Biology subjects

Yadav, A.

Publications and source records attributed to Yadav, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A broadly conserved phytoplasma α/β-hydrolase effector, SGP-7, is a catalytically active esterase that hydrolyses strigolactone analogues and alters shoot architecture

Phytoplasmas are obligate, phloem-limited bacterial pathogens that reprogram host development through secreted effector proteins. The effectors characterized to date, including SAP11, SAP54/PHYL1, SAP05 and SWP1, act principally by binding and destabilizing host transcription factors. Although /{beta}-hydrolase-fold phytoplasma proteins with catalytic activity toward lipid substrates have recently been described, no phytoplasma effector has been shown to possess enzymatic activity toward a plant hormone. The effector RY378 from rice yellow dwarf (RYD) phytoplasma induces excessive tillering and dwarfing in rice and alters strigolactone- and auxin-responsive gene expression; its predicted /{beta}-hydrolase domain was proposed to mediate these effects, but the protein was never expressed, purified or biochemically characterized, leaving the mechanism of this effector class unresolved. From the closed genome of 'Ca. P. sacchari' isolate SCGS, associated with sugarcane grassy shoot disease, we identified SGP-7, an orthologue of RY378 that is conserved across at least seven 16Sr phytoplasma groups. SGP-7 adopts a canonical /{beta}-hydrolase fold with a Ser116-Asp229-His261 catalytic triad, and its predicted substrate pocket is comparable in volume to that of the Arabidopsis strigolactone receptor AtD14. Recombinant SGP-7 is a bona fide esterase with a clear preference for short-chain acyl esters, and hydrolyses both the fluorogenic strigolactone probe Yoshimulactone Green (apparent Km 3.55 uM; kcat 9.38 x 10^-3 s^-1) and the strigolactone analogue rac-GR24. Both activities are abolished by substitution of the catalytic serine (SGP-7S116A). Transient expression of SGP-7 in Nicotiana benthamiana reduced plant height by 46%, increased axillary bud outgrowth approximately fourfold, and lowered transcript levels of the strigolactone-responsive marker NbBRC1, reproducing in a dicot host the architectural phenotype reported for RY378 in rice. Together, these results define the biochemical function of a conserved phytoplasma effector family and identify strigolactone-like molecules as direct enzymatic targets of a bacterial plant pathogen.

microbiology↗

bioRxiv2026

Association between breeding-habitat bacterial communities and oviposition behavior of Aedes aegypti: Insights from arboviral outbreak-affected areas of Pune, Western India

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

genomics↗

bioRxiv2026

Dendritic cell PD-L2 restrains intratumoral CD8+ T cell immunity

Blockade of inhibitory PD-1 signaling on T cells is a cornerstone of cancer immunotherapy, with current strategies targeting PD-1 or its ligand PD-L1. However, PD-1 engages an alternative ligand, PD-L2, whose role in tumor immunity remains poorly defined. Here, we show that PD-L2 is upregulated on intratumoral CCR7+ conventional dendritic cells (cDCs) in both mouse and human melanoma. Using genetic mouse models enabling selective ablation of PD-L1 or PD-L2 in cDCs, we identify a division of labor between these ligands: PD-L1 controls the size of the progenitor CD8 T cell pool in tumor-draining lymph nodes by modulating stem-like CD8 T cells, whereas PD-L2 limits progenitor exhausted CD8 T cell differentiation within the tumor microenvironment. Loss of PD-L2 in cDCs enhances cytotoxic CD8 T cell responses and suppresses tumor growth, particularly in tumors enriched for CCR7+ cDC1s. Consistent with this, increased CCR7+ cDC abundance is associated with poor prognosis in human cancers. Spatial transcriptomic analyses reveal co-localization of CCR7+ cDC1s and Tpex within CCL19hi niches, where cancer-associated fibroblasts serve as the predominant source of CCL19. Finally, intratumoral GM-CSF drives PD-L2 expression on CCR7+ cDCs, with Tpex and NK cells as major sources. Together, these findings establish cDC-associated PD-L1 and PD-L2 as spatially and functionally distinct checkpoints governing CD8 T cell differentiation. Our results suggest that the abundance of CCR7+ cDC1s may guide the choice between anti-PD-1 and anti-PD-L1 therapies and support the development of PD-L2-directed blockade.

