Biology subjects

Sharma, V.

Publications and source records attributed to Sharma, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Conformational dynamics of ND2 antiporter-like subunit in intact and truncated complex I

Respiratory complex I is a large redox-driven proton pump and contributes to the proton motive force required for ATP generation in mitochondria and bacteria. It has been extensively studied with structural and biochemical approaches leading to enhanced understanding of its three- dimensional architecture. Despite significant progress in deciphering its catalytic function, the redox-coupled proton pumping mechanism remains unclear and extensively debated. The three membrane-bound antiporter-like subunits ND2, ND4 and ND5 are responsible for pumping protons from the negatively charged side (N-side) of the membrane to the positively charged side (P-side). Emerging evidence supports the role of ND5 subunit in releasing pumped protons to the P-side of the membrane, but the question whether ND2 and ND4 also contribute to proton release remains unanswered. Here, we performed atomistic molecular dynamics (MD) simulations of an intact and a truncated model of complex I in which ND5 and ND4 subunits are removed in-silico. The simulation data reveals a previously unobserved conformational change in the P-side region of ND2 subunit that occurs much more easily in a truncated model of complex I, as also estimated based on free energy calculations. The conformational change, in part triggered by the artificial exposure of ND2 subunit to lipid bilayer, results in a local hydration of the region, similar to what has been observed in recent cryo-EM structure of plant CI:CIII2 supercomplex. We discuss the functional relevance of observed conformational transitions and identify residues that can be manipulated with biochemical approaches to shed light on proton pumping by complex I.

biophysics↗

bioRxiv2026

Fast prediction of acidic amino acid sidechain conformations for cryo-EM modeling

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.

biophysics↗

bioRxiv2026

Gram-negative-dominated polymicrobial microbiome of necrotizing soft tissue infections from North India: an integrated culture and 16S rRNA metagenomics prospective cohort study

Necrotizing soft tissue infections (NSTIs) carry 10 to 30% mortality. Current empirical antimicrobial guidance derives almost entirely from Western cohorts dominated by Streptococcus pyogenes and aerobic-anaerobic consortia, yet whether this microbial paradigm applies to tropical, high-antimicrobial-pressure settings has not been tested with culture-independent methods. We did a prospective cohort study of 169 patients with intraoperatively confirmed NSTI at a North Indian tertiary center (2021 to 2024). Wound tissue underwent aerobic and anaerobic culture, QIIME2-based 16S rRNA gene amplicon sequencing (V3-V4), and targeted SYBR Green quantitative PCR (qPCR) for Acinetobacter baumannii and S. pyogenes. The wound microbiota was overwhelmingly Gram-negative and polymicrobial, anchored by A. baumannii (culture, 33.7%; metagenomics, 49.1%; qPCR, 37.9%), Escherichia coli (32.0%), and Klebsiella pneumoniae (20.7%); S. pyogenes contributed only 4.7% of culture-positive cases. Polymicrobial wounds had higher Shannon diversity (2.59 versus 2.33; P = 0.048) and discrete community composition (PERMANOVA R2 = 0.511; P = 0.010). Culture-metagenomics agreement ranged from almost perfect for Escherichia ({kappa} = 0.849) to slight for Streptococcus ({kappa} = 0.131). North Indian NSTIs present a microbial picture distinct from the Western paradigm, with implications for empirical therapy. IMPORTANCENecrotizing soft tissue infections kill rapidly, and physicians must choose antibiotics before laboratory results are available. Globally, treatment guidelines assume the dominant pathogen is Streptococcus pyogenes, a Gram-positive organism, because nearly all molecular evidence comes from Western countries. We show that in a large Indian cohort, the infection is instead dominated by Gram-negative bacteria, especially Acinetobacter baumannii, with S. pyogenes rarely present. Polymicrobial status, not wound location or injury mechanism, most strongly determines community composition. These findings challenge the one-size-fits-all antibiotic approach and argue for region-specific empirical regimens prioritizing Gram-negative coverage in tropical, high-antimicrobial-pressure settings.

