Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Loss of B cell tolerance at the T2/T3a B cell transition is a convergent pathogenic mechanism in common variable immunodeficiency

Many patients with common variable immunodeficiency (CVID), including those with CTLA4 deficiency, NFKB1 variants and activated PI3K-delta syndrome (APDS), develop autoimmunity that is refractory to treatment. Despite this shared clinical phenotype, a unifying mechanism for the breakdown of B cell tolerance across monogenic forms of CVID has not been established. Here, we demonstrate that patients with loss-of-function NFKB1 variants, like those with CTLA4 variants and APDS, exhibit dysregulated CD4+ T cell expansion, accumulation of transitional B cells, and a relative lack of follicular B cells. In patients with monogenic CVID and clinical autoimmunity, we observed a relative expansion of transitional and activated naive (aN: CD21loCD11chi) B cells in peripheral blood accompanied by a marked increase in the frequency of VH4-34 expressing autoreactive 9G4+ B cells, which expanded between T2 and T3a transitional B cell stages. Single-cell transcriptomic and B cell receptor analysis further revealed a marked expansion of activated T1/2, T3 and extrafollicular activated naive and double negative (DN: IgD-CD27-) B cell subsets in APDS patients. Notably, one B cell subset appeared exclusively in the APDS disease state, characterized by high oxidative phosphorylation in transitional B cells, specifically. In APDS patients, we also observed a clonal expansion of specific extrafollicular class-switched DN B cells, which were clonally derived from activated transitional B cells. DN B cells were also identified in APDS lung tissue, consistent with the contribution of activated, extrafollicularly-derived B cells to tissue inflammation. Together, these findings suggest that in many patients with CVID and autoimmune features, premature activation of autoreactive transitional T2 and T3a B cells induces the survival and expansion, rather than the tolerization and elimination, of self-reactive B cells. This process leads to extrafollicular expansion of autoreactive B cells capable of tissue infiltration. One Sentence SummaryLoss of transitional B cell tolerance and extrafollicular expansion of autoreactive B cells drive autoimmunity in monogenic causes of CVID.

immunology↗

bioRxiv2025

Longitudinal multi-omic profiling uncovers immune escape and predictors of response in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of clonally expanded plasma cells shaped by complex interactions with the immune microenvironment. To investigate immune factors driving treatment response and resistance, we conducted multi-omics profiling including CD138neg single-cell RNA sequencing of 243 bone marrow samples from 102 patients (631,226 cells) and CD138pos bulk RNA and whole-genome sequencing from 209 samples. Longitudinal analyses revealed that interferon gamma signaling impairs T cell memory after autologous stem cell transplant, while naive B cell abundance and immunoglobulin diversity correlated with improved progression-free survival (HR = 0.48, p = 2.3e-4). At disease progression, MM cells upregulated cancer-testis antigens and immune effector genes, with concurrent B cell depletion, enrichment of myeloid-derived suppressor cell genes in monocytes, and T cell exhaustion. These findings highlight dynamic immune-tumor interactions, identifying naive B cell reconstitution as a biomarker of durable response, and cancer-testis antigens as potential targets for high-risk disease at progression. Statement of SignificanceLongitudinal profiling of multiple myeloma and the immune microenvironment revealed dynamic immune-tumor interactions across the disease course. Dysfunctional CD8 T cells limited memory formation post-transplant, while naive B recovery associated with sustained treatment response. At progression, cancer-testis antigen expression associated with immunosuppression, revealing novel mechanisms of immune escape.

cancer biology↗

bioRxiv2025

Stress-Induced Alteration of Small Extracellular Vesicles Drives Amyloid-Beta Sequestration and Exacerbates Alzheimer's Disease Pathogenesis

