Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

High prevalence of KPC-3 in carbapenem-resistant Pseudomonas aeruginosa across multiple clonal lineages in China

Background The epidemiology of Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa is rapidly evolving in China. While blaKPC-2 remains the predominant KPC variant in P. aeruginosa, blaKPC-3 has rarely been documented in this pathogen. This study investigated the molecular epidemiology, resistance and virulence characteristics, and plasmid features of blaKPC-3-producing CRPA isolates collected from a tertiary hospital in eastern China. Methods A total of 65 non-duplicate CRPA isolates collected in 2023 were subjected to whole-genome sequencing. Antimicrobial susceptibility testing, phylogenetic analysis, plasmid characterization, conjugation experiments, and virulence assays were performed. Results Among the 65 CRPA isolates, 37 (56.9%) carried blaKPC-3. These blaKPC-3-positive isolates belonged to four sequence types (STs), including ST1076 (62.2%), ST463 (21.6%), ST646 (10.8%), and ST3393 (5.4%). To our knowledge, this is the first report of blaKPC-3 in P. aeruginosa ST463, ST646 and ST3393. All isolates exhibited extensive drug resistance, and 51.8% were resistant to ceftazidime-avibactam. Phylogenetic analysis indicated that blaKPC-3 dissemination was driven by both clonal expansion and horizontal transmission. Comparative genomic analysis identified three kinds of blaKPC-3 -carrying plasmid. A transferable IncP-2 megaplasmid was widely distributed among ST1076, ST646, and ST3393 isolates, whereas non-transferable IncP-10 plasmids were primarily restricted to ST463. The genetic environments and plasmid backbones of blaKPC-3 were highly conserved and closely related to those of blaKPC-2 and its variants, suggesting evolution from pre-existing blaKPC-2-associated plasmids. Virulence analysis demonstrated marked heterogeneity across lineages. ST463 isolates co-harbored exoU and exoS, exhibited enhanced biofilm formation and pyocyanin production, and caused significantly higher mortality in the G. mellonella infection model, indicating a hypervirulent phenotype. Conclusions The blaKPC-3 is becoming an increasingly important determinant of carbapenem resistance in P. aeruginosa in China. The IncP-2 megaplasmid and IncP-10 plasmid derived blaKPC-3 spread across multiple lineages. Continuous genomic surveillance and enhanced infection control measures are urgently needed to prevent its further prevalence in clinical settings.

microbiology↗

bioRxiv2026

PPAR-γ/PCK1 metabolic pathway modulate synovitis and fibrosis in KOA rats

BackgroundKnee osteoarthritis (KOA) is a prevalent degenerative joint disease in which synovial inflammation and fibrosis are closely linked to pain, stiffness, and functional limitation. Growing evidence suggests that metabolic dysregulation, particularly in lipid metabolism, is involved in KOA pathogenesis, but the underlying mechanisms remain incompletely defined. MethodsSprague Dawley rats underwent bilateral anterior cruciate ligament transection to establish a KOA model; sham-operated rats served as controls. RNA sequencing of synovial tissues was performed to identify differentially expressed genes (DEGs) and enriched pathways, followed by GO/KEGG and GSEA analyses. In vivo, adeno-associated virus vectors were used to overexpress or knock down PPAR-{gamma} and phosphoenolpyruvate carboxykinase 1 (PCK1) via intra-articular injection. Ex vivo, primary rat fibroblast-like synoviocytes (FLSs) were stimulated with IL-1{beta} and transfected with PPAR-{gamma} or PCK1 siRNA/overexpression plasmids. synovitis and fibrosis were evaluated by HE, Masson, and Sirius Red staining, immunofluorescence, ELISA, RT-qPCR, and Western blotting. ResultsRNA-seq revealed 621 up-regulated and 228 down-regulated genes in KOA synovium versus sham, with DEGs significantly enriched in PPAR signaling, adipocytokine, and AMPK pathways. Metabolism-related genes including Fabp5, Plin1, Adipoq, Lep, and Pck1 were up-regulated. GSEA indicated downregulation of PPAR-{gamma} signaling in KOA synovium. In vivo and ex vivo, PPAR-{gamma} expression was reduced in KOA, whereas PCK1, FABP5, and ADIPOQ were increased. PPAR-{gamma} overexpression alleviated synovial inflammation, collagen I deposition, and fibrosis, and suppressed FABP5, ADIPOQ, and PCK1 expression; PPAR-{gamma} knockdown produced the opposite effects. Functional studies showed that PCK1 overexpression aggravated synovial inflammatory cell infiltration and fibrosis, elevated IL-1{beta}, IL-18, and TGF-{beta}, and decreased TIMP1 levels in serum, synovial tissue, and FLSs supernatants, whereas PCK1 silencing reversed these changes. ConclusionsThe PPAR-{gamma}/PCK1 metabolic axis modulates synovitis and fibrosis in KOA. Downregulation of PPAR-{gamma} and consequent upregulation of PCK1 promote synovitis and fibrotic remodeling. These findings identify the PPAR-{gamma}/PCK1 pathway as a potential therapeutic target for KOA.

molecular biology↗

bioRxiv2026

A Time-Resolved Single-Cell Atlas Reveals Infection-Status, Age-, and Sex-Dependent Immune Responses Drive Viral Disease Severity

The majority of mortality during viral infections occurs in older males; however, underlying mechanisms by which age and sex shape antiviral immunity and pathological inflammatory responses remain incompletely understood. Here, we performed time-resolved single-cell RNA sequencing across 16 conditions spanning age, sex, and four stages of influenza infection in mice, generating a high-resolution atlas. Aged mice demonstrate delayed antiviral and inflammatory responses in multiple myeloid cells, impairing viral clearance, which delays recovery. Similarly, endothelial cells from aging mice show prolonged inflammatory and antiviral gene signatures. Altered gene signatures in immune and endothelial cells result in a shift in endothelial-immune interactions in the aged lung. Further, the infection status of the cell is a major driver of transcriptional state, with infected myeloid cells exhibiting broad upregulation of genes, including interferon-stimulated, inflammatory, complement, and oxidative stress-related genes. To assess whether these age-associated transcriptional patterns are conserved in humans, we examined BAL cells obtained from healthy individuals and COVID-19 patients, and found that immune cells from aged COVID-19 patients had elevated antiviral and pro-inflammatory gene expression compared to cells from young patients. Our analyses of sex differences identified that multiple myeloid cell types in aged male mice, but not in young male mice, show persistent inflammatory responses at later stages of infection, a likely mechanism contributing to elevated mortality in older males. These data reveal how infection status of the cell, age, and sex interact to drive persistent inflammation and impaired resolution, providing a foundational resource for designing age- and sex-specific therapeutic strategies.

immunology↗

bioRxiv2026

Heart rate dynamics embed a shared representation of individual brain organization and cognitive function

Idiosyncratic brain functional organization shapes intricate cardiac dynamics through central-peripheral autonomic interactions, yet a comprehensive mapping between multi-scale heart rate dynamics and whole-brain functional architecture remains lacking. Here, combining highly comparative time-series analysis with multi-modal neuroimaging and intracranial electrophysiology, we establish heart rate dynamics as a physiological fingerprint that maps onto whole-brain functional architecture. Partial least squares analysis revealed a generalizable latent axis linking reduced heart rate temporal complexity and elevated micro-scale predictability to heightened resting-state functional connectivity across default mode, salience, and sensorimotor networks. This covariance aligns spatially with serotonergic, noradrenergic, and cholinergic neuromodulatory gradients, persists across physiological confound controls and cross-session validations, derives support from human intracranial electrophysiological recordings, and extends to active cognitive states. Furthermore, brain-covarying heart rate signatures underpin the predictive capacity of heart rate dynamics for individual fluid and crystallized intelligence, demonstrating a shared representational substrate. Our findings demonstrate a robust neurovisceral coupling architecture, establishing well-characterized heart rate dynamics as a scalable, neurobiologically anchored window into human brain functional organization and cognitive traits.

neuroscience↗

bioRxiv2026

Structure-aware deep learning predicts influenza antigenicity and guides vaccine strain recommendation

