Investigating the impact of silencing an RNA-binding protein gene SlRBP1 on tomato photosynthesis through RNA-sequencing analysis / 生物工程学报

Photosynthesis in plants directly affects the synthesis and accumulation of organic matter, which directly influences crop yield. RNA-binding proteins (RBPs) are involved in the regulation of a variety of physiological functions in plants, while the functions of RBPs in photosynthesis have not been clearly elucidated. To investigate the effect of a glycine-rich RNA-binding protein (SlRBP1) in tomato on plant photosynthesis, a stably inherited SlRBP1 silenced plant in Alisa Craig was obtained by plant tissue culture using artificial small RNA interference. It turns out that the size of the tomato fruit was reduced and leaves significantly turned yellow. Chlorophyll(Chl) content measurement, Chl fluorescence imaging and chloroplast transmission electron microscopy revealed that the chloroplast morphology and structure of the leaves of tomato amiR-SlRBP1 silenced plants were disrupted, and the chlorophyll content was significantly reduced. Measurement of photosynthesis rate of wild-type and amiR-SlRBP1 silenced plants in the same period demonstrated that the photosynthetic rate of these plants was significantly reduced, and analysis of RNA-seq data indicated that silencing of SlRBP1 significantly reduced the expression of photosynthesis-related genes, such as PsaE, PsaL, and PsbY, and affected the yield of tomato fruits through photosynthesis.

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).

Transcriptome analysis of Aedes aegypti larvae before and after treatment with fipronil / 中国热带医学

@#Abstract: Objective　In order to explore the application prospects of the phenyl pyrazole insecticide fipronil for mosquito control and identify potential target genes involved in the resistance of Aedes aegypti to fipronil, and lay the foundation for an in-depth study of the resistance mechanism of Aedes aegypti to fipronil. Methods　Using Aedes aegypti sensitive strains as experimental materials, Aedes aegypti larvae were treated with fipronil, and the differences in gene expression of Aedes aegypti larvae before and after drug administration were compared at the transcriptome level using transcriptome sequencing combined with bioinformatics analysis, and the differential genes were analyzed. Results　A total of 757 differentially expressed genes were identified between the fipronil-treated group and control group, including 217 and 540 up- and down-regulated genes, respectively. Among these, the expression of glutamate-gated chloride channel (GluCls) genes varied significantly before and after treatment. Gene ontology analysis revealed that differentially expressed genes were enriched in catalytic activity, binding, metabolic processes, and membrane-related functions, while KEGG pathway analysis indicated enrichment in biosynthesis, metabolism, and life regulation processes, while the glutathione metabolic pathway was enriched in 15 differentially expressed genes. Conclusions The transcriptome results revealed that GST gene expression was significantly upregulated in fipronil-treated Aedes aegypti larvae, indicating that GST gene is involved in the development of fipronil resistance in Aedes aegypti larvae. In addition, GluCls gene expression was also significantly different before and after treatment, suggesting that GluCls migh be a potential target receptor for fipronil resistance in Aedes aegypti. As GluCls is an ideal target receptor found only in invertebrates, this discovery provides a reference and basis for further exploration of the toxicological mechanism of fipronil on Aedes aegypti.

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.

Excessive iodine promotes the occurrence and development of experimental autoimmune thyroiditis in mice through p38 and intercellular adhesion molecule-1 / 中华地方病学杂志

