Biology subjects

Qiu, Y.

Publications and source records attributed to Qiu, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Divergent Minimum Inhibitory Concentrations of Clarithromycin and Azithromycin Against Clinical Isolates of Mycobacterium abscessus Complex

Current treatment guidelines for Mycobacterium abscessus complex (MAB) infections recommend macrolides (clarithromycin/CAM and azithromycin/AZM) as cornerstone therapies, considering them clinically interchangeable without distinction. This study evaluated the comparative in vitro activity of clarithromycin (CAM) and azithromycin (AZM) against 146 clinical isolates of Mycobacterium abscessus complex organisms. MIC distributions revealed significantly higher resistance to AZM (94.5% resistant at [≥]8 g/mL) compared to CAM (77.4% resistant at [≥]8 g/mL), with only 4.8% of isolates susceptible to both drugs. Notably, 26 strains (17.8% of total) showed AZM resistance despite being non-resistant to CAM (MIC <8 g/mL) among 146 clinical isolates. Among the 33 CAM-non-resistant isolates (including susceptible and intermediate strains), 78.8% (26/33) were AZM-resistant, demonstrating frequent discordance in susceptibility profiles. These findings contrast with current treatment guidelines that recommend using CAM and AZM interchangeably without distinction. Subspecies analysis showed three distinct susceptibility profiles: isolates susceptible to both antibiotics (n=3, M. massiliense), isolates resistant to both antibiotics (n=3, M. massiliense), and M. bolletii with CAM-susceptible/AZM-resistant phenotypes (n=2). This differential resistance pattern suggests that while both drugs face substantial resistance challenges, CAM retains meaningful activity against certain strains that are AZM-resistant--particularly in M. bolletii. These findings indicate that AZM and CAM should not be used interchangeably to treat MAB infections and emphasize the need to prioritize CAM over AZM in treatment regimens.

microbiology↗

bioRxiv2025

The Mycobacterium tuberculosis ESX effector promotes pyroptosis-dependent pathogenicity and dissemination

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/653593v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@8cd5e5org.highwire.dtl.DTLVardef@2ec22forg.highwire.dtl.DTLVardef@1ceb773org.highwire.dtl.DTLVardef@6dfba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic AbstractC_FLOATNO C_FIG Mycobacterium tuberculosis (Mtb) remains a major global health threat, partly due to the extensive cytotoxicity induced during infection. Although GSDMD-mediated excessive pyroptosis promotes pathogen dissemination and tissue damage in tuberculosis, the mechanism remains poorly understood. Here, we identify the effector EccB5, a component of the Mtb ESX-5 secretion system, as a key driver of pyroptosis and hyperinflammatory responses. EccB5 enhances Mtb virulence by inducing pyroptosis of macrophages, promoting bacterial dissemination and exacerbating lung pathology. Conditional knockdown EccB5 increases host cell viability. Mechanistically, EccB5 directly interacts with GSDMD, strengthens its association with caspase-1, and facilitates caspase-1-mediated cleavage of GSDMD both in vitro and in vivo. In summary, our findings uncover a precise mechanism by which Mtb modulates host responses and advances its pathogenicity.

microbiology↗

bioRxiv2025

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. HighlightsSubcellular proteomics of Paramecium tetraurelia revealed that glycolytic and gluconeogenic enzymes are mosaically distributed between the cytosol, mitochondrial matrix, and mitochondrial outer membrane. A distinct mitochondrial outer membrane compartment was identified with 105 classified proteins, including core mitochondrial biogenesis proteins and a putative Tom70-like protein. Phosphofructokinase, a key glycolytic enzyme, was found embedded in the mitochondrial outer membrane. Localization of biochemical pathways suggest ciliates favor gluconeogenesis over glycolysis. In total, over 9000 Paramecium proteins were identified using subcellular proteomics and classified into 16 different cellular compartments.

