Biology subjects

Liu, T.

Publications and source records attributed to Liu, T..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics

Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field for comprehensive characterization of cellular heterogeneity. However, most of the current SCP approaches employ sub-{micro}L to 1 {micro}L processing volume for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to the plate well to compensate water evaporation, which greatly limits their broad accessibility and analytical robustness. Here we report a robust convenient SCP method termed iSOP (improved Surfactant-assisted One-Pot processing) for processing of single cells at the low {micro}L processing volume using the 384-well plate with tight sealing to avoid sample drying loss. This iSOP SCP method was built upon our previously developed SOP method by the replacement of a PCR tube or 96-well plate with the low-volume 384-well plate and systematic optimization of the single-cell processing conditions. After optimization, 3 {micro}L was selected as the processing volume with a mixture of 2 ng trypsin and 2 ng Lys-C enzymes in terms of robustness, detection sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify [~]1,200-1,800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines has demonstrated that iSOP-MS enabled reliable identification of an average of [~]1,700 and [~]2,050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity between the two distinct cell types and within the same cell type. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective quantitative SCP analysis.

biochemistry↗

bioRxiv2025

To Predict is to Design: Unlocking Generative Capabilities in All-Atom Structure Predictors via Geometric Score Distillation

Current protein binder design largely relies on a decoupled paradigm: generating backbones via unconditioned diffusion followed by sequence filling or refilling with inverse folding models. This separation prevents the design process from accessing the holistic validation metrics of structure predictors during generation, wasting rich physical priors. While recent works like BindCraft have successfully inverted AlphaFold2 for protein design1, extending this inversion to state-of-the-art all-atom diffusion predictors (e.g., AlphaFold3, Boltz-2) remains a formidable challenge, particularly for modalities requiring non-standard residues such as cyclic peptides. In this work, we present DREAM (Differentiable Refinement via Energy-Anchored Manifolds), a model-agnostic framework that turns the passive predictive trajectory of diffusion models into an active, lucid design process. DREAM repurposes Boltz-22--a leading open-source all-atom predictor--via Geometric Score Distillation (GSD), a technique enabling explicit gradient-based optimization directly through the frozen diffusion network. Unlike previous methods constrained by standard amino acids, DREAM directly unlocks the models latent chemical vocabulary, allowing gradients to autonomously select the optimal building blocks up to 55 residue types (including D-amino acids and post-translational modifications) to minimize energy. We demonstrate this capability by designing cyclic peptide binders for diverse targets, including PD-L1, B7-H3, and the human -Opioid Receptor (hMOR). Our results suggest that the programmable design of chemically complex modalities is not a distant goal, but a latent capability of current all-atom predictors, waiting to be inverted. Ultimately, to predict is to design.

biophysics↗

bioRxiv2025

A high-throughput methods SNIPR-Nm reveals small RNA methylation ratio

Small RNAs regulate gene expression and genome stability, and their functions are shaped by 3'-terminal 2'-O-methylation (Nm). Although Nm is universal in plants and mainly marks animal piRNAs, recent evidence reveals Nm on mammalian miRNAs, highlighting broader regulatory roles. However, current sequencing underestimates Nm-modified RNAs due to ligation bias, and existing qPCR-based methods cannot simultaneously quantify abundance and modification, leaving Nm dynamics largely unexplored. We developed a unified enzymatic strategy that integrates poly(A) polymerase and a thermostable ligase to measure both small RNA abundance and 3'-end Nm status. Applied to mouse testis, it enables robust quantification of Nm ratios for piRNAs and miRNAs. Our method offers a versatile tool for dissecting small RNA methylation in development and disease.

genetics↗

bioRxiv2025

Identification of SASP-associated biomarkers and regulatory mechanisms in diabetic foot ulcers based on transcriptomics and experimental validation

