Biology subjects

Chen, W.

Publications and source records attributed to Chen, W..

At least 379 records · Page 21Linked to original sources

bioRxiv2022

The Receptor Kinase FER Mediates Phase Separation of Glycine-Rich RNA-Binding Protein 7 to Confer Temperature Resilience in Arabidopsis

Temperature fluctuations repress plant growth. Although glycine-rich RNA-binding proteins (GRPs) and cold shock proteins (CSPs) have been implicated in cold adaptation, their physiological roles in the response to temperature fluctuations are largely unknown. The receptor kinase FERONIA (FER), a master regulator of cell growth, phosphorylates GRP7 within its intrinsically disordered region to modulate mRNA alternative splicing in the nucleus. Here we show that natural variations at a GRP7 residue phosphorylated by FER influences GRP7 liquid-liquid phase separation (LLPS), aiding Arabidopsis to grow over a wider temperature range. LLPS of GRP7 in the cytoplasm leads to the formation of stress granules that recruits RNAs, along with the translation machinery component eIF4E1 and mRNA chaperones, CSP1 and CSP3, to inhibit translation. Mutations in FER and the GRP7-LLPS-recruited components attenuate root growth under temperature shift conditions. Our findings illustrate the roles of GRP7 LLPS in improving plant root capacity to withstand temperature fluctuations.

plant biology↗

bioRxiv2022

Bessel-droplet foci enable high-resolution and high-contrast volumetric imaging of synapses and circulation in the brain in vivo

Bessel beam has long been utilized in physics for its ability to maintain lateral confinement during propagation. When used for two-photon fluorescence microscopy, Bessel foci have enabled high-speed volumetric imaging of the brain. At high numeric aperture (NA), however, the substantial energy in the side rings of Bessel foci reduces image contrast. Therefore, a compromise between resolution and contrast has to be made, limiting Bessel foci in microscopy to low NA. Here, we describe a method of generating axially extended Bessel-droplet foci with much suppressed side rings. Shaping the excitation wavefront with novel phase patterns, we generated Bessel-droplet foci of variable NAs at high power throughput and scanned them interferometrically along the axial direction for continuous volume imaging. More resistant to optical aberrations than Bessel foci, Bessel-droplet foci enabled high-resolution and high-contrast volumetric imaging of synaptic anatomy and function as well as lymphatic circulation in the mouse brain in vivo.

bioengineering↗

bioRxiv2022

High coverage of single cell genomes by T7-assisted enzymatic methyl-sequencing

Conventional approaches to studying 5mC marks in single cells or samples with picogram input DNA amounts usually suffer from low genome coverage due to DNA degradation. Many methods have been developed to optimize the library construction efficiency for bisulfite-treated DNA. However, most of these approaches ignored the amplification bias of bisulfite-treated DNA, which leads to shallow genome coverage. In this study, we developed the T7-assisted enzymatic methyl-sequencing method (TEAM-seq), which adopts enzymatic conversion to minimize DNA degradation and T7 polymerase-assisted unbiased amplification. We demonstrate that TEAM-seq delivered, to the best of our knowledge, the highest reported coverage(70% for 100pg, 35% for 20pg) of single cell genomes in whole-genome 5mC sequencing.

genomics↗

bioRxiv2022

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

immunology↗

bioRxiv2022

The sialidase NEU3 promotes pulmonary fibrosis in mice

Sialic acid is often the distal sugar on glycoconjugates, and sialidases are enzymes that remove this sugar. In fibrotic lesions in human and mouse lungs, there is extensive desialylation of glycoconjugates, and upregulation of sialidases including the extracellular sialidase NEU3. In the bleomycin model of pulmonary fibrosis, mice lacking NEU3 (Neu3-/-) showed strongly attenuated bleomycin-induced weight loss, lung damage, inflammation, upregulation of TGF-{beta}1, and fibrosis. This indicates that NEU3 is necessary for the full spectrum of bleomycin-induced pulmonary fibrosis. To determine if NEU3 is sufficient for fibrosis, mice not treated with bleomycin were treated with recombinant murine NEU3 or inactive NEU3. Aspiration of NEU3 caused inflammation and fibrosis in the lungs, while inactive NEU3 caused inflammation but not fibrosis. Mice were also treated with NEU3 starting 10 days after bleomycin. In male but not female mice, NEU3 increased inflammation and fibrosis. Inactive NEU3 did not enhance bleomycin-induced lung fibrosis. These results suggest that NEU3 is sufficient to induce fibrosis in the lungs, and that this effect is mediated by NEU3s enzymic activity.