The continuous accumulation of genetic mutations in influenza A viruses (IAVs) drives antigenic drift, necessitating precise antigenic prediction for optimal vaccine strain selection. While sequence-based methods have advanced antigenic surveillance, they neglect the three-dimensional structural context that fundamentally dictates viral antigenicity. Here, we introduce Vir3D, which leverages ESMFold-derived structural information from amino acid sequences to precisely predict viral antigenicity and guide vaccine strain selection. Across both human H3 and highly pathogenic avian H5 subtypes, Vir3D not only accurately discriminates antigenic variants and infers pairwise antigenic distances, but also mechanistically delineates key structural residues driving viral immune evasion. In a decade-long retrospective analysis, Vir3D-prioritized vaccines consistently achieve broader antigenic coverage of circulating strains than World Health Organization (WHO) recommendations. Crucially, Vir3D successfully predicts that the emerging U.S. dairy cattle H5N1 virus (TX/24) remains antigenically stable relative to clade 2.3.4.4b vaccine strains, and subsequent wet-laboratory validation of hemagglutination inhibition (HI) assays definitively corroborates this finding. Overall, Vir3D establishes a powerful, structure-driven framework for proactive influenza surveillance and pandemic preparedness.

bioinformatics↗

bioRxiv2026

A curated human lactylome and protein language model framework enable accurate prediction and reveal local determinants of lysine lactylation

Lysine lactylation is a dynamic post-translational modification that can alter protein function and has been implicated in diverse physiological and pathological processes. Accurate identification of lactylation sites is therefore important for defining its regulatory landscape and for generating testable hypotheses about lactylation-associated mechanisms. Here, we introduce CLEAR-Lactyl and AttentionKla. CLEAR-Lactyl is a curated benchmark dataset of human lysine lactylation comprising 16,604 positive sites. AttentionKla is a deep learning framework trained on CLEAR-Lactyl that employs a pre-trained protein language model fine-tuned with LoRA; it significantly outperforms existing tools, and the factors contributing to its performance gain have been dissected through comprehensive ablation studies. Its utility in predicting novel lactylation sites and in sequence-directed modulation of lactylation levels has been experimentally validated in cellular assays. Together, CLEAR-Lactyl and AttentionKla provide a powerful platform for lysine lactylation research and offer an extensible framework for the precise modulation of other post-translational modifications.

bioinformatics↗

bioRxiv2026

Time Course and Microcircuit Mechanisms of Primary Motor Cortex Dysfunction in Progressive Parkinsonism

Dysfunction of the primary motor cortex (M1) has long been implicated in the pathophysiology of Parkinsons disease (PD), mostly as a link by which abnormal basal ganglia and thalamic activities are translated into motor symptoms. However, emerging evidence suggests that M1 neurons also exhibit maladaptive changes in advanced parkinsonism. Here, using a progressive mouse model of nigrostriatal neurodegeneration (i.e., the MitoPark mice, MP), we found that M1 neurons develop age- and striatal dopamine-dependent synaptic and cellular adaptations as parkinsonism progresses. The optogenetic, electrophysiological, pharmacological, and CRISPR-mediated genetic studies demonstrated that impaired 5-GABAA receptor-mediated inhibition and excessive activation of NMDA receptors of M1 pyramidal neurons are key microcircu it mechanisms underlying cortical circuit remodeling during progressive striatal dopamine (DA) loss. Furthermore, we found that treatment with L-DOPA at early or late stages of parkinsonism can prevent or rescue, respectively, the synaptic and cellular adaptations in M1. Together, the present study demonstrates the time course and the underlying molecular and microcircuit mechanisms of cortical network dysfunction during the development of parkinsonism.

neuroscience↗

bioRxiv2026

Nicotinamide N-methyltransferase couples inflammation and epigenetic remodelling to hepatic fibrosis

Background & aimsChronic inflammation is a key driver of progression from benign steatosis to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. The molecular mechanisms coupling inflammatory cytokine signaling to altered hepatocyte remodeling remain incompletely understood. Here, we report that nicotinamide N-methyltransferase (NNMT) is induced by specific inflammatory cytokines in hepatocytes and acts as a key hub to connect inflammation with fibrotic liver remodeling. Approach & resultsTranscriptomic profiling of primary human hepatocytes and human hepatoma cell lines identified NNMT as a selective downstream target of IL-1{beta} and IL-6, but not of TNF. Genetic silencing of NNMT markedly attenuated cytokine-induced inflammatory and fibrotic gene expression programs and partially reversed IL-6-mediated sensitization of IL-1{beta} responses. Integration of RNA-seq with ChIP-seq, CUT&Tag, and ATAC-seq revealed that inflammatory cytokines remodel the hepatocyte epigenome through coordinated changes in histone modifications. NNMT overexpression promoted selective remodeling of H3K4me2 chromatin landscapes, accompanied by activation of fibrosis-associated transcriptional programs. Nicotinamide supplementation partially suppressed inflammatory gene expression, supporting a role of NNMT-dependent NAD+ metabolism in this process. Importantly, cytokine-induced NNMT expression and its associated transcriptional program were conserved in primary human hepatocytes and 3D liver microtissues. ConclusionsNNMT functions as an inflammatory cytokine-inducible metabolic- epigenetic integrator that links IL-1{beta} and IL-6 signaling to chromatin remodeling and inflammatory transcriptional reprogramming in hepatocytes. These findings identify NNMT as a key regulator of inflammatory and profibrotic responses during MASLD progression and highlight NNMT as a potential therapeutic target for limiting liver inflammation and fibrosis. Impact and ImplicationsThis study identifies NNMT as a previously unrecognized metabolic-epigenetic integrator that selectively links IL-1{beta} and IL-6 signaling to chromatin remodeling and inflammatory transcriptional reprogramming in hepatocytes. Our findings establish a non-canonical mechanistic framework by which inflammatory cytokines drive profibrotic gene expression during MASLD progression through. By uncovering NNMT as a critical mediator of inflammatory-epigenetic crosstalk, this work broadens our understanding of hepatocyte-intrinsic mechanisms underlying liver fibrosis and highlights NNMT as a promising therapeutic target for preventing the transition from steatosis to progressive MASH and fibrosis. HighlightsO_LINNMT is selectively induced by IL-1{beta} and IL-6 in hepatocytes. C_LIO_LINNMT controls fibrogenic gene expression modules. C_LIO_LIIL-6 amplifies IL-1{beta} responses through an NNMT-dependent mechanism. C_LIO_LINNMT links inflammatory signaling to epigenetic remodeling in MASLD. C_LI

molecular biology↗

bioRxiv2026

Inflammatory suppression and cancer-associated bypass of the b0,+selenium-utilization program in the human intestine

The b0,+ transporter (SLC3A1/SLC7A9) imports cystine and selenocystine at the enterocyte apical membrane and defines a selenium-utilization program in the healthy human intestine (He et al., bioRxiv 2026); how this program is altered in disease is unknown. Using a multi-cohort single-cell framework with donor-level statistics, we find that inflammation and cancer remodel a single epithelial redox-currency axis in opposite directions. In adult Crohns disease, b0,+ co-expression in small-intestinal enterocytes was suppressed (median 4.9% vs 34% positive; P = 0.008) but preserved in paediatric IBD; this loss reflected replacement of b0,+-high mature enterocytes by a dedifferentiated state rather than transcriptional disruption, with the SLC7A9-SELENOP coupling intact. Downstream, GPX4 alone was selectively suppressed while the selenocysteine-incorporation machinery was preserved, priming the epithelium for ferroptosis in a severity-graded manner. Inflammatory cytokines (IFN{gamma}, TNF) suppressed the selenium pole in small-intestinal enteroids, and a network knockout indicated that b0,+ supports the selenoproteome through substrate supply rather than transcriptional control; an independent patient proteome and a Caco-2 polarization model corroborated the axis. Colorectal-cancer colonocytes showed the opposing pole, near-absent b0,+ with induction of the xCT/thiol program (P = 5.6 x 10-5) and a ferroptosis-resistant configuration. Thus b0,+ marks the supply node of a dual-pole selenium-thiol axis: inflammation collapses the selenium pole toward a ferroptosis-prone dedifferentiated state, whereas cancer bypasses it toward an xCT antioxidant pole. The baseline axis state is an exploratory correlate of anti-TNF response and a candidate point of disease-associated vulnerability.

molecular biology↗

bioRxiv2026

Intranucleolar Invasion of Cajal Body Remnants Suppresses Ribosomal Biogenesis in cis and Telomerase Functions in trans

Cellular processes are compartmentalized within immiscible heterotypic condensates, yet the functional consequences of losing their physical segregation remain unclear. Here, we show that genetic inactivation of the RNA chaperone SMN forces the aberrant intermixing of the two most prominent nuclear condensates: nucleolus and Cajal Body (CB). Upon SMN depletion, CB components invade the nucleolus and undergo reduced mobility and solubility consistent with a liquid-to-gel-like hardening transition. The CB-scaffold coilin aberrantly enriches at the nucleolar FC/DFC boundary and occupies rDNA chromatin, thereby locally suppressing rRNA production. Concurrently, this sequestration globally impairs coilin targeting to snRNA/snoRNA loci and limits telomerase access to telomeres, reducing telomeric synthesis. Crucially, genetic coilin depletion alone alleviates this mistargeting and rescues these functional impairments across condensates. Our findings reveal an inter-condensate rheostat model in which the loss of CB-nucleolar immiscibility is directly sensed, communicated, and executed by CB remnants, thereby proportionally coupling the functional outputs of otherwise distinct RNPs essential for splicing, translation, and genomic integrity.

molecular biology↗

bioRxiv2026

The Nematicide Tioxazafen Disrupts Proteasome Function via Cytochrome P450 Bioactivation

Tioxazafen is an effective nematicide whose commercialization was halted because handlers reportedly developed rashes after working with seeds coated with a tioxazafen-laced cocktail. Here, we show that tioxazafen is bioactivated into toxic products by nematode and human cytochrome P450s. Through systematic analyses, we found that bioactivated tioxazafen disrupts proteasome function, leading to the accumulation of the NRF1 transcription factor ortholog SKN-1A in the nematode C. elegans, and a bounce-back transcriptional up-regulation of proteasome components. Genetic upregulation of the C. elegans proteasome supresses tioxazafens lethality, indicating that proteotoxicity is a key contributor to death. A survey of human P450s revealed that skin-expressed CYP1A1 toxifies tioxazafen and may account for the reaction to tioxazafen-coated seeds. We also found that rabbit CYP1A1 fails to bioactivate tioxazafen, which may explain the pre-market failure to detect robust adverse skin reactions. Our work highlights vulnerabilities in pre-market toxicological assays and a provides potential solution to prospectively identifying P450 toxication events. One-Sentence SummaryTioxazafen is bioactivated by cytochrome P450s into a proteasome disruptor.

pharmacology and toxicology↗

bioRxiv2026

Lineage recording reveals hijacked hepatic progenitor states as a common origin of HCC and ICC

HighlightsCERAMIC enables continuous and high-capacity lineage tracing of liver tumor initiation Fatty liver-associated hepatocytes acquire regenerative and premalignant cell states before malignant transformation Lineage reconstruction identifies Hep_Bi-zonal cells as the cellular origin of liver tumor initiation Transcriptional and regulatory programs distinguish tumor-fated hepatocytes from failed-to-transform lineages Peroxisomal metabolism is required for progenitor-state formation and liver tumor initiation Spatial remodeling identifies a macrophage niche associated with tumor-fated hepatocytes Dual ontogenies and functional specialization of lipid-associated macrophages shape the tumor-fated hepatocyte niche Fatty liver disease predisposes to primary liver cancer, yet the lineage routes and niche mechanisms that select rare tumor-fated hepatocytes remain unclear. Here we developed CERAMIC, a high-capacity CRISPR-Cas9 lineage recorder that co-recovers editing scars and transcriptomes from single cells, and applied it to an AKT/NRAS-driven model of MASLD-associated liver tumor initiation. Longitudinal lineage, single-cell and spatial analyses revealed a hierarchical trajectory in which bipotential bi-zonal hepatocytes (Hep_Bi-zonal), rather than pericentral-like hepatocytes (Hep_CVlike), generated regenerative and neoplastic hepatocyte progenitor states that progressed toward both hepatocellular carcinoma and intrahepatic cholangiocarcinoma lineages. Tumor-fated cells preferentially expanded along a remodeled midlobular-periportal axis and depended on ACOX1-mediated peroxisomal beta-oxidation to withstand lipotoxic and oxidative stress. Spatial and lineage analyses further identified a sequential lipid-associated macrophage niche, in which monocyte-derived LAMs engaged tumor-fated hepatocytes through an LGALS9-P4HB axis, and P4HB inhibition suppressed tumor expansion. These findings define liver tumor initiation as a lineage-restricted process licensed by peroxisomal metabolic adaptation and macrophage-derived niche signals.

cancer biology↗

bioRxiv2026

Spatiotemporal biosensor profiling reveals an autonomous mitochondrial NAD+/NMN regulatory network centered on NMNAT3

Nicotinamide adenine dinucleotide (NAD+) and its precursor nicotinamide mononucleotide (NMN) are strictly compartmentalized, yet how individual organelles maintain local metabolic homeostasis remains unresolved. Here, we report FrNADS and FrNMNS1.0, a FRET-based biosensor toolkit that maps NAD+ and NMN dynamics in living cells with subcellular resolution, including the oxidizing lumen of the endoplasmic reticulum. We find that NAD+ recovery in the nucleus following PARP1 activation depends on NAMPT mediated salvage synthesis, while peroxisomes buffer NAD+ via NUDT12 and SLC25A17. In mitochondria, NMNAT3 acts as a NAD+ hydrolase that counterbalances import through SLC25A51; HINT2 functionally enhances this activity. Furthermore, SLC25A48 functions as a critical regulatory node that modulates the compartmental redistribution of the generated NMN. These findings establish a mitochondrial NAD+/NMN regulatory circuit and reveal how organelles independently resolve metabolic stress.

cell biology↗

bioRxiv2026

Muscle March5 restrains an ATF4-GDF15 endocrine stress axis that modulates feeding and body composition

Skeletal muscle can release endocrine stress signals during aging and wasting, but the upstream mechanisms that restrain this response remain incompletely defined. Here we identify March5 as a muscle proteostatic checkpoint that limits ATF4-dependent GDF15 production. March5 expression declined in aged and atrophic muscle, whereas muscle-specific March5 deletion induced ATF4 accumulation, marked GDF15 elevation, reduced food intake and progressive loss of body, muscle and bone mass. Restoration of feeding, GDF15 neutralization or muscle Atf4 deletion substantially attenuated the wasting phenotype. Mechanistically, March5 interacted with ATF4 and promoted its ubiquitination at K92, thereby limiting ATF4 stability and Gdf15 expression. Conversely, muscle March5 gain-of-function or pharmacological attenuation of ATF4 signaling improved feeding, body composition and physical performance in aged mice. These findings define a March5-ATF4-GDF15 endocrine stress axis linking muscle proteostatic control to feeding suppression and systemic body-composition remodeling.

physiology↗

bioRxiv2026

A nitrite-responsive regulatory RNA locus sustains commensal resilience against nitrosative stress

Commensal microbes in the gastrointestinal tract are central to host health, yet they must adapt to frequent perturbations such as intestinal inflammation that challenges microbial homeostasis. A major challenge during inflammation is exposure to host-derived reactive nitrogen species (RNS), which damage macromolecules and impair microbial fitness, but how commensals orchestrate defense against nitrosative stress remains poorly defined. Here, we show that Bacteroides thetaiotaomicron mounts a protective RNS-defense program centered on the hybrid cluster protein Hcp, which is required for fitness under nitrosative stress. We identify a nitrite-responsive SnoA locus (Stress-responsive Nitric Oxide regulator A) that promotes HcpR-dependent hcp expression. In vivo, this pathway promotes commensal resilience in both an antibiotic-perturbed, Nos2-dependent model of intestinal nitrosative stress and during Salmonella-induced gut inflammation. Together, our findings identify a regulatory pathway that enables a dominant gut commensal to withstand host-derived nitrosative stress and persist during intestinal inflammation.

microbiology↗

bioRxiv2026

New genes play a prominent role in evolution of new sperm classes of Drosophila

The widespread and regulated production of infertile parasperm assisting fertilization of eusperm provides a compelling model for elucidating the mechanisms of cellular innovation and altruism. Here we identify two specific parasperm lineages in Drosophila pseudoobscura that are segregated in development from the eusperm lineage by their single-cell transcriptomes. Most X-linked genes, except for species-specific new genes, show testis-specific repressive epigenetic modifications and become downregulated upon meiotic entry, supporting meiotic X chromosome inactivation. Between sperm classes, nearly half of the known meiotic arrest genes and their downstream targets are differentially transcribed, with over 200 genes displaying a gradient of transcription levels correlated with that of tail lengths. New genes are overrepresented throughout the hierarchical regulatory network of sperm divergence and over 60% of them exhibit a new cell-level expression compared to their parental copies. These results together indicate that new genes play a prominent role in the emergence of low-cost parasperm that shield the eusperm until fertilization.

evolutionary biology↗

bioRxiv2026

Kinome-wide CRISPR/Cas9-knockout screening reveals critical protein kinases in vasopressin V2-receptor signaling

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.

systems biology↗

bioRxiv2026

Single-Molecule Imaging Reveals Differential Stability of Alpha-Synuclein Aggregates

-Synuclein (-syn) aggregation is central to Parkinsons disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.

neuroscience↗