microbiology↗

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2026

How stimulation waveform shape affects collective oscillations in the brain networks

Brain oscillations emerge from nonlinear interactions across anatomically connected neural populations, alternating between transiently coordinated and desynchronised states. Transcranial alternating current stimulation (tACS) can modulate these dynamics, but most work has focused on frequency and amplitude, leaving waveform shape comparatively unexplored. Here we used a whole-brain model of delay-coupled Stuart-Landau oscillators constrained by empirical human structural connectivity to determine how sinusoidal, square, triangular, sawtooth and pulsed stimulation reshape spontaneous alpha-band activity. All waveforms were applied at the same frequency and amplitude to the posterior parieto-occipital regions. Network responses were quantified using the Kuramoto order parameter, spectral entropy and metastable oscillatory modes of transient alpha bursts. Sinusoidal and pulsed stimulation produced the strongest effects, increasing global synchrony while reducing metastability and spectral entropy, consistent with a transition from a fluctuation-rich regime to a coherent and spectrally concentrated state. These waveforms also transformed intermittent alpha bursts into prolonged or near-continuous oscillatory episodes. In contrast, square, triangular and sawtooth stimulation reduced synchrony while largely preserving metastability, producing weaker and more fragmented modulation. These findings identify waveform shape as a key determinant of rhythmic stimulation effects and a principled parameter for neuromodulation design.

neuroscience↗

bioRxiv2026

Amino acid repeat mosaics shape protein functional landscape

How combinations of functional units such as repeats and motifs within disordered regions influence protein functions remains poorly understood. Here, we investigate how co-occurring amino acid homorepeats of different types within the same protein (HR-mosaics) shape molecular outcomes. Using a novel evolutionary co-occurrence metric, human HR-mosaics were classified as segregated (each HR independently influences distinct outcomes), concerted-disjunct (both HRs jointly shape protein functionality without modulating each other), or concerted-conjunct (both HRs affect functionality through mutual influence). Molecular studies of naturally occurring polyGly-polyPro mosaic in DDX17 and chimeric mosaics of polyAla and polyHis demonstrate that HR-mosaics influence protein abundance, localisation, mobility and interaction landscape. Segregated HR-mosaics expand functional space, concerted-disjunct additionally confer biological context, and concerted-conjunct mosaics further enable rheostatic regulation between HRs. These design principles underscore amino acid type, length and relative positioning of HRs as central architects of HR-mosaic functionality, with implications in protein design and engineering and understanding repeat-associated pathologies.

Systems Biology↗

bioRxiv2026

Electrophysiological Signature of Stroke Recovery: Investigating EEG Biomarkers for Prognostic Insights

Stroke is a leading cause of long-term disability, often resulting in persistent motor impairments reflecting disruptions in large-scale brain networks. While electroencephalography (EEG) has long been used to monitor neurophysiological changes following stroke, an integrated framework capturing spatiotemporal dynamics would help understand changes over time. In this study, we analysed resting-state EEG from stroke patients at one week (Session 1) and three months (Session 2) post-stroke to investigate electrophysiological biomarkers of motor recovery, indexed by changes in the Fugl-Meyer scale ({Delta}FM ). We quantified spectral properties, focusing on the relative alpha band power, microstate metrics such as mean duration, complexity, and transition probabilities, and measures of metastability and synchrony derived from the Kuramoto Order Parameter. Among all the EEG measures, the longitudinal change in relative alpha power emerged as the strongest single correlate of motor improvement, accounting for the largest proportion of variance among the examined EEG measures. Although metastability and synchrony alone did not reach statistical significance, they showed moderate positive correlations with {Delta}FM, particularly in the alpha and theta ranges, and once combined with alpha power, added 26% in explaining the variance in {Delta}FM . Microstate parameters did not explain additional variance in recovery once alpha power and network-level dynamics were considered. A hierarchical model combining alpha power, metastability/synchrony, and microstates explained over 78% of the variance in {Delta}FM, indicating that stroke recovery involves restoring balanced alpha oscillations and flexible large-scale brain coordination. Future research with larger samples and more frequent longitudinal assessments is required to confirm the prognostic utility of integrated EEG biomarkers for guiding personalised stroke rehabilitation strategies.

