Biology subjects

Gu, W.

Publications and source records attributed to Gu, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Ratio-preserving super-resolution FLIM for quantitative live-cell lifetime imaging

Super-resolution (SR) fluorescence lifetime imaging promises nanoscale maps of molecular environment and function, but photon-limited acquisition and computational reconstruction can distort lifetime estimates, particularly for ratio-derived frequency-domain measurements under SR conditions. Here we introduce structured-illumination frequency-domain FLIM (SIM-fdFLIM), an SR lifetime imaging platform that combines commercial dual-tap fdFLIM detection with ratio-preserving reconstruction. SIM-fdFLIM uses direct-current (DC)-stabilized lifetime estimation to improve reconstruction robustness under low signal-to-noise conditions, followed by ratio-domain integrated variational estimation (RIVE) to correct channel misregistration and suppress noise amplification after SR reconstruction. The method achieves ~100 nm spatial resolution in lifetime maps and enables live-cell imaging at 2.8 Hz over a 56 x 56 {micro}m2 field of view. Across representative fluorophore standards, RIVE reduces lifetime standard deviation by approximately threefold relative to non-RIVE SIM-fdFLIM while preserving mean lifetime agreement with calibrated widefield fdFLIM. In living cells, SIM-fdFLIM resolves mitochondrial cristae-scale lifetime heterogeneity, mitochondrion-to-mitochondrion functional dynamics, nanoscale lipid-environment variations and perturbation-sensitive organelle-contact signatures. These results establish SIM-fdFLIM as a ratio-preserving platform for quantitative SR lifetime imaging, extending live-cell FLIM into a regime that combines nanoscale spatial resolution, Hz-rate acquisition, and preserved lifetime fidelity.

bioengineering↗

bioRxiv2026

Early Prenatal Cell-Free DNA Methylation Landscape Informs Epigenetic Purification of the Circulating Fetal Genome

Circulating fetal cfDNA is the analyte of a screening test used by millions of pregnant women annually. However, low fetal fraction limits current testing. Fetal cfDNA can be enriched via hypomethylation, but the informative sites require a first-trimester fetal methylome reference. Here we sequenced fetal cfDNA (median purity 94.7%) derived from discarded media from cultured chorionic villus biopsies (n=30) and assembled a first-trimester methylome across 26M CpG methylation sites at a median depth of 207x. We then built an analytical enrichment model that identified millions of CpG markers and assigned predicted enrichment scores. Guided by this model, we adapted epiSIFT as a genome-wide assay selective for unmethylated DNA molecules. In 28 prenatal plasma cases, epiSIFT raised the median fetal fraction from 5.1% to 28.4% (5.6-fold gain) using the top eighth of markers. These findings establish a first-trimester fetal cfDNA methylome reference and demonstrate epigenetic enrichment of the fetal genome.

genomics↗

bioRxiv2026

Spatiotemporal Heterogeneity of HPV Integration Architectures Drive Oncogenic Features of Oropharyngeal Cancer

Viral integration is common in human papillomavirus-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC), but its structural and clinical significance remains unclear. We integrated targeted capture sequencing of 197 HPV+ OPSCCs with long-read nanopore sequencing of 17 tumors and models to characterize HPV-host rearrangements. We developed a novel quantitative informatics framework that classifies integration structures as Type1 (simple, single/low-copy) or Type2 (clustered, high-copy, rearranged). HPV integration occurred in 83% of tumors, with recurrent hotspots including TP63, MYC, CD274, TRAF2, and KMT2C. Type2 events were enriched for HPV E2 disruption, focal human amplifications, preferential transcription, and noncanonical HPV-host RNA fusion isoforms. Type2 tumors showed increased HPV oncogene expression and activation of MYC, NF-{kappa}B/TNF, PI3K-AKT-mTOR, Hippo/YAP-TAZ, and epithelial stemness programs. Multi-region and longitudinal analyses revealed intratumoral heterogeneity and clonal selection of Type2 architectures during progression, supporting integration structure as a potential marker of aggressive behavior and risk stratification.

bioinformatics↗

bioRxiv2026

Structural basis of coiled-coil domain-mediated inhibition of A. baumannii AbTir NAD+ nucleosidase activity

