The Development of Chinese Herbal Formulae for Non-severe COVID-19 Based on Artificial Intelligence Technology and Investigation of Its Action Mechanisms / 中医杂志

ObjectiveTo develop traditional Chinese medicine （TCM） formulae for the treatment of nonsevere coronavirus disease 2019 （COVID-19） and to explore its anti-inflammatory mechanism. MethodsThe dysregulated signaling pathways were determined in macrophages from bronchoalveolar lavage fluid of COVID-19 patients and in lung epithelial cells infected with SARS-CoV-2 in vitro based on transcriptome analysis. A total of 102 TCM formulae for the clinical treatment of nonsevere COVID-19 were collected through literature. The pathway-reversing rates of these formulae in macrophages and lung epithelial cells were evaluated based on signature signaling pathways， and the basic formula was determined in conjunction with TCM theory. The commonly used Chinese materia medica for nonsevere COVID-19 were summarized from the 102 TCM formulae as abovementioned. And together with the screening results from the Pharmacopoeia of the People's Republic of China， a “Chinese materia medica pool” was esta-blished for the development of TCM formulae for COVID-19. The regulatory effects of each herb on signaling pathways were obtained based on targeted transcriptome analysis. Oriented at reversing dysregulated signaling pathways of COVID-19， the calculation was carried out， and the artificial intelligent methods for compositing formulae， that are exhaustive method and parallel computing， were used to obtain candidate compound formulas. Finally， with reference to professional experience， an innovative formula for the treatment of nonsevere COVID-19 was developed. The ethanol extract of the formula was evaluated for its anti-inflammatory effects by detecting the mRNA expression of interleukin 1b （Il1b）， C-X-C motif chemokine ligand 2 （Cxcl2）， C-X-C motif chemokine ligand 10 （Cxcl10）， C-C motif chemokine ligand 2 （Ccl2）， nitric oxide synthase 2 （Nos2）， and prostaglandin-endoperoxide synthase 2 （Ptgs2） using reverse transcription-quantitative polymerase chain reaction （RT-qPCR） in RAW264.7 cells treated with lipopolysaccharide （LPS）. ResultsIn macrophages and lung epithelial cells， 34 dysregulated signaling pathways associated with COVID-19 were identified respectively. The effects of the 102 formulae for clinical treatment of nonsevere COVID-19 were evaluated based on the dysregulated signaling pathways and targeted transcriptome， and the result showed that Yinqiao Powder and Pingwei Powder （银翘散合平胃散， YQPWP） ranked first， reversing 91.18% of the dysregulated signaling pathways in macrophages and 100% of the dysregulated signaling pathways in lung epithelial cells. Additionally， YQPWP had the function of scattering wind and clearing heat， resolving toxins and removing dampness in accordance with the pathogenesis of wind-heat with dampness in COVID-19. It was selected as the basic formula， and was further modified and optimized to develop an innovative fomula Qiaobang Zhupi Yin （翘蒡术皮饮， QBZPY） based on expert experience and artificial intelligence in composing formulae. QBZPY can reverse all the dysregulated signaling pathways associated with COVID-19 in macrophages and lung epithelial cells， with the reversing rates of 100%. The chief medicinal of QBZPY， including Lianqiao （Fructus Forsythiae）， Xixiancao （Herba Siegesbeckiae） and Niubangzi （Fructus Arctii）， can down-regulate multiple signaling pathways related with virus infection， immune response， and epithelial damage. RT-qPCR results indicated that compared with the model group， the QBZPY group down-regulated the mRNA expression of Il1b， tumor necrosis factor （Tnf）， Cxcl2， Cxcl10， Ccl2， Nos2 and Ptgs2 induced by LPS in RAW264.7 cells （P＜0.05 or P＜0.01）. ConclusionBased on targeted transcriptome analysis， expert experience in TCM and artificial intelligence， QBZPY has been developed for the treatment of nonsevere COVID-19. The ethanol extract of QBZPY has been found to inhibit mRNA expression of several pro-inflammatory genes in a cellular inflammation model.

