Biology subjects

Mishra, R.

Publications and source records attributed to Mishra, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

High-Fidelity Simulation of Cell Adhesion and Flow with Coarse Grain Modelling: A Numerical and Experimental Approach

A coupled CFD-DEM framework was developed to investigate red blood cell (RBC) transport and cellular adhesion in constricted microchannels, with a primary objective of reducing computational cost while preserving physiological accuracy. Initially, numerical softening factors were implemented, allowing greater overlap between particles to expedite computations, with an optimal value of 0.03 selected. Adhesion forces between RBCs and the channel wall were incorporated, with a minimum effective distance of 0.4{square}{micro}m identified as computationally feasible. However, RBC-RBC adhesion led to artificial clustering and was therefore omitted. Coarse-graining methods were applied up to a factor of 2 to further reduce simulation time, with a value of 1.8 chosen based on consistent cell-free layer (CFL) thickness and drag force characteristics. To correct for the increased drag in coarse-grained models, rolling resistance (Type C) with a coefficient of 0.8 was employed, bringing deviations within 10%. The validated model was then extended to simulate the detachment of adherent C2C12 myoblasts under physiological flow. Experimental validation showed increasing cell retention with seeding time, and simulations reproduced this trend with <5% deviation. The resulting Detachment Force Ratio (DFR) at 6 hours indicates near-equivalence between adhesion and hydrodynamic forces, establishing the models robustness for cell-substrate interaction studies in microfluidics.

cell biology↗

bioRxiv2025

Integrative Transcriptomic and miRNA Analysis Reveals Immune Suppression and Metabolic Reprogramming in FGFR3-TACC3 Fusion-Positive versus Fusion-Negative Bladder Cancer

FGFR3-TACC3 gene fusion drives a molecularly distinct subset of bladder cancer characterized by post-transcriptional immune evasion and metabolic rewiring. To elucidate the transcriptomic and regulatory consequences of this fusion, we performed an integrative analysis of mRNA and miRNA expression profiles in fusion-positive versus fusion-negative bladder tumors using TCGA-derived RNA-Seq and miRNA-Seq data. Fusion-positive tumors exhibited upregulation of mitochondrial oxidative phosphorylation and ribosomal genes (e.g., COX5B, ATP5F1D, RPS15) and marked downregulation of immune regulatory genes (e.g., CD4, STAT3, IL6, CD86, PTPRC). Pathway enrichment revealed suppression of JAK/STAT, PD-L1 signaling, and cytokine networks, consistent with a fusion-driven immunosuppressive phenotype. We identified fusion-specific miRNA-mRNA regulatory interactions, including upregulated miRNAs (miR-1226-3p, miR-149-5p, miR-301b-3p) that target immune transcripts and downregulated miRNAs (miR-199b-5p, miR-214-3p) that normally suppress mitochondrial and ribosomal genes. In contrast, fusion-negative tumors displayed an immunoreactive signature marked by elevated IFNG, CXCL10, and IRF1 expression and downregulation of immune-suppressive miRNAs (miR-125a-5p, miR-23b-3p). Minimum free energy analysis and AlphaFold3-based AGO2-RISC docking confirmed structural feasibility of 3D duplex formation for 13 miRNA-target pairs, supporting active silencing complexes. Immune deconvolution revealed reduced infiltration of cytotoxic T cells, NK cells, and dendritic cells in fusion-positive tumors, while fusion-negative tumors showed robust immune cell presence. Key gene-immune cell correlations further confirmed differential immune architecture: SRC and CD4 were strongly linked to suppressed infiltration in fusion-positive tumors, while IFNG and CXCL10 positively correlated with T and NK cell abundance in fusion-negative cases. ROC analysis highlighted COMT, RPS15, and miR-187-3p as robust classifiers of fusion-positive tumors (AUC > 0.9), and CXCL10, CD74, and miR-142 for fusion-negative tumors. Connectivity Map analysis highlighted Lofexidine and Lenalidomide as candidate compounds predicted to reverse fusion-specific transcriptional programs. Collectively, our findings define immune suppression and metabolic reprogramming as core features of FGFR3-TACC3 fusion-positive bladder cancer and nominate fusion-specific miRNA-mRNA interactions as candidate biomarkers and therapeutic targets.