Objective:To investigate the molecular mechanism of excessive iodine induced experimental autoimmune thyroiditis (EAT) in mice.Methods:Sixty female non-obese diabetic (NOD) mice were selected and divided into 5 groups according to body weight [(25 ± 3) g] via the random number table method, with 12 mice in each group: control group (group A), 10-fold high iodine group (group B), 100-fold high iodine group (group C), 1 000-fold high iodine group (group D) and 1 000-fold high iodine combined with polyinosinic acid-polycytidylic acid [Poly (I:C)] group (group E). The experiment period was 16 weeks. Mice in each group drank purified water with sodium iodine (NaI) content of 0.000, 0.005, 0.050, 0.500 and 0.500 mg/L, respectively; mice in group E were intraperitoneally injected with Poly (I:C) at week 7 and week 15, respectively. At the end of the 16th week, mice were dissected and blood samples and thyroid tissue were taken. The levels of serum thyroid function indexes [thyroid stimulating hormone (TSH), free triiodothyronine (FT 3), free thyroxine (FT 4), and thyroid peroxidase antibody (TPOAb)] were detected by enzyme-linked immunosorbent assay (ELISA); the pathological changes of thyroid tissue were observed after hematoxylin-eosin (HE) staining; differentially expressed genes in thyroid tissue were detected by RNA-sequencing (RNA-seq), and analyzed by KEGG pathway; mRNA and protein levels of p38, intercellular adhesion molecule-1 (ICAM-1) and chemokine 10 (CXCL10) in thyroid tissue were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting, respectively. Results:There were statistically significant differences in serum levels of TSH (ng/ml: 6.53 ± 0.86, 6.61 ± 0.82, 7.68 ± 0.55, 7.93 ± 0.60, 8.73 ± 1.60), FT 3 (pg/ml: 59.35 ± 10.16, 53.73 ± 10.96, 46.19 ± 8.03, 41.01 ± 8.67, 34.21 ± 11.75), FT 4 (pg/ml: 136.74 ± 10.06, 124.33 ± 14.34, 101.80 ± 6.78, 91.37 ± 6.75, 73.29 ± 17.31), and TPOAb (U/ml: 130.81 ± 24.53, 145.47 ± 28.89, 166.52 ± 41.59, 199.78 ± 42.19, 201.99 ± 44.03) among the 5 groups of mice ( F = 4.77, 4.96, 23.12, 3.68, P < 0.05). Compared with group A, the serum TSH levels of mice in groups C, D and E were higher, the levels of FT 3 and FT 4 in groups B, C, D and E were lower, and the levels of TPOAb in groups D and E were higher, and the differences were statistically significant ( P < 0.05). HE staining showed that the thyroid follicle lesion in groups D and E was serious, and the EAT phenotype appeared in both groups. The differentially expressed genes were analyzed by KEGG pathway. Compared with group A, 8 metabolic pathways related to thyroid autoimmunity and inflammation were found in groups B, C, D and E. Further analysis found that 3 genes appeared in multiple pathways, namely p38, ICAM-1 and CXCL10. There were significant differences in the mRNA levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 14.77, 12.76, 16.39, P < 0.05); compared with group A, the mRNA levels of p38 in groups B, C, D and E were higher, and the mRNA levels of ICAM-1 and CXCL10 in groups C, D and E were higher ( P < 0.05). There were significant differences in the protein levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 7.97, 73.86, 18.02, P < 0.05); compared with group A, the protein levels of ICAM-1 and CXCL10 in groups B, C, D and E were higher ( P < 0.05). Conclusion:Excessive iodine promotes the occurrence and development of EAT in mice by up-regulating the expressions of p38 and ICAM-1 genes that are closely related to thyroid autoimmune and inflammatory responses.

Screening, identification and functional analysis of differential expression genes in rat visual cortex after monocular deprivation by RNA-Seq technique / 中华实验眼科杂志

Objective:To screen differentially expressed genes (DEGs) in rat visual cortex after monocular deprivation by RNA sequencing technology, and to analyze the function of the DEGs.Methods:Eighteen 14-day-old SD rats were randomly divided into blank control group and monocular deprivation model group according to random number table method, with 9 rats in each group.The monocular deprivation model was established through lid suture of the right eye for 14 days.Patten visual evoked potential (PVEP) in the right eyes of the rats was recorded before and 14 days after modeling, respectively.Bilateral visual cortex tissues of the rats were dissected from the two groups, and specific genes associated with the pathogenesis of amblyopia were screened out for RNA-seq analysis.The biological functions of differentially expressed genes were evaluated by Gene Ontology (GO) enrichment analysis, and metabolic pathways involved were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.The use and care of the animals complied with ARVO statement.This study protocol was approved by an Ethics Committee of Gansu University of Chinese Medicine (No.2016-58).Results:Compared with blank control group, the latency of P 100 wave was significantly prolonged, and the amplitude was reduced in the eyes of monocular deprivation model group (both at P<0.05). Forty DEGs in the left visual cortex and 63 DEGs in the right visual cortex were determined, among which 9 genes were overlapped.GO analysis indicated that the DEGs were mainly involved in biological processes, such as DNA-templated transcription, glutamate secretion, transcriptional regulation of RNA polymerase Ⅱ promoter, protein phosphorylation etc., as well as molecular functions, such as DNA binding, ATP binding, protein serine/threonine kinase activity, calcium ion binding, zinc ion binding, phospholipase A 2 activity, nucleic acid binding and cell components involved in the formation of intracellular and membrane of endoplasmic reticulum.The abnormal expressions of Grm2 and Pla2g2a genes might be closely associated with visual function impairment. Grm2 gene was mainly involved in visual signaling pathway processes including glutamate synapse, long-term potentiation (LTP), long-term depression (LTD) etc. Pla2g2a gene was mainly involved in α-linolenic acid metabolism and arachidonic acid pathway. Conclusions:There are abnormal expressions of genes in the bilateral visual cortices of monocular deprivation rats in the sensitive period of visual development, mainly leading to the disorder of visual signal transduction pathway.Metabolic pathway changes based on specific response gene regulation may be one of the important molecular biological mechanisms in the pathogenesis of amblyopia.

