Biology subjects

Chen, G.

Publications and source records attributed to Chen, G..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Insights into the regulation of VPS13 family bridge-like lipid transfer proteins from the structure of VPS13C.

Bridge-like lipid transfer proteins (BLTPs) play central roles in redistributing lipids from their primary site of synthesis in the endoplasmic reticulum to other organelles. They comprise bridge-domains spanning between organelles at contact sites that allow lipids to transit the cytosol between adjacent membranes. The assembly of BLTPs into complexes with adaptor proteins enables their lipid transfer ability. To address the mechanisms underlying assembly and regulation of BLTP complexes, we used cryo-EM to resolve the structure of one such BLTP, the Parkinsons protein VPS13C, at near-atomic resolution. The structure identifies a lipid-transfer-nonpermissive conformation, where the built-in C-terminal VAB adaptor module blocks the end of the lipid transfer bridge, interfering with lipid delivery. We also identify calmodulin, central to calcium signaling, as a VPS13 partner, suggesting calcium regulation of VPS13 function. Altogether, this structure of intact VPS13C serves as starting point to understand its regulation and, more broadly, that of other BLTPs.

cell biology↗

bioRxiv2025

Dangling Ends of Third Strand and Duplex Drive Nucleic Acid Triplex Stabilization through Bimodal Association

Nucleic acid triplexes are crucial structural motifs in gene regulation and biotechnology, yet the kinetic principles governing their formation remain poorly understood. While a stability hierarchy of RNA*DNA-DNA > DNA*DNA-DNA > RNA*RNA-RNA, with no DNA*RNA-RNA triplex forming, is known, the kinetic roles of terminal residues remain poorly understood. Here, we employ bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy to demonstrate that dangling ends from both the third strand (triplex-forming oligonucleotide, TFO) and the duplex dramatically enhance triplex stability. Kinetic analysis reveals this stabilization is primarily driven by a marked increase in the association rate (k{square}{square}). Crucially, creating a single-base-pair dangling end at either terminus of the duplex enhanced triplex stability more effectively than blunt ends. For example, DNA TFO dTFO5 binding to d(HP5+TA) was enhanced compared to dHP5, and similarly RNA TFO rTFO5 binding to RNA duplex r(HP5+UA) and DNA duplex d(HP5+TA) showed stronger affinity and faster association than to blunt-ended rHP5 and dHP5. Interestingly, removal of a terminal base pair from the blunt-end duplex, generating a TFO dangling end, also enhances binding affinity and association rate. This indicates that both duplex and TFO dangling ends provide critical nucleation platforms, while blunt-ended terminal triples are dynamic and contribute minimally to stability. Thus, our work establishes that optimal triplex formation requires strategic optimization of both TFO and duplex terminal structures through a fundamental kinetic principle (bimodal nucleation). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/686076v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1dc6950org.highwire.dtl.DTLVardef@1861387org.highwire.dtl.DTLVardef@16c24a5org.highwire.dtl.DTLVardef@da0bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDangling ends at both third strand and duplex termini enhance triplex stability by accelerating association C_LIO_LITriplex formation can be nucleated from either end with a bimodal association mechanism C_LIO_LITerminal blunt-end base triples are dynamic and contribute minimally to stability compared to tailored overhangs C_LI

biophysics↗

bioRxiv2025

Synthesis of perfluorooctanoic acid-containing membrane lipids by human pathobionts