TIR (Toll/interleukin-1 receptor) domains are central components of innate immunity and cell-death pathways across diverse forms of life. In bacteria, TIR domain-containing proteins participate in diverse antiphage defence systems, several of which rely on NADase activity carried out by TIR domains. This nucleosidase activity is often regulated by additional domains that modulate activation and specificity in response to infection. AbTir, a TIR domain-containing protein from Acinetobacter baumannii, contains an N-terminal CC domain that negatively regulates its NADase activity, but the structural basis of this regulation remains unknown. Here, we used X-ray crystallography to investigate the molecular basis of CC domain-mediated regulation in AbTir. We determined the crystal structure of full-length AbTir in complex with a nanobody, revealing a symmetric AbTir TIR dimer in which only one of the two symmetry-related CC helices is accommodated at the dimer surface, breaking the overall symmetry of the full-length protein. Structural comparisons with other bacterial TIR domains suggest that such TIR dimers likely represent an inactive state, in which the NAD+-binding site has not yet formed. Phylogenetic analysis places AbTir within a family of bacterial TIR proteins associated with defence-related genomic loci, although phage-spot assays performed in the surrogate host Escherichia coli did not detect measurable antiphage activity. Together, these findings reveal an unusual structural mechanism of CC domain-mediated inhibition of a bacterial TIR NADase and expand our understanding of the mechanistic diversity of bacterial TIR proteins.

biochemistry↗

bioRxiv2026

Dietary fiber-type-specific gut microbiome preconditioning drives differential susceptibility to sporadic colon cancer development

Background: Dietary fiber intake is broadly associated with reduced colorectal cancer (CRC) risk, yet processed fiber supplements exert inconsistent and occasionally opposing effects on colon tumorigenesis. How structurally distinct dietary fibers differentially shape the gut microbiome-metabolome axis to influence carcinogenic susceptibility remains poorly understood. Objective: To determine whether structurally distinct dietary fibers differentially modulate colon tumor development and characterize the underlying gut microbiome and metabolic mechanisms. Design: Four-week-old male mice were maintained on low-fiber control or fiber-supplemented diets (7.5%w/w: cellulose, agar, pectin, or inulin) for 28 weeks and received eight intraperitoneal azoxymethane injections (AOM; 7.5 mg/kg). Temporal fecal microbiomes were profiled by 16S-rRNA sequencing, cecal metabolites quantified by H-NMR spectroscopy, and microbiome-metabolome interactions assessed by co-occurrence networks. Results: Refined inulin supplementation markedly elevated colon tumor incidence (~70%) compared with all other groups (~10-20%), and cecal extracts from inulin-fed mice promoted HT29 cancer-cell proliferation. Longitudinal microbiome profiling identified 36 inulin-specific microbial signatures converging on two axes: depletion of SCFAs and fermentation cross-feeding taxa and reprogramming of amino acid and nitrogen metabolism. Metabolomics confirmed succinate, fumarate, and lactate accumulation alongside propionate and amino acid depletion exclusively under AOM conditions, with no differences in carcinogen-naive animals. Two stable co-occurrence hubs were identified exclusively in AOM-treated animals, structurally integrating both functional axes. Conclusion: Inulin supplementation preconditions the gut microbiome toward functional vulnerability, characterized by disrupted fermentation throughput and reprogrammed amino acid metabolism, that is amplified by carcinogen exposure into a tumor-promoting ecosystem, providing a mechanistic basis for the disproportionately higher tumor prevalence in inulin-fed animals.

microbiology↗

bioRxiv2026

An in vitro System for Studying Osteochondrogenic Differentiation of Smooth Muscle Cells and Modeling Intimal Vascular Calcification

