Association of Toll-like receptor 7 and 8 gene polymorphisms with Graves' disease in Chinese Cantonese population.

Graves' disease (GD) is a common polygenic multifactorial autoimmune disease. Toll-like receptors (TLRs) play critical roles in the activation of innate and adaptive immune responses. This study investigated the association of TLR7 and TLR8 gene polymorphisms with susceptibility of GD. Five single nucleotide polymorphisms (SNPs), namely, rs179019, rs179010 and rs3853839 in TLR7 and rs3764880 and rs5744088 in TLR8, were evaluated in 332 GD patients and 351 controls using High-Resolution Melting analysis. After adjusting for age, SNP rs179010 was found to decrease the risk of GD in females (OR(T vs C) = 0.64, P = 0.004). In the additive model, the risk of GD decreased significantly as the number of T alleles increased in females [odds ratio (OR) = 0.67 (0.50-0.90), P = 0.007]. The multivariate logistic regression analysis confirmed the independent contribution of rs179010 to the protective effect against GD. This study indicates that rs179010 in TLR7 may be associated with the decreased susceptibility to GD in Chinese Cantonese.

Vacuum-assisted black liquor-recycling enhances the sugar yield of sugarcane bagasse and decreases water and alkali consumption.

Black liquor (BL) remains a critical problem during alkaline pretreatment. To solve this issue, a novel pretreatment strategy termed vacuum-assisted black liquor-recycling pretreatment, was established to pretreat sugarcane bagasse (SCB). Firstly, SCB was pretreated with 2% NaOH at 121 °C for 1 h under vacuum conditions. The produced BL was used for subsequent pretreatments after pH recovery with NaOH. The pretreated SCBs were subject to enzymatic hydrolysis and separate hydrolyzation and fermentation (SHF) without washing to neutral pH. BL was recycled on seven occasions. The results indicated that glucose yields did not significantly differ between pretreatment with NaOH and recovered BL. The enzymatic hydrolysis and the fermentation resulted in maximum 0.35 g/g of glucose yield and 116.5 g/kg of ethanol yield respectively. Compared with conventional pretreatment with NaOH, the VABLR method showed high conversion rates of cellulose into monosaccharaides, whilst preserving ~20% and ~46% of alkali and water usage, respectively.

Enhanced enzymatic saccharification of sugarcane bagasse pretreated by sodium methoxide with glycerol.

Sodium methoxide (CH3ONa) with glycerol pretreatment (CWGP) was performed to improve the enzymatic digestibility of sugarcane bagasse (SCB). Response surface methodology was utilized to optimize the CWGP parameters for pretreating SCB from the perspective of total fermentable sugar yield (TFSY) and total fermentable sugar concentration (TFSC). Under the optimal CWGP conditions, 0.5666g/g of TFSY (0.82% CH3ONa, 1.11h, 150°C) and 17.75g/L of TFSC (0.87% CH3ONa, 1.38h, 149.27°C) were achieved, corresponding to delignification of 79.05% and 79.34%, respectively. Compared the pretreatment using glycerol or CH3ONa alone, the CWGP has significant synergies to enhance the enzymatic efficiency of SCB. The physical and chemical characteristics of untreated and pretreated SCBs were analyzed using FT-IR, XRD, and SEM, and the results suggest that CWGP significantly increased the susceptibility of the substrates to enzymatic digestibility. Ultimately, CWGP might be a prospective candidate for the pretreatment process of enzyme-based lignocellulosic biorefineries.

Vacuum-assisted alkaline pretreatment as an innovative approach for enhancing fermentable sugar yield and decreasing inhibitor production of sugarcane bagasse.

Sodium hydroxide pretreatment of sugarcane bagasse under vacuum conditions was established and evaluated in this study. Compared to pretreatment under conventional moderate pressure conditions, only half of the total phenolic compounds and less than half of the formic acid were produced under vacuum conditions, while the yield of total fermentable sugar was significantly increased by 31.38%. The pretreatment parameters: NaOH concentration, pretreatment time, and pretreatment temperature, were optimized using response surface methodology based on the response values of the total fermentable sugar yield (TFSY) and the total fermentable sugar concentration (TFSC), respectively. Under the optimal conditions, the TFSY of 0.5146g/g and the TFSC of 17.37g/L were achieved, respectively. By adjusting the ratio of cellulases to xylanase, the TFSY reached a maximum of 0.5213g/g when the ratio was 1:1, while the maximum TFSC of 17.71g/L was achieved when the ratio was 1:4.

Characterization of Spodoptera litura multicapsid nucleopolyhedrovirus 38.7 k protein, which contains a conserved BRO domain.

