NGHIÊN CỨU TỐI ƯU ĐIỀU KIỆN BIỂU HIỆN INTERLEUKIN-3 NGƯỜI DUNG HỢP VỚI PelB TRONG E. coli

Interleukin-3 (IL-3) is a multifunctional cytokine which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Gene coding for IL-3 linked with pelB signal were incorporated into pET22b(+) for  expression of il3 gene in E. coli BL21; but IL-3 was synthesized at very low levels and still in fusion with PelB. To facilitate purification process, finding the appropriate fermentation conditions plays a key role in order to enhance gene il-3 expression and cleavage of PelB. In this study, we have optimized the conditions for the expression of IL-3, which included E. coli host strain JM109, LB cultivation medium, induction temperature was 25oC; induction with 0.05 mM IPTG at OD600 = 1. The cell biomass increases at about 2.3 times after optimization. IL-3 protein was expressed in the form of inclusion body and the PelB signal was cleaved. This result is conducive for purification of large amount of IL-3 to determine characteristics of protein. Interleukin-3 người (IL-3) là một cytokine đa chức năng tham gia vào các quá trình tự đổi mới, nhân lên, biệt hóa và trưởng thành của nhiều loại tế bào máu. Sau khi đưa gen il-3 gắn thêm tín hiệu tiết pelB vào vector pET22b(+) và tiến hành biểu hiện ở chủng E. coli BL21, chúng tôi nhận thấy IL-3 được tổng hợp ở mức rất thấp và còn gắn với PelB. Để thuận tiện cho khâu tinh sạch thì vấn đề then chốt là nghiên cứu tìm ra các điều kiện phù hợp làm tăng lượng IL-3 được tổng hợp, đồng thời cắt được PelB. Trong nghiên cứu này, chúng tôi so sánh khả năng sinh tổng hợp IL-3 của các chủng E. coli BL21, JM109, Soluble và Rossetta2; sau đó tối ưu hóa điều kiện biểu hiện gen il-3 về thành phần môi trường, nhiệt độ, nồng độ IPTG, thời điểm cảm ứng và kiểm tra trạng thái tồn tại của IL-3. Kết quả thu được cho thấy gen il-3 biểu hiện tốt và ổn định nhất ở chủng E. coli JM109. Dưới các điều kiện lên men thích hợp trong môi trường LB, ở 25oC, cảm ứng 0,05 mM IPTG tại OD600=1, IL-3 biểu hiện tốt, cắt khỏi PelB và tồn tại ở trạng thái không tan trong tế bào chất. Sinh khối tế bào tăng lên khoảng 2,3 lần sau khi tối ưu. Kết quả này là tiền đề cho bước tinh sạch lượng lớn IL-3 cho nghiên cứu tính chất của protein

Analysis of Antimicrobial Resistance in Non-typhoidal Salmonella Collected From Pork Retail Outlets and Slaughterhouses in Vietnam Using Whole Genome Sequencing.

Non-typhoidal salmonella (TS) remains a significant health burden worldwide. In Vietnam, pork accounts for 70% of the total meat consumed, and contamination with Salmonella is high. High levels of antimicrobial resistance (AMR) have emerged among porcine NTS and of particular concern is the emergence of colistin resistance, a "last defense" antibioic against multi-drug resistant (MDR) Gram-negative pathogens. This study aimed to investigate the antibiotic susceptibility of 69 NTS isolates collected from the pork retail outlets and slaughterhouses in Vietnam during 2014 a nd 2018/19. Phenotypic testing and whole genome sequencing was used to assess the serotype and AMR gene profiles of the 69 NTS isolates. Seventeen different serotypes were identified, of which S. enterica subsp enterica serotype Typhimurium was the most common followed by S. ser. Rissen, S. ser. London, S. ser. Anatum, and S. ser. Derby. Phenotype AMR was common with 41 (59.4%) isolates deemed MDR. MDR strains were most common in slaughterhouses (83%) and supermarkets (75%) and lowest in traditional markets (38%) and convenience stores (40%). Colistin resistance was identified in 18 strains (15 resistant, three intermediate) with mcr-1 identified in seven isolates (S. ser. Meleagridis, S. Rissen, S. Derby) and mcr-3 in two isolates (S. Typhimurium). This includes the first mcr positive S. Meleagridis to our knowledge. Surprisingly, boutique stores had high levels (60%) of MDR isolates including 5/20 isolates with mcr-1. This study demonstrates that pork from modern retail stores classed as supermarkets or boutique (with pork claiming to be high quality, traceable, environmentally friendly marketed toward higher income consumers) still contained NTS with high levels of AMR