Transcriptome Sequencing on Treatment of Kidney Deficiency and Blood Stasis-thin Endometrium in Rats with Bushen Huoxue Prescription Through Enema / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Bushen Huoxue enema in treating the rat model of kidney deficiency and blood stasis-thin endometrium （KDBS-TE） by transcriptome sequencing. MethodThe rat model of KDBS-TE was established by administration of tripterygium polyglycosides tablets combined with subcutaneous injection of adrenaline. The pathological changes of rat endometrium in each group were then observed. Three uterine tissue specimens from each of the blank group， model group， and Bushen Huoxue enema group were randomly selected for transcriptome sequencing. The differentially expressed circRNAs， lncRNAs， and miRNAs were screened， and the disease-related specific competitive endogenous RNA （ceRNA） regulatory network was constructed. Furthermore， the gene ontology （GO） functional annotation and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the mRNAs in the network. ResultCompared with the blank group， the model group showed endometrial dysplasia， decreased endometrial thickness and endometrial/total uterine wall thickness ratio （P<0.01）， and differential expression of 18 circRNAs， 410 lncRNAs， and 7 miRNAs. Compared with the model group， the enema and estradiol valerate groups showed improved endometrial morphology and increased endometrial thickness and ratio of endometrial to total uterine wall thickness （P<0.05）. In addition， 21 circRNAs， 518 lncRNAs， and 17 miRNAs were differentially expressed in the enema group. The disease-related specific circRNA-miRNA-mRNA regulatory network composed of 629 nodes and 664 edges contained 2 circRNAs， 34 miRNAs， and 593 mRNAs. The lncRNA-miRNA-mRNA regulatory network composed of 180 nodes and 212 edges contained 5 lncRNAs， 10 miRNAs， and 164 mRNAs. The mNRAs were mainly enriched in Hippo signaling pathway， autophagy-animal， axon guidance， etc. ConclusionBushen Huoxue enema can treat KDBS-TE in rats by regulating specific circRNAs， lncRNAs， and miRNAs in the uterus and the ceRNA network.

Indian food habit & food ingredients may have a role in lowering the severity & high death rate from COVID-19 in Indians: findings from the first nutrigenomic analysis

Background & objectives: During the COVID-19 pandemic, the death rate was reportedly 5-8 fold lower in India which is densely populated as compared to less populated western countries. The aim of this study was to investigate whether dietary habits were associated with the variations in COVID-19 severity and deaths between western and Indian population at the nutrigenomics level. Methods: In this study nutrigenomics approach was applied. Blood transcriptome of severe COVID-19 patients from three western countries (showing high fatality) and two datasets from Indian patients were used. Gene set enrichment analyses were performed for pathways, metabolites, nutrients, etc., and compared for western and Indian samples to identify the food- and nutrient-related factors, which may be associated with COVID-19 severity. Data on the daily consumption of twelve key food componentsacross four countries were collected and a correlation between nutrigenomics analyses and per capita daily dietary intake was investigated. Results: Distinct dietary habits of Indians were observed, which may be associated with low death rate from COVID-19. Increased consumption of red meat, dairy products and processed foods by western populations may increase the severity and death rate by activating cytokine storm-related pathways, intussusceptive angiogenesis, hypercapnia and enhancing blood glucose levels due to high contents of sphingolipids, palmitic acid and byproducts such as CO2 and lipopolysaccharide (LPS). Palmitic acid also induces ACE2 expression and increases the infection rate. Coffee and alcohol that are highly consumed in western countries may increase the severity and death rates from COVID-19 by deregulating blood iron, zinc and triglyceride levels. The components of Indian diets maintain high iron and zinc concentrations in blood and rich fibre in their foods may prevent CO2 and LPS-mediated COVID-19 severity. Regular consumption of tea by Indians maintains high high-density lipoprotein (HDL) and low triglyceride in blood as catechins in tea act as natural atorvastatin. Importantly, regular consumption of turmeric in daily food by Indians maintains strong immunity and curcumin in turmeric may prevent pathways and mechanisms associated with SARS-CoV-2 infection and COVID-19 severity and lowered the death rate. Interpretation & conclusions: Our results suggest that Indian food components suppress cytokine storm and various other severity related pathways of COVID-19 and may have a role in lowering severity and death rates from COVID-19 in India as compared to western populations. However, large multi-centered case?control studies are required to support our current findings.

