Biology subjects

Shi, Y.

Publications and source records attributed to Shi, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Dual histone methylation reader ZCWPW2 links histone methylation to initiation of meiotic recombination

Meiotic homologous recombination initiates with the formation of programmed DNA double-strand breaks (DSBs) by complexes comprising SPO11 and accessory proteins at discrete sites called recombination hotspots. In mammals, PRDM9-dependent H3K4me3 and H3K36me3 define recombination hotspots, but how these epigenetic characteristics determine the physiological DSB formation remains unknown. Here we show that dual histone methylation reader ZCWPW2 can recognize H3K4me3 and H3K36me3 marks in testis. The binding activity of ZCWPW2 to histone methylation is dependent on PRDM9 function. Moreover, we find the epigenetic writer-reader axis PRDM9-ZCWPW2 is essential for DSB formation at meiotic recombination hotspots, which may be partly explained by the finding that ZCWPW2 can physically interact with HORMAD1, IHO1, MEI4, and REC114. Finally, the absence of ZCWPW2 leads to disrupted chromosomal synapsis and recombination, thereby obstructing meiotic progression. Taken together, our findings provide new insights into how histone modifications and their associated regulatory proteins collectively regulate meiotic homologous recombination initiation.

cell biology↗

bioRxiv2026

Hierarchical Immune Suppressive Functions of Regulatory T Cells Built on Mechanical Force

Regulatory T (Treg) cells maintain immune tolerance via contact-dependent inhibition and soluble mediators. While canonical suppressive mechanisms are well characterized, their hierarchical spatiotemporal organization remains unclear. We previously showed Foxp3 represses ER Ca2+ channel RyR2, reducing cytoplasmic Ca2+ to inactivate m-Calpain (Calpain-2) and stabilize high-affinity LFA-1-mediated Treg-dendritic cell (DC) adhesion--an early step blocking DC antigen presentation. Using m-Calpain as a molecular switch, we generated mechanically deficient Tregs (TregFD) with constitutively active m-Calpain that abrogated high-force Treg-DC adhesion. TregFD retained a near-wild-type transcriptome (differential expression limited to adhesion pathways), yet failed to suppress autoimmunity or DC-driven T cell proliferation. Stable adhesion was required for localized TGF-{beta}/IL-10 delivery to DCs. Single-cell RNA sequencing of rescued Scurfy mouse lymphoid tissues revealed organ-specific division of labor: non-mechanical modules support homeostasis/tissue repair, while mechanical modules dominate epithelial barrier/antigen presentation suppression. Combined TregFD and RyR2-deficient Tconv transfer restored wild-type Treg activity, demonstrating module synergism. These findings establish a mechanical force-centered, two-tiered hierarchical model of Treg suppression, providing a framework for targeting Treg mechanics in autoimmunity and inflammation.

immunology↗

bioRxiv2026

Elongin B orchestrates chromatin and transcriptional programs in H3K27M-mutant diffuse midline glioma

Recurrent driver mutations in genes encoding histone H3 (H3.3K27M and H3.1K27M) are observed in [~]80% of diffuse midline gliomas (DMG), which lead to aberrant gene regulation, yet the specific RNA polymerase II (Pol2) regulators that induce transcriptional dysregulation in DMG are not fully defined. We identified multiple regulators of Pol2 elongation as DMG genetic dependencies in a chromatin-focused CRISPR screen. Additional studies confirm that knockout (KO) of the Pol2 SIII complex gene elongin B (ELOB) inhibits DMG cell proliferation in tissue culture and tumor growth in xenograft models. Further genomic analyses reveal that ELOB binding sites are enriched in H3K27M oncohistones and that ELOB KO alters H3K27me3 and H3K27M incorporation at thousands of genomic regions, implicating ELOB in the maintenance of dysfunctional chromatin states in DMG. Correspondingly, PRO-seq and RNA-seq profiling reveal that ELOB loss disrupts Pol2 transcriptional activity and alters the expression of transcripts involved in metabolism, proliferation, and brain development. These findings suggest that Pol2 elongation factors like ELOB cooperate with H3K27M oncohistones to maintain the epigenetic and transcriptional landscape driving DMG malignancy.

cancer biology↗

bioRxiv2026

CAD rewires nucleotide metabolism to drive oxidative stress in myocardial ischemia/reperfusion injury