HIV-Associated TB in An Giang Province, Vietnam, 2001–2004: Epidemiology and TB Treatment Outcomes

BACKGROUND: Mortality is high in HIV-infected TB patients, but few studies from Southeast Asia have documented the benefits of interventions, such as co-trimoxazole (CTX), in reducing mortality during TB treatment. To help guide policy in Vietnam, we studied the epidemiology of HIV-associated TB in one province and examined factors associated with outcomes, including the impact of CTX use. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively abstracted data for all HIV-infected persons diagnosed with TB from 2001-2004 in An Giang, a province in southern Vietnam in which TB patients receive HIV counseling and testing. We used standard WHO definitions to classify TB treatment outcomes. We conducted multivariate analysis to identify risk factors for the composite outcome of death, default, or treatment failure during TB treatment. From 2001-2004, 637 HIV-infected TB patients were diagnosed in An Giang. Of these, 501 (79%) were male, 321 (50%) were aged 25-34 years, and the most common self-reported HIV risk factor was sex with a commercial sex worker in 221 (35%). TB was classified as smear-positive in 531 (83%). During TB treatment, 167 (26%) patients died, 9 (1%) defaulted, and 6 (1%) failed treatment. Of 454 patients who took CTX, 116 (26%) had an unsuccessful outcome compared with 33 (70%) of 47 patients who did not take CTX (relative risk, 0.4; 95% confidence interval [CI], 0.3-0.5). Adjusting for male sex, rural residence, TB smear status and disease location, and the occurrence of adverse events during TB treatment in multivariate analysis, the benefit of CTX persisted (adjusted odds ratio for unsuccessful outcome 0.1; CI, 0.1-0.3). CONCLUSIONS/SIGNIFICANCE: In An Giang, Vietnam, HIV-associated TB was associated with poor TB treatment outcomes. Outcomes were significantly better in those taking CTX. This finding suggests that Vietnam should consider applying WHO recommendations to prescribe CTX to all HIV-infected TB patients

Genetic Comparison of ESBL-Producing <i>Escherichia coli </i>from Workers and Pigs at Vietnamese Pig Farms

We analyzed and compared genomes of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli from pigs and pig farm workers at 116 farms in Vietnam. Analyses revealed the presence of blaCTX-M-55, blaCTX-M-27, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-65, blaCTX-M-24, blaDHA-1, and blaCMY2 in both hosts. Most strains from pigs contained quinolones (qnr) and colistin resistance genes (mcr-1 and mcr-3). Isolates predominantly harbored more than one plasmid replicon and some harbored plasmid replicons on the same contigs as the ESBL genes. Five strains from farm workers of ST38 (2), ST69 (1), and ST1722 (2) were classified as either uropathogenic E. coli (UPECHM)/extraintestinal pathogenic E. coli (ExPECJJ) or UPECHM, and the remaining were genetically distinct commensals. A high heterogeneity was found among the ESBL-producing E. coli from pigs and workers, with most isolates belonging to unrelated phylogroups, serogroups, and sequence types with >4046 Single-Nucleotide Polymorphisms-(SNPs). In comparing the genomes of pig isolates to those from humans, it appeared that ESBL-producing E. coli in workers did not predominantly originate from pigs but were rather host-specific. Nevertheless, the occurrence of ESBL-producing E. coli carrying plasmid-mediated colistin and quinolone resistance genes in pigs could represent a potential source for horizontal transmission to humans through food rather than direct contact

De Novo Metagenomic Analysis of Microbial Community Contributing in Lignocellulose Degradation in Humus Samples Harvested from Cuc Phuong Tropical Forest in Vietnam

