Biology subjects

Chen, C.

Publications and source records attributed to Chen, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Super-Resolved Single Small Extracellular Vesicle Assay enabled by a Plasmonic Nanohole Array

The accurate quantification of biological nanoparticles, such as small extracellular vesicles (sEVs), is fundamentally hindered by a resolution-coincidence trade-off in digital assays. While physical confinement can isolate single particles, conventional optical readouts remain diffraction-limited, causing multi-particle occupancy to be miscounted as single events and thereby restricting the analytical dynamic range. Here, we report a nanoplasmonic platform that overcomes this limit by introducing a geometry-defined interface that uniquely unifies nanoscale compartmentalisation and near-field-assisted super-resolution imaging. Utilising a gold plasmonic nanohole array, the strict geometric periodicity of the lattice simultaneously serves as a template for single-vesicle confinement and a deterministic grid that generates an array of localised surface plasmon resonance near-field hotspots. This position-deterministic illumination pattern imposes known geometric priors on the excitation field, shifting high-spatial-frequency information into the detectable bandwidth to achieve sub-100 nm lateral resolution. This dual-purpose geometric determinism enables high-fidelity digital readout of individual vesicles with significantly fewer sub-images than stochastic, speckle-based metasurface structured illumination microscopy approaches. The assay achieves an analytical limit of detection of 143 sEVs/{micro}L, matching the performance of state-of-the-art single-EV counting technologies. It successfully differentiates distinct sEV subpopulations based on surface biomarker expression, establishing a clear pathway for future clinical liquid biopsy applications. By replacing stochastic loading and illumination with geometric design, this work establishes a robust framework for precise vesicle interrogation with broad implications for emerging translational applications and fundamental biology.

bioengineering↗

bioRxiv2026

A multimodal foundation model linking histopathology and DNA methylation

Hematoxylin and eosin (H&E) slides are routinely available in cancer care, but molecular profiling often requires additional tissue processing and turnaround time. We introduce HistoMethyl, a DNA methylation-aware pathology foundation model that aligns whole-slide histopathology with matched genome-scale methylation beta-value profiles during pre-training while requiring only an H&E slide at inference. We evaluated HistoMethyl across four task groups: gene mutation prediction, morphology-associated classification, overall survival prediction, and direct DNA methylation beta-value recovery. Evaluation spanned TCGA cross-validation, a disease-held-out lower-grade glioma cohort, and external cohort validation in CPTAC glioblastoma and SurGen rectal adenocarcinoma, totaling 14 cancer cohorts and 81 gene mutation tasks. The best-performing configuration improved mean mutation AUROC by 5.03 percentage points. By converting DNA methylation supervision into an H&E-only representation, His-toMethyl could support an early molecular triage layer that helps prioritize cases for confirmatory sequencing, methylation profiling, immunohistochemistry, and molecular tumor board review. These image-only predictions are intended to accelerate downstream molecular testing and tissue allocation while leaving final diagnosis and treatment selection anchored in validated molecular assays.

Cancer Biology↗

bioRxiv2026

Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol {zeta} complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4, LIG4, and NHEJ1, suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.

genetics↗

bioRxiv2026

Nucleoporin1 maintains male germ unit organization and transport in Arabidopsis pollen tubes, likely through shaping nuclear morphology

The male germ unit (MGU) in Arabidopsis pollen is comprised of one vegetative nucleus (VN) and two sperm nuclei (SN). It is evolutionarily specialized to deliver immotile sperm nuclei to an ovule for fertilization. Despite some progress in research on MGU, its organization and transport remain only partially understood. Here, we identified Nucleoporin1/136 as a new player in the structural organization and positioning of MGU in pollen tubes. We and others have previously reported the reduced fertility of nup1-1 plants; however, the mechanism remains unknown. In this work, we further examined the role of NUP1 in fertility using two mutant alleles, nup1-1 and nup1-2-/+. The reciprocal crosses between the nup1 mutants and the Col-0 wild type indicate that the nup1 mutant pollen is defective. To study the effect of a complete NUP1 knockout on pollen, we generated a transgenic line that produces pollen with and without NUP1 expression. This work led to the surprising discovery that the NUP1 protein is inherited from the pollen mother cell to the daughter cell during microgametophyte development. Subsequent in vitro experiments showed that NUP1 is required for pollen germination and pollen tube elongation. Further microscopic studies demonstrated that NUP1 is highly expressed in VN and essential for maintaining nuclear shape and size. We also demonstrated that NUP1 is required for proper MGU organization and transport, likely through maintaining VN morphology. Notably, our finding of nuclear morphology-mediated regulation of MGU may also explain the mechanistic details underlying the defective MGU movement in previously reported mutants such as kaku4, wit, and wip, which have abnormal nuclear morphology.

plant biology↗

bioRxiv2026

Repurposing of the assembly chaperone PAC1-PAC2 defines a specialized proteasome landscape in mature mammalian sperm

Mature mammalian sperm rely on active proteasomes containing the testis-specific subunit 4s for capacitation, acrosomal exocytosis and fertilization, but how these proteasomes are structurally specialized remains unclear. We determine the endogenous proteasome landscape of mature bovine spermatozoa and testis using cryo-electron microscopy. We resolve five sperm proteasome assemblies: PA200-20S, free 20S, a previously unrecognized PA200-20S-PAC1/2 hybrid, and PAC1/2-20S complexes capped at one or both ends. Unexpectedly, the PAC1/2-bound complexes are mature and POMP-free, selectively incorporated for 4s, lack 2, and adopt a PAC1/2 orientation and gate configuration distinct from known assembly intermediates. Together with elevated proteolytic activity, these features indicate that PAC1/2 is repurposed from a transient assembly chaperone into a stable post-assembly regulator in mature sperm. 4s-containing proteasomes assemble through the canonical PAC1-PAC4/POMP pathway yet may follow {beta}2-{beta}3-{beta}1 incorporation ordering; functionally, 4s incorporation remodels the sCP surface and enhances peptidase activities, definining the spermatoproteasomes as a hyperactive 20S variant. PAC1/2-bound proteasomes are absent from testis, where PA200-20S and 26S proteasomes are present, while 26S proteasomes are nearly lost in mature sperm. Our findings define a sperm-specific, ATP-independent proteasome landscape, expand the functional repertoires of 4s and PAC1/2, and provide a structural framework for proteasome specialization in male fertility.

cell biology↗

bioRxiv2026

Trustworthy ML/AI for Aging Clocks: Preventing Systematic Prediction Bias in Biological Age Estimation

Machine learning (ML)- and artificial intelligence (AI)-based aging clocks are increasingly used to quantify physiological and molecular aging from omics and medical imaging data as distinct from chronological age. Here, we characterize a fundamental but underappreciated statistical limitation of commonly used ML/AI regression models for continuous outcomes: systematic prediction bias and its propagation to downstream association estimates. This issue becomes more challenging when the true outcome, biological age, is latent and therefore unobserved during ML/AI model training. We demonstrate that systematic prediction bias can distort and, in some cases, even reverse downstream association analyses that use aging clocks as ML/AI-predicted outcomes to assess their associations with exposures or clinical factors. For example, it can produce spurious associations suggesting that older predicted brain age is linked to better cognitive performance, or that older epigenetic age is associated with better kidney function. To address this problem, we introduce a principled and broadly applicable ML/AI regression framework based on constrained optimization, yielding better calibrated aging-clock estimates and valid downstream inference.

bioinformatics↗

bioRxiv2026

Assessing and Optimizing Low-Frequency Somatic Mutation Detection: A Multi-Platform High-Throughput Sequencing Perspective

The availability of multiple commercial short-read sequencing platforms necessitates systematic cross-platform performance comparisons, particularly for challenging applications such as low-frequency somatic mutation detection. Here, a large-scale targeted sequencing dataset from five Genome in a Bottle (GIAB) human genomic DNA reference standards, HG001 to HG005, alongside Twist Biosciences cfDNA reference standards featuring 1% variant allele frequency (VAF), was generated by six platforms (NovaSeq 6000, NovaSeq X, FASTASeq 300, GenoLab M, SURFSeq 5000, and MGISEQ-T7). To build a realistic benchmark while keeping authentic sequencing backgrounds, we developed PosMix, a simulating tool that generates position-specific VAFs. To overcome the limitations of conventional variant callers (high recall with poor precision for VarScan2, higher precision with lower recall for Strelka2/Mutect2), we developed SomaticXGB, a machine learning-based caller. In this study, SURFSeq 5000 consistently exhibited the lowest error rates and achieved superior accuracy for VAFs as low as 0.5%, outperforming all other sequencing platforms. On the other hand, SomaticXGB attained F1 scores of approximately 0.92 on simulated datasets with VAFs ranging from 0.5% to 1.5% and 0.89 on Twist 1% standards, substantially outperforming conventional methods. This work delivers a valuable rich multi-platform data resource, offering a standardized pipeline for performance benchmarking and a machine learning-based strategy for optimized somatic mutation detection.

