Expression of the gene region prem-env of dengue virus type 2 in Pichia pastoris

The Dengue fever is one of the most serious illnesses in many tropical and subtropical countries. Laboratory tests are essential for diagnosis of the Dengue infection. The gene region preM-env of Dengue virus type 2 containing about 2000 bp was amplified by using the RT-PCR method from the infected cell and cloned in the pCR2.1 vector. This gene region was inserted in the pPIC9 vector for expression in the yeast cells. The electroporation was used to transfer the recombinant plasmid (pPIC-D2) into P. pastoris cells. The expressed full-length PrM-E gene in P. pastoris was identified by SDS-PAGE and Western-blot. It was shown that the recombinant protein with molecular weight of approximately 79 kDa excreted into the supernatant of culture when induced with 1% methanol after 3 days. The protein concentration expressed in P. pastoris was estimated of 0.1-0.2 mg/ml by the comparison with the molecular weight of the protein marker. The result of Western blot indicated that the recombinant protein PrM-E was able to bind with rabbit monoclonal antibody specific to Dengue virus type 2. The purified recombinant PrM-E protein will be applied for producing the diagnosis Kit of Dengue virus

STUDIES ON A NOVEL RECOMBINANT LIPASE A FROM BACILLUS SUBTILIS FS2

A strain of Bacillus subtilis FS2 isolated from a traditional fish source in Vietnam has been shown to produce an enzyme possessing both a gelatinase and a lipase A (LipA) activity when tested on agar plates containing 0.1% tributyrin and 0.3% gelatine. A gene encoding LipA  was isolated and expressed in E. coli BL21 (DE3). The enzyme has got an apparent Mr of 24 kDa, a pI of 9.2 and was purified to electrophoretical homogeneity by affinity chromatography. Both the lipase and the gelatinase showed optimum activity at pH 10 and at 30 0 C, with highest activity in the range 25-37°C. The metal ions Fe++, Co++, and Mn++ at 10 mM had strong inhibitory effect on both activities whereas Ca++ had no effect and Zn++  had a slight enhancing effect. The specific enzymatic activities obtained were 19814 U/mg for the lipase and 1245 U/mg for the gelatinase

Rapid detection and identification of nontuberculous mycobacterial pathogens in fish by using high-resolution melting analysis

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium

A novel electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas hydrophila, isolated from a microbial fuel cell

A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA-DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Using Response Surface Method to Optimize Conversion Reaction Conditions of Sucrose Into 5-hydroxymethyl-2-fufuraldehyde by A Combination of Heat and HCl as A Catalyst

5-Hydroxymethyl-2-furfuraldehyde is one of intermediate products of caramel reaction and it has a variety of applications in industry. Based on primary results, response surface method is employed to optimize conversion reaction conditions of sucrose into 5-HMF by a combination of heat and HCl as a catalyst and the target function is 5-HMF yield. The optimized conditions of conversion reaction is T = 17.4 (min), C = 1.81 (M), and R = 6.6:1 (mL:g); with the optimized conditions conversion reaction yield reaches the maximal value of 56,229 ± 2,519%. This research has provided important information for further research of 5-HMF and approach to large scale production and industrial production of 5-HMF

Metagenomic analysis of bacterial community structure and diversity of lignocellulolytic bacteria in Vietnamese native goat rumen