Objective: Smooth muscle cells (SMCs) undergo phenotypic transitions during atherosclerosis, including towards a chondromyocyte (CMC) state associated with intimal calcification. Although standard in vitro calcification assays robustly reproduce mineral deposition, it remains unclear how well they recapitulate these disease-associated SMC states. We sought to define the CMC transcriptional phenotype in atherosclerosis and develop an in vitro system that faithfully reproduces it. Approach and Results: We firstly identified a CMC transcriptional signature in murine and human atherosclerotic plaque through single-cell RNA-sequencing, and spatial transcriptomics. CMCs showed a conserved osteochondrogenic program which localized within plaques and adjacent to calcified regions. We then developed an osteochondrogenic differentiation (OCD) assay by combining well-established calcification components with a high-density SMC micromass culture and TGF-{beta}1 supplementation and benchmarked it against a standard calcification (SC) assay using calcium quantification and bulk RNA-sequencing. Despite comparable calcification, OCD and SC resulted in distinct transcriptional states, with OCD showing preferential upregulation of osteochondrogenic programs, and a higher CMC signature score. Additionally, OCD upregulated genes with a stronger enrichment near coronary artery disease (CAD)-associated loci. These responses were reproducible across several primary human SMC lines. Timecourse analysis also showed that chondrogenic programs preceded calcification and showed directional concordance with the inferred in vivo SMC-to-CMC trajectory. To interrogate regulatory pathways controlling this process, we overexpressed the chondrogenic regulator SOX9, which enhanced cartilage and extracellular matrix programs while repressing inflammatory pathways. Finally, we examined 552 CAD-associated genes nominated across five genome-wide association studies. Of these, 240 were differentially expressed by day 12, and included established SMC regulators as well as a number of candidates not previously characterized in osteochondrogenic SMC transition. Conclusions: The OCD assay results in a strong calcification phenotype together with a disease-associated CMC-like transcriptional state, providing a reliable in vitro model for mechanistic investigation of SMC phenotypic transition and prioritization of candidate regulators.

cell biology↗

bioRxiv2026

PromptBio: An Agentic Platform for End-to-End Computational Biomedical Research

Modern biomedical research increasingly depends on complex computational analyses, yet translating a scientific question into a reliable workflow still requires substantial technical expertise and manual coordination. PromptBio is a multi-agent AI platform available through a web portal at https://promptbio.ai that addresses this challenge. Through natural-language interaction, its agent harness, PromptGenie, translates a research objective into an inspectable plan, executes the required research and analysis, and adapts subsequent steps as evidence and results emerge. PromptBio integrates reasoning with managed execution while preserving human oversight and a traceable record of the research process. It can apply validated methods, construct custom analyses, and incorporate external workflows, enabling researchers to move beyond fixed pipelines while supporting reproducibility. We evaluate the platform through benchmarks of end-to-end bioinformatics analysis and biomedical deep research, validation of representative omics and machine-learning skills, and a hypothesis-driven regulatory-genomics case study. PromptGenie achieved higher analytical accuracy and stronger evidence retrieval and synthesis than the comparison agents, while the evaluated domain skills produced results consistent with established methods. The case study further demonstrates how PromptBio can integrate human-specific genomic and epigenomic features to investigate cortical development. Together, these findings show that PromptBio can coordinate complex biomedical analyses under researcher oversight, offering an accessible approach for accelerating scientific iteration and transforming research questions into transparent, reusable computational workflows.

bioinformatics↗

bioRxiv2026

Set mediates chromosome alignment and Cohesin cleavage independently of direct PP2A-B56-binding in oocyte meiosis

Set promotes cohesion removal in mitosis by evicting phosphorylated Histone H1 and counteracting Sgo1. In addition, Set promotes chromosome alignment by counteracting Aurora B activation. The underlying molecular mechanisms through which Set performs these activities remain insufficiently characterized, but roles of Set as a Histone chaperone and PP2A inhibitor have been proposed. Building on our previous observations that Set promotes pericentromeric Cohesin removal in oocyte meiosis II, we generated an oocyte-specific conditional knock-out of Set to address its functions in meiosis. Similar to mitosis, Set depletion caused chromosome alignment and cohesion defects. We found that Set is required for accurate error correction by localizing Aurora B/C, and for efficient cleavage of the meiosis-specific Cohesin subunit Rec8 by Separase. Paired chromosomes and sister chromatids were often incompletely separated, likely a primary cause of missegregation. Set performed both its roles in a Sgo2-dependent manner, but, unexpectedly, independently of interaction with PP2A-B56. In line with a role of Set as a Histone chaperone, accumulation of phosphorylated Histone H1 in Set knock-out oocytes occurs concomitantly with reduction of oocyte-specific H1foo on chromosome arms, indicating that Set is required to create the optimal chromatin environment for efficient Rec8 cleavage by Separase in meiosis.

