Biology subjects

Shi, X.

Publications and source records attributed to Shi, X..

At least 19 recordsLinked to original sources

bioRxiv2026

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

pharmacology and toxicology↗

bioRxiv2026

Mapping the Fragment-Binding Landscape of SARS-CoV-2 nsp14 for Antiviral Drug Discovery

Non-structural protein 14 (nsp14) is a bifunctional coronavirus drug target, combining a 3'-to-5' exoribonuclease domain with an N7-methyltransferase domain. Proof-of-concept data have demonstrated beneficial effects in animal models upon inhibition. Using microscale thermophoresis and X-ray crystallography, we screened full-length nsp14 for fragment hits and developed SAR-by-catalogue analogues with five- to eight-fold improved affinity. Several scaffolds showed pronounced overlap between distinct pockets, complicating structure-based optimisation. Analysis of mutational frequencies across over 14 million SARS-CoV-2 genome sequences identified nsp14 residues with markedly high and very low mutation rates, highlighting sites best suited to pan-coronavirus inhibitor design. Combining these data with all 19 publicly known fragment- and ligand-binding regions on nsp14, most of which have never been directly compared, we introduce a systematic site-naming system and rank every site by its potential for future structure-based development. Weighing criteria include proof-of-principle inhibitor data, cross-coronavirus conservation, mutation frequency, the number of fragments bound per site, and susceptibility to crystallographic artefacts from symmetry mates. This ranked, unified binding-site atlas resolves which of the many previously reported nsp14 sites merit prioritisation, providing a practical resource for future coronavirus antiviral drug development.

biochemistry↗

bioRxiv2026

FLASH X-rays preserve neutrophil chromatin to boost antitumor immunity

Ultrahigh dose-rate FLASH radiotherapy spares normal tissue while preserving antitumor activity, positioning it as a promising technology in radiation oncology, but its immunomodulatory mechanisms remain elusive. Here, using a bilateral subcutaneous tumor model in mice, we show that 8-Gy FLASH X-rays, versus an equal dose of conventional dose-rate (CONV) X-rays, elicit a robust abscopal antitumor effect with anti-PD-1 therapy. Single-cell transcriptome and T cell receptor (TCR) profiling reveal that FLASH X-rays plus anti-PD-1 enhance antitumor immune infiltration and TCR clonal expansion in both irradiated and abscopal tumor lesions. Mechanistically, the ultrashort temporal continuity of FLASH dose delivery conserves chromatin compaction in neutrophils during exposure and inhibits neutrophil extracellular trap (NET) formation, thereby dismantling NET-mediated CD8+ T-cell exclusion within primary tumors. Conversely, CONV or split-dose FLASH X-rays induce marked chromatin modifications prior to dose completion, leading to chromatin hyper-relaxation which promotes NETosis and consequently abrogates systemic antitumor immunity. Notably, this detrimental effect can be reversed by pharmacologically suppressing PARylation-mediated chromatin modification and decondensation in neutrophils. These findings identify radiation-induced aberrant chromatin relaxation in neutrophils as a critical determinant of radioimmunotherapy outcomes, providing a rationale for redefining the FLASH effect and advancing its clinical translation.

cancer biology↗

bioRxiv2026

Structural variants from donor-specific assemblies affect recurrent chromatin contact divergence in humans

Structural variants (SVs) are a major source of human genetic diversity, yet their contribution to three-dimensional (3D) genome organization remains difficult to resolve using a standard reference genome. Here, we integrate haplotype-resolved chromatin interaction maps across 354 human and 12 nonhuman ape haplotypes with matched genome assemblies and long-read transcriptomes to determine how inherited SVs influence chromatin organization. We observe significant improvements in contact mapping, with 13.2% of cis Hi-C contacts showing >25 kbp changes in inferred genomic separation and an additional 2.8% of valid contact pairs recovered when using donor-specific assemblies instead of standard references. We show that haplotype-specific chromatin differences are highly selective rather than pervasive, concentrating within 1,790 recurrent contact difference hotspots associated with a restricted subset of SVs. We identified 2,495 chromatin-structure-associated SVs that are enriched in regulatory genomic contexts and are linked to expression variation. Notably, more than 90% of these SVs are derived in the human lineage, consistent with recent remodeling of regulatory architecture at the population level. These findings highlight the importance of incorporating haplotype diversity from pangenomes into studies of chromatin organization and provide a framework for understanding how inherited genomic differences are linked to functional consequences across populations and evolution.

