Biology subjects

Li, T.

Publications and source records attributed to Li, T..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Alphaviral capsid proteins inhibit stress granule assembly via competitive RNA binding with G3BP1

Viral infection is one of the conditions that induces stress granule (SG) formation, a cellular defense mechanism that exerts antiviral effects. To counteract this host response, viruses have evolved a broad spectrum of strategies to inhibit SG formation. However, the molecular mechanisms underlying SG inhibition remain poorly understood. The nucleocapsid proteins play a critical role in virus replication and host interaction. Here, using Semliki Forest Virus (SFV) as a model, we uncover the function of the alphavirus nucleocapsid in SG inhibition. This inhibitory function depends on oligomerization mediated by an N-terminal -helix and with a positively charged intrinsically disordered region (IDR). We show that SFV capsid directly competes with G3BP1 for RNA binding, thereby disrupting G3BP1-RNA liquid-liquid phase separation (LLPS) in vitro and SG assembly in cells. This mechanism is conserved across the alphavirus family but is not shared by the nucleocapsid of SARS-CoV-2 or other endemic viruses examined. Notably, expression of a peptide from SFV capsid is sufficient to inhibit SG formation induced by Amyotrophic Lateral Sclerosis (ALS)-associated mutations, suggesting potential therapeutic applications. Our findings reveal mechanistic insight into SG modulation by the viral capsid protein and provide a possible bioengineering tool for probing SG dynamics in health and disease.

cell biology↗

bioRxiv2025

Visualization of photoreceptor outer segment renewal using AAV-delivered Dendra2-tagged rhodopsin

Visualization of photoreceptor outer segment (OS) renewal dynamics is essential for vision research but has proved difficult in mice due to the small size and dense packing of their photoreceptors. Lack of effective protein "trackers" and the time-consuming generation of transgenic reporter lines add to these challenges. In this study, we evaluated AAV-mediated delivery of photoconvertible Rhodopsin/Dendra2 and Peripherin2/Dendra2 fusion proteins as a means to track OS renewal in mouse photoreceptors. OS renewal was assessed by two approaches: (1) comparing the lengths of native (green) Dendra2 domains at two post-infection time points, and (2) using photoconversion of Dendra2 to distinguish newly synthesized discs from preexisting ones within the same cell. We validated this method in both wild type mice and a Tmem138-deficient ciliopathy mutant, in which a reduced OS renewal rate was observed. Taken together, this method offers a rapid genetic tool for "real-time" evaluation of OS renewal dynamics in mice, overcoming limitations posed by the compact and densely organized photoreceptor architecture.

cell biology↗

bioRxiv2025

Novel Dopamine 4 Receptor Ligands Differentially Ameliorate ADHD-like Behaviors in Spontaneously Hypertensive Rats

RationaleDopamine D4 receptors (D4Rs) have been implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD), yet their precise role and therapeutic relevance remain underexplored. Highly selective D4R compounds may provide a valuable tool to elucidate D4R function and assess their potential as non-stimulant ADHD treatments. ObjectivesThis study examined the behavioral effects of two novel D4R drugs, namely, FMJ-01-38 (high-efficacy partial agonist) and FMJ-01-54 (full antagonist) in adolescent spontaneously hypertensive (SHR/NCrl) rats, a validated ADHD model, and Wistar controls. MethodsRats received intraperitoneal FMJ-01-38 or FMJ-01-54 (5-10 mg/kg) or vehicle prior to behavioral assays assessing locomotor activity (open field tests), recognition memory (novel object preference), attention and working memory (Y-maze test), and impulsivity (delay discounting task). ResultsFMJ-01-38 dose-dependently reduced locomotor hyperactivity and improved spontaneous alternation behavior in SHR/NCrl; at 5 mg/kg it enhanced novel-object preference and decreased impulsive choice and action, indicating attenuation of ADHD-like symptoms and cognitive enhancement. FMJ-01-54 produced similar improvements in Y-maze and novel-object performance without altering locomotor activity or impulsivity of SHR/NCrl, suggesting selective cognitive improvement. In Wistar rats, FMJ-01-38 increased novel-object preference only at the 5 mg/kg dose, while FMJ-01-54 treatment did not produce any significant behavioral effects. ConclusionsThese findings demonstrate that D4R modulation, through either partial agonism or antagonism, differentially ameliorates ADHD-related behaviors. Both FMJ-01-38 and FMJ-01-54 produced minimal effects in control animals, suggesting pathology-specific efficacy and highlighting D4R ligands as promising non-stimulant therapeutic candidates for ADHD.

