Biology subjects

Shi, Q.

Publications and source records attributed to Shi, Q..

At least 19 recordsLinked to original sources

bioRxiv2026

Cell Surface Sialoglycan Engineering Through Exogenous mRNA

All human cells display a dense matrix of structurally diverse glycans that often terminate in monosaccharides belonging to the sialic acid family of sugars. While these sialoglycans are now recognized as critical regulators of human immunity, there is a lack of technologies that provide precise, transient control over their biosynthesis and presentation on a live cell surface. Here, we addressed this unmet need by developing an mRNA-based platform for delivery of transcripts coding for the enzymes that assemble sialoglycans - sialyltransferases - to human cells. We demonstrated that mRNA coding for the 2-6-specific sialyltransferase ST6Gal1 produced active, Golgi-resident enzyme that potentiated levels of 2-6-sialoglycans on the surface of human cell lines. Increased presentation of these sialoglycans was transient and tracked with the degradation kinetics of the delivered mRNA. Importantly, this approach minimally perturbed other classes of glycans and did not broadly alter physiological transcriptional networks beyond those involved in cellular responses to exogenous RNA. Similar on-target effects were observed for the O-linked 2-6-specific sialyltransferase ST6GalNAc4; however, the effects from mRNA coding for the 2-3-specific sialyltransferases ST3Gal1 and ST3Gal4 were more complex. Taken together, this mRNA platform provides a framework for cellular sialoglycoengineering endeavours, where precise and reversible control over glycocalyx composition is desired.

bioengineering↗

bioRxiv2026

IL-21-producing peripheral helper T cells associate with autoimmune bile duct injury in biliary atresia

BackgroundBiliary atresia (BA) is a severe neonatal liver disease characterized by progressive fibrosis and bile duct obliteration. ObjectiveAlthough immune dysregulation is implicated in the pathogenesis of BA, the specific mechanisms driving bile duct injury remain incompletely understood. This study aimed to characterize tertiary lymphoid structures (TLSs) within extrahepatic biliary remnants (EBRs), identify their cellular mediators, and evaluate the therapeutic potential of targeting IL-21 receptor signaling. DesignWe performed integrated bulk RNA sequencing, single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and flow cytometry on clinical samples from BA patients and non-BA cholestatic controls. TLS maturation was assessed by CD23 immunohistochemistry in EBRs from 148 BA patients and correlated with clinical parameters. Anti-IL-21R antibody treatment was evaluated in a rhesus rotavirus-induced BA mouse model, with treatment initiated on day 4 post-infection. ResultsTLSs were identified in BA EBRs with significantly higher prevalence than in matched liver tissues. Mature TLSs containing CD23 germinal centers were associated with elevated serum matrix metalloproteinase-7, more advanced hepatic fibrosis, and localized autoantibody deposition on injured bile ducts. Single-cell profiling revealed expanded CD4+ T peripheral helper (Tph) cells expressing IL-21 and CXCL13 within TLS-containing EBRs. Tph cells were enriched in peripheral blood of BA patients compared to non-BA cholestatic controls (P = 0.0025), and serum IL-21 was significantly elevated (P < 0.0001). Post-infection IL-21R blockade in the mouse model reduced jaundice incidence, improved weight gain, prevented extrahepatic biliary obstruction, and significantly improved long-term survival. ConclusionTLSs in BA extrahepatic biliary remnants harbor expanded Tph cells associated with IL-21-mediated B cell activation and bile duct injury. IL-21R blockade ameliorated disease in a murine BA model, identifying the IL-21/IL-21R axis as a potential therapeutic target warranting further investigation. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSImmune dysregulation contributes to biliary atresia (BA), with documented lymphocyte infiltration and defective B cell tolerance. However, the cellular mechanisms linking local immune activation to bile duct injury are unclear, and the roles of organized lymphoid structures and specific CD4 T cell subsets in orchestrating local humoral responses have not been characterized. What this study addsThis study demonstrates that mature tertiary lymphoid structures in extrahepatic biliary remnants are associated with disease severity markers and localized bile duct injury in BA. We identify T peripheral helper cells as an expanded IL-21-producing CD4 T cell population within these structures, and show that post-infection IL-21 receptor blockade prevents biliary obstruction and improves survival in a murine BA model. How this study might affect research, practice or policyThese findings identify the IL-21/IL-21R signaling axis as a candidate therapeutic target in BA warranting further preclinical and translational investigation. TLS maturation status in biliary remnants and serum autoantibody levels may serve as potential biomarkers of disease severity, meriting prospective evaluation in clinical cohorts.