neuroscience↗

bioRxiv2026

Elucidating the effect of a rationally designed nanostructured form-switching ASO (NaFASO) for targeting long non-coding RNA to alleviate Japanese encephalitis virus infection

RNA therapeutic modalities such as antisense oligonucleotides (ASOs) have emerged as promising tools to target previously "undruggable" targets. Despite their great promise as precision therapeutic agents, their clinical adoption remains limited due to production costs, sequence-length restrictions, limited structural heterogeneity, and the generation of environmentally hazardous waste during synthesis. Biocatalytic synthesis strategies provide a sustainable alternative; however, their reliance on specialized enzymes and precursors often limits sequence diversity and scalability. To address these limitations, we report the design and biocatalytic synthesis of a novel circular ASO: Nanostructured Form-switching Antisense Oligonucleotide (NaFASO) for targeting Japanese Encephalitis Virus (JEV) infection-associated host long non-coding RNA (lncRNA) JINR1 (JEV-induced non-coding RNA1) in SH-SY5Y cells. The novel modular architecture in NaFASO has been designed to have a metastable stem that separates the functional antisense domain from the splint-padlock circularizing region, ensuring both structural integrity and efficient target engagement. The serum- and nuclease-stable NaFASOs achieved knockdown of the lncRNA JINR1 during JEV infection, resulting in a reduction in JEV replication and neuronal cell death. NaFASO-mediated JINR1 depletion also resulted in downregulation of the JEV replication-associated gene GRP78. Together, these findings establish NaFASO as a first-of-its-kind structure-switching circular ASO platform for combating JEV infection, combining stability, efficacy, and environmental sustainability. Beyond the JEV, the generalizability of this design suggests broad applicability for targeting diverse RNA species implicated in genetic disorders, viral infections, and cancer, thus highlighting a promising paradigm for developing next-generation transformational nucleic acid therapeutics.

biochemistry↗

bioRxiv2026

Shape, Shrink, Spheroid: A DIY High-throughput Spheroid Generation Device

3D spheroids, which closely replicate three-dimensional cell-cell and cell-extracellular matrix interactions, offer superior predictive capabilities compared to conventional 2D monolayer cultures, positioning them as forward-looking platforms in drug testing, cancer biology, and regenerative medicine. However, high-throughput generation of uniform sized spheroids is still a technological challenge. In one hand, the use of conventional ultra-low attachment (ULA) multiwell plates for this purpose is labour intensive and complex. On the other hand, the use of microfabricated facilities demands cutting-edge infrastructure such as clean room, photolithography, and microfluidic setup which are often unavailable for the resource constrained laboratories. In this study, we addressed these problems by developing a low-cost Do-It-Yourself (DIY), polydimethylsiloxane (PDMS) and agarose-based spheroid generation device, capable of producing and maintaining hundreds of spheroids with minimal user intervention. We have demonstrated two variants based on their size, termed here as S1 and S2 devices which fit into 6-well and 12-well plates, and can generate 600 and 1200 uniform-sized spheroids respectively. We validated our device with various cell lines including primary and cancerous cell lines. We further demonstrated the drug testing capabilities of the device by estimating the IC50 value of the anticancer drug Temozolomide on U87-MG. The value was comparable with the same obtained from the spheroids generated using conventional ULA plates. Additional attachment of a perfusion system made the device suitable for long-term spheroid culture without much user intervention. Furthermore, the devices can also be used for the production of spheroids with gradually changing diameters in a controlled manner, resembling a size gradient. This feature is useful for checking the effect of drugs on different-sized spheroids and for co-culturing spheroids with varying cell densities, mimicking the disease architecture. We have co-cultured two types of the placental trophoblast cells, i.e., extravillous trophoblast (HTR-8) and syncytiotrophoblast (BeWo) with varying densities. In summary, this paper demonstrates a unique DIY method for a high-throughput uniform-sized spheroid generation at a fraction of cost which can be deployed to resource-constrained labs.