Arbuscular mycorrhizal fungi enhanced cadmium uptake in Photinia frase through altering root transcriptomes and root-associated microbial communities / 生物工程学报

As a non-essential metal, cadmium (Cd) pollution poses severe threats to plant growth, environment, and human health. Phytoextraction using nursery stocks prior to their transplantation is a potential useful approach for bioremediation of Cd contaminated soil. A greenhouse pot experiment was performed to investigate the growth, Cd accumulation, profiles of transcriptome as well as root-associated microbiomes of Photinia frase in Cd-added soil, upon inoculation of two types of arbuscular mycorrhizal fungi (AMF) Sieverdingia tortuosa and Funneliformis mosseae. Compared with the control, inoculation of F. mosseae increased Cd concentrations in root, stem and leaf by 57.2%, 44.1% and 71.1%, respectively, contributing to a total Cd content of 182 μg/plant. KEGG pathway analysis revealed that hundreds of genes involved in 'Mitogen-activated protein kinase (MAPK) signaling pathway', 'plant hormone signal transduction', 'biosynthesis of secondary metabolites' and 'glycolysis/gluconeogenesis' were enriched upon inoculation of F. mosseae. The relative abundance of Acidobacteria was increased upon inoculation of S. tortuosa, while Chloroflexi and Patescibacteria were increased upon inoculation of F. mosseae, and the abundance of Glomerales increased from 23.0% to above 70%. Correlation analysis indicated that ethylene-responsive transcription factor, alpha-aminoadipic semialdehyde synthase, isoamylase and agmatine deiminase related genes were negatively associated with the relative abundance of Glomerales operational taxonomic units (OTUs) upon inoculation of F. mosseae. In addition, plant cysteine oxidase, heat shock protein, cinnamoyl-CoA reductase and abscisic acid receptor related genes were positively associated with the relative abundance of Patescibacteria OTUs upon inoculation of F. mosseae. These finding suggested that AMF can enhance P. frase Cd uptake by modulating plant gene expression and altering the structure of the soil microbial community. This study provides a theoretical basis for better understanding the relationship between root-associated microbiomes and root transcriptomes of P. frase, from which a cost-effective and environment-friendly strategy for phytoextraction of Cd in Cd-polluted soil might be developed.

Identification of key pathways and genes involved in microglia inflammation by bioinformatics analysis of transcriptome sequencing / 中华骨科杂志

Objective:To explore the key pathways and genes involved in microglia inflammation through transcriptome sequencing and bioinformatics analysis.Methods:BV2 cells were stimulated by lipopolysaccharide to establish microglia inflammation model. The levels of IL-6 and TNF-α were detected by ELISA and RT-qPCR. The established microglia inflammation model was sequenced by transcriptome sequencing, and the differentially expressed genes were screened by bioinformatics method. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes were performed. The protein-protein interaction network of differentially expressed genes was constructed by using string database, and the protein-protein interaction network was visualized by using Cytoscape software. The protein interaction network module was extracted by using MCODE app. The hub gene was extracted by using cytohubba app and was verified through RT-qPCR. We conducted enrichment analysis of hub genes, predicted their targeted miRNAs and interacting drugs.Results:The microglia inflammation model was successfully established and verified by ELISA and RT-qPCR. We screened 434 differentially expressed genes by bioinformatics analysis of transcriptome sequencing results. GO analysis showed that these differentially expressed genes were mainly concentrated in cellular response to cytokine stimulus, inflammatory response, regulation of response to external stimulation. KEGG analysis showed that these differentially expressed genes were mainly concentrated in Chemokine signaling pathway, TNF signaling pathway, IL-17 signaling pathway. We constructed the protein interaction network of these differentially expressed genes, and carried out module analysis and extraction of hub genes. Most of hub genes are located in module 1, and the seed gene of module 1 is S1pr1. Hub genes include S1pr1, Cxcr4, Cx3cl1, Cx3cr1, Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9, Fpr1. RT-qPCR results showed that compared with the culture medium group, the mRNA expressions of S1pr1, Cxcr4, Cx3cl1 and Cx3cr1 were down-regulated, and the mRNA expressions of Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9 and Fpr1 were up-regulated in the LPS group. The enrichment analysis of hub genes mainly focused on chemokine-mediated signaling pathway, Class A/1 (Rhodopsin-like receptors), cell chemotaxis and so on. Drugs and miRNAs that may interact with hub genes were predicted. Conclusion:Through transcriptome sequencing and bioinformatics analysis of microglia inflammation model, differentially expressed genes were screened, hub genes and seed genes were extracted, which will help us further understand the molecular mechanism of microglia inflammation and provide potential targets for the treatment of related diseases.