While small extracellular vesicles (sEVs) are implicated in amyloid-beta (A{beta}) trafficking, the mechanisms governing their interaction with A{beta} aggregates and plaque formation remain unresolved. Here, we report a paradigm-shifting discovery: sEVs undergo dynamic structural remodelling in response to stress, enabling selective binding to A{beta} aggregates-a phenomenon absent under normal physiological conditions. Using multimodal stressors, including mechanical (ultrasonication/agitation), physical (hyperthermia), and biological (oxidative damage), we demonstrate that stress-modified sEVs exhibit high-affinity binding to small A{beta} aggregates (SA) through scaffold reorganization, as validated by super-resolution microscopy and quantitative colocalization assays. Crucially, these remodelled sEVs act as potent carriers, enhancing SA internalization by neuronal cells in vitro. Strikingly, in post-mortem Alzheimers disease (AD) brains and APP-PS1 transgenic mice, sEVs were spatially enriched at amyloid plaque margins, suggesting a direct role in A{beta} sequestration and plaque expansion. Consistent with clinical relevance, sEVs isolated from AD patients exhibited an intrinsic SA-binding capacity, recapitulating stress-induced interactions observed experimentally. Our findings reveal that stress-primed sEVs function as pathological chaperones, binding to and internalizing A{beta} aggregates, thereby accelerating plaque nucleation and disease progression. This study provides the first evidence of stress-mediated sEV plasticity as a critical driver of A{beta} pathology, redefining therapeutic strategies targeting extracellular vesicle biology in neurodegenerative disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/655679v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@107c76borg.highwire.dtl.DTLVardef@1b56848org.highwire.dtl.DTLVardef@d18636org.highwire.dtl.DTLVardef@1c26db4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

A novel molecular sexing method for Greater One-horned Rhinoceros (Rhinoceros unicornis) for reliable demographic assessments

Accurate assessment of demographic parameters is crucial for conserving endangered species. The Greater One-horned Rhinoceros (Rhinoceros unicornis), a sexually monomorphic species, lacks a validated molecular sexing protocol for diverse biological samples. This study established a multi-marker molecular sexing protocol using blood, tissue, horn, and dung samples from all wild populations in India. The optimized multiplex strategy that involves three sex-linked markers: ZF, AMESC, and TSRY, enabled simultaneous amplification of X-and Y-chromosome-specific fragments in degraded samples. Testing with 286 individual rhino samples from seven parks (5 blood, 180 tissue, seven horn, and 94 dung) led to a [~]92% (195 M: 69 F) success rate. Overall, this approach validates a molecular sexing protocol with increased reliability and fewer amplification failures, facilitating large-scale demographic studies. Future research should utilize this method to examine the sex ratio of all existing Indian rhino populations across their range, ensuring their future persistence and management.

ecology↗

bioRxiv2025

High-Throughput Machine Learning-Aided Antibody Discovery for Cell Surface Antigens

Machine learning (ML) has the potential to revolutionize antibody design and selection, but its success depends on access to extensive, well-curated datasets of antibody-antigen interactions. To address this need, we developed a synthetic Fab yeast display library optimized for seamless ML integration, focusing on sequence diversity within the CDRH3 loop. The library incorporates key sequence features derived from human B cell repertoires essential for efficient antibody generation captured in a compact antigen recognition module (ARM) format. Built using the VH1-69 heavy chain and four light chains, the library was evaluated against ten human and murine cell surface antigens, including PD-L1, TIGIT, and ROBO1. This approach yielded hundreds of antibodies with robust biophysical properties, validated for functional performance in flow cytometry and immunohistochemistry. Furthermore, ML analysis identified additional antibodies for ROBO2 and PD-L2 from the aggregate sequencing data, demonstrating utility for hybrid in silico and experimental workflows. We provide a publicly accessible dataset comprising more than 68,000 Fab sequences and 486 characterized antibodies. This study establishes an ML-compatible framework designed to accelerate and streamline antibody discovery and development.

biophysics↗

bioRxiv2025

Molecular plasticity in the flavin binding pocket of BLUF domain evolves first light-gated endonuclease in bacterial system