genomics↗

bioRxiv2026

An FEV-Associated Metastasis-Initiating-Like State Linked to Tumor Cell Plasticity and Immune Niche Remodeling in Prostate Cancer

Prostate cancer progression is accompanied by dynamic remodeling of malignant cell states, whose metastatic potential and regulatory programs remain poorly defined. Here, we integrated single-cell, spatial, bulk transcriptomic, epigenomic, and experimental data to characterize malignant cell state transitions and identify candidate regulators associated with metastatic progression. A distinct intermediate state exhibits increased copy-number alterations, stemness, proliferation, and MYC activity, together with the highest transcriptional similarity to metastatic tumor cells, and is therefore designated as a metastasis-initiating-like (MIC-like) state. Transcriptional and regulon dynamics along the malignant cell state trajectory highlight FEV as a prominent candidate associated with the MIC-like state, with concordant expression and regulon activity patterns. Functionally, FEV is associated with a hybrid epithelial-mesenchymal phenotype, and its perturbation disrupts this phenotype, accompanied by reduced epithelial and mesenchymal features as well as impaired prostate cancer cell migration and invasion. Beyond this tumor-intrinsic feature, FEV is also associated with reduced cytotoxicity and interferon responses in the tumor microenvironment through its regulation of MIF-family genes. In addition to its potential roles in tumor cell plasticity and immune remodeling, FEV shows a reciprocal association with androgen signaling and distinct expression patterns across treatment response groups, highlighting its potential relevance in therapeutic contexts.

bioinformatics↗

bioRxiv2026

BpLAZY1A Mediates Transcriptional Polarity and Drives Adaxial Tension Wood Like Tissue Formation in Silver Birch (Betula pendula) Branches

A defining feature of most trees is the architectural distinction between a vertically growing main stem and laterally growing branches. This growth habit increases fitness by increasing photosynthetically active surface area and shading competitors. Here, we studied the weeping birch cultivar Betula pendula Youngii to uncover the mechanisms that maintain lateral branch growth. We identified a loss-of-function mutation in BpLAZY1A, a core component of the gravitropic signalling pathway, as the cause of the weeping phenotype. Forward genetic analysis demonstrated that the weeping phenotype is recessive in silver birch. Transgenic BpLAZY1A RNAi lines phenocopied Youngii, confirming the functional role of BpLAZY1A. Reporter analysis revealed that BpLAZY1A is expressed predominantly in gravity-sensing starch sheath cells, with occasional expression in the main stem phloem. Time lapse imaging revealed two distinct gravitropic responses during branch development - an early response associated with establishment of the branch apex gravitropic set-point angle, and a later response associated with polar reinforcement at the branch base. The main stem retained normal gravitropic responses in BpLAZY1A RNAi lines, indicating a branch-specific role for BpLAZY1A. Integrative analyses combining transcriptomics, chemical profiling, and histochemistry indicated that BpLAZY1A establishes adaxial-abaxial polarity during early branch development. In wild-type branches, pectin-rich tension wood-like tissue formed preferentially in the adaxial xylem, accompanied by adaxial expression of pectin- and tension wood-associated genes, including BpRRT1 and BpCOBRA-LIKE4. Auxin-related genes, including BpIAA29 and BpSAURs, were preferentially upregulated in the abaxial side. This transcriptional asymmetry was reduced in BpLAZY1A RNAi line 1, where tension wood-like tissue formed in both adaxial and abaxial sides of the xylem, indicating a loss of polarity. Together, these findings demonstrate that BpLAZY1A functions specifically in branch gravitropism and links spatially asymmetric gene expression with branch growth orientation and biomechanical reinforcement. Our results identify BpLAZY1A as a key regulator coordinating gravitropic signalling, tissue polarity, and the developmental biomechanics underlying lateral branch growth.

plant biology↗

bioRxiv2026

The devil in citizen science data: observation processes invalidate the causal inference that photovoltaic policy reduces bird diversity