Analysis of Physiological Function of Escherichia coli Heme Peroxidase EfeB Based on Transcriptome / 中国生物化学与分子生物学报

Heme peroxidase EfeB in E. coli belongs to the dye-decolorizing peroxidase (DyP) superfamily. Peroxidases in this superfamily have a good ability in degradation of synthetic dyes, but their physiological functions in organisms are unclear. In order to further understand the physiological function of EfeB, the mutant strain EcoΔefeB was constructed by homologous recombination. The differences between parental strain E. coli BL21 and EcoΔefeB at genome transcription level as well as cell growth under different conditions were compared. The response of efeB to iron ion was also investigated. The results showed that the deletion of efeB gene caused the differential expression of 1 765 genes, which were mainly related to cell metabolic pathway, cell membrane synthesis and flagellum movement. There was no significant difference in cell growth between BL21 and EcoΔefeB at pH 7. 0, but the growth of BL21 was much better than that of EcoΔefeB at pH 4. 5. The functional expression of efeB may support the survival of E. coli at low pH. EfeB was significantly up-regulated when Fe

PTBP1 Promotes Growth, Migration and Invasion of Cholangiocarcinoma Cells by Regulating mRNA Alternative Splicing / 中国生物化学与分子生物学报

Cholangiocarcinoma (CCA) is a highly invasive type of cancer with insidious onset and high mortality. Polypyrimidine tract-binding protein 1 (PTBP1) is highly over-expressed in various types of tumor tissues, which contributes to cancer progression. But the role of PTBP1 in CCA has not been explored yet. In this study, we aim to investigate the function of PTBP1 in CCA. Therefore, we used publicly available data from the cancer genome atlas (TCGA) to evaluate the dysregulation of PTBP1 in CCA. The results showed that the PTBP1 is significantly up-regulated in CCA tissues compared to the matched non-tumor tissues (P < 0. 05). We assessed the effects of PTBP1 on the growth of CCA cell lines RBE and HuH28 by performing CCK-8 and plate colony formation assays. The results showed that overexpression of PTBP1 significantly promoted the growth (P < 0. 01) of CCA cells, whereas knockdown of PTBP1 exhibited opposite effects. Transwell and Invasion assays revealed that overexpression of PTBP1 significantly promotes the migration and invasion of CCA cells (P < 0. 001), whereas knockdown of PTBP1 exhibited opposite effects (P < 0. 001). The RNA sequencing (RNA-seq) analysis in PTBP1-depleted cells showed that the up-regulated genes are significantly enriched in p53 signaling pathway, while the down-regulated genes are represented by cholesterol metabolism, Rho GTPase and TGF-β pathways. Then, the alternative splicing analysis revealed that inhibition of PTBP1 led to series of aberrant alternative splicing events, including several cancer-associated ones, such as splicing events within the TGF-β regulator TGIF1 and the p53 activity-correlated gene GNAS. These results indicate that PTBP1 promotes the progression of CCA likely by regulating the transcriptome alternative splicing to influence multiple cancer-associated signaling pathways.

