Biology subjects

Liu, K.

Publications and source records attributed to Liu, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

CFMF: A Clustering-Free Cell Marker Finder for Single-Cell Transcriptomic Data

BackgroundAccurate identification of marker genes is essential in single-cell RNA sequencing (scRNA-seq) analysis. However, conventional clustering-based annotation is constrained by resolution parameter selection and predefined marker gene databases. MethodsWe developed Clustering-Free Cell Marker Finder (CFMF), a novel computational framework that enables the discovery of marker genes without clustering. CFMF was validated using multiple scRNA-seq datasets and systematically benchmarked against widely used gene selection methods. ResultsWe first validated CFMF using the PBMC3K dataset and found that it not only recovered canonical markers, but also uncovered novel marker genes. When validating with glioblastoma datasets, CFMF successfully recapitulated established subtype signatures. Using scRNA-seq data of human normal lung, we demonstrated its superior sensitivity in rare cell detection even at very low prevalence. We applied CFMF to perform integrative analysis on colorectal cancer scRNA-seq data and defined seven transcriptionally distinct subclones, which includes an LGR5+ population that is strongly associated with metastasis. ConclusionCFMF represents a versatile and robust strategy for marker gene discovery, rare cell detection, and integrative dissection of tumor heterogeneity, thereby advancing single-cell research and facilitating the identification of previously unrecognized cell types.

bioinformatics↗

bioRxiv2025

FLASH Radiotherapy Mitigates Radiation-Induced Lymphopenia and Prevents Immunosuppression via Chk1-STAT3 Axis Modulation in a Preclinical Thoracic Irradiation Model

Background and AimsRadiation-induced lymphopenia (RIL) is a frequent side effect of conventional radiation therapy (CONV RT), due to the high radiosensitivity of circulating lymphocytes. Ultra-high dose rate "FLASH" RT may preferentially spare normal tissue while maintaining tumor control. This study evaluates the impact of single-fraction and multi-fraction thoracic FLASH RT on lymphocyte preservation, apoptosis, and immunosuppressive signaling in mice. MethodsWe compared the immunological impact of thoracic FLASH RT and CONV RT in C57BL/6 mice using single-fraction (17 Gy) and multi-fraction (2 Gy x 5) regimens using the Mobetron (IntraOp). Longitudinal blood sampling was performed at multiple time-points post-irradiation through facial vein bleed with flow cytometry analysis for CD4+, CD8+, CD19+, and NK cells to assess lymphocyte counts, apoptotic lymphocytes through Annexin V staining, and immune suppression by examining regulatory T cells (Tregs) and PD-1/PD-L1 expression. Mechanistic studies included immunofluorescence and Western blot analyses of splenic tissues to evaluate Chk1 and STAT3 signaling pathways. ResultsIn single-fraction RT, FLASH significantly reduced lung and heart fibrosis (p < 0.0001) at 28 weeks post-RT. The FLASH effect was also seen acutely on circulating immune cells, with significantly reduced lymphocyte apoptosis and accelerated recovery of CD4, CD8, CD3, NK, and B cell populations compared to CONV RT in both single-fraction and multi-fraction regimens. Conversely, CONV RT induced long-lasting increases in Tregs and sustained PD-1 and PD-L1 expression on T- and B-cells at 2- and 5-months post-irradiation in both fractionation regimens. Within the spleen, we also found CONV RT induced sustained activation of the Chk1-STAT3 pathway in CD45+ immune cells, which correlates with increased PD-1/PD-L1 expression. ConclusionFLASH RT mitigates RIL, reduces lymphocyte apoptosis, and prevents long-term immunosuppression by reduced activation of the Chk1-STAT3 pathway. These findings suggest FLASH RT may confer immunological advantages over CONV RT to enhance therapeutic efficacy.

cancer biology↗

bioRxiv2025

Adjacent Rare-Earth Separation via a Protein Atomic Ruler

The separation of rare-earth elements (REEs) poses a formidable challenge due to their nearly identical ionic radii and chemical properties. Here, we present a transformative strategy for REE separation using a multimetal ion-stacking metalloprotein framework (MIF) that functions as an atomic ruler, effectively amplifying subtle differences in REE ionic radii. This approach achieves exceptional selectivity, enabling the separation of all 13 adjacent REE pairs with ultrahigh purity (up to 99.999%). Remarkably, the MIF retains its lanthanide-binding capacity over 410 consecutive absorption-desorption cycles, demonstrating outstanding stability. Structural insights reveal that the MIFs densely packed REE coordination environment magnifies minute radius variations--a key mechanism underlying its unparalleled separation efficiency. Our MIF-based strategy offers a sustainable, scalable solution to rare-earth separation in industrial applications.

biophysics↗

bioRxiv2025

Simultaneous Label-free Imaging of Nucleolar Dynamics and Subcellular Metabolic Shifts Across Tissue Contexts

The nucleolus is essential for ribosome biogenesis and for regulating cellular responses to growth and stress, and its relationship to cellular metabolic activity and functional state highlights its potential as a biomarker of cellular health. However, challenges in contrast multiplexing and high-resolution isotropic three-dimensional (3D) imaging hinder the non-invasive, simultaneous assessment of nucleolar activity and subcellular metabolic maps across different tissue contexts, especially in complex 3D environments. To fully harness the nucleoluss potential as a biomarker and diagnostic target, we present a multimodal imaging platform that combines third harmonic generation (THG) imaging with metabolic autofluorescence of NAD(P)H and FAD to study structural and metabolic nucleolar dynamics. Enabled by a high-power multimode fiber source and an axial deblurring network, we achieved [~] 400 nm isotropic resolution in deep 3D imaging and confirmed the high accuracy of our method for label-free nucleolus identification using co-registered immunostaining and electron microscopy. To establish the biological relevance of our approach, we demonstrate that nucleolar stress leads to an unexpected depletion of NADH across cellular compartments. Furthermore, in the human endometrium--where nucleolar dynamics are central to the tissues response to progesterone--our label-free imaging strategy delineated endometrial structures in freshly excised tissues and revealed that progesterone treatment induces distinct changes in nucleolar translocation and metabolic adaptation in organoids derived from diseased patients compared to controls. This capacity to non-invasively visualize and quantify features of the nucleolus and its local metabolic microenvironment at single-cell resolution in human tissues--and dynamically track these changes over time in patient-derived organoids--provides a powerful tool for uncovering the roles of the nucleolus in development, disease progression, and therapeutic response. Together, these findings establish our platform as a significant advance for both fundamental research and organelle-based tissue diagnostics.

cell biology↗

bioRxiv2025

Computational modeling of optogenetic control in human atrial tissue: The role of fibrosis type and illumination strategy

Atrial fibrosis promotes atrial fibrillation by stabilizing spiral waves, with focal fibrosis causing strong anchoring that resists termination. Optogenetics offers a low-energy alternative for rhythm control, but its efficacy in human atrial tissue remains under investigation. Here, we developed a two-dimensional computational model of human atrial tissue expressing the light-sensitive protein GtACR1, coupled with fibroblasts, to simulate spiral wave dynamics under varying degrees (0%-100%) and types (focal vs. diffuse) of fibrosis. We examined the effects of subthreshold illumination area (0%-100%) and spatial intensity profiles (uniform, parabolic, linear, exponential) on termination efficiency, and tested low-intensity periodic stimulation for wave drift induction. Results show that the minimum light area required for termination increases with fibrosis degree. At 25%-50% fibrosis, focal substrates require larger illumination areas than diffuse ones, confirming stronger anchoring. Crucially, the relationship between illumination area and termination time is non-monotonic. An "optimal light range" exists: smaller areas fail to eliminate the wave, while excessively large areas suppress wavefront collisions, prolonging termination. In diffuse fibrosis, low-intensity periodic illumination successfully induces drift and boundary collision, enabling self-termination. In contrast, in focal fibrosis, the spiral wave core remains anchored, and drift cannot be initiated by modulating stimulation frequency or intensity alone. Our findings demonstrate that fibrosis type and extent critically influence optogenetic control efficacy. The existence of an optimal illumination strategy highlights the need for spatially tailored interventions. These results provide a mechanistic basis for developing individualized, low-energy optogenetic defibrillation protocols. Author summaryAtrial fibrillation, a common heart rhythm disorder, is often sustained by rotating electrical waves in the heart muscle. Scarring (fibrosis) in the atria can anchor these waves, making them harder to eliminate. Optogenetics -- a technique that uses light to control genetically modified heart cells--offers a promising low-energy approach to stop these dangerous rhythms. However, how different patterns of scarring affect the success of optical interventions remains unclear. In this study, we developed a computational model of human atrial tissue expressing a light-sensitive protein (GtACR1) to investigate how two major types of fibrosis--localized "focal" scars versus widespread "diffuse" scarring -- influence the effectiveness of light-based rhythm control. We found that focal fibrosis strongly anchors spiral waves, requiring larger illuminated areas for termination, while diffuse fibrosis allows more efficient disruption with smaller light zones. Crucially, we discovered an "optimal light range": too little light fails to stop the wave, but too much can paradoxically prolong termination by suppressing wavefront collisions. Furthermore, we show that low-intensity periodic light can induce drift and self-termination in diffuse fibrotic tissue, but fails in focal substrates due to structural anchoring. Our findings highlight that one-size-fits-all optical strategies are suboptimal, and that personalized illumination protocols--tailored to individual fibrosis patterns--could enable safer, lower-energy defibrillation in the future.