Myocardial ischemia/reperfusion (I/R) injury is a major determinant of infarct size and clinical outcome, yet effective therapies remain limited. Although metabolic remodeling is central to I/R pathology, the contribution of nucleotide biosynthesis remains unclear. Here, we identify CAD, the multifunctional rate-limiting enzyme of de novo pyrimidine biosynthesis, as a previously unrecognized regulator of myocardial reperfusion injury. CAD activation exacerbated cardiomyocyte death during simulated I/R, whereas CAD knockdown or pharmacological inhibition was protective in vitro. Mechanistically, CAD enhanced dihydroorotate dehydrogenase (DHODH)-dependent electron transfer, increased the CoQH2/CoQ ratio, and promoted complex I reverse electron transport (RET), thereby amplifying mitochondrial ROS. In parallel, CAD suppressed de novo purine synthesis, causing purine insufficiency, DIS3L-dependent RNA decay, and cytosolic ROS. Importantly, cardiomyocyte-specific CAD deletion protected against cardiac I/R injury in vivo. Together, these findings establish CAD as a metabolic hub linking nucleotide flux to dual-compartment ROS signaling and identify nucleotide metabolism as a therapeutic vulnerability in myocardial I/R injury. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/725578v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@14c4fedorg.highwire.dtl.DTLVardef@1138ed2org.highwire.dtl.DTLVardef@1057d8borg.highwire.dtl.DTLVardef@17546bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Genomic, Clinical, and Spatial Predictors of Durable Response to BRAF/MEK Inhibition in BRAF-Mutant Melanoma

BRAF-targeted therapy (BRAFi/MEKi) and immune checkpoint blockade (anti-PD-1/anti-CTLA-4) have transformed the treatment of BRAF-mutant metastatic melanoma. While most patients who respond to targeted therapy eventually progress, a subset derives durable benefit, and biomarkers to identify this subset would inform optimal treatment selection. In this study, we analyzed pre-treatment tumor samples from a clinically annotated cohort of 155 patients with BRAF-mutant metastatic melanoma treated with first-line BRAFi/MEKi and followed for up to five years. We stratified patients into durable responders (PFS [≥] 24 months) and rapid progressors (PFS < 6 months with progression) and found that a global metric of tumor genomic heterogeneity, rather than individual gene alterations, distinguished these groups. Combining genomic heterogeneity with baseline tumor burden (e.g., lactate dehydrogenase (LDH) or radiographic lesion dimensions), we developed a parsimonious model that predicted durable responders with high precision and specificity. Notably, the analogous population of patients treated instead with immunotherapy were not durable responders, suggesting that the selected predictors of durable responders are targeted therapy specific. Spatial profiling of a subset of pre-treatment biopsies (n = 47) demonstrated that high intratumoral, but not peritumoral, CD8+ T-cell infiltration correlated with prolonged survival on BRAF-targeted therapy and served as an independent predictive factor when considered with genomic heterogeneity and features of clinical tumor burden. Together, these findings highlight the distinct baseline intrinsic and extrinsic features underlying durable response to BRAF-targeted therapy and support their potential implication in guiding treatment selection for patients with BRAF-mutant metastatic melanoma. One-Sentence SummaryIntegrated clinical, tumor genomic, and immune microenvironmental features predict durable responses to BRAF-targeted therapy.

cancer biology↗

bioRxiv2026

Single-cell RNA sequencing reveals that host Glutamine Metabolism Inhibition Enhances Macrophage Phagocytosis of Mycobacterium tuberculosis

Macrophages are crucial for host defense against the pathogen. However, pathogens such as Mycobacterium tuberculosis (Mtb) have evolved mechanisms to alter macrophage physiology and exploit these cells as their primary niche. Mtb-infected macrophages upregulate several metabolic pathways including glutamine metabolism. We previously showed that inhibiting glutamine metabolism with the pleiotropic glutamine metabolism antagonist prodrug JHU083 has dual antibacterial and immunomodulatory effects in a mouse model of tuberculosis. In the present study, using single-cell RNA sequencing and LS-MS/MS metabolomics, we showed that JHU083-mediated glutamine metabolism inhibition increased the population of interstitial macrophages in Mtb-infected lungs. JHU083 treatment also increased inflammatory signatures while lowering immunosuppressive markers on these macrophages. Metabolically, these macrophages exhibited marked depletion of complex lipids, accumulation of free fatty acids, and increased expression of transcripts associated with the {beta}-oxidation pathway. Additionally, JHU083-treatment also improved phagocytic activity of macrophages, as measured by using fluorescent E. coli as a bait. In conclusion, JHU083-mediated glutamine metabolism inhibition metabolically reprograms macrophages, increasing both their lipid utilization as well as phagocytic activity, potentially driving their antimycobacterial activity that we had observed earlier.

