Biology subjects

Sharma, K.

Publications and source records attributed to Sharma, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Vehiculation and functional delivery of lipophilic therapeutics and antibiotics via pulmonary surfactant in a lung-on-chip model

Pulmonary surfactant forms a dynamic proteolipid thin-film at the alveolar air-liquid interface. It is both a barrier to but also a "last-mile" carrier for inhaled particulate matter to the distal lung. The role of vehiculation in pulmonary delivery of inhaled therapeutics remains poorly understood, as it cannot be easily studied in animal models, and is not recapitulated in liquid cell culture. Here we adapted a thin-film bridge (TFB) delivery method from vehiculation experiments in acellular models to a lung-on-chip (LoC) platform under breathing-like stretch. Using confocal live-imaging, we demonstrate vehiculation and cellular uptake of fluorescently-labelled cargoes such as Tacrolimus or Beclomethasone. We systematically compared TFB vehiculation to existing in vitro delivery approaches. TFB vehiculation promoted sustained retention of functionally effective Tacrolimus, strong co-localization with surfactant lipids, and accumulation in macrophages in human LoCs reconstituted with in vitro-differentiated alveolar macrophage-like cells. This contrasted with the unphysiological dominant uptake of formulations in liquid by alveolar epithelial cells. Finally, in proof-of-concept experiments, TFB-vehiculated bedaquiline effectively supressed subsequent growth of Mycobacterium tuberculosis in a prophylactic manner. Overall, our work demonstrates an approach for studying drug vehiculation in models of the alveolar interface in vitro, and the feasibility of surfactant-containing therapeutic formulations for direct and effective pulmonary delivery.

bioengineering↗

bioRxiv2025

Multicenter HFpEF study identifies sex disparity linked with two discrete cardiac proteomic signatures

Heart failure with preserved ejection fraction (HFpEF) is epidemic, with an incidence exceeding that of heart failure with reduced ejection fraction (HFrEF). Sex differences in HFpEF phenotype have been observed, particularly linked to aging and metabolic comorbidities in women. Here, we report a multi-centre proteomic analysis of HFpEF and HFrEF cardiac samples. Using tissues and clinical data derived from multiple institutional biobanks, in-depth characterization of the proteome of ventricular samples was undertaken. Relative to HFrEF, the HFpEF cohort exhibited minimal proteome composition overlap and pronounced heterogeneity. A key finding of this investigation is that sex per se did not confer a distinctive proteomic HFpEF signature. Rather, two HFpEF proteomic profiles were identified, differing significantly in sex ratios. The identification of two Clusters revealed that HFpEF has two predominant proteomic signatures at the molecular level, marked by differences in the extent and nature of structural and contractile machinery and by local cellular and ECM communications. Upstream regulator analysis identified various molecular leads to be pursued in defining these two HFpEF profiles. Our findings offer specific opportunities for new exploration of therapeutic options to target the spectrum of HFpEF proteomic diversity and identify potential drug targeting prospects.

molecular biology↗

bioRxiv2025

Dapagliflozin mitigates hypoxia-induced metabolic stress, kidney tubular cell death and fibrosis

Sodium-glucose co-transporter 2 inhibitors (SGLT2-i) slow progression of kidney disease but therapeutic mechanisms remain elusive. Here we report the beneficial effect of dapagliflozin on hypoxia-mediated kidney tubular epithelial cell injury, a contributing factor to kidney disease progression, using a human pluripotent stem cell (hPSC)-derived hypoxic kidney organoid model. Hypoxic organoids showed increased expression of Hypoxia Inducible Factor (HIF)-associated transcriptional targets, decreased tricarboxylic acid (TCA) cycle metabolites and mitochondrial {beta}-oxidation protein expression, and activated unfolded protein response. A transcriptional signature derived from hypoxic organoids 1) identified a subgroup of individuals whose kidney disease subsequently progressed, and 2) correlated with worse tubular injury. Dapagliflozin enhanced mitochondrial stress response resulting in reversed hypoxia-induced tubular epithelial cell apoptosis, reactive oxygen species (ROS) accumulation, and organoid fibrosis. These results indicate that dapagliflozin may contribute to improved kidney disease outcomes by attenuating hypoxia-induced metabolic stress-mediated tubular epithelial cell injury.

cell biology↗

bioRxiv2025

Myeloid cell recruitment propels right ventricular dysfunction in HFpEF via sterile inflammation.