bioengineering↗

bioRxiv2026

Targeting lncRNA JINR1 with programmable Circular Active Nano DNAzyme (CANDe) suppresses Japanese Encephalitis Virus infection

Oligonucleotide therapeutics such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) enable sequence-specific gene silencing but rely on endogenous cellular machinery and often require extensive chemical modification for stability and efficacy. DNAzymes offer a mechanistically distinct alternative through intrinsic catalytic RNA cleavage; however, their therapeutic translation has been limited by nuclease susceptibility, structural constraints, and synthetic challenges. Here, we report the development of Circular Active Nano DNAzyme (CANDe), an enzymatically synthesized circular DNAzyme platform designed to enhance stability without backbone modification. The therapeutic potential of CANDe constructs was investigated against Japanese Encephalitis Virus (JEV) infection associated host long-noncoding RNA JINR1 (LINC01518). CANDe constructs were generated via splint-assisted ligation and incorporate modular elements, including catalytic cores (8-17 or 10-23), target-binding arms, and structural stems. Circularization conferred marked resistance to exonuclease-mediated degradation compared to linear DNA, maintaining structural integrity under nuclease-rich conditions.,CANDe targeting the lncRNA JINR1 achieved effective JINR1 knockdown in SHSY-5Y with and without JEV infection. This was accompanied by reduced expression JEV RNA and titers. In line with this, CANDe constructs attenuated of virus-induced cytotoxicity and apoptosis. Among the constructs, 10-23-based CANDe targeting the JINR1-1 site exhibited the strongest overall activity. These findings establish CANDe as a modular, modification-free DNAzyme platform that combines catalytic efficiency with enhanced stability, enabling effective host-directed antiviral intervention. This approach highlights topological engineering as a viable alternative to chemical modification for advancing DNAzyme-based therapeutics.

biochemistry↗

bioRxiv2026

Polysome Profiling Method for Low-Input Human Postmortem Brain

Polysome profiling is a powerful technique used to analyze the association of mRNA with ribosomes, providing insights into the translational status of a cell. It relies on the separation of ribosome-bound mRNAs through sucrose density gradient centrifugation, where the number of ribosomes on an mRNA correlates with its sedimentation rate. While numerous studies have successfully applied this method to cell line and mouse tissue, application to the human postmortem brain remains scarce due to challenges related to sample quality and low concentration of recoverable material. To overcome these challenges, we: O_LIImplemented a protocol specifically optimized for low-concentration human post-mortem brain tissue. C_LIO_LIImplemented a gradient-maker-free method to manually prepare sucrose gradients with tunable sensitivity for low-input samples. C_LIO_LIAdapted the human brain tissue protocol for neuronal cell lines and mouse brain with minimal modification. C_LI

neuroscience↗

bioRxiv2026

Molecular Characterization of the Progressive Landscape of Depression

Major Depressive Disorder (MDD) frequently follows a recurrent trajectory of episodes and remissions, often culminating in treatment-resistance. Molecular differences defining state-specific changes during episode and remission have been explored. However, progressive differences--defined here as cross-sectional linear trends across clinical stages from first to recurrent episodes or remissions, reflecting increasing illness burden over time--remain poorly understood, limiting sustained therapeutic outcomes. Here, we analyzed RNA-seq data from postmortem sgACC to identify progressive differences across MDD episodes or remission relative to state-specific differences, using an integrative assessment of molecular and cellular specificity, genetic-risk, disease-comorbidity and potential therapeutic targets. Differential expression analysis showed greater overlap between progressive and state-specific differences during remission than episode. Pathway enrichment highlighted disruptions in extracellular-matrix pathways shared by state-specific and progressive episodes, while metabolic and catalytic pathways were restored during remission. Cell-type-specific analyses showed that progressive changes were linked to superficial-layer intra-telencephalic neurons, whereas state-specific changes were enriched in pyramidal neuron subtypes and deeper layer SST-positive interneurons. Genome-wide association-informed enrichment analysis further linked these transcriptomic changes to genetic risk factors and symptom dimensions. Anhedonia was associated with both state-specific episode and progressive-remission signatures, suggesting that it is a persistent trait-like feature of MDD. Finally, an integrative pharmacological analysis revealed shared molecular mechanisms between pro-disease and therapeutic targets, highlighting pleiotropic effects of key pathways depending on disease state and dosage. Together, these findings provide a novel perspective on biological underpinnings of MDD progression over episodes or remissions and identify pharmacological targets that account for pathological and/or compensatory/therapeutic processes.

