Biology subjects

Kumari, R.

Publications and source records attributed to Kumari, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Validated LC-MS/MS quantification of kynurenine pathway metabolites and pathway-balance ratios in brain tissue and microglial culture supernatant: application to neonatal stroke

Kynurenine pathway (KP) metabolite imbalance is implicated in perinatal brain injury, and KP metabolites are measured in human plasma and cerebrospinal fluid as markers of neuroinflammation. Methods validated in neonatal brain tissue or glial culture supernatant to the same standard are scarce, limiting comparisons across preclinical and clinical measurements. We optimised LC-MS/MS detection of tryptophan (TRYP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), anthranilic acid and picolinic acid after protein precipitation and Enhanced Matrix Removal-Lipid clean-up, validated it against Clinical and Laboratory Standards Institute (CLSI) acceptance criteria in brain tissue homogenate and cell culture supernatant, and applied it to a rat model of neonatal stroke and immune-activated BV2 microglia, reporting KYN/TRYP, KYNA/KYN, 3-HK/3-HAA and QA/KYNA ratios. The lower limit of quantification was 6.53-34.17 nM; imprecision was 0.57-11.47%, accuracy was 84.9-108.3%, recovery was 83.2-112.3%, and matrix effect was 87.8-108.8%, with linear calibration (R2 > 0.99) over 6.5-2000 nM. KYN/TRYP and QA/KYNA ratios were higher in the stroke hemisphere than in control brain at 1, 7 and 14 days (p<0.001), and KYNA/KYN was lower; lipopolysaccharide- and cytokine-stimulated BV2 microglia showed the same change in all four ratios (p<0.01). The assay quantifies eight KP metabolites and four pathway-balance ratios in brain tissue and microglial supernatant within clinical acceptance limits, allowing direct comparison with KP ratios reported in human plasma and cerebrospinal fluid. Changes in these ratios after neonatal stroke identify KYN/TRYP and QA/KYNA as candidate targets for neonatal biofluid studies.

biochemistry↗

bioRxiv2026

BLTP3A dysfunction unleashes lysosomal stress-induced antitumor immunity through STING

High-grade serous ovarian cancer progresses within a lipid-rich ascites microenvironment that engages stress-adaptation programs supporting malignant growth, yet how failed stress resolution influences tumor progression remains incompletely defined. Here, we show that unresolved lysosomal stress restrains tumor progression by sustaining tumor cell-intrinsic STING activity and reorganizing antitumor immunity. We identify BLTP3A as a lysosomal stress-resolution factor in ovarian cancer and show that the common germline missense variant BLTP3A M1098T increases stress-induced lysosomal damage. Patients carrying M1098T had longer overall survival and tumors enriched for cytotoxic T cell-myeloid neighborhoods. In syngeneic tumors, Bltp3a loss or orthologous variant expression delayed progression and generated Cxcl9+ antigen-presenting macrophage niches; M1098T-expressing human tumors similarly promoted interferon-responsive myeloid and T-cell enrichment in humanized mice. Mechanistically, ascites-induced lysosomal stress activated a STING-ATF4-BLTP3A feedback program. BLTP3A dysfunction disrupted CASM-associated lysosomal stress resolution, resulting in prolonged STING activity and sustained tumor-derived IFN-{lambda} without increased cGAMP. Tumor-cell STING and IFN-{lambda} were required for immune remodeling and tumor control. Thus, BLTP3A-dependent lysosomal stress resolution constrains the persistence of stress-induced STING signaling, whereas failure of this response sustains a tumor-intrinsic inflammatory program that organizes protective antitumor immunity.

cancer biology↗

bioRxiv2026

Intratumoral dose heterogeneity promotes adaptive anti-tumor immunity and predicts clinical response to radiopharmaceutical therapy

Radiopharmaceutical therapies (RPT) deliver non-uniform radiation dose in tumors and the impact of this on response is poorly understood. Dose heterogeneity could engender treatment resistance in low dose regions, yet we hypothesize that a broader array of dose-dependent immuno-radiobiological mechanisms in tumor microenvironments (TME) and preservation of immune function in low-dose regions could promote adaptive anti-tumor immunity and response. In murine models, non-uniform lutetium-177 delivering <2.5 Gy to >20 Gy in a TME induced broader immunomodulatory effects and T cell-dependent survival improvement compared to more uniform distributions. Preserving low-dose regions promoted dendritic cell activation and TME infiltration of clonally expanded CD8+ T cells. In three independent cohorts of patients with prostate cancer, heterogeneous tumor dose distribution strongly correlated with improved clinical outcomes. These findings defy expected radiobiological dose-response and define a novel mechanism of action for RPT, supporting clinical investigation of dose distribution for optimizing patient selection and personalized dosing.

