Biology subjects

Jiang, M.

Publications and source records attributed to Jiang, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Multiscale transcriptomic organization of the human brain with DigitalBrain

The human brain varies across anatomical regions, cell types, development, aging and disease states, yet existing single-cell transcriptomic resources remain fragmented and difficult to integrate into a unified biological model. Here we present DigitalBrain, a human brain-specific atlas and foundation-model framework for organizing diverse and fragmented human brain transcriptomic data across scales. We first built DigitalBrain-Atlas, a harmonized whole-brain single-cell resource comprising 16.35 million transcriptomes from 2,143 donors across 165 brain regions, spanning the human lifespan and multiple neurological and clinical conditions. We then developed DigitalBrain-M1, a Transformer-based model that jointly encodes gene identity and expression magnitude to learn a shared embedding space for cells and genes. Across held-out datasets, DigitalBrain supported robust single-cell integration, clustering and cell-type annotation while preserving major biological structure and reducing technical fragmentation. Beyond these benchmarks, the learned embeddings revealed emergent large-scale hierarchical organization of the human brain, linking anatomically distinct regions into higher-order patterns consistent with known functional systems. Applied to human hippocampal aging, DigitalBrain identified cell-type-specific aging sensitive gene sets, highlighted dentate gyrus granule cells as a particularly age-sensitive population, and discovered selective reorganization of gene programs related to synaptic transmission, postsynaptic structure, membrane excitability and axon guidance during aging. Cross-dataset convergence was strongest at the level of functional modules and recurrent aging sensitive genes. Together, these results demonstrate that DigitalBrain is a brain-specific framework for mapping human brain organization across scales, and as an early step towards a complete virtual organ for the human brain.

bioinformatics↗

bioRxiv2026

HCF1 orchestrates O-GlcNAcylation and affinity-dependent transcription through extended molecular determinants and register-shifted binding

We recently identified Host Cell Factor 1 (HCF1), a transcriptional co-regulator discovered more than thirty years ago, as a cancer dependency. To further understand its molecular functions and expand its known interactome, here, we screened a proteome-wide library of candidate HCF1 binding peptides and identified previously uncharacterized canonical and non-canonical HCF1-binding partners. Through deep mutational scanning (DMS) screening, we uncovered an extended set of molecular determinants of binding and show how mutations outside its previously established interacting residues impact binding affinity. Next, we uncover non-canonical HCF1 binders with an extended register-shifted two-amino acid sequence between their "anchor" histidine and tyrosine amino-acid residues, which we show critically contributes, in an affinity-dependent manner, to the downstream transcriptional activity of IRF1. Our data also shows that HCF1 promotes O-GlcNAcylation of the majority of its transcriptional binders. Overall, our results significantly expand the number and diversity of HCF1 binders and propose an enhanced mechanistic understanding of how HCF1 orchestrates transcription and O-GlcNAcylation.

molecular biology↗

bioRxiv2026

Tumor-suppressor signature for robust prognosis and prediction in adenocarcinoma non-small cell lung cancer

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality, partly due to limited early detection strategies and incomplete understanding of tumor-suppressive mechanisms. In our previous work, we identified 26 tumor suppressor (TS) genes and characterized their biological functions and regulatory networks. We systematically evaluated these TS genes across multiple microarray datasets by analyzing differential expression patterns, correlations with oncogenes, tumor-associated genes, and PD-1-related immune genes, as well as somatic mutation frequencies. A weighted scoring algorithm was used to construct a TS gene signature. Patients were stratified using z-score normalization, and univariable and multivariable Cox proportional hazards models were applied across multiple adenocarcinoma (ADC) cohorts. Prognostic performance was assessed using Kaplan-Meier analysis and AUC metrics. The 26 TS genes were consistently down-regulated in tumors and showed strong negative correlations with oncogenes, particularly in advanced stages. TS genes also exhibited stage-dependent correlations with PD-1-associated immune genes, with chemotaxis/cytokine-signaling genes behaving TS-like, while PDCD1 and SIT1 showed oncogene-like patterns. Somatic mutations were detected in only 32% of LUAD samples for TS genes, compared with 68% for oncogenes. Across seven independent ADC cohorts, high TS-signature expression was associated with significantly reduced risk of death and recurrence/relapse. The TS signature outperformed several published prognostic signatures and demonstrated robust predictive accuracy, with AUC values exceeding 0.7 in multiple datasets and >0.8 for relapse prediction in GSE30219. Across seven independent cohorts, high TS signature expression was consistently associated with significantly reduced risk of death or recurrence/relapse in ADC patients. Patients with high TS signature expression exhibited markedly improved survival probabilities compared with those with low expression. When benchmarked against several established prognostic signatures using AUC metrics, our TS signature demonstrated superior robustness and predictive accuracy for ADC prognosis.

