Biology subjects

Xing, S.

Publications and source records attributed to Xing, S..

32 records · Page 2Linked to original sources

bioRxiv2023

Surf4 deficiency reduces intestinal lipid absorption and secretion and decreases metabolism in mice

BackgroundPostprandial dyslipidemia is a causative risk factor for cardiovascular disease. The majority of absorbed dietary lipids are packaged into chylomicron and then delivered to circulation. Previous studies showed that Surfeit4 (Surf4) mediates very low-density lipoprotein secretion from hepatocytes. Silencing hepatic Surf4 markedly reduces the development of atherosclerosis in different mouse models of atherosclerosis without causing hepatic steatosis. However, the role of Surf4 in chylomicron secretion is unknown. MethodsWe developed inducible intestinal-specific Surf4 knockdown mice (Surf4IKO) using Vil1Cre-ERT2 and Surf4flox mice. Metabolic cages were used to monitor mouse metabolism. Enzymatic kits were employed to measure serum and tissue lipid levels. The expression of target genes was detected by qRT-PCR and Western Blot. Transmission electron microscopy and radiolabeled oleic acid were used to assess the structure of enterocytes and intestinal lipid absorption and secretion, respectively. Proteomics was performed to determine changes in protein expression in serum and jejunum. ResultsSurf4IKO mice, especially male Surf4IKO mice, displayed significant body weight loss, increased mortality, and reduced metabolism. Surf4IKO mice exhibited lipid accumulation in enterocytes and impaired fat absorption and secretion. Lipid droplets and small lipid vacuoles were accumulated in the cytosol and the endoplasmic reticulum lumen of the enterocytes of Surf4IKO mice, respectively. Surf4 colocalized with apoB and co-immunoprecipitated with apoB48 in differentiated Caco-2 cells. Intestinal Surf4 deficiency also significantly reduced serum triglyceride, cholesterol and free fatty acid levels in mice. Proteomics data revealed that diverse pathways were altered in Surf4IKO mice. In addition, Surf4IKO mice had mild liver damage, decreased liver size and weight, and reduced hepatic triglyceride levels. ConclusionOur findings demonstrate that intestinal Surf4 plays an essential role in lipid absorption and chylomicron secretion and suggest that the therapeutic use of Surf4 inhibition requires highly cell/tissue-specific targeting. HighlightsO_LIIntestinal Surf4 deficiency reduces chylomicron secretion. C_LIO_LILack of intestinal Surf4 reduces absorption of dietary lipids. C_LIO_LISilencing intestinal Surf4 reduces serum lipid levels. C_LIO_LIIntestinal Surf4 deficiency leads to lipid accumulation in intestinal villi. C_LIO_LIIntestinal Surf4 is essential for dietary lipid absorption and secretion. C_LI

physiology↗

bioRxiv2023

Depletion-assisted multiplexing cell-free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicle

Cell-free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature. To investigate these fragmented cell-free RNAs (cfRNAs), we developed a cost-effective cfRNA sequencing method called DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing). DETECTOR-seq utilized a meticulously tailored set of customized guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimize plasma requirements. Using DETECTOR-seq, we conducted a comprehensive analysis of cell-free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs, and viral RNAs, while EVs showed enrichment in mRNAs and srpRNAs. Functional pathway analysis highlighted RNA splicing-related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration, and antigen receptor-mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types. Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA-based studies. We envision the cost-effectiveness and efficiency of DETECTOR-seq will empower transcriptome-wide investigations in the fields of extracellular vesicles and liquid biopsy.

molecular biology↗

bioRxiv2023

Cell-free multi-omics analysis reveals tumor status-informative signatures in gastrointestinal cancer patients' plasma

During cancer development, hosts tumorigenesis and immune signals are released to and informed by circulating molecules, like cell-free DNA (cfDNA) and RNA (cfRNA) in blood. However, these two kinds of molecules are still not systematically compared in gastrointestinal cancer. Here, we profiled 4 types of cell-free omics data from colorectal and stomach cancer patients, and assayed 15 types of genomic, epi-genomic, and transcriptomic variations. First, we demonstrated that the multi-omics data were more capable of detecting cancer genes than the single-omics data, where cfRNAs were more sensitive and informative than cfDNAs in terms of detection ratio, variation type, altered number, and enriched functional pathway. Moreover, we revealed several peripheral immune signatures that were suppressed in cancer patients and originated from specific circulating and tumor-microenvironment cells. Particularly, we defined a {gamma}{delta}-T-cell score and a cancer-associated-fibroblast (CAF) score using the cfRNA-seq data of 143 cancer patients. They were informative of clinical status like cancer stage, tumor size, and survival. In summary, our work reveals the cell-free multi-molecular landscape of colorectal and stomach cancer, and provides a potential monitoring utility in blood for the personalized cancer treatment.

systems biology↗

bioRxiv2022

Identification of the Metaphyseal Skeletal Stem Cell

Identification of novel regional skeletal stem cells (SSCs) will provide a new cellular paradigm for bone physiology and dysfunction. Several populations of SSCs have been identified at distinct skeletal sites. However, a bona fide SSC population has not yet been formally characterized in the bone marrow. Here, we identify a metaphyseal SSCs (mpSSCs) population whose transcriptional landscape is distinct from other bone mesenchymal stromal cells (bMSCs) in the bone marrow. These mpSSCs emerge at the postnatal stage and reside just underneath the growth plate, consistent with the fact that these mpSSCs are exclusively derived from hypertrophic chondrocytes (HCs). These mpSSCs possess SSC properties such as self-renewal and multipotency in vitro and in vivo, stand at the top of the HC de-differentiation path, and produce most HC progeny. Genetic block of the conversion from HCs to mpSSCs significantly compromises trabecular bone formation and bone regeneration. Thus, metaphysis houses a unique HC-derived SSC population, which is a major source of osteoblasts and bMSCs supporting postnatal trabecular bone formation.

developmental biology↗

bioRxiv2022

Molecular formula discovery via bottom-up MS/MS interrogation

A substantial fraction of metabolic features remains undetermined in mass spectrometry (MS)-based metabolomics. Here we present bottom-up tandem MS (MS/MS) interrogation to illuminate the unidentified features via accurate molecular formula annotation. Our approach prioritizes MS/MS-explainable formula candidates, implements machine-learned ranking, and offers false discovery rate estimation. Compared to the existing MS1-initiated formula annotation, our approach shrinks the formula candidate space by 42.8% on average. The superior annotation accuracy of our bottom-up interrogation was demonstrated on reference MS/MS libraries and real metabolomics datasets. Applied on 155,321 annotated recurrent unidentified spectra (ARUS), our approach confidently annotated >5,000 novel molecular formulae unarchived in chemical databases. Beyond the level of individual metabolic features, we combined bottom-up MS/MS interrogation with global peak annotation. This approach reveals peak interrelationships, allowing the systematic annotation of 37 fatty acid amide molecules in human fecal data, among other applications. All bioinformatics pipelines are available in a standalone software, BUDDY (https://github.com/HuanLab/BUDDY/).

bioinformatics↗

bioRxiv2022

A potent SARS-CoV-2 antibody neutralizes Omicron variant by disassembling the spike trimer

The continuous emergence of novel SARS-CoV-2 variants poses new challenges to the fight against the COVID-19 pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of antibody targeting conserved and universal epitope is urgently needed. A subset neutralizing antibody(nAbs) against COVID-19 from convalescent patients were isolated in our previous study. Here in this study, we investigated the accommodation of these nAbs to SARS-CoV-2 variants of concerns (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of SARS-CoV-2 Omicron variant. In addition, we determined the cryo-EM structure of SARS-CoV-2 spike complexed with three antibodies targeting different epitopes, including 553-49, 553-15 and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes virus by disassembling spike trimers. 553-15, an antibody that neutralizes all the other VOCs except omicron, cross-links two spike trimers to form trimer dimer, demonstrating that 553-15 neutralizes virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 49 and other antibody targeting this highly conserved epitope as promising cocktail therapeutics reagent for COVID-19. ImportanceThe newly emergence of Omicron strain caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 Omicron neutralizing antibody 553-49, which neutralizes Omicron variant by targeting a completely conserved novel epitope. Besides, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by crosslinking virions and 553-60 functions by blocking receptor binding. Comparison of different RBD epitopes revealed that the epitope of 553-49 is hidden in the S trimer and keeps high conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutics reagent to fight against the emerging Omicron and future variant of SARS-CoV-2.

microbiology↗

bioRxiv2022

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

Manuscript summaryThere are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks for 41 ASD-risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout reveal an association between mitochondrial activity and ASD-risk genes. The PPI network shows an enrichment of 112 additional ASD-risk genes and differentially expressed genes from post-mortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data reveal that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and reveal biological insight into disease mechanisms and sub-cohorts of ASD.

neuroscience↗

bioRxiv2022

Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Copy number variations (CNV) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

neuroscience↗

bioRxiv2021

Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZl was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism. IMPORTANCESinorhizobium meliloti infects the host alfalfa and induces formation of nitrogen-fixing nodules. Proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of symbiotic interactions. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

microbiology↗

bioRxiv2021

Disruption of the autism-associated gene SCN2A alters synaptic development and neuronal signaling in patient iPSC-glutamatergic neurons

SCN2A is an autism spectrum disorder (ASD) risk gene and encodes a voltage-gated sodium channel. However, the impact of autism-associated SCN2A de novo variants on human neuron development is unknown. We studied SCN2A using isogenic SCN2A-/- induced pluripotent stem cells (iPSCs), and patient-derived iPSCs harboring a p.R607* or a C-terminal p.G1744* de novo truncating variant. We used Neurogenin2 to generate excitatory glutamatergic neurons and found that SCN2A+/p.R607* and SCN2A-/- neurons displayed a reduction in synapse formation and excitatory synaptic activity using multielectrode arrays and electrophysiology. However, the p.G1744* variant, which leads to early-onset seizures in addition to ASD, altered action-potential dynamics but not synaptic activity. Proteomic and functional analysis of SCN2A+/p.R607* neurons revealed defects in neuronal morphology and bioenergetic pathways, which were not present in SCN2A+/p.G1744* neurons. Our study reveals that SCN2A de novo variants can have differential impact on human neuron function and signaling. HIGHTLIGHTS- Isogenic SCN2A-/- neurons display intrinsic hyperexcitability and impaired excitatory synapse function - SCN2A+/p.R607* variant reduces excitatory synapse function in patient neurons - C-terminal SCN2A+/p.G1744* variant enhances action potential properties but not synaptic transmission in patient neurons - SCN2A+/p.R607* variant display impacts on morphological and bioenergetic signaling networks through proteomic and functional analysis eTOC- Brown et al. examined Autism-associated SCN2A variants using patient-derived iPSC NGN2-neurons. They discover that genetic variants differentially impact neuronal development and synaptic function, and highlight neuronal and bioenergetic signaling networks underlying SCN2A loss-of-function.

neuroscience↗

bioRxiv2020

NtrX systemically controls transcription of the CtrA system genes to regulate Rhizobium cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by a signaling network centered on CtrA. The gene expression of regulatory proteins in the network can be regulated by nutrient signaling systems. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes in the CtrA signaling network. Three groups of S. meliloti ntrX mutants showed similar cell division defects, such as slow growth, abnormal morphology of some cells and delayed DNA synthesis. Quantitative RT-PCR assays indicated that in these mutants, the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZ1 was down-regulated. Western blotting showed that the CtrA and GcrA proteins were apparently increased in the mutants. The 53rd aspartate conserved in NtrX homologs can be phosphorylated in vitro and in vivo. The phosphorylated NtrX protein can bind directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN1-5TTG. Therefore, phosphorylation of NtrX is essential for cell cycle regulation of S. meliloti. We expressed the NtrX protein carrying a phosphorylation site substitution in Agrobacterium tumefaciens and found that the expressed strains had different growth phenotypes, suggesting that NtrX also regulates cell division in this bacterial species. Our findings reveal that NtrX acts as a transcriptional regulator that positively affects bacterial cell division, associated with nitrogen metabolism. IMPORTANCESinorhizobium meliloti infects the host legume alfalfa and induces the formation of nitrogen-fixing nodules. The proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of the interaction between symbiotic partners. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but the molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

molecular biology↗

bioRxiv2020

No endospore formation confirmed in members of the phylum Proteobacteria

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have documented endospore formation in species outside of Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens, members of the phylum Proteobacteria. Here, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes. ImportanceEndospore formation is a mechanism that allows bacteria to generate resilient dormant spores under harsh environmental conditions. Although this process has been traditionally restricted to the largely Gram-positive bacteria of the phylum Firmicutes, recent studies have also described endospores in some Proteobacteria. High complexity of endosporulation, reflected in extensive morphological transformations governed by hundreds of conserved genes, hinders its facile acquisition via horizontal gene transfer. Therefore, ability of distantly related bacteria to produce endospores would imply an ancient nature of this mechanism and potentially a pivotal role in species diversification and outer membrane biogenesis.

microbiology↗

bioRxiv2020

Human IgG cell neutralizing monoclonal antibodies block SARS-CoV-2 infection

The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development.

immunology↗

bioRxiv2018

Endogenous BioID elucidates TCF7L1 interactome modulation upon GSK-3 inhibition in mouse ESCs

Modulation of Wnt target gene expression via the TCF/LEFs remains poorly understood. We employ proximity-based biotin labeling (BioID) to examine GSK-3 inhibitor effects on the TCF7L1 interactome in mouse ESCs. We generated ESC lines with biotin ligase BirA* fused to TCF7L1 by knocking it into the endogenous TCF7L1 locus or by inserting a doxinducible BirA*-TCF7L1 transgene into the Rosa26 locus. Induction yielded BirA*-TCF7L1 levels 3-fold higher than in the endogenous system, but substantial overlap in biotinylated proteins with high peptide counts were detected by each method. Known TCF7L1 interactors TLE3/4 and {beta}-catenin, and numerous proteins not previously associated with TCF7L1, were identified in both systems. Despite reduced BirA*-TCF7L1 levels, the number of hits identified with both BioID approaches increased after GSK-3 inhibition. We elucidate the network of TCF7L1 proximal proteins regulated by GSK-3 inhibition, validate the utility of endogenous BioID, and provide mechanistic insights into TCF7L1 target gene regulation.\n\nHighlightsO_LIBirA*-TCF7L1 at single-copy physiological levels generates robust BioID data\nC_LIO_LICHIR99021 reduces TCF7L1 levels but increases detectable TCF7L1-proximal proteins\nC_LIO_LIThe TCF7L1 interactome of largely epigenetic/transcription factors fluctuates with GSK-3 inhibition\nC_LIO_LIJMJD1C, SALL4 and BRG1/SMARCA4 are validated as TCF7L-interacting proteins\nC_LI

cell biology↗