neuroscience↗

bioRxiv2026

Gating Mechanism of the Human Connexin 45 Gap Junction Channel

Gap junction channels formed by the 21-member human connexin family enable direct intercellular exchange of ions and small signaling metabolites, coordinating electrical coupling across cardiac, neural and epithelial tissues. Connexin 45 (Cx45), encoded by GJC1, mediates impulse conduction in the atrioventricular node, His bundle, and Purkinje fibers, where disease-linked mutations cause progressive atrioventricular block and familial atrial fibrillation, yet no experimental structure has been reported, and its regulatory mechanism remains undefined. Here, we determine the structural basis of Cx45 gating and Ca2+ regulation using cryo-electron microscopy, mutational analysis, and molecular dynamics simulations. Cryo-EM structures of the apo (2.76 [A]), Ca2+-bound (2.65 [A]), and E41A mutant (3.55 [A]) channel reveal a neck constriction formed by Y45, establishing a steric gate distinct from other connexins. Ca2+ associates with E41, stabilizing the neck via electrostatic remodeling without global conformational change. Together, these data define a dual steric-electrostatic mechanism for Cx45 regulation and provide a structural framework for isoform-specific connexin gating relevant to cardiac physiology and conduction disease.

biochemistry↗

bioRxiv2026

Genome-wide architecture of prolonged starvation adaptation in experimentally evolved Drosophila and comparative enrichment in human orthologs

Long-term starvation stress represents a strong evolutionary constraint across taxa, yet the genetic architecture underlying adaptation to sustained nutrient deprivation remains poorly resolved. We experimentally evolved Drosophila melanogaster under starvation stress for 60 generations, maintaining four starvation-selected populations and four matched controls, and then performed whole-genome resequencing. Starvation-selected populations exhibited extended survival and pronounced genome-wide restructuring, including the expansion of low-heterozygosity regions, reduced nucleotide diversity, and recurrent sweep signatures across replicates. Drift-aware allele-frequency modeling identified 3,578 single-nucleotide polymorphisms (SNPs) whose shifts exceeded neutral expectations, indicating widespread, parallel responses to sustained starvation. Mitochondrial pathways emerged as prominent targets: nuclear-encoded mitochondrial genes were significantly enriched among low-diversity, sweep-associated, and drift-exceeding loci, and the mitochondrial origin of replication harbored a sharply differentiated variant, suggesting a mito-nuclear component of starvation adaptation. Comparative analyses further showed that human orthologs of starvation-responsive fly genes were enriched for highly differentiated variants in selected 1000 Genomes populations, with regulators of TOR/S6K signaling recurring among loci in the extreme tails of population differentiation. Together, these results define a replicate-convergent and functionally coherent genomic response to prolonged starvation in Drosophila, comprising regional signatures of linked selection and distributed allele-frequency shifts exceeding neutral expectations, and connect laboratory selection in flies to population differentiation in human orthologs.

evolutionary biology↗

bioRxiv2026

Magnetic field-induced ER stress reprograms the tumor microenvironment to improve triple-negative breast cancer survival