We aimed to investigate the microbial diversity, mine lignocellulose-degrading enzymes/proteins, and analyze the domain structures of the mined enzymes/proteins in humus samples collected from the Cuc Phuong National Park, Vietnam. Using a high-throughput Illumina sequencer, 52 Gbs of microbial DNA were assembled in 2,611,883 contigs, from which 4,104,872 open reading frames (ORFs) were identified. Among the total microbiome analyzed, bacteria occupied 99.69%; the five ubiquitous bacterial phyla included Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Acidobacteria, which accounted for 92.59%. Proteobacteria (75.68%), the most dominant, was 5.77 folds higher than the second abundant phylum Bacteroidetes (13.11%). Considering the enzymes/proteins involved in lignocellulose degradation, 22,226 ORFs were obtained from the annotation analysis using a KEGG database. The estimated ratio of Proteobacteria/Bacteroidetes was approximately 1:1 for pretreatment and hemicellulases groups and 2.4:1 for cellulases. Furthermore, analysis of domain structures revealed their diversity in lignocellulose-degrading enzymes. CE and PL were two main families in pretreatment; GH1 and GH3-FN3 were the highest domains in the cellulase group, whereas GH2 and GH43 represented the hemicellulase group. These results validate that natural tropical forest soil could be considered as an important source to explore bacteria and novel enzymes/proteins for the degradation of lignocellulose

De Novo Metagenomic Analysis of Microbial Community Contributing in Lignocellulose Degradation in Humus Samples Harvested from Cuc Phuong Tropical Forest in Vietnam

We aimed to investigate the microbial diversity, mine lignocellulose-degrading enzymes/proteins, and analyze the domain structures of the mined enzymes/proteins in humus samples collected from the Cuc Phuong National Park, Vietnam. Using a high-throughput Illumina sequencer, 52 Gbs of microbial DNA were assembled in 2,611,883 contigs, from which 4,104,872 open reading frames (ORFs) were identified. Among the total microbiome analyzed, bacteria occupied 99.69%; the five ubiquitous bacterial phyla included Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Acidobacteria, which accounted for 92.59%. Proteobacteria (75.68%), the most dominant, was 5.77 folds higher than the second abundant phylum Bacteroidetes (13.11%). Considering the enzymes/proteins involved in lignocellulose degradation, 22,226 ORFs were obtained from the annotation analysis using a KEGG database. The estimated ratio of Proteobacteria/Bacteroidetes was approximately 1:1 for pretreatment and hemicellulases groups and 2.4:1 for cellulases. Furthermore, analysis of domain structures revealed their diversity in lignocellulose-degrading enzymes. CE and PL were two main families in pretreatment; GH1 and GH3-FN3 were the highest domains in the cellulase group, whereas GH2 and GH43 represented the hemicellulase group. These results validate that natural tropical forest soil could be considered as an important source to explore bacteria and novel enzymes/proteins for the degradation of lignocellulose

Enhanced soluble expression and effective purification of recombinant human interleukin-11 by SUMO fusion in <em>Escherichia coli</em>

579-585Human interleukin-11 is a multifunctional cytokine applied for the clinical treatment of thrombocytopenia. However, IL-11 has been considered a difficult protein in to express in an Escherichia coli expression system. Here, we demonstrate a suitable construction for high production of recombinant human interleukin-11 (rhIL-11) in E. coli. An optimized codon gene encoding human IL-11 was inserted in-frame with the small ubiquitin like modifier (SUMO) protein in the pE-SUMO3 vector. The SUMO IL-11 fusion protein was entirely expressed in soluble form and reached 31.6% of total soluble protein in E. coli. The rhIL-11 protein with a purity of over 99% was obtained at high protein yields of 320 mg rhIL-11 per liter of bacterial culture. Bioactivity of rhIL-11, as determined by proliferation of a TF-1 cytokine-dependent cell line, was 4.17 x 105 unit/mg, similar to the activity of the natural protein. Interestingly, the rhIL-11 was purified easily and effectively due to its selective precipitation from the reaction mixture. To the best of our knowledge, this is the first report demonstrating self-aggregating recombinant protein after cleavage from SUMO. Thus, expression of rhIL-11 fused with SUMO yielded greatly increased soluble production and convenient purification, and could offer a potential drug candidate for deployment in clinical trials