cancer biology↗

bioRxiv2025

Synthetic cargo adaptors reveal molecular features that can enhance dynein activation

Cytoplasmic dynein-1 (dynein) facilitates the microtubule-based retrograde trafficking of all cellular cargo. To become active, dynein binds dynactin and one of many cargo-specific adaptors to form the active transport complex. Despite having similar structures, active transport complexes assembled with different adaptors move with different properties in vitro. To explore how adaptors differentially activate dynein, we engineered a library of synthetic adaptors and characterized their ability to activate dynein using in vitro reconstitution and cell-based trafficking assays. We found that the apparent motility of dynein is highly plastic and tunable by the adaptor sequence and that it is possible to engineer adaptors that outperform endogenous adaptors ability to generate highly motile active transport complexes. We also found that different adaptors support distinct trafficking behavior and cargo movement in cells. These findings provide insight into how dynein motility is modulated to meet the unique trafficking requirements of all cellular cargo.

biophysics↗

bioRxiv2025

Regulators of Interferon-Responsive Microglia Uncovered by Genome-wide CRISPRi Screening

Microglia dynamically support brain homeostasis through the induction of specialized activation programs or states. One such program is the Interferon-Responsive Microglia state (IRM), which has been identified in developmental windows, aging, and disease. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference (CRISPRi) screen in human iPSC-derived microglia (iPS-Microglia) using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes (IFNAR2, TYK2, STAT1/2, USP18) and novel regulators. We uncovered a non-canonical role for the CCR4-NOT complex subunit CNOT10 in IRM activation, independent of its traditional function. This work provides a comprehensive resource for dissecting IRM biology and highlights both established and novel targets for modulating microglial interferon signaling in health and disease.

neuroscience↗

bioRxiv2025

Galectin-3 C-epitope, ANP, PPIA, and albumin function as drug-responsive panel biomarkers and therapeutic targets in aging and pressure overload-induced cardiac hypertrophy

Biological aging (BA) and pressure overload-induced cardiac hypertrophy (PO-CH) are marked by myocardial thickening, fibrosis, and functional decline, culminating in an increased risk of heart failure. Effective clinical management requires biomarkers that not only track disease progression but also monitor therapeutic response. Using Amalaki Rasayana (AR), a cardioprotective nutraceutical-based medicine, in rat models of BA and PO-CH, we identified a panel of serum biomarkers whose treatment-induced decline was associated with disease regression. Among them, galectin-3 emerged in both full-length and oligomeric C-epitope cleaved forms, with the latter closely linked to pathological surface remodeling. Treatment with AR and its bioactive component gallic acid (GA) suppressed extracellular galectin-3 C-epitope oligomer accumulation by inhibiting full-length galectin-3 secretion through phosphorylation-dependent mechanisms and promotion of intracellular retention. Concurrently, serum levels of atrial natriuretic peptide (ANP), cyclophilin A (PPIA), and albumin (ALB) were consistently modulated by therapy. Validation in sera from elderly individuals and cardiac hypertrophy patients responding to conventional treatments supported the translational relevance of this biomarker panel. These findings establish a novel, mechanistically grounded biomarker set--galectin-3 C-epitope, ANP, PPIA, and ALB--for monitoring therapeutic response in cardiac hypertrophy, and position AR as a promising medicinal intervention against age- and pressure-induced cardiac pathology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/657024v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@2498d8org.highwire.dtl.DTLVardef@aeb4fcorg.highwire.dtl.DTLVardef@1ec2f9eorg.highwire.dtl.DTLVardef@12a4392_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIGalectin-3 C-epitope, ANP, PPIA, and ALB are elevated in aging and pressure overload driven left ventricular hypertrophy. C_LIO_LIAmalaki rasayana (AR) and Gallic Acid (GA) reduce these markers in cardiac tissue and circulation. C_LIO_LIAR and GA inhibit galectin-3 secretion and oligomeric binding to cardiomyocyte surfaces. C_LIO_LIAR and GA regulation predominantly occurs via phosphorylation-dependent cytoplasmic retention of galectin-3. C_LIO_LIThese markers serve as potential therapeutic targets and diagnostic biomarkers. C_LI

pathology↗

bioRxiv2025

Colonization with Oxalobacter formigenes slows the progression of CKD and reduces cardiac remodeling in CKD