bioinformatics↗

bioRxiv2026

Glucocorticoid Programming of Erythrocyte Hypoxic Memory Enables Rapid High-Altitude Acclimatization

BackgroundRapid ascent to high altitude causes acute mountain sickness (AMS) and life-threatening pulmonary/cerebral edema, yet no prophylaxis enables immediate acclimatization. Intermittent hypoxia training (IHT) establishes a "hypoxic memory" that accelerates adaptation, to high altitude, but its cellular and molecular basis remains undefined, precluding effective pharmacological strategies. MethodsA human cohort of 18 sea-level inhibitants was equally divided into two groups, one group received IHT prior to ascent to 3,500 meters, the other group did not. Multi-omics profiling of erythrocytes and plasma, along with isotopic glucose tracing, was employed to examine the metabolic effects of IHT upon high altitude acclimatization. Preclinical studies with genetically engineered mice were used to further define the molecular and metabolic basis of IHT-induced hypoxic memory allowing rapid acclimatization to high altitude. ResultsMetabolomics revealed glucocorticoids as previously unrecognized endogenous erythroid hypoxic memory orchestrators induced by IHT that negatively correlated with AMS severity. Lipidomics and isotopic glucose tracing demonstrated that glucocorticoid signaling via the glucocorticoid receptor (GR) coordinately enhanced glucose metabolism and activated sphingosine kinase-1 (SPHK1)-driven sphingosine-1-phosphate (S1P) synthesis, pre-conditioning erythrocyte oxygen unloading and antioxidant capacity. Glucocorticoid supplementation enhanced erythrocyte SPHK1 activation and oxygen delivery, counteracting multi-tissue hypoxia and pulmonary and renal neutrophil infiltration. Conversely, erythrocyte-specific Sphk1 ablation abolished glucocorticoid-induced S1P production causing severe tissue hypoxia and exaggerated pulmonary neutrophil infiltration. ConclusionsWe establish a new function of glucocorticoids in erythrocyte metabolic plasticity to enhance oxygen delivery as a hypoxic memory mechanism for rapid adaptation to high altitude. This previously unrecognized GR-mediated reprograming of glucose and sphingolipid metabolism offers a transformative precision pharmacologic strategy for high altitude preconditioning, high altitude emergencies and hypoxia-driven diseases.

cell biology↗

bioRxiv2026

Human hindbrain organoids with segment-specific 5-HT neurons for motor modulation

The serotonergic system regulates human mood, reward, and motor control, and its impairment contributes to debilitating psychiatric disorders. Serotonergic neurons (SNs) arise predominantly in the hindbrain and acquire segment-specific identities and functions; however, the lack of human models remains a challenge to understanding the human serotonergic system. Here, we generate human serotonergic-enriched rostral and caudal hindbrain organoids (hsrHOs and hscHOs) that produce segment-specific SNs with regional identities corresponding to rhombomeres r2-r3 and r5-r8, respectively. hsrHOs and hscHOs produce SNs that recapitulate their in vivo counterparts in transcriptomic profiles, function, and ascending or descending projection preferences. Assembly with human neuromusculoskeletal organoids enables modeling of serotonergic modulation of motor control. This system further reveals that deficiency of the schizophrenia risk gene DISC1 disrupts human serotonergic development and impairs motor modulation. Together, our study establishes a human organoid platform for modeling development, circuit function and disease-associated dysfunction of the serotonergic modulatory system.

neuroscience↗

bioRxiv2026

A Novel Hydrogen Polysulfide Donor Alleviates Neuropathic Pain by Enhancing A-Type Potassium Currents via LIMK1-Cofilin Mediated Interaction between Filamin A and Kv4.2