physiology↗

bioRxiv2025

Integrated Multi-Omics Analysis for Molecular Subtyping in NSCLC: A Cohort Study

Lung cancer remains the leading cause of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of cases. Current histopathological classification and driver gene testing provide limited prognostic and therapeutic guidance due to intra-tumoral heterogeneity and incomplete characterization of the tumor microenvironment (TME). Here, we constructed single- and multi-omics molecular classification systems for NSCLC by integrating transcriptomic, genomic, epigenomic, proteomic, and TME data from TCGA LUAD and LUSC cohorts. Single-omics analyses revealed distinct molecular patterns, including DNA methylation subtypes associated with sex and histology. Multi-omics integration identified five consensus subtypes closely corresponding to histology, with three immune-activated (CS2-CS4) and two immunosuppressive (CS1, CS5) subtypes. While overall survival did not differ significantly, progression-free survival analysis highlighted CS4 as a high-risk subtype with KRAS-driven genomic alterations. These findings provide a framework for NSCLC molecular stratification and highlight molecular and immune features that could guide future research on targeted therapies and immunotherapy.

bioinformatics↗

bioRxiv2025

Structural insights and engineering of deep-sea halophilic PET hydrolytic enzymes

Pervasive use of polyethylene terephthalate (PET) poses tremendous challenges for global waste management and environmental sustainability, fueling growing interests in enzymatic degradation as an eco-friendly solution. While PET hydrolases hold significant promise, their industrial deployment is hindered by insufficient performance, particularly under high-salinity conditions raised from high substrate loads. Building on our previous discovery of three deep-sea PET hydrolases (dsPETase01, dsPETase05 and dsPETase06) with exceptional halophilicity and PET-degrading activity, we here present their three-dimensional structures and mechanistic characterization. Structural comparison, site-directed mutagenesis, and domain swapping reveal key structural features and the essential role of C-terminal acidic residues in salt tolerance. Integrating semi-rational protein design, Transformer-based modeling, and disulfide bond engineering, we synergistically enhance the thermostability and catalytic efficiency of dsPETase05. These findings elucidate the unique structural features and salt adaptation mechanisms of deep-sea halophilic PET hydrolases, and inform the future engineering of biocatalysts for application in harsh industrial environments.

molecular biology↗

bioRxiv2025

Pan-Cancer Single-Cell Profiling Uncovers the Biological Characteristics of Cancer-Testis Genes

Cancer-testis genes (CTGs) are attractive immunotherapeutic targets owing to their restricted testicular expression and aberrant activation in cancers. However, their regulatory mechanisms, spatial organization, and clinical utility remain incompletely understood. Here, we leveraged large-scale single-cell and spatial transcriptomic data to perform a comprehensive pan-cancer analysis of CTGs. We established a high-confidence pan-cancer CTG catalog and uncovered a heterogeneous epigenetic regulatory landscape in which X-linked CTGs are predominantly governed by DNA methylation, whereas autosomal CTGs are more strongly associated with chromatin regulators. Building on the observation that CTG activation is a robust pan-cancer hallmark of malignancy, we developed a computational framework that enables rapid malignant cell annotation with performance comparable to established copy number variation-based methods. Spatial transcriptomic analyses revealed that CTG expression in head and neck squamous cell carcinoma is preferentially enriched at the invasive tumor front. Clinically, we highlighted the underappreciated therapeutic potential of CTGs and prioritized CT83 and DCAF4L2 as promising candidate targets for T-cell receptor-engineered T-cell therapy in triple-negative breast cancer and liver cancer, respectively. Our study advances mechanistic understanding of CTG biology and provides a valuable resource for the development of CTG-based immunotherapies.

cancer biology↗

bioRxiv2025

Divergence of Bowman-Birk Protease Inhibitor Family into seed-specific and environmentally responsive subfamilies in Legume and Soybean: Implication for Legume Seed Composition Improvement.

