Biology subjects

Gupta, A.

Publications and source records attributed to Gupta, A..

At least 37 records · Page 2Linked to original sources

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

A 3D image atlas chronicling cellular and structural dynamics following lung injury identifies the aberrant expansion of endothelial cells that fail to form perfused vasculature

Intratracheally delivered bleomycin in mice is the most widely used in vivo model of pulmonary fibrosis, yet key aspects remain poorly defined, including sex-dependent responses and the temporal peak of injury. Standard 2D histology further overlooks regional heterogeneity and cannot resolve the 3D architecture or connectivity of endothelial cells (ECs). Here, we established a multi-scale 3D imaging pipeline integrating precision-cut lung slices from EC lineage-tracing mice, optical clearing, and AI-driven 3D segmentation to map cellular and structural dynamics from whole-lobe tile scans to single-cell resolution. We identified sex as a critical biological variable, with males exhibiting a delayed but more severe fibroproliferative response. Unsupervised K-means clustering identified three distinct tissue microenvironments: healthy parenchyma(KMC1), a myofibroblast-rich fibrotic core (KMC2), and a previously uncharacterized EC-dense perilesional region (KMC3) defined by massively expanded but non-perfused ECs that acquire a pro-inflammatory phenotype. This aberrant endothelial response precedes peak myofibroblast accumulation and persists beyond fibrotic resolution, leaving a vascular scar that extends into the large-vessel hierarchy. Together, this 3D image atlas, made publicly available as an interactive resource [https://mosaic-lung.com/], reveals the activated endothelium as an underexplored therapeutic target in pulmonary fibrosis.

bioengineering↗

bioRxiv2026

Ecological Cascades and Future Hantavirus Spillover Risk in a Changing Climate

Anthropogenic climate change is reshuffling global biodiversity and accelerating zoonotic spillover at the human-wildlife interface. The 2026 multi-country cruise ship outbreak of Andes virus highlights the unpredictable and globalized threat posed by rodent-borne orthohantaviruses, which cause severe, highly fatal human diseases worldwide. Despite this significant public health risk, macroecological synthesis linking environmental drivers to systemic shifts in hantavirus hazards has been lacking. Here, we implement an integrated ecological and machine learning framework combining 89 unique virus-host associations across 61 reservoir species with mechanistic Force-of-Infection (FOI) modeling to project global spillover hazards under current and future climate scenarios (SSP2-4.5 and SSP5-8.5) for 2041-2060. Our models demonstrate a spatially uneven but geographically extensive expansion of spillover hazards, with approximately 74.9% of modeled land area experiencing an increase in FOI. We identify three distinct high-hazard belts heavily concentrated in already-temperate reservoir ranges: western and central North America, central and northeastern Europe extending into western Russia, and discrete patches within central South America. Crucially, our findings reveal that hantavirus hazards are fundamentally shaped along virus-specific ecological axes. While thermal and precipitation anomalies predominate the transmission dynamics of Old and New World lineages like Sin-Nombre virus and Thailand virus, distinct urban-centric Profiles emerge for Seoul virus and Mamore virus, driven explicitly by fine-scale anthropogenic landscape modifications. These results reveal that climate change will not simply intensify viral spillover uniformly but will fundamentally restructure it across varied climatic and human-dominated environments. Our maps provide a critical baseline for targeted global surveillance and highlight the urgent need to integrate spatial conservation planning with public health preparedness.

ecology↗

bioRxiv2026

Endosulfan rewires PKA and GSK3β to disrupt primary cilia-dependent Hedgehog signalling