immunology↗

bioRxiv2026

Targeting Histamine H3 Receptors to Suppress NGF-Driven Hyperexcitability in Neuropathic Pain

Peripheral nerve injury induces long-lasting changes in sensory neurons that contribute to the development of neuropathic pain. Although the histamine H3 receptor (H3R) is best known for regulating neurotransmitter release in the central nervous system, its expression and role in dorsal root ganglia (DRG) remain poorly understood. In this study, we examined the expression, cellular distribution, and functional role of H3 R in DRG and implications in neuropathic pain. Sciatic nerve injury increased membrane-associated H3R protein expression in the DRG, suggesting enhanced receptor trafficking or stabilization at the neuronal membrane. Single-cell transcriptomic analysis revealed that H3R mRNA expression in DRG is predominantly restricted in peptidergic sensory neurons and C-LTMRs neurons. Fluorescence imaging and transcriptomic analysis also suggest that the majority of H3R co-express with TrkA (a NGF receptor) sensory neuron populations. These findings identify a subset of NGF-responsive nociceptors in which H3R may directly influence injury-induced sensitization. Pain behavior assessment demonstrated a paradoxical role of an H3R inverse agonist (GSK334429), which reduces mechanical hypersensitivity caused by chronic constriction injury (CCI) with no change in thermal pain. Patch-clamp recordings show that the GSK334429 attenuates NGF-induced hyperexcitability in DRG neuronal cultures. Overall, our work suggests a contributing role of H3R in modality-specific effects on sensory processing through its enrichment in specific neuronal populations in DRG.

neuroscience↗

bioRxiv2026

Bioactive Spermidine-Crosslinked DNA Hydrogel for rapid homeostasis and accelerated wound healing

Damage to the skin by trauma, burns, or surgical procedures often results in uncontrolled bleeding, which remains a leading cause of preventable death following injury, yet most conventional hemostatic materials are engineered solely to arrest bleeding and often adhere strongly to the wound bed, causing pain, rebleeding, and disruption of newly formed tissue upon removal. Here, we report a DNA hydrogel that structurally mimics neutrophil extracellular traps (NETs) and is crosslinked using a bioactive small molecule with potent autophagy-inducing, cardioprotective, anti-inflammatory, antioxidant, and mitochondria-protective properties, integrating rapid hemostasis with active support for tissue regeneration in a single biomaterial. The DNA network provides an intrinsically biocompatible, biodegradable scaffold capable of recruiting platelets and erythrocytes to achieve rapid clot formation, while the bioactive crosslinker is released as the network degrades, delivering a sustained cytoprotective and anti-inflammatory stimulus directly at the wound site. The hydrogel was characterised physiochemically and evaluated for cytocompatibility, hemolytic potential, hemostatic efficacy, and wound-healing performance in a murine model. Results demonstrate that the bioactive-crosslinked DNA hydrogel achieves rapid, effective hemostasis, while accelerating wound closure and supporting regenerative tissue remodelling. This dual-function platform offers a promising strategy for next-generation wound-care biomaterials that unite immediate bleeding control with accelerated, natural tissue healing.

bioengineering↗

bioRxiv2026

Deep Brain Stimulation Microelectrodes as a Source of Human Subcortical RNA: Validation of a Low-Input Transcriptomic Protocol