microbiology↗

bioRxiv2026

Chemical inactivation of a bacterial immune system de-domesticates a temperate phage and promotes its spread

Microbial communities shape hosts and ecosystems, yet the behaviors of these microbes are themselves controlled by mobile genetic elements. These mobile genetic elements (MGEs) compete and influence one anothers distributions. For example, many anti-phage immune systems are carried by prophages, plasmids, and integrative and conjugative elements (ICEs). It is unclear how important each of these interference mechanisms is for the spread of different mobile elements. Chemical inhibitors of these mechanisms of inter-MGE competition can clarify their mechanisms and significance in diverse environments. We discovered chemical inhibitors of SpbK--an antiphage defense encoded by an ICE in several strains of Bacillus subtilis. It is believed to prevent the dissemination of SP{beta} -like temperate phages, forcing SP{beta} to remain domesticated in its ICE-containing host. Chemical inhibition of SpbK dramatically improved the escape of infectious SP{beta} phages capable of disseminating and forming new lysogens. Furthermore, SpbK is part of a widespread class of Toll/interleukin-1 receptor (TIR)-domain containing enzymes that are prevalent both in prokaryotic antiphage immune systems and eukaryotic immune signaling. Our inhibitors reveal two distinct mechanisms of inhibiting TIR-domain enzymes. These models of inhibition may broadly apply across immune proteins, enabling inhibition of many TIR immune systems that influence phage spread as well as plant immunity and animal cellular signaling.

biochemistry↗

bioRxiv2026

Retest Reliability of Task-related fMRI BOLD Signals during Sequential Decision Making

IntroductionFunctional magnetic resonance imaging (fMRI) is widely used to study neural processes of behavior, but evaluations of test-retest reliability (TRR) of task-related blood-oxygen-level-dependent (BOLD) responses are scarce for many cognitive tasks. Such information is particularly important for longitudinal and intervention research. The ability to learn associations between choices and outcomes across decision stages is crucial for daily behavior. We assessed the measurement reliability of behavioral performance and fMRI BOLD signals during value-based sequential decision making to evaluate the TRR of task-relevant regions for future research on non-invasive brain stimulations. MethodsTwenty healthy adults (22 to 40 years) completed two task-fMRI sessions, at least 2 weeks apart. During scanning, participants performed two variants of a three-stage Markov decision task with conditions varied in temporal contingency (immediate vs. delayed) and magnitude of choice outcomes (high vs. low). Both sessions were conducted under sham tDCS via a focal 3 x 1 montage targeting left dorsolateral prefrontal cortex (DLPFC). TRR was assessed using intraclass correlation coefficients (ICC) with a two-way mixed-effects consistency model for decision performance and task-related fMRI signals at voxel level and summarized in key regions defined by the extended Human Connectome Project atlas (HCPex). ResultsDecision performance was lower with delayed than immediate outcomes (p < 0.001). Higher outcome magnitude improved performance (p < 0.001). Decision performance increased across learning bins (p < 0.001). The behavioral TRR was in the moderate to good level (ICC(3,1) = 0.744 for accuracy; ICC(3,1) = 0.788 for reaction time). At the whole-brain level, contrasting brain activities in delayed with immediate condition revealed suprathreshold cluster peaks in several frontal-parietal (e.g., orbitofrontal, bilateral dorsolateral prefrontal, and medial parietal cortices) and striatal regions (e.g., bilateral putamen). Voxel-wise ICCs revealed variable but often moderate-to-good TRR across task-relevant regions, with stronger reliability in several striatal, orbitofrontal, and left dorsolateral prefrontal parcels, and more variable reliability across anterior cingulate and medial prefrontal parcels. ConclusionThese results from a 2-session tDCS sham-sham stimulation study establish the validity of using the three-stage Markov decision task in future studies about intervention effects on the frontal-parietal-striatal network.

neuroscience↗

bioRxiv2026

Multispecific nanobody degraders co-deplete membrane receptors and enable targeted delivery of diverse payloads