cancer biology↗

bioRxiv2026

UniST: A Unified Computational Framework for 3D Spatial Transcriptomics Reconstruction

Spatial transcriptomics (ST) enables the measurement of gene expression in its native spatial context, yet most ST datasets are acquired as two-dimensional (2D) sections. Consequently, the underlying three-dimensional (3D) organization of tissues is only partially observed, and 3D ST data generated from serial sections are typically sparse and heterogeneous, with substantial tissue loss and missing measurements. These limitations pose major analytical challenges for reconstructing coherent 3D tissue architecture, rather than issues of experimental scalability alone. Here, we present UniST, a unified generative artificial intelligence (AI) framework designed to computationally reconstruct dense and continuous 3D ST landscapes from sparse serial sections, without altering the underlying experimental ST technologies. UniST integrates three complementary modules: kernel point convolution with cross-attention layers for point cloud upsampling, optical flow-based interpolation for continuous slice reconstruction, and a graph autoencoder with implicit neural representations for gene expression imputation. Together, these components densify sparse slices, resolve discontinuities, and map spatial coordinates to high-dimensional transcriptomics. Across multiple ST platforms and tissue contexts, UniST accurately restored structural continuity and biologically meaningful expression patterns. In a mouse embryo dataset, UniST reconstructed a dense 3D heart architecture from sparsely sampled slices. In two 3D human cancer tissues, UniST recovered critical spatial features, including tumor-immune boundaries and tertiary lymphoid structures, that were fragmented in the original data. By providing a generalizable computational solution that complements existing ST acquisition protocols, UniST facilitates cost-efficient and scalable reconstruction of 3D ST landscapes, enabling more faithful investigation of tissue organization and disease biology.

bioinformatics↗

bioRxiv2026

Proton sponge or membrane fusion? Endosomal escape of siRNA polyplexes illuminated by molecular dynamics simulations

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1d993forg.highwire.dtl.DTLVardef@1381f29org.highwire.dtl.DTLVardef@185faeborg.highwire.dtl.DTLVardef@11c8524_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Information Geometry Reconciles Discrete and Continuous Variation in Single-Cell and Spatial Transcriptomic Analysis

Single-cell and spatial transcriptomics provide high-resolution cellular characterization, yet standard analytical approaches remain theoretically misaligned with the probabilistic nature of the data. After UMI normalization, current pipelines rely on Euclidean or log-transformed Euclidean distance for similarity measurement. Both are fundamentally ill-suited to model the multinomial count data. Euclidean distance in normalized space overemphasizes high-variance genes, while log-transformation inverts this bias but at the cost of distorting subtle, continuous expression modulations. Neither approach naturally captures the dual nature of gene expression: both discrete presence/absence transitions and continuous quantitative variation. To overcome these limitations, we introduce GAIA (Geometric Analysis from an Information Aspect), an information-geometric framework for cell representation learning and inter-cell similarity measurement. By anchoring analysis in the true probabilistic model, treating cells as multinomial distributions over genes and projecting cells to a statistical manifold, GAIA organically reconciles both the presence/absence effect and the more continuous expression modulations. Mathematically, GAIA exploits the equivalence between Fisher-Rao distance in multinomial space and geodesic distance on the unit hypersphere, a property that enables both theoretical guarantees and computational efficiency. Experiments in synthetic and real scRNA-seq and spatial transcriptomic datasets demonstrate that GAIA preserves robust and consistent cell-to-cell relationships, delineates biologically nuanced sub-types, mitigates batch effects arising from sequencing depth variation, and eliminates the dependence on knowledge-restricted gene selection for learning meaningful cell representations. Overall, GAIA offers a knowledge-lean, variance-stabilizing framework for analyzing single-cell and spatial transcriptomic data, enhancing discrimination between nuanced cell sub-type and -states.