cancer biology↗

bioRxiv2026

A Paracrine Dietary Lipid Axis Constrains Antitumor Immunity in Liver Cancer

Overnutrition-related liver dysfunction and cancer are increasingly prevalent and highly resistant to immunotherapy. While metabolic dysregulation is a hallmark of hepatocellular carcinoma (HCC), how nutrient overload impairs antitumor immunity remains unclear. Here, we show that short-term Western diet (WD) exposure drives near-complete loss of CD8 T cell infiltration and antitumor function in HCC. We identify dietary linoleic acid (LA), the most abundant {omega}-6 fatty acid, as the dominant immunosuppressive driver. Cancer cell-restricted FADS2-mediated desaturation of LA to longer-chain {omega}-6 PUFAs drives their accumulation in the tumor interstitial fluid, suppressing infiltrating CD8 T cells via lipid peroxidation. FADS2 inhibition restores CD8 T cell function and sensitizes WD-driven HCC to PD-1-based immunotherapy. Further, the Parkinsons disease-associated deglycase DJ-1 protects LA-handling proteins from methylglyoxal-mediated glycation, sustaining tumoral immunosuppressive PUFA production. Across multiple independent human MASLD-HCC cohorts, LA metabolic activity correlates with CD8 T cell impairment, immune exclusion, and immunotherapy resistance. Overall, these studies identify a dietary lipid axis as a therapeutically actionable vulnerability in WD-associated HCC.

Cancer Biology↗

bioRxiv2026

Pathway-centric multi-omics and functional precision medicine reveal shared drug vulnerabilities in heterogeneous adult Wilms tumor

Wilms tumor, i.e., nephroblastoma, is rare in adults and lacks standardized treatment, complicating clinical decision-making. Within the functional precision medicine study (DEDUCER), we profiled two spatially distinct tumor regions (T1 and T2) of an adult Wilms tumor patient using integrated histopathology, whole-exome sequencing, FFPE transcriptomics, and ex vivo drug screening of short-term cultured patient-derived cancer cells (PDCs) with 528 compounds. Genomic profiling revealed a truncal ASXL1 frameshift and shared F7, UBA1, COL21A1, and ATM variants alongside region-specific alterations: a TP53 mutation and broad copy-number (CN) gains in T1, versus ARID1A and KMT2D stop-gains in copy-neutral T2. Transcriptomics of tumor areas identified convergent activation of the G2/M checkpoint, E2F targets, and mitotic spindle programs across regions, consistent with high proliferation and partially comparable biomarker signatures to those observed in an open-source pediatric Wilms tumor dataset (n = 130). Functional assays uncovered distinct and shared drug vulnerabilities: although ATM alterations were present in both tumors, T1 PDCs showed selective sensitivity to topoisomerase I and BCL-2 inhibition in the context of an additional T1-specific TP53 alteration, while broader single-agent sensitivity and stronger drug synergies were observed in T2. Pathway-centric data integration indicated that differential gene expression and copy-number gains, rather than single mutations alone, better predicted ex vivo drug responses, revealing actionable shared dependencies despite pronounced spatial heterogeneity and establishing a translational framework for individualized management in this rare disease. HIGHLIGHTS- In the adult Wilms tumor, multi-region genomics revealed a truncal ASXL1 frameshift together with F7, UBA1, COL21A1 and ATM mutations across two tumor regions (T1 and T2), as well as region-specific alterations: TP53 mutation and widespread copy-number gains in T1, versus ARID1A and KMT2D stop-gains in copy-neutral T2, illustrating spatial heterogeneity. - Transcriptomics showed convergent activation of E2F targets, G2/M checkpoint, and mitotic spindle programs in both regions, consistent with high proliferation and aligning with Wilms tumor signatures (TARGET dataset); these pathways were associated with higher ex vivo drug sensitivity scores. - Functional drug sensitivity testing of patient -derived cancer cells ex vivo uncovered distinct and shared vulnerabilities: Although both tumors shared an ATM mutation, T1-specific TP53 alteration and death-pathway/stress-response alterations may underlie selective sensitivity to topoisomerase I inhibitors and BCL-2 inhibition. - Clinically relevant combinations, including vincristine plus dactinomycin and doxorubicin plus dactinomycin, showed ex vivo synergy. These findings are consistent with the patients more than five-year relapse-free outcome following vincristine, doxorubicin, and dactinomycin treatment combined with surgery, supporting the translational relevance of the ex vivo drug testing approach. - Pathway-centric integration (copy-number gains and differential expression) predicted drug response better than single-gene biomarkers. Overall, pathway-level dependencies provide robust, actionable targets despite genomic and phenotypic heterogeneity in adult Wilms tumor.