cell biology↗

bioRxiv2026

IOBRpy enables agentic multi-omics decoding of anti-tumor immunity

Decoding the tumor immunity is pivotal for cancer immunotherapy, yet transcriptomic pipelines remain bottlenecked by fragmented tools and biased interpretations. Here we present IOBRpy, a Python toolkit driven by an innovative AI dual-agent layer for automated, highly standardized immuno-oncology workflows. Moving beyond conventional expression profiling, IOBRpy enables agentic multi-omics decoding. From raw FASTQ or TPM matrices, it seamlessly integrates upstream quality control, transcript quantification, and downstream TME parsing, encompassing signature scoring, ligand-receptor crosstalk, and cellular deconvolution. Crucially, IOBRpy expands data dimensions by incorporating complementary immunogenomic layers, empowering concurrent high-resolution SpecHLA typing and TRUST4-based TCR/BCR repertoire reconstruction from sequencing data. Deployed across two large-scale cohorts (IMvigor210 and OAKPOPLAR), IOBRpy successfully captured multi-dimensional prognostic insights. While broad HLA-I heterozygosity showed negligible impact, it precisely unmasked treatment-stratified, allele-specific survival associations (e.g., HLA-A*01 and HLA-DPA1*02) tightly coupled with distinct immunosuppressive ligand-receptor networks (such as HMGB1-THBD and EFNB2-EPHB6) and dynamic TCR clonal diversity shifts. Empowering this lifecycle is a paired agent framework: a workflow agent automatically audits project states to execute validated, path-aware commands, while a result agent evaluates tool provenance, handles method-aware adaptive visualizations, and organizes findings into evidence-constrained biological hypotheses. Collectively, IOBRpy provides a reproducible, scalable, and intelligence-augmented Python gateway to transform raw sequencing data into multi-omic, interpretation-ready discoveries for cohort-scale precision immunotherapy (https://iobr.github.io/IOBRpy/).

bioinformatics↗

bioRxiv2026

GluDs are ionotropic dopamine receptors tuned by G-proteins

Dopamine is a neurotransmitter essential for cognition, and its dysregulation is associated with neurological diseases1,2. Historically, dopamine has been understood to signal exclusively through metabotropic receptors3. Delta-type ionotropic glutamate receptors (GluDs), which have recently been established as ligand-gated ion channels4,5, are fundamental for synaptic maintenance, are implicated in neurological disorders, and co-localize with dopaminergic machinery. Here, we report that dopamine is a direct agonist of GluDs, eliciting ionotropic activity, as visualized by cryo-electron microscopy (cryo-EM), bilayer recordings, mutagenesis, and patch clamp recordings. Dopamine binds to the GluD ligand binding domain, inducing clamshell closure and channel activation through a distinct molecular interface. GluD channel activity is tightly regulated by G-proteins, which act as molecular switches to tune GluD activity: free G{beta}{gamma} inhibits ligand-gating, while G or inactive G-protein heterotrimers enable dopamine-induced GluD currents. This tuning of GluD activity by G-proteins is uncoupled in a point mutation associated with neurodegeneration. These findings expand mechanisms of neuronal dopaminergic signaling, uncover how G-proteins tune GluD channel activity, and provide a framework for targeting GluDs in neurological diseases.

biophysics↗

bioRxiv2026

Early-life mucosal T cells direct intestinal stem cell fate via a coordinated developmental program