BackgroundNon-invasive electromagnetic field (EMF)-based therapies offer a potential route to modulate local tumor-immune interactions but their mechanistic basis remains poorly defined. MethodsWe evaluated Asha therapy, a proprietary low-intensity (50khz, 2 mT, 25% duty cycle) alternating magnetic-field treatment in preclinical breast cancer models. Cellular responses in human triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) were evaluated using bulk RNA sequencing, quantitative proteomics, flow cytometry, and cytokine analysis and proteomics analysis. Tumor microenvironment responses in mouse 4T1 breast cancer model was characterized using single-cell CITE-seq analysis. Functional efficacy was assessed in vivo using the murine 4T1 triple-negative breast cancer model, both as monotherapy and in combination with anti-PD1 checkpoint blockade. Clinical relevance was assessed by deriving a 19-gene neutrophil activation signature from Asha-induced transcriptional changes and projecting it onto two independent TNBC patient cohorts (METABRIC n=338, SCAN-B n=874) for survival analysis. ResultsAsha therapy induced endoplasmic reticulum (ER) stress and activated an adaptive unfolded-protein response in tumor cells, triggering robust NF-{kappa}B and interferon signaling and time-dependent secretion of inflammatory cytokines. In vivo, these tumor-intrinsic changes propagated to the tumor microenvironment (TME), reprogramming fibroblasts from contractile states to immune-recruiting, interferon-responsive phenotypes and enriching for interferon-stimulated, metabolically active neutrophils and macrophages. These coordinated innate immune changes occurred without overt cytotoxicity and were associated with significant reductions in metastasis and improved survival. Combination with anti-PD1 therapy markedly enhanced efficacy, reducing lung metastasis and mortality by 88% compared with control. The neutrophil activation signature derived from Asha-treated tumors was associated with improved overall survival in both METABRIC (log-rank p=0.036) and SCAN-B (p=0.048) TNBC cohorts by Kaplan-Meier analysis, with pooled multivariable Cox regression confirming significant survival benefit (HR=0.75, 95% CI 0.59-0.94, p=0.01). ConclusionsAsha therapy triggers a controlled ER stress response in tumor cells that drives interferon-mediated cytokine release and immune reprogramming of the TME, resulting in anti-metastatic and survival benefits. These findings identify electromagnetic-field exposure as a potential non-pharmacologic strategy to activate innate immunity and sensitize tumors to checkpoint blockade, supporting further clinical development of EMF-based immunotherapy.

cancer biology↗

bioRxiv2026

Computational Design and Atomistic Validation of a High-Affinity VHH Nanobody Targeting the PI/RuvC Interface of Streptococcus pyogenes Cas9: A Bivalent Hub Strategy for CRISPR-Cas9 Enhancement

The CRISPR-Cas9 system has revolutionized genome engineering, yet its full therapeutic potential remains constrained by challenges in precisely modulating its activity and specificity. Here we report a fully computational end-to-end pipeline for the de novo design of a single-domain VHH nanobody (NbSpCas9-v1) targeting a structurally conserved, non-catalytic epitope at the PAM-interacting (PI) and RuvC-III interface of Streptococcus pyogenes Cas9 (SpCas9; PDB: 4UN3). Nanobody sequences were generated using BoltzGen, a generative diffusion binder design framework, and co-folded with SpCas9 using Boltz-2 to evaluate structural confidence and binding affinity. The top-ranked model (SpCas9_4UN3_Bivalent_Hub_v1) achieved a complex pLDDT of 0.8406, an aggregate score of 0.8016, and an ipTM of >0.8, indicating high confidence in the nanobody-antigen interface. The designed 1,616-residue quaternary complex (SpCas9 + sgRNA + DNA + nanobody) was subjected to 10 ns of all-atom molecular dynamics (MD) simulation using the AMBER14SB force field within the GROMACS/OpenMM framework. The complex stabilized at RMSD [~]6 [A] with a radius of gyration of 39-44 [A], confirming thermodynamic stability under physiological conditions (310 K, 0.15 M NaCl). A conserved 96.3 [A] inter-molecular distance between the nanobody centroid and the HNH catalytic residue H840 establishes NbSpCas9-v1 as a distal, non-inhibitory binder -- ideally suited for a Bivalent Hub architecture recruiting secondary effectors to the Cas9 ribonucleoprotein (RNP). The nanobody-Cas9 interface is stabilized by 8 hydrogen bonds, 4 salt bridges, and [~]1,850 [A]2 of buried solvent-accessible surface area. These results provide a rigorous structural and dynamic foundation for experimental validation of VHH-based CRISPR-Cas9 enhancers and modulators. GRAPHICAL ABSTRACTThe computational workflow proceeds from SpCas9 crystal structure acquisition (PDB: 4UN3) through BoltzGen nanobody design, Boltz-2 structural co-folding, 10 ns explicit-solvent MD validation, and Bivalent Hub functional characterization. The PyMOL rendering below shows the full quaternary complex at atomistic resolution.

