Biology subjects

Yin, C.

Publications and source records attributed to Yin, C..

4 recordsLinked to original sources

bioRxiv2018

A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

NS5A is a multi-functional phosphoprotein that plays a key role in both viral replication and assembly. The identity of the kinases that phosphorylate NS5A, and the consequences for HCV biology, remain largely undefined. We previously identified serine 225 (S225) within low complexity sequence (LCS) I as a major phosphorylation site and used a phosphoablatant mutant (S225A) to define a role for S225 phosphorylation in the regulation of genome replication, interactions of NS5A with several host proteins and the sub-cellular localisation of NS5A. To investigate this further, we raised an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was exclusively found in the hyper-phosphorylated NS5A species. Furthermore, using kinase inhibitors we demonstrated that S225 was phosphorylated by casein kinase 1 (CK1) and polo-like kinase 1 (PLK1). Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI we obtained the first direct evidence of bidirectional hierarchical phosphorylation initiated by phosphorylation at S225.\n\nUsing super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive clusters in HCV-infected cells - pS225 was concentrated on the surface of these clusters, close to lipid droplets. Pharmacological inhibition of S225 phosphorylation resulted in the condensation of NS5A-positive clusters into larger structures, recapitulating the S225A phenotype. Although S225 phosphorylation was not specifically affected by daclatasvir treatment, the latter also resulted in a similar condensation. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.\n\nImportanceNS5A has obligatory roles in the hepatitis C virus lifecycle, and is proposed to be regulated by phosphorylation. As NS5A is a target for highly effective direct-acting antivirals (DAAs) such as daclatasvir (DCV) it is vital to understand how phosphorylation occurs and regulates NS5A function. We previously identified serine 225 (S225) as a major phosphorylation site. Here we used an antiserum specific for NS5A phosphorylated at S225 (pS225-NS5A) to identify which kinases phosphorylate this residue. Using super-resolution microscopy we showed that pS225 was present in foci on the surface of larger NS5A-positive clusters likely representing genome replication complexes. This location would enable pS225-NS5A to interact with cellular proteins and regulate the function and distribution of these complexes. Both loss of pS225 and DCV treatment resulted in similar changes to the structure of these complexes, suggesting that DAA treatment might target a function of NS5A that is also regulated by phosphorylation.

microbiology↗

bioRxiv2018

Recovered and dead outcome patients caused by influenza A (H7N9) virus infection show different pro-inflammatory cytokine dynamics during disease progress and its application in real-time prognosis

The persistent circulation of influenza A(H7N9) virus within poultry markets and human society leads to sporadic epidemics of influenza infections. Severe pneumonia and acute respiratory distress syndrome (ARDS) caused by the virus lead to high morbidity and mortality rates in patients. Hyper induction of pro-inflammatory cytokines, which is known as \"cytokine storm\", is closely related to the process of viral infection. However, systemic analyses of H7N9 induced cytokine storm and its relationship with disease progress need further illuminated. In our study we collected 75 samples from 24 clinically confirmed H7N9-infected patients at different time points after hospitalization. Those samples were divided into three groups, which were mild, severe and fatal groups, according to disease severity and final outcome. Human cytokine antibody array was performed to demonstrate the dynamic profile of 80 cytokines and chemokines. By comparison among different prognosis groups and time series, we provide a more comprehensive insight into the hypercytokinemia caused by H7N9 influenza virus infection. Different dynamic changes of cytokines/chemokines were observed in H7N9 infected patients with different severity. Further, 33 cytokines or chemokines were found to be correlated with disease development and 11 of them were identified as potential therapeutic targets. Immuno-modulate the cytokine levels of IL-8, IL-10, BLC, MIP-3a, MCP-1, HGF, OPG, OPN, ENA-78, MDC and TGF-{beta} 3 are supposed to be beneficial in curing H7N9 infected patients. Apart from the identification of 35 independent predictors for H7N9 prognosis, we further established a real-time prediction model with multi-cytokine factors for the first time based on maximal relevance minimal redundancy method, and this model was proved to be powerful in predicting whether the H7N9 infection was severe or fatal. It exhibited promising application in prognosing the outcome of a H7N9 infected patients and thus help doctors take effective treatment strategies accordingly.

immunology↗

bioRxiv2018

Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging

Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides shadow-free contrast-enhanced imaging of fluorescently labeled samples.\n\nOne Sentence SummaryGlaser et al. describe a light-sheet microscopy architecture that enables passive multidirectional illumination with confocal line detection to enable both uniform fluorescence excitation and contrast-enhanced imaging of fluorescently labeled samples.

bioengineering↗

bioRxiv2017

The chromatin factor Gon4l regulates embryonic axis extension by promoting mediolateral cell polarity and notochord boundary formation through negative regulation of cell adhesion

Anteroposterior axis extension during vertebrate gastrulation requires cell proliferation, embryonic patterning, and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation. Although identified as a recessive enhancer of short axis phenotypes in planar cell polarity (PCP) mutants, we found that Gon4l functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. We identified direct genomic targets of Gon4l and found that it acts as both a positive and negative regulator of gene expression, including limiting expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrin3b. Excess epcam or itga3b in wild-type gastrulae phenocopied notochord boundary defects of udu mutants, while downregulation of itga3b suppressed them. By promoting formation of this anteroposteriorly aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis with the anteroposterior embryonic axis.

developmental biology↗