Citizen science data are increasingly used to infer biodiversity change, but causal claims based on such data are credible only if sampling effort and its temporal shifts are explicitly modeled. Zhang et al. (1) used citizen science data to conclude that greater photovoltaic policy stringency, measured using the photovoltaic policy stringency index (PSI), reduced county-level bird diversity in China. We reproduced their fixed effects and instrumental variable estimates. However, the observed Shannon diversity derived from pooled citizen science records reflects both bird communities and sampling effort, which the authors' controls do not adequately capture. Accounting for observer count changed the reported statistically significant 2.10% decline in Shannon index to a nonsignificant 0.58% increase (P = 0.288) per one-standard-deviation increase in PSI, and rendered the instrumental variable estimate statistically indistinguishable from zero (P = 0.912). Yet observer count is only one of many sources of sampling bias. PSI was also associated with multiple dimensions of sampling effort, consistent with sampling effort acting as a potential mediator in the PSI-diversity chain. The sampling domain also shifted markedly from 2014 to 2023: recorded county-months increased almost 24-fold, median observer count rose from one to three, and zero-duration records declined from 57.2% to 0.17%. Without adequate adjustment, these shifts confound estimates of temporal change in observed bird diversity. Beyond its inadequate treatment of sampling effort, the original study also misinterpreted its statistical results. Although the reported R{superscript 2} values are high, they are dominated by county and year-month fixed effects, with PSI contributing a partial R{superscript 2} of only 0.048% on observed Shannon index. The PSI-photovoltaic-area correlation is also weak (r = 0.0414) and vanishes after accounting for fixed effects (P = 0.977). Furthermore, the released bird observation data contain many erroneous outliers, raising significant concerns about insufficiently rigorous data preprocessing and quality control. These results show that the released data cannot properly distinguish ecological change from sampling effort change. Robust inference from citizen science data requires checklist-level effort metadata, explicit correction for spatiotemporal sampling shifts, and close collaboration among researchers with complementary methodological and ecological expertise.

ecology↗

bioRxiv2026

A virus co-opts vector's m⁶A machinery for adaptive compensation

Insect vectors harbor diverse viruses that threaten human health and agricultural production, yet the genetic basis of mutualistic virus-vector interactions remain elusive. This underscores a long-standing paradox in pest management: viruses that cause destructive diseases also facilitate vector adaptation in ways that benefit both parties. Here we show that the globally devastating tomato yellow leaf curl virus (TYLCV) acts as a Cooperative Partner, co-opting its vector's m6A epitranscriptomic machinery to offset the reproductive fitness costs of neonicotinoid resistance in the whitefly. In resistant vectors, TYLCV infection alleviates reproductive deficits via the m6A-dependent pathway, where the methyltransferase METTL14 and demethylase ALKBH4 coordinately stabilize vitellogenin (Vg) transcripts to boost female fecundity. Mechanistically, the viral C2 and CP proteins directly interact with METTL14 and ALKBH4, respectively, remodeling their RNA binding affinity to facilitate mA modification on Vg. Disrupting either of these viral-vector protein interactions restores a reproductive cost phenotype in viruliferous resistant vectors, offering novel targets for sustainable pest control. Our findings uncover a fundamental role of epigenetic marks in adaptive trait, reshaping beneficial virus-vector relationships and providing new insights for innovative public health and crop protection strategies to address global challenges.

microbiology↗

bioRxiv2026

The Maine Coon Cat Harboring the MYBPC3-A31P Mutation: A Genotype-Stratified Phenotypic Characterization of Hypertrophic Cardiomyopathy

