Biology subjects

Feng, H.

Publications and source records attributed to Feng, H..

At least 37 records · Page 2Linked to original sources

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↗

bioRxiv2025

A novel dephosphorylation peptide inhibits 17β-HSD1 enzyme activity in ovarian granulosa cells and breast cancer cells

Estradiol (E2), a pivotal mammalian reproductive hormone, is closely associated with estrogen-dependent diseases. 17{beta}-hydroxysteroid dehydrogenase type 1 (17{beta}-HSD1), a critical E2 synthesis enzyme, has been understudied for its post-translational regulation-despite its potential as a therapeutic target, no clinically approved inhibitors are currently available. In our investigation of porcine follicular atresia mechanisms, we identified five differentially phosphorylated residues in 17{beta}-HSD1. Using in vitro site-directed mutagenesis and transgenic mouse models carrying point mutations, we established that Ser30 and Ser274 are critical phosphorylation sites that robustly modulate 17{beta}-HSD1 enzymatic activity. We further demonstrated that activin A and insulin-like growth factor 1 (IGF-1) enhance 17{beta}-HSD1 activity by promoting its phosphorylation at these two sites. To target these regulatory residues, we designed and synthesized cell-penetrating peptides (CPPs) that specifically suppress 17{beta}-HSD1 phosphorylation. Functional assays conclusively demonstrated that these CPPs significantly suppressed 17{beta}-HSD1 activity in vitro. Notably, they also inhibited the proliferation, migration and invasion of MCF-7 cells, as well as tumor growth in a mouse model of breast cancer. Our study provides novel mechanistic insights into the regulation of 17{beta}-HSD1 and E2 synthesis, addressing a critical gap in steroid hormone biology. The identification of Ser30/Ser274 as functional phosphorylation sites and the development of CPP-based inhibitors offer both theoretical advances and translational potential, opening new avenues for the treatment of estrogen-dependent breast cancer.

cell biology↗

bioRxiv2025

A neuron type-specific microexon in Ank3/ankyrin-G modulates calcium activity and neuronal excitability

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

neuroscience↗

bioRxiv2025

Accelerated and High-Accuracy Variant Calling on Oxford Nanopore Technologies Sequencing Data with the Sentieon DNAscope LongRead and Hybrid Pipelines

Oxford Nanopore Technologies (ONT) has emerged as a clinically meaningful long-read sequencing platform, enabled by recent improvements in read accuracy and throughput. However, variant calling on ONT data remains challenging due to platform-specific error profiles, particularly indels in homopolymer regions. Here, we introduce description and comprehensive benchmarks of the Sentieon DNAscope LongRead and DNAscope Hybrid pipelines on ONT whole-genome datasets, demonstrating high accuracy and substantially reduced computational requirements across SNP, Indel, and Structural Variant detection. Benchmarked against multiple truth sets including GIAB v4.2.1, CMRG, and T2T-Q100, DNAscope LongRead reduced SNP errors by [~]50% relative to Clair3 across five reference samples and showed consistently higher accuracy in all non-long-homopolymer stratifications. Integration of ONT and Illumina short reads via DNAscope Hybrid further improved variant detection: with low depth ONT + Illumina datasets, the pipeline achieved F1 scores of 0.9992 (SNPs) and 0.9979 (Indels), outperforming alternative pipelines. In challenging benchmarks such as T2T-Q100 and CMRG genes, DS-Hybrid reduced SNP and Indel errors significantly, compared to next-best methods. For structural variants, DNAscope pipelines outperformed alternative (e.g. Sniffles2), highlighting the advantages of haplotype-resolved SV detection method. Despite its accuracy, the Sentieon software suite remains computationally efficient: complete ONT long read FASTQ-to-VCF analysis completed within [~]190 minutes on a 120-vCPU Azure instance, at costs less than $5 USD. Overall, the DNAscope LongRead and Hybrid pipelines deliver fast, accurate, and scalable germline variant calling for ONT datasets, providing a practical solution for comprehensive whole-genome analysis in both research and clinical genomics.