bioinformatics↗

bioRxiv2026

Accessible and reproducible mesoscale fMRI at 5.0 T: A Pulseq-based open framework for human laminar mapping

Mesoscale, layer-specific functional MRI (fMRI) enables noninvasive access to cortical microcircuitry, yet widespread adoption has been constrained by a reliance on ultra-high field ([≥]7.0 T) systems and proprietary pulse sequences. To bridge this gap and enhance accessibility, we developed an open-source framework at 5.0 T for mapping laminar brain activity. This framework integrates a Pulseq-based 3D vascular space occupancy (VASO) sequence with an end-to-end data acquisition and analysis pipeline. At matched sub-millimeter resolution (0.8 mm in-plane), the Pulseq-based 3D implementation increased slab coverage by [~]1.82-fold and improved temporal signal-to-noise ratio by [~]1.50-fold relative to a vendor-provided 2D-VASO sequence. Validated using a finger-tapping paradigm, individual cerebral blood volume-weighted (VASO) laminar activation profiles consistently revealed the canonical "double-peak" pattern, with distinct superficial and deep peaks in the primary motor cortex. These profiles exhibited excellent cross-visit reliability (r = 0.80), and peak depths showed good spatial reliability (ICC = 0.69 for deep layers; ICC = 0.58 for superficial layers). Between-subject reproducibility was high (r = 0.86). Deploying the identical Pulseq protocol at an independent imaging site reproduced the characteristic double-peak laminar profiles (r = 0.63). At the group level, 5.0 T laminar profiles closely matched established 7.0 T findings, robustly resolving both deep and superficial peaks despite the lower field strength. Notably, for each participant, a single 13-minute VASO run was sufficient to resolve reliable laminar activation patterns that exhibited high consistency with multi-run averages (r = 0.78), highlighting the potential for high-throughput population studies or clinical research settings. The Pulseq-based 3D VASO sequence file, image reconstruction pipeline, and data analysis scripts are openly available to facilitate the adoption of this framework. This work establishes a practical route towards more accessible and reproducible mesoscale fMRI for studying human laminar functional architecture.

neuroscience↗

bioRxiv2026

MtDNA heteroplasmy controls tumor immune reprogramming through mitochondrial translation

Mitochondrial DNA (mtDNA) mutations are among the most frequent genetic alterations in human cancers, yet their molecular mechanisms in shaping tumor progression and metastasis under physiological conditions remain poorly understood. Here, we show that mtDNA heteroplasmy acts as a tumor cell-intrinsic rheostat that orchestrates fate decisions between progression and suppression. Moderate heteroplasmy induces a ribosomal storm of mitochondrial translation and ribosome biogenesis, driven by leucyl-tRNA synthetase 2 (LARS2). This response enhances metabolic fitness and licenses the secretion of S100A8/A9, enabling tumor cells to actively remodel the immune microenvironment through recruitment of myeloid-derived suppressor cells and exclusion of CD8 T cells, thereby promoting immune evasion and metastatic progression. In contrast, high heteroplasmy impairs tumorigenesis due to oxidative phosphorylation collapse. Disruption of LARS2, mitochondrial translation, or S100A9 signalling abrogates tumor progression. Our findings establish mtDNA heteroplasmy as a key regulator of cancer evolution, linking mitochondrial genetic variation to immune modulation, and reveal the LARS2-mitochondrial translation-S100A8/A9 axis as a therapeutically targetable programme within the pro-tumorigenic window of mtDNA heteroplasmy.

cancer biology↗

bioRxiv2026

Native yeast kinetochore structures identify an essential inner kinetochore interaction

Kinetochores must accurately assemble on centromeres for faithful chromosome segregation. Although a conserved centromeric nucleosome is essential for kinetochore assembly, budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, perhaps due to its intrinsic rigidity. To better understand yeast inner kinetochore assembly, we developed a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We performed cryo-electron microscopy on the purifications and generated density maps of four separate inner kinetochore complexes, two of which had not previously been visualized and may represent intermediate assembly states. We identified an Ndc10 trimerization domain that engages centromeric DNA and a pair of CBF3 complexes and is associated with substantial bending of centromeric DNA. Ndc10 trimerization is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates centromeric DNA bending to stabilize the centromeric nucleosome and inner kinetochore.

biochemistry↗

bioRxiv2026

Kinetochore clustering is mediated by Mps1 phosphorylation of conserved MELT motifs in Stu1

Unattached kinetochores promote microtubule capture while preventing cell cycle progression during mitosis. The Mps1 kinase controls these events by mediating kinetochore assembly of the fibrous corona in animal cells and by triggering the spindle checkpoint. In budding yeast, which does not assemble a fibrous corona, the Stu1 and Slk19 spindle proteins promote microtubule capture by clustering unattached kinetochores, but the underlying mechanism is unclear. Here, we show that Mps1 controls this pathway by phosphorylating two conserved MELT motifs in Stu1 to recruit Slk19 and mediate kinetochore clustering. Structural analysis of the Stu1:Slk19 complex reveals long, string-like filaments and offers mechanistic insight into how kinetochores might cluster. Our findings suggest the regulation of kinetochore capture is a conserved Mps1 function across eukaryotes. SUMMARYUnattached kinetochores cluster in budding yeast to promote microtubule capture. Mallett et al. dissect the molecular mechanisms of this pathway, showing Mps1 kinase regulates an interaction between Stu1 and Slk19 to drive clustering.

cell biology↗

bioRxiv2025

Characterization and application of hyperthermia-evoked seizures in a mouse model of focal cortical dysplasia

Rodent models of drug-resistant epilepsy are widely used to uncover mechanisms of seizures and to test the efficacy of treatments; however, the stochastic and relatively infrequent nature of spontaneous seizures in these models makes it challenging to observe and manipulate peri-ictal activity, and to rapidly screen therapeutics. As fever has been identified as a clinical seizure trigger, we investigated the ability of induced hyperthermia to trigger seizures in a mouse model of focal cortical dysplasia (FCD), a common cause of drug-resistant epilepsy. Experimental mice were generated via in utero electroporation of a gain-of-function RHEB variant (RHEBp.P37L). In adult Rheb-FCD mice, hyperthermia elicited behavioral seizures [~]80% of the time, with a mean temperature threshold of [~]40 {degrees}C. Using this acute paradigm, as a proof-of-concept, we found that chemogenetic inhibition of a subset of excitatory neurons within the dysplasia significantly elevated the threshold for hyperthermia-evoked seizures. We then confirmed the relevance of this acute finding to spontaneous epileptiform activity, using repeated chemogenetic inhibition across chronic recordings to confirm a reduction of spontaneous seizures as well as interictal spikes. Overall, our data demonstrate that hyperthermia-evoked seizures can be readily elicited in Rheb-FCD mice, offering a tractable platform for evaluating therapeutic interventions targeting seizure susceptibility in focal epilepsy.

neuroscience↗

bioRxiv2025

TUPAD: A Tn5-Mediated, Pre-amplification/Adapter-Dimer-Free Platform for Ultra-Uniform, One-Step PGT Profiling with High Stability and Broad Compatibility

High-throughput sequencing stands as a cornerstone of clinical molecular diagnostics, particularly in preimplantation genetic testing (PGT)--a key application where the selection of chromosomally normal embryos is critical to enhancing in vitro fertilization (IVF) success rates. A fundamental challenge inherent to PGT, however, lies in the ultra-low gDNA input (typically <100 pg) from embryo biopsies. To mitigate this limitation, traditional workflows rely on whole genome amplification (WGA) technologies, such as MDA, PicoPlex, and MALBAC. Yet these WGA-based protocols inherently entail pre-amplification of gDNA prior to standard library construction---a step that introduces significant coverage bias and compromised uniformity. These are intrinsic flaws irreparable by downstream library preparation or bioinformatics analysis, ultimately leading to clinical uncertainty in the interpretation of copy number variations (CNVs). In this study, we developed TUPAD (Tn5-Mediated, Uniform Pre-amplification-free and Adapter-Dimer-Free Library preparation), a transformative technology that bypasses the limitations of traditional WGA. By engineering a novel adapter-dimer-free transposon system, TUPAD enables high-efficiency library construction from as little as 1 picogram (pg) of gDNA. This breakthrough allows for a pre-amplification-free workflow, effectively eliminating the primary source of amplification bias. Compared to existing WGA standards, these advantages are fully retained in embryo biopsy and positive sample testing, TUPAD consistently generates highly uniform library data, significantly enhancing the detection limit and accuracy of CNVs and other genomic variants. Beyond these performance advantages, TUPAD also cuts wet-lab processing time by 50%. Practical validation confirms TUPAD results are stable, reliable, with proven practical utility in PGT testing.

molecular biology↗

bioRxiv2025

An inherent T cell-activating mRNA delivery platform for in vivo CAR T generation

The clinical success of CAR T cell therapy has underscored the need for scalable, non-invasive strategies for in vivo T cell engineering. Although mRNA delivery offers a promising alternative, current lipid-based vectors lack intrinsic T cell tropism and often rely on antibody conjugation, complicating manufacturing and raising safety concerns. Here we present an inherent T cell-activating polymer-lipid nanoparticle (ERTLNPs) that enable ligand-free, efficient mRNA transfection and activation of T cells in vivo. ERTLNPs, composed of p-toluenesulfonyl arginine (RT)-functionalized polyethylenimine (denoted as PEI-RT) and helper lipids, preferentially accumulate in the spleen following systemic administration. Without exogenous stimulation, ERTLNPs intrinsically activate T cells, triggering robust mRNA expression and proliferation. Mechanistically, ERTLNPs engage the PI3K/AKT/mTOR signaling axis to reprogram T cell metabolism, promoting expansion while restraining exhaustion. Systemic delivery of mRNA encoding fibroblast activation protein chimeric antigen receptor (mFAP CAR) via ERTLNPs leads to in situ generation of functional CAR T cells, which efficiently eliminate pathological fibroblasts in models of cancer and fibrosis, with minimal off-target effects. This ligand-free, metabolically reprogramming mRNA delivery platform provides a clinically translatable approach for in vivo CAR T cell generation and broadens the landscape of non-viral immunotherapeutic engineering.

bioengineering↗

bioRxiv2025

Bio-synthesis of 15N-Labeled G-Quadruplexes to Investigate the Structure and Interactions in the Cell Lysate using Nuclear Magnetic Resonance

G-quadruplexes (G4) play key roles in biology, making it critical to understand their structure and ligand-binding behavior in cellular environments for advancing G4-targeted therapeutics. While in-cell nuclear magnetic resonance (NMR) is a powerful technique for studying G4 in situ, its application is limited by the challenge of producing isotope-labeled single-stranded DNA (ssDNA). Here, we introduce Restriction Endonuclease Digestion (RED), a simple and cost-effective method to generate 15N-labeled ssDNA. This approach combines molecular cloning and enzymatic design processing by propagating plasmids in E. coli cultured with 15NH4Cl, followed by double restriction digestion and isolation target 15N-ssDNA. Using RED, we produced milligram-scale quantities of 96%-enriched 15N-labeled human telomeric G4 ssDNA (wtTel23c, CTAGGG(TTAGGG)3), ideal for NMR analysis. The NMR spectra revealed that wtTel23c adopts G4 topology and undergoes multiple conformations of wtTel23c in potassium-containing solutions and in Xenopus laevis cell lysate. Interaction studies with the ligand TMPyP4 showed distinct binding profiles in cellular and dilute environments. In dilute solution, TMPyP4 binds to the top tetrad of wtTel23c, while it binds to the loop in cellular environments. The RED method offers an efficient strategy for producing stable isotope-labeled ssDNA, opening new avenues for studying G4 structures and their ligand interactions in complex biological contexts.

biochemistry↗

bioRxiv2025

Oncogenic and immunomodulatory functions of SUV420H1 in HPV-negative head and neck squamous cell carcinoma.