Bowman-Birk inhibitors (BBI) are an ancient class of serine protease inhibitors originating prior to the emergence of the angiosperms. While BBIs have been preserved in the legume (Fabaceae) and cereal (Poaceae) families, they have been lost in many other divergent lineages. However, their underlying molecular evolution and regulation of BBI remain largely uncharacterized. Our study shows that BBIs in legumes and cereals are encoded by two large and divergent gene families. BBI genes in legumes have further diversified into two subfamilies with distinct gene expression patterns. Genes in one legume BBI subfamily are specifically expressed in seeds while BBI genes in the other legume subfamily and cereal do not have significant expressions in any examined tissues including seed, root, leaf and flower. The soybean BBI gene family shows evidence of expansion via whole genome, segmental and tandem duplication. Protein sequence and structural analysis predicts that functional domains for double-headed inhibitory loops and binding abilities to trypsin and chymotrypsin are largely preserved within the soybean BBI family. The seed-specific subfamily genes are specifically expressed at maturation stages and not at embryogenesis stages. The other, non-seed BBI subfamily genes are highly responsive to a distinct spectrum of signals related to abiotic and biotic stresses. Their specific expression under non-essential biological processes for plant growth and development suggests that, although BBIs have been retained in both cereals and legumes, likely due to their role in enhancing plant fitness under natural selection pressures, they are not involved in core developmental processes. This may explain why BBIs were lost in many divergent plant lineages and support their well-established roles in plant adaptation to environmental stress. Having knocked out the seed-specific BBIs through a CRISPR/Cas9 approach, we have successfully generated soybeans which exhibited 69.4 - 73.7% reduction of trypsin inhibitor activity and 76.4 - 79.4% reduced chymotrypsin inhibitor activity. The edited soybean did not show significant changes in key agronomic traits, supporting that the functions of BBIs are not essential. While BBIs in soybean seeds may have a desirable function in natural selection, they are antinutrients from an applied perspective for their use in feed and food. It provides an opportunity to reduce BBIs in seeds for quality improvement. Our findings provide insights into molecular evolution, regulation, and function of BBI in plants, and successfully demonstrate engineering BBI in seeds to result in production of food and feed of higher nutritional value with minimal impacts on the agronomic performance of the plant.

plant biology↗

bioRxiv2025

Antibody immunogenicity prediction and optimization with ImmunoSeq

Therapeutic antibody development faces persistent immunogenicity challenges from anti-drug antibodies (ADA). Identifying peptide fragments presented by major histocompatibility complex is the central challenge in predicting immunogenicity. Here, we presented ImmunoSeq, an interpretable and applicable method for immunogenicity prediction. ImmunoSeq addresses this by deploying complementary k-mer (k=8-12) peptide libraries: a positive library of immunologically safe peptides from fragmented human proteins/antibodies, and a negative library of murine antibody fragments capturing evolutionary-selected immunogenic triggers. For candidate antibodies, we generate all possible k-mer peptides and compute hit rate by summing positive hits (+1.0) and negative hits (-0.2 penalty) normalized against total peptide number. Higher hit rate predicts lower ADA risk, with residue-level resolution enabling precise localization of immunogenic hotspots. ImmunoSeq demonstrated superior ADA correlation and humanness classification accuracy compared to deep learning models, while accurately predicts ADA reductions in humanization, enabling sufficient sequence optimization for humanness. By leveraging dual-library discrimination principles of self/non-self-peptide, ImmunoSeq provides a robust, interpretable solution for immunogenicity prediction and sequence optimization.

immunology↗

bioRxiv2025

RIG-I-dependent tumor-intrinsic type I interferon signaling restricts growth in breast cancer 3D culture