BackgroundUnderstanding the molecular basis of Parkinsons disease (PD) phenotypic heterogeneity maybe improved by in vivo access to deep brain tissue. Deep brain stimulation (DBS) surgery offers a unique opportunity: microelectrodes traversing the subthalamic nucleus (STN) carry adherent brain tissue upon withdrawal, providing a source of RNA from subcortical regions in living patients without additional invasive procedures. AimTo develop and validate a low-input RNA extraction and transcriptomic profiling protocol using DBS microelectrodes in post-mortem human brain, to confirm that recovered RNA is of brain rather than blood origin, and to characterise its sub-regional and cellular identity. MethodologyDBS microelectrodes were inserted without image guidance or guide tube into three unfixed post-mortem human brains targeting the STN trajectory. In clinical practice, a guide tube shields the electrode from cortical tissue; its absence here means tissue from the full insertion trajectory may contribute to recovered RNA. Thirty-eight microelectrodes were evaluated across single and pooled strategies. RNA was extracted using a modified RNeasy Micro low-input protocol; libraries prepared using NEBNext Single Cell/Low Input RNA Library Kit and sequenced on Illumina NovaSeq 6000 (paired-end, 2x150 bp). Tissue identity was validated against GTEx v10 (54 tissues) and Allen Human Brain Atlas (ABA, 19 subcortical regions including STN) using Pearson correlation with permutation testing (1,000 permutations) and BH-FDR correction. Transcriptional overlap between subcortical and cortical reference regions was quantified and subcortical-enriched gene filtering performed. ResultsTwenty-five RNA isolates were obtained from 38 microelectrodes; 54.5% of Bioanalyzer-assessed samples achieved RIN [≥]5 (median 7.1; range: 5.9-7.8). Fifteen libraries passed sequencing QC (mean depth 43.0 {+/-} 14.1 million read pairs; mean Q30 83.2 {+/-} 5.6 %). PCA of rlog transformed expression data resolved samples by donor identity. All samples confirmed brain tissue origin by GTEx {tau}-index tissue specificity correlation (n=996 brain and blood specific markers). Brain correlations were highest for frontal cortex (mean r= 0.683 {+/-} 0.072), anterior cingulate cortex (mean r= 0.658 {+/-} 0.069), amygdala (mean r= 0.615 {+/-} 0.068), and basal ganglia including caudate (mean r= 0.553 {+/-} 0.059), putamen (mean r= 0.546 {+/-} 0.059), and nucleus accumbens (mean r= 0.544 {+/-} 0.057); all BH-FDR < 0.05. Cerebellum showed the lowest brain correlations (cerebellar hemisphere: mean r = 0.327 {+/-} 0.039; cerebellum: mean r = 0.314 {+/-} 0.037). Whole blood correlation was strongly negative (mean r= -0.250 {+/-} 0.079), confirming non-haematological origin. ABA genome-wide analysis confirmed positive STN correlation (r=0.54-0.62; permutation p<0.001). MuSiC cell-type deconvolution against the Allen Brain Atlas HMBA-BG snRNA-seq reference identified oligodendrocytes (33.5 {+/-} 17.2%), frontal cortical neurons (9.1 {+/-} 7.0%), STR D1 MSNs (6.0 {+/-} 4.9%), and dopaminergic neurons (1.1{+/-} 2.7%) as the principal cell types, independently corroborating bulk transcriptomic findings at single-cell resolution. ConclusionThis study validates a low-input RNA extraction protocol for DBS microelectrodes, confirming brain-specific transcriptomic profiles consistent with STN-adjacent subcortical sampling. The 96% transcriptional overlap between cortical and subcortical regions, combined with absence of a guide tube in this post-mortem model, limits sub-regional specificity; clinical application with a guide tube would enrich the subcortical signal. These findings provide the methodological framework for in vivo molecular profiling of the human basal ganglia during DBS surgery in PD patients.

genetics↗

bioRxiv2026

Multivalent Surface Search Dynamics Shape Bacteriophage Adsorption Efficiency: A Stochastic Model of Tail Fiber Optimization

Phages must locate and bind to bacterial surface receptors to initiate infection. Their tail fiber configuration critically influences this process. We develop a stochastic model describing surface search as a renewal process, incorporating attachment, detachment, and target-finding steps. Using both numerical simulations and analytical calculations, we quantify how tail fiber number, attachment-detachment rates, and geometric constraints impact the mean and the distribution of time to successful adsorption. Notably, the search efficiency shows a nonmonotonic dependence on tail fibers number, governed by a trade-off between binding stability and diffusion-mediated mobility. This optimum shifts depending on the effective bacterial density, target radius, and fiber reach. Short fiber reach imposes severe geometric constraints, reducing mobility at high tail fiber counts and leading to performance degradation. Our findings suggest that phage adsorption strategies are shaped by a balance between anchoring and exploration, with evolutionary implications for tail fiber design and infection efficiency. SIGNIFICANCEBacteriophages typically use tail fibers to bind and explore bacterial surfaces. Using stochastic modeling and numerical simulations, we uncover how attachment-detachment kinetics and tail fiber geometry shape surface search efficiency. Our results reveal a trade-off: more tail fibers stabilize attachment but hinder exploration, especially with short reattachment ranges. These findings offer a mechanistic basis for understanding how phages optimize infection strategies and may explain the diversity of tail architectures observed in nature.

biophysics↗

bioRxiv2026

A p53-ΔNp73 signaling axis drives selective motor neuron degeneration in spinal muscular atrophy

Selective neuronal vulnerability is a hallmark of many neurodegenerative diseases, yet how ubiquitous genetic insults cause highly selective neuronal loss remains poorly understood. In spinal muscular atrophy (SMA), reduced SMN levels trigger degeneration of specific motor neuron pools. Although non-apoptotic, p53-mediated death pathways have been implicated, p53 is expressed in both vulnerable and resistant neurons, leaving the downstream determinants of selective vulnerability unresolved. Here, we identify a p53-{Delta}Np73 signaling axis as a previously unrecognized execution pathway driving motor neuron degeneration. Using differential transcriptional profiling of SMA motor neurons following pharmacological modulation of p53 activity, we uncover p73 as a critical downstream mediator of neuronal death. Notably, SMN deficiency induces cell-autonomous, p53-dependent expression of the {Delta}Np73 isoform selectively in vulnerable, but not resistant, motor neurons. {Delta}Np73 induction precisely parallels the spatial and temporal pattern of degeneration in mouse models and is also detected in motor neurons from SMA patients. Strikingly, despite its established role as a pro-survival antagonist of p53, depletion of {Delta}Np73 improves motor neuron survival and partially preserves neuromuscular junction integrity in SMA mice. These findings reveal a context-dependent, isoform-specific functional switch in p53 family signaling that redirects a canonical survival factor into a driver of neurodegeneration, identifying a novel molecular mechanism underlying selective neuronal vulnerability in SMA and a potential therapeutic target for neuroprotection.

neuroscience↗

bioRxiv2026

Plasmatic immune extracellular vesicle profiles identify prodromal and early stages of Parkinson's disease