Primary cilium-dependent Hedgehog signalling is essential for embryonic development, tissue patterning, and organ homeostasis, and its disruption causes a spectrum of developmental disorders collectively termed ciliopathies. Whether environmental toxicants can chemically induce ciliopathy-like states by targeting this pathway, however, remains poorly understood. Here we show that endosulfan, a banned organochlorine pesticide epidemiologically linked to severe congenital and reproductive defects in exposed human populations, disrupts Hedgehog signalling by driving GLI transcription factor processing into repressor forms and suppressing target gene expression at both transcriptional and protein levels. Having excluded direct effects on core ciliary receptors and GLI-DNA binding, we identify the pathway kinases PKA and GSK3{beta} as direct targets of endosulfan: endosulfan increases PKA activity through allosteric fine-tuning, and -- in a pharmacologically rare finding -- acts as the first reported small-molecule activator of GSK3{beta}, shifting the kinase toward a catalytically active conformation. We further identify Cetn3 and Cep250 as novel GLI-regulated genes required for centriole cohesion, both of which are repressed upon endosulfan exposure, providing a mechanistic link to the reproductive defects reported in exposed populations and animal models. These findings identify endosulfan as a candidate chemical inducer of ciliopathy and reveal how an environmental toxicant can hijack core kinase signalling to disrupt Hedgehog-dependent development.

cell biology↗

bioRxiv2026

A chemical-genetic approach for stress-independent activation of the fission yeast stress-activated protein kinase pathway

BackgroundThe fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors can have stressor-specific effects that may be difficult to separate from the effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA, in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This required combining multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactive. While this strain was useful, it had several limitations. ResultsHere we describe and validate a more rationally-designed strain, SISA4, that retains the features of the original SISA strain while overcoming its limitations. SISA4 is more stable genetically than SISA, easier to use in genetic crosses, and easy to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo, and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactive state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these are modest and generally do not affect interpretation of experiments. Finally, using SISA4, we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. ConclusionsSISA4 will be useful for investigating how SAPK pathway activation regulates diverse cellular processes.

cell biology↗

bioRxiv2026

The consequences of array size for human centromere performance in mitosis

Long-read sequencing and assembly of human genomes have revealed extreme variation in centromeric alpha-satellite array sizes across chromosomes and individuals. However, assessing the impact of centromeric array size on centromere function remains challenging. In this study, using quantitative FISH and microscopy assays, we established a framework for the systematic functional evaluation of naturally occurring variations in centromeric array size during mitosis. By comparing 8 pairs of homologous chromosomes exhibiting 1.24 - 4.6 -fold variation in centromeric array size, we uncovered size-based differences in function. We found that between a pair of homologous chromosomes, the chromosome harboring the smaller centromeric array is more prone to chromosome missegregation. Centromere size-based differences in chromosome missegregation rates cannot be explained by differential enrichment of molecular factors like centromeric histone CENP-A, CENP-B, or most kinetochore proteins. However, the smaller centromere out of a homologous pair consistently exhibits increased cohesion fatigue, suggesting the involvement of the cohesin complex in size-based differences in centromere cohesion. Consistent with our hypothesis, complete deprotection of the cohesin complex by depletion of shugoshin-1, removes the size-based bias in centromere cohesion. These results suggest that while CENP-A-enriched core centromeres are essential for kinetochore assembly, variation in overall centromeric array size has a significant impact on centromere performance and the fidelity of chromosome segregation during mitosis.

Cell Biology↗

bioRxiv2026

A Dual-Function Guanidinium Scaffold that Couples Copper Sequestration with Redox Protection in Wilson disease

Wilson disease (WD) is caused due to mutations in the copper ATPase gene ATP7B, that result in accumulation of labile copper pools and the consequent disruption of cellular redox homeostasis through uncontrolled copper-mediated reactive oxygen species generation. Current therapies mainly depend on high-affinity copper chelation to lower metal burden which sometimes also strip copper from cuproproteins and may disturb physiological copper-dependent processes. It also does not directly suppress pathological copper reactivity i.e., free radical generation, a major driver of WD progression. To overcome these limitations, we have rationally designed Gua-Cu-3, a C-symmetric guanidinium-based molecule that can chelate labile copper without metal stripping from cuproproteins due to moderate binding affinity and it has intrinsic antioxidant activity within a single nanosheet-forming supramolecular self-assembly. Spectroscopic, calorimetric, and computational analyses revealed controlled multivalent copper coordination (Kd = 95.4 M) while radical-scavenging and hydroxyl-radical inhibition assays further revealed potent redox-regulatory activity. In cellular models of copper overload, Gua-Cu-3 treatment reduces ATP7B trafficking from trans-Golgi network, confirming effective intracellular copper sequestration. This was accompanied by a marked reduction in oxidative stress, reduction of translocation of Nrf2 in nucleus and of HO-1 expression, thereby limiting lipid peroxidation which leads to restoration of cellular homeostasis, including improved lysosomal integrity, reduced endoplasmic reticulum stress, decreased mitochondrial superoxide levels, and diminished apoptosis. Importantly, the protective effects extended, beyond cultured cells, Gua-Cu-3 attenuates oxidative stress in ATP7B-homolog-deficient C. elegans and rescues copper-induced developmental defects in zebrafish, outperforming D-penicillamine, which is currently in use for Wilson disease management. These findings establish Gua-Cu-3 as a molecularly designed supramolecular copper-reactivity buffer that couples-controlled copper sequestration with redox regulation which is distinct from conventional copper depletion and provides a framework for treating Wilson disease and other disorders associated with metal dyshomeostasis and oxidative stress.

