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Inflammation plays pivotal role in the development of chronic diseases. Reducing chronic inflammation is an important strategy for preventing and managing many chronic diseases. In traditional Chinese medicine, the processed Buthus martensii Karsch (BmK) scorpion (also called "Quanxie") has been used to treat chronic inflammatory arthritis and spondylitis for hundreds of years suggests that "Quanxie" could potentially be utilized as a resource for identifying new anti-inflammatory compounds. However, the molecular basis and the underline mechanism for the anti-inflammatory effect of processed BmK scorpion are still unclear. The study aims to determine the potential involvement of macrophage-expressed Kv1.3 in the anti-inflammatory effect of processed BmK scorpion venom, as well as to identify new Kv1.3 blockers derived from processed BmK scorpion. In this study, the in vivo and in vitro anti-inflammatory activities were determined using carrageenan-induced paw edema, LPS-induced sepsis mouse models and LPS-induced macrophage activation model respectively. The effect of processed BmK scorpion water extract, processed BmK venom and BmKK2 on different potassium channels were detected by whole-cell voltage-clamp recordings on transfected HEK293 cells or mouse BMDMs. The cytokines were detected using Q-PCR and competitive enzyme-linked immunosorbent assay. High performance liquid chromatography, SDS-PAGE and peptide Mass Spectrometry analysis were used to isolate and identify the BmKK2. SiRNA, western blotting and flow cytometry were used to analysis the anti-inflammatory mechanism of BmKK2. Here we demonstrate that BmKK2, a thermostable toxin targeting Kv1.3 is the critical anti-inflammatory component in the processed BmK scorpion. BmKK2 inhibits inflammation by targeting and inhibiting the activity of macrophage Kv1.3, thereby inhibiting the activation of NF-κB-NLRP3 pathway and the subsequent release of inflammatory factors. These findings provide new insights into the molecular basis of the anti-inflammatory effects of "Quanxie" and highlight the importance of targeting Kv1.3 expressed on macrophages as an anti-inflammatory approach. [Display omitted] [ABSTRACT FROM AUTHOR]

RNA-sequencing, a powerful tool, yields a comprehensive view of whole transcriptome. Intracerebral hemorrhage (ICH) is a devastating form of stroke. To date, RNA-sequencing analysis of ICH has not been reported. Peripheral blood mononuclear cells (PBMCs) were used as a source of mRNA for gene expression profile analysis in stroke. In this study, we performed transcriptome analyses for PBMCs from four ICH patients and four healthy volunteers on Illumina platform. We identified 4040 significantly differentially expressed genes (DEGs). Functional annotation of DEGs with DAVID Bioinformatics Resources indicated that genes associated with cell apoptosis, autophagy, cell-cell adhesion, inflammatory response, protein binding, positive regulation of gene expression, and signal transduction were most significantly enriched by DEGs. Gene set enrichment analysis identified 40 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including chemokine signaling, cytokine-cytokine receptor interaction, oxidative phosphorylation, and glutathione metabolism processes. These data point to a complex mechanism for ICH pathogenesis. Overall, the present study demonstrated an altered gene expression profile of PBMCs in response to acute ICH. Our study provided important information for understanding the molecular mechanisms of ICH pathogenesis at system-wide levels. [ABSTRACT FROM AUTHOR]

The insulin and insulin-like signaling (IIS) pathway exists in a wide range of organisms from mammals to invertebrates and regulates several vital physiological functions. A phylogenetic analysis have indicated that insulin receptors have been duplicated at least twice among vertebrates, whereas only one duplication occurred in insects before the differentiation of Coleoptera, Hymenoptera, and Hemiptera. Thus, we cloned two putative insulin receptor genes, T.cas-ir1 and T.cas-ir2 , from T. castaneum and determined that T.cas-ir1 is most strongly expressed during the late adult and early pupal stages, whereas T.cas-ir2 is most strongly expressed during the late larval stage. We found that larval RNAi against T.cas-ir1 and T.cas-ir2 causes 100% and 42.0% insect death, respectively, and that parental RNAi against T.cas-ir1 and T.cas-ir2 leads to 100% and 33.3% reductions in beetle fecundity, respectively. The hatching rate of ds- ir2 insects was 66.2%. Moreover, RNAi against these two genes increased the expression of the pkc , foxo , jnk , cdc42 , ikk , and mekk genes but decreased erk gene expression. Despite these similarities, these two genes act via distinct regulatory pathways. These results indicate that these two receptors have functionally diverged with respect to the development and reproduction of T. castaneum , even though they retain some common regulatory signaling pathways. [ABSTRACT FROM AUTHOR]

