Akt contributes to activation of the TRIF-dependent signaling pathways of TLRs by interacting with TANK-binding kinase 1

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.     

Metformin and tenovin‐6 synergistically induces apoptosis through LKB1‐independent SIRT1 down‐regulation in non‐small cell lung cancer cells

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae

Yeast dehydrogenases and reductases were overexpressed in Saccharomyces cerevisiae D452-2 to detoxify 2-furaldehyde (furfural) and 5-hydroxymethyl furaldehyde (HMF), two potent toxic chemicals present in acid-hydrolyzed cellulosic biomass, and hence improve cell growth and ethanol production. Among those enzymes, aldehyde dehydrogenase 6 (ALD6) played the dual roles of direct oxidation of furan derivatives and supply of NADPH cofactor to their reduction reactions. Batch fermentation of S. cerevisiae D452-2/pH-ALD6 in the presence of 2 g/L furfural and 0.5 g/L HMF resulted in 20-30% increases in specific growth rate, ethanol concentration and ethanol productivity, compared with those of the wild type strain. It was proposed that overexpression of ALD6 could recover the yeast cell metabolism and hence increase ethanol production from lignocellulosic biomass containing furan-derived inhibitors.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Generation of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> Ghost by Coexpression of PhiX 174 Lysis <Emphasis Type="Italic">E</Emphasis> gene and Staphylococcal Nuclease <Emphasis Type="Italic">A</Emphasis> Gene

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-λP_R-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P _R /cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80–200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-λP_R-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.