Cell Biology↗

bioRxiv2026

Ultra-low biomass sequencing workflow (LBV-Seq) enables de novo metagenomic reconstruction of DNA and RNA viral genomes

Genome-resolved virome analysis remains inaccessible for many samples, including those with clinical relevance, because viral nucleic acid recovered after enrichment is often too scarce to support de novo genome assembly. As a result, many analyses are limited to sparse read-level detection, which cannot recover divergent viruses, resolve strains, or interpret gene-level variation. Here, we developed Low Biomass Viral Sequencing (LBV-Seq), a workflow that couples low-input viral sample handling with modified primary template-directed amplification and short- or long-read sequencing to enable de novo reconstruction of DNA and RNA viral genomes from sub-femtogram to nanogram inputs. LBV-Seq reproducibly captures the same relative community composition, amplifies diverse viruses, and achieved broad genome coverage across nearly all targets regardless of viral genome structure, Baltimore class, abundance, or input mass. Short-read assemblies recovered near-complete genomes from femtogram-scale inputs. Long-read sequencing provided orthogonal support for genome structure, with PacBio HiFi reads spanning large portions of viral genomes and, in some cases, complete small viral genomes. Applied to virus-enriched human duodenal biopsy eluates, LBV-Seq provided proof-of-feasibility for recovering both bacteriophage and eukaryotic viral genomes from low-input biopsy-derived material. In the eluates tested, LBV-Seq recovered co-occurring Alphatorquevirus and Betatorquevirus genomes estimated to be present at roughly 10 copies/{micro}L and at viral masses below 0.1 fg/{micro}L. LBV-Seq enables genome-resolved virome analysis in samples previously limited to detection-based viromics, supporting viral discovery and strain-resolved analyses in settings where viral mass is low, including viruses enriched from human tissue biopsies.

genomics↗

bioRxiv2026

Amplicon/Protein Bead Display enables quantitative in vitro biochemistry at scale

Scalable methods for producing and characterizing protein libraries are essential for generating standardized datasets needed to train models linking sequence to function. Here, we present Amplicon/Protein Bead Display (APB-Display), which expresses and purifies >100,000 protein variants in vitro in 1 day. APB-Display uses particle-templated emulsification to generate libraries of hydrogel beads that covalently display many copies of a given protein variant and its encoding DNA. By incubating beads with fluorescently-labeled ligands, sorting, and sequencing to generate titration curves (APB-TiteSeq), we simultaneously quantified expression levels and binding affinities (Kds) for >18,000 FLAG epitope variants interacting with M2 anti-FLAG antibody in 3 days, revealing chemistry-dependent epistasis between positions 4 and 5. Single-concentration binding measurements (APB-SortSeq) paired with neural network denoising further returned quantitative affinities for >88,000 variants. APB-Display requires only standard laboratory equipment and access to a FACS sorter, providing an accessible platform for quantitative in vitro biochemistry at scale.

biophysics↗

bioRxiv2026

Scinderin-driven Golgi Actin Remodeling coordinates GLP-1 and insulin secretion to regulate glucose homeostasis

