177 research outputs found

    Cytotoxic Effect of Recombinant Mycobacterium tuberculosis CFP-10/ESAT-6 Protein on the Crucial Pathways of WI-38 Cells

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    To unravel the cytotoxic effect of the recombinant CFP-10/ESAT-6 protein (rCFES) on WI-38 cells, an integrative analysis approach, combining time-course microarray data and annotated pathway databases, was proposed with the emphasis on identifying the potentially crucial pathways. The potentially crucial pathways were selected based on a composite criterion characterizing the average significance and topological properties of important genes. The analysis results suggested that the regulatory effect of rCFES was at least involved in cell proliferation, cell motility, cell survival, and metabolisms of WI-38 cells. The survivability of WI-38 cells, in particular, was significantly decreased to 62% with 12.5 μM rCFES. Furthermore, the focal adhesion pathway was identified as the potentially most-crucial pathway and 58 of 65 important genes in this pathway were downregulated by rCFES treatment. Using qRT-PCR, we have confirmed the changes in the expression levels of LAMA4, PIK3R3, BIRC3, and NFKBIA, suggesting that these proteins may play an essential role in the cytotoxic process in the rCFES-treated WI-38 cells

    Difference in the regulation of IL-8 expression induced by uropathogenic E. coli between two kinds of urinary tract epithelial cells

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    Bacterial adherence to epithelial cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of uropathogenic Escherichia coli (UPEC) have both been described to be important for the establishment of urinary tract infections (UTI). To explore the interactions between the host and bacterium responsible for the different environments of UPEC invasion, we examined the effect of pH and osmolarity on UPEC strain J96 fimbrial expression, and subsequent J96-induced interleukin-8 (IL-8) expression in different uroepithelial cells. The J96 strain grown in high pH with low osmolarity condition was favorable for the expression of type 1 fimbriae; whereas J96 grown in low pH with high osmolarity condition was beneficial for P fimbriae expression. Type 1 fimbriated J96 specifically invaded bladder 5637 epithelial cells and induced IL-8 expression. On the contrary, P fimbriated J96 invaded renal 786-O epithelial cells and induced IL-8 expression effectively. Type 1 fimbriated J96-induced IL-8 induction involved the p38, as well as ERK, JNK pathways, which leads to AP-1-mediated gene expression. P fimbriated J96-induced augmentation of IL-8 expression mainly involved p38-mediated AP-1 and NF-κB transcriptional activation. These results indicate that different expression of fimbriae in J96 trigger differential IL-8 gene regulation pathways in different uroepithelial cells

    Identification of activated cryptic 5′ splice sites using structure profiles and odds measure

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    The activation of cryptic 5′ splice sites (5′ SSs) is often related to human hereditary diseases. The DNA-based mutation screening strategies are commonly used to recognize the cryptic 5′ SSs, because features of the local DNA sequence can influence the choice of cryptic 5′ SSs. To improve the identification of the cryptic 5′ SSs, we developed a structure-based method, named SPO (structure profiles and odds measure), which combines two parameters, the structural feature derived from hydroxyl radical cleavage pattern and odds measure, to assess the likelihood of a cryptic 5′ SS activation in competing with its paired authentic 5′ SS. Compared to the current tools for identifying activated cryptic 5′ SSs, the SPO algorithm achieves higher prediction accuracy than the other methods, including MaxEnt, MDD, Markov model, weight matrix model, Shapiro and Senapathy matrix, Ri and ΔG. In addition, the predicted ΔSPO scores from the SPO algorithm exhibited a greater degree of correlation with the strength of cryptic 5′ SS activation than that measured from the other seven methods. In conclusion, the SPO algorithm provides an optimal identification of cryptic 5′ SSs, can be applied in designing mutagenesis experiments for various splicing events and may be helpful to investigate the relationship between structural variants and human hereditary diseases

    Ligand-Activated Peroxisome Proliferator-Activated Receptor-  Protects Against Ischemic Cerebral Infarction and Neuronal Apoptosis by 14-3-3  Upregulation

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    Thiazolidinediones (TZD) were reported to protect against ischemia-reperfusion (I/R) injury. Their protective actions are considered to be PPAR-γ (peroxisome proliferator-activated receptor γ)-dependent. However, it is unclear how PPAR-γ activation confers resistance to I/R