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

cancer biology↗

bioRxiv2026

Deep mapping of structural perturbations to energetics enables precise TCR design

Affinity optimization and cross-reactivity profiling are pivotal for T-cell receptor (TCR) engineering and immunotherapy, yet remain hindered by the vast diversity of the TCR repertoire and limited structural insights. Here, we present mpTCRai, a deep learning framework that captures the sequential characteristics of antigen presentation and recognition with high precision. Leveraging these precise structural predictions, we established a contact hotspot-based scoring mechanism that explicitly maps structural perturbations to energetic changes, yielding a strong correlation with experimental affinity (r = -0.88). In application, the model identified critical molecular switches, such as specific G-to-A substitutions, and revealed their dependence on structural context. Furthermore, it effectively distinguished functional variants from dangerous cross-reactive mutations like Y5W, thereby mitigating the risk of off-target toxicity. Guided by these insights, we computationally designed four novel A6-TCR variants, demonstrating a rational strategy for candidate selection in adult T-cell leukemia. This work establishes a unified platform integrating structural and energetic constraints to advance precise, safe TCR therapeutic design.

immunology↗

bioRxiv2026

Structural Mechanism of Electron Shuttling in Inducible Nitric Oxide Synthase

Nitric oxide (NO) signaling is pivotal in numerous physiological processes and is implicated in a spectrum of human diseases. Nitric oxide synthases (NOS) initiate NO signaling and govern its magnitude and duration, making them key drug targets. Despite decades of investigation, the structural mechanism by which NOS enzymes transfer electrons from NADPH to haem remains incompletely understood. Here, we report cryo-electron microscopy (cryo-EM) studies of the inducible NOS (iNOS) homodimer in complex with calmodulin (CaM) captured under catalytic turnover conditions, resolving two important functional states: the electron input state and output state. In the input state, the FMN-binding subdomain (FMND) docks onto the FAD/NADPH-binding subdomain (FNR), positioning the FMN cofactor to accept electrons from FAD. The FMND then undergoes a large rotational movement to engage the oxygenase domain (OXY) of the other protomer, adopting the output state, which enables electron transfer from FMN to the haem center via W366. This dynamic movement of the FMND shuttles electrons from the reductase domain (RED) to the OXY active site in iNOS. A point mutation (S594E) that disrupts the FMND-OXY interface markedly reduces the catalytic activity of iNOS and traps a fraction of enzymes in a non-productive intermediate conformation. Together, these findings elucidate the structural mechanism of FMND-mediated electron transfer in the iNOS catalytic cycle.

biochemistry↗

bioRxiv2026

Benchmarking scRNA-seq Copy Number Inference: A Comprehensive Evaluation and Practitioner Guide

Accurately inferring copy number variation (CNV) from scRNA-seq data is critical for identifying malignant cells, reconstructing tumor subclonal architecture, and uncovering the genomic drivers that dictate cancer cell biology. However, the performance of existing tools varies significantly, and current benchmarks lack the breadth of datasets and methods necessary to provide definitive guidance. We present a comprehensive benchmark of 12 CNV inference methods across 28 real datasets (>100,000 cells) and diverse synthetic datasets. By evaluating methods based on malignant cell classification accuracy, CNV inference accuracy, scalability, and robustness, we establish a definitive practitioners guideline: allele-aware methods like Numbat excel when high-quality allelic inference can be achieved, whereas expression-centric tools such as Clonalscope, CopyKAT, inferCNV, and SCEVAN remain reliable when raw sequencing data are unavailable. Our study provides both a practical decision-making framework for researchers and a public repository of standardized CNV profiles to catalyze further methodological innovation.

cancer biology↗

bioRxiv2026

Crystal structure and molecular dynamics simulations of rademikibart Fab-IL-4Rα complex reveal biochemical basis for next-generation potent IL-4Rα inhibition in type 2 allergic and inflammatory diseases

