Lipopolysaccharide Receptor, Toll -Like Receptor 4, Mediated Signaling Pathways Leading to Cyclooxygenase-2 Expression in Murine Macrophages.

Genetic evidence indicating that Toll-like receptor 4 (TLR4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine TLR4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through activation of NFkappaB. Thus, we determined whether murine TLR4 confers LPS responsiveness as evaluated by activation of NFkappaB and COX-2 expression. Expression of the constitutively active form (DeltaTlr4) of TLR4 in murine macrophage cell line (RAW 264.7) is sufficient to activate NFkappaB and COX-2 expression and to stimulate prostaglandin E2 synthesis. The truncated form [DeltaTlr4 (P712H)] of the missense mutant Tlr4 (P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NFkappaB activation and COX-2 expression. Inability of DeltaTlr4 (P712H) to activate NFkappaB and to induce COX-2 expression is rescued by a constitutively active adaptor protein (MyD88) which interacts directly with the cytoplasmic domain of TLR proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild type DeltaTlr4, but not with DeltaTlr4 (P712H) mutant. Together, these results indicate that TLR4 confers LPS responsiveness in RAW 264.7 cells, and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is due to disruption of TLR4-mediated signaling pathways resulting from inability of the mutant TLR4 (P712H) to interact with MyD88. In addition, the involvement of Lyn, Src-protein tyrosine kinase family, in LPS-induced signaling pathways leading to COX-2 expression was studied. All together, the results from this study may help pave the way to understanding the signal transduction pathways for COX-2 gene expression

Anti-oxidant and anti-inflammatory effects of rice bran and green tea fermentation mixture on lipopolysaccharideinduced RAW 264.7 macrophages

Purpose: To investigate the anti-inflammatory and anti-oxidant properties of an enzyme bath of Oryza sativa (rice bran) and Camellia sinensis O. Kuntz (green tea) fermented with Bacillus subtilis (OCB). Methods: The anti-oxidant effects of OCB were assessed by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay and flow cytometry. The anti-inflammatory effects of OCB were assessed by a nitric oxide (NO) assay. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were used to quantify expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The major compounds of OCB were identified using high performance liquid chromatography (HPLC) analysis. Results: OCB had no cytotoxic effect on LPS-stimulated macrophages or peripheral blood mononuclear cells up to 1 mg/mL. OCB displayed anti-oxidant effects comparable to those of ascorbic acid and reduced reactive oxygen species (ROS) levels in target cells. OCB treatment of LPSstimulated mavrophages decreased nitric oxide (NO), NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and key pro-inflammatory cytokine expressions, suggesting that OCB acts as an anti-oxidant and anti-inflammatory agent by reducing ROS levels and inhibiting pro-inflammatory mediators. The main effector compounds in OCB were epicatechin gallate, cathechin, synigrin acid, epicathechin, epigallocatechin gallate, rutin, and isoquercetin, which are known anti-oxidants. Conclusion: OCB fermentation product may be used as synergistic adjuvant therapy for inflammatory diseases. Keywords: Rice bran, Green tea, Bacillus subtilis, Enzyme bath, Anti-oxidant, Anti-inflammatio

Lipopolysaccharide: Basic Biochemistry, Intracellular Signaling, and Physiological Impacts in the Gut

Lipopolysaccharide (LPS), a main constituent of Gram-negative bacterial membrane, specifically activates Toll-like receptor 4, leading to the production of pleiotropic cytokines/chemokines which in turn regulate inflammatory and innate and subsequent adaptive immune responses. Given that human gut harbors a large collection of commensal bacteria, LPS released by gut microbes is able to make the great impact on gut homeostasis through the intracellular signaling pathways engaged by host-microbial interaction. Emerging evidence indicates that LPS in the gut has a potency to elicit the pathogenesis of intestinal inflammatory diseases such as inflammatory bowel disease and necrotizing enterocolitis. In this review, we discuss the current understanding of the basic biochemistry of LPS, LPS-induced intracellular signaling, and physiological impacts of LPS in the intestine

Implementing the Duty Trip Support Application

We are in the process of developing an agent and ontology-based Duty Trip Support application. The goal of this paper is to consider issues arising when implementing such a system. In addition to the description of our current implementation, which is also critically analyzed, other possible approaches are considered as well.software agents, agent systems, ontologies, transport objects, agent-non-agent integration.

Structural basis for the substrate specificity and catalytic features of pseudouridine kinase from Arabidopsis thaliana

RNA modifications can regulate the stability of RNAs, mRNA-protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/β protein with a protruding small β-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research

Semantic Segmentation Using Trade-Off and Internal Ensemble

The computer vision consists of image classification, image segmentation, object detection, and tracking, etc. Among them, image segmentation is the most basic technique of the computer vision, which divides an image into foreground and background. This paper proposes an ensemble model using a concept of physical perception for image segmentation. Practically two connected models, the DeepLab and a modified VGG model, get feedback each other in the training process. On inference processing, we combine the results of two parallel models and execute an atrous spatial pyramid pooling (ASPP) and post-processing by using conditional random field (CRF). The proposed model shows better performance than the DeepLab in local area and about 1% improvement on average on comparison of pixel-by-pixel