cell biology↗

bioRxiv2025

Widespread discordance between mRNA expression, protein abundance and de novo lipogenesis activity in hepatocytes during the fed-starvation transition

The mammalian liver plays a critical role in maintaining metabolic homeostasis during fasting and feeding. Liver function is further shaped by sex dimorphism and zonation of hepatocytes. To explore how these factors interact, we performed deep RNA-sequencing and label-free proteomics on periportal and pericentral hepatocytes isolated from male and female mice under fed and starved conditions. We developed a classification system to assess protein-mRNA relationship and found that gene products (mRNA or protein) for most zonation markers showed strong concordance between mRNA and protein. Although classical growth hormone regulated sex-biased gene products also exhibited concordance, [~]60% of sex-biased gene products showed protein-level enrichment without corresponding mRNA differences. In contrast, transition between feeding and starvation triggered widespread changes in mRNA expression without significantly affecting protein levels. In particular, key lipogenic mRNAs (e.g. Acly, Acaca, and Fasn) were dramatically induced by feeding, but their corresponding proteins (ACLY, ACC1, and FAS) showed little to no change even as functional de novo lipogenic activity increased [~]28-fold in the fed state. To facilitate further exploration of these findings, we developed Discorda (https://shinoda-lab.shinyapps.io/discorda/), a web database for interactive analysis. Our findings reinforce the principle that mRNA changes do not reliably predict corresponding protein levels (and vice versa), particularly in the context of sex and acute metabolic regulation of hepatocytes, and that de novo lipogenesis activity can be completely uncoupled from changes in protein expression.

bioinformatics↗

bioRxiv2025

Sustained cross-species transmission of gammacoronavirus in wild birds reveled by viral characterization in China

Gammacoronavirus ({gamma}-CoV) primarily infects poultry, wild birds, and marine mammals. The widespread distribution and circulation of {gamma}-CoV in the ecological environment may lead to sustained transmission and economic loss. To better understand the diversity of {gamma}-CoV in wild birds, we collect 482 wild-bird fecal samples from Yunnan, encompassing fourteen bird species. We detected twelve {gamma}-CoV positive samples in five bird species, with the characterization of five complete genomes - HNU5-1, HNU5-2, HNU5-3, HNU6-1, and HNU6-2-indicating that these genomes represent two viral species. The HNU5 strains were derived from Black-headed gull (Chroicocephalus ridibundus), while the HNU6 strains were came from Mallard (Anas platyrhynchos), and both of those were recombinant. The HNU5 strain exhibited the highest sequence identity (95.45%) with a {gamma}-CoV strain isolated from Numenius phaeopus (GenBank accession: PP845452). Similarly, the HNU6 strain showed 95.18% nucleotide identity with a {gamma}-CoV strain (GenBank accession: PP845437) derived from Anas platyrhynchos. Taxonomic analysis confirmed that HNU6s belong to the Gammacoronavirus anatis species, while HNU5s attributed to a new species. Cross-species analysis revealed active host-switching events among {gamma}-CoVs, indicating potential transmission of {gamma}-CoVs from marine mammals to wild bird, from wild bird to poultry, and inter-wild bird and inter-poultry transmission. In summary, we report five new {gamma}-CoV strains in wild birds and outline the cross-species transmission of {gamma}-CoVs. Our findings link {gamma}-CoV hosts across different natural environments and provide new insights for exploring {gamma}-CoVs.

microbiology↗

bioRxiv2025

Specific binding modes of SIRT6 C-terminal domain to the nucleosome core particle influence DNA unwrapping and H3K27 accessibility