Rademikibart (CBP-201) is a human monoclonal antibody with higher binding affinity to IL-4R compared to dupilumab. Dupilumab is a first-generation interleukin-4 receptor alpha (IL-4R) inhibitor for treating IL-4R-dependent inflammatory disorders, including several dermatologic and respiratory conditions. Rademikibart, however, demonstrated better inhibition of STAT6 intracellular signaling in vitro and similar potency in inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation. To further characterize the molecular function of rademikibart and its differentiation from dupilumab, we determined the crystal structure of the rademikibart fragment antigen binding (Fab) bound to IL-4R at 2.71 [A] resolution and compared this to the 2.82 [A] resolution structure of dupilumab Fab bound to IL-4R. The rotation angle between dupilumab and rademikibart bound to IL-4R is 54.88{degrees}. This rotation enables the binding epitopes of rademikibart, but not dupilumab, on IL-4R to overlap more closely with the conserved binding interface naturally utilized by IL-4 and IL-13 cytokines. Molecular dynamics (MD) studies on rademikibart and dupilumab bound to IL-4R examined the stability of the complexes and effects of amino acid mutations on receptor complex formation. MD simulations demonstrated that the third interface loop (residues 145 to 153 in domain 2) of IL-4R interacts directly with rademikibart, which is absent in the dupilumab/IL-4R complex. This finding is confirmed by increased hydrogen bond interactions at the interface between rademikibart and IL-4R, demonstrating superior binding energy for rademikibart. Through analysis of the x-ray crystallography structures, MD-equilibrated structures, and computational point-mutation analysis of rademikibart, we identified residue Y50 and R55 of the light chain and R97, R99, and Y101 of the heavy chain of rademikibart as key residues interacting with IL-4Rs third interface loop. Our data provides a molecular and structural rationale for the enhanced IL-4R inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4R inhibitor.

biochemistry↗

bioRxiv2026

The Ser83, Arg85, Tyr88, Asn124, Lys192 of C-terminal Lipid-associated membrane hemagglutinin affecting Mycoplasma synoviae agglutination of erythrocyte

Mycoplasma synoviae is an avian pathogen that causes respiratory disease and synovitis, and its hemagglutinin plays a critical role in host cell adhesion. However, the key residues and structural mechanisms underlying hemagglutination remain unclear. In this study, domain analysis of the hemagglutinin family of Mycoplasma synoviae revealed that it contains long-chain and short-chain types, among which LAM HA (VY93_RS01465) was selected as the bait protein due to its complete C-terminal conserved domain. Through yeast two-hybrid screening, 18 host proteins interacting with LAM HA were identified. Furthermore, five key amino acid residues S83, R85, Y88, N124, and K192 were found to mediate hemagglutination activity. Deletion of these residues reduced the hemagglutination titer of LAM HA under acidic conditions. Secondary structure analysis showed that the deletion mutation decreased the -helix content while increasing the proportions of {beta}-sheet and random coil. Molecular dynamics simulations revealed that the mutant exhibited generally higher root mean square deviation and root mean square fluctuation values than the wild-type under different pH conditions, with a marked decrease in structural stability particularly at pH 5.0 and 6.0. These findings indicate that LAM HA, as a critical adhesin, exerts its hemagglutination function dependent on specific key residues and pH-sensitive conformational stability. IMPORTANCEMycoplasma synoviae (M. synoviae) causes significant economic losses to the poultry industry worldwide. Lipid-related membrane protein hemagglutinin (LAM HA) is a surface adhesin essential for host cell attachment, but its precise amino acid residues and structural features have not been defined. In this study, five key residues (S83, R85, Y88, N124, and K192) were identified as critical for LAM HA-mediated hemagglutination activity. Deletion of these residues altered the secondary structure composition, reduced conformational stability under acidic pH conditions, and decreased hemagglutination activity. These findings reveal a previously unknown structure-function relationship of M. synoviae LAM HA, demonstrating that its hemagglutination activity depends on specific residues and pH-sensitive structural integrity. This provides new insights into the molecular mechanisms of M. synoviae adhesion and offers potential targets for the development of novel intervention strategies against avian mycoplasmosis.

microbiology↗

bioRxiv2026

Identification of a somatic H3K23me3 methyltransferase SET-19 in C. elegans

Histone methylation plays essential roles in modulating chromatin organization and gene expression. H3K23 methylation is a conserved histone modification, yet its biological roles and the enzymes responsible for its deposition remain poorly understood. Here, we show that the loss of set-19 leads to a pronounced reduction in H3K23 methylation in C. elegans, as revealed by quantitative mass spectrometry, western blotting, and immunofluorescence staining. In vitro biochemical assays show that recombinant SET-19 proteins purified from E. coli directly catalyze H3K23 methylation. Genome-wide chromatin immunoprecipitation assays reveal that H3K23me3 is enriched at heterochromatic regions and that loss of set-19 alters H3K23me3 levels, accompanied by derepression of gene expression. Genetic analyses indicate that SET-19 is dispensable for both germline and somatic RNAi as well as transgenerational epigenetic inheritance of RNAi. SET-19 is predominantly expressed in somatic cells and specifically mediates H3K23me3 deposition in somatic tissues. The loss of set-19 causes a developmental delay without affecting fertility. Together, our results identify SET-19 as a somatic H3K23 methyltransferase and link H3K23me3 to gene repression in C. elegans.