    Identification and prediction of RNA signal expression of species: splice site selections of RNA virus and human, and cytotoxic effect of recombinant Mycobacterium tuberculosis CFP-10/ESAT-10rotein on the crucial pathways of WI-38 cells

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    Identification and prediction of RNA signal expression is an important issue in genome research. Three important issues of RNA signal expression have been explored in this dissertation. The first one is identification of splice sites of RNA viruses, the second one is identification of cryptic 5’ splice site activation, and the last one is identification of crucial pathways. In the past, various methods have been developed for identifying splice sites of such species as Human, Drosophila, Arabidopsis thaliana, and so on, but not for RNA viruses. Splice site identification on an RNA virus has two potential difficulties seriously degrading the performance of most conventional splice site predictors. One is a limited number of genome strains available for a virus species and the other is the diversified sequence patterns around the splice sites caused by the high mutation frequency. Moreover, most splice site prediction methods have not taken into account the effect of mutations on splice sites. Therefore, they cannot effectively identify cryptic 5’ splice site activation when mutations occur around splice sites. For identification of crucial pathways, this dissertation focuses on the cytotoxic effect of recombinant Mycobacterium tuberculosis CFP-10/ESAT-10 protein on the crucial pathways of WI-38 cells, which has not been studied in depth previously. To overcome these three difficult issues, three new methods, called Genomic splice site prediction (GSSP algorithm), gapped-dinucleotide patterns with logarithmic frequency approach (GDLF algorithm), and crucial pathway analysis approach, have been proposed. GSSP algorithm used the eigen-patterns with cross-species strategy to identify splice sites for RNA viruses. The GSSP algorithm was shown to be effective and superior to NNsplice and SplicePredictor in predicting the splice sites of five RNA species in the Orthomyxoviruses family. The sensitivity and specificity achieved by the GSSP algorithm were all higher than 92% for splice sites. Furthermore, this method was successfully applied to identify the splice site prediction of human immunodeficiency virus type 1 (HIV1). GDLF algorithm combined gapped-dinucleotide patterns with logarithmic frequency to identify cryptic 5’ splice site activation when mutations occurred around splice sites. Based on the analyzed results, the GDLF algorithm was sufficiently shown to be a more efficient method than Ri value and free energy (△G) for identifying activated cryptic 5’ splice sites. The specificity achieved by the GDLF algorithm was 83% for cryptic 5’ splice sites when the sensitivity was fixed at 85%. Moreover, the GDLF algorithm was also successfully applied to the identification of alternative 5’ splice site selection of influenza A virus. rucial pathway analysis approach was an integrated analysis approach combining time-course microarray data and annotated pathway databases, and was proposed with the emphasis on identifying the potentially crucial pathways. The potentially crucial pathways were selected based on a composite criterion characterizing the average significance and topological properties of important genes. The analysis results suggested that the regulatory effect of rCFES was at least involved in cell proliferation, cell motility, cell survival, and metabolisms of WI-38 cells. The survivability of WI-38 cells, in particular, was significantly decreased to 62% with 12.5 μM rCFES. Furthermore, the focal adhesion pathway was identified as the potentially most-crucial pathway and 58 of 65 important genes in this pathway were down-regulated by rCFES treatment. Using qRT-PCR, we have confirmed the changes in the expression levels of LAMA4, PIK3R3, BIRC3, and NFKBIA, suggesting that these proteins may play an essential role in the cytotoxic process in the rCFES-treated WI-38 cells. The analysis results corroborate that the three proposed methods are effective in resolving the three underlying issues of RNA signal expression, respectively.口試委員會審定書………………………………………………………icknowledgments (Chinese)…………………………………………iibstract (Chinese)…………………………………………………iiibstract………………………………………………………………vontents………………………………………………………………viiiist of figures ……………………………………………………xiist of tables………………………………………………………xiiihapter 1 Introduction ……………………………………………1.1 Genomic project …………………………………………………1.2 RNA splicing ……………………………………………………2.3 Research status of mutations at splice sites …………3.4 Gene expression and gene regulation………………………4.5 Motivation…………………………………………………………6hapter 2 Related works …………………………………………11.1 Splice site identification ………………………………11.2 Activated cryptic 5’ splice site identification…14.3 Gene regulation pathway analysis…………………………16hapter 3 Materials and methods………………………………18.1 GSSP algorithm…………………………………………………18.1.1 Datasets………………………………………………………18.1.2 Proposed algorithm…………………………………………20.1.3 Sequence binarization………………………………………21.1.4 Consensus sequence…………………………………………23.1.5 Sequence pattern mining……………………………………23.2 GDLF algorithm…………………………………………………32.2.1 Datasets………………………………………………………32.2.2 Proposed algorithm…………………………………………33.3 Crucial pathway analysis approach………………………35.3.1 Cell cultures………………………………………………35.3.2 Expression and purification of rCFES…………………35.3.3 Cell survival assay………………………………………36.3.4 Microarray analysis………………………………………36.3.5 Significance analysis of gene expression…………37 .3.6 Pathway topology analysis………………………………38.3.7 RTQ-PCR analysis……………………………………………44hapter 4 Results and discussions……………………………45 .1 GSSP algorithm………………………………………………45.1.1 Performance analysis……………………………………45.1.2 Results………………………………………………………46.1.3 Comparisons with exist methods…………………………48.1.4 Application…………………………………………………57.2 GDLF algorithm…………………………………………………58.2.1 Performance analysis………………………………………58.2.2 Results………………………………………………………59.2.3 Comparisons with exist methods………………………60.2.4 Application…………………………………………………64.3 Crucial pathway analysis approach………………………68.3.1 Cytotoxic effect of rCFES on WI-38 …………………68.3.2 Identification of important genes……………………68.3.3 Potentially crucial pathways in rCFES-induced WI-38 cells…………………………………………………………………69.3.4 Validation of important genes by RTQ-PCR…………77.3.5 Application…………………………………………………82hapter 5 Conclusions and future works……………………88 eference……………………………………………………………92bbreviations………………………………………………………114ppendix………………………………………………………………116. List of test data for GDLF algorithm……………………116. List index of the 500 GDLF scores…………………………12

