Biology subjects

Chen, W.

Publications and source records attributed to Chen, W..

At least 433 records · Page 24Linked to original sources

bioRxiv2021

Development and Application of Cas13a-based Diagnostic Assay for Neisseria Gonorrhoeae Detection and Identification of Azithromycin Resistance

Gonorrhea caused by Neisseria gonorrhoeae has spread world-wide. Antimicrobial-resistant strains have emerged to an alarming level to most antibiotics including to the ceftriaxone-azithromycin combination, currently recommended as first-line dual therapy. Rapid testing for antimicrobial resistance will contribute to clinical decision-making for rational drug use and will slow this trend. Herein, we developed a Cas13a-based assay for N. gonorrhoeae detection (porA target) and azithromycin resistance identification (A2059G and C2611T point mutations). We evaluated the sensitivity and specificity of this method, and 10 copies per reaction can be achieved in porA detection and C2611T identification, with no cross-reactions. Comparison of the Cas13a-based assay (porA target) with Roche Cobas 4800 assay (n=23 urine samples) revealed 100% concordance. Isolated N. gonorrhoeae strains were used to validate the identification of A2059G and C2611T resistance mutations. All tested strains (8 A2059G strains, 8 C2611T strains, and 8 wild-type strains) were successfully distinguished by our assay and verified by testing MIC for azithromycin and sequencing the 23S rRNA gene. We adopted lateral flow for the SHERLOCK assay readout, which showed a visible difference between test group and NC group results. To further evaluate the capability of our assay, we tested 27 urethral swabs from patients with urethritis for N. gonorrhoeae detection and azithromycin-resistance identification. Of these, 62.96% (17/27) strains were detected with no mutant strains and confirmed by sequencing. In conclusion, the novel Cas13a-based assay for rapid and accurate N. gonorrhoeae detection combined with azithromycin drug resistance testing is a promising assay for application in clinical practice.

microbiology↗

bioRxiv2021

A novel cholinergic neural pathway and its role in the drug relapse

The lateral parabrachial nucleus (LPB) is critical hub implicated in the control of food intake, reward and aversion. Here, we identified a novel cholinergic projection from choline acetyltransferase (ChAT)-positive neurons in external portion of the lateral parabrachial nucleus (eLPBChAT) to {gamma}-aminobutyric acid (GABA) neurons in central nucleus of amygdala (CeAGABA), activation of which could block methamphetamine (METH)-primed conditioned place preference (CPP) in mice.

neuroscience↗

bioRxiv2021

Permeability profiling of all 13 Arabidopsis PIP aquaporins using a high throughput yeast approach

Plant aquaporins have many more functions than just transporting water. Within the diversity of plant aquaporins are isoforms capable of transporting signaling molecules, nutrients, metalloids and gases. It is established that aquaporin substrate discrimination depends on combinations of factors such as solute size, pore size and polarity, and post-translational protein modifications. But our understanding of the relationships between variation in aquaporin structures and the implications for permeability is limited. High-throughput yeast-based assays were developed to assess diverse substrate permeabilities to water, H2O2, boric acid, urea and Na+. All 13 plasma membrane intrinsic proteins (PIPs) from Arabidopsis (AtPIPs) were permeable to both water and H2O2, although their effectiveness varied, and none were permeable to urea. AtPIP2 isoforms were more permeable to water than AtPIP1s, while AtPIP1s were more efficient at transporting H2O2 with AtPIP1;3 and AtPIP1;4 being the most permeable. Among the AtPIP2s, AtPIP2;2 and AtPIP2;7 were also permeable to boric acid and Na+. Linking AtPIP substrate profiles with phylogenetics and gene expression data enabled us to align substrate preferences with known biological roles of AtPIPs and importantly guide towards unidentified roles hidden by functional redundancy at key developmental stages and within tissue types. This analysis positions us to more strategically test in planta physiological roles of AtPIPs in order to unravel their complex contributions to the transport of important substrates, and secondly, to resolve links between aquaporin protein structure, substrate discrimination, and transport efficiency. One sentence summaryYeast based high throughput assays were developed to assess the permeability of each Arabidopsis PIP aquaporin isoform to water, H2O2, boric acid, urea and sodium.

