Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 343 records · Page 19Linked to original sources

bioRxiv2025

Identification of the bona fide active center of influenza A virus polymerase acidic protein as the antiviral target

The influenza virus PA protein, a core subunit of RNA polymerase, is critical for viral replication and a key antiviral target. Contrary to the prevailing view that its endonuclease active site resides in the N-terminal domain (PAn, residues 1-319), we identified a C-terminal truncation (PAc, 320-716) of H1N1 PA that retained high endonuclease activity, while PAn showed minimal activity. Crystal structural analysis revealed that PAc binds substrate RNA via the YDS motif (residues 393-395), with mutations abolishing RNA association. Surprisingly, baloxavir acid (BXA), a known PA inhibitor, specifically targeted PAc rather than PAn, supported by molecular docking showing higher binding affinity to PAc. Mutations at PAc-BXA interaction sites identified F707A as a potential drug resistance mutation. Virtual screening targeting PAc identified methotrexate as potent inhibitors (EC50 < 100 nM), which effectively suppressed viral replication in vitro and alleviated symptoms in influenza-infected mice. Our findings redefine PAs catalytic architecture and establish PAc as a novel platform for antiviral discovery, offering strategies to combat drug resistance through structure-guided inhibitor design.

microbiology↗

bioRxiv2025

Tau deletion results in sex-dependent modulation of synaptic weakening in rat hippocampus

Tangles, a defining characteristic of Alzheimers disease (AD), are composed principally of hyperphosphorylated and misfolded tau species. However, dysregulation of tau precedes tangle formation by many years and is associated with cognitive decline, the best functional correlate of which is synaptic weakening and eventual synaptic loss. Although tau is present at synapses, its normal synaptic function is poorly understood - information vital to the rational development of tau-modifying therapies. Since rats have a greater cognitive repertoire than mice and display more robust signs of tau pathology in AD models, we developed a tau knockout (Mapt-/-) rat to investigate the impact of tau elimination on synaptic function. We observed that long-term depression (LTD), a form of synaptic weakening, is enhanced in the hippocampus of male, but not female, rats. This enhanced LTD was dependent on the synaptic activation of group I metabotropic glutamate receptors and involved altered synaptic actin polymerization. These studies therefore provide mechanistic insights into how tau modulates synaptic function via regulation of the actin cytoskeleton. Our findings are relevant to the understanding of the physiological roles of synaptic tau, the functional consequences of tau-lowering therapies, and the influence of sex in AD susceptibility.

neuroscience↗

bioRxiv2025

A platform for the systematic interrogation of genetic interactions in human cells: 26 gene by genome-wide double knockout screens as a proof of concept

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term "gene by genome-wide" knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca).

genetics↗

bioRxiv2025

Escape from antimicrobial CRISPR-Cas9 in E. coli ST131 depends on the genetic context of the target gene.

Escherichia coli (E. coli) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene blaCTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: blaCTX-M-15-positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a [~]10-5 frequency. Instead, blaCTX-M-15-negative escapers presented chromosomal deletions involving blaCTX-M-15 loss. The frequency of blaCTX-M-15 loss depended on the blaCTX-M-15 genetic context. Specifically, blaCTX-M-15-negative escapers emerged at low frequency ([~]10-5) in isolates where blaCTX-M-15 was located downstream of insertion sequence (IS) ISEcp1, while escapers emerged with high frequency ([~]10-3) in isolates where blaCTX-M-15 was flanked by IS26. This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS26 may drive increased frequencies of escape. IMPORTANCEIn the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene (blaCTX-M-15), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.

microbiology↗

bioRxiv2025

Reversible therapeutic resistance in EGFR mutant lung cancer caused by RB1 loss induced lineage plasticity