Extracellular vesicles (EVs) hold promise as minimally invasive biomarkers for neurodegenerative proteinopathies, but disease- and stage-specific profiles remain unclear. For this study, we enrolled 378 participants across five centers and the MJFF-BioFIND cohort: 100 healthy controls [HC], 64 isolated REM sleep behavior disorder [iRBD], 41 DeNovo Parkinsons Disease [PD], 89 Late PD, 32 other Synucleinopathies, and 52 Tauopathies. All participants underwent clinical evaluation and blood collection. The 77 subjects from the BioFIND cohort also provided CSF samples. EV concentration and size were assessed by nanoparticle tracking analysis; flow cytometry quantified tetraspanins (CD9/CD63/CD81) and 37 surface markers. Multivariable logistic regression, receiver operating characteristic analyses (ROC), and repeated random forest (rRF) classifiers evaluated diagnostic utility. Late PD showed the highest EV concentrations compared to HC and other disease groups. Participants exhibited distinct EV surface immunophenotypes, with the iRBD group displaying the most extensive immune activation signature vs HC, followed by PD patients. Multivariate logistic regression analysis identified diagnostic marker panels: CD3/CD9/CD25/CD56 for iRBD, SSEA4 for Late PD, CD146/CD209 for Synucleinopathies, and CD8/CD45/CD62P for Tauopathies. ROC confirmed good discriminatory performance, with CD56 emerging as the strongest single predictor for iRBD vs HC, SSEA4 showing high sensitivity for Late PD, and marker combinations providing optimal balance for Synucleinopathy/Tauopathy classification vs HC. In the CSF BioFIND subset, Late PD EVs exhibited increased myeloid (CD1c), adhesion (CD29), activation (CD69), and epithelial (CD326) markers compared to HC. Among these, CD326 was independently associated with Late PD diagnosis. Machine learning classifiers using all 37 surface antigens achieved excellent training performance (91.7-94.3% accuracy for iRBD/Synucleinopathies vs HC) and maintained robust validation accuracy, particularly for iRBD (77.8%) and DeNovo PD (76.6%) vs HC. EV immuno-phenotyping reveals distinct signatures across the neurodegenerative proteinopathies spectrum, with the highest diagnostic utility for prodromal iRBD detection. Longitudinal validation and cell-of-origin refinement represent key next steps toward clinical translation.

neuroscience↗

bioRxiv2026

Disrupted glial-mediated synaptic refinement in Fragile X syndrome

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the loss of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP is a translational regulator and is highly expressed in glial cells, where its role in neural circuit development remains poorly defined. Here, it was observed that Fmr1 knockout mice exhibit reduced synapse size and accelerated eye-specific segregation. To examine which cell-types participate in this process, a multi-omic framework was applied to FXS model mice at postnatal day 7, a critical window for synaptic remodeling in the retinogeniculate pathway, an established model system utilized to study synaptic pruning. Single-cell transcriptomics revealed coordinated alterations in microglia, astrocytes, and neurons in genes linked to synaptic pruning. Computational modeling further demonstrated enhanced astrocyte-to-microglia signaling, particularly through Ephrin A (EphA)- and semaphorin-mediated pathways, while lipidomic profiling revealed reductions in EphA-associated lipid species required for lipid raft stability and receptor localization. Consistent with these observations, a glial engulfment assay indicated that FXS microglia and astroglia over-engulf synaptic material in the lateral geniculate nucleus, supporting the transcriptomic profile. Together, these findings identify impaired glial-driven synaptic refinement as an early mechanistic feature of FXS pathogenesis, highlighting the genes involved in this process as potential therapeutic targets during circuit development.

neuroscience↗

bioRxiv2026

Cryo-EM structures of mycobacterial MCC reveal carrier-domain translocation between catalytic sites

3-Methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme complex that catalyzes an essential step in leucine degradation. In mycobacteria, MCC is formed by AccA1 and AccD1, but its structural organization has remained poorly defined. Here, we report two high-resolution cryo-EM structures of the endogenous, biotin-bound 6{beta}6 complex from Mycobacterium smegmatis. The MCC architecture comprises a central hexameric carboxyltransferase ({beta}/CT) core flanked by trimeric biotin carboxylase (/BC) modules. The structures capture BC- and CT-engaged conformations, in which the biotin carboxyl carrier protein (BCCP) engages either the BC or CT active site, providing structural evidence for long-range carrier-domain translocation. Conformational rearrangements within the BC module suggest a role in regulating BCCP engagement. Together, our findings provide insights into carrier-domain dynamics in mycobacterial MCC.

biochemistry↗

bioRxiv2026

Morphotype-Resolved 3D Morphometry Reveals a Structure-Density-Location Coupling in Mitochondrial Networks