Although early-life immunity was once considered immature, the human fetal immune system is dynamic and compartmentalized by the second trimester. By 21 weeks of gestation, T lymphocytes become a major immune population in the fetal small intestine (SI), yet their functional roles within this tissue remain largely undefined. To explore their unique contributions to intestinal development, we established an ex vivo co-culture system in which mucosal T cells isolated from fetal, neonatal, or adult SI donors were cultured with tissue-derived 3D SI organoids derived from various ages. Homeostatic early-life (fetal and neonatal) SI T cells uniquely promoted organoid generation, a metric of stem cell renewal, by upregulating cell cycle-associated gene programs. These early-life T cells also directed intestinal stem cell differentiation toward the secretory lineage in both growth and differentiation phase, highlighting that T cells poise stem cells to adopt secretory fates. T cells from infants with necrotizing enterocolitis (NEC) - an inflammatory intestinal disease affecting predominantly preterm infants - failed to activate these same programs, suggesting a pathologic role for T cells in NEC. T cells from the adult SI similarly failed to support organoid growth or differentiation, revealing developmentally specialized, nonimmune functions for early-life T cells in the intestine. Similarly, T cells derived from cord blood did not enhance organoid generation, indicating that this function is not necessarily a generalized feature of early-life T cells but rather is restricted to mucosal T cells. Organoids derived from adults or NEC, however, could re-enter regenerative states when co-cultured with fetal T cells, indicating that fetal T cells can restore stem cell self-renewal across developmentally and disease-imposed states. We further identified that T cell-derived soluble factors alone were insufficient to modulate intestinal stem cell fate, implying the need for physical interactions. Concordant with this finding, we report that T cells heavily localize to the stem cell niche during prenatal development, where they express factors involved in Notch, Wnt, and growth factor signaling to support fetal stem cell function. Collectively, these findings reveal a coordinated developmental program in which fetal SI T cells balance stem cell self-renewal and differentiation, identifying a developmental immune-epithelial axis that can be harnessed to restore intestinal regeneration.

immunology↗

bioRxiv2026

PromptBio-Bench: Benchmarking LLM-based Bioinformatics Agents for End-to-End Data Analysis

Large language model (LLM)-based agents hold transformative potential for automating bioinformatics workflows; however, systematic evaluations of their capabilities remain limited, hindering a clear assessment of their readiness for real-world application. We introduce PromptBio-Bench, a comprehensive evaluation suite of 244 expert-curated tasks spanning bioinformatics and data science at varied difficulty levels, and an evaluation framework for structured file comparison and scoring against expert reference answer files. Evaluation of three state-of-the-art bioinformatics agents revealed comparable performance between Biomni and ToolsGenie, with all agents showing a marked decline in accuracy as task difficulty increased. As foundation models and agent frameworks continue to evolve, PromptBio-Bench provides a valuable benchmark infrastructure for systematically tracking progress in agentic bioinformatics.

bioinformatics↗

bioRxiv2026

Generation and long-term expansion of human pancreatic islet organoids in vitro

The scarcity of expandable, functional human islet cells remains a major barrier to diabetes therapy. Here, we identify PROCR+ cells within adult human islets and establish a defined culture system to generate pancreatic islet organoids. These organoids self-organize into -, {beta}-, {delta}-, and PP cells at near-native ratios, exhibit regulated insulin and glucagon secretion, and support exponential in vitro expansion. Single-cell transcriptomics reveals a unique progenitor-like cell population that is transcriptionally primed for endocrine differentiation but shares molecular features with fetal trunk cells and endocrine progenitors. When transplanted, the organoids rapidly ameliorate hyperglycemia in diabetic mice. Importantly, in a non-human primate model, intraportal transplantation of these organoids reduced exogenous insulin requirements, restored glucose-stimulated C-peptide secretion, and achieved sustained glycemic control-representing a critical step toward clinical translation. This study provides a strategy for expanding human islet organoids, offering a scalable platform for diabetes treatment, disease modeling, and regenerative medicine.

cell biology↗

bioRxiv2026

HSC71 acetylation confers protection against Spiroplasma eriocheiris infection by inhibiting apoptosis and promoting ROS production in arthropods