Differential gene expression analysis in germinating and dormant teliospores of Tilletia indica using RNA seq approach

Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease.

Biofilm dysbiosis and caries activity: a surface or an individual issue?

Abstract Objective This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. Methodology A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Results Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Conclusion Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

Maackiain inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by inhibiting the MAPK/Ras signaling pathway / 中国天然药物

Nasopharyngeal carcinoma (NPC) is the third most common malignancy with a high recurrence and metastasis rate in South China. Natural compounds extracted from traditional Chinese herbal medicines have been developed and utilized for the treatment of a variety of cancers with modest properties and slight side effects. Maackiain (MA) is a type of flavonoid that was first isolated from leguminous plants, and it has been reported to relieve various nervous system disorders and exert anti-allergic as well as anti-inflammatory effects. In this study, we demonstrated that MA inhibited proliferation, arrested cell cycle and induced apoptosis in nasopharyngeal carcinoma CNE1 and CNE2 cells in vitro and in vivo. The expression of the related proteins associated with these processes were consistent with the above effects. Moreover, transcriptome sequencing and subsequent Western blot experiments revealed that inhibition of the MAPK/Ras pathway may be responsible to the anti-tumor effect of MA on NPC cells. Therefore, the effects of MA and an activator of this pathway, tertiary butylhydroquinone (TBHQ), alone or combination, were investigated. The results showed TBHQ neutralized the inhibitory effects of MA. These data suggest that MA exerts its anti-tumor effect by inhibiting the MAPK/Ras signaling pathway and it has the potential to become a treatment for patients with NPC.

Spatial transcriptome analysis of long non-coding RNAs reveals tissue specificity and functional roles in cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Long non-coding RNAs (lncRNAs) play a significant role in maintaining tissue morphology and functions, and their precise regulatory effectiveness is closely related to expression patterns. However, the spatial expression patterns of lncRNAs in humans are poorly characterized. Here, we constructed five comprehensive transcriptomic atlases of human lncRNAs covering thousands of major tissue samples in normal and disease states. The lncRNA transcriptomes exhibited high consistency within the same tissues across resources, and even higher complexity in specialized tissues. Tissue-elevated (TE) lncRNAs were identified in each resource and robust TE lncRNAs were refined by integrative analysis. We detected 1 to 4684 robust TE lncRNAs across tissues; the highest number was in testis tissue, followed by brain tissue. Functional analyses of TE lncRNAs indicated important roles in corresponding tissue-related pathways. Moreover, we found that the expression features of robust TE lncRNAs made them be effective biomarkers to distinguish tissues; TE lncRNAs also tended to be associated with cancer, and exhibited differential expression or were correlated with patient survival. In summary, spatial classification of lncRNAs is the starting point for elucidating the function of lncRNAs in both maintenance of tissue morphology and progress of tissue-constricted diseases.

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing / 生物工程学报

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Identification and expression analysis of MADS-box gene family in Docynia delavayi (Franch.) Schneid / 生物工程学报

