Biology subjects

Li, B.

Publications and source records attributed to Li, B..

At least 109 records · Page 6Linked to original sources

bioRxiv2026

Integrating temporal genomic and transcriptomic analyses to decipher genetic basis of feed intake in dairy cattle

BackgroundFood intake is a complex trait in living organisms, where the genetics of food intake have been widely studied in humans, mice, Drosophila, cattle, pigs, chicken, and fish. In dairy cattle, intake of feed is highly linked to individuals energy balance, health, production, efficiency, and the environmental footprint of the individual to the society. Recent studies have provided solid evidence of the genetic variation of feed intake (FI) in dairy cattle population, but the genetic basis and molecular mechanism of dairy feed intake is still far from clear especially considering the lactation cycles of dairy cattle. This study aims to integrate stage-dependent genome-wide association (GWA) analyses, regional heritability mapping (RHM), and RNA-seq gene expression analyses to identify temporal functional variants associated with cattle dry matter intake (DMI) across multiple stages in lactation cycles. A total of 750,000 daily DMI records from 7,500 lactations of 2,300 cows were available with animals genotype and pedigree information. Total RNA-seq from blood were generated for 121 individuals in this population from 2 lactation stages. Data were split into multiple lactations stages for GWA, RHM, and transcriptomic analyses. ResultsStage-dependent GWAS and RHM identified 21 significant loci associated with DMI across multiple lactation stages. A total of 45 candidate genes were identified from GWA and RHM. Among all the 45 genes, six genes were later found significantly differently expressed between high and low feed intake animal groups using gene expression information from RNA-seq data. These genes show links to sugar and adipose metabolism, milk production, body weight, dopamine-reward pathways and immune functions. ConclusionsOur multi-omics analyses provide molecular evidence that the genetic basis of cattle DMI across lactation is not static. Temporal genomic variants associated with FI were identified with their transcriptomic patterns investigated, decoding the molecular mechanisms underlying DMI. Overall, the associated variants and candidate genes uncovered herein decoded genetic architecture of dairy feed intake on a temporal and multi-omics basis, enhancing the understanding of basic biology of dairy feed intake and informing breeding strategies aimed at improving dairy feed efficiency.

genomics↗

bioRxiv2026

The genetic repertoire of the deep sea: from sequence to structure and function

The deep sea as the largest and maybe most hostile environment on Earth is still underexplored especially regarding its genetic repertoire. Yet, previous work has revealed significant habitat-specific deep-sea biodiversity. Here, we present an integrated deep-sea genetic dataset comprising 502 million nonredundant genes from 2,138 samples and 2.4 million predicted structures, and used it to link specific protein structures with genetic variants associated with life in the deep sea and to assess their biotechnology potential. Combining global sequence analysis with biophysical and biochemical measurements revealed unprecedented sequence diversity, yet substantial structural conservation of proteins. Especially proteins involved in replication, recombination, and repair were identified to be under rapid evolution and with specialized properties. Among these, a structurally divergent helicase exhibited advantages in controlling nanopore sequencing speed. Thus, our work positions the deep sea as a unique evolutionary engine that generates and hosts genetic diversity and bridges genetic knowledge with biotechnology.

bioinformatics↗

bioRxiv2026

Metabolite profiles distinguish exposure to Dengue and Zika flaviviruses in human induced pluripotent stem cells (hiPSCs)