Serine protease inhibitor Hespintor’s potential mechanism based on integrated transcriptomics in inhibiting the growth of transplanted hepatoma in nude mice / 西安交通大学学报(医学版)

【Objective】 To investigate the relationship between differential expressions of lncRNAs and mRNAs and Hespintor’s possible anti-tumor mechanism using transcriptome sequencing technology. 【Methods】 First， a nude mouse model of human hepatoma tumors was established. The tumor tissue mass was collected after 4 weeks of group treatment. The cDNA libraries of tumor tissues were established in the Hespintor treatment group and the solvent control group， and transcriptome sequencing was performed. Based on RNA-seq data， the differentially expressed lncRNA and mRNA genes were screened， and the functional enrichment and interaction analysis were performed. The network module division approach was utilized to identify the target genes of Hespintor and build its regulatory network. 【Results】 The Hespintor treatment group yielded a high-confidence differentially expressed gene collection of 2 003 lncRNAs （DEGs lncRNA） and 1 038 mRNAs （DEGs mRNA）. Target mRNAs regulated by DEGs lncRNA were mainly enriched in PIP3 to activate the Akt signal， p53 signal pathway， FOXO-mediated transcription， and cell cycle， among other things. DEGs mRNA were primarily enriched in chemokine signaling pathways， extracellular matrix receptor interactions， complement， and coagulation cascades. Both of them were related in three ways： cell behavior， transcriptional regulation， and cell cycle. 【Conclusion】 The PI3K/Akt signaling pathway may play a key role in the inhibitory effect of Hespintor on tumor， inducing G1/S phase cell cycle arrest and apoptosis.

Analysis on Antidepressant Mechanism of Verbascoside Based on RNA-Seq Technology / 中国实验方剂学杂志

Objetive： To investigate the mechanism of antidepressant effect of verbascoside based on high-throughput sequencing technology （RNA-Seq），and to explore the possible targets and signaling pathways. MethodsForty C57BL/6 mice were randomly divided into control group，model group，fluoxetine group and verbascoside group，10 mice in each group. Except for the control group，all the other three groups were constructed with chronic unpredictable mild stimulation （CUMS） combined with solitary feeding for four weeks. Control group，fluoxetine group and verbascoside group were administered by gavage once daily for three weeks during the second week of modeling. The mice were assessed by sugar-water preference test，forced swimming test，open field test，elevated cross maze test，and water maze test. Enzyme linked immunosorbent assay（ELISA） was performed to detect the levels of major neurotransmitters and inflammatory factors in mice serum，and mRNA high-throughput sequencing was performed in the nucleus accumben and colon to screen differentially expressed genes and perform pathway enrichment analysis. Real-time polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression of Gad，Slc32a1（VGAT） and brain-derived neurotrophic factor (BDNF） in nucleus accumbens. ResultCompared with the control group，the anxiety and depression-like behaviors in the model group increased，while the learning and memory ability decreased significantly. The content of neurotransmitter in serum decreased significantly. The content of pro-inflammatory factors increased significantly. The mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens decreased significantly，while that of BDNF increased significantly. Compared with the model group，the anxiety and depression-like behavior of mice in verbascoside group was significantly relieved. The neurotransmitter content increased significantly，and the pro-inflammatory factors decreased significantly. The mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens increased significantly，while that of BDNF decreased significantly.A total of 48 differentially expressed genes in nucleus accumbens and 43 differentially expressed genes in colon were screened by high-throughput sequencing. Differential genes in nucleus accumbens mainly focus on neuroactive ligand-receptor interaction， γ-aminobutyric acid（GABA）ergic synapse，synaptic vesicle cycle and other pathways. Colonic differential genes are mainly concentrated in GABAergic synapses，synaptic vesicle circulation，cyclic adenosine monophosphate（cAMP） signal pathway and other signal pathways. Compared with the control group，the mRNA expression of Gad and Slc32a1（VGAT） in nucleus accumbens of model group decreased significantly，while the mRNA expression of BDNF increased significantly. ConclusionVerbascoside has significant antidepressant effects. Its antidepressant effect may be related to the increase of monoamine neurotransmitters，the decrease of pro-inflammatory factors and the restoration of neurotransmitter homeostasis by increasing GABA，and it mainly acts through the signaling pathways such as neuroactive ligand-receptor interaction，GABAergic synapses，synaptic vesicle cycle and cAMP signaling pathway.

GC-MS Metabolites and Gene Expression Characteristics in Fibrous Roots of Dioscorea zingiberensis in Response to Low Phosphorus Stress / 中国实验方剂学杂志