Cancer Biology↗

bioRxiv2026

Systematic prediction and functional analysis of amino acid residues determining product specificity in the plant oxidosqualene cyclase superfamily

Oxidosqualene cyclases (OSCs) catalyse one of natures most intricate enzyme reactions, converting the linear precursor 2,3-oxidosqualene into an array of cyclic triterpene scaffolds through sequential carbocation cascades. Predicting OSC function based on sequence is challenging beyond broad family-level classification. Here, we develop a structure-based computational framework to identify amino acid determinants of OSC product specificity. Using 169 functionally characterised OSCs, we deploy a multifaceted approach combining differential conservation along with structural information, physico-chemical properties of amino acids and binding pocket electrostatics in order to understand the determinants of product specificity. Using Arabidopsis thaliana cycloartenol synthase AtCAS as a model, we then validate our predictions through targeted mutagenesis, achieving stepwise reprogramming towards the protosteryl-type products cucurbitadienol and lanosterol, including complete product switches. Molecular dynamics simulations support a mechanism in which subtle pocket remodelling alters active-site volume, water access and proton-elimination chemistry. These findings provide a blueprint for OSC engineering.

plant biology↗

bioRxiv2026

Non-enzymatic RNA Glycation is a Metabolic Sensor of Cellular Stress

Non-enzymatic RNA modifications expand the epitranscriptome, encoding a rapid and chemistry-driven response to cellular stress. While methylglyoxal, a reactive glycolytic byproduct of metabolic stress, has been shown to modify proteins and DNA, its impact on RNA has remained unexplored. Here, we identify mRNA as a dynamic substrate of MGO, whose modification is actively regulated by DJ-1 and the glyoxalase detoxification system. We show that mRNA glycation impairs translation and engages both the integrated stress response and the ribotoxic stress pathway, culminating in compromised pancreatic {beta}-cell function and reduced insulin secretion. Notably, this phenotype is alleviated by the frontline antihyperglycemic agent metformin. Together, our findings position mRNA as a direct sensor of metabolic stress and establish RNA glycation as a mechanistic link between glycolytic imbalance, translational stress and disease.

molecular biology↗

bioRxiv2026

Site-Dependent Decoupling of Drug-Biomarker Associations in Clear Cell Renal Cell Carcinoma Revealed by Functional Profiling of Patient-Derived Cell Models

Clear cell renal cell carcinoma (ccRCC) frequently exhibits primary and acquired resistance to standard-of-care therapies, underscoring the need for improved strategies to predict therapeutic response and prioritize patient-specific treatments. Although recent multi-omic and single-cell studies have provided insight into the molecular landscape of ccRCC, molecular alterations alone incompletely predict drug sensitivity. We prospectively profiled tumors from 28 patients with localized and metastatic ccRCC by integrating molecular characterization, functional drug sensitivity and resistance testing in patient-derived cell models, and longitudinal clinical data. Although genomic biomarkers suggested potentially actionable therapies in 27/28 patients, functional testing revealed discordance between genomic actionability and ex vivo drug sensitivity, whereas linear mixed-effects modelling uncovered 16 novel copy-number-based features associated with sensitivity to 11 drugs. Genotype-drug response associations were largely preserved between primary tumors and vena cava thrombi but frequently disrupted in distant metastases. Integrating functional drug testing with multi-omic profiling reveals vulnerabilities not apparent from genomic data alone, refines therapeutic actionability, captures interpatient and intersite heterogeneity in ccRCC, and provides a scalable framework for individualized treatment prioritization.

cancer biology↗

bioRxiv2026

Transitory enhancement of GATA2 chromatin engagement during early erythroid differentiation