Flavones from Combretum quadrangulare Growing in Vietnam and Their Alpha-Glucosidase Inhibitory Activity

Combretum quadrangulare Kurz is widely used in folk medicine in Eastern Asia and is associated with various ethnopharmacological properties including hepatoprotective, antipyretic, analgesic, antidysenteric, and anthelmintic activities. Previous phytochemical investigations reported the presence of numerous triterpenes (mostly cycloartanes, ursanes, lupanes, and oleananes) along with dozens of flavonoids. However, the extracts of C. quadrangulare and isolated flavonoids have not been evaluated for their alpha-glucosidase inhibition. In the frame of our efforts dedicated to the chemical investigation of Vietnamese medicinal plants and their biological activities, a phytochemical study of the MeOH extract of the leaves of C. quadrangulare using bioactive guided isolation was undertaken. In this paper, the isolation and structure elucidation of twelve known compounds, 5-hydroxy-3,7,4′-trimethoxyflavone (1), ayanin (2), kumatakenin (3), rhamnocitrin (4), ombuin (5), myricetin-3,7,3′,5′-tetramethyl ether (6), gardenin D (7), luteolin (12), apigenin (13), mearnsetin (14), isoorientin (15), and vitexin (16) were reported. Bromination was applied to compounds 2 and 3 to provide four new synthetic analogues 8–11. All isolated and synthesized compounds were evaluated for alpha-glucosidase inhibition and antibacterial activity. Compounds 4 and 5 showed moderate antibacterial activity against methicillin-resistant Staphylococcus aureus while others were inactive. All compounds failed to reveal any activity toward extended spectrum beta-lactamase-producing Escherichia coli. Compounds 2, 4, 6–9, and 11–14 showed good alpha-glucosidase inhibition with IC50 values in the range of 30.5–282.0 µM. The kinetic of enzyme inhibition showed that 8 and 11 were noncompetitive type inhibition against alpha-glucosidase. In silico molecular docking model indicated that compounds 8 and 11 were potential inhibitors against enzyme α-glucosidase

Assessing the performance of overseas tuberculosis screening programs: a study among US-bound immigrants in Vietnam.

BACKGROUND: Tuberculosis cases in foreign-born persons account for more than 50% of all tuberculosis cases in the United States. The Institute of Medicine has recommended enhancing overseas screening as one measure to support tuberculosis elimination efforts. We assessed the ability of overseas tuberculosis screening (chest radiograph followed by 3 acid-fast bacilli sputum smears for persons with abnormal chest radiographs [suggestive of active tuberculosis]) to detect pulmonary tuberculosis disease among US-bound immigrants with abnormal chest radiographs. METHODS: During October 1998 to October 1999, 14 098 US immigrant visa applicants were screened overseas in Vietnam. Adult applicants with abnormal chest radiographs were enrolled to assess screening test characteristics among this group using mycobacterial culture as the gold standard for pulmonary tuberculosis disease diagnosis. Risk factors for pulmonary tuberculosis disease were also evaluated. RESULTS: Among 1179 adult applicants with abnormal chest radiographs, 82 (7.0%) had positive acid-fast bacilli smear results, and 183 (15.5%) had positive Mycobacterium tuberculosis culture results (pulmonary tuberculosis disease). The sensitivity, specificity, and positive and negative predictive values of serial acid-fast bacilli screening among this group were 34.4% (63/183), 98.1% (977/996), 76.8% (63/82), and 89.1% (977/1097), respectively. Risk factors for pulmonary tuberculosis disease included younger age (18-34 years), no history of tuberculosis or treatment, reported symptoms, and cavitation or consolidation on chest radiograph. CONCLUSIONS: The ability of current overseas screening to detect tuberculosis among immigrants with abnormal chest radiographs is low. Improved diagnostic methods, enhanced screening measures, and postmigration follow-up are essential to control tuberculosis among immigrants and support US and global tuberculosis elimination