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.

Alterações sistêmicas na assinatura do ciclo celular de pacientes com COVID-19 / Systemic changes in the cell cycle signature of patients with COVID-19

Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Investigação de marcadores moleculares sanguíneos durante a infecção pelo vírus Chikungunya / Investigation of blood molecular markers during Chikungunya virus infection

A febre Chikungunya (CHIKF) é uma infecção viral causada pelo vírus Chikungunya (CHIKV). Os sintomas agudos incluem febre alta de início súbito, erupção cutânea, poliartrite e poliartralgia. Embora a infecção geralmente seja resolvida em menos de duas semanas, muitos pacientes experenciam recorrente dor e inflamação nas articulações, que podem persistir por anos. Esse estudo buscou marcadores moleculares no sangue de infectados pelo CHIKV que estejam associados a dor articular e cronicidade da CHIKF. O sequenciamento de receptores de células B (BCR) e T (TCR) demonstrou que a infecção por CHIKV diminui a diversidade desses receptores. Essa diversidade é ainda menor, durante a fase aguda da infecção, naqueles pacientes que irão desenvolver cronicidade. A menor diversidade de BCR em infectados está associada a um aumento na expressão de genes envolvidos na diferenciação e ativação de osteoclastos pela sinalização RANK/RANKL. Em adição, a cronicidade pode estar relacionada um aumento na expressão do gene ZBTB7A cuja expressão confere maior resistência a apoptose em precursores de osteoclastos naqueles pacientes que vão se tornar crônicos. Caso o envolvimento dos osteoclastos durante a patogênese de CHIKF seja confirmado, os pacientes poderão se beneficiar de abordagens terapêuticas já existentes como alternativas adicionais ao tratamento de CHIKF

Chikungunya fever (CHIKF) is a viral infection caused by the Chikungunya virus (CHIKV). Acute symptoms include sudden-onset high fever, rash, polyarthritis, and polyarthralgia. Although the infection usually resolves within two weeks, many patients experience recurrent joint pain and inflammation, which can persist for years. This study sought molecular markers in the blood of CHIKV-infected individuals that are associated with joint pain and chronicity of CHIKF. Sequencing of B (BCR) and T (TCR) cell receptors demonstrated that CHIKV infection decreases the diversity of these receptors. The diversity is even lower, during the acute phase of the infection, in those patients who will develop chronicity. The lower diversity of BCR in infected individuals is associated with an increase in the expression of genes involved in the differentiation and activation of osteoclasts by RANK/RANKL signaling. In addition, chronicity may be related to an increase in the expression of the ZBTB7A gene whose expression confers greater resistance to apoptosis in osteoclast precursors in those patients who will become chronic. If osteoclast role during CHIKF pathogenesis is confirmed, patients may benefit from existing therapeutic approaches as additional alternatives to CHIKF treatment

Similar hypothyroid and sepsis circulating mRNA expression could be useful as a biomarker in nonthyroidal illness syndrome: a pilot study

ABSTRACT Objective: Based on hypothetical hypothyroidism and nonthyroidal illness syndrome (NTIS) gene expression similarities, we decided to compare the patterns of expression of both as models of NTIS. The concordant profile between them may enlighten new biomarkers for NTIS challenging scenarios. Materials and methods: We used Ion Proton System next-generation sequencing to build the hypothyroidism transcriptome. We selected two databanks in GEO2 platform datasets to find the differentially expressed genes (DEGs) in adults and children with sepsis. The ROC curve was constructed to calculate the area under the curve (AUC). The AUC, chi-square, sensitivity, specificity, accuracy, kappa and likelihood were calculated. We performed Cox regression and Kaplan-Meier analyses for the survival analysis. Results: Concerning hypothyroidism DEGs, 70.42% were shared with sepsis survivors and 61.94% with sepsis nonsurvivors. Some of them were mitochondrial gene types (mitGenes), and 95 and 88 were related to sepsis survivors and nonsurvivors, respectively. BLOC1S1, ROMO1, SLIRP and TIMM8B mitGenes showed the capability to distinguish sepsis survivors and nonsurvivors. Conclusion: We matched our hypothyroidism DEGs with those in adults and children with sepsis. Additionally, we observed different patterns of hypothyroid-related genes among sepsis survivors and nonsurvivors. Finally, we demonstrated that ROMO1, SLIRP and TIMM8B could be predictive biomarkers in children's sepsis.

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.

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.