Mitochondrial architecture plays a critical role in cellular function, yet how organelle structure, metabolic density, and subcellular position are jointly remodeled across whole cells remains poorly understood. We applied quantitative 3D soft X-ray tomography to analyze intact INS-1E cells in a native, cryo-hydrated state. By integrating morphometric profiling with voxel-level linear absorption coefficients (LAC) and contour-based radial mapping, we tracked the structural, biochemical, and spatial remodeling of fragmented, intermediate, and interconnected mitochondrial morphotypes under high glucose and Exendin-4 stimulation. High glucose induces morphotype-specific hypertrophy, fission, and perinuclear redistribution of low-density fragments. Co-stimulation with Exendin-4 stabilizes interconnected networks and increases metabolic density at the cell periphery. Morphotype-resolved analysis uncovers a structure-density-location coupling in which mitochondrial shape, macromolecular packing, and radial position shift in concert. These results offer a quantitative framework and high-resolution spatial constraints for whole-cell modeling of organelle dynamics. HighlightsO_LIMitochondrial morphotypes occupy distinct regions during metabolic stimulation C_LIO_LIHigh glucose drives early mitochondrial swelling, fission, and spatial dispersion C_LIO_LIExendin-4 prevents structural collapse and preserves dense interconnected networks C_LIO_LIMorphotype-resolved analysis uncovers a structure-density-location coupling in mitochondrial networks. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/712811v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d495ecorg.highwire.dtl.DTLVardef@1bee80dorg.highwire.dtl.DTLVardef@74df9dorg.highwire.dtl.DTLVardef@104db6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Circulating extracellular vesicles drive microglial senescence and neurodegeneration in Parkinson`s disease

BackgroundExtracellular vesicles (EV), secreted membrane particles involved in cell-to-cell communication, carry important information on immunity and its dysregulation. Recent studies have demonstrated the crucial role of peripheral and central inflammation in causing Parkinsons disease (PD), as well as the involvement of EV in mediating neuron-glial interactions during neurodegeneration. However, the underlying mechanism of plasmatic EV in PD pathogenesis remains unknown. MethodsEV were isolated from pool of plasma of PD patients and age- and sex-matched healthy controls (HC) using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blot, and transmission electron microscopy. SH-SY5Y neurons and HMC3 microglia cells were treated with EV, and their impact was evaluated using flow cytometry and immunofluorescence. Conditioned medium (CM) from EV-treated HMC3 cells was applied to SH-SY5Y neurons to determine indirect neurotoxic effects. Cytokine profiling and senescence-like features of EV-treated HMC3 cells were assessed. Unbiased proteomic analysis of PD-EV and HC-EV were further performed. ResultsPD-EV induced axonal degeneration and cell death in SH-SY5Y neurons and increased levels of TNF-, IL-1{beta}, IFN-{gamma}, IL-8, and CCL11, accompanied by the expression of p16INK4a in HMC3 cells, suggesting a proinflammatory, senescence-associated secretory phenotype (SASP). Enrichment pathway analysis revealed that these changes were mainly related to inflammatory and immune responses. Moreover, CM from PD-EV-HMC3 cells increased apoptotic cell death in SH-SY5Y neurons more than direct PD-EV. Notably, proteomic analysis of PD-EV showed higher expression of proteins involved in complement cascades, immune response, phagocytosis, and post translational protein translation, further supporting the potential of EV to induce inflammatory changes in PD. ConclusionsThis study demonstrates that plasmatic PD-EV contributes to neuronal degeneration by reducing neuronal integrity and indirectly by activating microglia through the secretion of pro-inflammatory, senescence-associated mediators. Circulating EV exerts a role in bridging peripheral inflammation with microglia, modulating neuroinflammatory events.

neuroscience↗

bioRxiv2026

Electrophysiologically Targeted Biopsies Reveal the Transcriptional Landscape of Focal Epilepsy