Homology analysis revealed that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) 38.7 k protein has 22-83% amino acid identities with Ecotropis obliqua NPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, S. exigua MNPV and S. littoralis MNPV 38.7 k proteins. Analysis of the relationship of these 38.7 k proteins indicated that they contain a conserved BRO-N domain, and SpltMNPV and SpliMNPV 38.7 k proteins also contain a motif found in all known viral and prokaryotic single-strand DNA binding proteins. RT-PCR results showed that SpltMNPV 38.7 k gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter motif (ATAAG). Western blot analysis revealed that the 38.7 k was expressed in infected S. litura cells as a 41 kDa form and this protein distributed in the nucleus of infected cells. Using a histone extraction protocol, SpltMNPV 38.7 k could be detected in the histone H1 fraction. Micrococcal nuclease treatment released SpltMNPV 38.7 k protein from the chromatin fraction, suggesting that its involvement in nucleosome structures. Furthermore, column chromatography using DNA-cellulose showed that SpltMNPV 38.7 k protein interacted with nucleic acids. It was proposed that SpltMNPV 38.7 k might function as a DNA-binding protein.

Characterization of gp41 gene of Spodoptera litura multicapsid nucleopolyhedrovirus.

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) gp41 gene is 993 bp long and the protein encoded by this gene has 6-66% amino acid identities with other known baculovirus GP41 proteins. Slgp41 transcripts were detected from 12 to 96 h post-infection (p.i.) and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a 41 kDa protein, and this protein was present in the nucleus of infected cells from 12 to 96 h p.i., whereas in the cytoplasm from 24 to 96 h p.i. Structural localization confirmed that SlGP41 is associated with the envelope of occlusion-derived virus (ODV). Lectin-binding assay showed that three lectins erythrina cristaglli lectin (ECL), lycopersicon esculentum lectin (LEL), and bandeiraea simlicifolia lectin (BSL) recognizing N-acetylglucosamine were specifically bound to SlGP41. It was proposed that SlGP41 is an O-glycoprotein.

Polymorphisms in TLR1, TLR6 and TLR10 genes and the risk of Graves' disease.

Graves' disease (GD) is postulated to be caused by the combined effects of susceptibility genes and environmental triggers. Toll-like receptors (TLRs) play a role in the activation of innate and adaptive immune responses in mammalians. The aim of this study was to evaluate the potential association of polymorphisms in TLR1, TLR6 and TLR10 genes with GD in Chinese Cantonese population. Seven single nucleotide polymorphisms (i.e. rs4833095 and rs5743565 in TLR1; rs5743808 in TLR6; and rs4504265, rs11466655, rs11096957 and rs10856839 in TLR10) were evaluated in 332 GD patients and 351 unrelated controls from Chinese Cantonese population. SNP rs5743565 in TLR1 conferred a protective effect against GD. The minor allele G of rs5743565 decreased the risk of GD in all cases (odds ratio; ORG vs. A=0.72 (0.58-0.91); p=0.005; ptrend=0.004) and early onset patients (ORG vs. A=0.72 (0.56-0.91); p=0.007; ptrend=0.006). This study provided evidence that genetic variation rs5743565 in TLR1 might be associated with the decreased susceptibility of GD.

Characterization of a novel ubiquitin-fusion gene Uba256 from Spodoptera litura nucleopolyhedrovirus.

The complete nucleotide sequence of Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene, encoding ubiquitin fused to GP37 protein of 256 amino acids was determined. The first 76 amino acids of the SpltMNPV ubiquitin showed 78-88, 77 and 81-84% amino acid sequence identity to baculovirus, Melanoplus sanguinipes entomopoxvirus and eukaryotes ubiquitins, respectively. The deduced amino acid sequence of SpltMNPV GP37 protein was similar to other baculovirus GP37 proteins and to entomopoxvirus fusolin proteins. The GP37 protein also showed a distant similarity to Pseudaletia separata entomopoxvirus enhancing factor, bacterial chitinase B and chitin-binding protein 1, but the significance of this is unclear. The mRNA start site of Uba256 fusion gene was mapped within a consensus baculovirus late promoter sequence (ATAAG), commonly found for baculovirus late genes. Uba256 transcripts were present from 48 h p.i. and remained detectable until 72 h p.i. Western blot analysis of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed to free ubiquitin and GP37 protein. Whereas expression Uba256 gene in Escherichia coli did not result in processing of the fusion protein. Tunicamycin treatment of SpltMNPV-infected cells confirmed that SpltMNPV GP37 protein is N-glycosylated. These findings provide additional information on the evolution of ubi genes and insight into genomic variation in baculoviruses.

Characterization of a chitin-binding protein GP37 of Spodoptera litura multicapsid nucleopolyhedrovirus.