bioinformatics↗

bioRxiv2025

Dual-Channel Event Microscopy for Ultrafast Biological Imaging

Many fundamental biological processes--spanning immune-tumor interactions, neuronal signaling, and microvascular flow--exhibit fast, multiscale dynamics among diverse cell types within three-dimensional tissue environments. Capturing such activity requires imaging systems that simultaneously achieve high temporal resolution, multicolor capability, and volumetric coverage over large fields of view (FOVs). However, existing modalities remain limited by trade-offs among imaging speed, spectral capacity, depth of field (DOF), and spatial resolution. Here, we present Dual-Channel Event Microscopy (DEM), which integrates digital micromirror device (DMD)-based pulsed illumination, extended-DOF optics, and event-based sensing for ultrafast, dual-channel volumetric imaging across a 2.3 x 1.3 mm2 FOV with an effective 200 {micro}m DOF. Using dual-color fluorescent phantoms and microsphere flow assays, DEM achieves accurate spectral separation and reconstruction of rapid motion at kilohertz frame rates. In vivo, DEM enables simultaneous visualization of neutrophils and premalignant tumors in freely swimming zebrafish. In immobilized specimens, it provides robust, sensor-level optical sectioning near the heart, suppressing diffuse background to reveal fine vascular networks and active blood circulation into and out of the cardiac chambers. DEM further enables quantitative mapping of blood-flow dynamics in the zebrafish tail, resolving arterial-venous differences and capturing heartbeat-driven oscillations that reflect cardiac pumping with high temporal fidelity. By uniting ultrafast acquisition, dual-channel capability, volumetric coverage, and intrinsic optical sectioning within a single event-driven architecture, DEM offers a powerful platform for visualizing rapid multicellular interactions and physiological dynamics in living systems.

bioengineering↗

bioRxiv2025

SOFT METROPOLIS-HASTINGS CORRECTION FOR GENERATIVE MODEL SAMPLING

AO_SCPLOWBSTRACTC_SCPLOWMolecular diffusion models suffer from systematic sampling biases that prevent optimal structure formation, resulting in chemically suboptimal molecules with metastable conformations trapped in local energy minima. We introduce Metropolis-Hastings (MH) correction to molecular diffusion models, providing a principled framework to address these systematic sampling biases. The traditional hard accept-reject Metropolis-Hastings corrector creates discontinuous trajectories incompatible with the continuous nature of molecular potential energy surfaces, disrupting proper structure assembly. To address this, we develop a soft Metropolis-Hastings correction that replaces binary acceptance with continuous interpolation weighted by acceptance probabilities, maintaining smooth navigation in the chemical space while providing principled bias correction. We design three molecular-specific variants and demonstrate through extensive experiments on small molecules, drug conformations, and therapeutic antibody CDR-H3 loops that our method consistently improves chemical validity, structural stability, and conformational quality across diverse molecular families. Our method establishes MH correction as a powerful component for molecular generation.

bioinformatics↗

bioRxiv2025

Sarbecovirus Rc-o319 S-Protein Structures Reveal Highly Specialized Adaptation to Rhinolophus cornutus ACE2

Bat sarbecoviruses often exhibit species-dependent ACE2 specificity. Understanding the determinants of receptor specificity enables better assessment of the cross-species transmission potential of sarbecoviruses. Here, we characterize the S-protein of Rc-o319, a sarbecovirus identified in Japanese Rhinolophus cornutus bats. Featuring an unusual 9-amino-acid deletion within its receptor binding motif (RBM), Rc-o319 S-protein utilizes its cognate R. cornutus ACE2 (bACE2R.cor) but not human ACE2 (hACE2), demonstrating highly restricted receptor specificity. Cryo-EM structures reveal two locked prefusion conformations of the Rc-o319 S-trimer and define a novel type-1b receptor-binding domain (RBD), featuring a distinct beta-loop (BL) within the RBM due to the RBM-deletion. The Rc-o319-RBD:bACE2R.cor complex structure reveals unique interactions mediated by the specialized BL and RBM-loop of Rc-o319-RBD and by a bACE2R.corglycan. Structure-guided mutagenesis demonstrates that changes in BL and RBM-loop within the Rc-o319 S-RBD must occur simultaneously to allow medium-to-high-affinity hACE2 binding. Comparative assays further show that the bACE2R.cor receptor supports only a subset of sarbecoviruses, highlighting its restricted sarbecovirus compatibility. Our findings establish the Rc-o319 S-protein as a structurally and functionally specialized adaptation to R. cornutus ACE2 and identify the structural constraints limiting its cross-species transmission potential. Author SummarySARS-related viruses are widely found in horseshoe bats. Some can bind the human ACE2 receptor with variable affinities, whereas others bind only bat ACE2 receptors. Understanding the basis for this difference is critical for assessing spillover risk. We studied the spike protein of a bat sarbecovirus, Rc-o319, isolated from Japanese Rhinolophus cornutus horseshoe bats. Although Rc-o319 is genetically related to SARS-CoV-2, it is unable to bind the human ACE2 receptor. Structural analyses and functional experiments revealed that the Rc-o319 spike protein possesses a distinct receptor-binding motif (RBM). This RBM features a specialized "beta-loop" that replaces the "large-loop" found in human-infecting sarbecoviruses. The beta-loop enables high-affinity binding to its cognate Rhinolophus cornutus bat ACE2 receptor while simultaneously preventing optimal interaction with human ACE2. We further found that acquisition of high affinity for the human ACE2 receptor would require coordinated changes across multiple regions of the RBM, including changing the beta-loop into a large-loop structure. Together, our findings demonstrate that the Rc-o319 spike protein is highly adapted to its cognate bat ACE2 receptor and faces substantial structural constraints that limit its ability to switch to binding human ACE2.