The bacterium Rubellimicrobium mesophilum possesses a BLUF coupled endonuclease III (BLUF-EndoIII) with potential endonuclease activity. Interestingly, the crucial amino acid residues (tyrosine, histidine and tryptophan) responsible for BLUF photocycle and photodynamics are evolutionarily replaced by phenylalanine (Y5F), asparagine (H27N) and alanine (W87A) residues, respectively. In present communication, we have studied the impact of this evolutionary plasticity on the BLUF photodynamics and associated endonuclease activity. The results obtained showed that the evolutionary plasticity in the BLUF domain influenced various functional aspects of BLUF domain including FAD binding, domain stability, recovery kinetics, and spectral characteristics. The impact of amino acid plasticity on the C-terminal endonuclease (EndoIII) domain was also studied. The evolutionary plasticity induced changes in the flavin binding pocket of the BLUF domain elevated the light-gated endonuclease activity associated with EndoIII domain. The molecular docking analysis and spectroscopic studies also confirmed the substrate-binding ability of the BLUF-EndoIII. The elevated endonuclease activity suggested that the amino acid residues, which are crucial for BLUF photocycle are indeed dispensable and there might exists another electron transfer pathway for BLUF domain activation and regulation of associated endonuclease domain. Considering the role of endonucleases in bacterial defense, the understanding of the BLUF photodynamics, mechanism of signal transfer to the downstream endonuclease domain and associated endonuclease activity might elucidate the first naturally occurring light-gated endonuclease in bacterial system.

biochemistry↗

bioRxiv2025

The Association Between SLIT2 in Human Vitreous Humor and Plasma and Neurocognitive Test Scores

BackgroundSlit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. ObjectiveTo study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. MethodsSeventy-nine individuals with a mean age of 55.79 {+/-} 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. ResultsWe found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E (APOE) genotype. ConclusionsSLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.

neuroscience↗

bioRxiv2025

Lamin A/C Deficiency Drives Genomic Instability and Poor Survival in Small-Cell Lung Cancer through Increased R-loop Accumulation

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNAs role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging. Significance StatementLamin A/C, a key structural component of the nuclear envelope, is frequently lost or mutated in cancer, laminopathies, and aging-related disorders. Lamin A/C loss is associated with genomic instability, but the underlying mechanisms remain incompletely understood. We demonstrate that LMNA loss drives genomic instability by promoting R-loop accumulation through disrupted nuclear pore dynamics and impaired RNA export. These findings reveal a previously unrecognized link between LMNA loss, nuclear envelope dysfunction, and genome instability. Targeting this pathway could help mitigate genomic instability in aging and laminopathies, while leveraging R-loop accumulation may enhance the efficacy of DNA-damaging therapies in cancer.

cancer biology↗

bioRxiv2025

Polymer dynamics reveal stage-wise unfolding and homologous pairing of chromosomes in S. pombe

During meiosis, the exchange of genetic material ("recombination") is essential to bring up the genetic variation in offspring. To carry out the recombination, homologous pairs of DNA have to come in proximity and align. In fission yeast, this is accomplished by the extensive nuclear oscillation that applies pulling force on the chromosomes. In this work, we investigate the competition between the pulling force that tries to facilitate the alignment and the intra-chromatin interactions that assist the chromatin folding. The crucial role played by the intra-chromatin interaction for a range of pulling forces and loci location is highlighted here. Upon comparing our results with the experimental findings, we not only reaffirm the enhancement of contact probability with loci-dependent pulling but also get a meaningful interpretation of the chromosome unfolding and pairing in different stages of movement.

biophysics↗

bioRxiv2025

Stretchable, hair-compatible, and long-term stable wearable EEG system

Electroencephalography (EEG) is a cornerstone in both neuroscience research and clinical diagnostics. However, conventional EEG monitoring faces hardware limitations, particularly its adaptability and stability. Headsets either require complicated wiring or do not have enough stretchability and wearability to comply with the diverse head anthropometry and hair conditions of the user population. Additionally, there is an inherent tradeoff between wet and dry electrodes in capturing high-fidelity signals from hair-covered scalp regions while ensuring continuous and long-term recording quality. Here, we present a Mesh-integrated, Stretchable, and Hair-compatible EEG system engineered to overcome these limitations. By incorporating a kirigami-inspired mesh design and stretchable eutectic Gallium-Indium interconnects, MindStretcH adapts to various head sizes and allows for easy wearing and removal. Moreover, its uniquely designed porous, conical, soft 3D-printed mold, embedded with hydrogel electrodes, effectively penetrates hair layers to deliver low impedance and sustained signal integrity with minimal discomfort. We validate MindStretcH through offline and online EEG-based brain-computer interface tasks over a month, demonstrating its exceptional stability in continuous monitoring and dynamic applications. These results mark a promising advance toward non-invasive neural interfaces in both clinical and everyday use.