Bioinformatics analysis of gene expression profiles of retinoblastoma / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the key genes and molecular markers involved in the retinoblastoma development through bioinformatics.METHODS: The mRNA microarray datasets from the Gene Expression Omnibus(GEO)database were obtained, and the differentially expressed gene(DEG)between retinoblastoma cell lines and normal retinal pigment epithelial(RPE)cell lines were analyzed through gene ontology(GO)and KEGG enrichment analysis. To screen key genes, establish protein-protein interaction(PPI)network, and use receiver operating characteristic(ROC)curve to assess clinical diagnostic efficacy. The RNA expressions of key genes in retinoblastoma cell lines and normal RPE cell lines were compared by qRT-PCR.RESULTS: A total of 121 DEGs were obtained from the retinoblastoma dataset of GSE97508 and GSE110811. KEGG pathway analysis showed that DEG were enriched in phototransduction, cell cycle, and p53 signaling pathways. A total of 9 key genes, including MCM6, DTL, UBE2T, TOP2A, NUSAP1, CENPK, RRM2, RLBP1, and RHO, were obtained from the intersection of PPI network analysis and the top 30 DEG from each dataset. The differentially expressed 9 key genes were verified in GSE24673. ROC analysis showed that the area under the curve(AUC)for UBE2T, RRM2, and RHO was ≥80%, and there was a statistical significance(P>0.05). The mRNA level of UBE2T and RRM2 in retinoblastoma was significantly higher than APRE-19 cell line, while the mRNA level of RHO was significantly lower than that of ARPE-19 cell line.CONCLUSION: UBE2T, RRM2, and RHO may be served as potential molecular markers and potential therapeutic targets for retinoblastoma.

Expression pattern of MMP-12 in long-term optic nerve regeneration induced by lens injury / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression changes of MMP-12 during the long-term axon regeneration induced by the lens injury after the optic nerve clamp trauma in sprague-dawley(SD)rats.METHODS: The optic nerve injury model and lens injury model of SD rats were established, and the 24 experimental animals were divided into control group; lens injury group; optic nerve injury group; lens injury combined with optic nerve injury group, with 6 rats in each group. Reference transcriptome sequencing was used to analyze the expression changes of differentially expressed genes in the injured optic nerve region, and relevant differentially expressed genes with high expression were screened. Quantitative real-time polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to quantify the expression changes of matrix metalloproteinase-12(MMP-12)in the injured optic nerve region.RESULTS: The Principal Component Analysis of transcriptome sequencing indicated that lens injury combined with optic nerve injury was the principal component of gene expression change. Analysis of gene expression differences showed that the expression of MMP-12 gene was up-regulated in the lens injury combined with optic nerve injury group. The mRNA expression level of MMP-12 in the lens injury combined optic nerve injury group was up-regulated compared with the control group, the optic nerve injury group and the lens injury group at 14d and 21d after successful modeling(P<0.05). At 7, 28d, there was no difference in expression among all groups. The protein expression level of MMP-12 in the lens injury combined with optic nerve injury group was up-regulated compared with the control group and optic nerve injury group at 7, 14 and 21d after successful modeling(P<0.05), and it was up-regulated in the lens injury group combined with optic nerve injury group compared with optic nerve injury group at 21d(P<0.05). At 28d, there was no difference in expression among all groups.CONCLUSION: The up-regulated expression of MMP-12 may be involved in the long-term regeneration of the optic nerve after lens injury.

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.

Transcriptional analysis on biomarkers of liver injury induced by anti-tuberculosis drugs / 上海预防医学

ObjectiveThe study utilized human transcriptome microarray to explore biomarkers for diagnosing drug-induced liver injury （DILI） caused by anti-tuberculosis drugs. MethodsA 6-month follow-up study was conducted on 152 patients treated with anti-tuberculosis drugs in designated hospitals in Shanghai. The blood samples were collected at the 0， 2， 4， 8， 12 and 24 weeks after treatment. According to the clinical biochemical indicators， the research subjects were divided into DILI cases （34 cases） and Control cases （118 cases）. Single factor analysis was conducted on the influencing factors between the two groups. In a 1∶1 matched DILI-control study， RNA samples of 13 pairs of cases were sequenced by the whole transcript expression mRNA array. Differentially expressed genes （DEGs） were screened by Hotelling's T2 value sequencing and the expression trend analysis of genes by STEM （short-time series expression miner）， and the functional enrichment and pathway analysis of DEGs were carried out. ResultsIn total 152 clinical cases， weight of patients was a risk factor for the occurrence of hepatotoxicity caused by anti-tuberculous drugs. Based on the analysis results of mRNA array， 513 DEGs were screened by Hotelling's T2 value sequencing method， which were enriched in 32 annotations of GO （Gene Ontology） analysis and 10 pathways of KEGG （Kyoto encyclopedia of genes and genomes） analysis. One differential expression pattern was screened by STEM， which was enriched in 2 biological process notes of GO. Among them， the key genes AIM2， CD86， CXCL10 and non-coding RNAs SCARNA10， SNHG10 and SNORD105 are potential biomarkers of DILI caused by anti-tuberculosis drugs. ConclusionIn this research for biomarkers conducted on cases with liver injury caused by anti-tuberculosis drugs， biological pathways associated with hepatotoxicity are identified and a series of key genes related with drug-induced liver injury are found， which provides the basis for mechanism study and searching for earlier and more sensitive biomarkers.