MADS-box gene family is a significant transcription factor family that plays a crucial role in regulating plant growth, development, signal transduction, and other processes. In order to study the characteristics of MADS-box gene family in Docynia delavayi (Franch.) Schneid. and its expression during different stages of seed germination, this study used seedlings at different stages of germination as materials and screened MADS-box transcription factors from the transcriptome database of D. delavayi using bioinformatics methods based on transcriptome sequencing. The physical and chemical properties, protein conservative motifs, phylogenetic evolution, and expression patterns of the MADS-box transcription factors were analyzed. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of MADS-box gene family members during different stages of seed germination in D. delavayi. The results showed that 81 genes of MADS-box gene family were identified from the transcriptome data of D. delavayi, with the molecular weight distribution ranged of 6 211.34-173 512.77 Da and the theoretical isoelectric point ranged from 5.21 to 10.97. Phylogenetic analysis showed that the 81 genes could be divided into 15 subgroups, among which DdMADS27, DdMADS42, DdMADS45, DdMADS46, DdMADS53, DdMADS61, DdMADS76, DdMADS77 and DdMADS79 might be involved in the regulation of ovule development in D. delavayi. The combination of the transcriptome data and the qRT-PCR analysis results of D. delavayi seeds indicated that DdMADS25 and DdMADS42 might be involved in the regulation of seed development, and that DdMADS37 and DdMADS38 might have negative regulation effects on seed dormancy. Previous studies have reported that the MIKC* subgroup is mainly involved in regulating flower organ development. For the first time, we found that the transcription factors of the MIKC* subgroup exhibited a high expression level at the early stage of seed germination, so we speculated that the MIKC* subgroup played a regulatory role in the process of seed germination. To verify the accuracy of this speculation, we selected DdMADS60 and DdMADS75 from the MIKC* subgroup for qRT-PCR experiments, and the experimental results were consistent with the expression trend of transcriptome sequencing. This study provides a reference for further research on the biological function of D. delavayi MADS-box gene family from the perspective of molecular evolution.

The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

Potential value of differential expression of CDC42 gene in adult and umbilical cord blood reticulocytes / 中国输血杂志

【Objective】 To explore critical regulatory genes in the hemoglobin switch process by analyzing transcriptomic data from the GSE6236, GSE17639 and GSE35102 datasets. 【Methods】 The mRNA expression profiles of the three datasets were downloaded from the GEO database and gene annotation was performed using the AnnoProbe package.The remove-BatchEffect function of the Limma package was used to remove batch effects. Weighted gene co-expression network analysis (WGCNA) was used to explore the most relevant modular genes in reticulocytes. The receiver operating characteristic curve (ROC) was used to assess the value of differential genes in differentiating between cord blood and adult peripheral blood reticulocytes. The GSE35102 dataset was used to validate changes in differential gene expression during hemoglobin transformation. Finally, real-time quantitative PCR was used to verify differential gene expression in cord blood and adult peripheral blood reticulocytes. 【Results】 Twelve genes showed differential expression in reticulocytes from cord blood and adult peripheral blood ( |logFC|≥1.5, P<0.05). WGCNA found that genes in the blue module were most strongly associated with reticulocytes (R2 =0.76,P<0.001). Of the five genes that overlapped between the two, only CDC42 showed differential expression in the combined dataset (t =3.776, P<0.001) and was able to better differentiate between reticulocytes in cord blood and adult peripheral blood. The expression of CDC42 varied significantly during the hemoglobin transformation process (Z = -2.908, P<0.01), and was significantly lower in adult reticulocytes compared to reticulocytes from cord blood (t =7.824, P <0.001). 【Conclusion】 The CDC42 gene is involved in the hemoglobin switching of reticulocytes and could be a potential therapeutic target for sickle cell disease.

Transcriptome analysis and differential gene screening of Panax ginseng seed in different sand storage stages / 药学学报