microbiology↗

bioRxiv2025

SARS-CoV-2 NSP14 inhibitor exhibits potent antiviral activity and reverses NSP14-driven host modulation

The emergence of SARS-CoV-2 variants and drug-resistant mutants highlights the urgent need for novel antiviral therapeutics. SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective first-in-class non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and inhibits SARS-CoV-2 at the replication stage. C10 suppresses viral translation and exhibits immunostimulatory effect. Additionally, C10 specifically reversed NSP14-mediated alterations in host transcriptome. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases.

microbiology↗

bioRxiv2025

Learning from B Cell Evolution: Adaptive Multi-Expert Diffusion for AntibodyDesign via Online Optimization

Recent advances in diffusion models have shown remarkable potential for antibody design, yet existing approaches apply uniform generation strategies that cannot adapt to each antigens unique requirements. Inspired by B cell affinity maturation--where antibodies evolve through multi-objective optimization balancing affinity, stability, and self-avoidance--we propose the first biologically-motivated framework that leverages physics-based domain knowledge within an online meta-learning system. Our method employs multiple specialized experts (van der Waals, molecular recognition, energy balance, and interface geometry) whose parameters evolve during generation based on iterative feedback, mimicking natural antibody refinement cycles. Instead of fixed protocols, this adaptive guidance discovers personalized optimization strategies for each target. Our experiments demonstrate that this approach: (1) discovers optimal SE(3)-equivariant guidance strategies for different antigen classes without pre-training, preserving molecular symmetries throughout optimization; (2) significantly enhances hotspot coverage and interface quality through target-specific adaptation, achieving balanced multi-objective optimization characteristic of therapeutic antibodies; (3) establishes a paradigm for iterative refinement where each antibody-antigen system learns its unique optimization profile through online evaluation; (4) generalizes effectively across diverse design challenges, from small epitopes to large protein interfaces, enabling precision-focused campaigns for individual targets.

bioinformatics↗

bioRxiv2025

Single-Cell and Spatial Profiling of Tumor Microenvironment Heterogeneity in Human Osteosarcomas

Osteosarcoma, a highly heterogeneous malignant bone tumor, exhibits substantial interpatient and intratumoral heterogeneity. Utilizing integrated single-cell RNA sequencing and spatial transcriptomics, we uncovered distinct tumor microenvironment (TME) transcriptional profiles across patients, highlighting profound interpatient heterogeneity. Intratumorally, malignant cells primarily followed two differentiation trajectories, converging into osteoblastic and chondroblastic functional states, and hypoxia may be the influencer; notably, within individual patients, tumor cells exhibited greater transcriptional similarity driven by specific transcription factors, despite these divergent states. Spatial analysis revealed patient-specific TME cellular co-localization patterns, alongside conserved spatial relationships: vascular components (endothelial cells, pericytes) demonstrated strong co-localization, while immune cells (T cells, myeloid cells) clustered within shared regions. Crucially, these functional states occupied distinct microniches: osteoblastic regions were enriched with osteoclasts, vascular components, and immune cells, whereas chondroblastic regions displayed the opposite composition and were preferentially located in hypoxic, vascular-poor niches, exhibiting significant enrichment of hypoxia-related signaling pathways. Furthermore, our data suggest osteosarcoma cells may activate fibroblasts via the SPP1 signaling pathway, indicating fibroblasts act as key intermediaries in tumor-directed TME modulation. This study comprehensively delineates the intricate landscape of osteosarcoma heterogeneity, defining distinct functional cellular states, their spatially organized TME niches, and a potential SPP1-mediated tumor-fibroblast regulatory axis. This comprehensive analysis elucidates the intricate interpatient and intratumoral heterogeneity in osteosarcoma, revealing functional and spatial organization within the tumor and its microenvironment.