immunology↗

bioRxiv2026

Integrated multi-omics profiling of gut bacterial extracellular vesicles links cargo composition to host transcriptional responses

Bacterial extracellular vesicles (bEVs) enable gut microbiota to deliver bioactive cargo to host cells, yet the specific bacterial producers have not been systematically identified. Here, we profiled stool-derived bEVs from healthy individuals using metaproteomic profiling, revealing that vesiculation is widespread across gut bacterial phyla. We selected a subset of vesiculating species, showing that bEVs localize to distinct tissues in vivo, including extraintestinal sites such as lung, liver, kidney, and bone, suggesting roles beyond the gastrointestinal tract. We profiled intestinal epithelial cells and macrophages after endocytosing bEVs from various species, uncovering pronounced, cell-type-specific responses. For example, Bacteroides fragilis bEVs promote anti-inflammatory mitochondrial-telomeric regulation, while a set of commensal-derived bEVs contribute to epithelial survival and structural renewal. By combining proteomic, lipidomic, and metabolomic cargo profiling with transcriptional output, we find that specific Bacteroidota-derived protein cargo activates cytoprotective stress defenses while attenuating inflammatory signaling. Together, these findings establish a multi-layered comparative atlas of bEV composition, uptake, and host response, providing a framework for understanding bEV-mediated microbiome-host communication.

microbiology↗

bioRxiv2026

Somatic mutations reveal the ontogeny of human microglia

Microglia are the resident hematopoietic cells of the central nervous system1. In mice, microglia seed the brain during embryogenesis and can be maintained throughout life with minimal input from adult hematopoiesis2-4. The origins of human microglia are less clear, but recent evidence suggests that marrow-derived cells may be able to supplement the human microglial pool in certain individuals5,6. Here, to investigate the ontogeny of human microglia, we develop a method that uses the collection of accumulated somatic mutations which uniquely labels each clone of cells to track the infiltration of marrow-derived cells into the human brain. Applying this method to 20 aged individuals, we find evidence of an influx of marrow-derived cells into the brain in all examined individuals. Single cell analysis, including single cell lineage tracing using mitochondrial DNA variants, demonstrates that these infiltrating cells are nearly identical to microglia and can comprise a large fraction of the microglial pool. Analysis of large-scale sequencing cohorts demonstrates a protective association between most types of clonal hematopoiesis and Alzheimers disease. In sum, this work uncovers a widespread influx of myeloid cells into the healthy human brain which serves to reinforce the pool of human microglia and becomes common with aging.

immunology↗

bioRxiv2026

Towards molecular-based functional classification of fetal bovine serum

Quality and price of fetal bovine serum (FBS) are traditionally determined by geographical origin and parameters listed in the Certificate of Analysis (CoA). Despite its central role in cell culture, selecting suitable FBS batches remains costly and labor-intensive due to substantial batch-to-batch variation. We propose a molecular assessment strategy based on transcriptomic and cytokine profiling of cells cultured in different FBS batches to evaluate performance more reliably. Analysis of differential gene expression in three cell lines - MRC-5, Jurkat, and THP-1 - enables batch grouping and reveals pathway-specific effects, with immune-related pathways showing the most pronounced variability. Although CoA parameters can stratify batches by origin, they do not consistently correlate with cytokine secretion or gene expression across cell lines. These findings demonstrate that geographical origin is an inadequate predictor of functional FBS performance and that molecular profiling provides a more robust and informative assessment.

molecular biology↗

bioRxiv2026

A Long-Context Generative Foundation Model Deciphers RNA Design Principles

Programmable design of RNA sequences with defined functions remains a central challenge in biology. Despite recent advances, existing RNA generative models lack robust controllable design capabilities and are constrained by short context windows, limiting their capacity to model the complex evolutionary manifold of full-length transcripts. Here, we introduce EVA (Evolutionary Versatile Architect), a long-context generative RNA foundation model trained on over 114 million full-length RNA sequences spanning the full breadth of evolutionary diversity. By integrating a Mixture-of-Experts architecture with an 8,192-token context window, EVA learns a unified, evolution-consistent representation of RNA sequence space that captures both global transcript structure and fine-grained functional elements. EVA establishes a unified framework for a broad spectrum of RNA design tasks within a single architecture, ranging from mutational fitness prediction to context-aware sequence engineering. We demonstrate EVAs versatility through the de novo design and targeted optimization of diverse RNA classes, including tRNAs, aptamers, and CRISPR guide RNAs, as well as mRNA and circular RNA therapeutics. In systematic evaluations, EVA achieves state-of-the-art performance across seven of nine established benchmarks, improving structural modeling accuracy by up to an order of magnitude compared to existing approaches. EVA is fully open-source at the following repository: https://github.com/GENTEL-lab/EVA.