Objective In a previous study, analysis of Illumina sequenced metagenomic DNA data of bacteria in Vietnamese goats’ rumen showed a high diversity of putative lignocellulolytic genes. In this study, taxonomy speculation of microbial community and lignocellulolytic bacteria population in the rumen was conducted to elucidate a role of bacterial structure for effective degradation of plant materials. Methods The metagenomic data had been subjected into Basic Local Alignment Search Tool (BLASTX) algorithm and the National Center for Biotechnology Information non-redundant sequence database. Here the BLASTX hits were further processed by the Metagenome Analyzer program to statistically analyze the abundance of taxa. Results Microbial community in the rumen is defined by dominance of Bacteroidetes compared to Firmicutes. The ratio of Firmicutes versus Bacteroidetes was 0.36:1. An abundance of Synergistetes was uniquely identified in the goat microbiome may be formed by host genotype. With regard to bacterial lignocellulose degraders, the ratio of lignocellulolytic genes affiliated with Firmicutes compared to the genes linked to Bacteroidetes was 0.11:1, in which the genes encoding putative hemicellulases, carbohydrate esterases, polysaccharide lyases originated from Bacteroidetes were 14 to 20 times higher than from Firmicutes. Firmicutes seem to possess more cellulose hydrolysis capacity showing a Firmicutes/Bacteroidetes ratio of 0.35:1. Analysis of lignocellulolytic potential degraders shows that four species belonged to Bacteroidetes phylum, while two species belonged to Firmicutes phylum harbouring at least 12 different catalytic domains for all lignocellulose pretreatment, cellulose, as well as hemicellulose saccharification. Conclusion Based on these findings, we speculate that increasing the members of Bacteroidetes to keep a low ratio of Firmicutes versus Bacteroidetes in goat rumen has resulted most likely in an increased lignocellulose digestion

Impact of Infectious Disease after <i>Lactococcus lactis</i> Strain Plasma Intake in Vietnamese Schoolchildren: A Randomized, Placebo-Controlled, Double-Blind Study

Lactococcus lactis strain Plasma (LC-Plasma) is reported to have anti-viral effects via direct activation of plasmacytoid dendritic cells, which upregulate the production of type I and III interferons. A randomized, placebo-controlled, double-blind, parallel group study was designed for elementary schoolchildren, grades 1 to 3, in Vietnam. LC-Plasma or a control were administered to schoolchildren as a beverage (1.0 × 1011 count LC-Plasma/day/person). The primary endpoint was to determine the efficacy of LC-Plasma in reducing the cumulative days absent from school due to upper respiratory disease (URID) and gastrointestinal disease (GID), and the secondary endpoint was to evaluate the potency of LC-Plasma on URID/GID symptoms and general well-being scores. LC-Plasma intake significantly reduced the cumulative days absent from school due to URID/GID (Odds ratio (OR) = 0.57, p = 0.004) and URID alone (OR = 0.56, p = 0.005); LC-Plasma also significantly reduced the number of cumulative fever positive days during the first 4 weeks of intervention (OR = 0.58, p = 0.001) and cumulative days with diarrhea during the last 4 weeks of the intervention period (OR = 0.78, p = 0.01). The number of positive general wellbeing days was significantly improved in the LC-Plasma group compared with the control throughout the intervention period (OR = 0.93, 0.93, p = 0.03, 0.04 in the first and last 4 weeks of the intervention, respectively). These data suggest that LC-Plasma seems to improve the health condition of elementary schoolchildren and reduces school absenteeism due to infectious disease, especially URID

Resveratrol suppressed lps-induced cox-2 VIA miR-146a-5p inhibition in raw246.7 cells

Trans-resveratrol (Res) is a well-known natural stilbene frequently found in grapes which have been reported to possess antioxidant, anti-cancer activities and inhibited COX-2 expression. MicroRNAs (miRNAs) are short endogenous non-coding RNAs involved in the regulation of mRNA stability and protein synthesis. In our research, resveratrol isolated from Vitis heyneana Roem. & Schult Vitis heyneana was observed to suppress lipopolysaccharides (LPS)-induced COX-2 expression in Raw264.7 cells in a dose dependent manner. Using qPCR it was revealed that LPS induced the expression of miR-25, miR- 125a, miR-125b, miR-146a-5p, miR-146a-3p and miR-455. However, we only observed miR-146a-5p expression significantly decreased in resveratrol compared to untreated-control group. In addition, resveratrol abrogated the effect of miR-146a-5p mimic induced-COX-2 expression in Raw264.7 cells. Taken together, this study demonstrated for the first time the involvement of miR-146a-5p in resveratrol inhibited LPS-induced COX-2 expression in Raw264.7 cells

Metagenomic insights into lignocellulose-degrading genes through Illuminabased de novo sequencing of the microbiome in vietnamese native goats’ rumen

The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats’ rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes’ modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter