Biology subjects

Shi, H.

Publications and source records attributed to Shi, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Path-dependent recovery of the gut microbiome after antibiotics emerges from coupled ecological and evolutionary dynamics

Recovery of the gut microbiome after antibiotic exposure is often incomplete and variable, and the processes underlying this variation remain unclear. We performed longitudinal shotgun metagenomic sequencing of 2876 daily fecal samples from replicated humanized and conventional mouse cohorts exposed to controlled antibiotic perturbations. Metagenomic profiling recapitulated ecological trajectories previously observed by 16S sequencing, while revealing extensive strain-level dynamics, including reproducible sweeps of standing variants and de novo mutations in antibiotic target sites and regulatory loci. We also identified genetic changes whose effects depended on community composition, competitive release, and perturbation history. Cross-housing experiments revealed bidirectional strain transfer, with antibiotic-induced niche clearance enabling replacement of resident strains. In parallel, phage dynamics were heterogeneous and clustered by cage. Together, these findings show that post-antibiotic microbiome recovery is a path-dependent process shaped by selection, transmission, and phage activity, producing divergent outcomes even among closely matched communities exposed to the same perturbations.

microbiology↗

bioRxiv2026

CUPID-seq enables highly multiplexed amplicon sequencing via combinatorial in-line dual indexing

Targeted amplicon sequencing is widely used to profile genetic variation in defined genomic regions. In microbial ecology, for example, amplicon sequencing of the 16S and 18S ribosomal RNA genes has been transformative for characterizing microbial communities. However, on high-capacity sequencing platforms with patterned flow cells, throughput is constrained by the requirement for unique dual indexes (UDIs), which increases primer costs and limits the number of samples that can be pooled per sequencing run. Here, we introduce CUPID-seq (Combinatorial, Unique, Phased, In-line Dual-indexed sequencing), a highly multiplexed amplicon-sequencing strategy that increases scalability through combinatorial indexing across two rounds of PCR. CUPID-seq introduces phased, in-line UDIs during Round 1 gene-specific amplification, enabling multiple samples to share the same Illumina UDI during Round 2 PCR while remaining uniquely identifiable. This design reduces upfront costs by up to 85% and reduces library preparation time and reagent use by up to 40%. We develop and validate CUPID-seq primers targeting the 16S V4 region and provide a computational workflow for demultiplexing in-line indexes. Although optimized here for 16S-based profiling, CUPID-seq can be readily adapted to other user-defined amplicons. By reducing cost and increasing multiplexing capacity, CUPID-seq enables users to leverage high-throughput sequencing platforms more effectively across diverse biological contexts.

microbiology↗

bioRxiv2026

GeoEPred: A Multimodal Structure-Aware Geometric Deep Learning Framework for Gram-Negative Bacterial Secreted Effector Prediction with Sequence Semantics

Accurate prediction of effector proteins secreted by Gram-negative bacteria is important for elucidating bacterial pathogenic mechanisms and developing precise anti-infective strategies. Although existing methods have benefited from the strong sequence feature extraction capacity of pretrained protein language models, reliance on linear sequence information alone often fails to fully capture the three-dimensional conformational signals required for virulence functions. Meanwhile, conventional structure-based methods are limited by the scarcity of experimentally resolved protein structures. To address these challenges, We propose GeoEPred, a multimodal deep learning framework designed for the synergistic modeling of protein sequence and structure to identify Gram-negative bacterial effector proteins. Specifically, the model integrates sequence-contextual embeddings from a pretrained protein language model with three-dimensional structural representations predicted by ESMFold. A feature projection network refines fine-grained sequence signals associated with effector functions, while geometric vector perceptrons characterize inter-residue orientations, distances, and local spatial topology to capture potential structural conformational motifs. To further enable effective cross-modal fusion, we design a cross-modal alignment and feature-tokenized self-attention module. This module enhances consistency between the sequence-semantic and structural-geometric spaces through contrastive learning and models associations between linear functional motifs and spatial conformational patterns at a fine-grained token level. Extensive evaluations on multiple benchmark datasets show that GeoEPred achieves better predictive performance than existing leading models in T3SE, T4SE, and T6SE prediction tasks, while maintaining stable performance in remote homolog recognition scenarios. Moreover, the modular and extensible architecture of GeoEPred demonstrates strong generalization ability and substantial application potential for genome-scale effector protein discovery. Author summarySecreted effector proteins are central virulence factors used by many Gram-negative bacterial pathogens to execute infection strategies. Their functions are governed not only by secretion signals and short linear motifs in the amino acid sequence, but also by three-dimensional folds, local domains, and surface geometric patterns. However, current predictors mainly exploit sequence-contextual features, limiting their ability to model the correspondence between linear sequence signals and spatial conformational motifs, and thereby constraining accuracy and interpretability. Here, we present GeoEPred, a multimodal deep learning framework for secreted effector protein identification. GeoEPred couples sequence-semantic embeddings from a pretrained protein language model with structural representations learned by geometric vector perceptrons. A cross-modal alignment and interaction module uses contrastive learning to improve functional consistency between sequence and structure modalities, while feature-token attention captures fine-grained links between key linear and conformational motifs. Across benchmark datasets covering multiple effector types, GeoEPred outperforms existing state-of-the-art methods and provides interpretable evidence from sequence fragments, structural regions, and cross-modal associations, supporting functional annotation, pathogenic mechanism analysis, and experimental validation.

genomics↗

bioRxiv2026

Immunometabolic Remodeling of Perivascular Adipose Tissue in Murine Lupus: Implications for Lupus Vasculopathy

BackgroundPatients with systemic lupus erythematosus (SLE) face markedly increased cardiovascular disease (CVD) risk driven by mechanisms beyond traditional risk factors. Thoracic aortic perivascular adipose tissue (tPVAT) is dysfunctional in lupus and exacerbates endothelial dysfunction, yet the molecular basis of this dysfunction remains poorly defined. MethodsIntegrated multi-omics profiling, including bulk RNA-seq, untargeted proteomics, lipidomics, and high-dimensional spectral flow cytometry, was performed on tPVAT from 15-week-old MRL/lpr mice (active lupus, n = 4-6) and MRL control mice (n = 5-6). Adipogenic differentiation capacity of tPVAT adipose stromal and progenitor cells (ASPCs) from MRL/lpr was assessed by Oil Red O staining at 5 (pre-dieasea) and 15 weeks (active disease), with subcutaneous ASPCs used as depot controls. ResultsTranscriptomic profiling of tPVAT from MRL/lpr mice identified 2,742 upregulated and 1,494 downregulated genes (adjusted p < 0.001, |log2FC| > 1), with strong activation of interferon, IL6-JAK-STAT3, and TNFA signaling pathways together with suppression of fatty acid metabolism, oxidative phosphorylation, and adipogenic pathways. Proteomic and lipidomic analyses were concordant, revealing broad downregulation of mitochondrial bioenergetic machinery, depletion of cardiolipin and acylcarnitines, and enrichment of ceramide phosphoinositols and lysophosphatidylcholines. Cardiolipin strongly correlated with the mitochondrial/metabolic protein module (r = 0.95) and inversely with the immune/inflammatory protein module (r = -0.92). Spectral flow cytometry confirmed marked CD45+ leukocyte infiltration dominated by T cells, together with a significantly reduced Treg/CD4+ ratio indicating loss of local immunoregulatory balance. ASPCs derived from PVAT of 15-week-old MRL/lpr mice exhibited impaired white and beige adipogenic differentiation, while APCs from PVAT of 5-week-old MRL/lpr mice, and from subcutaneous adipose tissues of 15-week-old MRL/lpr mice, had normal white and beige differentiation, consistent with an acquired, depot-specific, disease-stage-dependent progenitor defect in PVAT of MRL/lpr mice. ConclusionsLupus tPVAT undergoes a concordant cross-platform molecular reprogramming of mitochondrial bioenergetic genes coupled with establishment of an interferon-dominant immune niche and acquired loss of ASPC adipogenic capacity. These findings provide a molecular framework for lupus PVAT dysfunction and identify restoration of mitochondrial function, suppression of interferon-driven inflammation, and renewal of progenitor differentiation as potential therapeutic strategies for lupus vasculopathy.

molecular biology↗

bioRxiv2026

Phage-based microbiome manipulation reveals ecological interactions within gut communities

Mechanistic understanding of gut ecology is limited by the availability of tools for precise manipulation of microbiome composition. Here, we isolate lytic phages to enable targeted removal of gut commensal Escherichia fergusonii (Ef) from complex, undefined stool-derived in vitro communities. A single phage drove resistance without fitness cost in monoculture, but resistant Ef exhibited reduced fitness in communities, enabling expansion of closely related Proteobacteria. Resistance arose via reversible promoter inversion linked to outer-membrane function. A phage cocktail overcame resistance to achieve Ef knockout across communities with minimal collateral effects. Using knockout communities, we show that Ef is necessary and sufficient for preventing Salmonella invasion. Replacement with an Ef transposon-mutant library revealed that community-specific fitness defects are enriched in genes involved in outer-membrane assembly. Disruption of these genes sensitized Ef to antagonistic community members, highlighting interspecies warfare as a key driver of microbiome ecology. These results establish phage-mediated perturbation as a framework for linking species to community-level function and for enabling precision microbiome engineering.

microbiology↗

bioRxiv2026

Microglial pruning of extinction-ensemble synapses preserves fear memory

Fear extinction suppresses learned fear without erasing the original memory, yet how competing fear and extinction ensembles are selectively updated remains unknown. Here, we show that microglia preserve fear memory by selectively editing extinction-ensemble synapses during retrieval. In medial prefrontal cortex, microglial processes, but not somata, expand, ramify, and tighten their engagement with extinction-ensemble dendrites and spines, where they preferentially engulf excitatory postsynaptic material and bias spine remodeling toward elimination. This selectivity is instructed by local find-me, eat-me, and dont-eat-me cues: purinergic signaling recruits microglial processes, phosphatidylserine exposure licenses engulfment of extinction-ensemble synapses, and CD47-SIRP protects fear ensembles from removal. Weakening microglial recruitment or engulfment, or removing this protection, accelerates extinction without impairing fear acquisition. These findings identify microglial processes as active gatekeepers of ensemble competition and reveal a neuroimmune mechanism that preserves fear memory by limiting extinction. In BriefDuring memory updating, microglial processes selectively engage extinction-ensemble synapses rather than globally pruning active circuits. Local recruitment, engulfment, and protection cues determine this choice, allowing microglia to preserve fear memory by limiting extinction. HighlightsO_LIExtinction retrieval selectively recruits microglial processes to extinction ensembles. C_LIO_LIMicroglia preferentially engulf excitatory postsynaptic material from extinction ensembles. C_LIO_LILocal "find-me," "eat-me," and "dont-eat-me" cues determine synapse selection. C_LIO_LIDisrupting microglial pruning facilitates extinction without impairing fear learning. C_LI

neuroscience↗

bioRxiv2026

A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR

CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.

molecular biology↗

bioRxiv2026

Impairing NK-mediated immune rejection through NKG2DL editing to improve CAR-T cell persistence

CAR-T immunotherapy has achieved remarkable efficacy in hematologic malignancies. However, the widespread clinical adoption of autologous CAR-T products remains constrained by high costs, lengthy manufacturing process, and limited accessibility. Universal or off the shelf CAR-T (UCAR-T) cells derived from healthy donors offer a promising alternative, enabling immediate treatment at a lower cost. However, the allogeneic nature of UCAR-T cells triggers immune rejection by the host immune system after infusion, thereby compromising their persistence and therapeutic efficacy. Current strategies to circumvent this rejection focus on disrupting HLA class I expression. Although this modification allows UCAR-T cells to successfully evade T cell mediated elimination, the loss of HLA class I molecules renders them vulnerable to attack by host natural killer (NK) cells. In contrast to previous approaches that attempt to retain certain non-classical HLA molecules (such as HLA-E or HLA-G) to inhibit NK cells, we directly focused on editing the ligands that mediate NK cell rejection. Through transcriptomic and in vitro validation analyses, we found that UL16 binding proteins (ULBP) 2/5/6 were substantially upregulated in UCAR-T cells compared with nontransduced donor T cells. Elevated ULBP expression effectively activates the NKG2D receptor on allogeneic NK cells and leads to killing of UCAR-T cells, thereby impairing UCAR-T function. To test whether abrogating this NK activating signal could improve UCAR-T persistence and antitumor efficacy, we generated ULBP knockout UCAR-T cells using CRISPR-Cas9 editing. Deletion of ULBP2/5/6 significantly reduced NK cell mediated killing in vitro without affecting CAR expression or T cell effector function. Compared with wild type UCAR-T cells, ULBP deficient UCAR-T cells exhibited enhanced tumor killing efficacy in the presence of NK cells. Collectively, our findings identify ULBP upregulation as one of the mechanisms underlying NK cell mediated rejection of HLA deficient UCAR-T cells. Targeted ablation of ULBP molecules provides a novel strategy to confer resistance to host NK cells, thereby improving the therapeutic potential of universal CAR T products.

immunology↗

bioRxiv2026

An oxytocin-gated circuit from the hypothalamus silences olfactory tubercle neurons to drive prosocial grooming

Spontaneous helping behaviors such as allogrooming are vital for survival in social species, yet their underlying neural mechanisms remain largely unknown. Although oxytocin (OXT) is known to modulate allogrooming, the precise neural circuits, molecular pathways, and sensory drivers remain unclear. Here, using a social emergency paradigm in mice, we show that observer mice selectively allogroom distressed, anesthetized conspecifics, a behavior that is guided by main olfactory system cues and mitigates the demonstrators anxiety. We identify an oxytocinergic circuit from the paraventricular hypothalamus to the medial olfactory tubercle (PVNOXT[->]mOT) that is necessary and sufficient for this helping behavior. In vivo fiber photometry reveals that PVNOXT[->]mOT activity and OXT release are temporally locked to allogrooming initiation. This behavioral effect requires oxytocin receptor (OXTR) signaling specifically on mOT dopamine D1 receptor-expressing neurons, where OXTRs suppress neuronal excitability via G protein-gated inwardly rectifying K+ (GIRK) channels. Disruption of this inhibitory mechanism induces neuronal hyperexcitability and impairs allogrooming, a deficit rescued by restoring GIRK function. These findings define a PVNOXT-mOT circuit for prosocial helping, revealing an oxytocinergic pathway with potential therapeutic relevance for neuropsychiatric disorders characterized by social deficits.

neuroscience↗

bioRxiv2026

MRN-ATM Pathway Activation in CD4 T-Cell Senescence during Chronic Hepatitis B Virus Infection

T-cell senescence is a hallmark of immune dysfunction in persistent viral infections, characterized by DNA damage accumulation and telomere erosion. However, the mechanisms driving CD4 T-cell senescence in the context of chronic hepatitis B virus (HBV) infection remain poorly defined. In this study, we demonstrated that people with chronic HBV infection exhibited CD4 T-cell senescence, marked by elevated KLRG1, along with increased DNA damage and telomere shortening, compared to HS. Notably, activation of the MRN-ATM (MRE11/RAD50/NBS1-Ataxia Telangiectasia Mutated Protein) pathway was prominent in CD4 T cells from HBV patients. Importantly, suppression of MRN attenuated ATM phosphorylation and its downstream signaling molecules, and inhibition of ATM reduced the production of proinflammatory cytokines in CD4 T cells derived from both HBV patients and HS. These results suggest that in chronic HBV infection, the virus induced CD4 T-cell senescence, telomere erosion, and DNA damage, while concurrent activation of the MRN-ATM pathway may serve as a compensatory mechanism to preserve CD4 T-cell function. Elucidating this relationship between T-cell senescence and DNA damage repair helps to understanding the mechanisms underlying HBV persistence and providing potential therapeutic targets against chronic HBV infection.

immunology↗

bioRxiv2026

Targeting phosphodiesterase 10A disrupts MAPK signaling pathways in the tumor microenvironment to unleash antitumor immunity

Phosphodiesterase 10A (PDE10A), a cyclic nucleotide-degrading enzyme, is overexpressed in various human cancers. While PDE10A inhibition using small-molecule inhibitors or gene silencing suppresses tumor growth in xenograft models, its precise mechanism of action and immunological impact remain unclear. Here, we report that ADT-030, a novel PDE10A inhibitor, exhibits potent cytotoxicity against a broad range of murine tumor cell lines. ADT-030 is orally bioavailable and effectively suppresses tumor growth across multiple syngeneic mouse models. Notably, its efficacy is diminished in immunodeficient mice or upon CD8+ T cell depletion, highlighting a critical dependence on host immunity. The immunostimulatory properties of ADT-030 are further supported by its ability to induce immunogenic tumor cell death and promote dendritic cell (DC) maturation, its reliance on Batf3-expressing DCs to elicit antitumor CD8+ T cell response, and its synergy with anti-PD-1 therapy. Comprehensive immune profiling in the 4T1 breast cancer model, both in orthotopic and metastatic settings, revealed that ADT-030 selectively reduces myeloid-derived suppressor cells (MDSCs) while normalizing the immune landscape within the tumor. Mechanistically, ADT-030 disrupts multiple components of the mitogen-activated protein kinase (MAPK) signaling network in both tumor cells and MDSCs, leading to induction of apoptosis in these populations. These findings highlight the multi-faceted impact of PDE10A inhibition as a therapeutic strategy that not only disrupts tumor-intrinsic oncogenic signaling to inhibit tumor progression but also reshapes the tumor immune microenvironment to unleash antitumor immunity.

immunology↗

bioRxiv2026

Optimal transport fate mapping resolves T cell differentiation dynamics across tissues

Immune responses evolve across time and tissues through coordinated programs of proliferation, differentiation, and migration, yet most single-cell measurements capture only static molecular snapshots. As a result, reconstructing how immune cells transition between alternative fates remains challenging, particularly for CD8 T cells, whose differentiation is highly dynamic and shaped by rapid expansion, contraction, and tissue trafficking. Here, we introduce an optimal transport-based fate mapping framework that reconstructs continuous CD8 T cell trajectories across time and tissues. Applied to longitudinal single-cell RNA-seq data from CD8 T cells responding to acute viral infection in mice, this approach accurately recapitulates population dynamics and resolves coherent effector and memory T cell differentiation trajectories. Extending the model to multiple tissues, we identify and experimentally validate temporally distinct waves of migration into the small intestine that give rise to divergent tissue-resident memory (TO_SCPLOWRMC_SCPLOW) fates, long-lived T cells crucial in immunosurveillance. By integrating optimal transport inference with time-resolved in vivo labeling, we demonstrate that CD52 marks recent tissue entrants and distinguishes them from TO_SCPLOWRMC_SCPLOW precursors. Finally, trajectory-guided analysis of transcription factor regulons reveals both shared and context-specific gene regulatory programs and identifies AP4 as a key regulator of circulating versus tissue-resident specification. These results establish optimal transport as a principled framework for reconstructing immune cell fate dynamics and provide a quantitative map of early events governing antiviral CD8 T cell differentiation across tissues.

bioinformatics↗

bioRxiv2026

Distinct and cooperative roles of host and tumor Osteopontin in colorectal cancer liver metastasis

Osteopontin (OPN) is a secreted phosphoprotein implicated in colorectal cancer liver metastasis (CRCLM), yet the distinct spatial contributions of host-and tumor-derived OPN in driving this disease remain unclear. Using a 2 x 2 genetic knockout mouse model targeting OPN in host and tumor compartments, combined with spatial transcriptomics, we investigated compartment-specific OPN functions in CRCLM. Tumor-derived OPN promotes tumor proliferation through MEK/ERK signaling. Host OPN licenses monocyte-to-macrophage differentiation, while tumor OPN polarizes macrophages towards an M2-like state. Both host and tumor OPN suppress T cells in the tumor microenvironment, whereas loss of host OPN reveals an interferon-driven, anti-tumor niche. Translational studies using OPN-blockade immunotherapy in syngeneic and patient-derived xenograft mouse models reduced tumor burden and enhanced T cell infiltration. Together, these findings redefine the OPN-myeloid paradigm in CRC and nominate OPN as a potential therapeutic target.

immunology↗

bioRxiv2026

A regulatory role of novel long non-coding RNA, BAZ1A-AS1, in vascular smooth muscle cell functions during neointima formation in human saphenous veins