Discovery of key growth drivers that can be targeted for therapy is a central goal in cancer research. While high-throughput CRISPR screens have revolutionized our ability to identify gene dependencies in cancer, most large-scale screens are conducted in two-dimensional (2D) culture systems that fail to recapitulate tumor organization and behavior. To uncover architecture-dependent vulnerabilities in breast cancer, we performed parallel CRISPR interference (CRISPRi) screens in 2D and three-dimensional (3D) cultures of MCF7 cells, an estrogen receptor-positive (ER+) breast cancer model representative of a high risk of relapse, luminal subtype. Knockdown of IFNAR2 and TYK2 conferred a growth advantage in 3D cultures, implicating type I interferon signaling as a tumor-intrinsic suppressor of proliferation in 3D spheroids. Transcriptomic and functional analyses demonstrated that type I IFN signaling is endogenously activated in 3D spheroids via RIG-I-mediated sensing of cytosolic double-stranded RNA, leading to TBK1 activation and induction of interferon-stimulated genes (ISGs). This tumor-intrinsic IFN response slowed proliferation in 3D culture, independent of exogenous stimuli or the presence of immune cells. Analysis of bulk, single-cell, and spatial transcriptomic datasets from breast cancer patients revealed that a subset of tumors exhibit elevated IFN signaling in cancer cells, including in immune-depleted tumor cores, consistent with a tumor-intrinsic IFN signature. Our findings uncover an IFN-mediated growth-suppressive program shaped by 3D tumor architecture, and contribute towards a better understanding of the role of tumor-intrinsic IFN activity.

cancer biology↗

bioRxiv2025

Purity and stability of modified nucleosides in the context of accurate quantification by LC-MS

Accurate LC-MS (liquid chromatography coupled mass spectrometry) analysis of RNA modifications relies on synthetic nucleoside standards whose chemical integrity determines both qualitative identification and quantitative measurements. While the purity of these standards is typically verified prior to use, their long-term chemical stability during storage has not been systematically investigated. Here, we evaluated the stability of 44 canonical and modified ribonucleosides in aqueous solution during storage at -80 {degrees}C and -20 {degrees}C. Initial quality control confirmed the identity of all tested standards and revealed purity issues in selected compounds, including contamination of 1-methyladenosine (m1A) with 6-methyladenosine (m6A) and the presence of S- and R-isomers of 5-(carboxyhydroxymethyl)-2'-O-methyluridine (mchm5Um). Long-term LC-UV-MS monitoring over 12 months showed that 30 nucleosides remained stable, two were stable for at least six months, and 12 exhibited substantial quantitative changes. Seven nucleosides formed detectable degradation products, most of which could be structurally assigned. Quantum-chemical calculations of reaction free energies for deglycosylation, deamination, deacetylation and desulfurization correlated with the experimentally observed stability trends. Based on these results, we propose a practical guideline for the preparation, storage and analytical quality control of nucleoside standards, including recommendations for purity verification by UV spectroscopy and quantitative NMR. These guidelines provide an experimental framework to improve the robustness and inter-laboratory comparability of LC-MS-based RNA modification analysis.

molecular biology↗

bioRxiv2025

FlyTomo: A streamlined software for on-the-fly cryo-ET data processing and diagnosis

Cryo-electron tomography (Cryo-ET) combined with subtomogram averaging (STA) enables the structural elucidation of macromolecular assemblies in their native environments. However, the widespread adoption of cryo-ET has been limited by its labor-intensive, expertise-dependent data processing workflow. Here we present FlyTomo, a software that streamlines data processing from frame alignment to STA with high-throughput for authentic cryo-ET scenarios. During data acquisition, FlyTomo performs real-time diagnosis, enabling prompt feedback on sample quality, microscope performance and structural features. After acquisition, it aggregates diagnostic metrics into an interactive overview, guiding users through data review and refinement. FlyTomo also curates raw and processed data into structured directories to simplify data management and archiving. We validated FlyTomo across a diverse set of authentic cryo-ET samples, including purified enveloped viruses and cryo-lamellae, on multiple microscopes and cameras, achieving structures at resolutions ranging from 3.4 to 5.7 [A]. Collectively, by integrating accuracy, scalability and usability, FlyTomo reduces the technical barrier for in situ structural biology using cryo-ET.

biophysics↗

bioRxiv2025

Impact of elevated temperature on immunity-related hormone signaling in tomato plants