cancer biology↗

bioRxiv2025

Sequential Amino Acid Mutagenesis-Driven De Novo Evolution of Adenine Deaminases Enables Efficient In Vivo Base Editing in Primate

Base editing allows for the precise modification of genetic information, providing new avenues for treating diseases1. The adenine base editor ABE8e is currently the most efficient and widely used tool for adenine base editing. ABE8e was developed through multiple rounds of directed evolution of Escherichia coli tRNA adenine deaminase, including phage-assisted continuous evolution (PACE)2,3. While PACE is highly effective, it is a complex system that poses challenges for implementation4. Despite its high efficiency, ABE8e is associated with limitations such as relatively higher bystander editing effects and elevated off-target activity5, which need to be addressed to further enhance its precision and safety. Here, we developed a novel method for the de novo discovery of evolved ABE components, particularly adenine deaminases. This process involves identifying candidate proteins through AI-based structural prediction and clustering, followed by the enhancement of deaminase editing activity through screening libraries created by sequential amino acid saturation mutagenesis. This evolutionary strategy simplifies the approach by employing saturation mutagenesis libraries tailored to specific segments, thereby enabling exploration of an expanded sequence space and increasing the likelihood of discovering adenine deaminases with superior capabilities. The newly developed hpABE5.20 here demonstrates a more refined editing window, reduced DNA off-target effects that are both sgRNA-dependent and -independent, and minimized RNA off-target activity, while maintaining robust editing efficiency relative to ABE8e. Furthermore, hpABE5.20 has been successfully applied for precise and effective therapeutic adenine base editing in cellular disease models, humanized mice, and non-human primates.

bioengineering↗

bioRxiv2025

Breakthrough infection elicits synergistic broadly neutralizing antibodies targeting non-dominant epitopes on RBD and NTD in an individual of inactivated SARS-CoV-2 vaccine

Background/ObjectivesIdentification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. MethodsThrough single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were respectively measured with ELISA and pseudovirus-based neutralization assay. Their epitopes were mapped by competition ELISA and site- directed mutation. ResultsAmong a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13, and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20, and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. ConclusionsThese four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection.

immunology↗

bioRxiv2025

Accelerated, Accurate, Hybrid Short and Long Reads Alignment and Variant Calling

BackgroundIntegrating short-read and long-read sequencing technologies has become a promising approach for achieving accurate and comprehensive genomic analysis. While short-read sequencing (Illumina, etc.) offers high base accuracy and cost efficiency, it struggles with structural variation (SV) detection and complex genomic regions. In contrast, long-read sequencing (PacBio HiFi) excels in resolving large SVs and repetitive sequences but is limited by throughput, higher Indel error rates, and sequencing costs. Hybrid approaches may combine these technologies and leverage their complementary strengths and different sources of error to provide higher accuracy, more comprehensive results, and higher throughput by lowering the coverage requirement for the long reads. MethodsThis study benchmarks the DNAscope Hybrid pipeline, a novel integrated alignment and variant calling framework that combines short- and long-read data sequenced from the same sample. We evaluate its performance across multiple human genome reference datasets (HG002-HG004) using the draft Q100 and Genome in a Bottle v4.2.1 benchmarks. The pipelines ability to detect small variants (SNPs/Indels), structural variants (SVs), and copy number variations (CNVs) is assessed using data from the Illumina and Pacbio sequencing systems at varying read depths (5x-30x). Benchmark results are compared to DeepVariant. ResultsThe DNAscope Hybrid pipeline significantly improves SNP and Indel calling accuracy, particularly in complex genomic regions. At lower long-read depths (e.g., 5x-10x), the hybrid approach outperforms standalone short- or long-read pipelines at full sequencing depths (30x-35x). Additionally, the DNAscope Hybrid outperforms leading open-source tools for SV and CNV detection, enhancing variant discovery in challenging genomic regions. The pipeline also demonstrates clinical utility by identifying disease- associated variants. Moreover, DNAscope Hybrid is highly efficient, achieving less than 90 minutes runtimes at single standard instance. ConclusionThe DNAscope Hybrid pipeline is a computationally efficient, highly accurate variant calling framework that leverages the advantages of both short- and long-read sequencing. By improving variant detection in challenging genomic regions and offering a robust solution for clinical and large-scale genomic applications, it holds significant promise for genetic disease diagnostics, population-scale studies, and personalized medicine.