BackgroundIn contrast to what has already been shown in HFpEF associated left ventricular (LV) diastolic dysfunction, leukocytes role in frequently occurring right ventricular dysfunction (RVD) secondary to HFpEF are so far missing, partially due to the lack of suitable small animal models. Here, we follow a translational research approach by establishing a murine HFpEF model developing manifest RVD and analyzed human HFpEF cohorts to study the mechanistic link between leukocytes and RVD in HFpEF. Methods8-week-old male and female C57BL/6J or Cx3cr1CreER/+R26tdTomato/+ mice were divided into four experimental groups: i) chow, ii) HFpEF (N[{omega}]-nitro-l-arginine methyl ester (L-NAME), 60% high-fat diet), iii) chronic hypoxia (10% O2) and iv) HFpEF and hypoxia (RV-HFpEF) to assess bi-ventricular function and myeloid cell dynamics. To test whether myeloid cells are causally involved in the development of RV remodeling in HFpEF, we additionally treated RV-HFpEF mice with the colony stimulating factor 1 receptor inhibitor PLX-5622 (PLX) to deplete myeloid cells. After 12 weeks, all experimental groups were subjected to transthoracic echocardiography, invasive hemodynamics or flow cytometry. ResultsRV-HFpEF resulted in LV diastolic dysfunction indicated by increased E/E ratio, reduced global longitudinal peak strain, smaller end-diastolic diameters and increased isovolumetric relaxation time compared to chow. RV-HFpEF animals developed RV hypertrophy and RVD evident as increased Fultons index and collagen content as well as elevated RV systolic pressures (RVSPs) and reduced tricuspid annular plane systolic excursion, respectively. Flow cytometric analyses revealed elevated total leukocyte, monocyte, and macrophage counts in RV tissue of RV-HFpEF compared to chow or LV tissue from RV-HFpEF animals. These data were confirmed by unbiased proteomic analyses of RV tissue from RV-HFpEF mice, demonstrating increased abundance of proteins involved in activation of the innate immune system, macrophage chemotaxis, cell adhesion and extracellular matrix organization when compared to LV tissue or other experimental groups. Fate mapping experiments revealed that recruited monocyte-derived macrophages became the main source of total cardiac macrophages in RV tissue from RV-HFpEF mice. Depletion of myeloid cells was associated with rescued RVSP profiles compared to RV-HFpEF control mice. In HFpEF patients, RV dilation was associated with an increased percentage of circulating monocytes. In RV biopsies from HFpEF patients, we found increased expression of adhesion molecules, fibrotic markers and inflammatory transcripts. ConclusionWe demonstrate that dysregulated myeloid cell dynamics are associated with, and directly contribute to, the pathogenesis of HFpEF-associated RVD in humans and mice. Clinical PerspectiveWhat is new: O_LIWe explore myeloid cell dynamics in a novel three-hit experimental HFpEF mouse model with RV hypertrophy, RV end-systolic pressure and RV dysfunction. C_LIO_LIIn this model, RV dysfunction was associated with macrophage expansion, monocyte recruitment and extracellular matrix deposition, whilst macrophage depletion partly reversed these changes and rescued RV hemodynamics. C_LIO_LIHFpEF patients with RV dilation or RV dysfunction exhibit unique leukocyte dynamics and inflammatory profiles when compared to HFpEF patients with normal RV function or diameters. C_LI Clinical implications: O_LIThere exists a major clinical discrepancy between high incidence of RV dysfunction associated to HFpEF and a lack of targeted treatment strategies. C_LIO_LIOur novel three-hit mouse model recapitulates many features of the clinical scenario of HFpEF patients with RV dysfunction, therefore representing an important step towards systematic testing and development of targeted treatment options. C_LIO_LISterile inflammation and dysregulation of innate immune cells may be suitable targets for therapeutic interventions against RV dysfunction in HFpEF. C_LI

physiology↗

bioRxiv2025

A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis and urinary tract infections (UTIs). Here, we report on a human mini-bladder model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonization of the bladder by uropathogenic E. coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy - prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributed to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.