Flaviviruses such as Dengue virus (DENV), Zika virus (ZIKV) and West Nile Virus (WNV) pose growing public health threats, exacerbated by asymptomatic infections and non-vector transmission. Current diagnostics, including nucleic acid tests and serology, are limited by transient viremia and antibody cross-reactivity, underscoring the need for improved detection methods. We utilized untargeted metabolomics to differentiate DENV and ZIKV infections in human induced pluripotent stem cells (hiPSCs), a model relevant to study virus-host interactions, drug screening, and therapeutic safety. LC-MSbased profiling revealed virus-specific metabolic reprogramming during both acute and long-term infections. DENV3 induced early and sustained activation of metabolic features, while ZIKV-MR766 triggered transient suppression. Long term infection with DENV2, DENV3, or ZIKV-PRV resulted in distinct metabolic signatures in hiPSCs, with ZIKV-PRV showing the greatest divergence. Shared metabolite changes across conditions included amino acids (e.g., tryptophan, glutamate), lipids, and nucleosides. Functional studies demonstrated that tryptophan metabolism regulates infection dynamics: inhibiting serotonin biosynthesis reduced viral load, whereas blocking kynurenine synthesis enhanced viral replication. These findings position metabolomics as a viable approach for flavivirus detection and highlight metabolic pathways as potential therapeutic targets. ImportanceAccurate diagnosis of flavivirus infections remains challenging due to short periods of viremia, high rates of asymptomatic infection, and extensive serologic cross-reactivity among related viruses such as DENV and ZIKV. These limitations complicate clinical diagnosis, surveillance, and screening of human donor tissues. In this study, we demonstrate that untargeted metabolomics can distinguish ZIKV and DENV infections by identifying virus-specific host metabolic signatures during both acute and long-term infection. Using human induced pluripotent stem cells as a clinically relevant model, we show that metabolic reprogramming persists even in the absence of overt cytopathic effects and can reveal functional pathways that regulate viral replication. Our findings highlight host metabolite profiling as a complementary diagnostic strategy that may improve detection of flavivirus exposure, particularly in asymptomatic individuals or settings where conventional molecular and serologic tests are insufficient.

microbiology↗

bioRxiv2026

Targeting Circulating FABP4 Ameliorates Obesity-Associated Hepatic Steatosis

Obesity is a major driver of hepatic steatosis, yet the molecular link between excess adiposity and hepatocellular lipid accumulation remains incompletely defined. Here, we identify circulating fatty acid-binding protein 4 (FABP4) as a key mediator of adipocyte-hepatocyte lipid crosstalk in obesity. Analyses of human liver specimens and mouse models reveal aberrant accumulation of FABP4 protein--but not transcript--in hepatocytes during steatosis, indicating an extrinsic source. Genetic deletion of FABP4, specifically in adipocytes, protects against high fat diet-induced hepatic steatosis without altering obesity or systemic lipid levels. Mechanistically, circulating FABP4 directly binds to hepatocytes, facilitating free fatty acid uptake. Furthermore, we developed a high-affinity humanized monoclonal antibody that selectively neutralizes circulating FABP4, blocks hepatocyte binding, suppresses fatty acid uptake, and markedly attenuates hepatic steatosis in multiple obese mouse models. These findings establish circulating FABP4 as a pathogenic lipid chaperone and a promising therapeutic target for obesity-associated hepatic steatosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/701451v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@5b91d5org.highwire.dtl.DTLVardef@1c34892org.highwire.dtl.DTLVardef@1d17ea9org.highwire.dtl.DTLVardef@d2b43c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHepatocytic accumulation of extrinsic FABP4 links adiposity to liver lipid deposition. C_LIO_LISpecific deletion of FABP4 in adipocytes prevents hepatic steatosis without affecting systemic lipid levels or obesity. C_LIO_LICirculating FABP4 derived from adipocytes directly binds hepatocytes to facilitate free fatty acid transfer. C_LIO_LIBlocking circulating FABP4 with a high-affinity anti-FABP4 monoclonal antibody inhibits hepatocyte lipid uptake and attenuates steatosis in multiple obese mouse models. C_LI

pathology↗

bioRxiv2026

Epigenetic Programming of Macrophage Phenotypes by STING-IRF3 Drives Inflammation in Ascending Thoracic Aortic Dissection

