Biology subjects

Xu, H.

Publications and source records attributed to Xu, H..

At least 433 records · Page 24Linked to original sources

bioRxiv2020

A novel full-reduced HMGB1 and kartogenin-containing bio-active scaffold for meniscus regeneration

A novel bio-active scaffold for enhancing wounded meniscus healing has been developed by combination of High mobility group box 1 protein (HMGB1) and kartogenin (KGN) with alginate gel. The properties of the bio-active scaffold were also investigated using an in vitro cell culture model and in vivo rat wounded meniscus healing model. This HMGB1-KGN-containing bioactive scaffold released HMGB1 and KGN into wound area and kept high concentrations of HMGB1 and KGN in the system for more than two weeks. This HMGB1-KGN-containing bioactive scaffold also activated rat bone marrow stem cells (BMSCs) from G0 to GAlert stage and promoted cell proliferation as evidenced by 5-bromo-2-deoxyuridine (BrdU) incorporation testing. Our results also demonstrated that the HMGB1-KGN-containing bioactive scaffold induced cell migration in vitro and recruited the cells to wound area to promote wounded rat meniscus healing in vivo.

bioengineering↗

bioRxiv2020

Neuron-glia Integrity: Functional Assessment, Molecular Underpinnings, and Implication for Higher Brain Functions

We developed a theory of neuron-glia integrity to underline the fact that neurons and glia cells work together in the central nervous system. Here we substantiated this theory and exemplified the implication of intact neuron-glia integrity for higher brain functions. An animal model of maternal separation with early weaning (MSEW) was applied to neonatal rats to mimic early life neglect and abuse in humans. Behavioral performance of rats was evaluated at adulthood, followed by functional assessments of neuron-glia integrity in living rats, and the demonstration of molecular underpinnings of impaired neuron-glia integrity in MSEW rats. MSEW rats showed higher levels of anxiety and explorative activity, higher glutamate level, but lower GABA level in PFC and hippocampus. MSEW procedure down-regulated protein levels of GLT-1 and ATP-, but up-regulated GAD65 and GS, while had no effects on GLAST and PAG. Moreover, it reduced the fractional anisotropy values in various brain regions, in addition to increasing NAA levels. Concurrently, MSEW led to hypomyelination in PFC as evidenced by relevant cellular and molecular changes.

neuroscience↗

bioRxiv2020

Assessing the statistical reporting quality in high-impact factor urology journals

BackgroundsObservational studies plays an important role in urology studies, But few studies have paid attention to the statistical reporting quality of observational studies. The purpose of this study was to investigate the frequency and evaluate the reporting quality of statistical methods of the published observational studies in urology. MethodsThe five urology journals were selected according to the 5-year impact factor. A systematic literature search was performed in PubMed for relevant articles. The quality of statistical reporting was assessed according to assessment criteria. ResultsA total of 193 articles were included in this study. The mean statistical reporting score of included articles was 0.42 (SD=0.15), accounting for 42% of total score. The items that must be reported with a reporting rate more than 50% were: alpha level (n=122, 65.2%), confidence intervals (n=134, 69.4%), name of statistical package (n=158, 84.5%) and exact P-values (n=161, 86.1%). The items with a reporting rate less than 50% were: outliers (n=2, 1.0%) and sample size (n=13, 6.7%). For multivariable regression models (liner, logistic and Cox), variables coding (n=27, 40.7%), validation analysis of assumptions (n=58, 40.3%), interaction test (n=43, 30.0%), collinearity diagnostics (n=5, 3.5%) and goodness of fit test (n=6, 5.9%) were reported. Number of authors more than 7(OR=2.06, 95%CI=1.04-4.08) and participation of statistician or epidemiologist (OR=1.73, 95%CI=1.18-3.39) were associated with the superior reporting quality. ConclusionThe statistical reporting quality of published observational studies in 5 high-impact factor urological journals was alarming. We encourage researchers to collaborate with statistician or epidemiologist. The authors, reviewers and editors should increase their knowledge of statistical methods, especially new and complex methods.

systems biology↗

bioRxiv2020

Learning from local to global - an efficient distributed algorithm for modeling time-to-event data

