Biology subjects

Shi, R.

Publications and source records attributed to Shi, R..

64 records · Page 4Linked to original sources

bioRxiv2019

The expression of YAP1 is increased in high-grade prostatic adenocarcinoma but is reduced in neuroendocrine prostate cancer

BACKGROUNDAfter long-term androgen deprivation therapy, 25-30% prostate cancer (PCa) acquires an aggressive neuroendocrine (NE) phenotype. Dysregulation of YAP1, a key transcription coactivator of the Hippo pathway, has been related to cancer progression. However, its role in neuroendocrine prostate cancer (NEPC) has not been assessed.\n\nMETHODSImmunohistochemistry was used to evaluate YAP1 protein levels during PCa initiation and progression. YAP1 knockdown and luciferase reporter assays were used to evaluate the ability of YAP1 to modulate Wnt/beta-Catenin signaling.\n\nRESULTSYAP1 expression was present in the basal epithelial cells in benign prostatic tissues, lost in low grade PCa, but elevated in high grade prostate adenocarcinomas. Interestingly, the expression of YAP1 was reduced/lost in both human and mouse NEPC. Finally, YAP1 knockdown in PCa cells activates Wnt/beta-Catenin signaling, which has been implicated in NE differentiation of PCa, supporting a functional involvement of the loss of YAP1 expression in NEPC development.\n\nCONCLUSIONSThe expression of YAP1 is elevated in high grade prostate adenocarcinomas while lost in NEPC. Reduced YAP1 activates Wnt/beta-Catenin signaling in PCa cells. These results suggest that when applied to PCa patients, YAP1 inhibitors shall be used with caution.

cancer biology↗

bioRxiv2019

Zinc-independent activation of Toll-like receptor 4 by S100A9

The homodimer formed by the protein S100A9 induces inflammation through Toll-like receptor 4 (TLR4), playing critical roles in both healthy and pathological innate immune responses. The molecular mechanism by which S100A9 activates TLR4 remains unknown. Previously, the interaction between purified S100A9 and TLR4 was shown to depend on Zn2+; however, the Zn2+ binding site(s) on S100A9 were not identified. Here, we investigated the role of Zn2+ binding in the pro-inflammatory activity of S100A9. We found that the S100A9 homodimer was prone to reversible, Zn2+-dependent aggregation in vitro. Using a combination of site-directed mutagenesis and Isothermal Titration Calorimetry (ITC), we identified multiple residues that contribute to Zn2+ binding in S100A9. We then used mutagenesis to construct a version of S100A9 with no detectable Zn2+ binding by either ITC or Inductively Coupled Plasma-Mass Spectrometry. This protein did not exhibit aggregation upon addition of saturating Zn2+. Further, despite the lack of Zn2+-binding, this protein was capable of activating TLR4 in a cell-based functional assay. We then modified the functional assay so the Zn2+ concentration was exceedingly low relative to the concentration of S100A9 added. Again, S100A9 was able to activate TLR4. This reveals that, despite the ability of S100A9 to bind Zn2+, S100A9 does not require Zn2+ to activate TLR4. Our work represents an important step in clarifying the nature of the interaction between S100A9 and TLR4.

biochemistry↗

bioRxiv2019

Transcriptome-based Analysis of Tomato Genotypes Resistant to Bacterial spot (Xanthomonas perforans) Race T4

Bacterial spot (BS) is one of the most devastating foliar bacterial diseases of tomato caused by multiple species of Xanthomonas. We performed the RNA-Seq analysis of three tomato lines with different level of resistance to Xanthomonas perforans race T4 to study the differentially expressed genes (DEGs) and transcript-based sequence variations.\n\nAnalysis between inoculated and control samples revealed that resistant line PI 270443 had more DEGs (834), followed by susceptible line NC 714 (373), and intermediate line NC 1CELBR (154). Gene functional analysis based on Gene Ontology (GO) terms revealed that more GO terms (51) were enriched for up-regulated DEGs in the resistant line PI 270443, and more down-regulated DEGs (67) were enriched in the susceptible line NC 714. The specific analysis for DEGs in biotic stress pathway using MapMan software showed more up-regulated biotic stress pathway DEGs (67) for PI 270443 compared to more down-regulated DEGs (125) for susceptible NC 714 line. One interesting feature was that resistant PI 270443 has three up-regulated DEGs for PR-protein, and susceptible line NC 714 has one down-regulated R gene, which is disease-related.\n\nAnalysis of sequence variations called from RNA-Seq reads against the reference genome of susceptible Heinz 1706 showed that chr11 which has multiple reported resistance QTLs to BS race T4 is identical between two resistant lines, PI 270443 and NC 1CELBR, suggesting that these two lines share the same resistance QTLs on this chromosome. Several loci for PR-resistance proteins with sequence variation between the resistant and susceptible tomato lines were identified near the known Rx4 resistance gene on chr11. These findings may be useful for further molecular breeding of tomato.

molecular biology↗

bioRxiv2019

A pipeline for multidimensional confocal analysis of mitochondrial morphology, function and dynamics in pancreatic β-cells

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software it is challenging to accurately extract features from dense multi-layered mitochondrial networks, such as those in insulin-secreting pancreatic {beta}-cells. Motivated by this, we developed a comprehensive pipeline, and associated ImageJ plugin, that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse {beta}-cells, and by extension other similarly challenging cell-types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photo-labelling and unsupervised cluster analysis, and is capable of morphological and functional analyses on a per-organelle basis, including in 4D (xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function, and provides a valuable resource to accelerate mitochondrial research in health and disease.

cell biology↗

bioRxiv2019

Immune Landscape of Invasive Ductal Carcinoma Tumour Microenvironment Identifies a Prognostic and Immunotherapeutically Relevant Gene Signature

BackgroundInvasive ductal carcinoma (IDC) is a clinically and molecularly distinct disease. Tumour microenvironment (TME) immune phenotypes play crucial roles in predicting clinical outcomes and therapeutic efficacy.\n\nMethodIn this study, we depict the immune landscape of IDC by using transcriptome profiling and clinical characteristics retrieved from The Cancer Genome Atlas (TCGA) data portal. Immune cell infiltration was evaluated via single-sample gene set enrichment (ssGSEA) analysis and systematically correlated with genomic characteristics and clinicopathological features of IDC patients. Furthermore, an immune signature was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression algorithm. A random forest algorithm was applied to identify the most important somatic gene mutations associated with the constructed immune signature. A nomogram that integrated clinicopathological features with the immune signature to predict survival probability was constructed by multivariate Cox regression.\n\nResultsThe IDC were clustered into low immune infiltration, intermediate immune infiltration, and high immune infiltration by the immune landscape. The high infiltration group had a favourable survival probability compared with that of the low infiltration group. The low-risk score subtype identified by the immune signature was characterized by T cell-mediated immune activation. Additionally, activation of the interferon- response, interferon-{gamma} response and TNF- signalling via the NF{kappa}B pathway was observed in the low-risk score subtype, which indicated T cell activation and may be responsible for significantly favourable outcomes in IDC patients. A random forest algorithm identified the most important somatic gene mutations associated with the constructed immune signature. Furthermore, a nomogram that integrated clinicopathological features with the immune signature to predict survival probability was constructed, revealing that the immune signature was an independent prognostic biomarker. Finally, the relationship of VEGFA, PD1, PDL-1 and CTLA-4 expression with the immune infiltration landscape and the immune signature was analysed to interpret the responses of IDC patients to immune checkpoint inhibitor therapy.\n\nConclusionTaken together, we performed a comprehensive evaluation of the immune landscape of IDC and constructed an immune signature related to the immune landscape. This analysis of TME immune infiltration patterns has shed light on how IDC respond to immune checkpoint therapy and may guide the development of novel drug combination strategies.

cancer biology↗

bioRxiv2018

Construction of an index system for big data application in health care enterprises: A study based on the Delphi method

This study explored the theory of medical enterprise management and big data. Based on the Delphi method, two rounds of expert opinions were consulted on the capability of a health care enterprise big data application index system covering 11 dimensions, 46 first-level indicators and 111 second-level indicators. The index system includes two categories of input and capacity. The input category includes five dimensions: human resources, material resources, financial resources, government policies, and social service system or social environment. The capabilities category includes six dimensions: data integration capabilities, service capabilities, data analysis capabilities, information security, profitability, and innovation capabilities. This index system aims to appraise the application capability of big data scientifically and systematically, and fills the gaps of such research in China so far, which has positive significance for further research in the future.

scientific communication and education↗

bioRxiv2018

Filter paper-based spin column for low throughput nucleic acid purification

We describe a method of recharging used spin column or assembling homemade spin column using filter paper as binding material for low throughput nucleic acid purification. We evaluated the efficiency of filter paper based spin columns in the purification of different type of nucleic acids. For instance, by following protocols of respective commercial kits, we found that filter paper to be a useful binding material for purification of many types of nucleic acids, including plant genomic DNA, plant total RNA, PCR product, and DNA from agarose gels. We also found that filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Also, we present the protocols of using filter paper recharged spin column or homemade spin column for low throughput purification of plant genomic DNA and plant total RNA with commercial kit buffer leftover and less expensive homemade buffer.

molecular biology↗

bioRxiv2018

Two-dimensional local Fourier image reconstruction via domain decomposition Fourier continuation method

The MRI image is obtained in the spatial domain from the given Fourier coefficients in the frequency domain. It is costly to obtain the high resolution image because it requires higher frequency Fourier data while the lower frequency Fourier data is less costly and effective if the image is smooth. However, the Gibbs ringing, if existent, prevails with the lower frequency Fourier data. We propose an efficient and accurate local reconstruction method with the lower frequency Fourier data that yields sharp image profile near the local edge. The proposed method utilizes only the small number of image data in the local area. Thus the method is efficient. Furthermore the method is accurate because it minimizes the global effects on the reconstruction near the weak edges shown in many other global methods for which all the image data is used for the reconstruction. To utilize the Fourier method locally based on the local non-periodic data, the proposed method is based on the Fourier continuation method. This work is an extension of our previous 1D Fourier domain decomposition method to 2D Fourier data. The proposed method first divides the MRI image in the spatial domain into many subdomains and applies the Fourier continuation method for the smooth periodic extension of the subdomain of interest. Then the proposed method reconstructs the local image based on L2 minimization regularized by the L1 norm of edge sparsity to sharpen the image near edges. Our numerical results suggest that the proposed method should be utilized in dimension-by-dimension manner instead of in a global manner for both the quality of the reconstruction and computational efficiency. The numerical results show that the proposed method is effective when the local reconstruction is sought and that the solution is free of Gibbs oscillations.

neuroscience↗

bioRxiv2018

PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing

BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.\n\nMethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform.\n\nResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones.\n\nConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.

genomics↗

bioRxiv2018

Programmable single and multiplex base-editing in Bombyx mori using RNA-guided cytidine deaminases

Standard genome editing tools (ZFN, TALEN and CRISPR/Cas9) edited genome depending on DNA double strand breaks (DSBs). A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks were developed recently, which have changed this status and have been quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites being knocked out by BE3 with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base-editing in an invertebrate model.

genetics↗