ObjectiveTo investigate the metabolites and gene expression characteristics in fibrous roots of Dioscorea zingiberensis in response to low phosphorus stress. MethodThe severe stress group， the moderate stress group， and the normal group were set up to stimulate the low phosphorus stress experiment. The fibrous roots of D. zingiberensis were collected during initial stress. The metabolites and transcriptomic characteristics were analyzed by gas chromatography-mass spectrometry （GC-MS） derivatization and RNA-seq techniques. Through multivariate statistical analysis of metabolites treated by different methods，functional analysis of differentially expressed genes， and data mining， the metabolism markers produced in fibrous roots of D. zingiberensis under low phosphorus stress were screened out， and the metabolic pathway characteristics of different genes were analyzed. ResultA total of 116 GC-MS metabolites were detected from the fibrous roots of D. zingiberensis. The metabolic characteristics of fibrous roots of D. zingiberensis under different low phosphorus treatments were obviously different. Orthogonal partial least squares discriminant analysis（OPLS-DA） model was used to screen six differential metabolites represented by sugars and alcohols from metabolites of fibrous roots treated with different methods，and these components were presumedly metabolism markers of fibrous roots of D. zingiberensis in response to low phosphorus stress. The differential genes screened out from the severe stress group and the normal group were mainly enriched in peroxidase pathway，phosphate and hypophosphate metabolism pathway，while the differential genes screened out from the severe stress group and the moderate stress group were mainly enriched in glutathione metabolism pathway and phosphopentose pathway. A total of 177 differential genes in response to low phosphorus stress were screened out from fibrous roots， involving many pathways such as terpenoid skeleton and inositol biosynthesis，which was consistent with the fact that the metabolic differential components in fibrous roots in response to low phosphorus stress were mainly saccharides and inositol. ConclusionThe metabolites and gene expression in fibrous roots of D. zingiberensis responded to low phosphorus stress，and the differential metabolites were closely related to differentially expressed genes. This study is expected to provide a theoretical basis for the research on the molecular mechanism of D. zingiberensis in response to low phosphorus stress.

Analysis of Crucial Transcription Factors of Berberine Against Cerebral Ischemia Based on Transcriptomics and Proteomics / 中国实验方剂学杂志

ObjectiveOn the basis of determining the protective effect of berberine （BBR） on cerebral ischemia， crucial transcription factors （TFs） of BBR against cerebral ischemia was identified by using transcriptome and proteome sequencing. MethodThe model of middle cerebral artery occlusion （MCAO） was established by thread embolization. The sham operation group， model group， low-dose group of BBR （dose of 37.5 mg·kg-1·d-1） and high-dose group of BBR （75 mg·kg-1·d-1） were set up. The rats were killed after continuous intragastric administration for 7 days. The pharmacodynamics was evaluated by Longa score and cerebral infarction rate， and the expressions of inflammatory cytokines， such as interleukin （IL）-1β， tumor necrosis factor （TNF）-α and monocyte chemotactic protein-1 （MCP-1） were measured by enzyme-linked immunosorbent assay （ELISA）. Then， RNA-Seq technique was used to detect the differentially expressed genes （DEGs） before and after BBR intervention， and DAVID 6.8 was used for enrichment analysis of DEGs. CatTFREs technique was used to detect differential TFs before and after BBR intervention， and DAVID 6.8 and STRING 11.0 were used for enrichment analysis and TFs association analysis. Finally， by integrating the activity of TFs and the changes of downstream target genes， crucial TFs were identified and the related regulatory network was constructed by Cytoscape 3.7.1. ResultCompared with the sham operation group， the neurological impairment was significant in the model group （P<0.01）， and compared with the model group， the low and high dose BBR groups could significantly reduce the neurological function damage （P<0.01） and decrease the rate of cerebral infarction （P<0.01）. Transcriptome data analysis showed that BBR was involved in the recovery process after cerebral ischemia mainly by affecting cell adhesion， brain development， neuron migration， calcium signaling pathway， cyclic adenosine monophosphate （cAMP） signaling pathway， inflammatory response and other related functions and signaling pathways. Proteomic data analysis showed that the differentially expressed TFs after BBR intervention interfered with cerebral ischemia mainly by regulating cell differentiation， immune system process， cell proliferation and other biological processes. In addition， integration analysis of TFs and DEGs revealed that transcription factor CP2-like 1 （TFCP2L1）， nuclear factor erythroid-2 like 1 （NFE2L1）， neurogenic differentiation protein 6 （NeuroD6） and POU domain， class 2， transcription factor 1 （POU2F1） were crucial TFs against cerebral ischemia-reperfusion injury mediated by BBR. ConclusionBBR has obvious protective effect on cerebral ischemia-reperfusion injury and its crucial TFs include TFCP2L1， NFE2L1， NeuroD6 and POU2F1.

