Lipopolysaccharide is an inflammatory agent and interleukin-1 is a cytokine. Their pro-inflammatory effects may be mediated by prostanoids produced by inducible cyclooxygenase-2. The aim of this study was to determine the prostanoids produced by lipopolysaccharide and interleukin-1 stimulated enterocytes through the cyclooxygenase-1 and 2 pathways. Cultured enterocytes were stimulated with lipopolysaccharide or interleukin-1beta with and without cyclooxygenase inhibitors. Low concentrations of indomethacin and valerylsalicylic acid (VSA) were evaluated as cyclooxygenase-1 inhibitors and their effects compared with the effects of a specific cyclooxygenase-2 inhibitor, SC-58125. Prostaglandin E2, 6-keto prostaglandin F1alpha, prostaglandin D2 and leukotriene B4 levels were determined by radioimmunoassay. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme (COX-2) was expressed by 4 h in LPS and IL-1beta treated cells while the constitutive COX-1 remained unaltered in its expression. Interleukin-1beta and lipopolysaccharide stimulated the formation of all prostanoids compared with untreated cells, but failed to stimulate leukotriene B4. Indomethacin at 20 microM concentration, and VSA inhibited lipopolysaccharide and interleukin 1beta stimulated prostaglandin E2, but not 6-keto prostaglandin F1alpha formation. SC-58125 inhibited lipopolysaccharide and interleukin-1beta stimulated 6-keto prostaglandin F1alpha but not prostaglandin E2 release. The specific cyclooxygenase-2 inhibitor also inhibited lipopolysaccharide produced prostaglandin D2 but not interleukin-1beta stimulated prostaglandin D2. While SC-58125 inhibited basal 6-keto prostaglandin-F1alpha formation it significantly increased basal prostaglandin E2 and prostaglandin D2 formation. As SC-58125 inhibited lipopolysaccharide and interleukin-1beta induced 6-keto prostaglandin F1alpha production but not prostaglandin E2 production, it suggests that these agents stimulate prostacyclin production through a cyclooxygenase-2 mediated mechanism and prostaglandin E2 production occurs through a cyclooxygenase-1 mediated mechanism. Prostaglandin D2 production appeared to be variably produced by cyclooxygenase-1 or cyclooxygenase-2, depending on the stimulus.
Motivation and Objectives Next generation sequencing has revolutionized genome research and marked the start of a new era. These new technologies present us with unprecedented amounts of data-but with this sequencing data come errors that are not only platform specific but also depend on the library preparation method and the type of sequencing (i.e. amplicon or metagenome). Illumina's sequencing platforms are currently among the most utilized platforms as they are able to generate millions of reads at relatively low cost-but Illumina error profiles are still poorly understood. A better knowledge of the error profiles is essential for sequence analysis and vital in order to draw valid conclusions. It has been reported that the major source of errors for Illumina are substitution-type miscalls (Archer et al., 2012). We developed a program that enables us to infer error profiles based on sequencing data from mock communities. This allows us to study and compare different errors and biases introduced by different sequencing machines, different library preparation methods as well as different types of sequencing. Here, we present the metagenome error profiles for a mock community that was sequenced on the Genome Analyzer (GA) II for the standard Illumina library preparation method (TruSeq). Being able to infer error profiles for individual sequencing runs has the potential to greatly improve our ability to correct errors and thus enhance further sequencing analysis.
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