molecular biology↗

bioRxiv2026

Hierarchical Anchoring Gating Dictates Specific H4K16 Acetylation by the Human MSL Complex

Histone acetylation is a versatile post-translational modification essential for diverse biological processes. While nearly all histone acetyltransferases (HATs) act promiscuously on multiple lysine residues, the human male-specific lethal (MSL) acetyltransferase complex mediates strictly site-specific acetylation of histone H4 at lysine 16 (H4K16ac), with its underlying mechanism remaining enigmatic. Here, we leveraged chemical protein synthesis to engineer a semisynthetic nucleosome probe that traps the MSL complex in a catalytically engaged state and determined its 3.12 [A] cryo-electron microscopy (cryo-EM) structure, which reveals a hierarchical anchoring-gating mechanism responsible for stringent H4K16 selectivity. The MSL complex adopts a dual-anchoring mode to bind nucleosomal DNA at superhelix location (SHL) 1.5 and the H2A-H2B acidic patch. Three spatially coordinated gating elements (the MSL1 anchor helix, KAT8 gating helix, and KAT8 gating hairpin) then conformationally constrain the H4 N-terminal tail, funneling H4K16 side chain exclusively into the active site while sterically occluding flanking lysines. Targeted mutations disrupting these interfaces impair H4K16ac in vitro and in cells, with concomitant defects in chromatin accessibility and transcription. Beyond the monomeric 1:1 complex, cryo-EM analysis identifies a 2:2 MSL-nucleosome assembly, wherein MSL3 dimerization mediates inter-nucleosomal contacts linked to genome-wide H4K16ac patterning. Collectively, our work delineates a residue-to-domain framework in which a chromatin acetylation writer hardwires specific catalysis into nucleosome engagement and scales this precision to higher-order chromatin architecture, providing a mechanistic foothold for understanding aberrant MSL-encoded chromatin programs in diseases.

biochemistry↗

bioRxiv2026

Anti-IL1RAP Antibodies for Pan-Inhibition of IL-1 Family Cytokine Signaling in Inflammatory Diseases and Oncology

The interleukin-1 (IL-1) superfamily of cytokines--comprising the IL-1, IL-33, and IL-36 subfamilies--orchestrates a vast array of innate and adaptive immune responses. While physiological activation of these pathways is essential for host defense and tissue repair, their chronic dysregulation is pathognomonic of a broad spectrum of autoimmune diseases and malignancies. Current therapeutic modalities largely rely on the neutralization of single cytokines, an approach often limited by the functional redundancy and compensatory signaling loops inherent to the IL-1 superfamily. The Interleukin-1 Receptor Accessory Protein (IL1RAP) serves as an obligatory co-receptor for IL-1, IL-33, and IL-36 subfamilies, functioning as a molecular bottleneck for pro-inflammatory signaling. Herein, we report the development and comprehensive characterization of two humanized monoclonal antibodies, DXP-006 and DXP-106, which target a unique epitope on domain 2 of IL1RAP. Cryo-electron microscopy studies at 3.55 [A] resolution reveal that these antibodies lock the critical c2d2 loop of IL1RAP, sterically occluding its recruitment to IL-1R1, ST2 and IL-36R binary complexes and potently blockade IL-1, IL33, and IL-36 signaling. DXP-006, engineered with a half-life-extended and effector-silenced Fc, exerts profound efficacy in murine models of psoriasis, superior to the clinical benchmark bimekizumab. Conversely, DXP-106, engineered with a defucosylated Fc to enhance antibody-dependent cellular cytotoxicity, exhibits potent antitumor activity in non-small cell lung cancer xenografts. These findings position DXP-006 and DXP-106 as ideal therapeutics for IL-1 family-driven autoimmunity and IL1RAP-positive cancers. One Sentence SummaryAnti-IL1RAP antibodies block multiple IL-1 family cytokines simultaneously, showing superior efficacy in inflammation and cancer models.

immunology↗

bioRxiv2026

Spatial multi-omics unveils the monoclonal origin, neuroendocrine plasticity, and microenvironment niches in combined small cell lung cancer