bioinformatics↗

bioRxiv2025

Palmdelphin facilitates R-spondin2 secretion to activate Wnt signaling and promote colorectal cancer stemness and tumorigenesis

The Wnt signaling pathway is a key driver of stemness and progression which contributes to mortality in colorectal cancer (CRC). R-spondins bind to LGR receptors to inhibit ubiquitin E3 ligases, thus protecting Frizzled from degradation and activating downstream Wnt signaling. Herein, we identify Palmdelphin (PALMD) as a functional marker of CRC stem cells, interspersed between intestinal and colonic crypt base epithelial cells, and predictive of aggressive CMS4 CRC and poor survival. Gene knockdown and overexpression studies revealed that PALMD initiates paracrine activation of Wnt/{beta}-catenin signaling via interacting with and facilitating the secretion of R-spondin2 (RSPO2), resulting in enhanced stemness and tumor growth in vitro and in vivo. Physiologic or pharmacologic inhibition of the PALMD-RSPO2 axis using R-spondin2-specific antibody or 6-methyl-1,3,8-trihydroxyanthraquinone (emodin), respectively, attenuates PALMD- mediated Wnt reporter activation, self-renewal, and tumorigenesis in cell and patient-derived organoid models. Together, these findings identify PALMD as a previously unknown player in Wnt signaling in CRC, and underscore the pro-tumorigenic role of R-spondin2 in this context.

cancer biology↗

bioRxiv2025

RPGR regulates motile cilia by interfering with actin dynamics

Cilia are highly conserved cellular organelles extruding from the surface of cell types carrying either sensory (signaling) or motile functions. These include photoreceptor cells and airway epithelia, where they function in light sensation and mucociliary clearance respectively. Retinitis pigmentosa GTPase regulator (RPGR) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and in some cases, primary ciliary dyskinesia (PCD), both debilitating conditions. Not all patients develop PCD and it is unclear which RPGR variants predispose patients to PCD and why this happens. In this study, using nasal biopsy samples of patients with RPGR-related RP, we leverage 2D organoid cell culturing, super-resolution microscopy, and live cell imaging to characterize the multiciliated cells from patients with different RPGR variants, healthy human nasal and bronchial multiciliated cells with CRISPR-modified RPGR function. We demonstrate for the first time that multiciliated cells with RPGR variants may have reduced ciliation, shorter cilia, significantly impaired cilia beat, or cilia beat incoordination, which could lead to defective mucociliary clearance and lung disease. In addition, we show the regulation of motile cilia by RPGR involves F-actin, as evidenced by temporarily reduced Gelsolin and undissolved condensed actin meshwork at the apical surface of RPGR-deficient multiciliated cells. In support, we show that the motile cilia defect can be ameliorated by treating with the actin polymerization inhibitor Latrunculin A. Though PCD was observed only in patients with variants that affect both main isoforms (RPGR1-19 and RPGRORF15), patients with variants affecting only RPGRORF15 also showed cilia and airway anomalies. Though all RPGR variants affected motile cilia in one way or another, RPGR loss of function variants affecting both isoforms are associated with more severe cilia and systemic phenotypes, the mechanisms of which involve the accumulation of apical F-actin. One Sentence Summary: Loss of RPGR, through the mechanism of apical F-actin accumulation in airway multiciliated cells, leads to reduced ciliation with short cilia that have an impaired beat, leading to defective mucociliary clearance.

cell biology↗

bioRxiv2025

Synergistic neuroprotective and cognitive-enhancing effects of Walnut Peptide and Theanine in human brain organoid and mouse stress models