immunology↗

bioRxiv2022

A Robust and Automated Spike Sorting Method based on the Density Peaks

ObjectiveSpike sorting is a fundamental step in extracting single-unit activity from neural ensemble recordings, which play an important role in basic neuroscience and neurotechnologies. A few algorithms have been applied in spike sorting. However, when noise level or waveform similarity becomes relatively high, their robustness still faces a big challenge. ApproachIn this study, we propose a spike sorting method combining Linear Discriminant Analysis (LDA) and Density Peaks (DP) for feature extraction and clustering. Relying on the joint optimization of LDA and DP: DP provides more accurate classification labels for LDA, LDA extracts more discriminative features to cluster for DP, and the algorithm achieves high performance after iteration. We first compared the proposed LDA-DP algorithm with several algorithms on one publicly available simulated dataset and one real rodent neural dataset with different noise levels. We further demonstrated the performance of the LDA-DP method on a real neural dataset from non-human primates with more complex distribution characteristics. Main resultsThe results show that our LDA-DP algorithm extracts a more discriminative feature subspace and achieves better cluster quality than previously established methods in both simulated and real data. Especially in the neural recordings with high noise levels or waveform similarity, the LDA-DP still yields a robust performance with automatic detection of the number of clusters. SignificanceThe proposed LDA-DP algorithm achieved high sorting accuracy and robustness to noise, which offers a promising tool for spike sorting and facilitates the following analysis of neural population activity.

neuroscience↗

bioRxiv2022

Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterised the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to other insect species, these methods could be applicable across agricultural insect pests.

evolutionary biology↗

bioRxiv2022

GLH/VASA helicases promote germ granule formation to ensure the fidelity of piRNA-mediated transcriptome surveillance

The ability to distinguish non-self from self is the key characteristic for any defense system. piRNAs function as guardians of the genome by silencing non- self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observed that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition was also observed in other mutants where perinuclear P granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA- based transcriptome surveillance in C. elegans and preserve self versus non-self distinction.

cell biology↗

bioRxiv2022

Encoding sensing functions into material interface for a rationally engineered integrated electrochemical liquid biopsy

Limited healthcare capacity highlights the needs of integrated and simple sensing systems for personalized health monitoring. However, only a limited set of sensors can be employed for point-of-care applications, emphasizing the lack of a generalizable engineering strategy for sensor construction. Here, we report a de novo rational engineering strategy for the construction of an integrated electrochemical liquid biopsy (ELB) platform capable of direct profiling cancer exosomes from blood. Using a bottom-up approach for sensor design, a series of critical sensing functions is considered and encoded into the material interface by programming the electrode material with different chemical and structure features. We present that the rationally engineered electrochemical liquid biopsy platform is able to achieve one-step sensor fabrication, target isolation, non-fouling and high-sensitivity sensing, direct signal transduction and multiplexed detection. Integrating the multiplexed sensing with principal component analysis, we demonstrate the capability of the programmed sensing system on differentiating cancerous groups from healthy controls by analyzing clinical samples from lung cancer patients.

bioengineering↗

bioRxiv2022

Catch bond models explain how force amplifies TCR signaling and antigen discrimination

Central to T cell biology, the T cell receptor (TCR) integrates forces in its triggering process upon interaction with peptide-major histocompatibility complex (pMHC)1-3. Phenotypically, forces elicit TCR catch-slip bonds with strong pMHCs but slip-only bonds with weak pMHCs4-10. While such correlation is commonly observed, the quantitative bond pattern and degree of "catchiness" vary. We developed two models based on the structure, elastic properties, and force-induced conformational changes of the TCR-pMHC-I/II complexes to derive from their bond characteristics more intrinsic parameters that underlie structural mechanisms, predict T cell signaling, and discriminate antigens. Applying the models to 55 datasets of 12 {beta}TCRs and their mutants interacting with corresponding pMHCs without coreceptor engagement demonstrated the ability for structural and physical parameters to quantitatively integrate and classify a broad range of bond behaviors and biological activities. Comparing to the generic two-state model for catch-slip bond that also fits the data, our models can distinguish class I from class II MHC systems and their best-fit parameters correlate with the TCR/pMHC potency to trigger T cell activation, which the generic model cannot. The models were tested by mutagenesis using structural analysis, bond profile measurement, and functional assay of a MHC and a TCR mutated to alter conformation changes. The extensive comparisons between theory and experiment provided strong validation of the models and testable hypothesis regarding specific conformational changes that control bond profiles, thereby suggesting structural mechanisms for the inner workings of the TCR mechanosensing machinery and plausible explanation of why and how force may amplify TCR signaling and antigen discrimination.

biophysics↗

bioRxiv2022

CHDbase: A Comprehensive Knowledgebase for Congenital Heart Disease-related Genes and Clinical Manifestations

Congenital heart disease (CHD) is the most common cause of major birth defects, with a prevalence of 1%. Although an increasing number of studies reporting the etiology of CHD, the findings scattered throughout the literature are difficult to retrieve and utilize in research and clinical practice. We therefore developed CHDbase, an evidence-based knowledgebase with CHD-related genes and clinical manifestations manually curated from 1114 publications, linking 1124 susceptibility genes and 3591 variations to more than 300 CHD types and related syndromes. Metadata such as the information of each publication and the selected population and samples, the strategy of studies, and the major findings of study were integrated with each item of research record. We also integrated functional annotations through parsing ~50 databases/tools to facilitate the interpretation of these genes and variations in disease pathogenicity. We further prioritized the significance of these CHD-related genes with a gene interaction network approach, and extracted a core CHD sub-network with 163 genes. The clear genetic landscape of CHD enables the phenotype classification based on the shared genetic origin. Overall, CHDbase provides a comprehensive and freely available resource to study CHD susceptibility, supporting a wide range of users in the scientific and medical communities. CHDbase is accessible at http://chddb.fwgenetics.org/.

genetics↗

bioRxiv2021

Biological characteristics of two mesenchymal stem cell lines isolated from the umbilical cord and adipose tissue of a neonatal common hippo (Hippopotamus amphibius)

Mesenchymal stem cells (MSCs) are multipotent adult stem cells and can be isolated from many tissues of the body. Due to their potentials to treat various diseases and be applied in animal breeding, MSCs have been isolated and identified regarding their biological properties. Common hippos (Hippopotamus amphibius) are a vulnerable species and yet the cryopreservation of their genetic materials is scare. In this study, we successfully established two MSC lines (UC-MSCs and AT-MSCs) from the umbilical cord and adipose tissue of a neonatal common hippo and comparatively described their features. Both UC-MSCs and AT-MSCs showed fibroblastoid morphology and could be continuously passaged for over 17 passages without dramatic signs of senescence. The cell cultures had normal chromosome composition, say, 17 pairs of autosomes and 1 pair of X chromosomes. UC-MSCs and AT-MSCs displayed similar gene expression profiles. They were positive for CD45, CD73, CD90 and CD105 and negative for HLA-DR. They demonstrated stemness maintenance by expression of classical stem cell markers. UC-MSCs and AT-MSCs manifested different differentiation potentials into other cell lineages. In summary, these two cell lines demonstrated the essential properties of mesenchymal stem cells and might play a role in the future research.

cell biology↗

bioRxiv2021

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0x10-12 M) and strong neutralizing activity (IC50 = 0.0017 nM). Together, we developed a tetravalent biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

microbiology↗

bioRxiv2021

Comprehensive Benchmarking of CITE-seq versus DOGMA-seq Single Cell Multimodal Omics

The recently developed transcription, epitopes, and chromatin accessibility by sequencing (TEA-seq) and similar DOGMA-seq single-cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent optimization, benchmarking and evaluation are lacking. We explored the utility, pros and cons of DOGMA-seq compared to the bimodal cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) assay in activated and stimulated human peripheral blood T cells. We identified an optimal incubation time and concentration of digitonin (DIG) for cell permeabilization and found that single-cell trimodal omics measurements after DIG permeabilization were generally better than after an alternative "low-loss lysis" (LLL) permeabilization condition. Next, we found that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, even though its mRNA and cell surface protein antibody-derived tag (ADT) libraries have slightly inferior quality, compared to CITE-seq. Finally, we recognized the additional value of DOGMA-seq for studying lineage-specific T helper cells.

genomics↗

bioRxiv2021

Comprehensive mapping of the alternative polyadenylation site usage and its dynamics at single cell resolution

Alternative polyadenylation (APA) plays an important role in post-transcriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at single cell level is largely unexplored. Here we developed single cell polyadenylation sequencing (scPolyA-seq), a strand-specific approach for sequencing 3 end of transcripts, to investigate the landscape of APA at single cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle was significantly enriched in genes showing high variation of polyA site usages. We further identified 414 genes showing polyA site usage switch after cell synchronization. Genes showing cell cycle associated polyA site usage switch were grouped into 6 clusters, with cell phase specific functional categories enriched in each cluster. Furthermore, scPolyA-seq could facilitate study of APA in various biological processes.

bioinformatics↗

bioRxiv2021

MAL suppresses OSCC tumorigenesis by maintaining epithelial cell differentiation

Oral squamous cell carcinoma (OSCC) is widely recognized as an optimal model for precise medicine guided molecular biomarkers of cancer, however, few clinical practices were applied till now. Based on the data from our own studies and published papers, it was found that the expression of MAL was significantly decreased in epithelial cancer as compared with normal tissues, and exhibited a opposite association with pathological grade. To study the molecular events related to deficiency of MAL during carcinogenesis, occurrence and development, a Mal knockout mouse model was constructed and consistently reproduced and bred. The Mal knockout mice are highly vulnerable to tumor induction by carcinogen of 4NQO, evidenced by their extremely earlier carcinogenesis, higher incidence, and more aggressive growth. Analysis of scRNA-seq data indicated that Mal knockout mice lost the ability in maintaining epithelial cell differentiation and get more prone to carcinogen with a remarkably higher incidence of epithelial malignancy. Further analyses identified putative co-functional genes of MAL, including DSG1, AQP3 and S100A8, which are key factors in maintaining epithelial cell differentiation. To conclude, the current study exhibits the clinical significance and explains the tumor suppressing function of MAL. The results also suggest the potential of MAL and its co-functional genes being biomarkers for designing the prevention and/or differentiation therapy strategies in OSCC. SignificanceMAL is found to be strongly opposite with tumor pathological grade from clinical and in vivo studies in OSCC. We propose MAL and its co-functional genes, including DSG1, AQP3 and S100A8, as key factors in maintaining epithelial cell differentiation and are valuable targets for designing prevention and differentiation therapy strategies in OSCC.

cell biology↗

bioRxiv2021

Repressing PTB is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease

Lineage reprograming of resident glia cells to induced dopaminergic neurons (iDAns) holds attractive prospect for cell-replacement therapy of Parkinsons disease (PD). Recently, whether repressing polypyrimidine tract binding protein 1 (PTBP1) could truly achieve efficient astrocyte-to-iDAn conversion in substantia nigra and striatum aroused widespread controversy. Although reporter positive iDAns were observed by two groups after delivering adeno-associated virus (AAV) expressing a reporter with shRNA or Crispr-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage tracing method. By adopting stringent lineage tracing strategy, two other studies showed that neither knockdown nor genetic deletion of quiescent astroglial PTBP1 fails to obtain iDAns under physiological condition. However, the role of reactive astrocyte might be underestimated since upon brain injury, reactive astrocyte could acquire certain stem cell hallmarks which may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage tracing method to investigate whether reactive astrocytes could be converted to DAns in the 6-OHDA PD model. However, we found that no astrocyte-originated DAn was generated after effective knockdown of astroglial PTBP1 either in the substantia nigra or in the striatum, while AAV "leakage" to nearby neurons was observed. Our results further confirmed that repressing PTBP1 is unable to convert astrocytes to DAns no matter in physiological or PD-related pathological conditions. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/468309v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@fa2fa5org.highwire.dtl.DTLVardef@14e328eorg.highwire.dtl.DTLVardef@d93faborg.highwire.dtl.DTLVardef@e32225_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAAV-shPtbp1 rapidly and efficiently induces viral-reporter-labeled DAns in mouse brain under physiological condition C_LIO_LIViral-reporter-positive DAns are not originated from PTBP1 repressed and lineage traced reactive astrocytes in a mouse PD model C_LI

neuroscience↗

bioRxiv2021

Integration of the Salmonella Typhimurium methylome and transcriptome following environmental or metabolic perturbation reveals DNA methylation and transcriptional regulation are largely decoupled

Despite being in a golden age of bacterial epigenomics, little work has systematically examined the plasticity and functional impacts of the bacterial DNA methylome. Here, we leveraged SMRT sequencing to examine the m6A DNA methylome of two Salmonella enterica ser. Typhimurium strains: 14028s and a {Delta}metJ mutant with derepressed methionine metabolism, grown in Luria Broth or a media that simulates the intracellular environment. We find that the methylome is remarkably static--over 95% of adenosine bases retain their methylation status across conditions. Integration of methylation with transcriptomic data revealed limited correlation between changes in methylation and gene expression. Further, examining the transcriptome in {Delta}yhdJ bacteria, lacking the m6A methylase with the most dynamic methylation pattern in our dataset, revealed little evidence of YhdJ-mediated gene regulation. Curiously, despite G(m6A)TC motifs being particularly resistant to change across conditions, incorporating dam mutants into our analyses revealed two examples where changes in methylation and transcription may be linked across conditions. This includes the novel finding that the {Delta}metJ motility defect may be partially driven by hypermethylation of the chemotaxis gene tsr. Together, these data redefine the S. Typhimurium epigenome as a highly stable system that has rare, but important, roles in transcriptional regulation. Incorporating these lessons into future studies will be critical as we progress through the epigenomic era. ImportanceWhile recent breakthroughs have enabled intense study of bacterial DNA modifications, limitations in current work have potentiated a surprisingly untested narrative that DNA methylation is a common mechanism of the bacterial response to environmental conditions. Essentially, whether epigenetic regulation of bacterial transcription is a common, generalizable phenomenon is a critical unanswered question that we address here. We find that most DNA methylation is static in Salmonella enterica serovar Typhimurium, even when the bacteria are grown under dramatically different conditions that cause broad changes in the transcriptome. Further, even when the methylation of individual bases change, these changes generally do not correlate with changes in gene expression. Finally, we demonstrate methods by which data can be stratified in order to identify coupled changes in methylation and gene expression.

microbiology↗