    Genomic Splicing Sites Prediction Algorithm Based on Nucleotide Sequence Pattern

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    Splicing sites prediction plays a major role in genomic research of biomedical science. If splicing sites of genomic sequence can be correctly predicted, numerous crucial problems in biomedical area may be resolved naturally. Some typical examples are finding pathogenic factors of serious diseases and developing new gene therapy and so on. The major difficulty in developing the method of predicting the splicing sites lies in th

    Inhibition of enterovirus 71- induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis

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    Enterovirus 71 infection causes significant morbidity and mortality in children, yet there is no effective treatment. In this study, a protein-bound pigment, allophycocyanin purified from bluegreen algae is first reported to exhibit anti-enterovirus 71 activity. Allophycocyanin neutralized the enterovirus 71-induced cytopathic effect in both human rhabdomyosarcoma cells and African green monkey kidney cells. The 50% inhibitory concentration of allophycocyanin for neutralizing the enterovirus 71-induced cytopathic effect was approximately 0.045 AE 0.012 mM in green monkey kidney cells. The cytotoxic concentrations of allophycocyanin for rhabdomyosarcoma cells and African green monkey kidney cells were 1.653 AE 0.003 mM and 1.521 AE 0.012 mM, respectively. A plaque reduction assay showed that the concentrations of allophycocyanin for reducing plaque formation by 50% were approximately 0.056 AE 0.007 mM and 0.101 AE 0.032 mM, when allophycocyanin were added at the state of viral adsorption and post-adsorption, respectively. Antiviral activity was more efficient in cultures treated with allophycocyanin before viral infection compared with that in the cultures treated after infection. Allophycocyanin was also able to delay viral RNA synthesis in the infected cells and to abate the apoptotic process in enterovirus 71-infected rhabdomyosarcoma cells with evidence of characteristic DNA fragmentation, decreasing membrane damage and declining cell sub-G1 phase. It is concluded that allophycocyanin possesses antiviral activity and has a potential for development as an antienterovirus 71 agent

    Additional file 1: of Inferring microbial interaction network from microbiome data using RMN algorithm

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    The table of OTUs for all the samples at the genus level with OTU assignment before filtering process. (XLS 81 kb
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