bioengineering↗

bioRxiv2025

Immunogenicity and Structural Features of a Stabilized Clade-C HIV-1 Env Derived from a Pediatric Elite-neutralizer

The envelope (Env) glycoprotein derived from circulating viruses in elite-neutralizers (who naturally develop broadly neutralizing antibodies (bNAbs)), serves as potential template for HIV-1 vaccine design. Herein, we report the structure and immunogenicity of soluble clade-C HIV-1 Env trimer (330) derived from a pediatric elite-neutralizer (AIIMS_330). Using SOSIP, NFL and ferritin-nanoparticle (NP) platforms, we engineered immunogens that preserved native-like Env conformation, exhibited high thermostability and nanomolar affinity for diverse bNAbs, with minimal reactivity to non-neutralizing antibodies. Cryo-EM structure solved at 5 [A] resolution of 330-SOSIP trimer-autologous bNAb 44m complex revealed interactions at GDIR motif, N332 supersite along with additional contacts at E293, K337, K446 and glycans N326, N442, N448, as compared to our previously reported BG505-44m structure. Rabbit immunizations with soluble and NP-displayed formats elicited autologous, and heterologous neutralization of tier-1 clade-C viruses. Herein we define a structurally resolved HIV-1 clade C Env that supports multivalent vaccine strategies and provides mechanistic insights toward rational HIV-1 immunogen design.

immunology↗

bioRxiv2025

Globally prevalent Kelch13 mutations increase partial artemisinin resistance and fitness in Bangladeshi Plasmodium falciparum parasites

Artemisinin partial resistance (ArtR), mediated by Kelch13 (K13) gene mutations in Plasmodium falciparum, has caused delayed parasite clearance and, together with partner drug resistance, treatment failures in the Greater Mekong Subregion (GMS). The Chittagong Hill Tracts (CHTs), located in southeastern Bangladesh and bordering Myanmar and India, regions with prevalent K13 mutations, account for approximately 90% of the countrys malaria infections but have not yet reported ArtR-causing K13 mutations. Importantly, however, some isolates from the CHTs have demonstrated moderate in vitro ArtR in the absence of K13 mutations. To assess the potential threat of K13-mediated ArtR in Bangladesh, we proactively evaluated the impact of three prevalent neighboring K13 mutations (F446I, R561H, and C580Y) on ArtR and parasite fitness in parasites isolated from the CHTs. We edited these mutations into two distinct genetic backgrounds: an artemisinin-sensitive strain (CHT-S) and a K13-independent, moderately resistant strain (CHT-R). In these edited lines, we then evaluated ArtR levels and fitness using ring-stage survival assays (RSA), post-drug treatment recovery assays, and competitive fitness assays against isogenic control strains. Prior to genome editing, both isolates were characterized for baseline drug susceptibility, resistance-associated mutations, and population structure. We found that C580Y and R561H mutations, but not F446I, confer increased in vitro ArtR in the CHT-R background already exhibiting K13-independent moderate resistance. All three mutations incurred minimal or no fitness costs in both genetic backgrounds. Notably, R561H, the dominant allele at the Thai-Myanmar border and currently expanding in Rwanda, mediates extreme resistance in the CHT-R background, with mean RSA survival rates of 30.9{+/-}1.9%, an unprecedented resistance level among K13-engineered lines. R561H also showed fitness advantages (0.5% per generation) and the highest growth recovery post-treatment. This represents the first experimental study modeling this K13 mediated ArtR risk in Bangladeshi parasites. In conclusion, we found that indigenous P. falciparum isolates in the CHTs possess inherent genetic potential to sustain high-level ArtR and fitness advantages if K13 mutations emerge, raising urgent concerns for containment and surveillance strategies during Bangladeshs malaria elimination phase.