BackgroundAscending thoracic aortic dissection (ATAD) is characterized by extensive macrophage (M{Phi}) accumulation and profound inflammation; however, the mechanisms sustaining pro-inflammatory M{Phi} activation remain incompletely defined. Emerging evidence indicates that epigenetically generated immune memory drives innate immune cells toward persistent inflammatory states. In this study, we investigated whether epigenetic reprogramming governs M{Phi} phenotypic fate and contributes to ATAD pathogenesis. MethodsWe performed single-cell RNA sequencing of human ascending aortic tissues from controls, patients with ascending thoracic aortic aneurysm (ATAA), and patients with acute ascending thoracic aortic dissection (ATAD). We also performed integrated single-cell RNA sequencing, single-cell ATAC sequencing, and spatial transcriptomics in an angiotensin II (Ang II)-infused mouse model. The role of the STING-IRF3 signaling axis in M{Phi} epigenetic programming was examined using M{Phi}-Sting -/- and M{Phi}-Irf3-/- mice. ResultsIn human and mouse aortic tissues, we identified multiple functional M{Phi} populations including pro-inflammatory, phagocytic/anti-inflammatory, proliferative, and reparative/healing M{Phi}s. Aortic M{Phi}s in both sporadic ATAD patients and Ang II-induced ATAD mice exhibited a pronounced pro-inflammatory bias with enhanced differentiation toward pro-inflammatory M{Phi}s and impaired differentiation toward phagocytic/anti-inflammatory states. Pro-inflammatory M{Phi}s were particularly abundant in dissection sites, whereas phagocytic M{Phi}s were enriched in discrete adventitial niches. Origin analyses revealed a substantial increase in CCR2 recruited M{Phi}s within the aortic wall, which preferentially differentiated into pro-inflammatory M{Phi}s. In contrast, LYVE1 resident M{Phi}s-- predominantly biased toward phagocytic phenotypes--were markedly depleted in ATAD. Single-cell ATAC sequencing identified coordinated chromatin remodeling with increased accessibility at pro-inflammatory gene loci and decreased accessibility at phagocytic gene loci. Among candidate transcriptional regulators identified, IRF family TFs, including IRF3 emerged as unique factors capable of simultaneously promoting pro-inflammatory gene programs while suppressing phagocytic gene expression. Mechanistically, STING-IRF3 signaling orchestrates this biased transcriptional state, likely through coordinated BRG1-dependent chromatin opening at pro-inflammatory gene loci and chromatin closing at phagocytic/anti-inflammatory gene loci. M{Phi} specific Sting -/- and Irf3-/- mice exhibited attenuated inflammatory reprogramming and reduced aortic destruction and dissection. ConclusionsThese findings identify STING-IRF3-mediated epigenetic programming of M{Phi}s as a fundamental mechanism driving aortic inflammation and ATAD development. Targeting M{Phi} epigenetic programming may represent a promising therapeutic strategy to prevent aortic dissection. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/701198v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@c97bcdorg.highwire.dtl.DTLVardef@1df0ca8org.highwire.dtl.DTLVardef@b7fd04org.highwire.dtl.DTLVardef@1443e16_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Multiomics-driven discovery of predictive biomarkers and strategies to overcome resistance to SFK-YAP inhibition in cholangiocarcinoma

The limited efficacy of current therapies against cholangiocarcinoma (CCA) necessitates the development of novel treatment strategies. Src family kinases (SFKs) contribute significantly to tumor progression and resistance in CCA. Therefore, we investigated the novel, first-in-class SFK OFF inhibitor NXP900 in diverse preclinical CCA models, including those with acquired resistance. This study evaluated the therapeutic effects of NXP900 and detailed adaptive molecular responses to SFK inhibitor therapy. We also aimed to identify biomarkers predictive of drug sensitivity using integrated multiomic profiling and develop strategies to overcome resistance. NXP900 inhibited YAP activity through direct inhibition of tyrosine phosphorylation and indirect activation of the Hippo pathway via LATS. These effects were associated with decreased tumor cell viability in CCA cell lines and several in vivo models. Notably, IDH-mutant patient-derived xenograft CCA models were particularly sensitive to NXP900. NXP900 also synergized with gemcitabine/cisplatin chemotherapy, enhancing antitumor efficacy in both in vitro and in vivo models. Multiomic analyses combining transcriptomics, global proteomics, and phosphoproteomics identified molecular features associated with primary response and acquired resistance. IL13RA-AKT signaling was upregulated in resistant models; NXP900 sensitivity could be restored with AKT or IL13RA2 inhibition. Together, these findings demonstrate the therapeutic potential of NXP900 as a novel YAP inhibitor in CCA and support further investigation in a clinical trial. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/699926v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1cb9e6eorg.highwire.dtl.DTLVardef@10e50e2org.highwire.dtl.DTLVardef@e04dfaorg.highwire.dtl.DTLVardef@1f7334_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

A Beneficial Endornavirus Enhances the Fitness of Phytopathogenic Fungus Rhizotonia solani

Although viruses are primarily characterized as pathogenic agents, certain viruses confer advantages to their hosts. The extent to which a virus can improve host biological performance, however, remains a fascinating topic in virology. An endornavirus, Rhizoctonia solani endornavirus IM (RsEV-IM), was identified as prevalent in Rhizoctonia solani isolates obtained from potato plants. Comparative analysis with a virus-free isogenic strain demonstrated that RsEV-IM infection enhances mycelial growth, sclerotium formation, stress tolerance, and fungal virulence across multiple plant species. Inoculation tests involving numerous R. solani strains confirmed that only RsEV-IM-infected strains exhibited high pathogenicity, independent of other mycovirus infections. Additionally, its secreted protein fraction of RsEV-IM-infected fungus contained elevated levels of various proteins, including those involved in cell wall degradation. This fraction not only facilitated R. solani infection but also suppressed the growth of other fungi and bacteria. These findings position RsEV-IM as a beneficial virus that widely enhances its hosts biological fitness. From both pathological and ecological perspectives, these observations are significant, as they reveal that a mycovirus can serve as a key virulence determinant in fungal populations and potentially shape microbial community dynamics in natural environments. ImportanceFungal pathogenicity and ecological traits have long been thought to be primarily governed by endogenous genetic factors. However, this study reveals a mutualistic relationship between an endornavirus (RsEV-IM) and Rhizoctonia solani, demonstrating that viral infection enhances fungal virulence and ecological fitness. RsEV-IM stimulates fungal growth and the secretion of cell wall-degrading enzymes, resulting in a severe disease phenotype. Ecologically, RsEV-IM-infected fungi potentially gain a competitive advantage over soil microbiota. These findings present a key example of a virus acting as an essential extrachromosomal determinant of fungal pathogenicity and ecosystem interactions. Our results advance the understanding of fungal virulence mechanisms and underscore the broader significance of beneficial virus-fungus associations in agriculture and microbial ecology.

microbiology↗

bioRxiv2026

Spatial Decoding of Tertiary Lymphoid Structure Maturation in Non-Small Cell Lung Cancer Using Deep Neural Networks

Understanding the role of tertiary lymphoid structures (TLS) is crucial in non-small cell lung cancer (NSCLC), as they are associated with patient prognosis and treatment outcomes. Specific cellular ecosystems that originate anti-tumor activity or predict immunotherapy response remain poorly characterized. To this end, we developed a high-resolution, multimodal spatial atlas jointly profiling transcriptomics, proteomics, and histology to characterize TLS maturation in NSCLC alongside secondary lymph organs as a baseline. Using this atlas, we proposed a pathologist-in-the-loop framework that combines a variational graph autoencoder (VGAE) with diffusion pseudotime to refine human expert annotations and characterize TLS maturation. These spatial molecular representations were extended to H&E whole-slide images via a vision transformer-based foundation model. Next, we resolved cellular composition, spatial organization, and cell-cell interactions within these data and defined two divergent spatial ecosystems. Clinical evidence suggests that these ecosystems are associated with distinct patient outcomes: a mature germinal center niche with favorable prognoses, and a tumor-macrophage-fibroblast niche with unfavorable prognoses. In summary, our work decodes key components of TLS heterogeneity, identifies hallmark spatial patterns involved in NSCLC adaptive immunity, and provides a framework for translating spatial omics insights into clinical applications.

genomics↗

bioRxiv2026

Progranulin deficiency induces lipid droplet accumulation in microglia via a STAT3-GPAT3 axis

Heterozygous mutations in the progranulin (PGRN) encoding gene GRN cause frontotemporal dementia (FTD), whereas homozygous GRN mutations lead to neuronal ceroid lipofuscinosis (NCL). However, the mechanisms underlying neurodegeneration due to PGRN deficiency remain unclear. In the aged brains or under neurodegenerative conditions, the accumulation of lipid droplets (LDs) in microglia contributes to cellular dysfunction and pro-inflammatory responses, exacerbating neurodegenerative pathology. Here, we investigated how PGRN deficiency induces LD accumulation in microglia. We found that PGRN ablation significantly upregulated triglycerol-3-phosphate acyltransferase 3 (GPAT3), the first and rate-limiting enzyme for triacylglycerol biosynthesis, and increased LD formation in both Grn-/- BV2 microglial cells and Grn-/-mouse brains. Mechanistic study revealed that PGRN deficiency upregulates GPAT3 via stimulating signal transduction and activator of transcription factor 3 (STAT3) signaling. Silencing Gpat3, inhibiting GPAT3 activity with the small molecule inhibitor FSG67, or PGRN restoration reduced LD accumulation and rescued the cytotoxicity of Grn-/- microglia conditional medium (MCM) toward N2a cells. Notably, intraperitoneal administration of FSG67 or AAV-MG1.2-mCx3cr1-mGrn-eGFP-mediated microglia-specific gene therapy reduced microglial LDs and ameliorated behavior phenotypes in Grn-/- mice. Our study elucidates the role of PGRN loss in microglial lipid homeostasis and identifies the STAT3-GPAT3 axis as a potential therapeutic target for LD-associated neurodegenerative diseases.

neuroscience↗

bioRxiv2026

Multiomics reveals epigenetic control of fibroblast activity after myocardial infarction and a key role for RUNX transcription factors

BackgroundAfter myocardial infarction (MI), cardiac fibroblasts proliferate and undergo a sequential differentiation process. They first transition into cardiac myofibroblasts, a transient and highly contractile state, and ultimately into matrifibrocytes, a more stable state that partially resembles chondrocytes. These dynamic transitions are essential for infarct healing and scar formation. While insufficient fibroblast activation can compromise infarct integrity, excessive activation promotes pathological fibrosis that impairs cardiac function. Despite its clinical importance, the transcriptional and epigenetic regulation of these transitions remain poorly understood. Elucidating underlying mechanisms is critical for developing strategies to fine-tune fibroblast activity during cardiac repair. MethodsWe performed bulk RNAseq, ATACseq, CUT&Tag, CUT&RUN, EMseq, and Hi-C on cardiac fibroblasts from uninjured and post-MI mouse hearts. In parallel, we conducted single-nucleus multiomic (snRNAseq and snATACseq) profiling across multiple time points after MI. Subsequent integrated analysis explored epigenetic mechanisms regulating cardiac fibroblast gene expression and activity. Using an improved computational strategy, we constructed gene regulatory networks to identify key transcription factors and biological processes regulated by these transcription factors. To assess the role of Runx1 specifically, we used tamoxifen-inducible, fibroblast-specific Runx1 knockout mice to evaluate transcriptional, epigenetic, and functional outcomes with the same genomic tools and additional complementary assays. ResultsCardiac fibroblasts undergo extensive chromatin remodeling after MI, which is highly correlated with changes in transcriptomic profiles. In contrast, the role of DNA methylation is relatively minor. Gene regulatory network analysis identified Runx1 as a central regulator of cardiac fibroblast proliferation and matrifibrocyte differentiation. In vitro and in vivo validation confirmed Runx1 as a key modulator of transcriptional and epigenetic changes in cardiac fibroblasts. Runx1 KO reduced cardiac fibroblast proliferation, disrupted the myofibroblast-to-matrifibrocyte transition, and affected macrophage cytokine expression through altered cardiac fibroblast-macrophage communication. Fibroblast-specific Runx1 knockout mice showed improved post-MI survival and reduced cardiac dilatation, especially in males. Simultaneous Runx2 deletion further enhanced the effects of Runx1 knockout. ConclusionsCardiac fibroblast activation and differentiation after MI are regulated by dynamic epigenetic changes. Runx1 plays a pivotal role in modulating cardiac fibroblast activities, and its deletion improves cardiac repair by mitigating maladaptive fibroblast responses. By illuminating the centrality of Runx1 in post-MI repair, this study identifies an actionable pathway for therapeutically steering fibroblast responses.

genomics↗

bioRxiv2026

A mediterranean-mimicking diet harnesses gut microbiota-derived 3-IAA to rejuvenate T cell

Diet profoundly shapes the tumor microenvironment through metabolite modulation, yet the mechanisms linking diet, gut microbiota, and antitumor immunity remain unclear. Here, we demonstrate that a mediterranean-mimicking diet (MedDiet) suppresses tumor growth by orchestrating a microbiota-metabolite-immune axis. MedDiet selectively enriched Bacteroides thetaiotaomicron (B. thetaiotaomicron) in the gut, whose administration recapitulated tumor suppression. Both MedDiet and B. thetaiotaomicron elevated plasma levels of the tryptophan metabolite indole-3-acetic acid (3-IAA). Mechanistically, 3-IAA enhanced CD8+ T cell cytotoxicity and alleviated exhaustion by inhibiting the integrated stress response, sustaining antitumor immunity in vivo. Importantly, 3-IAA synergized with anti-PD-1 therapy to further restrain tumor progression. Finally, we developed a MedDiet Score to predict patient survival and response to immunotherapy. These findings reveal that dietary modulation of gut microbiota can reshape systemic metabolites to potentiate T cell-mediated antitumor responses. Our study identifies 3-IAA as a metabolite-based adjuvant for immune checkpoint therapy and highlights the translational potential of diet-driven immunomodulation in cancer treatment.

cancer biology↗

bioRxiv2026

Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified Intra-tumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based TME classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13 CD4 T cells co-localize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudo-temporal maturation continuum: aggregated, activated, and post-activated. Intra-niche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12 macrophages, evolved dynamically with maturation. Single-nucleus RNA-sequencing combined with Tangram-based spatial mapping revealed CXCL12 macrophages and iCAFs forming a peripheral band in aggregated TLSs, while ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy. TeaserAn atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.

immunology↗

bioRxiv2026

A plug-and-play ROI imaging module and deep-learning denoising framework extend three-photon microscopy to 1.7 mm depth

Three-photon microscopy (3PM) has extended optical imaging beyond the reach of two-photon microscopy (2PM), but its practical depth remains far below theoretical predictions because of low photon efficiency and severe noise contamination. Here we present an integrated hardware and software solution that addresses these limitations. We developed a plug-and-play region-of-interest (ROI) imaging module that selectively excites neuron-occupied regions to improve power efficiency and reduce photothermal stress. The module incorporates frame-partitioned accumulative (FPA) imaging for high signal-to-noise ratio (SNR) structural acquisition, automated neuron segmentation, and motion-robust registration for stable recordings. Complementing this, OptiCal is a cascaded deep-learning framework that removes mixed noise including periodic, motion-induced, and random components. Together, these innovations enable high-fidelity imaging to 1.7 mm depth, extending the practical limit of 3PM by about 400 {micro}m while maintaining low excitation power. Our results reveal reliable calcium activity and behavior-correlated dynamics in deep medial prefrontal cortex of awake mice.

