had minimal effects on the survival of TNBC cells expressing relatively high levels of the protein. Together, these data suggest that AnxA6 facilitates the secretion of EVs and proinflammatory cytokines that may be critical for TNBC progression.

cancer biology↗

bioRxiv2024

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo, LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

cell biology↗

bioRxiv2024

Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments

Mitochondria are hubs of metabolism and signaling, playing crucial roles in tumorigenesis, therapeutic resistance, and metastasis in many types of cancer. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the full extent of the complexity of the mitochondrial network, nor its regulation upon exposure to therapeutic stressors. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Herein we present the first three-dimensional analysis of mitochondrial networks in human tumor tissues. Use of two experimentally tractable orthotopic patient-derived xenograft (PDX) models TNBC enabled us to conduct longitudinal analyses to construct mitochondrial networks in treatment-naive and residual tumors persisting after exposure to a variety of conventional chemotherapies. Further, we modeled lipid droplet (LD) structures and their physical contacts with mitochondria. In total, we reconstructed 3,750 mitochondria and 800 LDs in three dimensions using serial block-face scanning electron microscopy (SBF-SEM), providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Both carboplatin (CRB) and docetaxel (DTX) chemotherapies produced residual tumors that harbored mitochondria with significantly increased areas, volumes, and perimeters in both PDX models. Additionally, treatment with the conventional combinations DTX plus CRB or Adriamycin plus cyclophosphamide (AC), led to reduced mitochondrial branching and elongation. In contrast, DTX or CRB alone elicited model-specific changes in mitochondrial complexity. Further, the extensive intra-tumoral heterogeneity of mitochondrial structure in untreated PDX tumors significantly decreased in residual tumors. Analyses of LDs revealed significant and consistent elevation of the number and physical proximity of MLCs in residual tumors, congruent with our previous studies providing evidence for transcriptomic and proteomic rewiring of lipid metabolism in residual TNBC. These results highlight the potential for structure-based monitoring of chemotherapeutic metabolic reprogramming and suggest unique molecular mechanisms that may underlie chemoresistance in TNBC. Furthermore, our findings provide novel insights into a new type of intratumoral heterogeneity, that of mitochondrial intratumoral heterogeneity, which complements our understanding of the genomic, epigenomic, transcriptomic, and proteomic complexity of TNBC.

cancer biology↗

bioRxiv2024

Selection for early reproduction leads to accelerated aging and extensive metabolic remodeling in Drosophila melanogaster populations

Experimental evolution studies that feature selection on life-history characters are a proven approach for studying the evolution of aging and variation in rates of senescence. Recently, the incorporation of genomic and transcriptomic approaches into this framework has led to the identification of hundreds of genes associated with different aging patterns. However, our understanding of the specific molecular mechanisms underlying these aging patterns remains limited. Here, we incorporated extensive metabolomic profiling into this framework to generate mechanistic insights into aging patterns in Drosophila melanogaster. Specifically, we characterized metabolomic change over adult lifespan in populations of D. melanogaster where selection for early reproduction has led to an accelerated aging phenotype relative to their controls. Using these data we: i) evaluated evolutionary repeatability across the metabolome; ii) assessed the value of the metabolome as a predictor of "biological age" in this system; and iii) identified specific metabolites associated with accelerated aging. Generally, our findings suggest that selection for early reproduction resulted in highly repeatable alterations to the metabolome and the metabolome itself is a reliable predictor of "biological age". Specifically, we find clusters of metabolites that are associated with the different rates of senescence observed between our accelerated aging population and their controls, adding new insights into the metabolites that may be driving the accelerated aging phenotype. SignificanceWhile experimental evolution studies featuring Drosophila melanogaster have generated significant insights into the forces that shape aging and life history patterns, more recent efforts incorporating genomic and transcriptomic data have had comparatively little success identifying the molecular mechanisms underlying these patterns. Here we work to incorporate molecular phenotyping into this general framework as a way forward. Specifically, we characterize how the metabolome changes with age in populations of D. melanogaster where hundreds of generations of selection for early reproduction have led to enrichment for an accelerated aging phenotype. By comparing to control populations, we show that the metabolome does appear to capture true signals of "biological age" and provides a new avenue for understanding the factors that underlie complex trait variation in real populations.