Combined small-cell lung cancer (cSCLC) is a rare and aggressive subtype of small-cell lung cancer (SCLC) characterized by mixed histology comprising SCLC and non-small cell lung cancer (NSCLC) or large cell neuroendocrine carcinoma (LCNEC) components. Despite its histological heterogeneity and even poorer prognosis than de novo SCLC, cSCLC is clinically managed as pure SCLC, largely due to the lack of molecular insights into its biology, lineage plasticity, and tumor microenvironment (TME). Here, we perform multi-omics profiling, including spatially-resolved whole-exome sequencing (WES), spatial transcriptomics (ST) and single-nucleus RNA sequencing (snRNA-seq), across 19 treatment-naive cSCLC tumors spanning all major histological subtypes. Our analysis reveals that SCLC and NSCLC/LCNEC components share a monoclonal origin, with histological divergence characterized by distinct mutation and copy number alteration patterns. ST and snRNA-seq uncover spatially exclusive or interspersed tumor domains, with distinct TME compositions and immune landscapes. Notably, fibroblast-rich regions enriched for an aggressive fibroblast subtype form boundaries between tumor domains, potentially influencing immune TME and treatment responses. We identify extensive lineage plasticity within cSCLC, including active LUAD-to-SCLC transdifferentiation and SCLC subtype coexistence, suggesting transitional cellular states not captured by traditional diagnostics. Leveraging these insights, we developed the cSCLC Detector, a sensitive mutation-based diagnostic assay that improves the detection of cSCLC in tissue and liquid biopsy samples. Our findings offer critical insights into cSCLC lineage plasticity, cellular evolution, and microenvironmental interactions, underscoring the need for tailored treatment strategies and diagnostic frameworks for this aggressive cancer subtype.

cancer biology↗

bioRxiv2025

Tbx1 heterozygosity in the oligodendrocyte lineage has distinct effects on myelinated axons in the fimbria and on behaviors related to neurodevelopmental disorders in mice.

Constitutive heterozygosity of Tbx1, a T-box transcription factor gene located within the 22q11.2 deletion region, results in behavioral deficits and altered composition of myelinated axons in the fimbria, together with reduced levels of an oligodendrocyte precursor cell (OPC) marker, in mice. However, the cellular origins of these effects and the extent to which axonal changes causally contribute to behavioral impairments remain unclear. We hypothesized that Tbx1 deficiency specifically within the oligodendrocyte lineage contributes to myelin and behavioral phenotypes. To test this hypothesis, we first demonstrated through in vitro siRNA knockdown that Tbx1 regulates both OPCs and mature oligodendrocytes. Subsequently, we assessed the impact of Tbx1 heterozygosity initiated in OPCs on behavioral and myelin phenotypes in male conditional PdgfrCre;Tbx1+/flox mice. These mice exhibited Cre-mediated recombination in Pdgfr-expressing brain regions and in the OPC progeny within the fimbria. At one month of age, the mutants displayed a higher rate of spontaneous alternation at the longest inter-trial interval in the T-maze compared to their wild-type littermates--an effect that was dissipated at two months. No significant phenotypic abnormalities were observed in conditional PdgfrCre;Tbx1+/flox mice regarding neonatal ultrasonic vocalizations, social interaction, novel object approach, anxiety-like behavior (elevated plus maze), or open-field locomotion and thigmotaxis. Electron microscopic analysis revealed a compositional shift in myelinated axons within the fimbria of adult male mutants, characterized by an increased number of myelinated axons in the 300-800 nm diameter range and a decreased number in the [~]1,200 nm and [~]1,400 nm ranges, with myelin thickness remaining unchanged across diameters. These findings indicate that Tbx1 heterozygosity in the oligodendrocyte lineage leads to a selective shift towards smaller myelinated axons in the fimbria and a transiently higher level of capacity for working memory and cognitive flexibility. However, it does not replicate the full spectrum of myelination abnormalities or the broader cognitive and social deficits observed in constitutive Tbx1 heterozygotes, suggesting that Tbx1 deficiency in non-oligodendrocyte lineage cells may lead to altered myelination and neurodevelopmental behavioral impairments.

neuroscience↗

bioRxiv2025

DTCFinder: a bench-to-bits toolkit for label-free, whole-spectrum analysis of disseminated and circulating tumor cells in liquid biopsies