microbiology↗

bioRxiv2025

Estimating drivers of breast tissue transitions from normal to tumor state

Tumor tissues are characterized by dysregulated gene expression patterns leading to altered cellular pathways and molecular functions as a result of their transition from normal to tumor state. Further, tumor-adjacent normal tissues (TANTs), utilized as a control in cancer research, are not molecularly normal and differ from healthy normal tissues. These TANTs represent a distinctive transitional state betwixt normal and tumor states. However, the mechanism underlying this state transition, expression dysregulation, and perturbed regulation remain largely unexplored and hence elusive. Herein, the transitions of breast tissues from normal and TANT to tumor states were modeled using gene expression and regulation data to estimate key drivers underlying these transitions. As a result, we identified 645 shared driver genes underlying the transitions of breast tissues from the healthy normal state to the adjacent normal and tumor states. Besides, we identified 635 shared driver genes underlying the transitions of TANTs to different subtypes. When we intersected both lists of shared driver genes, a total of 615 commonly shared driver genes across the state transitions were observed. Subsequently, functional annotations of these driver genes revealed their involvement in the growth and maintenance-related activity of cells. Additionally, key pathways associated with cancer pathogenesis, such as Wnt signaling, Notch signaling, NF-kappa B signaling, and PD-L1 expression and PD-1 checkpoint pathway in cancer, were found significantly enriched with these shared driver genes. Thus, the shared driver genes identified across tissue transitions provide ways forward to devise more efficient diagnostic and therapeutic strategies for early and effective disease management.

bioinformatics↗

bioRxiv2025

Maternal obesity programs cardiac remodeling in offspring via epigenetic, metabolic, and immune dysregulations.

Maternal obesity during pregnancy predisposes the offspring to a high risk of developing cardiovascular and metabolic diseases later in life. This study investigated cardiac perturbations caused by maternal obesity by utilizing a mouse model of maternal high-fat diet (HFD)-induced obesity that recapitulates metabolic abnormalities observed in humans. Our study revealed that offspring of HFD-fed mothers (Off-HFD) exhibit a progression of obesity, dyslipidemia, and metabolic inflexibility when compared with offspring of regular diet-fed mothers (Off-RD). Deeper investigation of cardiac function revealed profound functional, metabolic, vascular, and immune perturbations in adult Off-HFD mice, with marked sex-specific differences. Although both male and female Off-HFD mice developed progressive cardiac hypertrophy, male offspring exhibited a more severe phenotype characterized by hypertension, increased vascular stiffness, impaired cardiac function, and myocardial fibrosis. To identify potential mechanisms underlying these changes, we performed DNA methylation analysis in collected hearts of newly weaned and adult offspring. This analysis revealed extensive, sex-dependent alterations in DNA methylation within or nearby genes involved in cardiac development, lipid metabolism, hypertrophic growth, and inflammatory signaling. Importantly, many of these epigenetic alterations persisted into adulthood, suggesting that maternal obesity establishes a durable molecular memory in the offspring heart. Consistent with these findings, transcriptome analysis of adult hearts revealed activation of gene programs associated with heart failure and pathological cardiac remodeling in male Off-HFD mice, whereas female Off-HFD mice showed activation of pathways consistent with adaptive or cardioprotective responses. Together, these findings demonstrate that maternal high fat diet induces early-life epigenetic remodeling in the offspring heart that persists into adulthood and is associated with sex-specific metabolic, functional, vascular, and immune dysregulations. By linking early epigenomic changes to adult cardiac disease susceptibility, this study identifies potential developmental windows for preventive and early therapeutic interventions aimed at reducing cardiovascular risk in offspring exposed to maternal obesity in utero.

molecular biology↗

bioRxiv2025

Development and validation of KASP markers for a novel powdery mildew resistance gene in wheat using BSR-Seq analysis