Accumulation of oxalate in patients with chronic kidney disease (CKD) is associated with CKD progression and increased risk of cardiac death. Whether reducing plasma or urine oxalate slows CKD progression and prevents cardiovascular complications remains unexplored. We colonized the intestines of control and CKD mice with Oxalobacter formigenes (Oxf), an oxalate-degrading microorganism. The mice were fed with the oxalate precursor hydroxyproline for 23 weeks at which time we assessed pathological changes in the kidney and heart. We demonstrate that Oxf reduces plasma oxalate (pOx) and creatinine levels, mitigates inflammation and fibrosis in the kidney, and reduces pathologic cardiac remodeling in the hearts of CKD mice. RNA-seq analysis of ventricular tissue of CKD mice reveals dysregulated expression of metabolic pathways while Oxf colonization reverses these changes. These findings demonstrate that oxalate accumulation plays a role not only in CKD progression but also in associated cardiovascular complications and suggest that strategies to reduce plasma oxalate levels may have therapeutic benefit. Translational statementChronic kidney disease (CKD) is a major health problem that can lead to kidney failure and which increases the risk of cardiovascular disease (CVD) mortality. Oxalate accumulation in advanced kidney disease contributes to further CKD progression and CVD complications. Intestinal colonization with Oxalobacter formigenes (Oxf) in a CKD animal model reduces plasma oxalate level and slows progression of both CKD and CVD. Strategies to reduce plasma oxalate levels may have therapeutic benefit in the setting of CKD.

microbiology↗

bioRxiv2025

miR-631 targets the oncogene RAB11A: implications for oral squamous cell carcinoma pathogenesis and therapeutics

Despite technological advancements, the five-year survival rate for oral squamous cell carcinoma (OSCC) remains dismally low. To develop more effective therapies, we intended to identify the potential therapeutic tumor suppressor microRNAs in OSCC. Previously, a microRNA microarray analysis of 5-Azacytidine treated SCC131 cells had identified 50 upregulated miRs. Of these, miR-631 that had no previously known roles in OSCC was selected for further analysis in the present study. We show that miR-631 binds directly to the oncogene RAB11A and reduces its transcript and protein levels. The upregulation of miR-631 occurs due to its gene promoter demethylation after 5-Azacytidine treatment. We demonstrate that miR-631 reduces proliferation and anchorage-independent growth of OSCC cells in soft agar and promotes apoptosis, in part, via targeting RAB11A. An inverse relationship between miR-631 and RAB11A levels observed across multiple cancer cell lines and in 58.33% of OSCC patient samples highlights the biological significance of their interaction. Further, miR-631 and RAB11A interaction reduces the Wnt signaling in OSCC. Additionally, the nude mice OSCC xenograft study proves the tumor suppressive nature of miR-631. Based on our results, we propose that miR-631 holds promise as a potential therapeutic agent for treating OSCC.

cancer biology↗

bioRxiv2025

Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis

Eradication of Mycobacterium tuberculosis (Mtb) requires strategies targeting bacteria inside the host. Mtb exhibits heterogeneity in redox metabolism inside macrophages to evade killing by anti-TB drugs. If and how macrophage physiology correlates with bacterial redox heterogeneity and drug tolerance remains unclear. Using a fluorescent reporter of mycobacterial redox potential, flow sorting, and RNA sequencing of infected macrophages, we characterized transcriptional and metabolic responses of macrophages harboring redox-diverse Mtb populations. We found that macrophages with suppressed glycolysis and elevated oxidative phosphorylation (OXPHOS) correlated with Mtb populations exhibiting reductive stress and drug tolerance. Conversely, macrophages with elevated glycolysis and suppressed OXPHOS displayed higher mitochondrial reactive oxygen species through reverse electron transport, resulting in oxidative stress in Mtb and enhancing drug efficacy. Computational and genetic approaches identified Nrf2 as a key regulator of macrophage bioenergetics driving redox heterogeneity and drug tolerance in Mtb. Redirecting macrophage metabolism from OXPHOS to glycolysis using an FDA- approved antiemetic drug, meclizine, subverted redox heterogeneity and diminished drug tolerance in macrophages and mice. The pharmacological profile of meclizine (Cmax and AUClast) indicated no adverse interactions with first-line anti-TB drugs in mice. Our data demonstrate the feasibility of reprogramming macrophage metabolism to reduce drug tolerance in Mtb infection.

microbiology↗

bioRxiv2024

FSTL3 is a biomarker of poor prognosis and is associated with immunotherapy resistance in ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC), is associated with high mortality rates due to late-stage diagnosis and limited treatment options. We investigated the role of FSTL3 in ovarian cancer progression both as a prognostic biomarker and as a potential therapeutic target. We measured levels of follistatin (FST) and follistatin-like 3 (FSTL3) in 96 ovarian cancer patient ascites samples and found that FSTL3 overexpression was more predominant than FST and associated with poorer survival outcomes. Mice implanted with an HGSOC syngeneic cell line bearing common alterations in ovarian cancer (KRASG12V, P53R172H, CCNE1oe, AKT2oe) had increasing levels of FST and FSTL3 in serum during tumor growth. Further alteration of this model to generate a knockout of FST (KPCA.FSTKO) and an overexpression of human FSTL3 (KPCA.FSTKO_hFSTL3), revealed that FSTL3 expression was associated with a more fibrotic tumor microenvironment, correlating with an increased abundance of cancer-associated myofibroblasts (myCAFs), and cancer cells with a more mesenchymal phenotype. Tumors overexpressing FSTL3 had less immunocyte infiltration and a significantly reduced intratumoral T-cell abundance (CD4+, CD8+). FSTL3 overexpression completely abrogated tumor response to PPC treatment (Prexasertib combined with PD-1 and CTLA-4 blockade) compared to controls, suggesting that FSTL3 may be involved in immunotherapy resistance. In conclusion, this study suggests a role for FSTL3 as a prognostic marker and as therapeutic target in HGSOC, where it may play a role in promoting a mesenchymal tumor phenotype, maintaining an immunosuppressive tumor microenvironment, and driving immunotherapy resistance. HighlightsHigh FSTL3 levels are associated with poor outcomes in ovarian cancer. Serum levels of FSTL3 increase during tumor growth and reflect tumor burden and therapy response. Overexpression of FSTL3 in cancer cells promotes a fibrotic tumor microenvironment and immunocyte exclusion. Overexpression of FSTL3 in tumors induces resistance to Chk1 and immune checkpoint inhibitor combination therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/627747v1_ufig1.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@16d0608org.highwire.dtl.DTLVardef@a70028org.highwire.dtl.DTLVardef@1fb4895org.highwire.dtl.DTLVardef@5a42b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

CD47 predominates over CD24 as a macrophage immune checkpoint in cancer.

Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity in mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors.

cancer biology↗

bioRxiv2024

LINC01432 binds to CELF2 in newly diagnosed multiple myeloma promoting short progression-free survival to standard therapy

Multiple Myeloma (MM) is a highly prevalent and incurable form of cancer that arises from malignant plasma cells, with over 35,000 new cases diagnosed annually in the United States. While there are a growing number of approved therapies, MM remains incurable and nearly all patients will relapse and exhaust all available treatment options. Mechanisms for disease progression are unclear and in particular, little is known regarding the role of long non-coding RNAs (lncRNA) in mediating disease progression and response to treatment. In this study, we used transcriptome sequencing to compare newly diagnosed MM patients who had short progression- free survival (PFS) to standard first-line treatment (PFS < 24 months) to patients who had prolonged PFS (PFS > 24 months). We identified 157 differentially upregulated lncRNAs with short PFS and focused our efforts on characterizing the most upregulated lncRNA, LINC01432. We investigated LINC01432 overexpression and CRISPR/Cas9 knockdown in MM cell lines to show that LINC01432 overexpression significantly increases cell viability and reduces apoptosis, while knockdown significantly reduces viability and increases apoptosis, supporting the clinical relevance of this lncRNA. Next, we used individual-nucleotide resolution cross-linking immunoprecipitation with RT-qPCR to show that LINC01432 directly interacts with the RNA binding protein, CELF2. Lastly, we showed that LINC01432-targeted locked nucleic acid antisense oligonucleotides reduce viability and increases apoptosis. In summary, this fundamental study identified lncRNAs associated with short PFS to standard NDMM treatment and further characterized LINC01432, which inhibits apoptosis. Key points: lncRNA expression was found to be dysregulated in patients with short PFS to standard multiple myeloma therapy. LINC01432-bound CELF2 inhibits apoptosis.

cancer biology↗

bioRxiv2023

Intronic miR-6741-3p is involved in oral squamous cell carcinoma pathogenesis by targeting the oncogene SRSF3

Epigenetic silencing through methylation is one of the major mechanisms for downregulation of tumor suppressor miRNAs in various malignancies. The aim of this study was to identify novel tumor suppressor miRNAs which are silenced by DNA hypermethylation and investigate the role of at least one of these in oral squamous cell carcinoma (OSCC) pathogenesis. We treated cells from an OSCC cell line SCC131 with 5-Azacytidine, a DNA methyltransferase inhibitor, to reactivate tumor suppressor miRNA genes silenced/downregulated due to DNA methylation. At 5-day post-treatment, total RNA was isolated from the 5-Azacytidine and vehicle control-treated cells. The expression of 2,459 mature miRNAs was analysed between 5-Azacytidine and control-treated OSCC cells by the microRNA microarray analysis. Of the 50 miRNAs which were found to be upregulated following 5-Azacytidine treatment, we decided to work with miR-6741-3p in details for further analysis, as it showed a mean fold expression of >4.0. The results of qRT-PCR, Western blotting, and dual-luciferase reporter assay indicated that miR-6741-3p directly targets the oncogene SRSF3 at the translational level only. The tumor-suppressive role of miR-6741-3p was established by various in vitro and in vivo assays. Our results revealed that miR-6741-3p plays a tumor-suppressive role in OSCC pathogenesis, in part, by directly regulating SRSF3. Based on our observations, we propose that miR-6741-3p may serve as a potential biological target in tumor diagnostics, prognostic evaluation, and treatment of OSCC and perhaps other malignancies.

cancer biology↗

bioRxiv2023

Intrarenal myeloid subsets associated with kidney injury are comparable in mice and patients with lupus nephritis

Resident macrophages and infiltrating monocytes in kidneys of patients with lupus nephritis are altered both in frequency and function relative to their counterparts in healthy kidneys. The extent to which mouse models might be useful in developing approaches to target these cells for treating lupus nephritis is poorly understood. Here, we studied four common lupus mouse models that share clinical, serologic, and histopathologic kidney changes with humans. Using single-cell profiling and multiplex spatial imaging to analyze the intrarenal myeloid compartment with the onset of clinical disease in these models, we identified monocyte and macrophage subsets that expand or contract in kidneys with clinical nephritis. A unique subset of classical monocytes expanded with the onset of disease and expressed genes such as CD9, Spp1, Ctsd, Cd63, Apoe, and Trem2 that were previously shown to be induced by tissue injury and play a role in inflammation, lipid metabolism and tissue repair in other organs. Resident macrophages transitioned from a pro-inflammatory to a similar injury-associated state with onset of disease. To test whether these findings in mouse models were also observed in humans, we re-analyzed monocytes and macrophages in a single-cell RNAseq dataset of kidney biopsies from 155 patients with lupus nephritis and 30 healthy donors, collected by the NIH AMP RA/SLE consortium. Human monocytes and macrophages showed conserved changes in gene expression programs associated with lupus nephritis disease indices, and localized to similar kidney microenvironments as in mice. By identifying myeloid subsets and disease-associated alterations in biological processes that are conserved across species, we provide a strong rationale for functional studies of these cells and pathways in mice to uncover mechanisms and find targets relevant to human lupus nephritis. One sentence summaryThis study characterizes intrarenal myeloid cells from four lupus mouse models and 155 patients with lupus nephritis using single-cell RNA-seq and imaging, and identifies novel infiltrating and resident myeloid subsets that are conserved between mouse and human lupus nephritis, thus providing a map and strong rationale for functional studies in mice with relevance to human disease.

immunology↗

bioRxiv2023

Augmentation of Histone Deacetylase 6 Activity Impairs Mitochondrial Respiratory Complex I in Ischemic/Reperfused Diabetic Hearts

BACKGROUNDDiabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor (TNF) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and {beta}-oxidation. Here we examined how HDAC6 regulates TNF production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts. METHODSHDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNF and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups. RESULTSMyocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNF levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNF with an anti-TNF monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNF levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNF levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown. CONCLUSIONSAugmenting HDAC6 activity inhibits mCI activity by increasing TNF levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIschemic heart disease (IHS) is a leading cause of death globally, and its presence in diabetic patients is a grievous combination, leading to high mortality and heart failure. C_LIO_LIDiabetes impairs assembly of mitochondrial complex I (mCI), complex III dimer, and complex IV monomer into the respiratory chain supercomplexes, resulting in electron leak and the formation of reactive oxygen species (ROS). C_LIO_LIBy oxidizing reduced nicotinamide adenine dinucleotide (NADH) and reducing ubiquinone, mCI physiologically regenerates NAD+ to sustain the tricarboxylic acid cycle and {beta}-oxidation. C_LI What New Information Does This Article Contribute?O_LIMyocardial ischemia/reperfusion injury (MIRI) and diabetes as comorbidities augment myocardial HDCA6 activity and generation of tumor necrosis factor (TNF), which inhibit myocardial mCI activity. C_LIO_LIGenetic disruption of histone deacetylase 6 (HDAC6) decreases mitochondrial NADH levels and augments mCI activity in type 1 diabetic mice undergoing MIRI via decreasing TNF production, leading to decreases in MIRI. C_LIO_LIPretreatment of type 2 diabetic db/db mice with a HDAC6 inhibitor, tubastatin A (TSA), decreases mitochondrial NADH levels and augments mCI activity by decreasing TNF levels, leading to improvements in cardiac function. C_LI Patients with diabetes are more susceptible to MIRI than non-diabetics with greater mortality and resultant heart failure. There is an unmet medical need in diabetic patients for the treatment of IHS. Our biochemical studies find that MIRI and diabetes synergistically augment myocardial HDAC6 activity and generation of TNF, along with cardiac mitochondrial fission and low bioactivity of mCI. Intriguingly, genetic disruption of HDAC6 decreases the MIRI-induced increases in TNF levels, concomitant with augmented mCI activity, decreased myocardial infarct size, and ameliorated cardiac dysfunction in T1D mice. Importantly, treatment of obese T2D db/db mice with TSA reduces the generation of TNF and mitochondrial fission and enhances mCI activity during reperfusion after ischemia. Our isolated heart studies revealed that genetic disruption or pharmacological inhibition of HDAC6 reduces mitochondrial NADH release during ischemia and ameliorates dysfunction of diabetic hearts undergoing MIRI. Furthermore, HDAC6 knockdown in cardiomyocytes blocks high glucose- and exogenous TNF-induced suppression of mCI activity in vitro, implying that HDAC6 knockdown can preserve mCI activity in high glucose and hypoxia/reoxygenation. These results demonstrate that HDAC6 is an important mediator in MIRI and cardiac function in diabetes. Selective inhibition of HDAC6 has high therapeutic potential for acute IHS in diabetes.

pharmacology and toxicology↗

bioRxiv2023

Profiling extracellular vesicles in circulation enables the early detection of ovarian cancer

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53, and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study (n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

cancer biology↗

bioRxiv2022

Cut homeodomain transcription factor is a novel regulator of cortical glia morphogenesis and niche maintenance around neural stem cells