evolutionary biology↗

bioRxiv2024

Myeloid Cell Glucocorticoid, Not Mineralocorticoid Receptor Signaling, Contributes to Salt-Sensitive Hypertension in Humans via Cortisol

BACKGROUNDSalt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality, yet the etiology is poorly understood. We previously found that serum/glucocorticoid-regulated kinase 1 (SGK1) and epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel (ENaC)-dependent sodium entry into monocyte-derived antigen-presenting cells (APCs) and activation of NADPH oxidase, leading to the formation of isolevuglandins (IsoLGs) in SSBP. Whereas aldosterone via the mineralocorticoid receptor (MR) activates SGK1 leading to hypertension, our past findings indicate that levels of plasma aldosterone do not correlate with SSBP, and there is little to no MR expression in APCs. Thus, we hypothesized that cortisol acting via the glucocorticoid receptor (GR), not the MR in APCs mediates SGK1 actions to induce SSBP. METHODSWe performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) analysis on peripheral blood mononuclear cells of humans rigorously phenotyped for SSBP using an inpatient salt loading/depletion protocol to determine expression of MR, GR, and SGK1 in immune cells. In additional experiments, we performed bulk transcriptomic analysis on isolated human monocytes following in vitro treatment with high salt from a separate cohort. We then measured urine and plasma cortisol, cortisone, renin, and aldosterone. Subsequently, we measured the association of these hormones with changes in systolic, diastolic, mean arterial pressure and pulse pressure as well as immune cell activation via IsoLG formation. RESULTSWe found that myeloid APCs predominantly express the GR and SGK1 with no expression of the MR. Expression of the GR in APCs increased after salt loading and decreased with salt depletion in salt-sensitive but not salt-resistant people and was associated with increased expression of SGK1. Moreover, we found that plasma and urine cortisol/cortisone but not aldosterone/renin correlated with SSBP and APCs activation via IsoLGs. We also found that cortisol negatively correlates with EETs. CONCLUSIONOur findings suggest that renal cortisol signaling via the GR but not the MR in APCs contributes to SSBP via cortisol. Urine and plasma cortisol may provide an important currently unavailable feasible diagnostic tool for SSBP. Moreover, cortisol-GR-SGK1-ENaC signaling pathway may provide treatment options for SSBP. Novelty and RelevanceO_ST_ABSWhat Is New?C_ST_ABSO_LIAlthough salt sensitivity is a major risk factor for cardiovascular morbidity and mortality, the mechanisms underlying the salt sensitivity of blood pressure (SSBP) are poorly understood. C_LIO_LIHigh salt modifies glucocorticoid-receptor expression in antigen-presenting cells (APCs), suggesting a critical role of glucocorticoids in SSBP. C_LIO_LIElevated glucocorticoid receptor (GR) expression compared to mineralocorticoid receptor (MR) expression in APCs provides evidence for a GR-dependent pathway to SSBP. Isolevuglandins (IsoLGs) increased in APCs in vitro after hydrocortisone treatment compared to aldosterone treatment, indicating that cortisol was the predominant driver of IsoLG production in these cells. C_LIO_LIOur studies suggest a mechanism for SGK1 expression through GR activation by cortisol that differs from the currently accepted mechanism for SSBP pathogenesis. C_LI What Is Relevant?O_LIAlthough aldosterone has been used to study SSBP, there has been no consideration of cortisol as a major driver of the condition. C_LIO_LIUnderstanding alternative inflammatory pathways that affect SSBP may provide insights into the mechanism of SSBP and suggest a range of therapeutic targets. C_LIO_LIOur studies may provide a practical approach to understanding and treating salt-sensitive hypertension. C_LI Clinical/Pathophysiological Implications?O_LIOur findings firmly support a GR-dependent signaling pathway for activating SSBP via SGK1 expression. A cortisol-driven mechanism could provide a practical approach for targeted treatments for salt-sensitive hypertension. Moreover, it could pave the way for a diagnostic approach. C_LI

physiology↗

bioRxiv2024

Cytokine and Chemokine Receptor Profiles in Adipose Tissue Vasculature Unravel Endothelial Cell Responses in HIV

Chronic systemic inflammation contributes to a substantially elevated risk of myocardial infarction in people living with HIV (PLWH). Endothelial cell dysfunction disrupts vascular homeostasis regulation, increasing the risk of vasoconstriction, inflammation, and thrombosis that contribute to cardiovascular disease. Our objective was to study the effects of plasma from PLWH on endothelial cell (EC) function, with the hypothesis that cytokines and chemokines are major drivers of EC activation. We first broadly phenotyped chemokine and cytokine receptor expression on arterial ECs, capillary ECs, venous ECs, and vascular smooth muscle cells (VSMCs) in adipose tissue in the subcutaneous adipose tissue of 59 PLWH using single cell transcriptomic analysis. We used CellChat to predict cell-cell interactions between ECs and other cells in the adipose tissue and Spearman correlation to measure the association between ECs and plasma cytokines. Finally, we cultured human arterial ECs (HAECs) in plasma-conditioned media from PLWH and performed bulk sequencing to study the direct effects ex-vivo. We observed that arterial and capillary ECs expressed higher interferon and tumor necrosis factor (TNF) receptors. Venous ECs had more interleukin (IL)-1R1 and ACKR1 receptors, and VSMCs had high significant IL-6R expression. CellChat predicted ligand-receptor interactions between adipose tissue immune cells as senders and capillary ECs as recipients in TNF-TNFRSF1A/B interactions. Chemokines expressed largely by capillary ECs were predicted to bind ACKR1 receptors on venous ECs. Beyond the adipose tissue, the proportion of venous ECs and VSMCs were positively plasma IL-6. In ex-vivo experiments, HAECs cultured with plasma-conditioned media from PLWH expressed transcripts that enriched for the TNF- and reactive oxidative phosphorylation pathways. In conclusion, ECs demonstrate heterogeneity in cytokine and chemokine receptor expression. Further research is needed to fully elucidate the role of cytokines and chemokines in EC dysfunction and to develop effective therapeutic strategies.

cell biology↗

bioRxiv2024

Strategies for Inducing and Validating Zinc Deficiency and Zinc Repletion

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases. New and NoteworthyThis methods paper details 1) dietary approaches to alter zinc homeostasis in rodents, and 2) qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/582542v3_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@15d4cddorg.highwire.dtl.DTLVardef@617214org.highwire.dtl.DTLVardef@188b38forg.highwire.dtl.DTLVardef@1c47526_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

physiology↗

bioRxiv2023

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/566502v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1d2be0eorg.highwire.dtl.DTLVardef@bba6caorg.highwire.dtl.DTLVardef@1c335e5org.highwire.dtl.DTLVardef@10789c9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Age-related skeletal muscle atrophy shows morphological alterations in mitochondrial structure associated with declining function. Our findings propose that exercise intervention may counteract these structural declines by reinstating levels of mitofusin 2, thus highlighting a potential mechanism by which exercise attenuates age-induced mitochondrial dysfunction. C_FIG

biophysics↗

bioRxiv2023

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/559135v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e5b7org.highwire.dtl.DTLVardef@e965e5org.highwire.dtl.DTLVardef@120c7e9org.highwire.dtl.DTLVardef@124bfb5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Workflow highlighting the process of murine interscapular BAT extraction, processing and imaging serial block facing-scanning electron microscopy and using Amira software for 3D reconstruction of LDs and mitochondrial lipid contact sites to elucidate their structure across aging. C_FIG

biophysics↗