Per- and polyfluoroalkyl substances (PFAS) are synthetic fluorinated compounds used widely in industrial and consumer products. They are unusually stable due to carbon-fluorine bonds and resistant to degradation, making them persistent contaminants in water, soil, and biota. PFAS are associated with adverse health effects in humans including cancer and liver disease. The effects of PFAS on human-associated bacteria are largely unexplored, a significant gap in knowledge because these bacteria are exposed to PFAS in vivo at sites including the colon and bladder. One of the best studied PFAS compounds is perfluorooctanoic acid (PFOA), an eight-carbon perfluorinated carboxylic acid whose structure is analogous to a fatty acid. Here, we cultured Enterococcus faecalis, a Gram-positive bacterium, and Pseudomonas aeruginosa, a Gram-negative bacterium, in growth medium supplemented with PFOA and corresponding control conditions and performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate lipid remodeling in response to PFOA exposure. Strikingly, novel fluoroalkyl-containing membrane lipids are synthesized by both of these bacteria, with each species synthesizing unique fluoroalkyl-lipids. Moreover, a high-level daptomycin-resistant strain of E. faecalis produces strikingly high levels of fluoroalkyl-lipids, demonstrating that prior antibiotic exposure and concomitant effects on bacterial evolution can alter bacterial interactions with PFAS. Because bacterial lipids are important immunomodulators in vivo, we propose that PFAS-containing bacterial lipids may be novel mediators of host-microbe-pollutant interactions. Our results also establish a novel mechanism for the bioaccumulation of PFOA and, potentially, for bioremediation of PFOA in biological systems such as the human gastrointestinal tract.

microbiology↗

bioRxiv2025

The Effective Inertia of the Lower Limb During Locomotion

Accurate modelling of inertial properties of human lower limbs is of great interest to many tasks, from gait analysis in biomechanics to motion tracking and control in computer animation. Previous work typically simplified the human musculoskeletal structure as a chain of rigid capsules, with muscle mass lumped with body segments. Such simplifications lead to errors in the inertia matrix, and the error propagates to torque and pose estimates. In this study, we developed a data-driven model to represent the joint-space inertia of the lower-body of a human in motion. The model does not make any assumptions other than that the estimated inertia matrices must be symmetric and positive definite. We show that a joint-space inertia matrix, estimated from synchronized motion and ground force data following foot strikes, reveals inertial coupling, and that estimated inertia matrices are bilaterally symmetric and motion-type dependent. These are properties which a rigid, mass-lumped inertia matrix fails to entail. Moreover, we show that the data-driven model fits to data better than the articulated rigid body inertia model, and that when used for reconstructing lower body kinematics estimated inertia yields more accurate and stable motion.

bioengineering↗

bioRxiv2025

A deep reinforcement learning platform for antibiotic discovery

Antimicrobial resistance (AMR) is projected to cause up to 10 million deaths annually by 2050, underscoring the urgent need for new antibiotics. Here we present ApexAmphion, a deep-learning framework for de novo design of antibiotics that couples a 6.4-billion-parameter protein language model with reinforcement learning. The model is first fine-tuned on curated peptide data to capture antimicrobial sequence regularities, then optimised with proximal policy optimization against a composite reward that combines predictions from a learned minimum inhibitory concentration (MIC) classifier with differentiable physicochemical objectives. In vitro evaluation of 100 designed peptides showed low MIC values (nanomolar range in some cases) for all candidates (100% hit rate). Moreover, 99 our of 100 compounds exhibited broad-spectrum antimicrobial activity against at least two clinically relevant bacteria. The lead molecules killed bacteria primarily by potently targeting the cytoplasmic membrane. By unifying generation, scoring and multi-objective optimization with deep reinforcement learning in a single pipeline, our approach rapidly produces diverse, potent candidates, offering a scalable route to peptide antibiotics and a platform for iterative steering toward potency and developability within hours.

molecular biology↗

bioRxiv2025

Resting-State fMRI and the Risk of Overinterpretation: Noise, Mechanisms, and a Missing Rosetta Stone