Treatment with EGFR tyrosine kinase inhibitors (EGFR TKI) is standard of care for patients with lung cancer initiated by activating mutations in the EGFR gene. While EGFR TKI treatment is effective, virtually all patients progress on therapy. Loss of function mutations in the RB1 tumor suppressor gene are associated with poor EGFR TKI outcomes, but underlying mechanisms remain unclear. Analysis of genetically engineered experimental models and human clinical specimens described here demonstrates RB1 loss has minimal impact on initial cell cycle responses to EGFR TKI but markedly accelerates acquired resistance over time. RB1 loss promotes transcriptional plasticity that enables rare cells to spontaneously assume reversible, resistance-conferring lineage states. These findings highlight the importance of non-genetic adaptations in driving acquired EGFR TKI resistance, advance our understanding of how RB1 impacts cancer, and identify opportunities to improve EGFR TKI treatment outcomes by targeting reversible epigenetic resistance mechanisms.

cancer biology↗

bioRxiv2025

Maternal IBD-related antibodies are associated with early life gut inflammatory status and microbiota composition: insights from cord blood

PurposeAntibodies in peripheral blood are used to aid in the diagnosis of inflammatory bowel disease (IBD), but their presence in neonatal cord blood and potential effects on early life development remain unknown. MethodsWe measured anti-CBir1, ANCA, anti-OmpC, ASCA IgA, and ASCA IgG levels in the cord blood of babies born to 78 mothers with or without IBD. Their association with fecal calprotectin (FC), and microbiota composition, characterized by 16S rRNA sequencing, was assessed throughout pregnancy and during the first 3 years of life using linear mixed-effects models. ResultsAntibodies were detected in cord blood, with significantly higher levels of anti-CBir1 and ASCA IgG in babies born to mothers with Crohns disease (p = 0.002) and higher abundance of ANCA and anti-OmpC in babies of mothers with ulcerative colitis (p = 0.002), compared to controls. ASCA IgG levels positively correlated with babies FC (p = 0.006), while babies microbiota Shannon diversity was negatively associated with ANCA, anti-OmpC, and ASCA IgA levels (p = 0.003, 0.04, and 0.008, respectively). Romboutsia spp., Citrobacter spp., Pseudomonas spp., Clostridiaceae, Clostridia, and Varibaculum spp. were positively correlated with either or both ANCA and anti-OmpC levels (all multiple testing adjusted q < 0.1). ConclusionOur findings suggest that maternal IBD-associated antibodies cross the placenta barrier and may be associated with intestinal inflammation and imbalanced microbiota colonization. Whether these serological profiles negatively influence the priming of the babys immune system or IBD risk later in life remains to be determined.

immunology↗

bioRxiv2025

An evolutionary leap in plant reproduction: From archegonia and motile sperm to embryo sacs and pollen tubes

The transition of plants from water to land marks a pivotal step in their evolutionary history, with the change in reproductive mode being the most fundamental. This change freed plant reproduction from aquatic dependence, fostering terrestrial lifes prosperity. Key to the shift from archegonial to embryo sac reproduction are pollen tube emergence and female gametophyte changes, though their evolutionary trajectories and mechanisms remain unclear. Ginkgo, a basal gymnosperm, occupies a pivotal position in this transition, retaining motile sperm, simplified archegonia, and primitive pollen tubes, making it an ideal model. To study how the transition between these two reproductive modes occurred, we collected transcriptome data from the reproductive organs of Ginkgo and evolutionarily representative species. Our findings reveal the MYB98-CRP-ECS module- critical for angiosperm pollen tube guidance--in Ginkgos mature archegonia, with pollen tubes containing guidance components. While egg cells are transcriptionally and functionally conserved across land plants, changes in the cell fate of other female gametophyte component cells drove the shift from archegonia to embryo sacs. Pollen tubes may originate from fern male gametophyte internalization and modification. These findings clarify the evolutionary path and molecular basis of this major reproductive transition.

plant biology↗

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

SYNPO2L Isoforms Regulate the Action Potential Characteristics and Contractility of Atrial Cardiomyocytes via YAP Signaling to Modulate Atrial Fibrillation Risk