BackgroundDiabetic foot ulcers (DFU) constitute a major complication arising from diabetes mellitus. Emerging research findings have underscored the pivotal contribution of cellular senescence to the pathophysiological development of wounds exhibiting delayed healing; nevertheless, the molecular regulatory networks governing this process in DFU pathogenesis remain incompletely elucidated MethodsDFU-related transcriptomic datasets were acquired from public databases. Biomarkers were identified through machine learning algorithms combined with expression validation. To evaluate the diagnostic predictive capacity of identified biomarkers for DFU, a nomogram prediction model was developed and validated. Further analyses included Gene Set Enrichment Analysis (GSEA), molecular network regulation, immune infiltration profiling, and drug prediction. Subsequently, the expression profiles of these biomarkers were quantitatively assessed through reverse transcription quantitative PCR (RT-qPCR) methodology. ResultsFour feature genes were identified using machine learning. Among them, CXCR2 and SIGLEC7 passed expression validation and were significantly up-regulated in DFU groups across two datasets (p < 0.05), confirming their potential as biomarkers. A nomogram constructed based on these two biomarkers demonstrated high discriminative accuracy and good calibration (decision curve net benefit > 0, AUC = 0.99). GSEA indicated that CXCR2 and SIGLEC7 were both enriched in the cytokine-cytokine receptor interaction pathway as well as other functionally associated pathways. Molecular regulatory network analysis indicated that both CXCR2 and SIGLEC7 were predicted to be regulated by transcription factors (TFs) such as SOX2. Immune infiltration analysis showed a significant strong positive correlation between CXCR2 and activated mast cells (correlation coefficient (cor) = 0.72, p < 0.001), and between SIGLEC7 and macrophages M0 (cor = 0.71, p < 0.001). Drug prediction identified 12 drugs targeting CXCR2 (e.g., Indoprofen) and one drug targeting SIGLEC7 (N-acetyl-alpha-D-glucosamine). Validation through Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) revealed that CXCR2 and SIGLEC7 expression levels were markedly increased in the DFU group, showing statistically significant differences (p<0.05), which aligned with the bioinformatics analysis outcomes ConclusionThis study identified CXCR2 and SIGLEC7 as SASPRG-related biomarkers in DFU through bioinformatic analysis, providing new theoretical support and a basis for early diagnosis and treatment of DFU.

genomics↗

bioRxiv2025

Graded BMP signals modulate yellow and red color in fishes impacting adult pigment pattern and behavior

Among the most interesting adult traits are those with roles in animal communication. Yet developmental mechanisms by which genes drive cell behaviors in building the final forms of such traits are rarely known. In this context, pigmentation is useful because colors and patterns often provide signals in mate choice, predation avoidance and other behaviors and pigmentation is unusually accessible to observation and manipulation. Here we focus on some of the most prominent signaling colors--red, orange and yellow--and show how BMP signaling at the cellular level allows for a very different kind of signal at the organismal level. Using pearl danio, Danio albolineatus, we find that spatially and temporally graded BMP signals promote development of yellow/orange xanthophores over red erythrophores in the fin of this species and a distantly related minnow, Tanichthys albonubes, and that conserved mechanisms, involving BMP co-receptor Rgmb, regulate differentiation of other pigment cell types in corresponding locations of zebrafish, D. rerio. We further use mutants of D. albolineatus with more red or more yellow cells than wild-type to demonstrate female responsiveness to carotenoid-based color differences between males in shoaling preference assays, and we show the existence of polygenic standing variation for this pigmentary trait. Our findings illustrate a chain of function spanning hierarchical levels and provide a deeper understanding of pigmentary form and function and its evolution. HIGHLIGHTSO_LIFate specification of alternative red or yellow pigment cell types in the fin of a minnow, pearl danio, depends on thresholds and gradients in BMP signaling during fin outgrowth. C_LIO_LIGenetic loss of BMP signaling leads to production of red over yellow carotenoids with resulting "super-red" fish preferred by females in shoaling assays. C_LIO_LIBMP-dependence of pigmentary traits is conserved across species with standing, polygenic variation for fin pattern and color in pearl danio. C_LI

developmental biology↗

bioRxiv2025

3' to 5' Translation of Circular RNAs?

For decades, the 5[->]3 direction of translation has been a central dogma of molecular biology(1). Here we present evidence that eukaryotic circular RNAs (circRNAs) can serve as templates for 3[->]5 backward translation (BT), yielding polypeptides with distinct sequence and structural features not found in canonical proteomes. Mining of more than 6,000 multi-omics datasets identified [~]1 million candidate BT proteins across eukaryotes, including 59,000 high-confidence human BT proteins supported by mass spectrometry. Genetic combinatorial experiments (KO and KI) and cell-free translation of synthetic circRNAs establish BT as a conserved mechanism. Loss-of-function studies of hs.circCAPN15 in human and dm.circROLS in Drosophila underscore the functional importance of BT-derived proteins. Our work challenges the long-standing unidirectional translation paradigm, expands the functional landscape of the genetic code, and reveals a hitherto hidden layer of proteomic complexity with broad implications for biology and therapeutics.

molecular biology↗

bioRxiv2025

RBM15 promotes lung adenocarcinoma progression and palbociclib sensitivity via m6A-mediated STIL activation and downstream cyclin D1/CDK4 signaling

RNA-binding motif protein 15 (RBM15), an N6-methyladenosine (m6A) methyltransferase, plays a crucial role in the progression of lung adenocarcinoma (LUAD); however, the underlying mechanisms remain insufficiently defined. In this study, we investigated the contribution of RBM15-mediated m6A modification to LUAD pathogenesis. Our data showed that RBM15 was highly expressed in LUAD tissues and cell lines. Notably, its expression levels were positively correlated with tumor TNM staging and negatively correlated with differentiation status. The overexpression of RBM15 enhanced cell proliferation, migration, invasion, and cell cycle progression, while simultaneously inhibiting apoptosis. Conversely, RBM15 knockdown reversed these phenotypes and induced G2/M-phase arrest. In vivo experiments further confirmed that RBM15 knockdown led to the suppression of tumor growth. RNA sequencing, MeRIP sequencing, and bioinformatics analyses identified STIL as a gene directly regulated by RBM15 through m6A modification, a finding subsequently validated by luciferase reporter assays. Western blot and rescue experiments demonstrated that RBM15 and STIL cooperatively activate the cyclin D1/CDK4 signaling pathway, thereby promoting LUAD progression. However, this effect was markedly attenuated by CDK4/6 inhibitor application. Collectively, these findings indicated that the RBM15-STIL-cyclin D1/CDK4 axis represents a promising therapeutic target for LUAD.

cancer biology↗

bioRxiv2025

BrainBridge Characterizes Key Factors affecting Alzheimer's Disease and Associated Phenotypes

Single-cell RNA sequencing (scRNA-seq) has significantly advanced our understanding of Alzheimers disease and aging by revealing cellular heterogeneity and shifts in cell-type composition between diseased/old and healthy/young individuals. However, few existing studies utilize the rich information in single-cell transcriptomic atlases for robust patient-level modeling and biological feature selection. To address this gap, we present BrainBridge, a deep learning-based framework designed to integrate atlas-scale single-cell transcriptomic data with phenotypic information to model the biomolecular complexity of the human brain. BrainBridge functions both as a powerful predictor and an embedding model for representing sample-level expression profiles and covariates through comprehensive benchmarking. We also demonstrate its effectiveness in prioritizing key genes and cell types associated with disease progression, aging, and sex differences. We further validate our findings using external resources, including genome-wide and epigenome-wide association studies (GWAS and EWAS), spatial transcriptomics, and perturb-seq experiments. Finally, we deploy BrainBridge within an interactive, agent-powered interface that enables intuitive and user-friendly model interactions, promoting broader accessibility and application in biomedical research.

bioinformatics↗

bioRxiv2025

PIWIL4 regulates gene expression and piRNA levels in RSV-infected airway epithelial cells

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections with significant morbidity and mortality in young children, the elderly and immunocompromised hosts. Despite its clinical burden, no effective RSV vaccine or therapy exists for infants, only prophylactic treatment. Small non-coding RNAs have emerged as important regulators of host-pathogen interactions. PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs known for maintaining the genome complexity and integrity in gonadal cells. However, there is growing evidence of their role in controlling gene expression in somatic cells. The biogenesis and function of piRNAs is associated with P-element Induced Wimpy testis (Piwi) proteins, whose function in the respiratory epithelium in response to infections remains largely unexplored. Here, we characterize the expression and function of the Piwi-like protein PIWIL4 in the context of RSV infection. We found that PIWIL4 is expressed in both primary and immortalized small airway epithelial cells and is significantly induced at the mRNA and protein levels following RSV infection or poly I:C stimulation, a proxy of viral infection. Immunofluorescence microscopy revealed that PIWIL4 was primarily nuclear in uninfected cells but translocated to the cytoplasm upon RSV exposure. While siRNA-mediated knockdown of PIWIL4 did not significantly affect RSV replication, it led to decreased secretion of several cytokines, chemokines and growth factors, indicating a role in modulating host innate immune responses. Transcriptomic analysis of PIWIL4-silenced iSAE cells showed significant changes in gene expression both in basal conditions and upon RSV infection. Ingenuity Pathway analysis of differentially expressed genes underscored the role of PIWIL4 in modulation of interferon signaling, cytokine production, stress and metabolic responses, as well as airway remodeling pathways. Silencing of PIWIL4 also resulted in global alteration of piRNA expression both in uninfected and infected cells. However, the predicted targets of the differentially expressed piRNAs had limited overlap with the differentially expressed genes identified by transcriptomics, suggesting a function of PIWIL4 in regulating airway epithelial cell responses at least in part independent of piRNAs. Taken together, our study uncovers an important role for PIWIL4 in somatic cells and position it as a key regulator of airway epithelial innate immunity. A better understanding of the mechanisms by which PIWIL4 affects host cells responses following a pathogen exposure may identify novel therapeutic strategies for RSV, as well as other viral respiratory infections.

microbiology↗

bioRxiv2025

Structural basis of Retron-Eco8-mediated anti-phage defense

Retrons represent a novel class of bacterial defense systems that employ reverse transcriptase (RT), non-coding RNA, and effector proteins to counteract phage infections. In this study, we elucidate the molecular mechanism of a retron system, Retron-Eco8. Biochemical experiments reveal that the Retron-Eco8 holocomplex, rather than the effector alone, exhibits double-stranded DNA cleavage activity in an ATP-independent manner, triggering abortive infection and effectively halting phage propagation. Cryo-electron microscopy (cryo-EM) analysis reveals a supramolecular assembly comprising four RT subunits, four msDNA (multicopy single-stranded DNA) molecules, and four OLD (overcoming lysogenization defect) nucleases--a configuration critical for anti-phage defense. Structural comparisons between apo and ATP-Mg2+-bound states demonstrate a local conformational change. Notably, we identify the phage SSB (single-stranded DNA-binding) protein as an activator of Retron-Eco8, and phylogenetic analysis of SSB proteins further elucidates the phage resistance specificity. Collectively, our findings delineate the structural architecture of the Retron-Eco8 defense complex and provide mechanistic insights into retron-mediated bacterial immunity.

biophysics↗

bioRxiv2025

Longitudinal analysis of influenza A virus deletion-containing viral genomes reveals key determinants of co-evolutionary dynamics and interference

The substantial genetic diversity generated during influenza A virus replication facilitates both evasion of pre-existing host immunity and cross-species emergence. A major contributor to this diversity is the ubiquitous production of deletion-containing viral genomes (DelVGs) - viral RNAs with large internal deletions that arise during replication. DelVGs directly compete with wild-type (WT) genomes, and their accumulation has been implicated in modulating disease severity. However, the specific functional and genetic interactions between DelVGs and WT genomes remain poorly understood. To examine how DelVGs and WT genomes may co-evolve in mixed populations, we serially passaged influenza A virus under sustained high multiplicity-of-infection (MOI) conditions and used next-generation sequencing to build a longitudinal profile of DelVG emergence and dynamics. Early passages yielded a highly diverse repertoire of DelVGs across multiple segments, followed by a sharp contraction in overall diversity and the rise of one to two PB2-derived DelVGs that persisted at sustained high frequency. We identified a single-nucleotide substitution within PB2-derived DelVGs that significantly enhanced their replication and interference, whereas the same mutation proved lethal in the WT background. Collectively, these findings indicate that DelVGs are not mere byproducts of replication but dynamic genomic elements shaped by selection; their capacity to acquire adaptive mutations and outcompete WT genomes underscores their active role in shaping within-population viral dynamics.

microbiology↗

bioRxiv2025

The roles of Transposable Elements and Gene Family Dynamics in Shaping Diversity and Evolution in Diatoms

Diatoms are key players in aquatic ecosystems, having evolved through secondary endosymbiosis. Using long-read sequencing, we investigated how transposable elements (TEs) and gene family dynamics have shaped diatom diversification from inter-lineage to intra-species scales. Across diatom lineages, we identified ecological adaptation-linked expansions, including polyamine synthesis genes for silicification and glutathione S-transferases for oxidative stress resistance. Centric diatoms showed lineage-specific expansion of flotation-associated microtubule genes, while pennate diatoms expanded motility-related actin and myosin genes. At the intra-species level, distinct Phaeodactylum tricornutum strains revealed genomic adaptations correlated with their unique features, including strain-specific expansion and contraction in the cruciform strains morphological genes and the Baltic Sea isolates amine metabolism genes. Our estimates of major lineage divergence times in diatoms ([~]202 Ma and [~]173 Ma) were highly consistent with the two deep WGD events ([~]200 Ma and [~]170 Ma). At these evolutionary nodes, gene families showed extensive lineage-specific expansions and contractions, likely linking ancient polyploidy to subsequent gene content evolution. Substantial TE expansions occurred more recently (0.5-5 Ma), with most diatoms showing recent bursts of Long Terminal Repeat Retrotransposons (LTR-RTs) and araphid pennate diatoms displaying more ancient TE insertion peaks. This likely reflects the progressive loss of ancient TE copies, leaving only recent TE insertions detectable. Our findings provide genomic evidence for the adaptive evolution of diatoms, highlighting the crucial roles of TEs and gene family dynamics in shaping their morphological diversity and environmental adaptations, and suggesting a potential connection between WGDs, gene family dynamics, and TE insertions in genome evolution.

genomics↗

bioRxiv2025

Single-cell RNA sequencing and large-scale bulk combination with machine learning reveal gastric cancer-related macrophage heterogeneity

BackgroundThe tumor microenvironment (TME) significantly impacts cancer progression and overall patient survival. However, the complexity of tumor cell-TME interactions in gastric cancer (GC) and their underlying molecular basis remain to be systematically elucidated. MethodsWe performed single-cell RNA sequencing (scRNA-seq) on paired tumor and adjacent tissues from 3 treatment-naive GC patients. We conducted an in-depth characterization of the cellular composition and molecular features of the GC TME, with a particular focus on tumor-associated macrophages (TAMs) subsets and their mediated intercellular communication. Investigating the effect of C1q on the polarisation of THP-1 induced macrophages through migration and invasion experiments ResultsTranscriptomic analysis of 56,151 single cells identified a key TAM subset - C1QA{square} TAMs. This subset is characterized by high expression of genes including C1QA, C1QB, C1QC and FN1, exhibiting a distinct M2-like macrophage phenotype. We developed a LASSO-based predictive model, C1Q-LASSO, to accurately stratify patients based on survival outcomes and chemotherapy responses, independently of established prognostic parameters. In vitro functional experiments further confirmed that C1q directly promotes malignant phenotypes in GC cells. Additionally, C1q significantly enhances the malignant progression of gastric cancer induced by M2 macrophages. ConclusionsUtilizing scRNA-seq technology, this study systematically delineated the heterogeneous landscape of the GC immune microenvironment and its complex cellular interaction network at single-cell resolution. We first identified and functionally validated the C1QA{square} TAM subset, demonstrating its association with poor prognosis and pro-tumorigenic functions.

cancer biology↗

bioRxiv2025

Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

Cancer cells evolve to increase fitness and evade the immune system, but it is not clear if tumor infiltrating lymphocytes (TILs) undergo selection for somatic mutations that augment anti-cancer immunity. Using single molecule whole exome sequencing in TILs, we identified somatic mutations in Tyrosine-protein kinase 2 (TYK2), several of which increased TYK2 phosphorylation, JAK-STAT signaling, and interferon gamma signaling. Among these mutations, TYK2D810V was recurrently observed in patients with diverse cancer types. In vitro, TYK2D810V enhanced T cells effector functions and cytokine production. We generated mice with a germline Tyk2D807V mutation that recapitulated the human TYK2D810Vmutation. These mice were healthy and did not develop autoimmune disorders. However, they possessed enhanced anti-tumor effects in the context of syngeneic and autochthonous cancer models. Adoptive therapy with Tyk2D807V CD8 T cells also decreased cancer growth. Thus, naturally occurring mutations in non-malignant lymphocytes can antagonize cancer and inspire new immunotherapies strategies.

cancer biology↗

bioRxiv2025

Intravenous Administration of Serotonergic Psychedelics Produce Short-lasting Changes in Sleep-Wake Behavior and High Gamma Functional Connectivity in Rats

Background and PurposeGiven the increase in recreational psychedelic use and ongoing efforts to explore psychedelics as therapeutic agents for mental health disorders, there is an urgent need to understand the effect of psychedelics such as psilocybin and N,N-dimethyltryptamine (DMT) on sleep-wake states, which share a bidirectional relationship with mental health. Here, we investigated the effects of intravenous psilocybin and DMT on sleep-wake states and EEG spectral power and functional connectivity in rats. Experimental ApproachSprague Dawley rats (n=25, 13 male) were surgically instrumented to record high-density EEG (27 electrodes) and EMG during 12-h light and 12-h dark cycle after intravenous psilocybin (2.5 mg/kg, 10 mg/kg), DMT (3.75 mg/kg, 7.5 mg/kg) or 0.9% saline. The EEG/EMG data were scored in 4-second epochs into wake, slow-wave sleep (SWS), and rapid eye movement (REM) sleep. EEG spectral power and corticocortical coherence, a surrogate for functional connectivity, were computed in 12-second epochs. Key ResultsPsilocybin and DMT delayed the onset of SWS and REM sleep, and caused a short-lasting increase in wakefulness and decrease in SWS. Psilocybin also produced a 1) decrease in REM sleep, 2) decrease in theta power and coherence and increase in high gamma power and coherence during wake and SWS, and 3) increase in high gamma coherence during REM sleep. DMT increased gamma coherence only during wakefulness. Conclusions and ImplicationsSerotonergic psychedelics have minimal effects on sleep-wake states. The enhanced high gamma functional connectivity suggests that the psychedelic-induced changes in EEG/neural dynamics can occur independent of the arousal states.

neuroscience↗

bioRxiv2025

Arhgef18 is a component of the outer limiting membrane required for retinal homeostasis

Biallelic ARHGEF18 mutations cause human adult-onset retinal degeneration. We now find that Arhgef18 associates with the retinal outer limiting membrane (OLM), an adherens junction between Muller glia and photoreceptors. Arhgef18 knockout in Muller glial cells led to OLM disruption and vision loss by P60. While mice developed morphologically normal retinas, retinal rosettes started to form by P8, and retinas then progressively degenerated with OLM disintegration, retinal thinning, and vascular leakage. ARHGEF18/p114RhoGEF depletion in Muller cells in culture confirmed disruption of junctional recruitment of OLM proteins. Depletion also induced activation of NF-{kappa}B and {beta}-catenin signalling, activation of the multifunctional kinase Tank-binding kinase 1 (TBK1) and reduced mitochondrial activity. TBK1 inhibition or directly supporting mitochondrial activity with nicotinamide attenuated NF-{kappa}B and {beta}-catenin signalling and rescued mitochondrial activity. Thus, Arhgef18 is essential for OLM maintenance, and its disruption leads to activation of mechanisms that are targetable for possible therapeutic approaches.

cell biology↗

bioRxiv2025

Elucidating the potential carcinogenic molecular mechanisms of parabens in head and neck squamous cell carcinoma through network toxicology and molecular docking

ObjectiveThis study aims to systematically investigate the molecular mechanisms through which parabens may contribute to head and neck squamous cell carcinoma (HNSCC) carcinogenesis using integrated network toxicology and molecular docking. Materials and MethodsSix commonly used parabens (ethyl-, propyl-, methyl-, heptyl-, butyl-, and benzylparaben) were selected for toxicity prediction via ProTox 3.0 and ADMETlab2.0. Their potential targets were retrieved from Swiss Target Prediction, ChEMBL, and the Similarity Ensemble Approach (SEA). HNSCC-related targets were collected from GeneCards, OMIM, and CTD, while differentially expressed genes (DEGs) were identified using TCGA-HNSC data. Functional enrichment, protein-protein interaction (PPI) network construction, hub gene identification, molecular docking, and immune infiltration analyses were performed using DAVID, STRING, Cytoscape, CB-DOCK2, and TIMER2.0. ResultsWe identified 80 common targets through which parabens may exert toxic effects in HNSCC. Among these, 12 hub genes (CCNB1, CDK1, CCNA2, CDK2, CDK4, TYMS, AURKA, CCNA1, CHEK1, CCNB2, PLK1, CDC25A) were significantly overexpressed in HNSCC tissues and were primarily enriched in cell cycle regulation, p53 signaling, and viral carcinogenesis pathways. CCNA1 was further validated as an independent prognostic factor associated with poor survival. Molecular docking revealed strong binding affinities between parabens and hub proteins. Immune infiltration analysis indicated a negative correlation between CCNA1 expression and CD8+ T cell and B cell infiltration. ConclusionsParabens may promote HNSCC progression by disrupting cell cycle regulation and immune responses via direct interactions with key hub genes. These findings provide a novel mechanistic basis for the carcinogenic potential of parabens in HNSCC and underscore the need for further experimental and epidemiological validation.

pharmacology and toxicology↗

bioRxiv2025

Hepatitis E ORF2 blocks trophoblast autophagy to induce miscarriage via LC3B binding rather than PI3K/Akt/mTOR pathway suppression

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection.. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR show that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we proposed that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage.

pathology↗