Resting-state fMRI has generated influential insights into large-scale brain organization and contributed to clinically relevant applications, largely through correlation-based measures of cross-regional association in BOLD responses. At the same time, interpreting these statistical associations as reflecting underlying neural interactions requires careful consideration of fundamental methodological constraints. Here, we distinguish two fundamental but often conflated limitations. The first is measurement distortion: the fMRI signal is an indirect and heterogeneous measurement of neural activity, arising from neurovascular coupling, physiology, and measurement-related processes, which can introduce systematic and incompletely characterized biases into estimated correlations. The second is causal non-identifiability: even if correlations perfectly reflected neural synchrony, the resulting correlation structure would not uniquely determine the underlying neural interactions. Using causal reasoning, simulations, and analytic arguments, we distinguish these limitations and examine their consequences for interpretation. Although both apply broadly to fMRI, their implications are particularly important in resting-state analyses, where correlation structure is the primary object of inference in the absence of experimental perturbation. We show that measurement-related biases can distort estimated correlations (e.g., attenuating or in-flating associations and affecting group comparisons) and can produce reproducible patterns that do not necessarily reflect underlying neural relationships, highlighting that statistical reliability does not guarantee biological validity. We further show that graph-theoretic, geometric, and other higher-order representations derived from these correlations do not, by themselves, justify mechanistic interpretation. We argue not against the utility of resting-state fMRI, but for greater precision in interpretation. Our conclusions concern the interpretation of correlation-based analyses rather than their methodological utility. Distinguishing measurement distortion from causal non-identifiability clarifies the inferential boundaries separating descriptive association, predictive utility, and mechanistic interpretation.

neuroscience↗

bioRxiv2025

Decoder-FFPE-seq enables sensitive, genome-wide spatial transcriptomics of archival tissues at single-cell resolution

Formalin-fixed paraffin-embedded (FFPE) tissue-compatible spatial transcriptomics (ST) is transforming biology and histopathology, but achieving high sensitivity, genome-wide coverage, single-cell resolution and researcher-accessibility remains challenging. Here, we present a dendrimeric DNA coordinate-barcoding design for FFPE-adapted spatial RNA sequencing (Decoder-FFPE-seq), by integrating RNA-to-DNA probe conversion with blotting-assisted barcoding. Decoder-FFPE-seq provides single-cell resolution with a 2.54-4.25-fold sensitivity increase in mouse brain transcriptomic profiling over similar assays with an available protocol, successfully mapping rare genes and sparse cell types. It robustly resolves tumor microenvironments (TMEs) in clinical archives preserved 6-20 years. With expanded probe libraries, Decoder-FFPE-seq captures long non-coding and viral RNAs, revealing spatial co-expression networks with mRNAs in oral squamous cell carcinomas (OSCCs). In an OSCC immunotherapy cohort, it delineates TME heterogeneity across pathology response groups and tracks spatial cellular dynamics pre- and post-immuno-chemotherapy. Decoder-FFPE-seq establishes a robust, high-resolution framework for sensitive ST profiling in fundamental and translational studies of archival tissues.

genomics↗

bioRxiv2025

Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis

Genetic mutations affecting proteasome function can result in multi-organ diseases, such as Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome. Neurological symptoms associated with CANDLE suggest that proteasomal mutations may impact neuronal development and/or function. We generated cerebral organoids (COs) from CANDLE patient induced pluripotent stem cells (iPSCs), which exhibited impaired neuronal development when compared to COs from healthy control iPSCs. Impaired neuronal maturation in CANDLE COs was correlated with increased polyamines, which were also elevated in CANDLE patient CSF. The proteasome-regulated Ornithine decarboxylase (ODC), a rate limiting enzyme for polyamines, was elevated in CANDLE neurons. Inhibition of ODC reversed polyamine overproduction and repaired neuronal maturation in CANDLE COs, suggesting a potential therapeutic avenue for intervention. These findings demonstrate that dysfunction of the proteasome affects neuronal development through overproduction of polyamines via dysregulation of ODC and offer insight into potential therapeutic strategies for CNS-related proteasomal dysfunction.

neuroscience↗

bioRxiv2025

Neural representation of emotional valence in human amygdala

The amygdala is a core structure for encoding the affective value of external stimuli. Animal studies suggest that positive and negative emotions are separately encoded by distinct neuronal populations within the amygdala; however, this hypothesis has rarely been tested in humans. The current study examined this hypothesis by comparing the distributed emotion encoding model, as proposed in animal studies, with the univariate emotion encoding model using functional magnetic resonance (fMRI) imaging. More specifically, we applied univariate regression, using average amygdala activation to represent global activation level, and multivariate regression, using distributed voxel-level pattern within the amygdala, to predict normative valence of affective images from the IAPS library. In the core amygdala, the multivariate models prediction performance was not better than that of the univariate model, with weight map analysis revealing an overwhelming predominance of voxels selectively responsive to negative stimuli. When the region of interest was expanded to include voxels with lower anatomical probability of belonging to the amygdala as well as voxels from adjacent areas, the multivariate model significantly outperformed the univariate model, with the voxels selectively responsive to positive valence primarily located in regions surrounding the core amygdala. These findings suggest that in the human amygdala, the core region encodes emotional valence primarily through a global activation signal, rather than distributed patterns consisting of separate clusters of positive and negative voxels, and a more distributed valence representation emerges when voxels surrounding the amygdala are taken into consideration.

neuroscience↗

bioRxiv2025

A new dual-affinity peptide nucleic acid for targeting miRNA-21 precursor rescues tumor repressor PTEN expression

MicroRNAs (miRNAs) have multiple functions in cells and are related to many diseases including cancer by regulating posttranscriptional gene expression. The microRNA precursors (pre-miRs), which can be cleaved by Dicer endonuclease to produce mature miRNAs, often have a cleavage site consisting of both double-stranded (ds) and single-stranded (ss) RNA structures. Peptide nucleic acid (PNA) is a kind of analogue of DNA which can hybridize to DNA or RNA through Watson-Crick and Hoogsteen pairing. We previously reported a novel dual-affinity PNA (daPNA) platform that can simultaneously form a duplex and a triplex with a target RNAs ssRNA-dsRNA junction region. The PNA-RNA complex structure is stabilized by forming antisense PNA (asPNA)-ssRNA duplex immediately adjacent to a chemically modified dsRNA-binding PNA (dbPNA)-dsRNA triplex. In this study, we further explored the application of the daPNA platform to target the precursor of miR-21, which is considered an oncogene. We have designed a set of PNAs including asPNAs, dbPNAs, and daPNAs. The nondenaturing polyacrylamide gel electrophoresis (PAGE) and biolayer interferometry (BLI) data reveal that daPNA-21-10 can strongly bind to pre-miR-21 with high specificity. The data show that pre-miR-21 is easily targeted by traditional antimir, asPNA or dbPNA and optimization of the dsRNA-ssRNA junction position is needed for identifying a tightly binding daPNA. Furthermore, daPNA-21-10 not only inhibits the Dicer activity on pre-miR-21 in cell-free assays, but also down-regulates the expression of miR-21, rescuing PTEN protein expression in cells. Taken together, the biofunction and programmability of daPNA make it a promising platform for probing and regulating miRNA biogenesis and many other RNA-involved biological processes. HighlightsO_LISimultaneous recognition of dsRNA and ssRNA regions C_LIO_LIRNA targeting by dividing and conquering through a dsRNA-ssRNA junction construction C_LIO_LISubstrate-specific inhibition of Dicer acting on pre-miR-21 C_LIO_LIDerepressing PTEN expression by inhibiting miR-21 maturation C_LI O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/668835v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@c8af64org.highwire.dtl.DTLVardef@172d2bdorg.highwire.dtl.DTLVardef@1698ea1org.highwire.dtl.DTLVardef@112fa16_HPS_FORMAT_FIGEXP M_FIG C_FIG eToc BlurbLian et al. show that the miR-21 precursor structure containing the Dicer cleavage site can be targeted by a new type of dual-affinity peptide nucleic acids (daPNAs) through the creation of a dsRNA-ssRNA junction, energetically optimized for the simultaneous duplex and triplex formation. Pre-miR-21 structure, which is hardly accessible to traditional antisense strategies and triplex formation alone, can be targeted by a daPNA with high sequence/structure specificity and strong binding affinity. daPNA platform has a great potential in probing many other RNA structures and broad therapeutic applications.

molecular biology↗

bioRxiv2025

Cytokine Regulation of Human Antibody Responses to Influenza Vaccines

Studies show that human vaccine responses widely vary. Here, we analyzed data measuring 66 cytokines from 4 different inactivated influenza vaccine (IIV) cohorts over 5 seasons (N=581) and identified a significant correlation between baseline/day 0 serum IL-18 and IFN-{beta} concentrations and the vaccine-specific antibody response on day 28, suggesting these factors may have an adjuvant-like effect. To investigate this further, we tested the impact of 19 cytokines on the development of anti-influenza antibody response when administered together with the IIV vaccine in human tonsil and spleen organoids. We found that Type I IFNs (IFN-{beta} and others), IL-21, IL-12, IL-10, but not IL-18 or IFN-{gamma}, enhanced the antibody response. The live attenuated influenza vaccine (LAIV) induced a stronger antibody response than the inactivated one in organoids. Adding a single cytokine, IFN-{beta}, to IIV stimulation recapitulated most of the live vaccine-specific cytokine activation program. It increased the antibody response of the inactivated vaccine to that of the LAIV. Two other antibody-boosting cytokines, IL-12 and IL-21, were induced by LAIV but not by Type I IFNs, indicating different cytokines can affect different pathways leading to a robust antibody response. We then generated cytokine mRNA lipid nanoparticles (LNPs) to test the effect of cytokines on IIV response in a mouse model of immunization. We found that IL-21-LNPs augmented the quantity and breadth of the antibody responses, while IFN-{beta} LNPs enhanced durability. These findings identified parallel cytokine pathways regulating human vaccine responses and provide a rationale for using cytokines as adjuvants to mimic the effectiveness of live-attenuated vaccines without the risk of viral replication.

immunology↗

bioRxiv2025

Exogenous Photoreceptor-Specific N-Glycosylated PROM1 Rescues Retinal Degeneration in Patient and Mouse Models

PROM1 is widely expressed across various tissues. However, its pathogenic mutations are exclusively associated with inherited retinal dystrophy (IRD). The mechanisms underlying this retina-specific vulnerability remain poorly understood, and no effective treatment currently exists for PROM1-IRD. Here, we utilized urine cells, hiPSCs, hiPSC-RPE cells, retinal organoids (ROs) and Prom1-/- mice to address these challenges. During photoreceptor development in ROs, PROM1 co-localized with ciliary marker ARL13B and outer segment (OS) marker PRPH2. It exhibited photoreceptor-specific mRNA splicing isoforms and unique N-glycosylation. In IRD patient-specific models with the PROM1 c.619G>T (p.E207X) homozygous mutation, we observed nonsense-mediated mRNA decay and altered splicing, leading to complete loss of PROM1 protein and OS-like structure disruption, faithfully recapitulating PROM1-IRD pathology. To rescue these defects, we engineered a photoreceptor-specific AAV7m8-CRXp-hPROM1, which successfully restored PROM1 expression and OS-like structures in patient-derived ROs. Therapeutic efficacy was further validated in Prom1-/- mice, where subretinal delivery of AAV8-CRXp-hPROM1 led to photoreceptor-specific expression of human PROM1, significantly preserving OS morphology and improving visual function. These findings not only provide the first solid preclinical evidence supporting gene therapy for PROM1-IRD, but also reveal photoreceptor-specific vulnerability to PROM1 mutations, offering a novel conceptual framework for investigating and treating related IRDs.

neuroscience↗

bioRxiv2025

Walking Selectively Modulates Behaviorally Relevant Auditory Responses According to Stride Cycle Phase

Perception in natural environments unfolds while the body is in motion, yet how locomotion shapes cortical computations for fine-grained and dynamic scene analysis remains unclear. Here, we combined over-ground walking with mobile EEG and a transitional click-train paradigm to test how natural locomotion influences auditory processing in humans. Participants listened to two-segment click trains in which a subtle inter-click-interval (ICI) change signaled a pitch shift and performed a task-engaged change-detection task while either sitting or walking; an independent cohort also completed a passive-listening version. Walking elevated behavioral detection thresholds and slowed responses, indicating reduced sensitivity to fine temporal changes. Neurally, locomotion produced a response-component-specific attenuation: activity time-locked to the ICI transition--the change response previously linked to temporally integrative updating of an ongoing auditory object--was strongly reduced, whereas the offset response was largely preserved and onset responses were only modestly affected. Critically, this modulation persisted during passive listening and was selectively amplified near the perceptual threshold during task-engaged detection, revealing an interaction between motor state and cognitive demand. Thus, natural locomotion does not uniformly degrade hearing; instead, it preferentially down-weights a behaviorally critical, temporally demanding stage of auditory processing, offering a framework for understanding how motor-cognitive state shapes auditory scene analysis in the moving brain.

neuroscience↗

bioRxiv2025

Physiological Reconstitution of Microtubule Doublets

The formation of microtubule doublets (MTDs) is a foundational step in cilia biogenesis, yet how B-tubule nucleation is initiated at the molecular level remains elusive. Here, we identify FAP53 as a crucial factor that promotes B-tubule assembly. Molecular dynamics simulations revealed that FAP53 alleviates steric hindrance from the -tubulin C-terminal tail and stabilizes B-tubule docking at the A-tubule surface. In vitro reconstitution demonstrated that recombinant FAP53 is sufficient to drive MTD formation under physiological tubulin conditions. In cultured HeLa cells, co-expression of CFAP53 and CFAP20--an inner junction protein--induced ectopic MTD-like structures in the cytoplasm. Furthermore, a structurally homologous protein in C. elegans, WFAP-53, localized to sensory cilia; however, when overexpressed, it triggered ectopic MTD formation in neuronal dendrites and concomitantly led to sensory cilia disassembly. These findings uncover a conserved mechanism of B-tubule initiation and underscore the necessity for spatially restricted expression of MTD assembly factors during ciliogenesis.

cell biology↗

bioRxiv2025

Representation Transfer via Invariant Input-driven Neural Manifolds for Brain-inspired Computations

Domain adaptation is a core challenge for embodied AI deployed in unpredictable, noisy environments. Conventional deep models degrade under domain shifts and require costly retraining. Inspired by biological brains, we propose a modular framework where each module is a recurrent neural network pretrained via a simple, task-agnostic protocol to learn robust, transferable features. This shapes stable yet flexible representations as invariant input-driven continuous attractor manifolds embedded in high-dimensional latent space, supporting robust transfer and resilience to noise. At deployment, only a lightweight adapter needs training, enabling rapid few-shot adaptation. Evaluated on the DVS Gesture benchmark and a custom RGB rehabilitation dataset, our framework matches or surpasses leading C3D and ViViT models while using ten times fewer parameters and only one training epoch. By unifying biologically inspired attractor dynamics with cortical-like modular composition, our approach offers a practical path toward robust, continual adaptation for real-world embodied AI. TeaserInvariant input-driven attractors enable representation transfer and domain adaptation in brain-inspired AI.

neuroscience↗

bioRxiv2025

Loss of vitamin C biosynthesis protects from a parasitic infection

The ability to synthesize essential molecules is sometimes lost in evolution. A classic example is ascorbate (Vitamin C), which is synthesized in most animals by L-Gulonolactone Oxidase (GULO), an enzyme lost multiple independent times in animal evolution. This event is thought to be evolutionarily neutral, however, GULO-deficient animals including humans need to obtain ascorbate from their diet and are susceptible to ascorbate deficiency and scurvy. We therefore hypothesized that this disadvantage of GULO loss is offset by physiological benefits. Here we show that ascorbate deficiency protects mice from schistosomiasis, a debilitating parasitic disease which afflicts 250 million people. Schistosoma mansoni worms required host ascorbate to produce eggs in vivo. Consequently, ascorbate-deficient mice were protected from schistosomiasis pathologies and transmission. Intermittent ascorbate deficiency protected Gulo-deficient mice from both scurvy and schistosomiasis mortality. The effects of ascorbate on schistosome reproduction were mediated by ascorbate-dependent histone demethylation which promoted vitellocyte development in female schistosomes. We propose that vitamin deficiencies are not always detrimental but can protect animals from pathogens which need to obtain vitamins from their host.