pharmacology and toxicology↗

bioRxiv2025

Determining gene specificity from multivariate single-cell RNA sequencing data

An important application of single-cell genomics experiments is to identify genes specific to biological categories or experimental conditions. Although numerous approaches have been proposed to identify such genes, we consider an axiomatic approach based on defining properties that a specificity measure should have. This leads us to develop ember (Entropy Metrics for Biological ExploRation), which we show is the only method satisfying four key desired properties for a specificity measure. Applying ember to eight tissues from eight founder mouse strains, we find that gene specificity is often unintuitive: canonical markers can be supplanted, housekeeping genes are context-dependent, and mouse strain can drive unexpected cell type switching. Unsupervised learning on entropy metrics uncovers shared genes specialized to male gonads and kidney, as well as genes specific to non-consecutive developmental stages in the kidney. To facilitate further exploration of gene specificity in mice, we have also developed a comprehensive specificity database, along with a web interface, API and MCP server. Extending ember to a human PBMC dataset collected from 255 diverse individuals, we find that variation in PBMCs is largely localized to classical monocytes. We also find genes with unique specificity by sex, age and ancestral background. Together, these applications establish ember as a powerful tool and provide a roadmap for elucidating the impact of human genetic variation using the murine model.

bioinformatics↗

bioRxiv2025

ecDNA-driven oncogene super-expressors shape immunoevasive tumor microenvironment

ecDNA contributes to cancer genetic heterogeneity through random segregation during mitosis. Emerging evidence links ecDNA to immune evasion, but the mechanism remains elusive. Using genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC), we show that Kras and Myc oncogenes are amplified either on ecDNAs or as homogeneously staining regions (HSRs) on chromosomes. ecDNA-driven tumors are more aggressive in immunocompetent mice. Single-cell transcriptomic and histological analyses reveal that ecDNA-driven tumors rapidly establish an immunoevasive tumor microenvironment (TME), marked by increased myofibroblastic cancer-associated fibroblasts (myCAFs) and reduced T cell infiltration. Mechanistically, ecDNA heterogeneity generates a subset of cancer cells with extremely high Kras expression, termed super-expressors, which secrete amphiregulin to promote myCAF expansion and suppress T cell infiltration. Clonally organized super-expressors establish an immunoevasive niche in the TME from patients with PDAC. Our findings demonstrate a causal role of ecDNA in TME remodeling, offering insights into cancer heterogeneity and immune evasion.

cancer biology↗

bioRxiv2025

Decorin promotes nascent proteoglycan retention in cartilage matrix by strengthening collagen II-aggrecan integration

Cartilage extracellular matrix (ECM), a hydrated collagen II-aggrecan composite, undergoes dynamic turnover during both normal homeostasis and disease-associated remodeling. This study elucidates a crucial role for decorin in promoting the retention and stability of nascent aggrecan within this matrix. By applying bio-orthogonal click-labeling, we demonstrate that loss of decorin accelerates the release of nascent aggrecan under both physiological and inflammatory conditions, without affecting its preferred localization to the pericellular matrix. Conversely, supplementation with exogenous decorin mitigates inflammation-induced loss of nascent aggrecan, supporting its potential as a therapeutic target. At the molecular level, decorin exhibits strong binding affinity for aggrecan, and enhances aggrecan-aggrecan and aggrecan-collagen II interactions, reinforcing its direct role in integrating cartilage matrix constituents. Also, by binding to collagen II, decorin stiffens the collagen II fibril network, thereby strengthening the confinement effect that limits the diffusive loss of entrapped aggrecan. Notably, decorin does not alter chondrocyte transcriptomic profiles in vivo, emphasizing its primary role in maintaining matrix integrity through biophysical mechanisms rather than cell signaling. Together, these findings provide a mechanistic foundation for developing decorin-based biomaterials or gene therapies aimed at preserving or regenerating the cartilage matrix for improved outcomes in osteoarthritis.

biophysics↗

bioRxiv2025

FAM19A4 Enhances Neutrophil Respiratory Burst via p38 MAPK in Lethal Sepsis

Sepsis causes high mortality and resource strain, with neutrophil-derived reactive oxygen species (ROS) contributing to excessive inflammation. The secretory protein FAM19A4 modulates ROS release, but its role in sepsis was unclear. We measured elevated FAM19A4 levels in septic patients and cecal ligation and puncture (CLP) mice, correlating with increased mortality. Fam19a4-/- mice subjected to CLP showed significantly improved survival and attenuated multiorgan injury without impaired peritoneal bacterial clearance or altered circulating neutrophil counts. Deficiency reduced the cell counts of neutrophil (Ly6G+) and macrophage (F4/80+) in lungs and liver, diminished systemic ROS production tracked by bioluminescence, and decreased neutrophil extracellular trap (NET) formation in serum and lung tissue. In vitro, FAM19A4 enhanced neutrophil phagocytosis and ROS generation but did not affect lipopolysaccharide-induced chemotaxis. Mechanistically, bulk RNA sequencing, western blotting, and the p38 inhibitor SB203580 demonstrated that FAM19A4 drives neutrophil ROS release specifically through p38 MAPK signaling activation. These results indicate that FAM19A4 is upregulated during sepsis and exacerbates outcomes by enhancing neutrophil ROS production via p38 MAPK, representing a promising therapeutic target for this condition.

molecular biology↗

bioRxiv2025

A spatial transcriptomic atlas of autism-associated genes identifies convergence in the developing human thalamus

Autism is a highly heritable neurodevelopmental condition that manifests across a wide phenotypic spectrum. Rare and de novo loss-of-function mutations strongly predispose to autism and co-occurring developmental and intellectual disabilities in over 10% of autistic individuals. Understanding whether these variants converge on specific regional brain circuits or widely alter human brain development is crucial to understanding the etiology of profound autism. To date, transcriptomic atlases have mainly implicated the developing cerebral cortex, yet other brain areas have received relatively little attention. Here, we present a single-cell resolution spatial transcriptomic atlas of 250 autism susceptibility genes during human brain development. Profiling over 10 million cells across the midgestation forebrain, we found convergence of these genes across a small number of regional programs. The developing thalamus showed the most prevalent expression of autism susceptibility genes, followed by germinal zones throughout the brain. Within the thalamus, excitatory neurons showed the most enriched expression, which varied across thalamic nuclei harboring distinct circuits. Across the germinal zones, neural progenitors in the medial ganglionic eminences that generate parvalbumin- and somatostanin-positive interneurons showed highest expression. Our findings reveal the prevalent expression of autism associated genes beyond the developing cerebral cortex and implicate the developing human thalamus as a major hub of autism susceptibility.

neuroscience↗

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

Accurate protein structure determination from cryo-EM maps using deep learning and structure prediction

Cryo-electron microscopy (cryo-EM) has become the main-stream technique for macromolecular structure determination. However, due to intrinsic resolution heterogeneity, accurate modeling of all-atom structure from cryo-EM maps remains challenging even for maps at near-atomic resolutions. Addressing the challenge, we present EMProt, a fully automated method for accurate protein structure determination from cryo-EM maps by efficiently integrating map information and structure prediction with a three-track attention network. EMProt is extensively evaluated on a diverse test set of 177 experimental cryo-EM maps with up to 54 chains in a case at <4.0 [A] resolutions, and compared with state-of-the-art methods including DeepMainmast, ModelAngelo, phenix.dock_and_rebuild, and AlphaFold3. It is shown that EMProt significantly outperforms the existing methods in recovering the protein structure and building the complete structure. In addition, the built models by EMProt also exhibit a high accuracy in model-to-map fit and structure validations. EMProt is freely available at http://huanglab.phys.hust.edu.cn/EMProt/.

bioinformatics↗

bioRxiv2025

Gut microbial communities of velvet worm Euperipatoides rowelli (Onychophora) across deadwood microhabitats in southeastern Australia.