Maintenance of glucose homeostasis requires coordinated hormone secretion from intestinal enteroendocrine cells and pancreatic {beta}-cells, yet the intracellular mechanisms that couple nutrient sensing to endocrine output remain poorly defined. Here, we identify the actin remodeler Scinderin (SCIN) as a shared regulator of hormone secretion across these systems. SCIN is selectively expressed in enteroendocrine L-cells and pancreatic {beta}-cells, where it localizes to phosphatidylinositol-4-phosphate (PI(4)P)-enriched Golgi membranes and controls Golgi-associated actin dynamics. Loss of SCIN disrupts Golgi organization, impairs prohormone trafficking, and reduces secretory granule formation, resulting in defective nutrient-stimulated GLP-1 and insulin secretion while preserving cAMP-dependent amplification pathways. In vivo, tissue-specific deletion of Scin compromises incretin responses, {beta}-cell insulin secretion, and systemic glucose homeostasis. Consistent with these findings, SCIN expression is reduced in human diabetic {beta}-cells and associates with stress-related loss of {beta}-cell maturity. Transcriptomic analyses reveal a conserved Golgi stress program upon SCIN loss, linking intracellular trafficking defects to endocrine dysfunction. Together, our results identify SCIN-dependent Golgi actin remodeling as a rate-limiting intracellular mechanism coordinating enteroendocrine and pancreatic hormone secretion. This work uncovers a shared, targetable node controlling endocrine output, providing a mechanistic link between secretory pathway dysfunction and diabetes.

cell biology↗

bioRxiv2026

High-throughput engineering of ligand-activated splicing ribozyme through domain insertion

Domain insertion is an established method to engineer ligand-mediated control of activity in protein scaffolds. Whether this strategy can be systematically applied to large, structured RNAs remains unclear. In this study, we investigated the feasibility of engineering ligand-activated splicing ribozymes (LASRs) from group I catalytic introns. Using domain-insertion profiling coupled with high-throughput screening, we mapped the nucleotide-resolution landscape of aptamer insertion across the ribozyme and identified sites that support robust ligand-dependent control. We showed LASRs function across multiple kingdoms of life, including diverse species of bacteria and even fungi, and can be used to regulate various genetic outputs. Finally, we integrated LASRs with a genetic recorder that writes information into ribosomal RNA, enabling sequencing-based recovery of intracellular chemical signals from microbial consortia. This work establishes LASRs as an RNA-based inducible control platform for sensing diverse chemical inputs, regulating the expression of diverse genes of interest, and recording intracellular information.

synthetic biology↗

bioRxiv2026

Promiscuous RNA binding by WDR5 remodels the KMT2A (MLL1) histone methyltransferase complex to an inactive state

Chromatin-modifying complexes are critical in gene regulation, yet their proposed interactions with RNA remain poorly understood. Here, we show that WDR5, an essential subunit of the MLL1 (KMT2A) histone methyltransferase complex, binds RNA with high affinity but without sequence specificity. Using a stringent approach, we demonstrate that WDR5 directly engages a diverse pool of RNAs in cells, predominantly as a function of RNA abundance rather than specific motifs. Equilibrium binding assays further show that RNA length, rather than sequence, dictates WDR5 affinity. We map multiple RNA-binding regions on WDR5, some of which overlap with surfaces used in MLL1 complex subunit interfaces. Strikingly, we find that RNA binding disrupts the MLL1 complex by competitively displacing WDR5 from these critical protein-protein interactions, leading to a marked inhibition of MLL1 catalytic activity. This regulatory mechanism provides a layer of control over histone methylation, potentially integrating transcriptional activity with chromatin state.

molecular biology↗

bioRxiv2026

Copper-transporting ATPase ATP7B and the lysosomal exocytosis pathway synergise to detoxify cadmium

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

cell biology↗

bioRxiv2026

Bio-BLIP: A Multimodal Architecture for Transferable Reasoning in Genomic Variant Interpretation

Developing scientific hypotheses in biology requires integrating heterogeneous evidence across DNA sequence, gene context, protein function, and prior literature. Existing multimodal AI systems expose biological evidence to reasoning models through textification or by projecting biological embeddings into fine-tuned language models. However, these models are typically highly optimized the specific set of tasks for which they are fine-tuned. Here we present Bio-BLIP, a multimodal Q-former based architecture which leverages biological embeddings and a LLM to generalize to complex reasoning tasks without task-specific fine-tuning. The key to Bio-BLIP is a new neural network architecture that integrates four data modalities - DNA, genes, proteins, and text - through a master Qformer model, which integrates the modality-specific information into a fixed-length prefix for the LLM backbone. Bio-BLIP is pretrained on the task of human genetic variant annotation and achieves a 29.8% increase in generating accurate variant features over frontier LLMs. We evaluate Bio-BLIP zero-shot on downstream genomic tasks of variant prioritization and target gene prediction. Bio-BLIP outperforms two alignment-free genomic language models on regulatory variant prioritization for Mendelian disease. Across the target gene prediction task, Bio-BLIP improves accuracy over LLMs by leveraging learned genomic variant knowledge in difficult cases. Our model produces rich, transparent reasoning traces. In biological domains characterized by multiple scales of data and varied downstream tasks, Bio-BLIP offers a step toward natively multimodal, generalizable reasoning.

genomics↗

bioRxiv2026

Reparative and regenerative immature neutrophil-like population derived from HL-60 cells

Unlike the conventional mature neutrophils, immature neutrophils have been investigated for their regenerative properties; however, their limited availability necessitates alternative generation strategies. Here, we used a combination of dimethylsulfoxide (DMSO) and 1,25-dihydroxyvitamin D3 (D3) to differentiate myeloid leukemia (HL-60) cells into immature neutrophil-like cells. Differentiated cells exhibited reduced cell size, loss of uniformity, decreased nuclear-to-cytoplasmic ratio, band-shaped nuclei, increased proportion of CD11b+CD14+ cells (indicative of immature neutrophils), decreased proportion of CD11b+CD16+ cells (indicative of mature neutrophils), higher levels of arginase 1, TGF{beta}1 (markers of immature neutrophils), and no expression of CD16, MRC1 (markers of mature neutrophils and M2 macrophages, respectively). Proteomic analysis revealed enrichment of proteins associated with immature neutrophils and wound healing. Functionally, these cells supported limbal stem cell growth and wound closure in vitro, indicating relevance for corneal regeneration. Administration of these cells to ex-vivo and in-vivo alkali-injured corneas, resulted in significant effect on promotion of wound healing, with epithelial regeneration and decreased fibrotic markers, proving that such cells hold promise for clinical translation as a therapeutic tool for tissue repair.

cell biology↗

bioRxiv2026

Interlayer Communication Integrates Genetic and Mechanical Signals for Robust Leaf Morphogenesis

Leaf development requires coordinated growth across distinct tissue layers, integrating genetic regulation with mechanical coupling to achieve proper organ shaping. However, the mechanisms coordinating differentiation and growth across layers to ensure coherent leaf morphogenesis remain poorly understood. Therefore, we investigated the role of TCP4 as a potential central regulator of interlayer communication coupling genetic and mechanical signals during Arabidopsis leaf development. Three-dimensional analyses revealed pronounced asymmetry in the expression of TCP4 and its negative regulator, MIR319C, across developing primordia, establishing spatially patterned differentiation cues. Tissue-specific perturbations demonstrated that TCP4 activity in either epidermis or subepidermis modulates cell proliferation and expansion across layers through both protein mobility and mobility-independent signalling mechanisms. Strong epidermal TCP4 induction arrested leaf development by prematurely suppressing proliferation and preventing symmetry breaking, thereby locking primordia in cylindrical states. Live imaging revealed fundamental reprogramming of growth anisotropy, with TCP4 driving cortical microtubule alignment, increasing cell wall stiffness, and establishing layer-specific pectin patterns. Together, these findings establish TCP4 as an integrator of genetic and mechanical signals across leaf tissue layers, coupling transcriptional programmes to cytoskeletal organisation, cell wall remodelling, and cross-layer mechanical feedback.