Hydrogen persulfide (H2S2) is an important endogenous signaling molecule, holding significant therapeutic potential across diverse disease models due to its potent antioxidant and redox-regulating properties. Herein, we report the synthesis, characterization, and in vivo evaluation of an esterase responsive H2S2 donor, HPD1. It reduced mechanical and cold allodynia at 14 mg/kg (i.p.) in chronic constriction injury and paclitaxel-induced neuropathic pain models. Moreover, HPD1 exhibited negligible systemic toxicity and behavioral side effects even at 28 mg/kg. Electrophysiological tests showed that HPD1 suppressed PTX-induced hyperexcitability in dorsal root ganglion (DRG) neurons by specifically potentiating A-type potassium currents (IA). Mechanistically, we demonstrate that HPD1 activates LIMK1, which inactivates cofilin and stabilizes F-actin, thereby promoting the interaction between the actin-binding protein Filamin A and Kv4.2. Furthermore, both the HPD1-induced increase in Filamin A-Kv4.2 co-localization and the subsequent restoration of IA density in DRG neurons, as well as the analgesic effect of HPD1 were dampened by pharmacological inhibition of LIMK1 with BMS-5. This work has developed a new generation of H2S2 donors, demonstrated the analgesic efficacy of HPD1, and uncovered the novel LIMK1-cofilin-Filamin A-Kv4.2 dependent mechanism that restores IA, thus providing a reliable therapeutic strategy for neuropathic pain based on H2S2 donors.

pharmacology and toxicology↗

bioRxiv2026

Wheat MYB transcription factor TaMYB83-7B regulates seed dormancy by influencing the balance between abscisic acid and gibberellin

In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor (TaMYB83-7B) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at -907 bp and -1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

molecular biology↗

bioRxiv2026

Calcium-dependent synaptic proteomics reveals EGFR signaling at active synapses

Activity-dependent remodeling of the synaptic proteome is essential for circuit maturation, plasticity, and adaptation. However, these molecular changes are transient and spatially sparse in intact neural circuits, making them difficult to resolve under physiological conditions. To address this challenge, we developed synaptic Cal-ID, a synapse-targeted, calcium-dependent proximity labeling strategy that enables activity-resolved proteomic profiling under native physiological conditions. Using synaptic Cal-ID, we uncover an unexpected mechanism by which synaptic activity locally engages epidermal growth factor receptor (EGFR) signaling. We identify two novel synaptic proteins, Anks1a and Ubash3b, that are rapidly recruited following activity and cooperatively promote postsynaptic enrichment and signaling of EGFR. Disruption of this pathway impairs synaptic maturation, plasticity, and memory. Together, these findings reveal how neuronal activity establishes local signaling competence at active synapses by concentrating the molecular machinery required to engage a broadly acting growth factor pathway.

neuroscience↗

bioRxiv2026

Membrane proteomics of the Drosophila circadian neural network

Circadian behaviors are controlled by dedicated brain pacemaker neurons, whose activity oscillate during the day and the night. The Drosophila brain contains ca. 240 such neurons. Their molecular clock is synchronized, but the phase of their rhythmic neural activity differs dramatically between functional groups. This explains how specific circadian neurons can for example promote morning or evening locomotor activity. To understand in depth how the circadian network functions, we surveyed its membrane proteome in the morning and evening, using in situ protein labeling and mass spectrometry. In addition to detecting known regulators of circadian behavior, we identified novel membrane or membrane-associated proteins present in circadian neurons. Through genetic screens, we found that many of these proteins regulate circadian behavior. In particular, Piezo regulates morning activity specifically under short photoperiod, and its loss compromises the structural plasticity of the clock neurons controlling locomotion at dawn. Our work thus illustrates the power of proteome-guided genetic screens to understand the mechanisms underlying circadian behavior.

neuroscience↗

bioRxiv2026

Diet-dependent sleep modulation by the Drosophila amino acid transporter ANIDRA

Sleep is a conserved animal behavior necessary for survival. It is under tight circadian and homeostatic control, and modulated by diet. Here, we identify the amino acid transporter ANIDRA (ANID) as an important sleep regulator in Drosophila. Flies lacking ANID show decreased and poorly consolidated daytime and nighttime sleep. Contrary to wild-type controls, anid mutant flies are unable to adjust their sleep to their diet, behaving as if they were constantly on a complete diet rich in amino acids. ANID is expressed in ensheathing and cortex glia, where it inhibits mTOR activity in a diet-dependent manner. Moreover, pharmacological inhibition of mTOR attenuates the anid mutant sleep phenotypes. Interestingly, DH44-expressing brain neurons, which promote arousal and sense amino acids, are constantly active in ANIDs absence. We therefore propose that ANID mediates detection of dietary amino acids by ensheathing and cortex glia to regulate the activity of arousal-promoting neurons.

neuroscience↗

bioRxiv2026

Tumor-derived mitochondria enhance CD8+ T cell cytotoxicity through SPHK2-dependent S1P signaling

Intercellular organelle exchange is increasingly recognized as a feature of the tumor microenvironment, but whether tumor-derived mitochondria functionally shape anti-tumor T cell immunity remains unclear. Here we show that tumor-infiltrating CD8+ T cells acquire functional mitochondria from tumor cells through a contact-dependent, TCR-independent mechanism requiring the mitochondrial trafficking machinery Trak1-Miro1. Transferred tumor mitochondria enhanced CD8+ T cell effector activity, increasing cytotoxic molecule expression and tumor-cell killing. Mechanistically, tumor-derived mitochondria carried sphingosine-1-phosphate (S1P), which engaged S1PR1 signaling in recipient T cells. Tumor-specific deletion of Sphk2 diminished mitochondrial transfer-associated T cell activation, impaired CD8+ T cell effector function, and accelerated tumor progression in vivo. These findings reveal tumor-to-T cell mitochondrial transfer as an unexpected immunostimulatory circuit in the TME and identify mitochondrial SPHK2-S1P signaling as a regulator of CD8+ T cell anti-tumor function.

cell biology↗

bioRxiv2026

Development and characterisation of an optimised in vitro differentiation protocol for deriving hepatocyte-like cells from mouse embryonic stem cells

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

cell biology↗

bioRxiv2026

Membrane-anchored influenza neuraminidase vaccine drives human-like broadly protective B cell responses

Influenza neuraminidase (NA) is a promising target for universal flu vaccines, yet eliciting potent B-cell responses against its conserved epitopes remains challenging. Here, we developed a membrane-anchored, folding-domain-free NA (mNA) that elicited superior head-specific germinal center B cell and antibody responses compared to soluble tetrameric NA. In non-human primates, mNA immunization induced cross-reactive memory B cell (MBC) responses, expanding clones with the conserved "DR" motif in HCDR3, a hallmark of human broadly reactive NA antibodies. These MBCs conferred cross-inhibitory activity against diverse NA variants and in vivo cross-protection. Cryo-EM analysis revealed that the 554-C2 clone targets the conserved enzymatic pocket via the "DR" motif, while the 554-C1 clone recognizes previously uncharacterized epitopes at the interface between two adjacent N2 monomers, effectively reducing plaque formation by contemporary H3N2 strains. Our findings highlight the immunological advantages of membrane-anchoring, providing a robust strategy for designing next-generation vaccines against influenza and other pathogens.

immunology↗

bioRxiv2026

A compact Druantia defense clears phage infections via single-stranded DNA recognition and directional duplex unwinding

Bacteria encode diverse anti-phage defense systems triggered by invader-specific molecular cues. Here, we report that the compact type III-A Druantia system recognizes exposed single-stranded DNA to drive phage clearance. Using representative systems from Escherichia coli, we show that the two encoded proteins, DruE and DruH, together clear restriction-sensitive or recombination-prone phages without affecting cell growth or viability. DruE dimerizes and engages DNA at exposed single-stranded regions to unwind DNA with 3'-to-5' directionality, resorting to unique molecular lock, wedge, and clamp elements that aid strand separation and processive translocation. DruH is a monomer in isolation and indirectly interacts with DruE and other host proteins under uninfected conditions, with an infection resulting in the dissociation of the complex. Taken together, our results reveal that exposed single-stranded DNA can trigger bacterial immunity through the directional helicase activity of type III-A Druantia. HighlightsO_LIThe E. coli Druantia III-A defense, comprising DruE and DruH, clears infecting phages C_LIO_LIDruE dimers bind exposed single-stranded DNA and unwind the upstream DNA duplex C_LIO_LI3'-to-5' DNA unwinding is aided by molecular lock, wedge, and clamp elements C_LIO_LIDruE interacts with DruH and host proteins, which are displaced upon infection C_LI

microbiology↗