plant biology↗

bioRxiv2021

Kaposi's Sarcoma-associated Herpesvirus vFLIP Promotes MEndT to Generate Hybrid M/E State for Tumorigenesis

Kaposis sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposis sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers PDGFRA/Nestin and endothelial markers PDPN/CD31. The hybrid M/E cells have acquired high tumorigenic properties in vitro and the potential to form KS-like tumors after transplanted in mice under renal capsules. These results faithfully recapitulate Kaposis sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit a hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposis sarcomagenesis. Author SummaryKaposis sarcoma manifests as multifocal lesions with spindle cell proliferation, intense angiogenesis, and erythrocyte extravasation. Although the origin and true malignant nature of KS remains contentious, it is established that KSHV infection with concomitant viral oncogene expression in normal cell progenitors causes KS. The mechanism of KSHV oncogenesis could be revealed through reproduction of KS by infection of normal cells. This study reports that the KSHV infection of mesenchymal stem cells initiates mesenchymal-to-endothelial transition (MEndT) that generates mesenchymal/endothelial (M/E) hybrid state cells. The hybrid M/E cells acquired high tumorigenic properties, including tumor initiation, angiogenesis, migration, and the potential to form KS-like tumors after transplanted in mice. This finding faithfully recapitulates Kaposis sarcoma where proliferating KS spindle cells and the cells that line KS-specific aberrant vessels are also found to exhibit the hybrid M/E state. We also found that KSHV-encoded viral FLICE inhibitory protein (vFLIP) plays a crucial role in promoting MEndT and the generation of M/E state cells. These results provide a new layer of evidence for MSCs being the cell source of KS spindle cells and reveal novel insight into KS pathogenesis and viral tumorigenesis.

microbiology↗

bioRxiv2021

Myofibroblast transcriptome indicates SFRP2+ fibroblast progenitors in systemic sclerosis skin

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells that arise from a variety of cell types in murine fibrosis models. Utilizing single cell RNA-sequencing to examine the transcriptome changes, we show that SSc dermal myofibroblasts arise from an SFRP2/DPP4-expressing progenitor fibroblast population that globally upregulates expression of transcriptome markers, such as PRSS23 and THBS1. Only a fraction of SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. The myofibroblast transcriptome implicates upstream transcription factors that drive myofibroblast differentiation.

cell biology↗

bioRxiv2021

Kaposi's Sarcoma-associated Herpesvirus Promotes Mesenchymal-to-Endothelial Transition by Resolving the Bivalent Chromatin of PROX1 Gene

Human mesenchymal stem cells (MSCs) are highly susceptible to Kaposis sarcoma-associated herpesvirus (KSHV) infection, and the infection promotes mesenchymal-to-endothelial transition (MEndT) and acquisition of Kaposis sarcoma (KS)-like phenotypes. Increasing evidence suggests that KS may arise from KSHV-infected MSCs. To understand how KSHV induces MEndT and transforms MSCs to KS cells, we investigated the mechanism underlying KSHV-mediated MSC endothelial lineage differentiation. Like embryonic stem cells, MSC differentiation and fate determination are under epigenetic control. Prospero homeobox 1 (PROX1) is a master regulator that controls lymphatic vessel development and endothelial differentiation. We found that the PROX1 gene in MSCs harbors a distinctive bivalent epigenetic signature consisting of both active marker H3K4me3 and repressive marker H3K27me3, which poises expression of the genes, allowing timely activation upon differentiation signals or environmental stimuli. KSHV infection effectively resolved the bivalent chromatin by decreased H3K27me3 and increased H3K4me3 to activate the PROX1 gene. vIL-6 signaling leads to the recruitment of MLL2 and Set1 complexes to the PROX1 promoter to increase H3K4me3, and the vGPCR-VEGF-A axis is responsible for removing PRC2 from the promoter to reduce H3K27me3. Therefore, through a dual signaling process, KSHV activates PROX1 gene expression and initiates MEndT, which renders MSC tumorigenic features including angiogenesis, invasion and migration.

microbiology↗

bioRxiv2021

Personalized genome assembly for accurate cancer somatic mutation discovery using cancer-normal paired reference samples

The use of personalized genome assembly as a reference for detecting the full spectrum of somatic events from cancers has long been advocated but never been systematically investigated. Here we address the critical need of assessing the accuracy of somatic mutation detection using personalized genome assembly versus the standard human reference assembly (i.e. GRCh38). We first obtained massive whole genome sequencing data using multiple sequencing technologies, and then performed de novo assembly of the first tumor-normal paired genomes, both nuclear and mitochondrial, derived from the same donor with triple negative breast cancer. Compared to standard human reference assembly, the haplotype phased chromosomal-scale personalized genome was best demonstrated with individual specific haplotypes for some complex regions and medical relevant genes. We then used this well-assembled personalized genome as a reference for read mapping and somatic variant discovery. We showed that the personalized genome assembly results in better alignments of sequencing reads and more accurate somatic mutation calls. Direct comparison of mitochondrial genomes led to discovery of unreported nonsynonymous somatic mutations. Our findings provided a unique resource and proved the necessity of personalized genome assembly as a reference in improving somatic mutation detection at personal genome level not only for breast cancer reference samples, but also potentially for other cancers.

bioinformatics↗

bioRxiv2021

CDCA-seq resolve the different chromatin structure on integral circular DNAs

Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In the majority of conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Here, we developed the sequencing of enzyme-accessible chromatin in circular DNA (CCDA-seq) method, which uses methylase to label open chromatin without fragmentation and exonuclease to enrich the ecDNA sequencing depth, followed by long-read nanopore sequencing. Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding in ecDNA at a single-molecule resolution. These results deepen the understanding of ecDNA regulatory mechanisms.

genomics↗

bioRxiv2021

New metabolic alterations and predictive marker pipecolic acid in sera for esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a major histological subtype of esophageal cancer with dismal prognosis. Although several serum metabolomic investigations have been reported, ESCC tumor-associated metabolic alterations along with predictive biomarkers in sera were not defined. Here we enrolled 34 treatment-naive ESCC patients and collected their pre-and post-esophagectomy sera together with sera from 34 healthy volunteers for metabolomic survey. Our comprehensive analysis discerned ESCC tumor-associated metabolic alterations as represented by a panel of 12 serum metabolites. Notably, postoperative abrosia and parenteral nutrition significantly perturbed the serum metabolome. Furthermore, we performed examination using sera from carcinogen-induced mice at dysplasia and ESCC stages, and identified three ESCC tumor-associated metabolites conserved between mice and humans. Notably, among these metabolites, pipecolic acid was progressively increased in mouse sera from dysplasia to cancerization, and it could accurately discriminate between mice at dysplasia stage and healthy control mice. Furthermore, this metabolite was essential for ECSS cells to oppose oxidative stress-induced DNA damage and cell proliferation arrest. Together, this study uncovered 12 ESCC tumor-associated serum metabolites with potential for monitoring therapeutic efficacy and disease relapse, presented evidence for refining parenteral nutrition composition, and highlighted serum pipecolic acid as an attractive biomarker for prediction of ESCC tumorigenesis.

cancer biology↗

bioRxiv2021

Genome-wide molecular recording using Live-seq

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity in health and disease. However, scRNA-seq requires lysing cells, which makes it impossible to link the individual cells to downstream molecular and phenotypic states. Here, we established Live-seq, an approach for single-cell transcriptome profiling that preserves cell viability during RNA extraction using fluidic force microscopy. Based on cell division, functional responses and whole-cell transcriptome read-outs, we show that Live-seq does not induce major cellular perturbations and therefore can function as a transcriptomic recorder. We demonstrate this recording capacity by preregistering the transcriptomes of individual macrophage-like RAW 264.7 cells that were subsequently subjected to time-lapse imaging after lipopolysaccharide (LPS) exposure. This enabled the unsupervised, genome-wide ranking of genes based on their ability to impact macrophage LPS response heterogeneity, revealing basal NFKBIA expression level and cell cycle state as major phenotypic determinants. Furthermore, we show that Live-seq can be used to sequentially profile the transcriptomes of individual macrophages before and after stimulation with LPS, thus enabling the direct mapping of a cells trajectory. Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

molecular biology↗

bioRxiv2021

In vivo co-registered hybrid-contrast imaging by successive photoacoustic tomography and magnetic resonance imaging

Magnetic resonance imaging (MRI) and photoacoustic tomography (PAT) are two advanced imaging modalities that offer two distinct image contrasts: MRI has a multi-parameter contrast mechanism that provides excellent anatomical soft tissue contrast, whereas PAT is capable of mapping tissue physiological metabolism and exogenous contrast agents with optical specificity. Attempts have been made to integrate these two modalities, but rigid and reliable registration of the images for in vivo imaging is still challenging. In this paper, we present a complete hardware-software solution for the successive acquisition and co-registration of PAT and MRI images in in vivo animal studies. Based on commercial PAT and MRI scanners, our solution includes a 3D-printed dual-modality animal imaging bed, a 3-D spatial image co-registration algorithm with bi-model markers, and a robust modality switching protocol for in vivo imaging studies. Using the proposed solution, we successfully demonstrated co-registered hybrid-contrast PAT-MRI imaging that simultaneously display multi-scale anatomical, functional and molecular characteristics on healthy and cancerous living mice. Week-long longitudinal dual-modality imaging of tumor development reveals information on size, border, vascular pattern, blood oxygenation, and molecular probe metabolism of the tumor micro-environment at the same time. Additionally, by incorporating soft-tissue information in the co-registered MRI image, we further show that PAT image quality could be enhanced by MRI-guided light fluence correction. The proposed methodology holds the promise for a wide range of pre-clinical research applications that benefit from the PAT-MRI dual-modality image contrast.

bioengineering↗

bioRxiv2021

A feedback mechanism mediated by myosin II-dependent apical targeting of Rab11 vesicles reinforces apical constriction

During tissue morphogenesis, cell shape changes resulting from cell-generated forces often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin dependent mechanism involving Rab11- mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.

developmental biology↗

bioRxiv2021

Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-{kappa}B from DNA is achieved through accelerated dissociation by the inhibitor protein I{kappa}B. Using single-molecule FRET, we observe a continuum of conformations of NF-{kappa}B in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-{kappa}B is partially bound, allowing I{kappa}B invasion to facilitate DNA dissociation. I{kappa}B induces a locked conformation where the DNA-binding domains of NF-{kappa}B are too far apart to bind DNA, whereas the loss-of-function I{kappa}B mutant retains the NF-{kappa}B conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

biophysics↗

bioRxiv2021

Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

Differences in gene expression, which can arise from divergence in cis-regulatory elements or alterations in transcription factors binding specificity, are one of the most important causes of phenotypic diversity during evolution. By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent. Author summaryOur study 1) represented a first systematic analysis of species-specific adaptation in DNA binding pattern of transcription factor. Although the mouse-specific amino acid changes did not manifest functional impact in our system, several explanations may account for it (See Discussion part for the detail); 2) represented a first study of cis-regulation between two reproductively isolated species by using a novel allodiploid system; 3) demonstrated a higher conservation of transcriptional output than that of DNA binding, suggesting the evolvability/plasticity of the latter; 4) finally provided a rich data resource for Cdx2 mediated regulation, including gene expression, chromatin accessibility and DNA binding etc.

evolutionary biology↗

bioRxiv2021

Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study

With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

bioinformatics↗

bioRxiv2021

Comprehensive evaluation of ACE2 expression in female ovary by single-cell RNA-seq analysis

Pneumonia induced by severe acute respiratory coronavirus 2 (SARS-CoV-2) via ACE2 receptor may affect many organ systems like lung, heart and kidney. An autopsy report revealed positive SARS-Cov-2 detection results in ovary, however, the developmental-stage-specific and cell-type-specific risk in fetal primordial germ cells (PGCs) and adult women ovary remained unclear. In this study, we used single-cell RNA-sequencing (scRNA-seq) datasets spanning several developmental stages of ovary including PGCs and cumulus-oocyte complex (COC) to investigate the potential risk of SARS-CoV-2 infection. We found that PGCs and COC exhibited high ACE2 expression. More importantly, the ratio of ACE2-positive cells was sharply up-regulated in primary stage and ACE2 was expressed in all oocytes and cumulus cells in preovulatory stage, suggesting the possible risk of SARS-CoV-2 infection in follicular development. CatB/L, not TMPRSS2, was identified to prime for SARS-CoV-2 entry in follicle. Our findings provided insights into the potential risk of SARS-CoV-2 infection during folliculogenesis in adulthood and the possible risk in fetal PGCs.

developmental biology↗

bioRxiv2021

Comprehensive Assessment of Somatic Copy Number Variation Calling Using Next-Generation Sequencing Data

Copy number variation (CNV) is a common type of mutation that often drives cancer progression. With advances in next-generation sequencing (NGS), CNVs can be detected in a detailed manner via newly developed computational tools but quality of such CNV calls has not been carefully evaluated. We analyzed CNV calls reported by 6 cutting-edge callers for 91 samples which were derived from the same cancer cell line, prepared and sequenced by varying the following factors: type of tissue sample (Fresh vs. Formalin Fixed Paraffin Embedded (FFPE)), library DNA amount, tumor purity, sequencing platform (Whole-Genome Sequencing (WGS) versus Whole-Exome Sequencing (WES)), and sequencing coverage. We found that callers greatly determined the pattern of CNV calls. Calling quality was drastically impaired by low purity (<50%) and became variable when WES, FFPE, and medium purity (50%-75%) were applied. Effects of low DNA amount and low coverage were relatively minor. Our analysis demonstrates the limitation of benchmarking somatic CNV callers when the real ground truth is not available. Our comprehensive analysis has further characterized each caller with respect to confounding factors and examined the consistency of CNV calls, thereby providing guidelines for conducting somatic CNV analysis.

bioinformatics↗

bioRxiv2021

B/tMSI-CAST: a systematic approach for microsatellite instability detection in both tumor tissue and plasma specimens from next-generation sequencing data

Microsatellite instability (MSI) is a well-established prognostic and predictive biomarker in certain types of cancers. MSI detection using tumour tissue is often limited by the availability of specimens. Next generation sequencing (NGS)-based MSI detection in plasma cell-free DNA (cfDNA) is challenged by a much lower signal-to-noise ratio. We developed a highly accurate cfDNA MSI detection method called bMSI-CAST (blood MSI Caller Adjusted with Sequence duplicaTes), with improvement on three features including a set of locus selection principles ensuring loci with high robustness and compatibility across sequencing platforms, an MSI-specific duplicate removal strategy, and a calling algorithm that dynamically matches baselines with a broad range of duplication levels. Analytical validation via MSI-high (MSI-H) cell gDNA showed an LOD of 0.15%. Furthermore, in an analysis of 95 evaluable cfDNA samples from patients with gastrointestinal cancers, bMSI-CAST exhibited a positive predictive agreement (PPA) of 92.9% (39/42) and negative predictive agreement (NPA) of 100% (53/53) with tissue MSI-PCR. In conclusion, bMSI-CAST provides novel and advanced solutions to key aspects fundamental to cfDNA MSI calling but not sufficiently addressed by existing methods, and it is a validated method ready to be applied to aid clinical decisions for cancer patients.

bioinformatics↗