The GP37 amino acid sequence of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) was compared with other baculovirus GP37, entomopoxvirus fusolin, the enhancing factor of Pseudaletia separata entomopoxvirus, and Alteromonas sp. chitin-binding protein 1. In these proteins, five 'conserved regions' previously reported constitute a chitin-binding domain. SpltMNPV GP37 effectively bound to purified crab shell chitin and the dissociation constant (Kd) for binding was 0.28 microM. Immunofluorescence analysis indicated that SpltMNPV GP37 was located in both cytoplasm and nucleus. Immunoblot analysis revealed that this protein was present in the envelopes of both occlusion body-derived virus and budded virus. Further analysis suggested that GP37 may bind to the chitin component of the peritrophic membrane of S. litura larvae.

[Cloning and expression of a novel ubiquitin fusion gene Uba256].

Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene is the only Ub-gp37 fusion gene in the genome of insect viruses. With the specific primers designed for Uba256 gene that was reported recently, the coding regions of Uba256, N-terminal ubiquitin and C-terminal GP37 that lacking the signal sequence, were amplified from SpltMNPV genomic DNA by PCR. The Uba256 coding region was expressed using the expression vector pBV220, and a band of 38 kD was detected with Western blot analysis, indicating that ubiquitin-GP37 fusion protein did not undergo post-transcriptional processing in E. coli. The ubiquitin and GP37 coding region were highly expressed, respectively, using pQE30 expression vector. Antibody to the purified ubiquitin reacted not only to the recombinant ubiquitin but also to bovine ubiquitin, and an antibody to the purified GP37 also reacted to the recombinant GP37. The results indicated that the purified ubiquitin and GP37 retained their antigenicity. Western blot analysis results of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed in this insect cell line to yield free ubiquitin and GP37 protein.

Synergism of cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting from different pretreatment technologies.

Sugarcane bagasse (SCB) resulting from different pretreatments was hydrolyzed by enzyme cocktails based on replacement of cellulase (Celluclast 1.5 L:Novozym 188=1FPU:4pNPGU) by xylanase or pectinase at different proportions. Lignin content of NaOH pretreated SCB and hemicellulose content of H2SO4 pretreated SCB were the lowest. NaOH pretreatment showed the best for monosaccharide production among the four pretreatments. Synergism was apparently observed between cellulase and xylanase for monosaccharide production from steam exploded SCB (SESB), NaOH, and H2O2 pretreated SCB. No synergism was observed between cellulase and pectinase for producing glucose. Additionally, no synergism was present when H2SO4 pretreated SCB was used. Replacement of 20% of the cellulase by xylanase enhanced the glucose yield by 6.6%, 8.8%, and 9.5% from SESB, NaOH, and H2O2 pretreated SCB, respectively. Degree of synergism between cellulase and xylanase had positive relationship with xylan content and was affected by hydrolysis time.

Ethanol production from xylan-removed sugarcane bagasse using low loading of commercial cellulase.

Xylan was always extracted as the feedstock for xylooligosaccharides production. The xylan-removed residue may contain high content of cellulose and thus had a possibility to be converted into ethanol. After soaked in 12% of NaOH at room temperature overnight, solubilization of cellulose, xylan, and lignin was 4.64%, 72.06%, and 81.87% respectively. The xylan-removed sugarcane bagasse (XRSB) was enzymatically hydrolyzed by using decreased cellulase loadings. The results showed that 7.5 FPU/g cellulose could obtain a cellulose conversion yield of 82%. Increasing the cellulase loading did not result in higher yield. Based on this, bioethanol production was performed using 7.5 FPU/g cellulose by employing fed-batch fermentation mode. The final ethanol concentration reached 40.59 g/L corresponding to 74.2% of the theoretical maximum. The high titer ethanol and low cellulase loading may reduce the overall cost.

Direct fermentation of amorphous cellulose to ethanol by engineered Saccharomyces cerevisiae coexpressing Trichoderma viride EG3 and BGL1.

Direct ethanol fermentation from amorphous cellulose was achieved using an engineered industrial Saccharomyces cerevisiae strain. Two cellulase genes endoglucanase (eg3) and ß-glucosidase (bgl1) were obtained from Trichoderma viride and integrated into the genome of S. cerevisiae. These two cellulases could be constitutively coexpressed and secreted by the recombinant strain S. cerevisiae-eb. The enzyme activities were analyzed in the culture supernatants, with the highest endoglucanase activity of 2.34 units/ml and ß-glucosidase activity of 0.95 units/ml. The effects of pH, temperature and metal ions on enzyme activities were analyzed. The coexpression strain S. cerevisiae-eb could grow in carboxymethyl cellulose (CMC) and utilize it as the single carbon source. The 20 g/L CMC as a model substrate of amorphous cellulose was used in fermentation. The ethanol production reached 4.63 g/L in 24 h, with the conversion ratio of 64.2% compared with the theoretical concentration. This study demonstrated that the engineered industrial strain S. cerevisiae-eb could convert amorphous cellulose to ethanol simultaneously and achieve consolidated bioprocessing (CBP) directly.

