Biology subjects
Publications and source records attributed to Wu, L..
Recent advances in large-scale single cell RNA-seq enable fine-grained characterization of phenotypically distinct cellular states within heterogeneous tissues. We present scScope, a scalable deep-learning based approach that can accurately and rapidly identify cell-type composition from millions of noisy single-cell gene-expression profiles.
Probiotic candidate L. reuteri was screened out for in vivo experiments based on a relatively higher gastrointestinal tolerance and moderate adhesiveness. As results shown in in-vivo experiments, a significantly higher level of IL-12 at low-dose group was found both in females and males. Higher levels of T-lymphocytes were also observed in females compared to control group, however, males displayed a reduction expcept for CD8-positive cells in ileum. In comparison to the control group, the relative abundance of phylotypes in the phylum Bacteroidetes (genus of Bacteroides, Prevotella) and Firmicutes (genus of ClostridiumIV) exihibited a reserve shift between sexes after L. reuteri intervened. Meanwhile, the relative abundance of several taxa (Acetobacteroides, Lactobcaillus, bacillus) also differed markedly in sexes at low-dose group, together with microbiota diversity, as indicated by Shannon index.\n\nImportanceSexual dimorphism has triggered researchers attention. However, the relationship between immune parameters and gut microbiota caused by Lactobacillus at different dosage are not fully elucidated. In present research, the possible probiotic role of L. reuteri DMSZ 8533 on immunomodulation and effect on fecal microbiota composition were investigated. Our findings demonstrate the importance of L. reuteri DMSZ 8533 as a potential probiotic strain with an immunomodulatory effect, which also alters the microflora composition depending on the sex of the host.
ObjectivesCycloserine is crucial in multidrug-resistant tuberculosis (MDR-TB) treatment. Although extensive research has been carried out on MDR-TB, most researchers have not treated cycloserine in much detail. Therefore, we evaluate the efficacy and safety of cycloserine and seek to clarify the role of cycloserine for treatment of simple MDR-TB, pre-extensively drug-resistant tuberculosis (pre-XDR-TB), and extensively drug-resistant tuberculosis (XDR-TB).\n\nPatients and methodsA retrospective observational study was performed in China. We determined the treatment outcome as the primary outcome for 144 cycloserine-treated and 181 cycloserine-nontreated patients according to the definitions of WHO. The proportion of patients with sputum-culture conversion and the frequency of adverse drug reactions related to cycloserine were assessed as well.\n\nResultsAmong 325 MDR-TB patients, 144 were treated with cycloserine and 100 (69.4%) out of 144 successfully completed treatment. Compared with patients in non-cycloserine group, the hazard ratio of any unfavorable treatment outcome was 0.53 (95%CI: 0.35-0.81, P=0.003). Culture conversion rate at the intensive phase was similar whether cycloserine was administered or not (P=0.703). Of the 144 patients treated with cycloserine, a total of 16 (11.1%) patients experienced side-effects related to cycloserine, including 7 patients who discontinued cycloserine permanently.\n\nConclusionsCycloserine could be an attractive agent to treat MDR-TB. Its safety profile warrants use in the most of MDR-TB cases. Cycloserine significantly improved the chance of favorable outcome for patients with simple MDR-TB but not pre-XDR-TB and XDR-TB. More aggressive regimens might be required for pre-XDR-TB or XDR-TB patients.
Shotgun proteomics has grown rapidly in recent decades, but a large fraction of tandem mass spectrometry (MS/MS) data in shotgun proteomics are not successfully identified. We have developed a novel database search algorithm, Open-pFind, to efficiently identify peptides even in an ultra-large search space which takes into account unexpected modifications, amino acid mutations, semi- or non-specific digestion and co-eluting peptides. Tested on two metabolically labeled MS/MS datasets, Open-pFind reported 50.5-117.0% more peptide-spectrum matches (PSMs) than the seven other advanced algorithms. More importantly, the Open-pFind results were more credible judged by the verification experiments using stable isotopic labeling. Tested on four additional large-scale datasets, 70-85% of the spectra were confidently identified, and high-quality spectra were nearly completely interpreted by Open-pFind. Further, Open-pFind was over 40 times faster than the other three open search algorithms and 2-3 times faster than three restricted search algorithms. Re-analysis of an entire human proteome dataset consisting of [~]25 million spectra using Open-pFind identified a total of 14,064 proteins encoded by 12,723 genes by requiring at least two uniquely identified peptides. In this search results, Open-pFind also excelled in an independent test for false positives based on the presence or absence of olfactory receptors. Thus, a practical use of the open search strategy has been realized by Open-pFind for the truly global-scale proteomics experiments of today and in the future.
Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in non-dividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T-cells that are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 LTR activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T-cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 long terminal repeat (LTR)-driven gene expression at the level of transcription. SAMHD1 overexpression also suppressed LTR activity from human T-cell leukemia virus type 1 (HTLV-1), but not from murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D/R207N, or HD/RN) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression contributes to regulation of viral latency in CD4+ T-cells.\n\nIMPORTANCEA critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T-cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in non-dividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T-cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T-cells.
BackgroundThe internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3, or Y1-3) bind to m6A-modified cellular mRNA and modulate its metabolism and processing, thereby affecting protein translation in cells. We previously reported that HIV-1 RNA contains m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription. Here we extended our studies to further understand the mechanisms of Y1-3-mediated inhibition of HIV-1 infection and viral production.\n\nResultsOverexpression of Y1-3 proteins in HIV-1 target cells decreased viral genome RNA (gRNA) levels and inhibited early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5 leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5 leader sequence of gRNA demonstrated significantly lower infectivity compared with wild-type HIV-1, confirming that these sites are important for viral infection. HIV-1 produced from cells with knockdown of endogenous Y1, Y3, or Y1-3 proteins together showed increased viral infectivity compared with HIV-1 produced from control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag formed a complex with RNA in HIV-1-infected target cells.\n\nConclusionsOur results suggest the inhibitory effects of Y1-3 proteins on HIV-1 infection and provide new insight into the mechanisms of m6A modification of HIV-1 RNA in regulating viral replication, which clarify some discrepancies in the previously published studies in this area.
Integrating genome-wide association (GWAS) and expression quantitative trait locus (eQTL) data into transcriptome-wide association studies (TWAS) based on predicted expression can boost power to detect novel disease loci or pinpoint the susceptibility gene at a known disease locus. However, it is often the case that multiple eQTL genes colocalize at disease loci, making the identification of the true susceptibility gene challenging, due to confounding through linkage disequilibrium (LD). To distinguish between true susceptibility genes (where the genetic effect on phenotype is mediated through expression) and colocalization due to LD, we examine an extension of the Mendelian Randomization Egger regression method that allows for LD while only requiring summary association data for both GWAS and eQTL. We derive the standard TWAS approach in the context of Mendelian Randomization and show in simulations that the standard TWAS does not control Type I error for causal gene identification when eQTLs have pleiotropic or LD-confounded effects on disease. In contrast, LD Aware MR-Egger regression can control Type I error in this case while attaining similar power as other methods in situations where these provide valid tests. However, when the direct effects of genetic variants on traits are correlated with the eQTL associations, all of the methods we examined including LD Aware MR-Egger regression can have inflated Type I error. We illustrate these methods by integrating gene expression within a recent large-scale breast cancer GWAS to provide guidance on susceptibility gene identification.
Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.
Human SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular nucleotides required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory SAM domain. In this study, we determined the first crystal structure of full-length SAMHD1 by capturing mouse SAMHD1 (mSAMHD1) structures in three different nucleotide bound states. Although mSAMHD1 and hSAMHD1 are highly similar in sequence and function, we found that mSAMHD1 possesses a more complex nucleotide-induced activation process, highlighting the regulatory role of the SAM domain. Our results provide new insights into the regulation of SAMHD1 activity, thereby will facilitate the improvement of HIV mouse models and the development of new therapies for certain cancers and autoimmune diseases.
Based on a novel kind of single strand adaptor (SSA), this study developed a new method to construct next-generation sequencing (NGS) library, named as SALP, representing Single strand Adaptor Library Preparation. The key creativity of the method lies in the design and verification of a special adaptor that can be efficiently linked to the 3' end of single-stranded DNA, which is a double-stranded oligonucleotide with a 3' overhang of 3 random nucleotides. This method can start with the denatured DNAs or chromatins fragmented by different methods such as Tn5 tagmentation, enzyme digestion and sonication. When applied to Tn5-tagmented chromatin, SALP overcomes the key limitation of the current ATAC-seq method and develops a high-throughput NGS library construction and sequencing approach, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells simply and unbiasly. In this way, the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, were successfully characterized. This study also demonstrated that SALP-seq could characterize the chromatin openness states with 105 to 500 cells, indicating the high sensitivity of SALP-seq in characterizing chromatin state of cells. SALP should have wide applications in the future biological sciences and biomedicine.
Chronic exposure to intraperitoneal asbestos triggered a marked response in the mesothelium well before tumor development. Macrophages, mesothelial precursor cells, cytokines and growth factors accumulated in the peritoneal lavage. Transcriptome profiling revealed YAP/TAZ activation in inflamed mesothelium with further activation in tumors, paralleled by increased levels of cells with nuclear YAP/TAZ. Arg1 was one of the highest upregulated genes in inflamed tissue and tumor. Inflamed tissue showed increased levels of single nucleotide variations, with an RNA-editing signature, which were even higher in the tumor samples. Subcutaneous injection of asbestos-treated, but tumor-free mice with syngeneic mesothelioma tumor cells resulted in a significantly higher incidence of tumor growth when compared to naive mice supporting the role of the environment in tumor progression.\n\nHighlightsO_LIAsbestos increases levels of cytokines and growth factors in mesothelium environment\nC_LIO_LIRecruitment of macrophages and mesothelial precursor cells prior to tumor development\nC_LIO_LIYAP/TAZ signaling upregulated in pre-neoplastic tissues and cancer\nC_LIO_LIIncreased RNA-editing and somatic mutations as early steps in tumor development\nC_LI
Hybridization is a key molecular process in biology and biotechnology, but to date there is no predictive model for accurately determining hybridization rate constants based on sequence information. To approach this problem systematically, we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (subsequences of the CYCS and VEGF genes) at temperatures ranging from 28 {degrees}C to 55 {degrees}C. Next, we rationally designed 38 features computable based on sequence, each feature individually correlated with hybridization kinetics. These features are used in our implementation of a weighted neighbor voting (WNV) algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants (a.k.a. labeled instances). Automated feature selection and weighting optimization resulted in a final 6-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 2 with {approx}74% accuracy and within a factor of 3 with {approx}92% accuracy, based on leave-one-out cross-validation. Predictive understanding of hybridization kinetics allows more efficient design of nucleic acid probes, for example in allowing sparse hybrid-capture panels to more quickly and economically enrich desired regions from genomic DNA.
A hallmark of systemic lupus erythematosus is high titers of circulating autoantibody. A novel CD11c+ B cell subset has been identified that is critical for the development of autoimmunity. However, the role of CD11c+ B cells in the development of lupus is unclear. Chronic graft-versus-host disease (cGVHD) is a lupus-like syndrome with great autoantibody production. In the present study we investigated the role of CD11c+ B cells in the pathogenesis of lupus in the cGVHD model. Here, we found the percentage and absolute number of CD11c+ B cells and titer of sera anti-chromatin IgG and IgG2a antibody were increased in cGVHD mice. CD11c+ plasma cells from cGVHD mice produced large amounts of anti-chromatin IgG2a upon stimulation. Depletion of CD11c+ B cells reduced anti-chromatin IgG and IgG2a production. T-bet expression was further shown to be upregulated in CD11c+ B cells. Knockout of T-bet in B cells alleviated cGVHD. The percentage of T-bet+ CD11c+ B cells was elevated in lupus patients and positively correlated with serum anti-chromatin levels. Our findings suggest T-bet+ CD11c+ B cells contribute to the pathogenesis of lupus and provides potential target for therapeutic intervention.