This study investigated the gut microbial community of the velvet worm Euperipatoides rowelli, a saproxylic invertebrate residing in deadwood microhabitats within historical refugia in the Tallaganda forest, New South Wales, Australia. The eucalyptus-dominated temperate forest is marked by unique topography, hydrology, and historical influences from Pleistocene glacial cycles. We described patterns in gut microbial community structure and composition across eight sites. The velvet worms were unfed for two weeks prior to dissection to lessen the influence of transient prey-associated microorganisms. Amplicon sequencing of the V4-V5 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform, with positive and negative controls integrated to assess contamination and ensure data quality. Alpha and beta diversity metrics demonstrated minimal overall variation among sites, suggesting broadly similar community structure. However, a closer look at individual taxa revealed contrasting patterns. Proteobacteria dominated the gut microbiome, with genus A37b (Rickettsia) forming part of the core microbiome in 50% of individuals; its presence varied among sites. In contrast, Spiroplasma displayed substantial site-specific variation in relative abundance. These findings suggest that, while overall diversity remained relatively stable, particular taxa displayed heterogenous distributions across historical refugia. Collectively, our findings suggest that gut microbiomes of velvet worms are shaped more by environmental filtering within deadwood microhabitats than by host geographic differences, reflecting a combination of stochastic and niche-driven assembly processes. Impact StatementWe present the first characterization of the gut microbiome in the saproxylic velvet worm Euperipatoides rowelli from temperate Australian forests. Despite marked host genetic divergence, microbial communities showed minimal spatial structuring, suggesting substantial environmental filtering within deadwood microhabitats. The limited variation and transient taxa may indicate microbial associations are more likely environmentally acquired rather than host-specific. These findings broaden our understanding of the microbiome assembly in underexplored invertebrates and underscore the influence of ecological context over site-based spatial differentiation in shaping gut microbial communities.

microbiology↗

bioRxiv2025

Lymphatics unload VEGFR3 in response to the endothelial stress upon VEGFR2 insufficiency

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for regulating the endothelial cell lineage and angiogenesis. It is also expressed by lymphatic endothelial cells (LEC) while its participation in the process of lymphangiogenesis remains inadequately characterized. We show in this study that VEGFR2 is highly expressed in dermal initial lymphatic vessels and valves. The induced deletion of pan-endothelial Vegfr2 at the neonatal stage produced a potent suppression of dermal lymphatic growth, including the thinner lymphatic diameter, the decrease of LEC number and lymphatic valves. Mechanistically, the VEGFR2 insufficiency led to a dramatic decrease of lymphatic VEGFR3, a key regulator mediating signals for lymphatic growth and remodeling. The RNA sequencing analysis revealed that GO terms enriched for the downregulated genes included biological processes related to the EC development while pathways related to the hematopoiesis and immune responses were upregulated in the skin of Vegfr2 mutants compared with the littermate controls. This was further confirmed by the RNA-seq analysis of dermal tissues 48 hours after the endothelial Vegfr2 deletion. Consistently, an inhibitory effect on the dermal lymphatic growth was also observed by targeting Vegfr2 in PROX1+ cells, manifesting a similarly altered transcriptome signature. The alteration of lymphatic gene expression was further validated by the siRNA mediated knockdown of Vegfr2 in primary lymphatic endothelial cells, showing a transcriptional trend of LEC to hematopoietic transition. Findings from this study imply that VEGFR2 is required for the maintenance of endothelial identity and its insufficiency may trigger the alteration of lymphatic transcriptional programs to diminish the VEGFR3-mediated lymphangiogenesis.

developmental biology↗

bioRxiv2025

SLOGEN: A Structure-based Lead Optimization Model Unifying Fragment Generation and Screening

Lead optimization plays an important role in preclinical drug discovery. While deep learning has accelerated this process, structure-based approaches that leverage 3D protein-ligand information remain underexplored. Existing models could improve predicted affinity but often yield synthetically inaccessible compounds, whereas screening-based methods limit chemical novelty by relying on fixed fragment libraries. To bridge the gap, we introduce Slogen--a Structure-based Lead Optimization algorithm unifying fragment Generation and screENing. To achieve this, Slogen integrates a transformer-based variational autoencoder, pretrained on the BindingNet v2 dataset, with an E(3)-equivariant graph neural network that models 3D protein-fragment interactions. This unified framework enables both fragment generation and similarity-based screening, simultaneously addressing synthetic tractability and structural diversity. Benchmarking study shows that Slogen matches or surpasses state-of-the-art methods while exploring broader chemical space. Case studies on the Smoothened and D1 dopamine receptors demonstrate its capacity to design high-affinity, drug-like molecules, providing a practical method for structure-guided lead optimization.

bioinformatics↗

bioRxiv2025

AI-guided design of common light chains to enable manufacturable bispecific antibodies