bioengineering↗

bioRxiv2025

Moving Beyond Binary Biomarkers: Machine Learning Model Resolves Concurrent and Molecularly Heterogeneous Mismatch Repair and Homologous Recombination Deficiencies in Prostate Cancer

Current DNA damage repair (DDR) biomarkers employ binary classifications that fail to capture the molecular complexity of tumors with concurrent repair deficiencies. We used genomics analysis to stratify 672 metastatic prostate cancer patients into 11 DDR subgroups, identifying 51 molecular signatures with weighted roles in class identity. We identified a tumor-mutational-burden very-high subset, characterized by 19 mutations/Mb or more, as a molecularly distinct group characterized by preserved genomic integrity and enhanced immunogenicity. Critically, 2.3 percent of tumors exhibited concurrent TMB-High and HRR mutant phenotypes, while 1.5 percent harbored MMR bi-allelic loss without MMRd (mismatch-repair-deficiency) signatures. Clinical validation in 130 patients demonstrated superior immunotherapy responses in tumors with very high TMB levels. We developed CHIMERA DDR, a probabilistic machine learning tool that integrates these 51 genomic features using a nested Random Forest architecture to infer seven clinically relevant DDR subgroups. After negating model overfit concerns, CHIMERA-DDR showed exceptional classification performance (AUCs 0.919-0.999) to accurately detect MMRd and HRR mutant molecular subtypes with or without concurrent DDR deficiencies, resolving admixed phenotypes to enable precision therapeutic stratification beyond binary methods.

genomics↗

bioRxiv2025

Quinolinic acid metabolism may mitigate AKI to CKD transition

The transition from acute kidney injury (AKI) to chronic kidney disease (CKD) remains a significant clinical problem with unclear underlying mechanisms. Emerging evidence suggests that alterations in tryptophan metabolism, particularly in the production of downstream metabolites such as quinolinic acid (QA), play a role in renal pathophysiology. QA is a NAD biosynthesis intermediate metabolized by the enzyme quinolinate phosphoribosyltransferase (QPRT). In this study, we investigated the role of QA in the AKI-to-CKD transition using experimental mouse models and clinical observations and leveraging multiple omics approaches. Systematic metabolomic profiling identified endogenous QA as one of the most significantly elevated metabolites following folic acid-(FA) induced injury. Exogenous QA exacerbated FA-induced kidney dysfunction. Conversely, aged mice deficient in QPRT showed worsened expression of kidney fibrosis markers even in absence of kidney injury, while younger littermates exhibited worsened induced kidney injury. Mice lacking QA-producing enzymes resisted experimental AKI and AKI-to-CKD progression. Multimodal spatial metabolomics analysis of human AKI kidney biopsies revealed QA accumulation in regions of inflammatory infiltration. Finally, children with CKD exhibited higher urinary QA levels compared to healthy controls. These findings underscore QA as a potential mediator of kidney injury and a therapeutic target for preventing the progression from AKI to CKD. One Sentence SummaryQuinolinic acid promotes kidney damage and fibrosis, suggesting it as a contributor of AKI-to-CKD progression and a potential therapeutic target.

pathology↗

bioRxiv2025

Characterization of the cardiac proteome of wild-type transthyretin amyloidosis cardiomyopathy

IntroductionMyocardial accumulation of the protein transthyretin (TTR) can result in amyloid TTR cardiomyopathy (ATTR-CM), a form of restrictive heart disease with limited therapies and still generally poor clinical outcomes. The mechanisms by which TTR fibril accumulation elicits cardiac toxicity at the protein level remain largely unknown. Accordingly, we performed untargeted proteomics of ventricular myocardium from patients with ATTR-CM versus controls. MethodsMyocardial tissue from non-failing (NF) controls (n=7) and ATTR-CM (n=4) were assayed by mass spectrometry. HFrEF, HCM, and HFpEF proteomics were acquired from published databases. ResultsA total of 539/7093 (7.6% of total) proteins were found to be differentially expressed in ATTR-CM, 227/359 (42%) upregulated and 312/539 (58%) downregulated. Gene ontology pathway analysis found that downregulated proteins were enriched for oxidative phosphorylation and mitochondrial protein translation pathways, while upregulated proteins were enriched for enhanced endocytosis and intracellular vesicle mediated transport. The latter is not observed in other forms of heart failure. We further identify a profound downregulation of sarcomere protein content, which is also not seen in other cardiomyopathies. ConclusionThe ATTR-CM myocardial proteome identifies endocytosis and intracellular transport as uniquely upregulated processes, whereas sarcomere protein content is uniquely downregulated. Both maybe potential therapeutic targets.