Transcriptome analysis of primary human oral keratinocytes stimulated with Prevotella melaninogenica / 口腔疾病防治

Objective@# To investigate the transcriptomic changes in primary human oral keratinocytes (pHOKs) after coculture with Prevotella melaninogenica (P.m) and to verify the changes in human oral keratinocyte (HOK) cell lines.@*Methods@# pHOK was isolated and cocultured with P.m for 0, 4 and 24 h. Total RNA was extracted, a gene library was constructed, transcriptional sequencing was performed, differentially expressed genes (DEGs) were analyzed, gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and the validation of DEGs was performed by qRT-PCR and Western Blot in the HOK and P.m coculture cell model. @*Results @#1 788 DEGs were detected between the 4 h group and control group, including upregulated DEGs such as lymphocyte cytosolic protein 1(LCP1), keratin 7 (KRT7) and Cilia and flagella associated protein 251(CFAP251) and downregulated DEGs such as FERM, ARH/RhoGEF and Pleckstrin domain protein 1 (FARP1), WW domain containing transcription regulator 1(WWTR1) and Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2). 1 832 DEGs were detected between the 24 h group and control group, including upregulated DEGs such as LCP1, complement C1s(C1S), kynureninase (KYNU) and downregulated DEGs such as phosphoserine aminotransferase 1 (PSAT1), FARP1 and FKBP prolyl isomerase 10 (FKBP10). There were 1 090 common differentially expressed genes (cDEGs) in the 4 h and 24 h groups, including LCP1, KYNU and long intergenic nonprotein coding RNA 958 (LINC00958). The GO pathways were mainly enriched in response to lipopolysaccharide and the molecules of bacterial origin and apical part of the cell. KEGG pathway analysis revealed enrichment in the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor (TLR) pathway, etc. We verified the expression of a cDEG, Myosin1B (MYO1B), and qRT-PCR and Western Blot analysis showed that MYO1B expression was significantly upregulated between the control group and the P.m cocultured group (P<0.001), and its expression followed a time-dependent and concentration-dependent manner. @*Conclusion@#P.m played an important role in the transcriptome of oral keratinocytes.

Quantifying the state of cell differentiation based on the gene networks entropy / 生物工程学报

Studies of cellular dynamic processes have shown that cells undergo state changes during dynamic processes, controlled mainly by the expression of genes within the cell. With the development of high-throughput sequencing technologies, the availability of large amounts of gene expression data enables the acquisition of true gene expression information of cells at the single-cell level. However, most existing research methods require the use of information beyond gene expression, thus introducing additional complexity and uncertainty. In addition, the prevalence of dropout events hampers the study of cellular dynamics. To this end, we propose an approach named gene interaction network entropy (GINE) to quantify the state of cell differentiation as a means of studying cellular dynamics. Specifically, by constructing a cell-specific network based on the association between genes through the stability of the network, and defining the GINE, the unstable gene expression data is converted into a relatively stable GINE. This method has no additional complexity or uncertainty, and at the same time circumvents the effects of dropout events to a certain extent, allowing for a more reliable characterization of biological processes such as cell fate. This method was applied to study two single-cell RNA-seq datasets, head and neck squamous cell carcinoma and chronic myeloid leukaemia. The GINE method not only effectively distinguishes malignant cells from benign cells and differentiates between different periods of differentiation, but also effectively reflects the disease efficacy process, demonstrating the potential of using GINE to study cellular dynamics. The method aims to explore the dynamic information at the level of single cell disorganization and thus to study the dynamics of biological system processes. The results of this study may provide scientific recommendations for research on cell differentiation, tracking cancer development, and the process of disease response to drugs.

Transcriptome Analysis of Inhibitory Effect of Astaxanthin Against HepG2 Cell Lines / 肿瘤防治研究

Objective To investigate the gene expression differences of hepatocellular carcinoma (HCC) cells treated with astaxanthin and to analyze its biological information. Methods After treated with astaxanthin, the total RNA of HCC cells was extracted with TRIzol reagent. Illumina TruseqTM RNA sample Prep Kit was used for RNA-seq library construction and sequencing. We analyzed the differentially-expressed genes and function enrichments. Results Transcriptomic analysis showed that there were 39 642 566 and 497 155 920 reads in the control group and treatment group, respectively; the proportion of clean reads obtained by filtration were 94.89% and 93.56%, respectively. A total of 77 344 transcripts were detected, with 4 997 genes with significant differences in expression, among which 1 564 genes were up-regulated and 3 433 genes were down-regulated. Conclusions Astaxanthin may participate in several biological processes and signaling pathways of tumors. Significant repression of translation process by astaxanthin may result in the growth inhibition of HCC.