Heat shock proteins (HSP) are increasingly recognized as pivotal regulators of host innate immunity. However, the role of HSP70 post-translational modifications in modulating the immunologic functions during pathogenic infection remains poorly understood. Here, we demonstrated that heat shock cognate 71 kDa protein (HSC71), a member of the HSP70 family, was deacetylated following infection with intracellular pathogen Spiroplasma eriocheiris. HSC71 deficiency exacerbated hemocyte apoptosis and increased host susceptibility to S. eriocheiris infection. Conversely, ectopic expression of hyperacetylation-mimetic HSC71(K579Q) in Drosophila S2 cells conferred robust protection against S. eriocheiris by suppressing apoptosis and augmenting reactive oxygen species (ROS) production. Mechanistically, carnitine O-acetyltransferase (Crat) was identified as the specific acetyltransferase for HSC71. Crat acetylated HSC71 at lysine 579 (K579), which in turn impeded its interaction with the E3 ubiquitin ligase CHIP, thereby stabilizing HSC71 by attenuating ubiquitin-mediated degradation. Crat deficiency induced apoptosis and reduced ROS levels in hemocytes, thereby exacerbating S. eriocheiris intracellular proliferation. Furthermore, acetylation of HSC71 at K579 weakened its binding to superoxide dismutase (SOD), resulting in impaired SOD activity and consequent accumulation of microbicidal ROS. Pharmacological inhibition of the deacetylase SIRT1 with EX-527 enhanced HSC71 acetylation and ROS production, conferring resistance to S. eriocheiris infection in crabs. Notably, EX-527 similarly enhanced the acetylation of Drosophila HSC71 homolog, HSPA8, which in turn impaired its interaction with SOD. This led to elevated ROS levels and restricted intracellular proliferation of S. eriocheiris in S2 cells, demonstrating evolutionary conservation of this mechanism among arthropods. Therefore, this study established the modulation of HSC71 acetylation as a promising strategy to enhance arthropod immunity. Author summaryMembers of the HSP70 family are indispensable components of host innate immunity, and their functions are finely tuned by post-translational modifications. This study investigated the mechanisms that underlie acetylation modification of HSC71 mediated resistance to S. eriocheiris infection in crab. Our results revealed that the acetyltransferase Crat and the antioxidant enzyme SOD directly interacted with HSC71. Mechanistically, Crat acetylated HSC71 at K579, which prevented its ubiquitination by promoting the disassociation of E3 ubiquitin ligase CHIP, thereby improving HSC71 stability. In HSC71-deficient or Crat-deficient crabs, hemocyte apoptosis was markedly enhanced, leading to higher host mortality upon S. eriocheiris challenge. Meanwhile, K579 acetylation on HSC71 weakened the interaction between HSC71 and SOD, resulting in the accumulation of intracellular ROS and thereby restricting S. eriocheiris propagation. Administration of the SIRT1 inhibitor EX-527 enhanced HSC71 acetylation, elevated ROS production and reduced host susceptibility to S. eriocheiris infection. These findings revealed a novel role for Crat-mediated acetylation of HSC71 in orchestrating host defense through two synergistic mechanisms: suppressing apoptosis and promoting ROS accumulation. Thus, targeting HSC71 acetylation may represent a promising avenue to combat S. eriocheiris infection.

immunology↗

bioRxiv2026

Structural basis of canonical TIR-NLR activation in plant innate immunity

In plants, intracellular NLRs (nucleotide-binding leucine-rich repeat receptors) detect pathogen effector proteins, form oligomeric resistosomes, and activate ETI (effector-triggered immunity). NLRs contain N-terminal signaling, central NB-ARC (nucleotide-binding) and C-terminal LRR (leucine-rich repeat) domains. NLRs with N-terminal TIR (Toll/interleukin-1 receptor) domains (TNLs) hydrolyze NAD+ (nicotinamide adenine dinucleotide) to generate signaling molecules. We determined cryo-EM structures of flax M, a canonical non C-JID (C-terminal jellyroll/Ig-like domain) TNL, in both monomeric autoinhibited conformation, and tetrameric resistosome after activation by its rust fungal effector AvrM-A. AvrM-A homodimers dissociate into monomers to bind directly to the LRR and NB-ARC domains in the M resistosome. The resistosome structure includes a non-hydrolyzable NAD+ analogue, revealing the substrate NAD+ recognition mechanism by the TIR domains. M cleaves NAD+ and generates the same signaling compounds as the related flax TNL, L6. Our findings explain the mechanism of TNL signaling, and provide a basis for rational engineering of disease-resistant crops.

plant biology↗

bioRxiv2026

Leptin Receptor+ cells create a perisinusoidal niche for thrombopoiesis in the bone marrow by synthesizing CXCL14