Erythroid differentiation requires precise regulation of transcription factor binding to chromatin targets as hematopoietic progenitors relinquish multipotency and activate lineage programs. GATA2 maintains progenitor identity and is thought to be progressively silenced as GATA1 levels rise; however, the precise changes in GATA2 chromatin binding kinetics during this transition remain undefined. Here, we combined live-cell single-molecule imaging in cell lines and primary mouse progenitors with CUT&Tag chromatin profiling to define GATA2 activity during erythropoiesis. Single-molecule tracking resolved two interaction modes: short-lived (<1 s) searching interactions and long-lived (>5 s) binding. Surprisingly, early erythroid differentiation was characterized by a transitory strengthening of long-lived GATA2 chromatin engagement. This manifested as increased residence time of GATA2 bound to chromatin in G1E-ER4 cells and an expansion of the long-lived bound population in HPC7 cells and primary mouse progenitors. This transitory phase of enhanced engagement declined upon further differentiation. Genome-wide mapping identified regulatory elements selectively occupied by GATA2 during this early transition state, revealing promoter-proximal sites enriched for GATA/RUNX motifs and distal elements containing composite GATA/E-box signatures. Together, our imaging and chromatin profiling indicate that GATA2 chromatin engagement is kinetically remodeled at the onset of differentiation, with early recruitment targets partitioning into distinct promoter- and enhancer-associated subclasses. These results support a model in which transcription factor kinetics constitute a dynamic chromatin engagement layer that characterizes the GATA2-to-GATA1 transition.

cell biology↗

bioRxiv2026

A human-in-the-loop explanation framework for morphologically transparent AI predictions from whole-slide images

Deep learning models enable the prediction of clinical endpoints from whole-slide images (WSIs), but many such models function as "black boxes", lacking transparency about whether and which histomorphological patterns drive their predictions, hindering interpretability and clinical adoption. Here we propose a human-in-the-loop explanation framework, MorphoXAI, which provides both local and global interpretability for deep learning models by incorporating human-expert interpretations. At the global level, it reveals the histomorphological patterns on which the model consistently relies to distinguish between classes of WSIs, as well as the patterns associated with confusion between classes. At the local level, it indicates which of these patterns are used in the prediction of an individual WSI and which regions within the slide correspond to such patterns. We validated our method on a deep learning model trained for ovarian tumor histologic subtype prediction. The results show that our framework generates explanations that accurately reflect the histomorphology underlying the models predictions at both global and local levels. For interpretability and clinical utility in diagnostic contexts, human evaluation results showed that our explanations were easy to interpret, rich in diagnostic features, and directly helpful for diagnostic decision-making, thereby enhancing pathologist-AI collaboration. Our work highlights that unifying global and local explanations and grounding them in expert-interpreted morphology enhances the interpretability and verifiability of deep learning models, thereby facilitating the transparent deployment of such models in clinical practice.

pathology↗

bioRxiv2026

Deeper neuronal and glial proteomic insights using an optimized pipeline for proximity labeling proteomics

Proximity-based proteomics using TurboID has enabled cell-type-specific profiling without the need for cell purification, although major bottlenecks in sample lysis, biotinylated protein enrichment, digestion, and mass spectrometry (MS) parameters have limited depth of proteome coverage. Here, we systematically optimized these variables using TurboID-based labeling of BV2 microglia in vitro and brain astrocytes in vivo to define conditions that maximize proteome coverage. In microglia, the optimized protocol using 8 M urea lysis with on-bead S-Trap digestion and data-independent acquisition MS (DIA-MS) identified 4,016 proteins, double the depth of prior studies, and revealed metabolic, ribosomal, lipid-processing, autophagy, and trafficking signatures. Brain astrocyte proteomes were best recovered using SDS lysis with S-Trap digestion and DIA-MS, yielding a proteome of over 3,600 highly enriched proteins, twice the depth of prior astrocyte-TurboID studies. The expanded astrocyte proteomes captured canonical astrocyte markers as well as membrane-associated, vesicular trafficking, and presynaptic protein signatures, consistent with labeling of astrocyte-neuron interface regions, including proteins involved in receptor signaling, lipid metabolism, and plasticity at tripartite synapses, and several AD risk proteins. The increased peptide recovery following S-Trap digestion allowed the reduction of starting material to 20 {micro}g protein for DIA-MS, and enabled multiplexed tandem mass tag (TMT-MS) proteomics using even smaller samples. When applied to synaptosomes enriched from mouse brains with neuronal TurboID labeling, our pipeline identified a synapse-specific proteome of 2,529 proteins, revealing synaptic, mitochondrial and disease-relevant signatures not detectable in prior studies. By tackling critical bottlenecks from tissue processing to MS, our optimized pipelines enable cell-type and compartment-specific proximity-labeling proteomics to obtain comprehensive biological and disease-relevant insights across various biological fields.

