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Of up or down regulation were well preserved K162 site between the array and validation studies 15900046 (Table S1). Ctse, Foxp1, IL-6, and Muc1 were modulated in the array but were not validated. Other genes such as Ifng, IL-3, Jak1, Stat6, and Vwf were not modulated in either study. SignificantIngenuity Pathways AnalysisAll significantly modulated genes were submitted to Ingenuity Pathways Analysis. IPA has some advantages over DAVID in that it takes into account the fold change associated with each modulated transcript when determining biological significance.Tick-Host InterfaceFigure 2. Diagram showing the interactions between genes and the temporal increase in representation of the acute inflammation and immune cell recruitment pathway from the IPA analysis. Gene expression data was I-BRD9 entered into ingenuity pathway analysis software to identify potential canonical pathways modulated during tick feeding. Upregulated genes are orange/pink, downregulated genes are green, and unchanged or unsignificant genes are grey. doi:10.1371/journal.pone.0047301.gincreases in chemokine gene expression begin at 1 hr postinfestation and intensify as time progresses, supporting the importance of chemotaxis seen in the array. Upregulation of IL1b, IL-10, and IL-4ra along with the general inflammation group supports early inflammatory changes at the bite site. While genes in the viral reproduction group showed some significant modulation, changes between time points were not consistent, casting some doubt on the biological significance of these differences.HistopathologyConcurrently with gene expression measurements, we pursued histopathological analysis of the bite site to see if morphological changes could be correlated with array data. Resident cells such as keratinocytes, fibroblasts, dendritic cells, and mast cells initially sense cutaneous tissue injury or antigenic stimuli. These cells release rapidly synthesized or pre-formed pro-inflammatory and chemotactic molecules including histamine and eicosanoids.Tick-Host InterfaceFigure 3. Temporal changes in the immune genes expression during tick feeding. Gene expression data was entered into ingenuity pathway analysis software. All genes significantly modulated at any time point in the inflammatory response pathway from the IPA analysis were plotted showing temporal changes in gene expression. Genes significant at each time point are marked with an asterisk. doi:10.1371/journal.pone.0047301.gTick-Host InterfaceFigure 4. Real time PCR validation of array data. Validation targets were chosen based on significant fold change in the array study at 12 hpi and/or genes previously identified as important in host anti-tick responses [13]. Pre-optimized primers were purchased from Qiagen, and real-time PCR was performed as described in the methods section. All significantly modulated genes at any time point in the validation study are plotted. Significant changes in gene expression at individual time points are marked with an asterisk. Significance was assessed using the delta-delta Ct method for gene expression normalization and measurement, and the linear models in microarray analysis (LIMMA) package for statistical comparisons between infested and tick-free mice, as previously described [13]. doi:10.1371/journal.pone.0047301.gThese molecules can stimulate cytokine production and endothelial cells to mobilize Weibel-Palade bodies containing pre-formed selectins to the cell surface. While these early events cannot be measured a.Of up or down regulation were well preserved between the array and validation studies 15900046 (Table S1). Ctse, Foxp1, IL-6, and Muc1 were modulated in the array but were not validated. Other genes such as Ifng, IL-3, Jak1, Stat6, and Vwf were not modulated in either study. SignificantIngenuity Pathways AnalysisAll significantly modulated genes were submitted to Ingenuity Pathways Analysis. IPA has some advantages over DAVID in that it takes into account the fold change associated with each modulated transcript when determining biological significance.Tick-Host InterfaceFigure 2. Diagram showing the interactions between genes and the temporal increase in representation of the acute inflammation and immune cell recruitment pathway from the IPA analysis. Gene expression data was entered into ingenuity pathway analysis software to identify potential canonical pathways modulated during tick feeding. Upregulated genes are orange/pink, downregulated genes are green, and unchanged or unsignificant genes are grey. doi:10.1371/journal.pone.0047301.gincreases in chemokine gene expression begin at 1 hr postinfestation and intensify as time progresses, supporting the importance of chemotaxis seen in the array. Upregulation of IL1b, IL-10, and IL-4ra along with the general inflammation group supports early inflammatory changes at the bite site. While genes in the viral reproduction group showed some significant modulation, changes between time points were not consistent, casting some doubt on the biological significance of these differences.HistopathologyConcurrently with gene expression measurements, we pursued histopathological analysis of the bite site to see if morphological changes could be correlated with array data. Resident cells such as keratinocytes, fibroblasts, dendritic cells, and mast cells initially sense cutaneous tissue injury or antigenic stimuli. These cells release rapidly synthesized or pre-formed pro-inflammatory and chemotactic molecules including histamine and eicosanoids.Tick-Host InterfaceFigure 3. Temporal changes in the immune genes expression during tick feeding. Gene expression data was entered into ingenuity pathway analysis software. All genes significantly modulated at any time point in the inflammatory response pathway from the IPA analysis were plotted showing temporal changes in gene expression. Genes significant at each time point are marked with an asterisk. doi:10.1371/journal.pone.0047301.gTick-Host InterfaceFigure 4. Real time PCR validation of array data. Validation targets were chosen based on significant fold change in the array study at 12 hpi and/or genes previously identified as important in host anti-tick responses [13]. Pre-optimized primers were purchased from Qiagen, and real-time PCR was performed as described in the methods section. All significantly modulated genes at any time point in the validation study are plotted. Significant changes in gene expression at individual time points are marked with an asterisk. Significance was assessed using the delta-delta Ct method for gene expression normalization and measurement, and the linear models in microarray analysis (LIMMA) package for statistical comparisons between infested and tick-free mice, as previously described [13]. doi:10.1371/journal.pone.0047301.gThese molecules can stimulate cytokine production and endothelial cells to mobilize Weibel-Palade bodies containing pre-formed selectins to the cell surface. While these early events cannot be measured a.

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Author: Ubiquitin Ligase- ubiquitin-ligase