molecular biology↗

bioRxiv2025

PINK1 regulates cholesterol homeostasis via SCAP phosphorylation in human dopaminergic neurons

Cholesterol is a key lipid enriched in neuronal membranes and essential for signaling and synaptic transmission. An imbalance in cholesterol levels may affect synaptic plasticity and contribute to neurodegeneration. Here, we identify in human dopaminergic neurons a mechanism linking loss of function of the Parkinsons disease (PD) gene PINK1 to altered cholesterol homeostasis. Loss of functional PINK1 impaired SCAP phosphorylation at Ser822 and Ser838, stabilizing SCAP and driving excess cholesterol biosynthesis. Cholesterol accumulated at the plasma membrane and in flotillin-rich lipid rafts, causing reduced neurotransmitter uptake and altering the distribution of dopamine transporter (DAT). Restoring PINK1 expression normalized cholesterol biosynthesis and levels. Moreover, the cholesterol-lowering drugs simvastatin and {beta}-cyclodextrin rescued DAT distribution and neurotransmitter uptake defects. These findings demonstrate that PINK1 influences cholesterol homeostasis through SCAP phosphorylation at Ser822 and Ser838 and that restoring cholesterol levels mitigates phenotypes observed in PINK1 PD neurons. These findings further highlight the cross-talk between mitochondria and lipid homeostasis in PD models, underscoring the relevance of cholesterol levels to dopaminergic functions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/672574v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@165b4d4org.highwire.dtl.DTLVardef@42e3a2org.highwire.dtl.DTLVardef@16ea6f7org.highwire.dtl.DTLVardef@193e170_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

neuroscience↗

bioRxiv2025

VitriFlex: An Open-Source, Modular, and Customizable Robotic Platform for Cryo-EM Grid Preparation

Specimen preparation remains the principal bottleneck in cryo-electron microscopy (cryo-EM). Achieving uniformly thin ice without denaturation, preferred particle orientation, or air- water-interface damage is challenging. The demands of time-resolved experiments and sub-nanoliter sample volumes raise the bar even further. Commercial plungers are closed, high-cost platforms that limit experimental flexibility, constraining users to a few vendor-defined "custom" routines. By contrast, an open, modular and automated system would allow researchers to tailor experimental conditions by tweaking mixing, dispensing, and freezing parameters while lowering barriers to adoption. We present VitriFlex, an open-source cryo-EM grid preparation platform built around an industrial-grade SCARA robot and 3D-printed components. Designed for modularity and ease of assembly, the system enables programmable control of grid handling, sample deposition, and blotting, supporting both standard and time-resolved workflows. VitriFlexs modularity allows flexible implementation of sample delivery strategies, including pre-mixing and on-grid mixing modes, combined with acoustic-assisted spray application. The system allows for spray-to-plunge delays as short as [~]130 ms, enabling initiation of biochemical reactions within sub-second timescales prior to vitrification. Protocols are easily modifiable, and new components can be integrated with minimal redesign. We validated the platform using standard single-particle samples including apoferritin and dGTPase. Additionally, we demonstrate effective mixing capabilities by generating spike-ACE2 and 7-bungarotoxin complexes. In all cases, VitriFlex reproducibly yielded high-quality, collectable grids, allowing us to achieve high-resolution reconstructions. These results highlight VitriFlex as a flexible, accessible solution for both routine and time-resolved cryo-EM applications.

biophysics↗

bioRxiv2025

Molecular characterization of unique multi-domain harbouring fungal rhodopsin for establishing their novel opto-synthetic biological usages

Organisms employ light as an external stimulus for regulating cellular functions. The light-sensitive photoreceptors detect light at varying wavelengths, activating signaling cascades and triggering a range of physiological responses. Rhodopsin is a transmembrane heptahelical protein that functions as an ion channel, or a pump, and sensory receptor, respectively. It consists of a light-sensing chromophore, a retinal that upon absorbing light, initiates a series of signaling pathways of sensory perception, growth and survival. Modular rhodopsin (Different from Rhodopsin-Cyclase Module) has been reported in lower eukaryotes, its identification, characterisation and functional significance in the Fungal Kingdom largely unknown. Here, we report the identification of novel modular rhodopsins in fungi, which highlights their potential usages towards the unexplored opto-biotechnological applications (e.g., biomanufacturing of terpenoids, cytoskeleton regulation, DNA metabolism, light-controlled acetyltransferase, etc.) simply by illumination. Furthermore, identification of novel modular rhodopsins augments the expansion of the new optogenetic tools for a wide range of relevant applications. The structural and homology analysis of these identified domains sheds light on their evolutionary lineage and relatedness with the well-characterised bacteriorhodopsin, sensory and channelrhodopsin. The interactome analysis effector domain coupled with the microbial rhodopsin (Rh) reveals RPEL-mediated gene expression and metabolite regulation, which further modulates the retinol synthesis pathway. The role of the fungal Rh-RPEL effector domain in modulating the terpenoid and sphingolipid metabolism in response to light was successfully elucidated via protein-protein interaction and Biosynthesis Gene Cluster (BGC) analysis. This highlights the potential of these novel opto-synthetic biological usages that can induce the light-dependent production of commercially relevant fungal bioactive(s).

bioinformatics↗

bioRxiv2025

Random survival forests identify myocardial gene signatures associated with survival in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) continues to be poorly understood at the molecular level. While previous studies have identified gene expression signatures unique to HFpEF, genes associated with clinical decompensation have not been determined. Here, we performed exploratory analysis of myocardial RNA-seq data to identify genes associated with event-free survival in HFpEF. We analyzed previously published RNA sequencing data of right ventricular septal endomyocardial biopsies from HFpEF patients (n=41) with paired clinical, echocardiographic, and outcome data (including mortality and heart failure hospitalizations). We constructed random survival forests with forward stepwise regularization (the "variable hunting" method) to determine genes associated with time to first event using a combined end-point of heart failure hospitalization or all-cause death. Selection of candidate forest variables was tested with both random and weighted sampling methods. We identified 33 genes that are predictive of survival in HFpEF. This set includes genes previously implicated in heart failure, including ADAMTSL2, ADRB1, BMP6, and METRNL. Survival forests constructed using these genes outperform those constructed from clinical and hemodynamic parameters alone (out-of-bag C-index 0.894 vs 0.545). Moreover, in survival forests constructed from both gene data and hemodynamic measurements, individual survival genes consistently showed higher variable importance than clinical/hemodynamic parameters. Our study shows that random survival forests identify myocardial gene signatures that may better model HFpEF prognosis than clinical measurements. Further studies are warranted to validate findings in independent cohorts.

genomics↗

bioRxiv2025

Empirical validation of ephaptic coupling in printed human neural circuits

Ephaptic coupling is a phenomenon describing the influence of endogenous electric fields on neuronal activity1,2. Although ephaptic coupling is deemed to contribute to computations in the brain1,3,4, the olfactory system5, the retina6,7, and to cardiac conduction8,9, and being associated with diseases like epilepsy10,11 and arrhythmia12,13, it is still poorly understood since it is notoriously difficult to investigate in vivo and in vitro. In vitro electrophysiology allows accessible and flexible experimentation, but circuits form randomly, leading to a lack of precision and reproducibility. Here, we present a method for reproducibly constructing human neuronal networks in vitro with single-cell precision. We constructed neuronal circuits from the bottom up by bringing their axons into close contact. This enabled us to measure the effects of ephaptic coupling and validate theoretical predictions, such as reduced action potential velocity, increased activity synchronization14-16 and reduced stimulation threshold17. Our precise measurements of electrophysiological activity support the importance of ephaptic coupling in neuronal circuit function. Printed neuronal circuits allow detailed in vitro studies of neuronal interactions and may serve as a platform for disease modeling related to synaptic, ephaptic, or myelination processes.

bioengineering↗

bioRxiv2025

Bisdemethoxycurcumin mitigates Alzheimer disease pathology through autophagy-mediated reduction of senescence and amyloid beta

AD pathology is accompanied by increased senescence and reduced levels of autophagy in the brain. We investigated whether pharmacologically inducing autophagy could alter the senescent phenotype and help ameliorate AD pathology. We discovered that Bisdemethoxycurcumin (BDMC), a natural compound found in Curcuma longa, stimulates autophagy in primary astrocytes. We found that autophagy and senescence exhibit an inverse relationship in aging astrocytes, with increased expression of senescent proteins and downregulation of autophagic proteins. However, treatment of aged astrocytes with BDMC reversed the senescent phenotype by ameliorating the impaired autophagy. Interestingly, the senescent phenotype persisted when autophagy was downregulated by knockdown of AMPK. Additionally, BDMC-induced autophagy aided in the removal of amyloid beta that was administered externally to the astrocytes. Further, to validate these results in a mouse model of AD, we confirmed that BDMC can significantly penetrate the blood-brain barrier (BBB) in mice. Therefore, we administered 50 and 100 mg/kg b.w. of BDMC to transgenic 3xTg-AD mice for two months. In their hippocampus, the Control 3xTg-AD animals showed more senescent cells and lower autophagy levels. In contrast, autophagic proteins were significantly upregulated while senescence indicators, such as senescence-associated secretory phenotype (SASP) proteins, were sharply downregulated in the brain of treated animals. Additionally, we discovered that the treated mices hippocampus had a significantly lower amyloid beta load. These molecular changes in the brain were ultimately reflected in the improved working memory and neuromuscular coordination behavior of mice treated with BDMC. This study warrants further evaluation of BDMC for the management of AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/654834v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1176bbforg.highwire.dtl.DTLVardef@a2e2cdorg.highwire.dtl.DTLVardef@1d824f5org.highwire.dtl.DTLVardef@1628a30_HPS_FORMAT_FIGEXP M_FIG This illustration was created by using biorender.com C_FIG

pharmacology and toxicology↗

bioRxiv2025

Single cell transcriptomic analyses of human heart failure with preserved ejection fraction

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a poorly understood, multi-system disease with high morbidity and mortality. To improve our understanding of its underlying biology, we used single-nucleus RNA sequencing (snRNA-seq) to characterize cell-specific gene expression patterns in human HFpEF myocardium. MethodsSeptal myocardial biopsies (2-3 mg) from 30 HFpEF patients and 29 non-failing donor controls were analyzed using the 10X Genomics platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression quantified with CellRanger and CellBender. After quality control, nuclei were clustered and annotated by cell types based on specific marker genes. Differential expression (DE) by cell-type in HFpEF vs controls was performed using limma-voom and functional analysis performed using Gene Set Enrichment Analysis. Data were compared to dilated cardiomyopathy (DCM) using prior snRNA-seq in DCM vs respective controls. ResultsWe successfully demultiplexed pooled myocardial biopsies, assigning >75% of droplets to individual patients. From eight pooled samples (19 HFpEF, 24 controls), we recovered 48,886 nuclei and identified 14 cell types. Cardiomyocytes (5159 differentially expressed [DE] genes, 36%) and fibroblasts (5905 DE genes, 49%) showed the most DE genes, while endothelial cells (2143), pericytes (1812), and macrophages (1405) had fewer. Enriched pathways common to multiple cell types included transcription/translation, immune activation, metabolism, and protein quality control. Of 7848 DE genes identified via pseudo-bulk snRNA-seq, 51% were DE in fibroblasts and 47% in cardiomyocytes, compared to <20% in other cell types. Unlike dilated cardiomyopathy (DCM), sub-clustering fibroblasts did not reveal an activated fibroblast population in HFpEF. Comparative analysis between HFpEF and DCM identified transcriptional differences primarily in cardiomyocytes. ConclusionsThis study demonstrates the power of genotype-based demultiplexing for single-cell transcriptomic analyses of small endomyocardial biopsies and identifies cardiomyocytes as the principal cell type with distinct transcriptional changes in HFpEF versus DCM. These findings, coupled with differential gene expression and functional pathway analyses, illuminate HFpEF pathways and may nominate compelling targets for future mechanistic studies and therapeutic efforts for HFpEF. Clinical PerspectiveWhat is new? We successfully used genotype-based demultiplexing to perform single nucleus RNA-seq from myocardial biopsies. The snRNA-seq analysis revealed distinct enrichment of pathways related to transcription/translation, immune activation, metabolism, and protein quality control across multiple cell types in HFpEF. In contrast to DCM, HFpEF is distinguished by the absence of an activated fibroblast population and a predominance of transcriptional differences within cardiomyocytes, highlighting a distinct disease mechanism. What are the clinical implications? The cell-selective HFpEF myocardial transcriptional landscape highlights altered metabolism, protein translation and quality control, immune activation and growth/matrix organization. These pathways provide compelling new targets for developing effective treatments.

bioinformatics↗

bioRxiv2025

β-Hydroxybutyrate enhances brain metabolism in normoglycemia and hyperglycemia, providing cerebroprotection in a mouse stroke model.

Hyperglycemia in poorly controlled diabetes is widely recognized as detrimental to organ dysfunction. However, the acute effects of hyperglycemia on brain metabolism and function are not fully understood. The potential protective benefit of ketone bodies on mitochondrial function in the brain has also not been well characterized. Here, we evaluated the acute effects of hyperglycemia and {beta}-hydroxybutyrate (BHB) on brain metabolism by employing a novel approach leveraging adenosine triphosphate (ATP)-dependence of bioluminescence originating from luciferin-luciferase activity. Oxygen consumption rate was measured in ex vivo live brain punches to further evaluate mitochondrial function. Additionally, we investigated the functional relevance of BHB using an in vivo photothrombotic stroke model to assess its cerebroprotective effects. Our data demonstrate that brain metabolism in mice is affected by acute exposure to high glucose, at a level similar to consuming food or a beverage with high sucrose. This short-term effect of glucose exposure was reduced by co-administration with the ketone body BHB. Moreover, BHB significantly reduced infarct size in the brain stroke model, providing evidence for its functional protective role in the brain. These findings suggest that BHB may effectively mitigate the adverse effects of metabolic stress and ischemic events on brain metabolism and function.

neuroscience↗

bioRxiv2025

Inhibition of methylthioadenosine phosphorylase provides protection from experimental acute kidney injury

Acute kidney injury (AKI) increases mortality risk and predisposes individuals to chronic kidney disease. Metabolic pathways play a crucial role in AKI pathophysiology. Here, we investigate the potential of methylthioadenosine phosphorylase (MTAP) inhibition as a novel renoprotective strategy in AKI. Using AKI mouse models, we demonstrate that a small molecule MTAP inhibitor significantly reduces kidney injury markers and improves renal histology. RNA sequencing reveals that MTAP inhibition modulates pathways associated with inflammation, oxidative phosphorylation, and cell survival. Additionally, analysis of human single-cell RNA sequencing data links MTAP expression to kidney injury marker in AKI. This study provides evidence of MTAP inhibition as a potential therapeutic strategy for AKI, highlighting metabolic dysregulation as a target for future clinical interventions.

pathology↗

bioRxiv2025

Clonal memory of colitis accumulates and promotes tumor growth

Chronic inflammation is a well-established risk factor for cancer, but the underlying molecular mechanisms remain unclear. Using a mouse model of colitis, we demonstrate that colonic stem cells retain an epigenetic memory of inflammation following disease resolution, characterized by a cumulative gain of activator protein 1 (AP-1) transcription factor activity. Further, we develop SHARE-TRACE, a method that enables simultaneous profiling of gene expression, chromatin accessibility and clonal history in single cells, enabling high resolution tracking of epigenomic memory. This reveals that inflammatory memory is propagated cell-intrinsically and inherited through stem cell lineages, with certain clones demonstrating dramatically stronger memory than others. Finally, we show that colitis primes stem cells for amplified expression of regenerative gene programs following oncogenic mutation that accelerate tumor growth. This includes a subpopulation of tumors that have exceptionally high AP-1 activity and the additional upregulation of pro-oncogenic programs. Together, our findings provide a mechanistic link between chronic inflammation and malignancy, revealing how long-lived epigenetic alterations in regenerative tissues may contribute to disease susceptibility and suggesting potential therapeutic strategies to mitigate cancer risk in patients with chronic inflammatory conditions.

cancer biology↗