neuroscience↗

bioRxiv2025

Kv1.3 inhibition alleviates neuropathology via neuroinflammatory and resilience pathways in a mouse model of Aβ pathology

Inhibition of voltage-gated potassium channel Kv1.3 is a therapeutic strategy to curb microglia-mediated neuroinflammation in neurodegeneration, although the cellular and signaling mechanisms of disease-modification by Kv1.3 blockers are unclear. In this study, we delineate protective mechanisms of Kv1.3 blockade in a mouse model of Alzheimers disease (AD) pathology using comprehensive transcriptomics and proteomics profiling of brain, corresponding with neuropathological effects of two translationally relevant Kv1.3 blockers, namely small molecule PAP-1 and peptide ShK-223. Following 3 months of treatment, both molecules reduced Ab plaque burden. Single nuclear RNA seq (snRNA seq) of brain nuclei showed that PAP-1 disproportionately impacted oligodendrocytes and microglia and increased crosstalk between neurons and astrocytes with endothelial cells. In contrast, ShK-223 had pronounced effects on glutamatergic neurons and astrocytes. Both blockers increased expression of myelination genes in oligodendrocytes and synaptic genes in neurons. Neuroprotective effects of PAP-1 were further confirmed by bulk brain transcriptomics and proteomics whereby PAP-1 increased levels of synaptic, cognitive resilience and mitochondrial proteins, while decreasing glial and immune pathways including STAT1/3 phosphorylation. Using proximity labeling and co-immunoprecipitation, we found that Kv1.3 interacts with STAT1/3 in microglia. Using microglial cell lines and primary microglia, we discovered a preferential functional coupling between Kv1.3 and type 2 but not type 1 IFN signaling. Brain-level disease modification by Kv1.3 blockade was reflected in the cerebrospinal fluid (CSF) via reduced levels of neurofilament-light (NEFL) and resilience protein RPH3A, both of which are increased in human AD CSF. Together, this study demonstrates functional links between Kv1.3 channels and type 2 IFN signaling and reveals distinct cellular effects of Kv1.3 blockers in AD pathology that correspond with reduced neuropathology and neuroinflammation, augmentation of resilience and neuro-vascular pathways, along with biomarkers of therapeutic effect.

neuroscience↗

bioRxiv2025

A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury

BackgroundNeonatal brain injuries such as stroke cause focal ischemic lesions that often result in lifelong neurological disabilities, as treatment options are limited. To speed up the discovery of potential therapies, early-phase screening with models that reliably reproduce brain injury, with scalable injury volume and minimal confounders, such as varying anaesthesia duration and painful procedures, is essential. MethodsPostnatal day 10 Sprague-Dawley rats of both sexes, with four litters per group and timepoint, were randomly allocated to delivery of intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and 10 minutes of light-emitting diode illumination through the intact scalp and skull. Infarct progression and reproducibility were assessed at 24 hours, 7 days, and 14 days post-injury. Outcomes included infarct volume and sensorimotor function, and cleaved caspase-3, glial fibrillary acidic protein (GFAP), and ionised calcium-binding adaptor molecule 1 (Iba1) immunoreactivity, with analysis of sex differences. Data were analysed using one-way or two-way ANOVA with Sidaks post-hoc tests. ResultsThere was no mortality due to the infarct, and procedure time was approximately 19 minutes across all groups; the lesion was consistent and supported scalability. The 25 mg/kg dose produced a reproducible cortical infarct (3.74 {+/-} 0.58 mm3; CV = 31%). Lesion size increased with dose and decreased over time (11.15 {+/-} 0.63 mm3 at 60 mg/kg versus 0.05 {+/-} 0.007 mm3 at 14 days; p < 0.0001). Cleaved caspase-3 and glial activation persisted for 14 days, indicating ongoing apoptosis and gliosis. No sex-dependent effects were observed in lesion volume, behaviour, or gliosis. ConclusionsThis refined neonatal photothrombotic ischaemia model is reproducible, scalable, and ethically improved, requiring no skin incision. Its minimal surgical burden, absence of mortality, consistent histopathology, and measurable functional outcomes make it an ideal platform for preclinical screening of neuroprotective and reparative interventions in the developing brain.

developmental biology↗

bioRxiv2025

SNHG10 promote tumorigenesis via miR-150/VEGFA/EGFR/AKT/ERK/mTOR axis and gemcitabine resistance in PDAC