Sinoatrial node pacemaker cells share dominant biological properties with glutamatergic neurons

Activation of the heart normally begins in the sinoatrial node (SAN). Electrical impulses spontaneously released by SAN pacemaker cells (SANPCs) trigger the contraction of the heart. However, the cellular nature of SANPCs remains controversial. Here, we report that SANPCs exhibit glutamatergic neuron-like properties. By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse, we found that SANPCs co-clustered with cortical neurons. Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system, expressing genes encoding glutamate synthesis pathway (Gls), ionotropic and metabotropic glutamate receptors (Grina, Gria3, Grm1 and Grm5), and glutamate transporters (Slc17a7). SANPCs highly expressed cell markers of glutamatergic neurons (Snap25 and Slc17a7), whereas Gad1, a marker of GABAergic neurons, was negative. Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca

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.

Analysis of the Mechanism of Yam Protein in the Prevention and Treatment of Diabetes-induced Erectile Dysfunction Based on RNA-Seq Technology / 中国药房

OBJECTIVE：To study the potential mechanism of yam protein （DOT） in the prevention and treatment of diabetes-induced erectile dysfunction （DIED）. METHODS ：DIED model was induced by high-glucose and high-fat diet and intraperitoneal injection of streptozotocin （40 mg/kg）. The experiment was set up in the normal control group （normal saline ）， model group （normal saline ），DOT low-dose ，medium-dose and high-dose groups （0.3，0.6，0.9 mg/kg），sildenafil group （positive control ，4.4 mg/kg），with 9 rats in each group. In the stage of successful establishment of diabetes model and initiation of inducing DIED ，rats in each group were given relevant solution intragastrically ，once a day ，for consecutive 11 weeks. Body weight，fasting plasma glucose （FPG），the times and rate of penile erection ，fasting insulin （FINS），insulin resistance index （IR），the contents of endothelial nitric oxide synthase （eNOS）and cyclic guanosine monophosphate （cGMP）in penile cavernous tissue were determined so as to evaluate the intervention effects of DOT on DIED model rats. High-glucose damaged mice cavernous endothelial cells （MCECs）model was induced by 30 mmol/L glucose for 48 h，and then give DOT 125，250，500 μg/mL. The cell viability was detected so as to evaluate the effects of DOT on high-glucose damaged MCECs model. RNA-Seq mail：xingxin0902@163.com technology was adopted to screen the differentially expressed genes between normal MCECs and high-glucose damaged MCECs，high-glucose damaged MCECs and MCECs treated with 250 μg/mL DOT. Gene ontology（GO）function enrichment analysis and KEGG pathway enrichme nt analysis were performed for differentially expressed genes. The common differentially expressed genes between 2 groups were analyzed ，and mRNA expressions of six key genes were validated. RESULTS ：Different doses of DOT could reverse the reduction of body weight ，the increase of FINS and IR ，the reduction of the times and rate of penile erection ，the decrease of eNOS and cGMP contents in penile cavernous tissue of DIED model rats ；above indexes of DIED model rats were reversed significantly after treated with high-dose of DOT（P＜0.05 or P＜0.01）. 125，250，500 μg/L DOT could significantly improve the activity of high-glucose damaged MCECs （P＜0.05 or P＜0.01）. RNA-Seq technology showed that compared with normal MCECs ，a total of 48 differentially expressed genes were found in high-glucose damaged MCECs. Compared with high-glucose damaged MCECs ，a total of 779 differentially expressed genes were found in MCECs treated with DOT. The differentially expressed genes of 2 groups were mainly cellular process in biological process annotation ，cellular part in cell component annotation and binding molecular function in molecular function annotation ，which were mainly enriched in extracellular matrix receptor interaction pathway ，mismatch repair pathway ， phosphatidylinositol 3 kinase-protein kinase B （PI3K-Akt）signal pathway and so on. Among differentially expressed genes of 2 groups，13 common differentially expressed genes such as Aldh1a1，Abcc5，Tac1 were found. DOT could significantly reverse the expression of the above common differentially expressed genes in high-glucose damaged MCECs. After validation ，DOT could significantly reverse the mRNA expression of TGF-β3，Txnip，Aldh1a1，Loxl1，Mt1 and Mt2 in high-glucose damaged MCECs. CONCLUSIONS：DOT could improve the symptom of DIED model rats ，the mechanism of which may be related to biological pathway of inhibiting fibrosis and reducing oxidative stress ，so as to improve the endothelial function of cavernous body.