BackgroundSYNPO2L is a component of the sarcomere Z-disk with two distinct isoforms (SYNPO2L_A and SYNPO2L_B) described to regulate actin bundle size. Common and rare variations in SYNPO2L have been implicated in risk for heart failure and atrial fibrillation (AF); however, little is known about the function or pathophysiological mechanism upon disease risk. MethodsThe role of SYNPO2L was explored with human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) models of a rare splicing mutation rs766868752 differentiated into both atrial and ventricular lineages. Electrophysiology was assessed via multi-electrode arrays (MEAs) and contractility was assessed using engineered heart tissues (EHTs). Genetic survival analyses were performed to explore the potential therapeutic role of the SYNPO2L_A isoform in AF risk after myocardial infarction. ResultshiPSC-CMs carrying the splice mutation rs766868752 resulted in preferential expression of the SYNPO2L_B isoform and absence of SYNPO2L_A isoform, and atrial hiPSC-CMs displayed significant differences in action potential durations when unpaced, and spontaneous extra-systolic beats when paced. Contractility of atrial hiPSC-CMs carrying the splice mutation was severely compromised. Electrophysiological differences were normalized and contractility was partially restored by overexpression with AAV:SYNPO2L_A. Absence of SYNPO2L_A in the mutant led to an observed decreased phosphorylation of YAP along with significant downstream transcriptional effects. A direct interaction between SYNPO2L_A and LATS2, a known regulator of YAP phosphorylation was found to occur in hiPSC-CMs. Additionally, a common variant time-to-event analysis may suggest a beneficial effect of the SYNPO2L_A isoform to lower the risk of atrial fibrillation after myocardial infarction. ConclusionsA rare splice mutation conferring isoform predominance of SYNPO2L_B appears to disturb the electrophysiology and contractility of atrial hiPSC-CMs via LATS2 phospho-regulatory effects upon YAP signaling. Supplementation of the SYNPO2L_A isoform can restore many functional deficits and supports a specific gene regulatory role for SYNPO2L in modulating the risk of AF and heart failure.

cell biology↗

bioRxiv2025

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches to demonstrate that CDK11 is a critical regulator of cancer cell survival that is required for RNA splicing and the expression of homologous recombination genes. Inhibition of CDK11 disrupts genome stability, promotes the retention of intronic sequences in mature mRNAs, and induces synthetic lethality with PARP inhibitors. Through integrative analysis of functional genomics datasets, we identify heterozygous deletions of chromosome 1p36 - which encompasses CDK11 and its activating cyclin CCNL2 - as a recurrent and predictive biomarker of sensitivity to CDK11 inhibition. To assess the therapeutic potential of CDK11, we develop MEL-495R, a selective and orally bioavailable CDK11 inhibitor. Additionally, we establish a genetically-engineered mouse model that allows us to differentiate between the on-target and off-target effects of CDK11 inhibitors in vivo. Using this platform, we demonstrate that MEL-495R induces widespread on-target toxicity, revealing a narrow therapeutic index. Together, these findings define CDK11 as a core cancer dependency, uncover a chromosomal deletion that sensitizes tumors to CDK11 inhibition, and provide a generalizable strategy for deconvolving drug efficacy and toxicity in vivo for novel oncology targets.

cancer biology↗

bioRxiv2025

Iterative delivery of mRNA by MEFI

Conventional therapeutic mRNA delivery systems are inherently single-use, limiting expression potency, duration, and penetration across biological barriers. To address this, we engineered the mRNA Exporting and Ferrying Implement (MEFI), a platform that enables iterative cell-to-cell transfer of cargo mRNA, allowing a single mRNA molecule to be utilized multiple times across various cells. MEFI encodes a fusion protein that assembles virus-like particles (VLPs) and encapsulates both itself and cargo mRNA into these VLPs, orchestrating reproduction of VLPs from targeted cells and continuous intercellular spreading. MEFI significantly amplified cargo protein expression in the transfected cell populations ([~]20-fold) and in organs ([~]500-fold in lung) from mice administered systemically. Notably, through intramuscular injection of naked plasmids, MEFI promoted robust ([~]20-fold) and durable ([~]2-month) cargo expression, interorgan mRNA transfer, and successful crossing of the blood-brain barrier. Moreover, modified MEFI demonstrated cell-type-specific targeting, mediating the elimination of antigen-expressing cells. Collectively, MEFI establishes a modular platform for enhancing mRNA delivery, leveraging the concept of iterative delivery of mRNA. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/667827v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@14522c2org.highwire.dtl.DTLVardef@1ecfa7dorg.highwire.dtl.DTLVardef@1314347org.highwire.dtl.DTLVardef@1380cc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Interpretable spatial multi-omics data integration and dimension reduction with SpaMV

Spatial multi-omics technologies have revolutionized our understanding of biological systems by providing spatially resolved molecular profiles from multiple perspectives. Existing spatial multi-omics integration methods often assume that data from different modalities share a common underlying distribution, aiming to project them into a single unified latent space. This assumption, however, can obscure the unique insights offered by each modality, thereby limiting the full potential of multi-omics analyses. To address this limitation, we present the Spatial Multi-View (SpaMV) representation learning algorithm, which captures both the shared information across modalities and the distinct, modality-specific information, enabling a more comprehensive and interpretable representation of spatial multi-omics data. Through extensive evaluation on both simulated and real-world datasets, SpaMV demonstrates superior spatial domain clustering performance and provides users with more interpretable dimension reduction for downstream analysis. Moreover, SpaMV effectively annotates cell types within clusters of a mouse thymus dataset, highlighting its effectiveness in interpretable dimensionality reduction.

bioinformatics↗

bioRxiv2025

Virtual Screening-Guided Discovery of Small Molecule CHI3L1 Inhibitors with Functional Activity in Glioblastoma Spheroids

Chitinase-3-like protein 1 (CHI3L1), a glycoprotein implicated in inflammation, fibrosis, and cancer, has emerged as a potential therapeutic target for glioblastoma (GBM). CHI3L1 contributes to tumor progression and immune evasion by promoting STAT3 signaling and mesenchymal transition. To identify small molecule CHI3L1 inhibitors, a structure-based 3D pharmacophore model was developed and applied to virtually screen over 4.4 million compounds from the Enamine collection. Following multi-tiered filtering, 35 candidates were selected for experimental evaluation. Binding validation via microscale thermophoresis (MST) confirmed dose-dependent CHI3L1 interactions for two compounds, 8 and 39, with dissociation constants (Kd) of 6.8 {micro}M and 22 {micro}M, respectively. These affinities were further supported by surface plasmon resonance (SPR), which yielded Kd values of 5.69 {micro}M for compound 8 and 17.09 {micro}M for compound 39. In 3D GBM spheroid models, compound 8 significantly reduced spheroid viability and attenuated phospho-STAT3 levels, consistent with CHI3L1 pathway disruption. These findings identify two promising scaffolds and support the utility of pharmacophore-guided virtual screening for discovering functionally active ligands targeting CHI3L1 in GBM. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/667816v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@291464org.highwire.dtl.DTLVardef@f9d224org.highwire.dtl.DTLVardef@1535eeaorg.highwire.dtl.DTLVardef@7c596e_HPS_FORMAT_FIGEXP M_FIG Table of Contents artwork C_FIG

pharmacology and toxicology↗

bioRxiv2025

A structure-informed evolutionary model for predicting viral immune escape and evolution

Persistent emergence of viral variants capable of evading host immunity constitutes a significant threat to public health. This antigenic evolution frequently outpaces the development of vaccines and therapeutics, highlighting the necessity of predictive models surveilling the immune escape potential of emerging variants. However, existing models suffer from two key limitations: they inadequately incorporate protein structural information and neglect the importance of distinguishing large-impact mutations from neutral ones given multiple mutations. To address these gaps, we presented KEScape, a deep learning model designed to predict viral immune escape and evolution. KEScape integrates evolutionary context with protein structural information, introduces a novel top-K L2-differential pooling mechanism to prioritize mutations with large functional effects, and incorporates a supervised L2 margin loss to facilitate the L2-distance-based ranking of high-impact mutations. We demonstrated that KEScape significantly outperformed state-of-the-art models on the most comprehensive benchmark to date, comprising eleven deep mutational scanning experiments spanning diverse viruses. Furthermore, KEScape exhibited outstanding performance in the practical applications of identifying immune escape hotspots and variants in emerging lineages and real-time surveillance of lineages associated with WHO-designated variants of SARS-CoV-2. These results show that KEScape is an effective model to predict viral immune escape and evolution. Its capacity for early warning can directly inform public health interventions and guide the development of countermeasures, thereby mitigating the threat of future viral pandemics.

microbiology↗

bioRxiv2025

Caffeic acid promoted deep vein thrombosis resolution in mice by suppressed macrophage M1 polarization through Keap1/Nrf2 pathwayv

BackgroundDeep vein thrombosis (DVT) carries significant health risks, with macrophages playing a key role in thrombus resolution. Caffeic acid (CA) has been shown to inhibit thrombosis, but its role in DVT resolution remains unclear. PurposeThis study aimed to investigates the effects and mechanisms of CA in accelerating DVT resolution. MethodsA stasis-type DVT model was established in mice via inferior vena cava ligation. The effects of CA were analyzed. Thrombus resolution was assessed using histological staining, gelatin zymography, immunoblotting, and immunofluorescence. Macrophage depletion was conducted with chlorophosphate liposomes, and bone marrow-derived macrophages (BMDMs) were used for polarization studies. RNA sequencing identified potential molecular pathways. Nrf2 gene-deficient mice were used to verify the role of Nrf2 in deep vein thrombus resolution. ResultsCaffeic acid reduced thrombus size, enhanced collagenolysis via increased MMP-2 activity, promoted neovascularization, increased macrophage infiltration, and suppressed M1 polarization and inflammation. CA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by inhibiting Keap1, leading to enhanced antioxidant responses in both BMDMs and thrombus tissue. Macrophage depletion negated CAs benefits, confirming the central role macrophages. ConclusionCA promoted early thrombus resolution via macrophage recruitment, M1 polarization suppression, and Nrf2 activation, highlighting its potential as a preventive strategy for DVT.

pharmacology and toxicology↗

bioRxiv2025

Regulatory Plasticity and Metabolic Trade-offs Drive Adaptive Evolution of Alternative Flagellar Configurations in Pseudomonas aeruginosa

Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related Pseudomonas species are hyperflagellated, P. aeruginosa maintains strict monoflagellation through the FleQ-FleN regulatory circuit. Here, we demonstrate that FleN dosage is essential for maintaining monoflagellation and bacterial fitness. Wild-type P. aeruginosa consistently displayed unipolar monoflagellation, while {Delta}fleN mutants developed over two-to-five flagella per cell in uni- or bipolar arrangements. Hyperflagellated {Delta}fleN cells exhibited severe fitness defects including reduced growth rates, attenuated virulence in nematode infection models, and competitive disadvantages in co-culture experiments. Remarkably, {Delta}fleN cells rapidly evolved suppressor mutations in fleQ that partially restored growth and motility without always restoring monoflagellation. Five independent suppressor alleles mapped to critical FleQ functional domains (four in the AAA+ ATPase domain, one in the DNA-binding domain), suggesting reduced protein activity that rebalances the disrupted regulatory circuit. Single-cell motility analysis revealed that suppressor strains exhibit heterogeneous swimming dynamics, with subpopulations achieving wild-type speeds despite carrying multiple flagella. Proteomic analysis demonstrated that hyperflagellation triggers extensive cellular reprogramming beyond flagellar components, affecting metabolic pathways, stress responses, and signaling networks. While hyperflagellated cells suffered complete loss of pathogenicity in animal infection models, environmental selection under viscous conditions could drive wild-type cells to evolve enhanced motility through specific fleN mutations. These findings suggest that bacterial flagellar regulatory circuits function as evolutionary capacitors, normally constraining phenotypic variation but enabling rapid adaptation to alternative motility configurations when environmental pressures exceed the performance limits of standard monotrichous flagellation. SIGNIFICANCE STATEMENTBacterial flagella are extracellular appendages that rotate to propel the cell and enable swimming motility. While some bacteria have multiple flagella, many pathogenic species like Pseudomonas aeruginosa have just one. Surprisingly, mutants of P. aeruginosa with multiple flagella performed worse, i.e., they grew more slowly, were less infectious in laboratory animals, and were outcompeted by wild-type bacteria. Even when some mutant bacteria evolved compensatory changes, they still struggled compared to single-flagellum bacteria. This reveals an important evolutionary trade-off: while multiple flagella might seem advantageous for movement, having just one flagellum allows bacteria to grow faster and cause more severe infections. This plasticity likely explains why P. aeruginosa is so successful both in the environment and as a human pathogen.

microbiology↗

bioRxiv2025

Rpl13a snoRNAs Downregulate Smooth Muscle Cell COX4I2 and Promote Neointimal Hyperplasia

BACKGROUNDReactive oxygen species (ROS) augment the activation of vascular smooth muscle cells (SMCs) and promote neointimal hyperplasia evoked by arterial injury or atherogenesis. We have previously shown that small nucleolar RNAs (snoRNAs) from the Rpl13a locus are key regulators of cellular ROS levels. METHODSUsing mice deficient in the Rpl13a snoRNAs, we tested whether these snoRNAs regulate SMC activation in vitro and in vivo. Carotid endothelial denudation was used to provoke neointimal hyperplasia in wild-type (WT) and snoRNA knockout (snoKO) mice, which lack all four intronically-encoded Rpl13a snoRNAs. Primary SMCs from WT and snoKO mice were used for in vitro functional and proteomic analyses. HEK293T cells with specific snoRNA deletions were used to test for snoRNA-guided 2-O-methylation of mRNA. RESULTSArterial ROS levels, inflammation, and carotid artery neointimal hyperplasia were reduced in snoKO compared with WT mice. In vitro, snoKO SMCs demonstrated lower ROS levels and less migration, proliferation, and inflammatory signaling than WT SMCs. Reduced ROS levels in snoKO SMCs and aortas correlated with upregulation of the mitochondrial protein COX4I2, which is associated with reduced mitochondrial ROS under normoxic conditions. Deleting the snoRNA U32A in human HEK293T cells decreased 2-O-methylation of COX4I2 mRNA and upregulated COX4I2 protein without changing COX4I2 mRNA levels. Silencing Cox4i2 in snoKO SMCs upregulated SMC ROS to WT levels. CONCLUSIONSRpl13a snoRNAs are important drivers of SMC activation and neointimal hyperplasia. Rpl13a snoRNAs augment SMC ROS levels, at least in part, by post-transcriptional downregulation of COX4I2 expression.

molecular biology↗

bioRxiv2025

C1q limits cystoid edema by maintaining basal beta-catenin-dependent signaling and blood-retina barrier function

Macular edema (ME) causes significant vision impairment and occurs in several prevalent retinal diseases including diabetic retinopathy (DR), choroidal neovascularization (CNV), retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterize Tspan12 KODBM (Disrupted Barrier Maintenance) mice, a new mouse model of cystoid edema based on genetically and pharmacologically manipulating beta-catenin-dependent norrin/frizzled4 (FZD4) signaling. We assess BRB function, cystoid edema, ERG, and microglia activation outcomes in an aging study with WT, C1qa KO, Tspan12 KODBM, and Tspan12 KODBM; C1qa KO compound mutant mice. Phenotypic analyses and cell-based experiments indicate that C1QA contributes to maintaining basal beta-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12 KODBM; C1qa compound mutant mice. Activation of beta-catenin-dependent signaling by a FZD4/LRP5 agonist antibody modality achieves complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/frizzled4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides novel insights into the contribution of C1QA to BRB maintenance.

neuroscience↗