Abstract Background: Most animal models of HCM are mouse-based, but the thin interventricular septum in mice makes it difficult to clearly distinguish pathological hypertrophy, which introduces substantial errors and constrains basic HCM research. Cats develop HCM spontaneously, and the common MYBPC3-A31P variant in cats is homologous to human mutations in both genetics and pathology, with a larger body size that makes them suitable as large-animal models. This study examines how heterozygosity or homozygosity for the p.A31P mutation (c.91G>C) in the MYBPC3 gene affects the phenotype and severity of HCM in affected cats, with the aim of establishing an ideal large-animal model for clinical risk stratification and precision diagnosis and treatment of human HCM. Methods: Forty-nine Maine Coon cats were enrolled and stratified into homozygous mutant (HOM, n=8), heterozygous mutant (HET, n=26), and wild-type (WT, n=15) groups. All cats underwent echocardiography, blood pressure measurement, physiological assessment, hematological and biochemical analyses, and cross-species sequence conservation analysis. Results: No significant differences in baseline characteristics including age and body weight were observed among groups (P>0.05). HOM cats exhibited significantly higher left ventricular outflow tract pressure gradients and greater basal septal thickness compared to WT cats (P<0.05), with HET cats showing intermediate values. Analysis of hematological and serum biochemical parameters revealed no evidence of systemic inflammation or hepatic injury. Sequence conservation analysis confirmed that the A31 residue is highly conserved across mammalian species. Conclusions: This study provides a phenotypic characterization of Maine Coon cats carrying the MYBPC3-A31P mutation, revealing marked gene-dose effects on cardiac structure and function, with homozygous individuals exhibiting more severe phenotypic features. This model serves as a large-animal translational platform that not only clarifies genotype-phenotype correlations but also supports risk stratification and precision therapeutic strategies in human HCM. Its spontaneous nature and genetic homology to human disease make it particularly valuable for bridging preclinical findings to clinical application.

genetics↗

bioRxiv2026

Functional and evolutionary insights into the emerging tet(X4)-carrying non-O1/O139 Vibrio cholerae from retail pork

The tigecycline resistance gene tet(X4) is prevalent in Enterobacteriaceae, particularly in Escherichia coli. To our knowledge, no study has reported the dissemination dynamics of tet(X4) in Vibrio spp. Herein, we isolated and characterized a first tet(X4)-positive non-O1/O139 Vibrio cholerae isolate from retail pork. Genomic sequencing identified a novel tet(X4) variant in the V. cholerae chromosome, harboring a G568A nucleotide substitution that resulted in an Ala190Thr (A190T) amino acid substitution in Tet(X4). While this Tet(X4)-A190T variant conferred lower phenotypic resistance to tetracyclines (including tigecycline) than the wild-type Tet(X4), its overall catalytic efficiency against these antibiotics was paradoxically enhanced despite a reduced substrate affinity. Genomic comparisons revealed that two copies of ISCR2 flanked the variant gene, and the structure was ISCR2-hp-hp-abh-tet(X4)G568A -ISCR2, which is highly homologous to the reported E. coli plasmids carrying tet(X4). In addition, it confirmed the presence of an ISCR2-mediated circular intermediate, proving this modules capacity for horizontal transfer of the tet(X4)G568A variant. Furthermore, the ISCR2-tet(X4) genetic structure carrying the G568A substitution was integrated within a chimeric SXT/R391-like integrative and conjugative element (ICE), which is also serving as a vehicle for genetic dissemination. As per our knowledge, this is the first report on the emergence of SXT/R391-like ICE carrying tet(X4) in Vibrio strains. Our finding demonstrates that the clinically relevant tigecycline resistance gene tet(X4), previously confined mainly to Enterobacterales from humans and livestock, is now actively spreading into environmental Vibrio populations. This cross-species transfer highlights a previously underappreciated ecological and public health concern in aquatic ecosystems.

microbiology↗

bioRxiv2026

De novo transformer modeling improves recovery of genetic cell types from sparse single-cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) simultaneously provides gene-expression profiles and genetic variants from individual cells, creating an opportunity to relate cellular phenotypes to their somatic evolutionary histories. However, delineation of genetic type (GTs) from scRNA-seq remains difficult because most variant positions are unobserved in individual cells and the observed base calls contain substantial false-positive and false-negative errors. We evaluated some existing phylogenetic and imputation methods using one simulated dataset and two empirical tumor datasets. We found that extreme sparsity prevented reliable recovery of known or independently inferred GTs when multiple GTs were present. This led us to adapt the STICI transformer architecture to train a separate model de novo on each sparse cell-variant (CV) matrix. These data-specific models predicted millions of missing bases, greatly reducing matrix sparsity. Phylogenetic analyses of the imputed CV matrices showed substantially improved recovery of GTs in both simulated and empirical datasets. In the empirical dataset, transformer-based analysis also suggested finer-scale genetic structure within some previously reported GTs that was not apparent with the existing methods. These results demonstrate that highly sparse scRNA-seq datasets contain substantially more recoverable lineage information than previously appreciated and that de novo transformer modeling provides an effective approach for recovering much of this hidden information. Nevertheless, sequencing errors persisted, limiting reconstruction of cellular lineage structure and leaving significant room for methodological improvement before expression phenotypes can be examined reliably in the context of their cellular evolutionary relationships.

bioinformatics↗

bioRxiv2026

5β-Dihydrotestosterone reveals a mutant androgen receptor vulnerability in prostate cancer

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5{beta}-dihydrotestosterone (5{beta}-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5{beta}-DHT and related 5{beta}-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5{beta}-DHT and 3{beta}-etiocholanediol (3{beta}-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2, with weaker activity than testosterone, whereas other 5{beta} metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5{beta}-DHT and 3{beta}-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5{beta}-DHT and 3{beta}-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5{beta}-DHT, but not 3{beta}-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5{beta}-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects. HighlightsO_LI5{beta}-DHT and 3{beta}-ecdiol promote AR-dependent prostate cancer cell growth C_LIO_LIBoth are weaker AR agonists than testosterone by RNA-seq and qPCR C_LIO_LISupraphysiologic 5{beta}-DHT suppresses growth via AR-mediated senescence C_LIO_LIGrowth suppression extends to AR mutants W742C and H875Y C_LIO_LI5{beta}-DHT may be a lower-androgenicity testosterone surrogate for BAT C_LI

cancer biology↗

bioRxiv2026

Toward pharmacologic therapy for glioblastoma: Characterization of the very long-chain acyl-CoA synthetase 3 (ACSVL3) inhibitor Grassofermata

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 {micro}M were toxic. Treatment at the lower dose of 3 {micro}M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5-treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 {micro}M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 {micro}M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5-treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 {micro}M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

cancer biology↗

bioRxiv2026

Unearthing a fungal giant: Dianjunaceae fam. nov., a novel Paleocene lineage of Xylariales harbouring Dianjunus rex gen. et sp. nov.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene ([~]65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxons phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

microbiology↗

bioRxiv2026

Establishing a Retron-Based Cytosine Base Editor for Targeted Hypermutation in Escherichia coli

Current cytosine base editors (CBEs) are limited to unidirectional C to T conversions, restricting their applications. Retrons, bacterial genetic elements, encode a reverse transcriptase that generates multicopy single-stranded DNA (msDNA) by reverse transcribing specific non-coding RNA (ncRNA). This msDNA mimics Okazaki fragments during DNA replication, making retrons promising for gene editing. Here, we developed a retron-based cytosine base editor (RCBE) by fusing cytosine deaminase with reverse transcriptase (RT-CDA) within the retron system. RCBE first transcribes ncRNA, allowing RT-CDA to deaminate cytosine on the ncRNA. The modified ncRNA is then reverse transcribed into msDNA, where RT-CDA induces further cytosine deamination. This mutant msDNA introduces specific mutations into target gene sequences, enabling both C to T and G to A conversions. Using RCBE, we demonstrated accelerated molecular evolution of the rpoB gene in Escherichia coli. High-throughput sequencing confirmed that RCBE achieves a mutation rate of up to 0.2% in regions with high GC content. Our findings establish RCBE as a versatile tool, particularly suitable for directed evolution in GC-rich regions, with broad potential applications across various bacterial and eukaryotic hosts.

Synthetic Biology↗

bioRxiv2026

Accurate de novo design of peptides from programming biophysical landscape

Peptides regulate virtually every cellular process and emerge as transformative modalities in bioengineering and therapeutics. However, the de novo design of functional peptides remains challenging, as peptide-protein interactions are governed by delicate and dynamic biophysics that are difficult to model, and experimental datasets are scarce. Here we present NeoPep, a generative deep-learning framework that encodes biophysical principles to accurately design functional peptides de novo. By integrating over 5 million peptide-protein complexes spanning experimentally determined, sequence mimics, and structure ensembles, NeoPep learns the complex biophysical landscape governing peptide function and enables its programmable control. In prospective application across 10 diverse and challenging targets, NeoPep generated potent peptide binders, agonists, and antagonists with hit rates of 12.5-66.7%, even in the absence of defined binding sites or structure information. Beyond de novo co-design, NeoPep supports standalone structure or sequence redesign, readily discriminating subtle context differences. In the structure redesign mode, it generates highly selective peptides with atomic-level conformational accuracy (C RMSD < 2.0 [A] to our solved cryo-EM structure). This structural precision circumvents the need for experimental structure determination, further accelerating iterative sequence redesign to yield a 43.3-fold improvement in potency. These findings establish a general framework for translating biophysical principles into actionable peptide functions, with broad implications for basic research, bioengineering, and medicine.

molecular biology↗

bioRxiv2026

Population-scale Y chromosome assemblies reveal recurrent remodeling within constrained architectures

The human Y chromosome is among the most structurally dynamic chromosomes in the human genome, yet much of its diversity remains unresolved because of extensive palindromes, ampliconic gene families, satellite-rich heterochromatin and large segmental duplications. What remained unclear was how these diverse forms of variation fit together across the full chromosome, how often similar structures recur in different lineages, and which aspects of organization remain constrained despite rapid sequence turnover. Here, we generated and analyzed 142 nearly complete human Y chromosome assemblies from 17 major haplogroups spanning approximately 180,000 years of evolution, creating a population-scale resource for studying Y chromosome biology and diversity. These assemblies show that structural change on the Y chromosome is recurrent but constrained, even in its most repetitive regions. In the fertility-associated azoospermia factor c (AZFc) region, recurrent inversions, deletions, and complex rearrangements generate a limited repertoire of structural haplotypes. Multicopy ampliconic gene families follow distinct evolutionary paths: DAZ paralogues differ in structural constraint, RBMY evolves within a modular array, and TSPY copy number varies mainly through local expansion and contraction. The centromere and Yq12 heterochromatin vary greatly in size but retain a stable higher-order organization, including a single hypomethylated centromeric core and conserved Yq12 repeat composition and orientation. Methylation across palindromic and ampliconic regions is likewise structured by repeat class, copy order and local architecture. Together, these results provide a population-scale resource for the human Y chromosome and show that its rapid structural evolution is repeatedly funneled into a limited set of architectural outcomes.

genomics↗

bioRxiv2026

STID: A Standardized Spatial Transcriptomic Analysis Framework for Infectious Diseases

Spatial transcriptomic studies of infectious diseases still rely on fragmented data analysis processes. Here, we developed STID, a standardized framework for spatial transcriptomic analysis of infectious diseases that leverages the Seurat ecosystem and incorporates Python-based modules. STID provides an extensible infection-specific data structure and supports a full suite of analyses, such as pathogen background correction, infection-associated spot and niche identification, single-sample niche characterization, and multi-sample comparative and temporal analyses. Moreover, STID is broadly applicable to spatial transcriptomic data from infectious diseases caused by bacteria, viruses, and parasites, and enables systematic characterization of the structural features, cellular composition, molecular functions, and host-pathogen interactions within pathogen-infected and/or host-responsive niches. Overall, STID provides an accessible, reproducible, and extensible framework for analyzing infection-associated spatial transcriptomic data and for dissecting host-pathogen interactions in their native spatial microenvironments. MotivationSpatial transcriptomics technologies have emerged as powerful approaches for dissecting the structural and functional features of spatial microenvironments. However, the current general-purpose tools remain fundamentally inadequate for resolving the spatial heterogeneity of infectious disease samples, where the intricacies of host-pathogen interactions render spatial microenvironments both challenging to dissect and largely inaccessible. Tools tailored to infectious diseases are critically lacking, including those for reducing pathogen-derived background noise, identifying and isolating infection{square}associated spots or niches, dissecting host-pathogen interactions, and supporting systematic multi-sample analyses. We therefore developed STID, a unified framework that integrates standardized workflows and addresses the analytical bottlenecks in spatial transcriptomic analysis of infectious diseases. HighlightsO_LISTID standardizes spatial transcriptomic analysis in infectious diseases C_LIO_LISTID improves pathogen-infected spot detection by correcting pathogen background C_LIO_LISTID distinguishes pathogen-infected and host-responsive niches C_LIO_LISTID supports multi-sample comparative and temporal analyses of niches C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/727492v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@167d351org.highwire.dtl.DTLVardef@1628848org.highwire.dtl.DTLVardef@1e157aforg.highwire.dtl.DTLVardef@143ca1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