Abstract: A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine A Proliferation-Inducing Ligand belonging to TNF family (bAPRIL). The open reading frame (ORF) of this cDNA covers 753bp, encoding 250 amino acids. The functional soluble part of bAPRIL (bsAPRIL) shows 97% and 92% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bAPRIL genomic sequence consists of six exons and five introns, is approximately 1.8kb in size, and maps to bovine chromosome 19q. Real-time quantitative PCR (qPCR) analysis revealed that bAPRIL is predominantly expressed in bovine lymphoid tissues spleen. The predicted three-dimensional (3D) structure of the bsAPRIL monomer analyzed by “comparative protein modelling” revealed that it is very similar to its mouse counterpart. The bsAPRIL and EGFP/bsAPRIL were efficiently expression in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, the EGFP/bsAPRIL fusion protein obtained similar fluorescence spectrum to those of EGFP. Laser scanning confocal microscopy analysis showed EGFP/bsAPRIL could bind to its receptor. In vitro, bsAPRIL could promote the proliferation of bovine or mouse splenic B cells together with/without SAC or anti-mouse IgM. Furthermore, compared to mouse soluble APRIL, the bovine soluble APRIL has the similar proliferation to mouse B cell. Those findings indicated that bsAPRIL plays an important role in proliferation of bovine B cells and has functional cross-reactivity among cow and other mammalians. Therefore, APRIL may be a potential immunologic factor for enhancing immunological efficacy in animals. [Copyright &y& Elsevier]

The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in promoting the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens. It is also involved in MHC I-restricted antigens catalyzing disulfide bond reduction in fishes' adaptive immunity. The open reading frame of tongue sole (Cynoglossus semilaevis) GILT (tsGILT) gene is 771 bp long, encoding 257 amino acids, with a calculated molecular weight of 28.465 kDa and isoelectric point (pI) of 5.35. After induction with lipopolysaccharide, the expression of tsGILT mRNA was upregulated in spleen and kidney and recombinant tsGILT protein transferred to late endosomes and lysosomes in HeLa cells. The refolded tsGILT was capable of catalyzing the reduction of the interchain disulfide bonds against an IgG substrate depend on the active site CXXC motif at residues 75–78. The process of immune response to bacteria challenge needs GILT to catalyze the reduction of disulfide bond and unfolding native protein antigens, promoting their hydrolysis by proteases. Whether a single mutation or a double mutation of active site CXXC at residues75–78, the 3D structure of tsGILT protein has undergone major changes and lost its activity of catalyzing the reduction of the interchain disulfide bonds. • tsGILT is constitutively expressed in a tissue-specific manner. • tsGILT expression is up-regulated in spleen and kidney cells following LPS stimulation. • tsGILT translocated to late endosomes and lysosomes under the condition of LPS stimulation. • tsGILT exhibited thiol reductase activity in vitro. • Mutation of one or double cysteine residues of CXXC active site abolished thiol reductase activity. [ABSTRACT FROM AUTHOR]

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli . To improve the expression level of ABP-dHC-cecropin A in E. coli , tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors ( n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins ( n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs are now recognized as key post-transcriptional regulators in animal ontogenesis and phenotypic diversity. Eupolyphaga sinensis Walker (Blattaria) is a sexually dimorphic insect, which is also an important source of material used in traditional Chinese medicine. The male E. sinensis have shorter lifecycles and go through fewer instars than the female. Furthermore, the males have forewings, while the females are totally wingless. Results: We used the Illumina/Solexa deep sequencing technology to sequence small RNA libraries prepared from the fourth-instar larvae of male and female E. sinensis. 19,097,799 raw reads were yielded in total: 7,817,445 reads from the female library and 11,280,354 from the male, respectively. As a result, we identified 168 known miRNAs belonging to 55 families as well as 204 novel miRNAs. Moreover, 45 miRNAs showed significantly different expression between the female and the male fourth-instar larvae, and we validated 10 of them by Stem-loop qRT-PCR. Some of these differentially expressed miRNAs are related to metamorphosis, development and phenotypic diversity. Conclusions/Significance: This is the first comprehensive description of miRNAs in E. sinensis. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs. These findings not only enrich miRNAs for hemimetabolans but also lay the foundation for the study of post-transcriptional regulation on the phenomena of sexual dimorphism. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, the cDNA encoding the extracellular soluble domain of rabbit BAFF (rsBAFF) was subcloned into the pET28a vector and the recombinant rsBAFF protein was solubly produced in Escherichia coli. Recombinant rsBAFF protein of 17.1kDa was then purified to a purity of 95% by using Ni2+-IDA resin. In vitro, purified rsBAFF protein was able to promote the survival/proliferation of B-lymphocytes of rabbits, mice and humans in the presence of anti-IgM antibodies. These findings indicate that the recombinant rsBAFF produced in E. coli have the bioactivity, and functional cross-reactivity occurs between rabbit and other mammalians BAFF proteins. [Copyright &y& Elsevier]