Disseminated (DTCs) and circulating tumor cells (CTCs) are rare but pivotal in understanding cancer metastasis and advancing liquid biopsy-based diagnostics. Traditional detection approaches, hindered by their dependency on epithelial markers or morphological features, impair the accurate identification of varied DTC/CTC phenotypes within liquid biopsies. We describe DTCFinder, a bench-to-bits, label-free method integrating high-throughput single-cell RNA sequencing and machine learning, enabling unbiased detection, in-depth molecular characterization, and precise tissue-of-origin tracing of DTCs/CTCs across various types of liquid biopsies, even with ultra-scarce DTC/CTC presence in peripheral blood and low sequencing depth. DTCFinder offers a robust and versatile toolkit for liquid biopsy-based cancer diagnostics, enhancing our understanding of cancer metastasis.

cancer biology↗

bioRxiv2025

Amaranth: Enhanced Single-Cell Transcript Assembly via Discriminative Modeling of UMI Reads and Internal Reads

MotivationSingle-cell RNA sequencing has transformed transcriptome profiling at cellular resolution, yet accurate reconstruction of full-length transcripts for individual cells remains a central challenge. Emerging scRNA-seq protocols can produce reads that span entire transcripts, enabling isoform-level expression analysis. For example, Smart-seq protocols combine UMI-linked reads that index and stitch together multiple reads from the same molecule, with internal reads filling coverage gaps. We demonstrate that these read types exhibit markedly different biological and statistical properties in strandness, 5/3 coverage bias, and genomic locality. Existing assemblers fail to leverage these distinctions, yielding suboptimal assembly. ResultsWe developed Amaranth, a novel single-cell assembler that discriminatively models UMI and internal reads. Amaranth implements heuristics specifically designed to address the distinct biases of UMI-linked and internal reads, enabling accurate strandness assignment for internal reads, reliable splicing graph refinement, and precise transcript start site determination. We also developed Amaranth-meta, which integrates information across cells to enhance individual cell assemblies. Benchmarked on Smart-seq3 datasets from human HEK293T and mouse fibroblast cells, Amaranth outperformed other state-of-the-art assemblers in assembling individual cells and in meta-assembly. Amaranth advances isoform-level analysis in single-cell transcriptomics, facilitating detailed studies at cellular resolution. Availability and ImplementationAmaranth is implemented in C++ and is freely available at https://github.com/Shao-Group/amaranth under the BSD-3-Clause license. Scripts, documentation, and data for reproducing experiments in this manuscript are available at https://github.com/Shao-Group/amaranth-test.

bioinformatics↗

bioRxiv2025

Spatial Conformation of Pancreatic Cancer Is Associated with Disease Recurrence after Curative-Intent Total Neoadjuvant Therapy

Pancreatic ductal adenocarcinoma (PDAC) frequently recurs after total neoadjuvant therapy (TNT) and curative-intent resection. Traditional histopathologic response assessments demonstrate that major pathologic response (<5-10% viable residual cancer) is associated with favorable outcomes. However, most TNT cases achieve only a minor response. We investigated whether the spatial organization of residual PDAC encodes clinically meaningful biology beyond residual tumor burden. In a retrospective cohort of 203 resected PDAC patients, all with minor pathologic response, H&E whole-slide images were segmented into cancer and stroma to subsequently quantify spatial composition (e.g., patch size/density, edge density) and configuration (e.g., patch complexity/compactness, spatial intermixing) to model treatment-resistant tumor architecture. Non-response was associated with a more fragmented interface-rich ecology, higher edge density and diversity, and reduced homotypic aggregation, independent of conventional clinicopathologic features. This demonstrates a link between emergent tissue architecture of treated PDAC and therapeutic resistance. Further, two multivariable, spatial risk models were independently associated with disease-free survival (DFS): (1) cancer mean shape index and stromal shape-index variability (high-risk median DFS 7.23 vs 11.57 months; adjusted HR 1.75, p=0.007) and (2) mean stromal area with edge density (high- vs low-risk adjusted HR 1.94, p=0.006), outperforming traditional treatment response assessments. Quantifying residual cancer-stroma topology thus yields independent, prognostic signals in post-TNT PDAC and motivates prospective, spatially informed adjuvant strategies and mechanistic studies of edge habitats and mixing as therapeutic vulnerabilities.