plant biology↗

bioRxiv2026

SNAC-DB: An ML-Ready Database for Antibody and NANOBODY(R) VHH-Antigen Complexes with Expanded Structural Diversity and Real-World Benchmarking

Predicting antibody and NANOBODY(R) VHH-antigen complexes remains a critical challenge for state-of-the-art structure prediction models, limiting their impact in therapeutic discovery pipelines. We introduce SNAC-DB, an ML-ready database and curation pipeline enriched with structural biology expertise, designed to accelerate model accuracy and generalization by providing 31-37% expanded structural diversity over existing resources like SAbDab through comprehensive re-curation that extracts maximum value from available experimental structures. SNAC-DB expands coverage by capturing often-overlooked complexes and accurately identifying complete multi-chain epitopes through improved biological-assembly-based logic. Built for ML practitioners, SNAC-DB provides standardized formats with multi-threshold structure-based clustering to enable principled sample weighting during training. Using a rigorous benchmark of public PDB entries deposited post-May 2024 plus confidential therapeutic structures, we evaluate seven leading models (Protenix-v1, OpenFold-3p2, RosettaFold-3, Boltz-2, Boltz-1x, Chai-1, and AlphaFold2.3-multimer) with evaluation methodology tailored to antibody/NAN-OBODY(R) VHH-antigen complexes to ensure correct handling of multi-chain epitopes, revealing systematic performance gaps: success rates rarely exceed 25%, confidence-based ranking fails to identify best predictions even when accurate structures exist in ensembles, and all models consistently struggle with therapeutically relevant NANOBODY(R) VHHs. Systematic evaluation of sampling strategies demonstrates that while generating 1000 samples per target substantially increases the likelihood of producing accurate structures (oracle selection improves from 11.9% to 50.5%), confidence-based ranking remains nearly flat (between 10.9% and 14.9%), revealing that improved ranking mechanisms represent a more tractable path to performance gains. Finally, fine-tuning GeoDock on SNAC-DB yields higher success rates than training on SAbDab (11.0% vs. 7.1% for antibodies; 7.0% vs. 4.0% for NANOBODY(R) VHHs), suggesting that SNAC-DBs expanded structural diversity translates to improved model generalization. Significance StatementComputational antibody/NANOBODY(R) VHH design shows promise but remains unreliable for therapeutic development. SNAC-DB provides 31-37% expanded structural diversity through comprehensive data curation, immediately accelerating model development. Benchmarking seven leading AI models reveals accuracy rarely exceeds 25% on therapeutic targets, with confidence-based ranking failing to identify correct structures even when they exist in model outputs. Training on SNAC-DB increases prediction accuracy, validating that high-quality, diverse training data is critical for advancing computational methods toward clinical impact.

bioinformatics↗

bioRxiv2026

Hypothalamic Interleukin 6 linked to sex-specific behavioral deficits following adolescent social isolation

Social isolation refers to an extreme form of social deprivation that has enduring effects on the brain and behavior. Adolescents show selective vulnerability to such heightened social stress, displaying aberrant behavior and psychiatric ailments. The post-weaning social isolation rodent model has been widely used to recapitulate such behavioral anomalies and delineate their mechanistic bases. Here, we aim to identify how prolonged social isolation during adolescence affects neuroimmune responses in both sexes and the implications for behavioral outcomes, particularly aggression. While males subjected to adolescent isolation were hyper-aggressive with pathological signs, females showed reduced social exploration and inactivity. Cytokine profiling in core brain regions implicated in aggression revealed reduced interleukin 6 (IL6) levels, specifically in the hypothalamus, in both sexes. Other proinflammatory cytokines, including interferon-gamma and interleukin-1beta, were unaltered. IL6-responsive genes, SOCS3 and TIMP1, were also downregulated in the hypothalamus of both socially isolated males and females. The hypothalamus is crucial for stress responsiveness and the expression of excessive aggression. Despite behavioral dimorphism, reduced IL6 levels in both sexes may indicate differences in downstream signaling and roles beyond classical immune responses. Our findings suggest that hypothalamic IL6 may be a key mediator of adolescent social isolation, which is associated with aberrant behavior, including aggression.

neuroscience↗