Cortex glia in Drosophila central nervous system forms a niche around neural cells for necessary signals to establish cross-talk with their surroundings. These cells grow and expand their thin processes around neural cell bodies. Although essential for the development and function of the nervous system, how these cells make extensive and intricate connected networks remain largely unknown. Here we show that Cut, a homeodomain transcription factor, directly regulates the fate of the cortex glia, impacting NSC homeostasis. Focusing on the thoracic ventral nerve cord (tVNC), we found that Cut is required for normal growth and development of cortex glia and timely increase in DNA content to undergo endomitosis. Knockdown of Cut in cortex glia significantly reduces the growth of cellular processes, the network around NSCs, and their progenys cell bodies. Conversely, overexpression of Cut induces overall growth of the main processes at the expense of side ones. Whereas the Cut knockdown slowdown the timely increase of DNA, Cut overexpression results in a significant increase in nuclear size and volume and a threefold increase in DNA content of cortex glia. Further, we note that constitutively high Cut also interfered with nuclei separation during endomitosis. Since cortex glia form syncytial networks around neural cells, the finding identifies Cut as a novel regulator of glial growth and endomitosis to support a functional nervous system. Article SummaryCut homeodomain transcription factor is crucial for cortex glia growth and the formation of complex cellular processes around neural cells. This regulation ensures a timely increase in DNA content, allowing the cells to enter endomitosis. Constitutively high Cut levels increase the DNA content of these cells to several folds. The finding emphasizes the need to investigate if activated CUX1, the human homolog of Cut, in glioma enhances chromosomal instability and, in conjunction with other mutations, enhances their tumorigenic potential.

developmental biology↗

bioRxiv2022

Inhibition of hyperactive cyclin dependent kinase 5/p25 is protective in the 6-hydroxydopamine model of Parkinson's disease

BackgroundCyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme involved in neuronal development, maturation and survival. CDK5 activity is tightly regulated by association with regulatory proteins p35 and p39. Upon neuronal insults, increased intracellular calcium activates calpain, cleaving p35 into p25, which has a higher affinity for CDK5. p25 hyperactivates CDK5, initiating apoptotic cascades that lead to significant dopaminergic (DAergic) loss that can leads to neurodegenerative disorders, such as Parkinsons disease (PD). ObjectiveThis study investigates hyperactivation of CDK5/p25 in the 6-hydroxydopamine (6-OHDA) rat model of PD and specific inhibition of CDK5/p25 by truncated peptide 5 (TP5). TP5 was investigated for amelioration of 6-OHDA induced behaviour impairments and significant protection of dopamine neurons through tyrosine hydroxylase (TH). Methods6-OHDA induced motor impairments and reduced TH. Motor assessments included locomotor activity, beam transversal, fixed speed rotarod and amphetamine-induced rotations. Immunohistochemistry investigated DAergic neurodegeneration using TH levels and immunoprecipitation and assay investigated CDK5 activity. ResultsPre-administration of TP5 maintained locomotor activity, preserved beam transversal scores, protected motor coordination and attenuated amphetamine induced rotations in 6-OHDA lesioned rats, all indicative of neuroprotection by TP5. 6-OHDA without pretreatment of TP increased CDK5 activation. CDK5 activity in TP5+6-OHDA animals was not significantly different from artificial cerebrospinal fluid (aCSF) treated sham surgery controls. Immunohistochemistry revealed significant TH protection within the substantia nigra (SN) of TP5 pretreated animals. Conclusions6-OHDA increases CDK5 activity. Hyperactive CDK5/p25 inhibition in the 6-OHDA model has neuroprotective capability, protecting against the development of a toxin-based induction of PD-like motor phenotypes and pathology. This supports CDK5/p25 specific inhibition as a target for further neuroprotective therapeutic development.

pharmacology and toxicology↗

bioRxiv2022

Mapping protein-DNA interactions with DiMeLo-seq

We recently developed Directed Methylation with Long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype specific protein-DNA interactions, and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule, DNA sequencing platforms such as Nanopore sequencing. Here, we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed, or frozen cells. The protocol requires 1-2 days for performing in situ targeted methylation, 1-5 days for library preparation depending on desired fragment length, and 1-3 days for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data. Key papersAltemose, N., Maslan, A., Smith, O.K., Sundararajan, K., Brown, R.R., Mishra, R., Detweiler, A.M., Neff, N., Miga, K.H., Straight, A.F. and Streets, A., 2022. DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nature Methods, pp.1-13. (https://doi.org/10.1038/s41592-022-01475-6)

genomics↗