bioinformatics↗

bioRxiv2026

Mutant ATXN1 impacts human and mouse microglia and contributes to cognitive, mood, and motor deficits in SCA1 mice

Microglia, resident immune cells of the brain, are important players in neurodegeneration. While microglial activation is a hallmark of many neurodegenerative diseases, the specific role of microglia intrinsic factors in microglial activation and disease pathogenesis remains unknown. Spinocerebellar ataxia type-1 (SCA1) is an inherited autosomal dominant neurodegenerative disease characterized by severe neuronal loss and early microglial activation in the cerebellum. SCA1 is caused by CAG repeat expansion in the ubiquitously expressed ATAXIN1 (ATXN1) gene. Using human microglia differentiated from SCA1 patient derived iPSCs, we found that mutant ATXN1 is sufficient to alter morphology, gene and protein expression in human microglia in a cell-autonomous manner. Moreover, compared to controls, human SCA1 microglia exhibited increased phagocytosis and pro-inflammatory cytokine production, indicating an immune priming. To determine the extent to which mutant ATXN1 in microglia contributes to SCA1 pathogenesis and behavioral symptoms, we removed mutant ATXN1 from microglia and macrophages in a novel conditional SCA1 mouse model, f-ATXN1146Q/2Q mice. Microglial mutant ATXN1 reduction led to a marked correction in microglia phenotype, in particular in the transcriptomic signature of interferon type 1 mediated immune response, reduced microglial density and resulted in smaller microglia with reduced branching in the cerebellum. Pathology of Purkinje neurons and cerebellar astrogliosis were also ameliorated. Utilizing a battery of behavioral tests, we found that microglia and macrophage mutant ATXN1 reduction ameliorated cognitive, mood, and motor deficits in SCA1 mice. Together, these results indicate that mutant ATXN1 directly impacts microglial phenotype in SCA1, contributing to SCA1 pathology and behavioral deficits.

neuroscience↗

bioRxiv2026

Metabolic starvation-induced cell swelling drives solid stress in tumors

Solid stress shapes tumor growth, invasion, and therapeutic response, yet its physical origin and clinical relevance remain unclear. Here, we develop a mechano-electro-osmotic model integrating metabolic gradients, ion transport, and cellular mechanics to explain residual solid stress emergence in tumor spheroids, common models of solid tumors. We show that solid stress arises predominantly from osmotic cell swelling driven by metabolic deprivation and ion accumulation, rather than proliferation. This mechanism generates a characteristic stress architecture: isotropic compression in the hypoxic core balanced by peripheral tangential tension, causing pronounced cell and nuclear deformation. The resulting nuclear strain provides a mechanical basis for DNA damage and genomic instability implicated in disease progression and treatment resistance. We validate these predictions in breast cancer using MDA-MB-231 spheroids and patient-derived ductal carcinoma in situ lesions, and corroborate them across published spheroid models and in vivo and ex vivo tumors spanning additional cancer types. Our findings link tumor metabolism to clinically relevant mechanical stresses, suggesting opportunities to target osmotic and metabolic pathways to mitigate solid stress and improve therapeutic outcomes.

bioengineering↗