Biology subjects

Ai, Y.

Publications and source records attributed to Ai, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

PFOS aggravates atherosclerosis via Bacteroides caecimuris expansion-driven bile acid remodeling and subsequent intestinal FXR-TLR3 signaling cascade

BACKGROUNDPerfluorooctane sulfonate (PFOS) is a widely distributed persistent organic pollutant in the environment and has been associated with an increased risk of atherosclerosis. However, the underlying pathogenic mechanisms remain largely unclear. This study aimed to investigate the effects of PFOS on atherosclerosis and its associated gut-vascular axis. METHODSPseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to determine the role of the gut microbiota in PFOS-induced atherosclerosis. Metagenomic sequencing was performed to characterize alterations in gut microbial composition following PFOS exposure, and targeted metabolomics was used to assess bile acid profiles in the ileum and plasma. Transcriptomic analysis of Bacteroides caecimuris (B.caecimuris) was conducted to explore the reasons for the increased abundance of B.caecimuris after PFOS exposure. In addition, intestinal transcriptomics and ChIP-qPCR were performed to validate transcriptional regulation within the FXR-TLR3 signaling axis. RESULTSAmong 127 participants with paired serum and fecal samples, including 82 patients undergoing coronary angiography with Gensini scores (GS score), fecal PFOS levels were significantly associated with lipid profiles and GS score, whereas serum PFOS showed no such association.Mechanistically, PFOS exposure promotes intestinal enrichment of B.caecimuris by upregulating its tolC gene, thereby enhancing efflux capacity.This microbial shift was accompanied by reduced levels of tauro-ursodeoxycholic acid (TUDCA) and aberrant activation of intestinal FXR signaling.Further analyses demonstrated that FXR activation upregulated TLR3 expression and promoted inflammatory responses and atherosclerosis progression via the TLR3-NF-{kappa}B signaling axis. Both intestinal epithelial-specific FXR deficiency (Fxr{Delta}IE) and TUDCA supplementation significantly suppressed pathway activation and alleviated disease phenotypes.Functional experiments identified TLR3 as a key downstream effector of FXR. Overexpression of TLR3 abolished the protective effects observed in Fxr{Delta}IE mice. Moreover, pharmacological inhibition of TLR3 using CU CPT-4a significantly improved established atherosclerotic lesions in vivo. CONCLUSIONSThis study identifies a gut microbiota-driven FXR-TLR3 signaling axis that mediates PFOS-induced atherosclerosis. These findings provide new mechanistic insights into environmentally induced cardiovascular disease and suggest potential targets for risk assessment and therapeutic intervention.

pathology↗

bioRxiv2026

Species-Specific Roles of RIPK1 and TRADD in TNF-Induced Cell Death Reveal a Translational Gap Between Mouse Models and Human Biology

Mouse models have historically been central to studies of TNF-induced cell death and guided pharmaceutical translation into clinic, based on the assumption that TNF signaling is conserved between human and mouse. Here, our work uncovers critical species-specific differences between the two. By systematically dissecting the roles of RIPK1, TRADD, and sensitivity to RIPK1 inhibitors in TNF signaling--including RIPK1 kinase-dependent and-independent apoptosis--we found that both apoptosis modalities diverge between human and mouse cells. In mouse cells, RIPK1 suppresses TRADD-mediated kinase-independent apoptosis, whereas in human cells, RIPK1 and TRADD act redundantly. Moreover, RIPK1 inhibitors block kinase-dependent apoptosis in mouse but not human cells, despite effectively inhibiting RIPK1 S166 phosphorylation. Cross-species complementation revealed that these discrepancies stem not from RIPK1 itself but from cell-context differences. These findings echo the clinical failures of RIPK1 inhibitors despite efficacy in mouse models and underscore the need for humanized models and therapeutics that more faithfully predict clinical outcomes.

Cell Biology↗

bioRxiv2026

mTORC1 Directs TNF-Induced Life-or-Death Decisions via Complex I Destabilization

The transition from tumor necrosis factor (TNF)-induced plasma membrane-bound complex I to cytosolic death-inducing complex II switches cells from survival to death. However, the precise regulation of this fatal decision is incompletely understood. Here, we show that mTORC1 promotes this transition by destabilizing later-stage complex I without affecting its initial assembly. Inhibition of mTORC1 unleashes ATG9A and FIP200 activity, thereby promoting the accumulation of CHUK (IKK) in complex I. CHUK scaffolds the kinase-active IKK{beta} to stabilize complex I and prevent complex II formation. Activation of this ATG9A/FIP200-CHUK/IKK{beta} axis protects against TNF-induced fulminant hepatitis while compromises antibacterial defense against Staphylococcus aureus. This mTORC1-governed life-or-death transition provides therapeutic insight into TNF-related pathologies--including cancer, metabolic tissue injury, and microbial infections--where mTORC1 activity is frequently suppressed.