Up to 30% of patients with epilepsy have intractable seizures, yet the mechanisms of focal ictogenesis remain unclear. Tissue involvement in ictal regions is heterogeneous, with different regions playing distinct roles in ictogenesis, seizure propagation, and resistance to spread. These roles are reflected in electrophysiologic differences between the seizure focus and the ictal penumbra, where evidence of synaptic spread is present but excitatory firing is constrained by largely intact inhibition. Investigation of the disruption of the normal interplay between excitatory and inhibitory activity, thought to underlie ictogenesis across a range of epilepsy etiologies, is limited by network complexity and cellular heterogeneity in human tissue samples. In this study, we relate cellular and molecular alterations to excitatory-inhibitory disruption in network dynamics defined by electrophysiologic features. This work may aid in the identification of clinically relevant tissue biomarkers and support novel preclinical therapeutic approaches for treatment-resistant focal epilepsy disorders. We developed a novel intracranial EEG guided, MRI-localized approach to sample paired biopsies from 11 patients with drug-resistant focal epilepsy with diverse etiologies, which were then studied using single-nucleus RNA sequencing (snRNAseq) and immunohistochemistry (IHC). EEG recorded from stereotactically implanted depth arrays (sEEG) was used to identify regions of epileptic involvement, based on findings from prior simultaneous clinical and microelectrode recordings. This approach addresses the intrinsic heterogeneity due to etiology and cortical architecture through paired, within-patient comparisons. We identified distinct cell-type specific transcriptional signatures that differentiate cellular populations in the seizure focus and ictal penumbra in intractable focal epilepsies. Our findings provide a link between tissue composition and gene expression that correlate with electrographic features in a heterogeneous seizure landscape. Our findings support common pathways of seizure generation and spread that are conserved across disease etiologies. Relative depletion of interneuron populations in the seizure focus supports the hypothesis of disrupted inhibition as a driver of epileptiform activity in the seizure focus. The enrichment of plasticity-associated gene signatures in the penumbra suggests a complex interaction of these regions with the seizure focus, as well as the role of the penumbra in enabling or limiting seizure expansion. This study provides a novel methodology for tissue sampling in epilepsy and uncovers biologically relevant tissue signatures that provide grounds for future work in targeting cellular and molecular alterations present in focal epilepsies.

neuroscience↗

bioRxiv2026

Evaluating Limits of Machine Learning-Assisted Raman Spectroscopy in Classification of Biological Samples

Machine learning (ML)-assisted Raman spectroscopy has become a powerful analytical tool for the classification and identification of analytes; however, technical challenges impacting its detection accuracy have not been investigated. This study explores experimental factors affecting classification performance. Among the evaluated ML models, ML algorithms show minimal impacts on classification accuracy. Instead, experimental factors, including spectral similarity between tested samples and the data quality, dominate detection performance. Increases in spectral noises and spectral similarity significantly reduce classification accuracy. In well-controlled samples with low experimental noise, ML-assisted Raman spectroscopy can discriminate lipid mixtures with a composition difference of 1.85 mol%. To assess the effect of biological heterogeneity, we analyzed single-cell Raman spectra from Saccharomyces cerevisiae strains carrying single, double, or triple gene mutations. Intrinsic cell-to-cell variability introduced substantial spectral differences, severely reducing the accuracy of multiclass classification of these genetically similar strains at the single-cell level. Averaging Raman spectra across multiple cells improved classification accuracy by reducing this spectral variability. We also assess the effectiveness of transfer learning across different Raman spectrometers, specifically by applying a ML model trained on one instrument to another Raman spectrometer. Transfer learning can be improved with proper instrument calibration, highlighting the importance of instrument standardization. Overall, our results demonstrate that data quality and spectral similarity are the primary bottlenecks in ML-assisted Raman spectroscopy. Careful attention to sample preparation, data acquisition, measurement conditions, and instrument calibration is critical to achieving robust and reliable classification performance.

bioinformatics↗

bioRxiv2026

Structural Basis of Substrate Selectivity and Catalysis in the Mycobacterial Long-Chain Acyl-CoA Carboxylase