neuroscience↗

bioRxiv2025

Kilohertz-rate two-photon voltage imaging of population dynamics in vivo

Understanding how neural circuits compute requires capturing voltage dynamics across large neuronal populations with millisecond resolution in vivo. However, two-photon voltage imaging remains fundamentally limited by a trade-off among imaging speed, field of view, and excitation efficiency. We introduce HS2PM, a hybrid scanning two-photon microscope that overcomes this bottleneck, enabling kilohertz-rate imaging over a 650 x 524 m2 field of view at single-cell resolution while preserving the photon efficiency of single-point excitation. HS2PM stably records deep-layer membrane potentials from over 160 neurons simultaneously, with minimal photobleaching and phototoxicity. It resolves both spikes and subthreshold voltage dynamics in vivo, revealing how these jointly shape sensory adaptation and population coding. Beyond voltage imaging, HS2PM supports high-speed fluorescence lifetime and vascular flow imaging, establishing a multimodal platform for dissecting fast circuit dynamics with precision previously inaccessible to optical methods.

neuroscience↗

bioRxiv2025

OmniCell: Unified Foundation Modeling of Single-Cell and Spatial Transcriptomics for Cellular and Molecular Insights

A cells transcriptional programme is not fully defined by gene expression alone, but by the tissue context in which that programme is enacted. Single-cell RNA sequencing resolves molecular identity after dissociation, whereas spatial transcriptomics preserves tissue architecture but remains constrained by assay-specific sparsity and gene coverage. Here we present OmniCell, a tissue-contextual transcriptomic foundation model pretrained on 67 million dissociated and spatially resolved profiles. By integrating gene identity, expression magnitude and tissue context, OmniCell links transcriptional programmes to the cellular neighbourhoods and anatomical contexts in which they operate. OmniCell organised transcriptomes across molecular, cellular and tissue scales. It recovered cell-type-specific programmes and tissue-aligned gene modules, preserved robust cell-state structure across batches, species and rare populations, and improved the reconstruction of spatial cell identity, anatomical domains and cell-type composition. In human liver cancer Stereo-seq data, OmniCell resolved a tumour-margin transition zone characterised by immune infiltration, acute-phase inflammation, coagulation/complement activity and metallothionein-linked metal-ion detoxification. Contextual gene-embedding similarity analysis showed that gene relationships differed across tumour core, transition-zone and paratumour/adjacent non-malignant niches, indicating that OmniCell captures tissue-dependent gene function rather than expression similarity alone. In mouse brain development and macaque cortex, spatial virtual perturbations mapped regulatory genes onto stage- and region-specific anatomical programmes. Together, these results establish tissue context as a primary axis of transcriptomic representation and provide a framework for studying how cellular programmes acquire context-dependent biological meaning in intact tissues.

bioinformatics↗

bioRxiv2025

PocketX: Preference Alignment for Protein Pockets Design through Group Relative Policy Optimization

Designing protein pockets that target specific ligands is crucial for drug discovery and enzyme engineering. Although deep generative models show promise in proposing high-quality pockets, they are usually trained purely to match the data distribution and therefore overlook key biophysical properties, such as binding affinity, expression, and solubility, that ultimately determine developability and success. We introduce PocketX, an online reinforcement learning framework that explicitly aligns a generative model with desired biophysical properties. The framework first trains a base model that co-designs pocket structures and sequences conditioned on a target ligand, and then fine-tunes this model with Group Relative Policy Optimization (GRPO) to reward the desired attributes. Because GRPO employs group-relative rewards, it produces lower-variance policy updates, resulting in more stable and efficient learning than competing alignment strategies. Evaluated on the CrossDocked2020 benchmark, PocketX surpasses existing methods in metrics such as binding energy and evolutionary plausibility. Ablation studies further show that GRPO outperforms alternative alignment strategies, including Direct Preference Optimization (DPO), confirming GRPOs effectiveness for biophysical property alignment.

bioinformatics↗

bioRxiv2025

PERFECT PCR: Advancing DNA Data Storage to Near-Maximal Density

DNA-based data storage offers unprecedented storage density and durability compared to traditional media, but it faces challenges in long access latency and limited encoding efficiency. Current DNA storage methods achieve only 8% of the theoretical maximum storage density (TMSD) due to biological constraints and error rates. Here, we demonstrate an approach that improves both synthesis speed and encoding density, bringing DNA data storage closer to practical implementation. We describe Polymerase-Enabled Rapid and Frugal Error Correcting Technology (PERFECT) PCR, which integrates PCR with hyperthermophilic EndoNucS enzymes for efficient DNA mismatch detection and cleavage, enabling accurate and fast DNA synthesis using overlapping oligonucleotides. This approach reduced latency while streamlining the overall DNA synthesis process, achieving significantly lower error rates in amplicons compared to state-of-the-art methods, thereby reducing physical redundancy. Specifically, we achieved a substantially reduced error rate of 0.06 errors per kilobase, with 95% of sequences verified as accurate. As a result, our technique reached 94% TMSD, representing a twelve-fold improvement over current systems and approaching the theoretical limit for existing sequencing technologies. We further validated the versatility of PERFECT PCR by successfully encoding and retrieving digital images, music files, and multilingual text files with near-perfect information recovery. By addressing the core challenges of latency, density, and accuracy, our approach brings DNA-based data storage significantly closer to practical implementation. In addition, the accurate DNA synthesis methods described here may be applied to de novo genome assembly and therefore have broad implications for the synthesis and analysis of biological systems. SUMMARYLow cost, rapid, and error free de novo DNA synthesis for DNA-based data storage and gene synthesis applications.

bioengineering↗

bioRxiv2025

Elevated neuronal TAF15 expression leads to FTD-like phenotype

TAF15, a DNA/RNA-binding protein whose dysfunction in alternative splicing impairs NMDA receptor signaling, can form amyloid fibrils that contribute to neurodegeneration in the neurodegenerative diseases like FTD and ALS, indicating a close involvement of TAF15 in both neuronal physiological function and activation of pathological pathways. However, the relationship between TAF15 expression levels and neurodegeneration, as well as the specific downstream pathways mediating its neurotoxicity, remains unclear. Here, we found a consistent upregulation of TAF15 in prefrontal cortex neurons from patients across multiple FTD and ALS subtypes. Both in vitro and in vivo experiments demonstrated that neuronal TAF15 overexpression triggered oxidative stress, evidenced by reactive oxygen species (ROS) production, leading to neurotoxicity and gliosis. Mice overexpressing TAF15 in medial prefrontal cortex (mPFC) neurons exhibited heightened anxiety and impaired fear memory, mimicking some key features of FTD behavioral abnormalities. Notably, these pathological and behavioral phenotypes are rescued by the antioxidant N-acetylcysteine amide (NACA), indicating that elevated TAF15 level exacerbates neurodegeneration primarily by activating oxidative stress pathways. Together, this work elucidates a novel TAF15-oxidative stress axis in FTD-associated neurodegeneration, providing a conceptual framework for future therapeutic development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/694105v1_ufig1.gif" ALT="Figure 1000"> View larger version (35K): org.highwire.dtl.DTLVardef@cc581eorg.highwire.dtl.DTLVardef@1ad2129org.highwire.dtl.DTLVardef@15c0fb5org.highwire.dtl.DTLVardef@10b57a6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsTAF15 is upregulated in prefrontal cortex neurons of patients with FTD and ALS across different subtypes. Elevated TAF15 expression provokes ROS elevation, leading to neurotoxicity and gliosis. Neuronal TAF15 overexpression in the mouse mPFC results in FTD-like anxiety and impaired fear-memory phenotype. Reducing oxidative stress can rescue FTD-like phenotype caused by TAF15 upregulation.

neuroscience↗