Cell Biology↗

bioRxiv2026

A U1-U3 snRNA-snoRNA interaction couples SF3B1 mutation to chromatin-state rewiring and genome instability

Mutations in spliceosome factors such as SF3B1 are recurrent across human diseases, including myelodysplastic syndromes and leukemia1-4, yet splicing defects alone do not fully explain the widespread chromatin alterations and genome instability in mutant cells5. Here, by comprehensively mapping snRNA-directed RNA-RNA interactions, we identify two previously unrecognized interaction motifs in U1 snRNA beyond canonical 5' splice-site pairing6,7. These motifs enable U1 RNA to i) bind intronic and other chromatin-associated RNA (caRNA) regions outside of splice sites, and ii) base pair specifically with snoRNA. We uncover a U1-U3 snRNA-snoRNA interaction that recruits the H3K36 methyltransferase SETD2 to caRNA, promoting gene-body H3K36me3 and antagonizing H3K27me3 to modulate chromatin accessibility. The snRNA-snoRNA interface is essential for this previously unrecognized layer of chromatin and transcriptional regulation mediated through SETD2. SF3B1 mutation enhances U1-U3 binding and increases the association of the U1-U3 complex with caRNA, driving chromatin-accessibility remolding, R-loop formation, DNA damage, and copy-number abnormalities that promote tumorigenesis. A U1-specific 2'-O-methoxyethyl antisense oligonucleotide that selectively blocks U1-U3 pairing suppresses these genomic abnormalities, reduces leukemic infiltration, and prolongs survival in xenograft and patient-derived models, establishing pathological snRNA-snoRNA rewiring as a critical driver of SF3B1-mutant leukemogenesis.

cell biology↗

bioRxiv2026

Humanization of N-glycan-dependent protein quality control system in Kluyveromyces marxianus promotes glycoprotein secretion

Human N-glycoproteins constitute a market worth hundreds of billions of dollars. However, their production in yeast is often limited by misfolding and subsequent degradation, largely due to differences in N-glycan-dependent protein quality control (QC) systems between humans and yeast. Notably, yeast lacks the UGGT-mediated reglucosylation-refolding cycle that rescues misfolded glycoproteins, and its degradation pathway involves fewer rate-limiting steps. To address this, we engineer the glycoprotein QC system in Kluyveromyces marxianus, a promising host for protein production, by introducing key human components and modifying native pathways. Expression of human UGGT1 or UGGT2 enhances the soluble and secretory production of glycoproteins in an activity-dependent manner. This effect is further improved by co-expression of the UGGT cochaperone SEP15 and by reducing native glucosidase II trimming activity. In addition, introduction of human EDEM2, a rate-limiting enzyme in glycoprotein degradation, delays ER-associated degradation and increases secretion. Integration of these engineering strategies substantially enhances the production of several high-value human-derived glycoprotein therapeutics, including etanercept, dulaglutide, and abatacept, with up to a [~]12-fold increase. These findings demonstrate that engineering a human-like glycoprotein QC network in yeast is an effective strategy to improve glycoprotein folding and secretion.

bioengineering↗

bioRxiv2026

A post-translational regulatory map of chronic antigen-driven human T cell dysfunction.

T cells exposed to persistent antigen in the context of chronic viral infections or cancer lose self-renewal and cytotoxic capacity. Several transcriptional, epigenetic, and metabolic drivers of this process have been identified. However, the post-transcriptional regulatory mechanisms influencing the proteome of dysfunctional T cells are not well understood. Here we present a time-resolved molecular landscape of human T cells during the development of chronic antigen-driven dysfunction. Persistent T cell receptor stimulation significantly remodeled the proteome, including changes in canonical T cell exhaustion-associated proteins and proteins related to mitochondrial function, redox homeostasis, nucleotide metabolism, and cell-cycle progression. Dysfunctional T cells displayed activation of stress response pathways that were recapitulated in vivo; targeting these pathways altered the cytotoxic capacity of T cells during persistent tumor exposure. Our comprehensive proteomic resource reveals unique post-transcriptional changes in dysfunctional T cells and lays the groundwork for novel cysteine-directed therapeutics to enhance cancer immunotherapy.

immunology↗

bioRxiv2025

Super-giga and tiny orchid genomes illuminate evolution of Orchidaceae

Orchidaceae (orchids) is commonly known as one of the largest families of seed plants, and grow in an extensive range of habitats worldwide. In the present study, we generated chromosome-level reference genomes for two orchids using a combination of PacBio, Illumina, and Hi-C sequencing, Cypripedium singchii has the largest genome and chromosomes among the sequenced species so far, with a genome size of 43.19 Gb (1C) with ten chromosomes, and Apostasia fujianica has the smallest known genome and chromosomes in Orchidaceae, with a genome size of 340.90 Mb (1C) with 35 chromosomes. We predicted a total of 32,412 and 21,724 protein-coding genes for C. singchii and A. fujianica, respectively. The overall BUSCO score was 85.01% for C. singchii and 91.80% in A. fujianica. Based on protein-coding sequences from 55 conserved single-copy families across 21 plant species, we constructed a high-confidence phylogenetic tree and estimated the divergence times. The high-quality genomes of super-giga and tiny orchids offer key insight for future evolutionary researches.

genomics↗

bioRxiv2025

The genomic and transcriptional landscape of the spinal cord H3K27-altered diffuse midline glioma suggests the potential therapeutic strategy

Diffuse midline glioma with H3K27-altered (DMG) is a lethal pediatric malignancy that primarily occurs in the brainstem of children. In adults, DMG arising in the spinal cord is rarely reported and remains poorly understood. The cellular architecture and clinical outcomes of DMG vary depending on the tumors location. To dissect the genomic and transcriptional architecture of a cohort of spinal cord DMG patients, we conducted whole-exome sequencing and single-cell RNA sequencing on 19 tumor samples from 7 patients. We identified previously underappreciated stem-like cells associated with hypoxia in malignant tumor cells, which were present in all samples of spinal cord DMG but absent in published intracranial DMG, corroborating cellular heterogeneity based on tumor location. We discovered that tumor-associated myeloid cells constituted the largest population of non-malignant cells in adult spinal cord DMG. Our research revealed a transition from microglia to macrophages, characterized by enhanced cell-to-cell interactions mediated through the VEGF signaling pathway network. Notably, we found that epigenetic agents can repress the expression of hypoxia-related gene programs in patient-derived spinal cord DMG cultures and inhibit cell proliferation in vitro. Collectively, our study reveals a crucial anatomical dimension that DMG exhibits a location-specific genomic and transcriptional landscape despite shared H3K27 alterations, providing the paradigm for developing precision therapeutic strategies that extend beyond DMG to other molecularly defined yet anatomically divergent malignancies.

cancer biology↗

bioRxiv2024

SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

Upregulation of protein SUMOylation is associated with various diseases, and SUMOylation inhibitors are promising drug candidates. We performed the first virtual screening of SUMO E1 covalent allosteric inhibitors (CAIs) and identified two SUMO E1 CAIs with new scaffolds and covalent warheads. We further demonstrated that these new CAIs perturbed the SUMOylation pathway and protein SUMOylation. Specifically, these CAIs affected the SUMOylation of the methionine adenosyltransferases MAT2A. The inhibition of MAT2A SUMOylation unexpectedly stimulated polyamine synthesis. Lastly, we showed that the combination of SUMO E1 CAIs with a polyamine synthesis inhibitor had synergistic effects in inhibiting T47D cells. Our work demonstrated the first cost-effective virtual screening of SUMO E1 CAIs, found that the downregulation of MAT2A SUMOylation increases polyamine synthesis, and SUMO E1 CAIs can synergize with polyamine synthesis inhibitors. This work would be of great value to the study of SUMOylation, covalent/allosteric drugs, and the polyamine metabolism network.

pharmacology and toxicology↗

bioRxiv2024

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3 untranslated region (3 UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3UTR APA sites, leading to mRNA isoforms that differ substantially in 3UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRASG12V. Perturbations of isoform expression with long 3UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3UTR isoform contributes to the secreted Timp2 protein much more than the short 3UTR isoform. The short and long 3UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRASG12V. Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

molecular biology↗

bioRxiv2024

High-efficiency discovery and structure-activity-relationship analysis of non-substrate-based covalent inhibitors of S-adenosylmethionine decarboxylase

Targeted covalent inhibitors (TCIs) form covalent bonds with targets following initial non-covalent binding. The advantages of TCIs have driven a resurgence in rational TCI design over the past decade, resulting in the approval of several blockbuster covalent drugs. To support TCI discovery, various computational methods have been developed. However, accurately predicting TCI reactivity remains challenging due to interference between non-covalent scaffolds and reactive warheads, leading to inefficiencies in computational screening and high experimental costs. In this study, we enhanced the SCARdock protocol, a validated computational screening tool developed by our lab, by incorporating quantum chemistry-based warhead reactivity calculations. By integrating these calculations with non-covalent docking scores, docking ranks, and bonding-atom distances, non-covalent and covalent inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC) were correctly classified. Using the optimized SCARdock, we successfully identified twelve new AdoMetDC covalent inhibitors from 17 compounds, achieving a 70.6% hit rate. From these novel inhibitors, we analyzed the contributions of non-covalent interactions and covalent bonding, enabling a structure-activity relationship (SAR) analysis for AdoMetDC covalent inhibitors, which was previously unexplored with substrate-based inhibitors. Overall, this work presents an efficient computational protocol for TCI discovery and offers new insights into AdoMetDC inhibitor design. We anticipate that this approach will stimulate TCI development by improving computational screening efficiency and reducing experimental costs.

pharmacology and toxicology↗

bioRxiv2024

Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions

The class I PI3K-AKT signaling pathway is the master regulator of cell survival, growth, and proliferation, and among the most frequently mutated pathways in cancer. However, where and how the PI3K-AKT signaling is spatially activated and organized in mammalian cells remain poorly understood. Here, we identified focal adhesions (FAs) as the subcellular signaling hubs organizing the activation of PI3K-PI(3,4,5)P3-AKT signaling in mammalian cells. We found that class IA PI3Ks are preferentially and dynamically recruited to FAs for activation, resulting in localized production of the critical signaling lipid PI(3,4,5)P3 around FAs. As the effector protein of PI(3,4,5)P3, AKT molecules are dynamically recruited around FAs for activation. Mechanistically, the spatial recruitment/activation of PI3K-PI(3,4,5)P3-AKT cascade are regulated by the activated FAK. Furthermore, combined inhibition of class I PI3K and FAK results in a more potent inhibitory effect on cancer cells. Thus, our results unveil a growth-factor independent, compartmentalized organization mechanism for PI3K-PI(3,4,5)P3-AKT signaling.

cell biology↗

bioRxiv2023

Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosomes, initiating an endosome-mediated pathway of exosome biogenesis. Contrary to this model, we show here that syntenin drives the biogenesis of CD63 exosomes by blocking CD63 endocytosis, thereby allowing CD63 to accumulate at the plasma membrane, the primary site of exosome biogenesis. Consistent with these results, we find that inhibitors of endocytosis induce the exosomal secretion of CD63, that endocytosis inhibits the vesicular secretion of exosome cargo proteins, and that high-level expression of CD63 itself also inhibits endocytosis. These and other results indicate that exosomes bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, that syntenin and CD63 are expression-dependent regulators of exosome biogenesis, and that syntenin drives the biogenesis of CD63 exosomes even in Alix knockout cells.

cell biology↗

bioRxiv2022

Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat generated by CRISPR/Cas9

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-{kappa}B signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-{kappa}B-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

immunology↗

bioRxiv2022

Tissue-Specific Control of Ribosomal RNA Synthesis Revealed by Transcription Factor Profiling

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA Polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2200 ChIP-Seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA, and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of CEBPA (C/EBP alpha), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced Pol I occupancy. Our work identifies numerous potential rRNA regulators, and provides a template for dissection of TF roles in rRNA transcription. HIGHLIGHTSO_LIMultiple cell-type-specific transcription factors (TFs) bind canonical motifs on rDNA. C_LIO_LIThe hematopoietic TF CEBPA binds to active rDNA alleles at a conserved site. C_LIO_LICEBPA promotes Polymerase I occupancy and rRNA transcription in myeloid progenitors. C_LIO_LIWe present 47S-FISH-Flow, a sensitive assay to quantify nascent rRNA. C_LI

molecular biology↗

bioRxiv2021

CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.

molecular biology↗

bioRxiv2020

The D614G Mutation Enhances the Lysosomal Trafficking of SARS-CoV-2 Spike

The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutations effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.

cell biology↗

bioRxiv2020

Casein Kinase 1G2 Suppresses Necroptosis-Promoted Testis Aging by Inhibiting Receptor-Interacting Kinase 3

Casein kinases are a large family of intracellular serine/threonine kinases that control a variety of cellular signaling functions. Here we report that a member of casein kinase 1 family, casein kinase 1G2, CSNK1G2, binds and inhibits the activation of receptor-interacting kinase 3, RIP3, thereby attenuating RIP3-mediated necroptosis. The binding of CSNK1G2 to RIP3 is triggered by auto-phosphorylation at serine 211/threonine 215 sites in its C-terminal domain. CSNK1G2-knockout mice showed significantly enhanced necroptosis response and pre-maturing aging of their testis, a phenotype that was rescued by either double knockout of the RIP3 gene or feeding the animal with a RIP1 kinase inhibitor-containing diet. Moreover, CSNK1G2 is also co-expressed with RIP3 in human testis, and the necroptosis activation marker phospho-MLKL was observed in the testis of old (>80) but not young men, indicating that the testis-aging program carried out by the RIP3-mediated and CSNK1G2-attenuated necroptosis is evolutionarily conserved between mice and men.

cell biology↗