Analyze the changes in gene expression profiles during the process of Panax ginseng seed dormancy release, and screen for differential genes, providing a basis for analyzing the mechanism of P. ginseng seed dormancy release. Comparative transcriptome analysis was conducted by using RNA-Seq sequencing technology in P. ginseng seeds stored at different low temperature. A total of 80.97 Gb of Raw reads and 80.19 Gb of Clean reads were obtained from the transcriptome. Principal component analysis and correlation analysis showed that there were significant differences in gene expression patterns at different developmental stages. Upset results showed that 46 248 unigenes were co-expressed in four stages, and 414, 445, 400 and 389 unigenes were specifically expressed in 0, 8,14 and 28 days, respectively. Gene Ontology functional annotation showed that the differentially expressed genes were mainly involved in nsaturated fatty acid biosynthetic process, nuclear body and oxidoreductase activity. Encyclopedia of Genes and Genomes metabolic pathway showed that differentially expressed genes were mainly involved in peroxisome, mitogen-activated protein kinase signaling pathway-plant, plant hormone signal transduction, ribosome, biosynthesis of unsaturated fatty acid, circadian rhythm-plant and other metabolic pathways. In the process of P. ginseng seed dormancy release, multiple biological processes, such as unsaturated fatty acid biosynthesis and plant hormone signal transduction, are required to coordinate regulation, which constitutes a complex dormancy release regulation network. Transcriptome analysis and differential gene screening of P. ginseng seeds at different sand storage time laid a foundation for the analysis of P. ginseng seed dormancy release mechanism and molecular breeding.

Mechanism of Polysaccharides from Two Spleen-invigorating Chinese Medicine Prescriptions in Regulating Growth of Bacteroides fragilis in vitro： Based on Differential Expression of Polysaccharide Utilization Loci / 中国实验方剂学杂志

ObjectiveTo investigate the effects of using the polysaccharides from two Chinese medicine compound prescriptions as the carbon source on the growth of Bacteroides fragilis and to decipher the mechanism from the perspective of differential expression of polysaccharide utilization loci （PULs） based on transcriptomics. MethodThe media with different carbon sources ［20% polysaccharides of Lizhongtang， polysaccharides of Shenling Baizhusan， glucose， and brain heart infusion （BHI） Broth］ were used for the anaerobic culture of B. fragile ATCC25285. The effects of different carbon sources on the growth of B. fragilis ATCC25285 were determined by continuous sampling and spectrophotometry. Then， transcriptome sequencing was performed for the cultures obtained with different carbon sources to study the mechanism of different carbon sources in regulating bacterial growth. ResultThe concentration of bacteria with the polysaccharide of Lizhongtang， polysaccharide of Shenling Baizhusan， BHI Broth， and glucose as the carbon sources peaked at 26， 32， 26， 38 h， respectively， and the bacteria in all the four groups achieved robust growth. Gene ontology （GO） enrichment indicated that the differentially expressed genes in the Lizhongtang polysaccharide group and Shenling Baizhusan polysaccharide group were concentrated in the transport and transmembrane transport of dicarboxylic acid. The Shenling Baizhusan polysaccharide and BHI Broth groups showed high expression of PUL 4 and 27， glycoside hydrolase 13 （GH13）， and glycosyl transferases 5 （GT5）. PUL9 was highly expressed in Shenling Baizhusan polysaccharide group， and PUL 17， 19， and 20， GH3， and GH144 in the BHI Broth group. PUL27 and GT5 were highly expressed in Shenling Baizhusan polysaccharide and glucose groups. PUL 4 and 9 and GH13 were only highly expressed in Shenling Baizhusan polysaccharide group， and PUL 2， 17， and 19 and GH2 in the glucose group. Both Lizhongtang polysaccharide group and BHI group highly expressed PUL 4， 17， 19， 20， and 27， GH3， and GH144. PUL 2， 8， 23， and 27， GH2， and GH57 were highly expressed in Lizhongtang polysaccharide group， while GH13 showed high expression in the BHI group. Both the glucose and Lizhongtang polysacharride groups showed high expression of PUL 4 and 27 and GH2. PUL 4， 8， 20， and 23， GH3， and GH144 were highly expressed in Lizhongtang polysaccharide group， while PUL30 was highly expressed in the glucose group. ConclusionThe in vitro experiments and transcriptome sequencing results confirmed that the expression of PULs and GH may provide benefits or costs to the adaptive growth of Bacteroides fragilis ATCC25285 cultured with different carbon sources， which may be one of the mechanisms by which polysaccharides from Chinese medicine compound prescriptions regulate the growth of B. fragilis ATCC25285. The findings can provide a reference for further research on the relationship between B. fragilis metabolic pathway and polysaccharides of Chinese medicine compound prescriptions.

Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus type Ⅲ and its antibody dependent enhancement / 中国生物制品学杂志

@#ObjectiveTo establish models of Dengue virus type Ⅲ（DENV-3,DV-3）infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation / 中华眼底病杂志

Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

Gene expression profile in human cervical epithelial cells infected with Chlamydia trachomatis / 中华微生物学和免疫学杂志

Objective:To compare gene expression profiles in normal human cervical epithelial cells (HcerEpic) before and after Chlamydia trachomatis ( Ct) infection. Methods:HcerEpic cells that were pretreated with DEAE-D were infected with Ct serotype E standard strain and then cultured for 44 h. Uninfected HcerEpic cells were used as the control group. Total RNA was extracted from the cells in each group and reverse transcribed to construct a cDNA library. Differences in gene expression profiles between the two groups were analyzed by high-throughput sequencing and the representative genes were selected for verification by qPCR. Results:A total of 23 997 genes were detected, including 125 differentially expressed genes. Among the 125 genes, 119 were up-regulated and six were down-regulated. GO analysis showed that the differentially expressed genes were enriched in several biological processes including defense response to virus, typeⅠinterferon signaling pathway and cellular responses to typeⅠinterferons. KEGG enrichment analysis showed the differentially expressed genes were mainly enriched in the pathways related to virus infections, such as influenza A virus, herpes simplex virus, EB virus and HPV, and NOD-like receptor pathway.Conclusions:There were significant differences in transcriptome profiles of HcerEpic cells before and after Ct infection. The differentially expressed genes were mainly enriched in the interferon pathway, which was closely related to the antiviral processes in cells. qPCR verified the differentially expressed genes and the genes closely related to the interferon pathway, such as ISG15, IFIT2, OASL and UBE2L6.

Mechanism of Huashi Baidu Prescription in Treating Influenza Viral Pneumonia Based on Transcriptome / 中国实验方剂学杂志

ObjectiveTo clarify the therapeutic effect of Huashi Baidu prescription on pneumonia in mice caused by influenza A （H1N1） virus and explore its mechanism based on the transcriptome. MethodA mouse influenza viral pneumonia model was built by intranasal infection with influenza A virus， and mice were continuously administered the drug for five days， so as to investigate the general condition， lung index， viral load， pathological morphology of lung tissue， survival time， and prolongation rate of survival time of mice and clarify the therapeutic effect of Huashi Baidu prescription on influenza viral pneumonia. Transcriptome technology was used to detect the differentially expressed genes in the lung tissue of mice in the model group and the normal group， as well as the Huashi Baidu prescription group and the model group， and the potential core target of the Huashi Baidu prescription for the treatment of influenza viral pneumonia was screened. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to verify the effect of Huashi Baidu prescription on the mRNA expression level of core target genes. ResultCompared with the normal group， the lung index and viral load in the lung tissue of the model group were significantly increased （P<0.05， P<0.01）. Compared with the model group， the high-dose group of Huashi Baidu prescription significantly prolonged the survival time of mice infected with influenza A virus （P<0.05） and significantly reduced the lung index value of mice （P<0.05） and the viral load of lung tissue. The high-dose， medium-dose， and low-dose groups of Huashi Baidu prescription could significantly reduce lung tissue inflammation， blood stasis， swelling， and other pathological changes in mice （P<0.05， P<0.01）. Transcriptome analysis of lung tissue showed that core genes were mainly enriched in the nuclear transcription factor-κB （NF-κB） signaling pathway， interleukin-17 （IL-17） signaling pathway， cytokine-cytokine receptor interaction， and other pathways after the intervention of Huashi Baidu prescription. TRAF6， NFKBIA， CCL2， CCL7， and CXCL2 were the top five node genes with combined score values. Real-time PCR validation showed that Huashi Baidu prescription significantly downregulated the mRNA expression of key genes TRAF6 and NFKBIA in the NF-κB signaling pathway， as well as chemokines CCL2， CCL7， and CXCL2 （P<0.05， P<0.01）. ConclusionHuashi Baidu prescription has a therapeutic effect on influenza viral pneumonia， possibly by inhibiting the expression of key nodes TRAF6 and NFKBIA in the NF-κB signaling pathway and that of chemokines CCL2， CCL7， and CXCL2， reducing the recruitment of inflammatory cells and viral load， and exerting anti-influenza viral pneumonia effects.

Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology / 药学学报

Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. Firstly, the effects of artesunate on cell activity, proliferation and apoptosis of U87 and U251 human glioma cells were explored. It was found that artesunate exerted stronger inhibitory effects on the activity and proliferation of U87 cells than U251 cells. It could significantly promote apoptosis in U87 cells (P < 0.05), while only high dose of artesunate can promote that of U251 cells (P < 0.01), detected by Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained through transcriptome sequencing and differential data analysis by using the lysates of U87 and U251 cells before and after artesunate treatment, aiming to explore the molecular mechanism of distinct artesunate sensitivity to two types of cells. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which apoptosis-related activating transcription factor 4 (ATF4)-DNA damage induced transcript 3 (DDIT3)- polyadenosine diphosphate ribose polymerase 1 (PARP1) signaling axis was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that ATF4-DDIT3-PARP1 signaling axis is the target pathway of artesunate intervening with U87 glioma cells, and PARP1 may be an important gene for U251 cells to develop resistance to artesunate. Our results not only provide fundamental experimental evidence for artesunate as a potential therapeutic drug in glioma treatment, but shed light into overcoming drug resistance in its clinical therapy.

Effect of Zishenwan on Intestinal Barrier and Transcriptome of Skeletal Muscle of db/db Diabetic Mice / 中国实验方剂学杂志

ObjectiveTo explore the effect of Zishenwan on glucose and lipid metabolism in spontaneous type 2 diabetes （db/db） mice and investigate the underlying mechanism for improving diabetes based on intestinal barrier function and skeletal muscle transcriptome sequencing results. MethodLiquid chromatography-tandem mass spectrometry （LC-MS/MS） was used to analyze the components of Zishenwan. Sixteen 6-week-old db/db mice were divided into a model group and a Zishenwan group， while eight wild-type mice were assigned to the normal group. The Zishenwan group received oral administration of drugs for six weeks， during which fasting blood glucose， body weight， and food intake were measured. Serum total cholesterol （TC） and triglyceride （TG） levels were determined， and fasting insulin levels were measured to calculate the homeostatic model assessment of insulin resistance （HOMA-IR）. After the treatment， skeletal muscle and ileum tissues were collected， followed by hematoxylin-eosin （HE） staining. Immunohistochemistry was used to detect the expression of tight junction proteins occludin and zonula occludens-1 （ZO-1） in the ileum. Transcriptome sequencing was performed to detect the skeletal muscle transcriptome， and enrichment analysis was conducted for differentially expressed genes. ResultMultiple active components were identified in Zishenwan. Compared with the normal group， the model group showed increased fasting blood glucose， body weight， TC， TG， and HOMA-IR （P<0.01）. Compared with the model group， Zishenwan significantly reduced fasting blood glucose， body weight， TC， TG， and HOMA-IR in db/db mice （P<0.01）， while there was no statistically significant difference in food intake. Compared with the normal group， the model group exhibited lipid deposition in skeletal muscle， as well as structural changes in the ileum， with significant decreases in the protein expression levels of intestinal occludin and ZO-1 （P<0.01）. Compared with the model group， Zishenwan improved the pathological changes in skeletal muscle and ileum， and increased the protein expression of occludin and ZO-1 in the ileum （P<0.01）. Transcriptome analysis suggested that Zishenwan might improve skeletal muscle metabolism and increase insulin sensitivity in mice. ConclusionZishenwan can improve glucose and lipid metabolism in db/db mice， and this effect may be related to its protection of intestinal barrier function and transcriptional regulation of skeletal muscle metabolism-related genes.