Integrated strategy for biomarkers of stable coronary heart disease with phlegm and blood stasis syndrome based on RNA-seq and network pharmacology / 中国中药杂志

This study aimed to analyze the biological foundation and biomarkers of stable coronary heart disease(CHD) with phlegm and blood stasis(PBS) syndrome based on RNA-seq and network pharmacology. Peripheral blood nucleated cells from five CHD patients with PBS syndrome, five CHD patients with non-PBS syndrome, and five healthy adults were collected for RNA-seq. The specific targets of CHD with PBS syndrome were determined by differential gene expression analysis and Venn diagram analysis. The active ingredients of Danlou Tablets were screened out from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the "component-target" prediction was completed through PubChem and SwissTargetPrediction. The "drug-ingredient-target-signaling pathway" network of Danlou Tablets against CHD with PBS syndrome was optimized by Cytoscape software. After the target biomarkers were identified, 90 participants were enrolled for diagnostic tests, and 30 CHD patients with PBS syndrome were included in before-and-after experiment to determine the therapeutic effect of Danlou Tablets on those targets. As revealed by RNA-seq and Venn diagram analysis, 200 specific genes were identified for CHD with PBS syndrome. A total of 1 118 potential therapeutic targets of Danlou Tablets were predicted through network pharmacology. Through integrated analysis of the two gene sets, 13 key targets of Danlou Tablets in the treatment of CHD with PBS syndrome were screened out, including CSF1, AKR1C2, PDGFRB, ARG1, CNR2, ALOX15B, ALDH1A1, CTSL, PLA2G7, LAP3, AKR1C3, IGFBP3, and CA1. They were presumably the biomarkers of CHD with PBS syndrome. The ELISA test further showed that CSF1 was significantly up-regulated in the peripheral blood of CHD patients with PBS syndrome, and was significantly down-regulated after Danlou Tablets intervention. CSF1 may be a biomarker for CHD with PBS syndrome, and it is positively correlated with the severity of the disease. The diagnostic cut-off of CSF1 for CHD with PBS syndrome was 286 pg·mL~(-1).

Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies / 中国结合医学杂志

OBJECTIVE@#To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF).@*METHODS@#Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting.@*RESULTS@#The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs.@*CONCLUSIONS@#STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

Brain Tumor Heterogeneity

Tumor heterogeneity is the concept that different tumor cells provide distinct biomorphological lesions, gene expressions, proliferation, microenvironment and graduated capacity of metastatic lesions. Brain tumor heterogeneity has been recently discussed about the interesting interaction of chronic inflammation, microenvironment, epigenetics and glioma steam cells. Brain tumors remain a challenge with regards to medication and disease, due to the lack of treatment options and unsatisfactory results. These results might be the result of the brain tumor heterogeneity and its multiple resistance mechanisms to chemo and radiotherapy.

Differences between male and female leaves of Schisandra sphenanthera: based on RNA-Seq / 中国中药杂志

Schisandra sphenanthera is dioecious and only the fruits of female plants can be used as medicine and food. It is of great significance for the cultivation and production of S. sphenanthera to explore the differences between male and female plants at the non-flowering stage and develop the identification markers at non-flowering or seedling stage. In this study, the transcriptome of male and female leaves of S. sphenanthera at the non-flowering stage was sequenced by Illumina high-throughput sequencing technology and analyzed based on bioinformatics. A total of 236 682 transcripts were assembled by Trinity software and 171 588 were chosen as unigenes. Finally, 1 525 differentially expressed genes(DEGs) were identified, with 458 up-regulated and 1 067 down-regulated in female lea-ves. The down-regulated genes mainly involve photosynthesis, photosynthesis-antenna protein, carbon fixation in photosynthetic or-ganisms, and other pathways. Real-time quantitative PCR(qPCR) identified two genes between male and female leaves and one of them was a HVA22-like gene related to floral organ development and abscisic acid(ABA). Enzyme linked immunosorbent assay(ELISA) was applied to determine the content of ABA, auxin, gibberellin, and zeatin riboside(ZR) in leaves of S. sphenanthera. The results showed that the content of ABA and ZR in male leaves was significantly higher than that in female leaves. The involvement of down-regulated genes in female leaves in the photosynthesis pathway and the significant differences in the content of endogenous hormones between male and female leaves lay a scientific basis for analyzing the factors affecting sex differentiation of S. sphenanthera.