biochemistry↗

bioRxiv2025

A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

A family of prokaryotic antiviral systems collectively known as the cyclic oligonucleotide-based signaling system (CBASS) is found in many bacterial species. Phage defense in CBASS is mediated by the action of a specific effector activated by the cognate cyclic oligonucleotide, often resulting in abortive infection. In the prototypical Type II-A CBASS, phage defense is initiated by the production of cyclic GMP-AMP (cGAMP) by enzymes known as cGAS/DncV-like nucleotidyltransferases (CD-NTases) within the bacterial host, and subsequent activation of the CapV phospholipase by cGAMP leads to cell lysis, thereby limiting phage replication within the host population. Phage proteins that degrade cGAMP, sequester cGAMP, or prevent cGAMP synthesis have been identified in phages to evade CBASS protection. However, some phages are resistant to CBASS protection despite not encoding these proteins, suggesting that additional counter-defense mechanisms exist. To identify new anti-CBASS mechanisms in phages, we exploited bacterial host sensitivity to the antimicrobial compound sulfamethoxazole (SMX) as an indirect read-out for CBASS activity. Bacteria with active CBASS are more sensitive to SMX; therefore, we postulated that any viral proteins produced inside the bacterial host that target CBASS could increase bacterial SMX resistance. Using this screen, we identified that the Gp15 protein from the vibriophage ICP3 increases SMX resistance in Vibrio cholerae El Tor biotype that encodes an active CBASS. Gp15 is an essential virion protein involved in DNA ejection and conserved in many phages. Expression of Gp15 of another coliphage increases SMX resistance in Escherichia coli expressing an active CBASS, and it allows CBASS-sensitive phage to infect the bacterial host with CBASS, demonstrating an important role of Gp15 in phage defense evasion. Unlike other known anti-CBASS proteins, Gp15 does not degrade or sequester cGAMP, and it does not reduce the cellular level of cGAMP. Gp15 also does not interfere with CapV binding to cGAMP. Instead, Gp15 directly inhibits cGAMP-activated CapV serine hydrolase activity in a stoichiometric manner. Together, our study reveals a new mechanism for CBASS antagonism, in which a phage DNA ejection protein moonlights to inhibit a CBASS effector, thereby evading anti-phage defense.

microbiology↗

bioRxiv2025

Biomechanical Phenotyping Reveals Unique Mechanobiological Signatures of Early-Onset Colorectal Cancer

While both incidence and mortality of sporadic average-onset colorectal cancer (AO CRC, above 50 years of age) are in constant decline, sporadic early-onset colorectal cancer (EO CRC, under 50 years of age) is rising rapidly. Yet, the causes behind this rise remain poorly understood. Epidemiological studies indicate that lifestyle and environmental exposures may result in chronic inflammation, which is known to trigger tissue fibrosis. This study tests the hypothesis that fibrotic remodeling and biomechanical stiffening of colorectal tissues represent measurable hallmarks and potential drivers of EO CRC. Using primary human tissues, this work shows that EO CRC is associated with changes in collagen microstructure, increased stiffness and elevated viscosity of primary tumors. Spatial transcriptional profiling and immunostaining reveal pro-fibrotic signatures in stromal cells, alongside enhanced Yes-associated protein (YAP) mechanotransduction and proliferation in epithelial cells of EO CRC tissues. Mechanistically, increasing matrix stiffness in vitro promotes proliferation of epithelial cells in 2D and 3D colorectal cancer models. Together, these findings establish EO CRC as a disease marked by early and widespread biomechanical remodeling, suggesting that a fibrotic and stiffened tissue microenvironment may orchestrate EO CRC tumor initiation.

bioengineering↗