cancer biology↗

bioRxiv2025

Evolutionary landscape of oral microbiome over 100,000 years

The human oral microbiome represents an intimate and enduring partnership between host and microbial communities, yet its evolutionary trajectory across deep time remains largely uncovered. Here we reconstruct the global history of the oral microbiome over 102,400 years using a global collection of 1854 oral samples spanning 61 countries. We reveal that major cultural transitions of the Neolithic Revolution, industrialization, and recent medical advances have driven a directional, globally coordinated co-evolution of oral microbial ecology, function, and population structure. Network analysis identifies two universal microbial modules of dental calculus modules: the commensal-enriched DCM1 module and the pathogen-enriched DCM2, both exhibiting progressive ecological polarization. DCM2 surged around 4,000 years ago with agricultural intensification but subsequently declined in modern European populations while persisting at high levels in African and some American groups, a geographic divide mirroring socioeconomic inequities. Species-level analyses demonstrate widespread directional changes, with obligate anaerobes declining as aerotolerant taxa expand, reflecting a carbohydrate-associated functional remodeling toward starch metabolism. Phylogenomic reconstruction reveals contrasting evolutionary trajectories. Pauljensenia mediterranea experienced near-extinction following a severe bottleneck, while Actinomyces israelii underwent a selective sweep with rapid population expansion 1,000 years ago. Critically, the oral resistome has accelerated in the antibiotic era, resulting in [~]33-50-fold surge of highly mobile, clinically relevant antimicrobial resistance genes in the last century, with Streptococcus acting as a central hub of dissemination. Collectively, these results position the oral microbiome as an active participant that has co-evolved with human subsistence, urbanization and medical practice, with direct implications for global oral health, antimicrobial resistance and interventions aimed at restoring resilient microbial states.

microbiology↗

bioRxiv2025

DNA Framework Nanoreactor for Programmable Membrane Fusion

Membrane fusion is a fundamental yet transient process that has long resisted direct structural and kinetic dissection. Here we introduce a DNA framework vesicle (DFV) nanoreactor that transforms this elusive biological phenomenon into a programmable, visualizable, and quantifiable process-a nanoreaction confined in space yet extended in time. By embedding lipid membranes and SNARE proteins within precisely defined DNA apertures, DFVs convert stochastic vesicle collisions into geometry-controlled fusion events with tunable kinetics. Cryo-electron microscopy resolves a complete sequence of six intermediates, revealing how nanoscale confinement reshapes the energetic landscape of bilayer merger. Quantitative fluorescence and nano-flow cytometry further establish a direct link between spatial design and fusion probability. Extending this concept to living cells, DFVs enable controllable membrane fusion and augmentation on VAMP2-expressing membranes, achieving direct cytosolic delivery of functional siRNA via fusion-driven transfer rather than endocytosis. This framework bridges structural precision and functional mimicry, offering a unified platform to reconstruct, quantify, and harness membrane fusion as a programmable process for synthetic biology and nanomedicine.

bioengineering↗

bioRxiv2025

Genetic elements promote retention of extrachromosomal DNA in cancer cells

Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division3-6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

cancer biology↗

bioRxiv2025

Cryoprotectants-assisted plunge freezing of thick brain tissue specimens for targeted physiologically relevant cryo-imaging in situ

In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. As a result, cryo-imaging reports obtained from non-trivial specimens including mammalian brain tissues are scarce and their physiological relevance remains to be determined. Here, we benchmarked plunge freezing with a variety of cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 microns thick and across several brain regions while keeping the tissue functional. By utilizing the knock-in (KI) mouse model with fluorescent astrocytes we have performed targeted cryo-FIB/SEM volume-EM imaging as well as targeted high-resolution cryoET imaging. Prior to cryoET, we have successfully generated lamellae in a semi-automated fashion on both LMIS (liquid metal ion source)- and plasma-based cryo-FIB/SEM instrumentation thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and astrocytes processes and validated the physiological relevance of our outputs based on the morphology of the corresponding cellular and subcellular features. The pipeline utilizes common vitrification setups and can be potentially extended toward alternative tissue specimens. Ultimately, we expect our approach to become an important step towards democratization of physiologically relevant in situ cryo-imaging studies.

biophysics↗

bioRxiv2025

Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar

BackgroundClimate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels. ResultsWe identified 28 KTIs in the haploid genome of Populus x canescens, 21 of them were responsive to herbivory. The greatest induction was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri017G153200 orthologue), a protein mainly localized in the xylem sap. Mechanical wounding and methyl-jasmonate resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE (AOS), whereas exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR/Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400+kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced and their double knock-out lines in increased weight gain of the larvae. In contrast, overexpressing or knock-out lines of KTI_53200 had no effect on larval weight gain compared with controls. ConclusionKTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against herbivores in trees and presents new options to investigate growth-defense theories.

plant biology↗