Bispecific antibodies (BsAbs) offer therapeutic advantages but face manufacturing bottlenecks from light chain mispairing, which can generate a substantial fraction of incorrect products and increase manufacturing complexity and cost of goods. Common light chains (cLC) eliminate mispairing, yet existing approaches require screening thousands of variants per target. We present an AI-driven framework that computationally designs cLCs through structure-guided pairing of non-cognate VH-VL interfaces, reducing experimental screening by three orders of magnitude. The platform successfully engineers therapeutic antibodies lacking experimental structures, expanding applicability beyond crystallographic databases. Among 10 therapeutic targets, we successfully generated designs for 7 targets, comprising 55 unique BsAb pairs. Of these, 43.6% (24/55) were successfully as BsAb cLCs. Three bispecific antibodies reached production-ready specifications: >90% purity and 1.6-1.8 g/L titers. This platform democratizes bispecific antibody development, expanding access beyond well-resourced programs.

bioengineering↗

bioRxiv2025

Chronic kidney disease exacerbates hyperlipidemia and atherosclerosis via miR-148a-3p targeting SIK1/AMPKα1 in liver

BackgroundCardiovascular complications are the leading cause of death in patients with chronic kidney disease (CKD). As a major risk factor for atherosclerotic cardiovascular disease (ASCVD), hyperlipidemia is a common sequela of CKD, which is linked to increased lipid synthesis, impaired lipoprotein clearance, and maladapted reverse cholesterol transport. Because exosomes can transport miRNAs to modulate intercellular and tissue communication, we hypothesized that exosomal miRNAs can act as endocrine-like molecules to modulate the CKD-associated hyperlipidemia and ASCVD. MethodsSmall RNA-seq and qPCR were used to determine the differential plasma level of exosomal miR-148a-3p in ASCVD patients with and without CKD. RNA-seq and in silico analysis were used to establish the regulation of lipid metabolism via miR-148a-3p/salt-inducible kinase 1 (SIK1)/AMP-activated protein kinase alpha 1 subunit (AMPK1) in the liver. Inhibition of miR-148a-3p or overexpression of SIK1/AMPK1 in ApoE-/- mice with 5/6 nephrectomy was used to elucidate the role of the miR-148a-3p/SIK1/AMPK1 axis in CKD-related hyperlipidemia and atherosclerosis. ResultsThe plasma level of exosomal miR-148a-3p was significantly elevated in patients with ASCVD and CKD (ASCVD/CKD) and ApoE-/- mice with 5/6 nephrectomy. Via circulating exosomes, kidney-generated miR-148a-3p was delivered to the liver, where it targeted SIK1/AMPK1 transcripts, thereby upregulating SREBP2/PCSK9 and reciprocally downregulating LDLR. Levels of SIK1 and AMPK1 were decreased in hepatocytes transfected with miR-148a-3p or treated with plasma exosomes from ASCVD/CKD patients, with attendant SREBP2 activation and attenuated LDL-C binding. The decreased LDL-C binding was rectified by SIK1/AMPK1 or LNA-miR-148a-3p overexpression. LNA-miR-148a-3p or AAV8-SIK1/AMPK administration in 5/6 nephrectomy/ApoE-/- mice significantly ameliorated hyperlipidemia and atherosclerosis. ConclusionIn ASCVD/CKD patients and nephrectomy/ApoE-/- mice, kidney-originating miR-148a-3p targeted hepatic SIK1/AMPK1 mRNA. This exosome-mediated endocrine effect upregulated SREBP2/PCSK9 and reciprocally reduced LDLR level, which elevated plasma LDL-C level and exacerbated ASCVD. These findings underscore a kidney-liver-artery axis involved in cardiovascular-kidney-metabolic syndrome.

pathology↗

bioRxiv2025

Cortex-Wide Substrates for Body Schema and Action Awareness

The concept of the body schema originated from studies of patients with cortical lesions who were unable to localize their body parts in space or perceive passive movements (Head & Holmes, 1911). Current consensus defines the body schema as the brains internal representation of the body configuration in space used for both embodied perception and voluntary action (de Vignemont, 2010; Berlucchi & Aglioti, 2010; Vallar et al., 2025). Despite extensive research, how the cortex efficiently encodes the vast repertoire of body postures to support natural behavior remains poorly understood. Here, we reveal the representational format underlying the body schema by combining large-scale electrophysiological recordings with full-body 3D tracking and joint angle computation in freely behaving mice. Across cortical regions, neurons encoded diverse postural features in distinct reference frames: posterior parietal cortex (PPC) populations preferentially encoded body midline-referenced features, while sensorimotor cortices utilized a gravity-referenced frame. Strikingly, during performance of different stereotyped actions, distinct cortical ensembles were selectively activated. These action ensembles fired in phase-locked sequences that tiled the full action cycle, providing a neural substrate for action awareness. A network model recapitulated these dynamics and identified low-dimensional action subspaces within population activity. Together, these findings reveal that the cortex organizes the body schema around actions, compressing high-dimensional kinematics into an efficient, body-midline-and gravity-anchored, action-aware neural code.