SNHG10 emerged as a key regulator in progression and metastasis of cancers. However, potential of SNHG10 in PDAC tumorigenesis, gemcitabine resistance, and underlying mechanisms remains poorly understood. We observed significant upregulation of SNHG10 in 179 PDAC cases, revealing a positive correlation with clinical stages. Our results showed a significant SNHG10 overexpression in several PDAC cells. Downregulation of SNHG10 significantly decreased the proliferation, clonogenicity, EMT, tumor growth in the xenograft model, and the induction of cell cycle arrest and apoptosis of PDAC cells. Mechanistically, SNHG10 knockdown significantly inhibited the expression of vimentin, N-cadherin, survivin, CDK4, CDK6, cyclin B1, cyclin D1, aurora kinase A, and B, with an increased expression of E-cadherin and p21. RNA Immunoprecipitation data displayed physical interaction among SNHG10, miR-150-5p, and VEGFA in PDAC cells. SNHG10 silencing led to the significant induction of miR-150-5p, which repressed VEGFA expression in PDAC cells. SNHG10 downregulation enhanced gemcitabine sensitivity in PDAC cells. SNHG10 silencing suppressed the phosphorylation of EGFR, AKT, ERK1/2, mTOR, and c-MET pathways. Silencing SNHG10 and its regulated signalling offers a novel prospective therapeutic strategy.

cancer biology↗

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

Simultaneous profiling of native-state proteomes and transcriptomes of neural cell types using proximity labeling

Deep molecular phenotyping of cells at transcriptomic and proteomic levels is an essential first step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcriptome level abundances only modestly correlate with each other, complementary profiling of both is needed. We report a novel method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously from both in vitro and in vivo experimental model systems. This method leverages the ability of biotin ligase, TurboID, to biotinylate cytosolic proteins including ribosomal and RNA-binding proteins, which allows enrichment of biotinylated proteins for proteomics as well as protein-associated RNA for transcriptomics. We validated this approach first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the ground truth, bulk proteomes and transcriptomes. We also show that the effect of a biological stimulus (e.g., neuroinflammatory activation by lipopolysaccharide) can be faithfully captured. We also applied this approach to obtain native-state proteomes and transcriptomes from two key neural cell types, astrocytes and neurons, thereby validating the in vivo application of SPARO. Next, we used these data to interrogate protein-mRNA concordance and discordance across these cell types, providing insights into groups of molecular processes that exhibit uniform or cell type-specific patterns of mRNA-protein discordance.

neuroscience↗

bioRxiv2025

Identification of novel Kv1.3 channel-interacting proteins using proximity labelling in T-cells

Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1) although majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T- cell Kv1.3 interactors included 335 cell surface, T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors of T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. Our studies reveal novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate immune (microglia), providing a foundation for how Kv1.3 channels may regulate immune mechanisms in autoimmune and neurological diseases.

cell biology↗

bioRxiv2024

Non-resolving neuroinflammation regulates axon regeneration in chronic spinal cord injury

Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse, facilitate axon retraction away from lesion boundaries, as well as play a key role in orchestrating the growth-inhibitory glial scar. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects repair and regeneration. We performed transcriptional analysis using the Nanostring Neuropathology panel to characterize the resolution of inflammation into chronic SCI, to characterize the chronic SCI microenvironment, as well as to identify spinal cord responses to macrophage depletion and repopulation using the CSF1R inhibitor, PLX-5622. We determined the ability for macrophage depletion and repopulation to augment axon growth into chronic lesions both with and without regenerative stimulation using neuronal-specific PTEN knockout (PTEN-KO). PTEN-KO was delivered with spinal injections of retrogradely transported adeno associated viruses (AAVrgs). Both transcriptional analyses and immunohistochemistry revealed the ability for PLX-5622 to significantly deplete inflammation around and within chronic SCI lesions, with a return to pre-depleted inflammatory densities after treatment removal. Neuronal-specific transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with macrophage depletion alone. Axon densities significantly increased within the lesion after PLX-5622 treatment with a more consistent effect observed in mice with inflammatory repopulation. PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. We identified that PLX-5622 increased axon densities within the lesion that are histologically identified as 5-HT+ and CGRP+, both of which are not robustly transduced by AAVrgs. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI. HighlightsO_LIMacrophages and microglia repopulate the chronically injured spinal cord after depletion. C_LIO_LICSF1R antagonism in chronic SCI augments the growth of specific axon types in the lesion. C_LIO_LICSF1R antagonism does not augment a PTEN-knockout-induced functional recovery. C_LI

neuroscience↗