Effects of vitrification and cryostorage duration on single-cell RNA-Seq profiling of vitrified-thawed human metaphase II oocytes / 医学前沿

Oocyte cryopreservation is widely used for clinical and social reasons. Previous studies have demonstrated that conventional slow-freezing cryopreservation procedures, but not storage time, can alter the gene expression profiles of frozen oocytes. Whether vitrification procedures and the related frozen storage durations have any effects on the transcriptomes of human metaphase II oocytes remain unknown. Four women (30-32 years old) who had undergone IVF treatment were recruited for this study. RNA-Seq profiles of 3 fresh oocytes and 13 surviving vitrified-thawed oocytes (3, 3, 4, and 3 oocytes were cryostored for 1,2, 3, and 12 months) were analyzed at a single-cell resolution. A total of 1987 genes were differentially expressed in the 13 vitrified-thawed oocytes. However, no differentially expressed genes were found between any two groups among the 1-, 2-, 3-, and 12-month storage groups. Further analysis revealed that the aberrant genes in the vitrified oocytes were closely related to oogenesis and development. Our findings indicated that the effects of vitrification on the transcriptomes of mature human oocytes are induced by the procedure itself, suggesting that long-term cryostorage of human oocytes is safe.

Transcriptome analysis of mild hypothermia protection against radiation / 中南大学学报(医学版)

OBJECTIVES@#To analyze the differentially expressed genes (DEGs) with radiation-induced rat lung injury, and to reveal the protective mechanism for mild hypothermia in the radiation-induced lung injury in rats at the transcriptome level.@*METHODS@#A total of 10 male SD rats aged 6-8 weeks were randomly divided into 2 groups to establish a rat model of radiation-induced lung injury, and one group was treated with mild hypothermia. RNA was extracted from left lung tissue of each group, and sequenced by BGISEQ-500 platform. Significance analysis of DEGs was carried out by edgeR software. Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyze the gene function. Then 5 key DEGs were verified by real-time reverse transcription PCR (real-time RT-PCR).@*RESULTS@#There were 2 790 DEGs (false discovery rate<0.001, |log@*CONCLUSIONS@#The DEGs and pathways related to mild hypothermia protection against radiation-induced lung injury in rats are obtained, which provides an experimental basis for the protection of mild hypothermia against radiation-induced lung injury.

Transcriptome profiling reveals differential expression of genes potentially involved in muscle and adipose tissue development of cattle

BACKGROUND: To identify differentially expressed genes (DEGs) between muscle and adipose in cattle, we analyzed the data from the RNA sequencing of three Angus×Qinchuan crossbred cattle. RESULTS: Searched the Gene Expression Omnibus (GEO) for a microarray dataset of Yan yellow cattle, GSE49992. After the DEGs were identified, we used STRING and Cytoscape to construct a protein­protein interaction (PPI) network, subsequently analyzing the major modules of key genes. In total, 340 DEGs were discovered, including 21 hub genes, which were mainly enriched in muscle contraction, skeletal muscle contraction, troponin complex, lipid particle, Z disc, tropomyosin binding, and actin filament binding. CONCLUSIONS: In summary, these genes can be regarded as candidate biomarkers for the regulation of muscle and adipose development.

Cichoric acid from extracted Echinacea purpurea induces the proliferation and apoptosis of peripheral blood mononuclear cells from yaks

BACKGROUND: Cichoric acid (CA) is extracted from Echinacea purpurea. It is well known and widely used for its immunological function. However, the effect of CA on peripheral blood mononuclear cells (PBMCs) from yaks is still unclear. This study investigated the potential influences of CA on the proliferation, cytokine induction, and apoptosis of PBMCs from Datong yak in vivo, and aimed to provide a basis for exploring the pharmacological activities of CA on yaks. RESULTS: In this study, CA promoted PBMCs proliferation by combining concanavalin A (Con A) and exhibited a dose-dependent effect as demonstrated by a Cell Counting Kit-8. The concentration of 60 µg/ml CA was the best and promoted the transformation from the G0/G1 phase to the S and G2/M phases with Con A. Furthermore, 60 µg/ml CA significantly increased IL-2, IL-6, and IFN-γ levels and PCNA, CDK4 and Bcl-2 expression levels, but it significantly inhibited the TP53, Bax, and Caspase-3 expression levels. Transcriptome analysis revealed a total of 6807 differentially expressed genes (DEGs) between the CA treatment and control groups. Of these genes, 3788 were significantly upregulated and 3019 were downregulated. Gene Ontology and pathway analysis revealed that DEGs were enriched in cell proliferation and immune function signaling pathways. The expression level of some transcription factors (BTB, Ras, RRM_1, and zf-C2H2) and genes (CCNF, CCND1, and CDK4) related to PBMCs proliferation in yaks were significantly promoted after CA treatment. By contrast, anti-proliferation-associated genes (TP53 and CDKN1A) were inhibited. CONCLUSIONS: In summary, CA could regulate the immune function of yaks by promoting proliferation and inhibiting inflammation and apoptosis of PBMCs.

