MiR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses.

BACKGROUND: Microglial cells, which are resident macrophages of the central nervous system, play important roles in immune responses and pathogenesis. Japanese encephalitis virus (JEV) is a neurotropic virus that infects microglial cells in brain. Several microRNAs including miR-155 and miR-146a play an important role in defining the microglia inflammatory profile. In this study, we have investigated the effect of miR-155 and miR-146a modulation on JEV infection as well as innate immune responses in human microglial cells. METHODS: In vitro studies were performed in JEV-infected human microglial CHME3 cells. miR-155 or miR-146a were overexpressed and total RNA and protein were extracted following JEV-infection. Expression of genes involved in innate immune responses was studied by PCR array, quantitative real-time PCR (qPCR), western blot and Fluorescence activated cell sorter (FACS). JEV replication was monitored by studying the viral RNA by qPCR, protein by western blot, and titres by plaque assay. RESULTS: Overexpression of miR-155 in CHME3 cells resulted in significantly reduced JEV replication whereas miR-146a overexpression had an insignificant effect. Additionally, interferon regulatory factor 8 (IRF8) and complement factor H (CFH) were induced during JEV infection; however, this induction was attenuated in miR-155 overexpressing cells following JEV infection. Further, JEV-induced NF-κB regulated downstream gene expression was attenuated. Interestingly, an increased level of CD45, a negative regulator of microglia activation and a reduced phosphorylated-Signal Transducers and Activators of Transcription (p-STAT1) expression was observed in miR-155 overexpressing cells upon JEV infection. CONCLUSION: Induction of miR-155 in human microglial cells may negatively modulate JEV-induced innate immune gene expression and may have a beneficial role in limiting JEV replication in human microglial cells.

Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Stearic acid mediated growth of edge-on oriented bilayer poly(3-hexylthiophene) Langmuir films.

The growth and structural evolution of stearic acid (SA) blended poly(3-hexylthiophene) [P3HT] Langmuir and Langmuir-Blodgett (LB) films were studied using complimentary surface and interface sensitive techniques to understand the possibility of ordering and layering of promising charge carrier mobility polymers, at the air-water interface and on the transferred solid substrate. SA-induced and subsequent compression-induced transitions in P3HT structure, from aggregated-3D to soft-2D and from in-plane mixed to unmixed layer, are evident at low and high pressures, respectively. The blending of SA molecules enhances the amphiphilic character of P3HT, which reduces the extent of the out-of-plane aggregation to form edge-on oriented (EO) bottom side-chain folded-bilayer (f-BL) islands (of size ~60 nm) within SA monolayer (ML), of commensurate thickness (~2.6 nm). Further compression, gradually rejects the less hydrophilic f-BL islands from the mixed layer to form EO P3HT BL islands (of coverage in-tune with starting composition) on top of SA ML. The formation of nearly covered P3HT(BL)/SA(ML) structured film on solid substrate is evident for the first time, which (even of limited P3HT thickness) has immense importance in the device properties, as the current in the bottom-gated organic thin-film transistors is known to travel only within few ML region near gate-dielectric.

Novel xylanases from Simplicillium obclavatum MTCC 9604: comparative analysis of production, purification and characterization of enzyme from submerged and solid state fermentation.

The production of extracellular xylanase by a newly isolated fungus Simplicillium obclavatum MTCC 9604 was studied in solid-state and submerged fermentation. Multiple xylanases and endoglucanases were produced by the strain during growth on wheat bran in solid state fermentation (SSF). A single xylanase isoform was found to be produced by the same fungus under submerged fermentation (SF) using wheat bran as sole carbon source. Enzyme activity, stability and the protein yield were much higher in SSF than SF. The two dimensional zymogram of the crude enzyme indicated the presence of six isoforms with different pI values starting from pH 3-10. The optimum temperature and pH for the partially purified xylanase activity were 50°C and pH 5.0 respectively; xylanase enzymes exhibited remarkable stability over a broad pH range and the temperature range of 30-60°C which has great potential to be used in biofuels, animal feed and food industry applications.

Enhanced expression and fucosylation of ficolin3 in plasma of RA patients.

OBJECTIVES: The aim of this work was to detect low abundant proteins, which may be potential biomarkers of rheumatoid arthritis (RA) at the early stage. We compared plasma protein profiles of RA patients with healthy individuals in two dimensional gel electrophoresis after removal of abundant proteins (albumin and IgG) using depletion kit and Aleuria aurantia lectin (AAL) affinity chromatography. DESIGN AND METHODS: Forty plasma samples each from healthy control individuals and RA patients were used in this study. RESULTS: We found ficolin 3, haptoglobin alpha chain, IgM chain, alpha-1-antitrypsin and hemopexin precursor to be up regulated in the plasma of RA patients. These proteins were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) from several reproducible 2D gels. Ficolin 3, which was not at all visible in albumin and IgG depleted gels, but detected in AAL bound fractions, was further verified by immunobloting and enzyme immunoassay. Elevated fucosylation in ficolin 3 was detected using high performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD), lectin blotting and enzyme linked lectin binding assay. CONCLUSIONS: Altered fucosylation and elevated level of Ficolin 3 might be exploited to be a potential marker for diagnosis of RA.

Suramin ameliorates collagen induced arthritis.

Suramin, a polysulfonated polyaromatic symmetrical urea is known for multiple therapeutic effects including antineoplastic activity. It is known as an antagonist of ATP at P2X purinergic receptors. Suramin is also found to inhibit protein synthesis affecting both initiation and elongation of the polypeptide chain. As a growth factor blocker, it is reported to suppress experimental myocardial inflammation. Here, we describe the anti-arthritic property of suramin in the collagen induced arthritic (CIA) rat, a model of human rheumatoid arthritis (RA). Intraperitoneal (i.p) injection of suramin (10 mg/kg/day) for 3 weeks was found to reduce inflammation and repair joint destruction in CIA rats. Recovery of body weight (p<0.0001), reduction in splenic (p<0.05) and arthritic indices (p<0.0001) and reappearance of smooth synovial lining after suramin treatment to CIA rats were found to be significant. Levels of pro-inflammatory cytokines such as TNF-α, IL-1ß and IL-6 in plasma and joint extracts were reduced (p<0.0001) significantly in response to suramin treatment. Several acute phase proteins were normalized after suramin administration.