Molecular mechanisms governing the plant-pathogen-environment "disease triangle" are starting to emerge, although less so in agriculturally important species like tomato (Solanum lycopersicum). Here we analyzed defence hormone responses of tomato plants infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 under two different temperatures. Our results showed that tomato plants exhibited temperature-sensitive expression of marker genes associated with salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) pathways, but not ethylene (ET). Our findings highlight the complexity of plant- microbe interactions and the importance of considering environmental conditions when studying plant defence responses.

plant biology↗

bioRxiv2025

Multi-omics evaluation of cell lines as models for metastatic prostate cancer

Cell lines are indispensable tools in prostate cancer research yet their suitability as models for distance metastasis remains incompletely characterized. Here, we conduct a systematic evaluation study using large-scale public multi-omics data. We reveal substantial genomic differences between cell lines and metastatic patient samples, and meanwhile pinpoint cell lines which more closely resemble metastatic prostate cancer. Notably, hypermutation significantly influences the tumor microenvironment, underscoring the importance of considering mutational burden in model selection. Surprisingly, the widely used PC3 cell line exhibits poor transcriptomic similarity to any prostate cancer subtype, revealing a previously unrecognized limitation. Furthermore, we find existing engineered stem-like cell lines fail to faithfully recapitulate the transcriptomic profiles of mesenchymal stem-like prostate cancer, whereas selected organoids exhibit higher fidelity. Our study provides guidance for cell line selection in metastatic prostate cancer research and highlights the urgent need to develop improved cell lines for the mesenchymal stem-like subtype.

bioinformatics↗

bioRxiv2025

Curcumin activates distinct programming of redox metabolism towards differential regulation of gene expression mediated by Nrf1 and Nrf2

AbstractionCurcumin (CUR), a naturally occurring phenolic small molecule, has been extensively applied in the treatment of diverse diseases for over half a century. Currently, this chemical has also been verified to exhibit a wide array of its biopharmacological activities, such as enhancing immunity, possessing antiviral, anti-cardiovascular, and anti-cancer properties. Besides, also as a nutritional supplement, CUR enables inflammatory prevention and enhance the combined efficacy of it with chemotherapy. Such characteristic of safeguarding normal organisms while treating or alleviating diseases is manifested as a common bidirectional regulation and its hormetin effect of phenolic compounds, particularly in response to stress. In this redox process, two antioxidant transcription factors Nrf1 and Nrf2 (encoded by Nfe2l1 and Nfe2l2, respectively) play distinct and crucial roles. Thereby, we investigated their expression profiles of genes regulated by Nrf1 and Nrf2 in different signaling responses modulated by CUR. The resulting evidence has been provided, demonstrating that distinctive cellular metabolisms, molecular pathways, and signaling mechanisms account for Nrf1 and Nrf2, as drug targets. Both factors also play diverse roles in the anticancer effects of CUR on HepG2 cells and xenograft mice. However, the effect of CUR on xenograft tumors in vivo is not entirely satisfactory, although such anti-cancer effect was achieved by promoting Nrf1 expression, it appeared more reliant on Nrf2 (particularly in the absence of Nrf1). As such, we unexpectedly discovered there is not a simple regulatory relationship between CUR and Nrf2. This is supported by substantial inhibition of Nrf2 by CUR in the aberrantly proliferating Nrf1-/- cells, even albeit this chemical stimulates the expression of Nrf1 and Nrf2 in wild-type cells.

cancer biology↗

bioRxiv2025

Spleen-derived Small Extracellular Vesicles Protect against Myocardial Infarction via Mediating Spleen-heart Crosstalk

Background and AimsAcute myocardial infarction (AMI) triggers systemic responses that influence cardiac injury and repair, but protective mediators within the cardiosplenic axis remain incompletely understood. This study aimed to investigate whether spleen-derived small extracellular vesicles (sEVs) exert cardioprotection after AMI, identify critical cargo, and evaluate their clinical relevance. MethodsThe effects of splenectomy, spleen-derived sEVs and pharmacological inhibition of sEVs biogenesis on cardiac injury were evaluated in mice with AMI. Parabiosis, donor splenectomy, and fluorescent labelling traced the origin and myocardial recruitment of circulating sEVs. 4D-proteomics profiled sEVs cargo with a focus on pyruvate carrier-1 (MPC1). ResultsSplenectomy worsened survival, ventricular function, infarct size, and fibrosis in mice with AMI. AMI upregulated splenic extracellular-vesicle pathways, with sEVs release peaking at day 3 (M3D-sEVs). Labelled splenic sEVs preferentially accumulated in ischemic myocardium, confirmed by parabiosis experiments. M3D-sEVs improved survival and cardiac function and reduced infarct size, fibrosis, apoptosis, inflammation, and hypertrophy. In addition, anti-apoptotic effects were reproduced in vitro. Inhibition of sEVs biogenesis decreased circulating sEVs, aggravated injury, and was rescued by M3D-sEVs. M3D-sEVs were enriched in MPC1, and MPC1 neutralization or pharmacological blockade abrogated sEVs-mediated restoration of respiration, ATP generation, and reduction of reactive oxygen species. Plasma sEVs-associated MPC1 was highest in patients with AMI, intermediate in coronary heart disease, and lowest in controls. ConclusionsThe spleen responds adaptively to AMI by releasing MPC1lenriched sEVs that travel to injured myocardium, preserve mitochondrial energetics, and reduce damage, supporting cargolspecific sEVs augmentation and sEVslMPC1 as a potential therapeutic target and biomarker in ischemic heart disease. Structured graphical abstractAcute myocardial infarction activates a spleen-heart axis in which the spleen releases mitochondrial pyruvate carrier 1 (MPC1) enriched small extracellular vesicles that home to the infarcted myocardium, preserve mitochondrial oxidative phosphorylation, reduce ROS, and limit infarct size, apoptosis, fibrosis, and hypertrophy. Circulating sEVs-associated MPC1 might serve as a potential biomarker for estimating risk of ischemic heart disease. Translational perspectiveO_LIOur findings reveal a novel heart-spleen communication axis mediated by splenic small extracellular vesicles (sEVs) following AMI. These vesicular messengers predominantly deliver mitochondrial pyruvate carrier 1 (MPC1), enhancing cardiac mitochondrial energy metabolism in the injured myocardium, ultimately improving post-AMI functional recovery in experimental models. C_LIO_LIA higher plasma sEVs-associated MPC1 level was observed in AMI patients when compared with non-AMI patients. These findings support the clinical relevance of the spleen-heart axis, introduce sEVs-associated MPC1 as a potential circulating biomarker for early myocardial injury and therapeutic target. C_LI

molecular biology↗

bioRxiv2025

Elucidating the Contribution of OVLT Glutamatergic Neurons to Mineralocorticoid Hypertension in TASK-/- Mice

BackgroundAldosterone overactivity intensifies central sodium sensitivity and sympathetic output, driving salt-sensitive hypertension, but specific mechanisms remain incompletely defined. Herein, we aimed to explore the role of organum vasculosum of the lamina terminalis glutamatergic neurons (OVLTGlut) and their hyperexcitability mechanisms in hyperaldosteronism-associated hypertension. MethodsAdult age matched male TASK-/- mice (primary aldosteronism model) and wild-type controls (TASK+/+) mice were used. Neuronal excitability was assessed via patch-clamp techniques. Arterial blood pressure (BP) monitored via telemetry or carotid catheterization. Chronic drug delivery used minipumps. RNA-seq/qPCR profiled gene expression, and intracerebroventricular hypertonic saline tested sodium sensitivity. ResultsIn TASK-/- mice, heightened OVLTGlut activity increased sympathetic outflow and hypertension, mitigated by OVLTGlut neuron ablation. Optogenetic activation of these neurons or their paraventricular nucleus (PVN) / rostral ventrolateral medulla (RVLM) projections acutely elevated BP, with ablation reducing BP selectively in TASK-/- mice. Aldosterone dependence of OVLTGlut-PVN/RVLM neuron hyperactivity was evident in both TASK-/- mice and TASK+/+ mice with chronic aldosterone infusion. Aldosterone chronic infusion enhanced central sodium pressor effects, that were nullified by OVLTGlut-PVN/RVLM neuron lesioning. RNA-seq indicated that aldosterone-induced ion channel expression spectrum changes, including potassium channels and the epithelial sodium channel, underlie the neuronal hyperexcitability. ConclusionOveractivation of OVLTGlut neurons contributes to hypertension in TASK-/- mice through regulation of OVLTGlut-PVN/RVLM circuits. The hyperexcitability of these neurons, possibly due to aldosterone-induced changes in ion channel expression spectrum, contribute to hypertension by amplifying central sodium sensitivity.

neuroscience↗