Association of TLR4 and TLR5 gene polymorphisms with Graves' disease in Chinese Cantonese population.

Graves' disease (GD) is postulated to be caused by the combined effects of susceptibility genes and environmental triggers. Toll like receptors (TLRs) play a role in the activation of innate and adaptive immune responses in mammalians. The aim of this study was to evaluate the potential association of TLR4 and TLR5 gene polymorphisms with GD in Chinese Cantonese population. Four single nucleotide polymorphisms (SNPs), rs11536889 and rs7873784 in TLR4, rs2072493 and rs5744174 in TLR5, were evaluated in 332 GD patients and 351 unrelated controls from Chinese Cantonese population. The minor allele C of TLR5 rs5744174 decreased the risk to GD in females (ORC vs. T=0.63; p=0.003; ptrend=0.003). Under a dominant model, rs5744174 conferred a protective effect in all cases (ORCC/CT vs. TT=0.65; p=0.009) or female subset (ORCC/CT vs. TT=0.57; p=0.002). Under a co-dominant model, rs5744174 also conferred a protective effect in all cases (ORTC vs. TT=0.64; p=0.008) and females (ORTC vs. TT=0.57; p=0.002). The haplotype A-C of TLR5 (rs2072493-rs5744174) decreased the risk of GD in females (OR=0.62; p=0.002). The other three SNPs were not found associated with GD. This study provided evidence that polymorphisms in TLR5 might be associated with decreased susceptibility of GD in females.

One-pot simultaneous saccharification and fermentation: a preliminary study of a novel configuration for cellulosic ethanol production.

Combination of size reduction and mild alkali pretreatment may be a feasible way to produce bioethanol without rinsing and detoxifying the solid substrate. Based on that, a fermentation configuration named one-pot SSF in which pretreatment and fermentation steps were integrated was developed. Additionally, the effect of laccase on fermentation performance was investigated. Delignification was the major effect of the alkali pretreatment at 121°C for 60min. The highest glucose and xylose yield, which obtained from the smallest particle at a substrate loading of 2%, was 6.75 and 2.71g/L, respectively. Laccase improved the fermentation efficiency by 6.8% for one-pot SSF and 5.7% for SSF. Bioethanol from one-pot SSF with laccase supplementation reached 67.56% of the theoretical maximum, whereas that from SSF with laccase supplementation reached 57.27%. One-pot SSF might be a promising configuration to produce bioethanol because of 100% solid recovery, and rinsing water and detoxification elimination.

Solvent extraction of antioxidants from steam exploded sugarcane bagasse and enzymatic convertibility of the solid fraction.

Solvent extraction of steam exploded lignocellulosic biomass may be a potential way to obtain antioxidative extracts and to enhance the enzymatic convertibility of the solid residue. Boiling solvent extraction (BSE) showed higher solid and phenolic yields than room temperature extraction. Solubilities of phenolics and sugars were higher in anhydrous ethanol (AE) and deionized water (DW) than in ethyl acetate under each individual extraction condition. The antioxidant activities of the AE and DW extract obtained under BSE were better than those of 10mM vitamin C. Conversion of the solid fractions into reducing sugar using Celluclast 1.5L and Novozym 188 after AE and DW extraction was 95.13% and 92.97%, respectively, higher than that obtained with SESB (88.95%).

Rupture of plasma membrane under tension.

We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions.

Identification and characterization of a novel thermostable gh-57 gene from metagenomic fosmid library of the Juan de Fuca Ridge hydrothemal vent.

A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu¹54 and Asp²6³. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90 °C, and its thermostability at 90 °C could remain over 50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg²âº while an inhibitory effect was observed with EDTA, ATP, and Ca²âº. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms.

Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid).

Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10(-7) J m(-2)) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

Identification of the apoptosis inhibitor gene p49 of Spodoptera litura multicapsid nucleopolyhedrovirus.

Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). Computer-assisted analysis indicated that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) ORF55 (designated as the p49 gene) display 79 and 31% amino acid identity with Spodoptera littoralis (Spli)MNPV P49 and Autographa californica (Ac)MNPV P35, respectively, Splt MNPV putative P49 contains a peptide cleavage site TVTDG recognized by death caspases. In marker rescue assay, Splt-p49 was able to suppress apoptosis induced by infection of a mutant AcMNPV deficient in p35 and rescue the mutant virus replication from apoptosis in Sf-9 cells.