Stress is a prevalent mental health concern emerging predominantly in late adolescence or early adulthood. Since 2007, the Food and Drug Administration (FDA) has not approved any novel anxiolytic pharmaceuticals, fueling interest in nutritional supplements as alternative therapies for stress management. Building on prior zebrafish research, this study investigates the synergistic effects of Theanine (Th) and Walnut Peptide (WP) on stress mitigation and cognitive enhancement. Utilizing the human brain organoid stress (BO-stress) model, WP+ Th were observed to reduce stress and regulate expression of neurotransmitters, including Gamma-Aminobutyric Acid (GABA), serotonin (5-HT), dopamine (DA), and acetylcholine (Ach), as well as brain-derived neurotrophic factor (BDNF) and serotonin transporter (SERT). Subsequent in vivo study using C57BL/6J mouse-stress model demonstrated that the treatment (Th 85 mg/mL + WP 200 mg/mL), or administrated with vehicles, significantly improved their performance in stress and cognitive assessments, partially normalized neurotransmitter imbalances by modulating SERT and BDNF expression. These findings highlighted the potential of using WP + Th, particularly when delivered with vehicles (eg: powder/yogurt/milk), as a novel combined therapeutic approach for stress management and cognitive enhancement. We performed a correlation analysis between BO-stress model and mouse-stress model, revealing a alignement in the SERT levels.In addition, SERT was highly correlated with other markers in the mouse hippocampus and may represent a key target for modulating the balance between stress and cognition. Significance StatementThis study established an innovative human brain organoid-stress model and, in conjunction with mouse-stress model, elucidated the synergistic actions of Theanine and Walnut Peptide in mitigating stress and augmenting cognitive function. Their beneficial effects were mediated through the regulation of SERT and BDNF, presenting a promising non-pharmacological avenue for mental health care. In addition, species differences between humans and mice were also examined through correlation analysis of brain organoids and mouse models. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/640260v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1a81886org.highwire.dtl.DTLVardef@e2281dorg.highwire.dtl.DTLVardef@1b7b167org.highwire.dtl.DTLVardef@dff9aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Glycosylphosphatidylinositol biosynthesis restricts coronavirus infection via the regulation of LY6E

Coronaviruses, including SARS-CoV-2, rely on host factors for their replication and pathogenesis, while hosts deploy defense mechanisms to counteract viral infections. Although numerous host proviral factors have been identified, the landscape of host restriction factors and their underlying mechanisms remain less explored. Here, we conducted genome-wide CRISPR knockout screens using three distinct coronaviruses--SARS-CoV-2, HCoV-OC43 (a common cold human virus from the genus Betacoronavirus) and porcine epidemic diarrhea virus (Alphacoronavirus) to identify conserved host restriction factors. We identified glycosylphosphatidylinositol (GPI) biosynthesis as the pan-coronavirus host factor that restrict viral entry by disrupting spike protein-mediated membrane fusion at both endosomal and plasma membranes. GPI biosynthesis generates GPI moieties that covalently anchor proteins (GPI-anchored proteins [GPI-APs]) to the cell membrane, playing essential roles in various cellular processes. Through focused CRISPR knockout screens targeting 193 GPI-APs, we identified LY6E as the key downstream effector mediating the antiviral activity of the GPI biosynthesis pathway. These findings reveal a novel role for GPI biosynthesis as a conserved host defense mechanism against coronaviruses and highlight LY6E as a critical antiviral effector. This study provides new insights into virus-host interactions and the development of host-directed antiviral therapies.

microbiology↗

bioRxiv2024

Base-modified nucleotides mediate immune signaling in bacteria

Signaling from pathogen sensing to effector activation is a fundamental principle of cellular immunity. While cyclic (oligo)nucleotides have emerged as key signaling molecules, the existence of other messengers remains largely unexplored. Here, we reveal a bacterial anti-phage system that mediates immune signaling through nucleobase modification. Immunity is triggered by phage nucleotide kinases, which, combined with the system-encoded adenosine deaminase, produce deoxyinosine 5-triphosphate (dITP) as immune messengers. The dITP signal activates downstream effector to mediate cellular NAD+ depletion, resulting in population-level defense through the death of infected cells. To counteract immune signaling, phages deploy specialized enzymes that deplete cellular dAMP, the precursor of dITP messengers. Our findings uncover a nucleobase modification-based anti-phage signaling pathway, establishing noncanonical nucleotides as a new type of immune messengers in bacteria.

microbiology↗