Despite the advent of immunotherapy, human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) carries a high morbidity and mortality rate, thus novel therapies are urgently needed. Suppressor Of Variegation 4-20 Homolog 1 (SUV420H1) is a protein lysine methyltransferase that writes H4K20me3. Approximately 35% of HPV-negative HNSCC tumors carry gains/amplifications of SUV420H1. Gene Set Enrichment Analysis (GSEA) showed enrichment of proliferation-, epithelial-mesenchymal transition (EMT)- and immune-response pathways in SUV420H1-overexpressing HPV-negative HNSCC tumors. Depletion of SUV420H1 led to decreased proliferation, cell cycling and invasion in human HPV-negative HNSCC cell lines, while enzymatic inhibition decreased the invasive capacity but not the proliferation and cell cycling of HPV-negative HNSCC cell lines, supporting the presence of catalytically-independent and -dependent functions of SUV420H1. In a syngeneic mouse model of mouse oral carcinoma 1 tumors (MOC1), Suv420h1 knockout (KO) in MOC1 cancer cells halted tumor growth and synergized with anti-PD-1 therapy. In the tumor immune microenvironment (TIME) of Suv420h1 KO MOC1 tumors, a significant increase in the macrophage compartment with a concurrent decrease in the protumorigenic granulocytic myeloid derived suppressor cells (gMDSCs) was observed. Genome-wide mapping of SUV420H1-mediated H4K20me3 revealed enrichment of EMT-, IFN-response, and myeloid-attracting chemokines. This work provides rationale for the depletion/degradation of SUV420H1 as a novel therapeutic strategy to hinder proliferation and invasion, and to impart sensitization to anti-PD-1 immunotherapy for patients with HPV-negative HNSCC tumors with SUV420H1 gain/amplification.

cancer biology↗

bioRxiv2025

Butyrate blocks specific histone acetylation by preventing recruitment of p300 to acetylated histones

Butyrate, a short-chain fatty acid (SCFA) produced by microbial fermentation of dietary fiber, exerts beneficial metabolic and immunomodulatory functions through hyperactivation of the histone acetylase (HAT) p300 and inhibition of histone deacetylases (HDAC). These effects are widely believed to result in the acetylation of distinct histones that regulate specific genes, including at transcription starting sites (TSS) and enhancers. However, we show that this is not the case, as butyrate dose-dependently increases the acetylation of histones throughout the entire genome. Molecular dynamics simulations suggest that the RING-loop prevents the recruitment of p300 to acetylated histones through its bromodomain, and thereby cannot maintain the acetylation of specific histones through positive feedback. This was confirmed by showing that only catalytically inactive p300 stably binds to acetylated histones. Thus, the epigenetic regulation of specific genes by butyrate is limited, but butyrate instead increases histone acetylation globally resulting in opening of the entire chromatin structure.

molecular biology↗

bioRxiv2025

Mitochondrial Dysfunction in Endothelial Cells Drives Greater Vascular Impairment in Females with Diabetes-Associated Peripheral Artery Disease

BackgroundWomen with peripheral artery disease (PAD) experience poorer clinical outcomes than men, particularly in the setting of diabetes. However, the mechanistic basis for these sex- specific disparities remains unclear. MethodsHere, we investigated endothelial cell (EC) function(s) in diabetes-associated PAD, with a focus on sex differences. Limb tissues from patients with diabetes and chronic limb-threatening ischemia (CLTI) undergoing amputation, and a diabetes mouse model of hindlimb ischemia (HLI), were assessed for vasodilatory capacity, angiogenesis, oxidative stress and changes to expression of mitochondrial complex genes. ECs exposed to a hyperglycemic environment in vitro were assessed for mitochondrial function. The therapeutic potential of the mitochondrial-targeted antioxidant MitoQ was investigated. ResultsECs from females with diabetes-associated PAD have altered responses compared to males. Specifically, limb vessels and skeletal muscle from females exhibit reduced arterial relaxation, angiogenesis and increased oxidative stress in response to HLI in mice, and in tissues from patients. Single-cell RNA sequencing of murine limbs revealed marked suppression of EC mitochondrial complex genes in females with diabetes. Female human ECs exposed to high glucose had reduced respiration, reduced expression of mitochondrial genes and increased oxidative stress. Remarkably, MitoQ restored arterial relaxation and the angiogenic response in female diabetes- associated PAD. ConclusionOur findings uncover a striking sex-specific vulnerability involving oxidative stress and mitochondrial dysfunction in EC health in diabetes-associated PAD. These results highlight the need for sex-specific therapeutic strategies in diabetic PAD, which might include mitochondrial targeted antioxidant strategies.

pathology↗

bioRxiv2025

Developing and Benchmarking One Health Genomic Surveillance Tools for Influenza A Virus in Wastewater

Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool. However, IAV is at low abundance in wastewater, making it difficult to sequence. We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes. However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.

microbiology↗