Leptin Receptor-expressing (LepR+) stromal cells in the bone marrow are a critical source of growth factors for the maintenance of hematopoietic stem cells (HSCs) and most restricted hematopoietic progenitors. An important unresolved question is whether they also regulate terminal differentiation in some hematopoietic cells. We found that LepR+ cells promote thrombopoiesis by synthesizing the chemokine CXCL14, which is expressed in the bone marrow by a subset of LepR+ cells. Cxcl14-expressing LepR+ cells extend fine processes that wrap around perisinusoidal megakaryocytes. Deletion of Cxcl14 from LepR+ cells did not significantly alter HSC function or most aspects of bone marrow hematopoiesis, including megakaryocyte generation; however, it significantly reduced the numbers of proplatelet-forming megakaryocytes in the bone marrow and platelets in the blood. CXCL14 promoted platelet formation by remodeling lipid metabolism in megakaryocytes, increasing fatty acid transporter expression and enabling megakaryocytes to take up more polyunsaturated fatty acids from circulation. A high fat diet rescued the formation of proplatelet-forming megakaryocytes and platelets in Lepr-cre; Cxcl14 fl/fl mice. CXCL14 protein was sufficient to promote platelet formation by megakaryocytes in vitro and in vivo. LepR+ cells thus create a perisinusoidal niche for thrombopoiesis by producing CXCL14, which regulates lipid metabolism and terminal differentiation in megakaryocytes. Key pointsO_LILeptin Receptor+ stromal cells regulate terminal differentiation in megakaryocytes in addition to maintaining stem and progenitor cells C_LIO_LICXCL14 from Leptin Receptor+ cells promotes the formation of platelets by remodeling lipid metabolism in megakaryocytes in the bone marrow C_LI

cell biology↗

bioRxiv2026

ToolsGenie 2.0: A Scalable and Extensible Multi-Agent System for Bioinformatics Automation

The rapid expansion of biomedical data necessitates efficient bioinformatics tools, yet conventional workflows rely heavily on manual dependencies, hindering scalability and broader adoption. Building on the foundation of ToolsGenie 1.0, we introduce ToolsGenie 2.0, a multi-agent AI framework that automates bioinformatics analyses through natural language queries and file inputs. ToolsGenie 2.0 addresses the growing need for customizable analyses by offering extensibility, reproducibility, improved accuracy, and ease of use. It incorporates a ReAct-based architecture with dual-layer extensibility for sub-agents and specialized tools, along with dynamic Docker image selection for automated, sandboxed, and secure environment management. Rigorous benchmarking shows that ToolsGenie 2.0 achieves 68.6% accuracy on an in-house dataset and 48.3% accuracy on BixBench, demonstrating competitive performance across diverse evaluation settings. These innovations position ToolsGenie 2.0 as a versatile platform for broadening access to bioinformatics in both research and clinical contexts. ToolsGenie 2.0 is available on the PromptBio platform (platform.promptbio.ai).

bioinformatics↗

bioRxiv2026

DNA sequence quantitatively encodes CTCF-binding affinity at genome scale

CTCF is a central architectural protein that shapes 3D genome organization through sequence-specific DNA binding, but how DNA sequence quantitatively determines CTCF-binding strength remains poorly understood. Progress has been hampered by the lack of large, high-quality measurements of binding affinity. Here, we experimentally determine in vitro CTCF-binding affinity for 276,765 DNA sequences derived from the human genome, generating a comprehensive quantitative landscape of CTCF-DNA interactions. Leveraging these data, we develop DeepCTCF, a deep learning model that predicts CTCF-binding strength directly from DNA sequence and enables quantitative interpretation of CTCF motif grammar. Using this framework, we systematically dissect how specific sequence features modulate CTCF-binding affinity and generate quantitative predictions for disease-associated variants that alter CTCF binding. Together, this study defines general principles by which DNA sequence encodes CTCF-binding affinity and provides a quantitative framework for interpreting regulatory sequence variation. TeaserMapping how DNA encodes CTCF binding reveals quantitative rules across over 1{per thousand} of the human genome.

genomics↗