Wheat powdery mildew occurs as a devastating infection caused by the pathogenic fungi Blumeeria graminis f. sp. tritici (Bgt). The management of disease becomes much more effective when host resistance methods are employed. The WL711-2U/2B line served as the introgressed line to identify genes linked with powdery mildew resistance. This allowed us to look into the potential resistance components in response to powdery mildew infection. The resistance status of the line towards powdery mildew developed from a single dominant gene named PmAT. Bulked segregant RNA-Seq (BSR-Seq) together with previous QTL-Seq SNP markers helped identify strong candidate regions in chromosome arm 2B extending from 531114848 bp to 530235618 bp. The LOD score from this region reached 72.4 with a PVE% of 64.7, thus proving its main role in resistance. The physical position was subsequently locked up, which was different from the previously identified genes on the same chromosome arm in its position, suggesting that it is most likely a new Pm gene. The QTL interval contained five potential genes that we identified during this research. The five candidate genes directs the protein production, which performs functions linked to the pentatricopeptide repeat family as well as B-box-type zinc finger domain, P-loop-containing nucleoside triphosphate hydrolase, and plant peroxidase. HighlightThe identification of a new wheat powdery mildew resistance gene (PmAT) using a combination of BSR-Seq and QTL-Seq analysis, which located five candidate genes specifically on chromosome 2B.

genetics↗

bioRxiv2025

M6A reader protein YTHDF3 regulates cardiomyocyte death and atrophy by modulating the alternative splicing program

BackgroundThe functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. ObjectiveWe aim to uncover the function of Ythdf3 in the hearts. Methods& ResultsHere, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKII{delta}, and its knockdown leads to an increase in CaMKII{delta}A and CaMKII{delta}C isoforms while a decrease in CaMKII{delta}9 isoform. ConclusionCollectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program.

cell biology↗

bioRxiv2025

VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.

cell biology↗

bioRxiv2025

Bimodal genomic approach predicting Semaphorin 7A (SEMA7A) as prognostic biomarker in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy with high mortality and poor prognosis. To elucidate the genetic underpinnings of ACCs, we have analyzed the transcriptome data of 112 ACC tumor samples from patients enrolled in the TCGA and NCI. Among 72 bimodally expressed genes stratifying patients into prognostic groups, we focused on SEMA7A, as it encodes a glycosylphosphatidylinositol-anchored membrane glycoprotein (Semaphorin 7a) regulating integrin-mediated signaling, cell migration and immune responses. We find that high SEMA7A gene expression is associated with poor prognosis (hazard ratio = 4.27; p-value < 0.001). In hormone-producing ACCs, SEMA7A expression is elevated and positively correlated with genes driving steroidogenesis, aldosterone and cortisol synthesis, including CYP17A1, CYP11A1, INHA, DLK1, NR5A1 and MC2R. Correlation analyses show that SEMA7A is co-expressed with the integrin-{beta}1, FAK (focal adhesion kinase) and MAPK/ERK (mitogen-activated protein kinase/extracellular signal regulated kinases) signaling pathways. Immunohistochemistry (IHC) staining demonstrates the feasibility of evaluating SEMA7A in ACC tissues and shows significant correlation between gene expression (RNA-Seq) and protein expression (IHC). These findings suggest SEMA7A as a candidate for further research in ACC biology, a candidate for cancer therapy, as well as a potential prognosis biomarker for ACC patients. Translational relevanceAdrenocortical cancer (ACC) remains a challenging disease primarily due to the scarcity of reliable biomarkers for predicting patient outcomes and informing innovative therapeutic strategies, as well as its rarity, which restricts the scope of clinical trials. In our study, we performed RNAseq and IHC analyses of ACC samples sourced from The Cancer Genome Atlas (TCGA), tissue microarray slide, and National Cancer Institute (NCI) cancer patient samples. Our findings indicate that a substantial proportion of ACC tumors exhibit expression of SEMA7A, a glycoprotein involved in Semaphorin cell surface signaling. Notably, elevated levels of SEMA7A were identified as a poor prognostic biomarker and were associated with activation of the integrin-ERK-MAPK kinase signaling pathways. These results suggest that ACC tumors with high SEMA7A expression should be considered at elevated risk, and SEMA7A may serve as a potential target for immunotherapeutic strategies, including antibody-drug conjugates, T-cell engagers, and/or small molecule inhibitors targeting the MAPK pathway.

cancer biology↗