BackgroundNeointimal proliferation (NP) is a major cause of stenosis and occlusion in arteries and veins. Human saphenous veins (HSV), often used for coronary artery bypass grafting, frequently fail in part due to NP. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of vascular smooth muscle cell (VSMC) phenotype, yet lncRNAs governing NP in human veins remain largely uncharacterized. Methods and ResultsUsing an ex vivo model of NP in human SV, we performed bulk RNA sequencing on SV tissues with and without NP. Among differentially expressed lncRNAs, we identified a previously uncharacterized transcript, Bromodomain Adjacent to Zinc Finger A1 antisense 1 (BAZ1A-AS1), and its predicted cis-regulatory partner gene BAZ1A, as markedly upregulated during NP and predominantly enriched in VSMCs. Both transcripts were induced by genotoxic stimuli, with BAZ1A-AS1 showing transient induction preceding sustained BAZ1A upregulation, consistent with a priming role in the DNA damage response. Silencing of either BAZ1A-AS1 or BAZ1A attenuated VSMC proliferation and migration, accompanied by upregulation of contractile markers and suppression of proliferative and inflammatory transcriptional programs. ChIRP-qPCR demonstrated that BAZ1A-AS1 physically interacts with 3' distal exons of BAZ1A at the DNA level while selectively engaging CNN1, CCND1, and IL6 mRNAs, indicating mechanistically distinct chromatin- and RNA-level regulatory functions. Baz1a haploinsufficiency attenuated neointima formation and preserved VSMC contractile identity in a mouse carotid artery ligation model. ConclusionsWe identify BAZ1A-AS1 and BAZ1A as stress-responsive regulators of VSMC phenotype, operating through dual mechanisms of cis-regulatory chromatin interaction and selective mRNA engagement, and demonstrate a novel role for the BAZ1A-AS1/BAZ1A axis in promoting NP.

physiology↗

bioRxiv2026

Radiosynthesis and Evaluation of Novel Cholesterol 24-Hydroxylase Positron Emission Tomography Tracers

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a pivotal enzyme in brain cholesterol metabolism and has emerged as a therapeutic and imaging target in neurodegenerative disorders. Although [18F]Cholestify ([18F]CHL-2205) has shown promise as a positron emission tomography (PET) tracer for imaging of CYP46A1, the impact of cyclopropyl moiety conformation on binding and imaging performance remains unexplored. Here, we report the rational design and preliminary evaluation of novel CYP46A1 PET tracers, in which the left-side cyclopropyl group was modified into bridged, spirocyclic, and fused bicyclic architectures to probe steric and conformational effects. All compounds 9-11 exhibited high CYP46A1 affinity (IC50 = 0.19-0.28 nM). Radiosynthesis of [18F]9-11 was achieved via copper-mediated [18F]fluorination, providing practical non-decay-corrected radiochemical yields of 10-34% with excellent radiochemical purity (>98%). In vitro autoradiography in rat brain sections demonstrated specific and regionally selective binding, comparable to that observed for [18F]CHL-2205. These cyclopropyl-derived scaffolds establish a scaffold-driven strategy for PET tracer development, providing a robust framework for further structure-activity relationship studies and the rational optimization of CYP46A1 PET tracers.

pharmacology and toxicology↗

bioRxiv2026

Efficient transgene-free multiplexed genome editing via viral delivery of an engineered TnpB.

Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform.

plant biology↗

bioRxiv2026

MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein

The global outbreak of SARS-CoV-2 has resulted in a renewed focus on coronaviruses with the potential for cross-species transmission and mutation risks. In particular, swine acute diarrhea syndrome coronavirus (SADS-CoV), potentially originating from intermediate horseshoe bat (Rhinolophus affinis), is a novel coronavirus that causes acute diarrhea, vomiting, and high mortality in suckling piglets. Innate immunity plays a crucial role in defending the host against invading pathogens. Previous studies have identified several host factors that inhibit SADS-CoV replication through innate immunity; however, research on SADS-CoV remains insufficient compared to that on other coronaviruses. In this study, the host factor Moloney leukemia virus 10 protein (MOV10) inhibited SADS-CoV replication by promoting interferon (IFN) production. Mechanistically, MOV10 exerted its antiviral effect primarily through its N-terminal domain, which regulated the TNF receptor-associated factor 3 (TRAF3)-mediated innate immune pathway. Specifically, we reveal that MOV10 enhanced tripartite motif-containing 24 (TRIM24)-mediated K63-linked ubiquitination of TRAF3, thereby promoting IFN production and inhibiting SADS-CoV replication. Furthermore, we identified the strategies by which SADS-CoV evaded innate immunity. The viral N protein inhibited the antiviral effect of MOV10 by disrupting interaction between MOV10 and TRAF3, and by promoting K48-linked polyubiquitination of TRAF3, leading to TRAF3 degradation via the ubiquitin-proteasome pathway. Collectively, our findings reveal the role of MOV10 in antiviral immunity and how SADS-CoV evades host immune defense.

molecular biology↗

bioRxiv2026

Stepwise DNA unwinding gates TnpB genome-editing activity

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

biochemistry↗