Analysis on transcriptionomic characteristics of Naoxintong Capsules in prevention of post-ischemic inflammation based on RNA-Seq technology / 中国中药杂志

In this research, high-throughput sequencing was used to investigate the mechanism of Naoxintong Capsules(NXTC) in prevention of post-ischemic inflammation. First, microglia BV-2 inflammatory model was induced by 1.0 μg·mL~(-1) LPS to investigate the effect of intestinal absorption solution of NXTC(NXTCIA) at different concentrations(62.5, 31.25, 15.63, 7.81 μg·mL~(-1)) on LPS-induced BV-2 inflammatory factors in microglia. Then, an RNA-Seq high-throughput sequencing method was performed to identify the differentially expressed mRNAs in microglia BV-2 after pre-treatment with NXTC. GO and KEGG enrichment analysis was used to screen the potential biological processes and related signaling pathways of NXTC in inhibiting inflammation. The results showed that four NXTCIA concentrations could significantly inhibit the release of LPS-induced inflammatory mediators in BV-2 in a dose-dependent manner. Furthermore, high-throughput sequencing results showed that 392 mRNA transcripts were reversed following pre-treatment with NXTC. GO enrichment analysis showed that the transcripts reversed by NXTC were mainly involved in Toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway. Taken together, our findings showed that NXTC treatment could provide protective effects against inflammatory response and the mechanism might be related to the regulation of Toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway.

New avenues for systematically inferring cell-cell communication: through single-cell transcriptomics data

For multicellular organisms, cell-cell communication is essential to numerous biological processes. Drawing upon the latest development of single-cell RNA-sequencing (scRNA-seq), high-resolution transcriptomic data have deepened our understanding of cellular phenotype heterogeneity and composition of complex tissues, which enables systematic cell-cell communication studies at a single-cell level. We first summarize a common workflow of cell-cell communication study using scRNA-seq data, which often includes data preparation, construction of communication networks, and result validation. Two common strategies taken to uncover cell-cell communications are reviewed, e.g., physically vicinal structure-based and ligand-receptor interaction-based one. To conclude, challenges and current applications of cell-cell communication studies at a single-cell resolution are discussed in details and future perspectives are proposed.

RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

A whole-transcriptome approach to evaluate reference genes for quantitative diurnal gene expression studies under natural field conditions in Tamarix ramosissima leaves

Background: Tamarix ramosissima is a desert forest tree species that is widely distributed in the drought-stricken areas to sustain the fragile ecosystem. Owing to its wide usage in the desert restoration of Asia, it can be used as an ecophysiological model plant. To obtain reliable and accurate results, a set of reference genes should be screened before gene expression. However, up to date, systematical evaluation of reference genes has not been conducted in T. ramosissima. Results: In this study, we used eigenvalues derived from principal component analysis to identify stable expressed genes from 72,035 unigenes from diurnal transcriptomes under natural field conditions. With combined criteria of read counts above 900 and CV of FPKM below 0.3, a total of 7385 unigenes could be qualified as candidate reference genes in T. ramosissima. By using three statistical algorithm packages, geNorm, NormFinder, and BestKeeper, the stabilities of these novel reference genes were further compared with a panel of traditional reference genes. The expression patterns of three aquaporins (AQPs) suggested that at least UBQ (high expression), EIF4A2 (low expression), and GAPDH (moderate expression) could be qualified as ideal reference genes in both RT-PCR and RNA-seq analysis of T. ramosissima. Conclusions: This work will not only facilitate future studies on gene expression and functional analysis of genetic resources of desert plants but also improve our understanding of the molecular regulation of water transport in this plant, which could provide a new clue to further investigate the drought adaptation mechanism of desert plant species under harsh environments.

Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence