The 1.6 ? Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.     

Design Method of a Broadband Wide-Angle Plasmonic Absorber in the Visible Range

A new design method of a broadband wide-angle metal-dielectric-metal plasmonic absorber is presented based on the cavity mode theory. The broadband absorption is implemented by filling a unit cell with multi-size square metal patches resonant at adjacent wavelengths, with the widths of the patches and thickness of the dielectric layer optimized with the presented method. A broadband plasmonic absorber working in the visible range is designed, the absorption of which is insensitive to the azimuth angle of incident field and keeps over 0.7 at incident angle up to 60° for p polarization and above 0.6 at up to 40° for s polarization.     

3,3′,4,5′-Tetramethoxy-trans-stilbene Improves Insulin Resistance by Activating the IRS/PI3K/Akt Pathway and Inhibiting Oxidative Stress

The potential anti-diabetic effect of resveratrol derivative, 3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and its underlying mechanism in high glucose (HG) and dexamethasone (DXMS)-stimulated insulin-resistant HepG2 cells (IR-HepG2) were investigated. 3,3′,4,5′-TMS did not reduce the cell viability of IR-HepG2 cells at the concentrations of 0.5–10 µM. 3,3′,4,5′-TMS increased the potential of glucose consumption and glycogen synthesis in a concentration-dependent manner in IR-HepG2 cells. 3,3′,4,5′-TMS ameliorated insulin resistance by enhancing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), inhibiting phosphorylation of insulin receptor substrate-1 (IRS-1), and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells. Furthermore, 3,3′,4,5′-TMS significantly suppressed levels of reactive oxygen species (ROS) with up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. To conclude, the beneficial effect of 3,3′,4,5′-TMS against insulin resistance to increase glucose consumption and glycogen synthesis was mediated through activation of IRS/PI3K/Akt signaling pathways in the IR-HepG2 cells, accomplished with anti-oxidative activity through up-regulation of Nrf2.     

HIV-1 glycoprotein 41 ectodomain induces activation of the CD74 protein-mediated extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to enhance viral infection

Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library and identified a novel gp41-binding protein, CD74 (the MHC class II-associated invariant chain). Here, we report possible biological effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could bind directly to host CD74 in HIV-1-infected cells, and the peptide 6358 derived from gp41 loop region (aa 597-611) could effectively block the gp41-CD74 interaction. As a result of this binding, recombinant soluble gp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1 infection in vitro. Conversely, the enhancing effect could be suppressed by the recombinant CD74 extracellular domain. These results reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.     

Metabolomic approach to optimizing and evaluating antibiotic treatment in the axenic culture of cyanobacterium Nostoc flagelliforme

The application of antibiotic treatment with assistance of metabolomic approach in axenic isolation of cyanobacterium Nostoc flagelliforme was investigated. Seven antibiotics were tested at 1–100 mg L^?1, and order of tolerance of N. flagelliforme cells was obtained as kanamycin > ampicillin, tetracycline > chloromycetin, gentamicin > spectinomycin > streptomycin. Four antibiotics were selected based on differences in antibiotic sensitivity of N. flagelliforme and associated bacteria, and their effects on N. flagelliforme cells including the changes of metabolic activity with antibiotics and the metabolic recovery after removal were assessed by a metabolomic approach based on gas chromatography–mass spectrometry combined with multivariate analysis. The results showed that antibiotic treatment had affected cell metabolism as antibiotics treated cells were metabolically distinct from control cells, but the metabolic activity would be recovered via eliminating antibiotics and the sequence of metabolic recovery time needed was spectinomycin, gentamicin > ampicillin > kanamycin. The procedures of antibiotic treatment have been accordingly optimized as a consecutive treatment starting with spectinomycin, then gentamicin, ampicillin and lastly kanamycin, and proved to be highly effective in eliminating the bacteria as examined by agar plating method and light microscope examination. Our work presented a strategy to obtain axenic culture of N. flagelliforme and provided a method for evaluating and optimizing cyanobacteria purification process through diagnosing target species cellular state.     

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.