ObjectivesWe developed and evaluated a privacy-preserving One-shot Distributed Algorithm to fit a multi-center Cox proportional hazard model (ODAC) without sharing patient-level information across sites. MethodsUsing patient-level data from a single site combined with only aggregated information from other sites, we constructed a surrogate likelihood function, approximating the Cox partial likelihood function obtained using patient-level data from all sites. By maximizing the surrogate likelihood function, each site obtained a local estimate of the model parameter, and the ODAC estimator was constructed as a weighted average of all the local estimates. We evaluated the performance of ODAC with (1) a simulation study, and (2) a real-world use case study using four datasets from the Observational Health Data Sciences and Informatics (OHDSI) network. ResultsOur simulation study showed that ODAC provided estimates nearly the same as the estimator obtained by analyzing, in a single dataset, the combined patient-level data from all sites (i.e., the pooled estimator). The relative bias was less than 0.1% across all scenarios. The accuracy of ODAC remained high across different sample sizes and event rates. On the other hand, the metaanalysis estimator, which was obtained by the inverse variance weighted average of the sitespecific estimates, had substantial bias when the event rate is less than 5%, with the relative bias reaching 20% when the event rate is 1%. In the OHDSI network application, the ODAC estimates have a relative bias less than 5% for 15 out of 16 log hazard ratios; while the meta-analysis estimates had substantially higher bias than ODAC. ConclusionsODAC is a privacy-preserving and non-iterative method for implementing time-to-event analyses across multiple sites. It provides estimates on par with the pooled estimator and substantially outperforms the meta-analysis estimator when the event is uncommon, making it extremely suitable for studying rare events and diseases in a distributed manner.

bioinformatics↗

bioRxiv2020

Tracing the origin of a new organ by inferring the genetic basis of rumen evolution

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We used 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen origin and evolution. Comparative analyses reveal that the rumen and the first-chamber stomachs of camels and cetaceans shared a common tissue origin from the esophagus. The rumen recruited genes from other tissues/organs and up-regulated many esophagus genes to aquire functional innovations involving epithelium absorption, improvement of the ketone body metabolism and regulation of microbial community. These innovations involve such genetic changes as ruminant-specific conserved elements, newly evolved genes and positively selected genes. Our in vitro experiements validate the functions of one enhancer, one positively selected gene and two newly evolved antibacterial genes. Our study provides novel insights into the origin and evolution of a complex organ.

evolutionary biology↗

bioRxiv2020

Building a robust chromatin immunoprecipitation (ChIP) method with substantially improved efficiency

Chromatin immunoprecipitation (ChIP) is the gold-standard method to detect the interactions between proteins and chromatin, and is a powerful tool to identify epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting inefficient ChIP enrichment. There is an active demand for a highly efficient ChIP protocol. In the present study, we employed Betula platyphylla (birch) and Arabidopsis thaliana as the research materials, and five factors closely associated with ChIP efficiency were identified, including crosslinking, chromatin concentration using centrifugal filter, using new immunoprecipitation buffer, rescue DNA with proteinase K, and using sucrose to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, a robust ChIP protocol was developed, for which the average fold enrichments were 16.88 and 6.43 fold of that gained using standard ChIP in birch and Arabidopsis, respectively. As this built ChIP method works well in both birch and Arabidopsis, it should be also suitable for other woody and herbaceous species. In addition, this ChIP method make it is possible to detect low-abundance TF-DNA interactions, and may extend the application of ChIP in plant kingdom. One sentence summaryBuilding a ChIP method that increases fold enrichment of birch by 16 folds in average and is adapted for both woody and herbaceous plants.

plant biology↗

bioRxiv2020

Transcriptome analysis reveals the effects of transgenic expression of the Gal4 protein on normal gene expression in silkworm tissues

The Gal4/upstream activating sequence(UAS) system, a well-known genetic tool, has been widely used to analyze gene function in many organisms, including the silkworm (Bombyx mori), a model lepidopteran insect. Several studies have suggested that Gal4 protein activation in tissues can negatively affect transgenic individuals; however, whether and to what extent the Gal4 protein affects normal endogenous gene expression have rarely been studied. Here, we analyzed the transcriptomes of transgenic silkworms expressing the Gal4 protein at high levels in both the wing disc (WD) and epidermis (EP) and investigated gene expression changes in both tissues. Overall, 24,593 genes were identified in the WD and EP libraries, and 2,025 and 2,488 were identified as significant differentially expressed genes(DEGs) in the WD and EP between the transgenic and control groups, respectively. These DEGs were further annotated by gene function classification and pathway assessment using public databases. In addition, 506 DEGs were shared (common) between both tissues. Of these, 97 genes were commonly upregulated, and 234 were commonly downregulated; many of them were annotated to be involved in metabolic processes such as "fat digestion and absorption", "glycine, serine and threonine metabolism" and "glutathione metabolism" and in signal transduction pathways such as the "Rap1 signaling pathway", "MAPK signaling pathway" and "Hippo signaling pathway". Overall, this work enhances understanding of the effects of transgenic Gal4 protein expression on normal gene expression in silkworm tissues and suggests that researchers should pay attention to unexpected effects when using the Gal4/UAS system to study gene function.

genomics↗

bioRxiv2020

Suppression of canonical TGF-β signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis

Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-{beta}) inhibition efficiently promotes reprogramming. However, the mechanisms by which TGF-{beta} blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) - demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-{beta} signaling regulates the interaction between GATA4 and JMJD3. TGF-{beta} activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) exhibits reduced binding to JMJD3 and impaired cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-{beta} pathway activation impairs cardiac gene programming by interfering with GATA4-JMJD3 interactions.

cell biology↗

bioRxiv2020

Mitochondria regulate Drosophila intestinal stem cell differentiation through FOXO

Stem cells often rely on glycolysis for energy production, and switching to oxidative phosphorylation is believed to be essential for their differentiation. To explore the link between mitochondrial respiration and stem cell differentiation, we genetically disrupted electron transport chain (ETC) complexes in the intestinal stem cells (ISCs) of Drosophila. We found that ISCs carrying impaired ETC proliferated much more slowly than normal, produced very few intestinal progenitors, or enteroblasts, and failed to differentiate into enterocytes or enteroendocrine cells. One of the main impediments to ISCs differentiation appeared to be abnormally elevated forkhead box O (FOXO) signaling in the ETC-deficient ISCs, as genetically suppressing the signaling pathway partially rescued the differentiation defect. Contrary to common belief, neither reactive oxygen species (ROS) accumulation nor adenosine triphosphate (ATP) reduction appeared to mediate the ETC mutant phenotype. Our results demonstrate that ETC is essential for Drosophila ISC proliferation and differentiation in vivo, and acts at least partially by repressing endogenous FOXO signaling. They also raise the possibility that ETC complexes have a role in stem cell differentiation beyond electron transfer and ATP production.

developmental biology↗

bioRxiv2020

The transmembrane serine protease inhibitors are potential antiviral drugs for 2019-nCoV targeting the insertion sequence-induced viral infectivity enhancement

At the end of 2019, the SARS-CoV-2 induces an ongoing outbreak of pneumonia in China1, even more spread than SARS-CoV infection2. The entry of SARS-CoV into host cells mainly depends on the cell receptor (ACE2) recognition and spike protein cleavage-induced cell membrane fusion3,4. The spike protein of SARS-CoV-2 also binds to ACE2 with a similar affinity, whereas its spike protein cleavage remains unclear5,6. Here we show that an insertion sequence in the spike protein of SARS-CoV-2 enhances the cleavage efficiency, and besides pulmonary alveoli, intestinal and esophagus epithelium were also the target tissues of SARS-CoV-2. Compared with SARS-CoV, we found a SPRR insertion in the S1/S2 protease cleavage sites of SARS-CoV-2 spike protein increasing the cleavage efficiency by the protein sequence aligment and furin score calculation. Additionally, the insertion sequence facilitates the formation of an extended loop which was more suitable for protease recognition by the homology modeling and molicular docking. Furthermore, the single-cell transcriptomes identified that ACE2 and TMPRSSs are highly coexpressed in AT2 cells of lung, along with esophageal upper epithelial cells and absorptive enterocytes. Our results provide the bioinformatics evidence for the increased spike protein cleavage of SARS-CoV-2 and indicate its potential target cells.

microbiology↗

bioRxiv2020

An isothermal method for sensitive detection of Mycobacterium tuberculosis complexes using CRISPR/Cas12a cis- and trans-cleavage

Tuberculosis is still one of the most serious infectious diseases resulting in lethal death worldwide. The traditional method is still not enough to meet the clinical requirements of rapid diagnosis, high specificity and sensitivity. Fast, sensitive and accurate detection of mycobacterium tuberculosis (MTB) is an urgent need for the treatment and control of tuberculosis disease. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas12a) exhibits strongly nonspecific degradation ability of exogenous single-strand nucleic acid (trans-cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA (gRNA) based on conserved sequences of MTB from gRNA library we designed. Then, we proposed a novel method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay based on fluorescence detection pattern showed 4.48 fM of limit of detection (LOD) and good linear correlation of concentration and fluorescence value (R2=0.9775). Also, it showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% confidence interval, respectively, compared with culture method. The CRISPR/Cas12a system showed good specificity, excellent sensitivity and accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

molecular biology↗

bioRxiv2020

The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes

Since December 2019, a newly identified coronavirus (2019 novel coronavirus, 2019-nCov) is causing outbreak of pneumonia in one of largest cities, Wuhan, in Hubei province of China and has draw significant public health attention. The same as severe acute respiratory syndrome coronavirus (SARS-CoV), 2019-nCov enters into host cells via cell receptor angiotensin converting enzyme II (ACE2). In order to dissect the ACE2-expressing cell composition and proportion and explore a potential route of the 2019-nCov infection in digestive system infection, 4 datasets with single-cell transcriptomes of lung, esophagus, gastric, ileum and colon were analyzed. The data showed that ACE2 was not only highly expressed in the lung AT2 cells, esophagus upper and stratified epithelial cells but also in absorptive enterocytes from ileum and colon. These results indicated along with respiratory systems, digestive system is a potential routes for 2019-nCov infection. In conclusion, this study has provided the bioinformatics evidence of the potential route for infection of 2019-nCov in digestive system along with respiratory tract and may have significant impact for our healthy policy setting regards to prevention of 2019-nCoV infection.

microbiology↗

bioRxiv2020

Maturation of human induced pluripotent stem cell-derived cardiomyocytes for modeling hypertrophic cardiomyopathy

RationaleHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. ObjectiveWe sought to develop a simple method which could drive cultured hiPSC-CMs towards maturity across a number of phenotypes. Methods and resultsCells were cultured in fatty acid-based media and plated on micropatterned surfaces to promote alignment and elongation. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, enhanced maturity of sarcomeric structures, metabolic behavior, and increased myofibril contractile force. Most notably, hiPSC-CMs cultured under optimal maturity-inducing conditions recapitulate the pathological hypertrophy caused by either a pro-hypertrophic agent or genetic mutations. ConclusionsThe more mature hiPSC-CMs produced by the methods described here will serve as a useful in vitro platform for characterizing cardiovascular disease.

cell biology↗

bioRxiv2020

A sample preparation protocol for high throughput immunofluorescence of suspension cells

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, nonadherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines and peripheral blood mononucleated cells (PBMC) and human platelets. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

cell biology↗

bioRxiv2019

Alpha 6 Integrins Regulate the Expression of Laminin-511 and CXCR4 to Promote Endothelial Tubular Morphogenesis

During angiogenesis, endothelial cells engage components of the extracellular matrix through integrin-mediated adhesion. Endothelial expression of laminin-411 and laminin-511 are known to promote vessel stability. However, little is known about the contribution of these laminins to endothelial morphogenesis. We used two organotypic cell culture angiogenesis assays in conjunction with RNAi approaches to demonstrate that depletion of either the 4 chain of laminin-411 or the 5 chain of laminin-511 from endothelial cells inhibits sprouting and tube formation. Depletion of 6 integrins resulted in similar phenotypes. Gene expression analysis indicated that loss of either laminin-511 or 6 integrins inhibited the expression of CXCR4, a gene previously associated with angiogenic endothelial cells. Pharmacological or RNAi-dependent inhibition of CXCR4 suppressed endothelial sprouting and morphogenesis. Importantly, expression of recombinant CXCR4 rescued endothelial morphogenesis when the 6 integrin expression was inhibited. Additionally, the depletion of 6 integrins from established tubes resulted in the loss of tube integrity and laminin-511. Taken together, our results indicate that 6 integrins and laminin-511 can promote endothelial morphogenesis by regulating the expression of CXCR4 and suggest that the 6-dependent deposition of laminin-511 protects the integrity of established endothelial tubes. Summary statementEndothelial-secreted laminin-511 and 6 integrins promote endothelial morphogenesis by regulating the expression of the chemokine receptor, CXCR4. The depletion of 6 integrins from established tubes results in the loss of tube integrity and laminin-511.

cell biology↗

bioRxiv2019

Escape from nonsense mediated decay associates with anti-tumor immunogenicity

Frameshift insertion/deletions (fs-indels) are an infrequent but potentially highly immunogenic mutation subtype. Although fs-indel transcripts are susceptible to degradation through the non-sense mediated decay (NMD) pathway, we hypothesise that some fs-indels escape degradation and lead to an increased abundance of tumor specific neoantigens, that are highly distinct from self. We analysed matched DNA and RNA sequencing data from TCGA, and five separate melanoma cohorts treated with immunotherapy. Using allele-specific expression analysis we show that expressed fs-indels were enriched in genomic positions predicted to escape NMD, and associated with higher protein expression, consistent with degradation escape ("NMD-escape"). Across four independent cohorts, fs-indel NMD-escape mutations were found to be significantly associated with clinical benefit to checkpoint inhibitor (CPI) therapy (Pmeta=0.0039), a stronger association than either nsSNV (Pmeta=0.073) or fs-indel (Pmeta=0.064) count. NMD-escape mutations were additionally shown to have independent predictive power in the "low-TMB" setting, and may serve as a biomarker to rescue patients judged ineligible for CPI based on overall TMB, but still with a high chance of response (low-TMB cohort: NMD-escape-positive % clinical benefit=53%, NMD-escape-negative % clinical benefit=16%, P=0.0098). Furthermore, in an adoptive cell therapy (ACT) treated cohort, NMD-escape mutation count was the most significant biomarker associated with clinical benefit (P=0.021). Analysis of functional T-cell reactivity screens from recent personalized vaccine and CPI studies shows direct evidence of fs-indel derived neoantigens eliciting patient anti-tumor immune response (n=15). We additionally observe a subset of fs-indel mutations, with highly elongated neo open reading frames, which are found to be significantly enriched for immunogenic reactivity in these patient studies (P=0.0032). Finally, consistent with the potency of NMD-escape derived neo-antigens and ongoing immune-editing, NMD-escape fs-indels appear to be under negative selective pressure in untreated TCGA cases. Given the strongly immunogenic potential, and relatively rare nature of NMD-escape fs-indels, these alterations may be attractive candidates in immunotherapy biomarker optimisation and neoantigen ACT or vaccine strategies.

cancer biology↗

bioRxiv2019

Alpha 6 integrins promote cytokinesis by regulating the expression of RSK2 and MKLP1

The integrin-mediated interaction of cells with components of the extracellular matrix (ECM) regulates many cellular processes including cell division. Cytokinesis is the last step of cell division and is critical for normal development and tissue homeostasis as it ensures the proper segregation of genetic and cytoplasmic material between daughter cells. Cytokinesis failure leads to defects in development and tissue differentiation, as well as tumorigenesis. Abscission of intercellular bridge that connects presumptive daughter cells is the last step of cell division. The mitotic kinesin-like protein 1 (MKLP1) plays a central role in positioning the abscission machinery. Here, we show that 6 integrins promote successful cytokinesis in salivary gland epithelial cells by regulating the expression of MKLP1. RNAi-mediated depletion of 6 integrins inhibits cytokinesis and the expression of MKLP1 and p90 ribosomal-S6-kinase 2 (RSK2). Depletion of RSK2 results in similar defects in cytokinesis and also inhibits the expression of MKLP1, suggesting that the expression of RSK2 is required downstream of integrins to promote MKLP1 expression and successful cytokinesis. RNAi-mediated depletion of RSK2 in embryonic salivary glands in organ culture also results in the inhibition of cytokinesis and MKLP1 expression, indicating the physiological significance of this pathway.

cell biology↗

bioRxiv2019

Stochastic simulation platform for visualization and estimation of transcriptional kinetics

We present an implementation of the Gillespie algorithm that simulates the stochastic kinetics of nascent and mature RNA. Our model includes two-state gene regulation, RNA synthesis initiation and stepwise elongation, release to the cytoplasm, and stepwise degradation, a granular description currently tractable only by simulation. To facilitate comparison with experimental data, the algorithm predicts fluorescent probe signals measurable by single-cell RNA imaging. We approach the inverse problem of estimating underlying parameters in a five-dimensional parameter space and suggest optimization heuristics that successfully recover known reaction rates from simulated gene expression turn-on data. The simulation framework includes a graphical user interface, available as a MATLAB app at https://data.caltech.edu/records/1287.

biophysics↗