genetics↗

bioRxiv2026

MetaReact: A Reaction-Aware Transformer for End-to-End Prediction of Drug Metabolism

Accurate prediction of drug metabolites and enzyme selectivity is essential for rational drug design and safety assessment. However, existing computational approaches are often limited to specific enzyme families or reaction types, lacking the capacity to model enzyme-subtype specificity and prioritize major metabolites. Here, we present MetaReact, an end-to-end generalizable Transformer-based model that unifies the prediction of metabolic enzymes, metabolites, and sites of metabolism (SOM). By integrating structure-aware encoding ReactSeq, a chemistry reaction-based pretraining, MetaReact consistently outperforms state-of-the-art methods across multiple benchmarks under three settings: enzyme-agnostic, enzyme-completion, enzyme-conditioned. Notably, it achieves 60% Top-3 accuracy in identifying major metabolites and superior CYP450 enzyme-subtype prediction/SOM recognition. Case studies validate its applicability to complex natural products, synthetic cannabinoids, and clinical candidates, facilitating toxicity assessment and molecular optimization. This scalable, rule-free solution advances human metabolism modeling, with potential for computational pharmacokinetics and early drug discovery.

biochemistry↗

bioRxiv2026

Isoform-resolved spatial transcriptomics on a lab-made high-density array via a single-chip NGS-TGS workflow

High-resolution spatial transcriptomics is still cost-prohibitive and dominated by short-read sequencing, limiting in situ detection of transcript structures. Here we present a low-cost, lab-made, high-density bead-in-microwell chip assembled by routine centrifuge and decoded by a tri-part combinatorial long-barcode strategy. The design provides 5.6x107 barcode combinations across 4.1x10{square} capture sites, reducing collisions while remaining tolerant to third-generation sequencing raw errors. Using a single-chip, dual-platform workflow, we split full-length cDNA for next-generation and third-generation sequencing within the same spatial coordinates. In an incompatible tomato-pepper graft, we identified interface-associated splicing reprogramming, including thousands of unannotated isoforms, enriched novel CaGRP1 isoforms, and SlPIP2 intron retention, supported by individual long-read sequences. In mouse embryos, we further demonstrate cross-species applicability by resolving unannotated Col1a2 isoforms in oligodendrocyte progenitors and spatially restricted Dalrd3 intron retention. This cost-efficient system broadens access to spatial full-length transcriptomics and may support diverse biological studies.

plant biology↗

bioRxiv2026

Molecular genetic characterization of bacterial KH-domain proteins

Post-transcriptional gene regulation is a key mechanism for bacterial stress responses and virulence, and RNA-mediated regulation frequently relies on global RNA-binding proteins (RBPs). A pair of interacting KH-domain proteins (KhpA and KhpB) have recently been identified as global RBPs in several bacterial species. To better understand their molecular functions, we employed bacterial two- and three-hybrid (B2H/B3H) assays in an E. coli reporter system to analyze protein-protein and protein-RNA interactions of KhpA/B orthologs from three human pathogens: Campylobacter jejuni, Helicobacter pylori, and Clostridioides difficile. Protein-protein interactions were conserved across all species, with KhpA-KhpB heterodimers forming more robustly than either homodimer and KhpA homodimerizing more readily than KhpB. On the other hand, protein-RNA interactions were more varied across species: C. jejuni and C. difficile KhpA bound both species-specific and non-specific RNAs, but H. pylori KhpA--and KhpB orthologs from all species--showed no RNA interaction in B3H assays. Site-directed mutagenesis experiments demonstrated that residues in the GXXG motif of KhpA are critical for RNA interaction and differences in these residues account for the distinct RNA-binding behaviors of KhpA orthologs. Collectively, these findings provide a cross-species, molecular view of how KhpA and KhpB recognize one another and RNA ligands to regulate gene expression.

molecular biology↗