neuroscience↗

bioRxiv2025

CDCA7 facilitates MET1-mediated CG DNA methylation maintenance in centromeric heterochromatin via histone H1

DNA methylation is a conserved epigenetic modification essential for maintaining genome stability. However, how methyltransferases maintain CG methylation within compact chromatin, including centromeres, remains unclear. In humans, CDCA7 is necessary for the inheritance of DNA methylation at juxta-centromeres. Mutations that impair its ability to bind chromatin result in Immunodeficiency, Centromeric Instability, and Facial Anomalies (ICF) syndrome, characterized by centromeric instability. To investigate whether CDCA7 function is conserved, we identified two Arabidopsis thaliana orthologs, CDCA7A and CDCA7B. The loss of both copies results in CG hypomethylation at pericentromeric regions and centromeric satellite repeat arrays. Machine learning analysis suggested that heterochromatic nucleosomes, with enrichment of H1, H2A.W, and H3K9me2 levels, depend heavily on CDCA7 proteins for CG methylation maintenance of the associated DNA. Loss of H1 restores heterochromatic DNA methylation in cdca7a cdca7b mutants, indicating that CDCA7A and CDCA7B mainly remodel H1-containing nucleosomes for methyltransferases to access DNA. Notably, in h1.1 h1.2 mutants, CG methylation shows a significant increase in centromeres, which reveals a new inhibitory role of H1 in DNA methylation maintenance within satellite repeat arrays. Centromeric DNA hypermethylation is lost in h1.1 h1.2 cdca7a cdca7b quadruple mutants, demonstrating that CDCA7A and CDCA7B can act independently of H1 to enhance MET1 activity. Overall, these findings establish CDCA7A and CDCA7B as conserved regulators of DNA methylation within heterochromatin and centromeric satellite repeat arrays.

molecular biology↗

bioRxiv2025

Nanoscopy Reveals Heparan Sulfate Clusters as Docking Sites for SARS-CoV-2 Attachment and Entry

Virus entry is thought to involve binding a unique receptor for cell attachment and cytosolic entry. For SARS-CoV-2 underlying the COVID-19 pandemic, angiotensin-converting enzyme 2 (ACE2) is widely considered the receptor for cell-surface attachment and cytosolic entry. Using advanced light microscopy to resolve individual virions and receptors, we found instead that heparan sulfate (HS), not ACE2, mediates SARS-CoV-2 cell-surface attachment and subsequent endocytosis. ACE2 functions only downstream of HS to enable viral genome expression. Instead of binding single HS molecules that electrostatically interact with viral surface proteins weakly, SARS-CoV-2 binds clusters of [~]6-137 HS molecules projecting 60-410 nm above the plasma membrane. These tall, HS-rich clusters, present at about one per 6 m{superscript 2}, act as docking sites for viral attachment. Blocking HS binding with the clinically used HS-binding agent pixantrone strongly inhibited an authentic pathogen, the SARS-CoV-2 Omicron JN.1 subvariant, from attaching to and infecting human airway cells. This work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting downstream, thereby identifying HS interactions as a key anti-COVID-19 strategy. This paradigm and its therapeutic implications may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator. Statement of SignificanceViral entry, a crucial antiviral target, is typically thought to involve binding its unique receptor for the cell surface attachment and subsequent entry. We examined this concept with advanced microscopies to resolve individual receptors and SARS-CoV-2 virions responsible for the COVID-19 pandemic. We discovered two receptors for viral entry: heparan sulfate, a polysaccharide that may bind many viruses, mediates viral attachment and subsequent endocytosis, whereas angiotensin-converting enzyme 2 (ACE2), the generally assumed SARS-CoV-2 receptor, acts only downstream to facilitate viral infection. This model suggests perturbation of HS binding as a more effective anti-COVID-19 strategy than previously recognized. It may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator.

microbiology↗