Long-chain acyl-CoA carboxylase (LCC) is an essential enzyme complex in mycobacteria that generates acyl-CoA precursors for mycolic acid and complex lipid biosynthesis, yet its architecture and mechanism of substrate selection have remained unclear. Here we determine pre- and post-reaction states of the endogenous 868-kDa LCC complex from Mycobacterium smegmatis by cryo-electron microscopy at 2.1-3.7 [A] resolution. These structures visualize ATP-dependent redistribution of the biotin carboxyl carrier protein. LCC assembles into an asymmetric 8:2:4:2 organization of AccA3, AccD4, AccD5, and AccE5, with two biotin carboxylase modules flexibly tethered to a heterohexameric carboxyltransferase core. We define the structural basis of substrate selectivity within the CT core: AccD5 selectively binds the short-chain substrate C3-CoA, whereas AccD4 accommodates the long-chain substrate C16-CoA. In addition, we resolve AccD5-centered assemblies that associate with biotin carboxylase modules yet lack AccD4, providing structural evidence that distinct carboxyltransferase cores can engage shared modules to generate alternative holoenzyme architectures. Together, these findings define LCC and AccD5-centered assemblies as elements of a combinatorial acyl-CoA carboxylase platform and establish the structural principles governing assembly-specific function in mycobacteria.

biochemistry↗

bioRxiv2026

Vitamin B12 Improves Skeletal Muscle Mitochondrial Biology in Aged Mice

Age-related skeletal muscle deterioration is a commonly reported disability among older adults, attributed to several factors including mitochondrial dysfunction, a major hallmark of aging. Therapies to attenuate or reverse mitochondrial decline are limited. Despite identified positive relationships between vitamin B12 (B12) and mitochondrial biology, the impact of B12 supplementation on skeletal muscle mitochondria, in advanced aged, has not been examined. Thus, the impact of B12 supplementation on skeletal muscle mitochondrial biology was examined in (i) aged female mice, given 12 weeks of B12 supplementation (SUPP) or vehicle control, and (ii) in human primary myotubes. In the mouse model, mitochondrial DNA and content were measured with PCR and citrate synthase activity, respectively; mitochondrial morphology was examined using transmission electron microscopy; mitochondrial function was examined using extracellular metabolic flux analysis; and proteins and pathway enrichment was identified with proteomics. In the cell model, ROS and glutathione was measured using luminescent assays. The results demonstrated that SUPP in aged mice increased muscle mitochondrial content and improved morphology. Further, differentially expressed proteins were enriched in TCA cycle, OXPHOS, and oxidative stress pathways. In the cell model, B12 supplementation reduced ROS levels. This is the first study, to our knowledge, examining the impact of B12 supplementation on skeletal muscle mitochondrial biology in aged female mice. Results suggest that B12 supplementation improves mitochondrial biology in aged female mice.

molecular biology↗

bioRxiv2025

Robust ciliary flows protect early Xenopus embryos from pathogens independent of multiciliated cell patterning

During the early stages of development, the skin of the Xenopus embryo is covered by around two thousand evenly distributed multiciliated cells (MCCs). This striking spatial distribution is believed to maximise the generation of superficial flows at the scale of the embryo. However, the specific role of regular MCC distribution and the physiological function of vigorous ciliary activity remain elusive. We investigate the extent to which superficial flows provide protection against external pathogens before the immune system matures, by combining experimental and computational approaches. First, we cultivated epithelial explants in order to quantify the distribution of MCCs, beating frequencies, and three-dimensional fluid flows. We then use these data to validate a computational fluid dynamics model. Using this model, seeded with structural data from whole embryos, our simulations reveal that the collective ciliary beatings create a robust liquid shield along the embryonic flank, which is highly effective at clearing pathogens from the vicinity of the epithelial surface. Through parametric analyses, we further demonstrate that this protective function is remarkably resilient. Indeed, the effectiveness of pathogen clearance is primarily governed by the overall characteristic velocity of the cilia and is less affected by moderate variations in MCC density and spatial organisation. Our findings suggest that, rather than optimising energy consumption, the biological system prioritises functional robustness to ensure reliable protection.

biophysics↗