Sirtuins are a class of NAD-dependent histone deacetylases that regulate important biological pathways in prokaryotes and eukaryotes. This enzyme family comprises seven members, named SIRT1 to SIRT7. Among them, Sirtuin 6 (SIRT6) is a human sirtuin that deacetylates histones and plays a key role in DNA repair, telomere maintenance, carbohydrate and lipid metabolism, and lifespan. SIRT6s structure consists of a zinc finger domain, a Rossmann fold domain containing the NAD+ binding site, and disordered N-terminal and C-terminal (CTD) extensions. The specific role of the CTD on SIRT6 interaction with nucleosomes for histone deacetylation remains unclear. Here, we resort to extended molecular dynamics simulations to uncover the dynamical behavior of the full-length SIRT6 bound to a nucleosome core particle. Our simulations reveal that the CTD preferentially interacts with DNA at the entry/exit near the enzymes docking site, exhibiting a variety of different binding modes. In specific cases, the CTD participates to the promotion of DNA unwrapping and promotes H3K27 accessibility to SIRT6s active site, suggesting a pivotal role of this domain for H3K27 deacetylation. This work provides new structural insights into the binding process of the full-length SIRT6 to a nucleosome core particle, shedding light on the conformational behavior and functional role of its CTD. It constitutes an important step towards the understanding of SIRT6 deacetylation mechanisms and specificity.

biochemistry↗

bioRxiv2025

Direct RNA sequencing of primary human T cells reveals the impact of immortalization on mRNA pseudouridine modifications.

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine ({Psi}), one of the most abundant mRNA modifications, and compare {Psi} profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of {Psi}-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in {Psi} levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels. Significance StatementIt is widely believed that RNA modification machinery and modification sites could be significantly altered in immortalized cells, yet this has never been tested. Focusing on pseudouridine ({Psi}), we map {Psi} in the transcriptomes of primary and immortalized human T cells. Surprisingly, most sites are conserved in the primary and immortalized T cells, with several important examples of cases with cell type specificity and should be considered on a case-by-case basis. Furthermore, we evaluated RNA-modification machinery levels in primary and immortalized T cells, finding high conservation across the cell lines. Our findings demonstrate that RNA modifications are largely conserved between primary and immortalized cells, and the edge cases can be considered individually.

genomics↗

bioRxiv2025

Nuclear speckle proteins form intrinsic and MALAT1-dependent microphases

Nuclear speckles are enriched in serine / arginine rich splicing factors (SRSFs), such as SRSF1. Splicing factors and proteins such as TDP-43 concentrate into distinct speckle territories to enable pre-mRNA processing. We have discovered that SRSFs and TDP-43 are block copolymers and the protein-specific interplay of inter-block repulsions and attractions drives spontaneous microphase separation. This gives rise to size-limited, ordered assemblies, that are 30 - 45 nm in diameter. Depending on the protein, each microphase comprises several tens to hundreds of molecules. The sub-micron scale territories observed in cells are shown to be clusters of microphases. The regulatory lncRNA MALAT1 binds preferentially to SRSF1 microphases to enhance microphase separation and alter microphase structures. Microphase separation enables the concentration of finite numbers of splicing factors into assemblies with distinct nanoscale structures that can be modulated by MALAT1. Our findings provide a structural framework for the functional organization of splicing factors.

biophysics↗

bioRxiv2025

Circulating extracellular vesicles from HIV-1 gp120-treated mice act as endogenous algogens, mediating and maintaining HIV-associated chronic pain

HIV-associated chronic pain (HIV-PAIN) remains prevalent in the post combined antiretroviral therapy era, affecting 30-60% of HIV patients worldwide. The underlying mechanism responsible for the development and maintenance of chronic pain remains unclear. gp120 is a causal factor of the HIV-PAIN and functions as an exogenous algogen. The pain experienced by human HIV-PAIN has been modeled in mice (referred to as mHIV-PAIN) using intrathecal (i.t.) injections of gp120. gp120 is a relatively short-term, static, exogenous algogen that is exhaustible in vivo. In authentic infection, HIV virions serve as the primary source of exogenous gp120, which initiates the early phase of clinical HIV-PAIN. Interestingly, while the source of replenishing gp120 decreases after antiretroviral therapy by suppressing viremia, the prevalence of chronic HIV-PAIN remains stable. To induce chronic pain in mice, gp120 needs to be repeatedly applied by the i.t. route. This raises a key question: Is an endogenous inexhaustible algogen responsible for maintaining the chronicity of HIV-PAIN? In the present study, we isolated circulating small extracellular vesicles (sEV) from mice using our mHIV-PAIN model that is i.t. injected with gp120. We refer to such sEV as gp120-sEV herein. We observed that gp120 is absent in gp120-sEV. Following transfusion of gp120-sEV intrathecally, naive recipient mice exhibit an extensive pain phenotype, including cold pain tested with we newly invented dry ice vapor cold test. RNA-sequence analysis suggests that gp120-sEVs induced expression of genes related to nociception and neuroinflammation pathways. These findings provide direct evidence that circulating sEV function as endogenous long-term "dynamic" algogens that enhance initial pain and extend the chronification of HIV-PAIN in mice, suggesting that chronic HIV-PAIN requires an exogenous algogen (gp120) paired with endogenous algogen (gp120-sEV), and that these components work synchronically to initiate and extend pain chronification. This double algogen concept provides a new insight into the pathogenesis of HIV-PAIN chronification. Our new mechanistic understanding will also assist in identifying new therapeutics to alleviate HIV-PAIN by targeting pathological gp120-sEV.

neuroscience↗

bioRxiv2025

Microscopic Control of Cortical Flows in Polarized C. elegans Zygotes

Cell polarization, migration, and cytokinesis rely on flows of the cell cortex, a network of actin filaments, cross-linkers, and motors beneath the plasma membrane of animal cells. While actin network architecture, assembly dynamics, and motor activity are known to be important for cortical flows, how their modulation tunes macroscopic flow dynamics remains poorly quantified in vivo. Here, we use quantitative microscopy to constrain agent-based simulations that account for filament assembly, crosslinking, and motor activity. We calibrate the model to reproduce steady-state flows in polarized C. elegans zygotes and then challenge it to predict the results of RNA interference (RNAi) experiments. Our model predicts, and experiments largely confirm, a biphasic dependence of flow speed on microscopic rates of actin filament assembly and crosslinking. This biphasic dependence reflects a competition between the tendencies of perturbations to disrupt both transmission of and resistance to cortical forces. Our results provide new insights into how variations in microscopic features shape the emergent dynamics of the cell cortex. By establishing a well-calibrated model of cortical flow in a highly tractable model cell, we also provide a foundation for future studies of microscopic origins and biological control of cortical contractility and flow in vivo.

cell biology↗

bioRxiv2024

Leukemia escapes immunity by imposing a Type-1 regulatory program on neoantigen-specific CD4+ T cells.

The significance of endogenous immune surveillance in acute lymphoblastic leukemia (ALL) remains controversial. Using clinical B-ALL samples and a novel mouse model, we show that neoantigen-specific CD4+ T cells are induced to adopt type-1 regulatory (Tr1) function in the leukemia microenvironment. Tr1s then inhibit cytotoxic CD8+ T cells, preventing effective leukemia clearance. Leukemic cells induce Tr1s by phenocopying hematopoietic stem cells, which normally are subject to effective surveillance by this CD4+ subset. This mechanism effectively redirects Tr1 cells from a role in preventing cancer to maladaptively promoting clinical relapse. In mouse models, inhibition of Tr1 expansion with IL10 receptor (IL10R) blockade is insufficient to improve leukemia control. In contrast, combined therapy with a cytotoxic agent and anti-PDL1 blockade eradicated measurable residual disease. This correlates with polarization of the neoantigen-specific CD4+ T-cell population from Tr1 towards Th1 states. Our findings uncover a mechanism that enables leukemic relapse and resolves existing controversies on the role of immune surveillance towards this cancer type. Therapeutic polarization of neoantigen-specific CD4+ T cells towards Th1 states may improve contemporary immune therapies by reshaping the immune microenvironment towards states permissive for cytotoxic attack of residual leukemia. Key PointsO_LIB-ALL induces neoantigen-specific CD4+ T cells to adopt Type-1 regulatory states, which protect leukemic cells from immune pressure. C_LIO_LIRepolarizing neoantigen-specific CD4+ T-cells towards Th1 states eradicates measurable residual disease. C_LI

cancer biology↗

bioRxiv2024

Deciphering Colorectal Cancer-Hepatocyte Interactions: A Multiomic Platform for Interrogation of Metabolic Crosstalk in the Liver-Tumor Microenvironment

Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to adapt to and exploit their microenvironment for sustained growth. The liver is a common site of metastasis, but the interactions between tumor cells and hepatocytes remain poorly understood. In the context of liver metastasis, these interactions play a crucial role in promoting tumor survival and progression. This study leverages multiomics coverage of the microenvironment via liquid chromatography and high-resolution, high-mass accuracy mass spectrometry-based untargeted metabolomics, 13C-stable isotope tracing, and RNA sequencing to uncover the metabolic impact of co-localized primary hepatocytes and a colon adenocarcinoma cell line, SW480, using a 2D co-culture model. Metabolic profiling revealed disrupted Warburg metabolism with an 80% decrease in glucose consumption and 94% decrease in lactate production by hepatocyte-SW480 co-cultures relative to SW480 control cultures. Decreased glucose consumption was coupled with alterations in glutamine and ketone body metabolism, suggesting a possible fuel switch upon co-culturing. Further, integrated multiomic analysis indicates that disruptions in metabolic pathways, including nucleoside biosynthesis, amino acids, and TCA cycle, correlate with altered SW480 transcriptional profiles and highlight the importance of redox homeostasis in tumor adaptation. Finally, these findings were replicated in 3-dimensional microtissue organoids. Taken together, these studies support a bioinformatic approach to study metabolic crosstalk and discovery of potential therapeutic targets in preclinical models of the tumor microenvironment.

bioinformatics↗

bioRxiv2024

Human stem cell-derived GABAergic interneuron development reveals early emergence of subtype diversity followed by gradual electrochemical maturation

Medial ganglionic eminence-derived inhibitory GABAergic pallial interneurons (MGE-pINs) are essential regulators of cortical circuits; their dysfunction is associated with numerous neurological disorders. We developed human (h) MGE-pINs from pluripotent stem cells for the treatment of drug-resistant epilepsy. Here, we analyzed xenografted hMGE-pINs over the lifespan of host mice using single nuclei RNA sequencing. Comparative transcriptomics against endogenous human brain datasets revealed that 97% of grafted cells developed into somatostatin (SST) and parvalbumin (PVALB) subtypes, including populations that exhibit selective vulnerability in Alzheimers disease. Transplanted hMGE-pINs demonstrated rapid emergence of subclass features, progressing through distinct transcriptional states sequentially involving neuronal migration, synapse organization, and membrane maturation. We present molecular, electrophysiological, and morphological data that collectively confirm the derivation of diverse bona-fide human SST and PVALB subtypes, providing a high-fidelity model to study human MGE-pIN development and functional maturation as well as a compositional atlas for regenerative cell therapy applications.

neuroscience↗

bioRxiv2024

Nucleosome condensate and linker DNA alter chromatin folding pathways and rates

Chromatin organization is essential for DNA packaging and gene regulation in eukaryotic genomes. While significant progresses have been made, the exact atomistic arrangement of nucleosomes remains controversial. Using a well-calibrated residue-level coarse-grained model and advanced dynamics modeling techniques, particularly the non-Markovian dynamics model, we map the free energy landscape of tetra-nucleosome systems, identify both metastable conformations and intermediate states in folding pathways, and quantify the folding kinetics. Our findings show that chromatin with 10n base pairs (bp) DNA linker lengths favor zigzag fibril structures. However, longer linker lengths destabilize this conformation. When the linker length is 10n + 5 bp, chromatin loses unique conformations, favoring a dynamic ensemble of structures resembling folding intermediates. Embedding the tetra-nucleosome in a nucleosome condensate similarly shifts stability towards folding intermediates as a result of the competition of inter-nucleosomal contacts. These results suggest that chromatin organization observed in vivo arises from the unfolding of fibril structures due to nucleosome crowding and linker length variation. This perspective aids in unifying experimental studies to develop atomistic models for chromatin. SignificanceAtomic structures of chromatin have become increasingly accessible, largely through cryo-EM techniques. Nonetheless, these approaches often face limitations in addressing how intrinsic in vivo factors influence chromatin organization. We present a structural characterization of chromatin under the combined effects of nucleosome condensate crowding and linker DNA length variation--two critical in vivo features that have remained challenging to capture experimentally. This work leverages a novel application of non-Markovian dynamical modeling, providing accurate mapping of chromatin folding kinetics and pathways. Our findings support a hypothesis that in vivo chromatin organization arises from folding intermediates advancing toward a stable fibril configuration, potentially resolving longstanding questions surrounding chromatin atomic structure.

biophysics↗

bioRxiv2024

Dystrophin interacts with Msp300 to Regulate Myonuclear Positioning and Microtubule Organization

During Drosophila myognesis, myonuclei are actively moved during embryogenesis, and their spacing is maintained through an anchoring mechanism in the fully differentiated myofiber. While we have identified microtubule associated proteins, motors, and nuclear envelope proteins that regulate myonuclear spacing, the developmental time during which each gene functions has not been tested. Here we have identified a Dystrophin as required only for the maintenance of myonuclear spacing. Furthermore, we demonstrate that Dystrophin genetically interacts with the KASH-domain protein Msp300 to maintain myonuclear spacing. Mechanistically, both Dystrophin and Msp300 regulate microtubule organization. Specifically, in animals with disrupted expression of both Dystrophin and Msp300, microtubule colocalization with sarcomeres is reduced. Taken altogether, these data indicate that the peripheral membrane protein Dystrophin, and the outer nuclear membrane protein Msp300, together regulate the organization of the microtubule network which then acts as an anchor to restrict myonuclear movement in contractile myofibers. These data are consistent with growing evidence that myonuclear movement and myonuclear spacing are critical to muscle development, muscle function, and muscle repair and provide a mechanism to connect disparate muscle diseases. Summary StatementHere we show that Dystrophin is required to maintain the spacing of nuclei in differentiated myofibers. Furthermore, Dystrophin achieves this function via a genetic interaction with Msp300 which regulates microtubule organization.

cell biology↗

bioRxiv2024

RING1 dictates GSDMD-mediated inflammatory response and host susceptibility to pathogen infection

RING1 is an E3 ligase component of the polycomb repressive complex 1 (PRC1) with known roles in chromatin regulation and cellular processes such as apoptosis and autophagy. However, its involvement in inflammation and pyroptosis needs to be better characterized. Here, we found that human RING1, but not RING2, promoted K48-linked ubiquitination of Gasdermin D (GSDMD) and acted as a negative regulator of pyroptosis and bacterial infection. Ring1 knockout mice increased the bacterial loads and mortality rate by treatment of S. typhimurium. Upon infection by M. tuberculosis (Mtb) H37Rv strain, RING1 deletion initially reduced bacterial loads but later increased lung inflammation and impaired immune defense responses in mice. Also, Ring1 knockout exacerbated acute sepsis induced by lipopolysaccharide (LPS). Mechanistically, RING1 directly interacts with GSDMD and ubiquitinates the K51 and K168 sites of GSDMD for K48-linked proteasomal degradation, thereby inhibiting pyroptosis. Inhibition of RING1 E3 ligase activity by mutation or small molecular compound increased GSDMD level and cell death during pyroptosis. Our findings reveal that RING1 dictates GSDMD-mediated inflammatory response and host susceptibility to pathogen infection, highlighting RING1 as a potential therapeutic target for combating infectious diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/622030v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@e6d4a6org.highwire.dtl.DTLVardef@92f137org.highwire.dtl.DTLVardef@11a69c4org.highwire.dtl.DTLVardef@115dc0b_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG HighlightsO_LIE3 ubiquitin ligase RING1 targets GSDMD for proteasomal degradation. C_LIO_LIMice lacking RING1 are more vulnerable to S. typhimurium infection and LPS- induced sepsis. C_LIO_LIIn the early stages of M. tuberculosis (Mtb) infection, Ring1-/- mice exhibit efficient bacterial clearance. C_LIO_LIChronic inflammation in Ring1-/- mice impairs the ability to control Mtb in the late stages of infection. C_LI

cell biology↗

bioRxiv2024

Stacked Human Arterial Endothelial Cells Generate Atherosclerotic Fatty Streaks and Release Proinflammatory Cytokines

Fatty streaks are the first sign of atherosclerosis. They consist of lipid-containing foam cells, which were believed to be derived from the monocytes in the blood through the leaky endothelium, and from the vascular smooth muscle cells migrating into the intima from the media. Here, we showed that fatty streaks can also be formed by the stacked human arterial endothelial cells (HAECs) cultured in vitro. Via SEM we revealed a novel cell phenotype (coralthelial) that forms a streak/coral-like structure. We observed accumulation of lipids in the coralthelial cell and increased Golgi and coat protein II markers in its nucleus. Additionally, proinflammatory cytokines were upregulated in these cells, likely due to Golgi nuclear translocation and subsequently increased expression of the ribosomal protein RPL23 in the nucleus. We demonstrated, for the first time, that the atherosclerotic fatty streak-like structure can be generated from the stacked HAECs, which also create a proinflammatory microenvironment. Significance StatementOur work presents a novel perspective on atherosclerotic fatty streak formation, a critical early event in atherosclerosis development. We demonstrate, for the first time, that human arterial endothelial cells (HAECs) can transform into a novel phenotype, termed "coralthelial cells". These cells contribute to fatty streak formation, and produce proinflammatory cytokines through the relocation of RPL23 with the Golgi apparatus to the nucleus. This finding provides a new cellular mechanism by which endothelial cells initiate atherosclerosis, independent of traditional monocyte-derived foam cells. By enriching established paradigms, our research not only opens new avenues for therapeutic intervention targeting endothelial cells but also enhances understanding of vascular diseases and provides potential diagnostic and prognostic markers for atherosclerosis.

cell biology↗

bioRxiv2024

NuclampFISH enables cell sorting based on nuclear RNA expression for chromatin analysis

Transcriptional bursts refer to periods when RNA polymerase interacts with a DNA locus, leading to active gene transcription. This bursting activity can vary across individual cells, and analyzing the differences in transcription sites can help identify key drivers of gene expression for a specific target. Scaffolding methods based on fluorescence in situ hybridization (FISH) have been widely used to amplify the fluorescent signal of mRNAs and sort cells based on mRNA expression levels. However, these methods are inefficient at targeting nuclear RNA, including transcription sites, due to the probes limited accessibility through cellular compartment membranes and crosslinked proteins. Additionally, the required formaldehyde fixation interferes with downstream analysis of chromatin and protein-binding interactions. To address these challenges, a platform that integrates amplified FISH with reversible crosslinkers and allows access to the nucleus is needed. In response, we developed nuclear clampFISH (nuclampFISH). This method amplifies the fluorescent signal of mRNAs using a reversible crosslinker, enabling the sorting of cells based on nuclear RNA expression and compatible with downstream biochemical analysis. This assay demonstrates that transcriptionally active cells have more accessible chromatin for a respective gene. Notably, the tools developed are highly accessible and do not require specialized computation or equipment.

genomics↗