Isolation and selection of bacteria inhibiting Streptococcus agalactiae causing dark-body disease on snakeskin gourami fish (Trichogaster pectoralis)

Streptococcus agalactiae is one of the microbial pathogens causing the dark-body disease on snakeskin gourami fish (Trichogaster pectoralis) that affects the growth and quality of fish. This research aimed to isolate and select bacteria inhibiting S. agalactiae which are able to use for controling pathogenic bacteria instead of antibiotics. Fourteen bacteria strains were isolated and screened from healthy fishes, sediment and water samples at fish ponds in Dong Thap province. Among these strains, L7 strain showed the highest inhibition ability with the clear zone diameter was 9,3 mm. The results of the 16S rRNA sequence analysis indicated that the L7 strain belonged to Bacillus subtilis. The experiment to evaluate the inhibition capacity and fish disease control of selected B. subtilis in experimental conditions was conducted by challenging fish with S. agalactiae. Fishes in the control treatment was infected with S. agalactiae at 106 CFU/mL had survival rate 41,7%. The experimental treatments NT1, NT2, NT3 which were treated with B. subtilis at concentrations of 105 CFU/mL, 106 CFU/mL, and 107 CFU/mL gave higher survival rates compared with the non-treated control, with the rates of 60%, 76,7%, and 81,7%, respectively. These results revealed that the isolated B. subtilis is potential used in control dark-body disease on snakeskin gourami fish.

Direct induction of adventitious root in Schefflera octophylla (Lour.) Harms from leaf explants cultured in vitro

Schefflera octophylla (Lour.) Harms is a precious plant species belonging to the Araliaceae family. All parts of plant have been used to create products for human health. In tissue culture of medicinal plants, the induction and multiplication of adventitious root of Schefflera octophylla for biomass collection have been studied. In this report, results on induction of adventitious root from leaf explants cultured in vitro of this plant species were presented. Leaf disks (~ 10 x 10 mm), leaf transverse - thin cell layers (t-TCLs) (~ 3 x 10 mm) were cultured on different mineral media MS, ½MS, B5, SH with NAA (0 - 5 mg/L), sucrose (0 - 50 g/L) and light intensity (0 - 4,000 lux). The results showed that, 30 days after culturing on ½MS solid medium plus 3 mg/L NAA, and 30 g/L sucrose in 4,000 lux light condition, direct formation of adventitious root was best from leaf disks, t-TCLs with rooting rate (%) 100, 100; root number/sample 68.80, 21.96; root lenght (mm) 16.53, 15.53, respectively. Leaf disk culture resulted in better rooting than t-TCL culture in two criteria of root number and root length. Morphological and histological observations of adventitious root primordia formation in the leaf disk were also performed. This is the first report on direct formation of adventitious root by in vitro culture of leaf disks/t-TCLs in Schefflera octophylla with very high efficiency, creating basis for further studies on root biomass multiplication for production of bioactive compounds.

Effect of metal ions and chemical agents on the activity of endoglucanase GH5 exploited from goats-rumen bacterial metagenomic DNA data

A gene coding for GH5 endoglucanase exploited from metagnomic DNA data of bacteria in Vietnamese goats’ rumen was modularity structure including a catalytic module, a fibronectin-3 like module and an X module. The recombinant enzyme was sucessfully expressed in E. coli and purified. To study the effect of some metal ions and chemicals on enzyme activity, in this study, we used some tools including Swiss-Prot, ProFunc, COFACTOR for prediction of enzyme structure and ligands interaction. The obtained results indicated that the most similar structure with enzyme had two conserved residues (Asp-190 và Asp-192) linked with Mn2+ within a radius of ~ 3.5 Å from the center of ion Mn2+ and enzyme molecule contained a disulphide bond. Experimental results for essessment of the effect of some metal ions (Ca2 +, Mn2 +, Mg2+, Ni2+, K+, Co2+, Cu2+, Zn2+, Fe3+) at the final concentration of 10 mM and of six common chemicals including SDS (1%), urea (1 µM), 2-mercaptoethanol (1 µM), EDTA (1 µM), tween 80 (1mM), triton X-100 (1 µM) showed that only Mn2+ increased enzyme activity slightly at concentration of 10 mM and two times at the concentration of 40 mM Mn2+. The Mn2+ has been identified as a specific binding agent may increase the stability and activity of endoglucanase GH5.

Study on biological characteristics and biomass production of the green microalgae (Nannochloris atomus) isolated from Vietnam for the extraction of bioactive compounds

Microalgae are known to be a nutrient-rich feed source for many aquatic animals. It is also an important raw material source to exploit high biological activity substances for humans. This is the first study on biological characteristics and algae biomass production from the green microalgae Nannochloris atomus being carried out in Vietnam. In this study, scientific name of the strain N. atomus NT12 based on morphological characteristics and 18S rRNA gene sequence (with accession number MW007766 on the GenBank) was identified. At the best conditions for the growth (i.e. Walne medium, 3 x 106 cells/mL initial cell density, 25 -30oC growth temperature, 60 - 100 µmol/m2s light intensity, pH 7, 30‰ salinity), highest NT12 strain cell density of 30 x 106 cells/mL was obtained after 30 days of culture. The microalgae N. atomus NT12 was also successfully cultured on a pilot scale in the plastic bottle 10 L and closed photobioreactors 20 – 50 L resulting in a high biomass productivity of 209 mg/L/day and a biomass rich in polyunsaturated fatty acids such as oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and α-linolenic acid (C18:3n-3) qualified for the purpose of extraction of value bioactive compounds.

Development of an in vitro hairy root induction system in different soybean cultivars for gene expression and genome editing studies

Hairy root induction system has been widely applied for studies of gene function, gene expression, and genome editing in numerous plant species. In this study, we developed and evaluated the performance of an in vitro hairy root induction system via Agrobacterium rhizogenes on several Vietnamese and international soybean cultivars. The efficacy of in vitro hairy root induction and of transformation using this system was varied and depended on soybean cultivars as well as transgenic constructs. The hairy root induction frequency of different soybean cultivars ranged from 61.67% to 100% after 5 days on culture medium. From 43.8% to 79.8% of hairy roots transformed with GFP-expressing construct showed transgene expression, while that for the construct with the gus gene was from 38.07% to 72.33%. Among tested soybean cultivars, DT26 demonstrated the highest transformation and gene expression efficacy with both investigated vectors. This hairy root induction system was further utilized for targeted knockout mutagenesis via CRISPR/Cas9 of two genes which are G03 and G09 in the DT26 soybean cultivar. Successful mutagenesis in the regions of targeted genes was confirmed by shifted and multiple bands compared to which of non-transgenic hairy root in PAGE analysis. Sequencing results of targeted regions presented various nucleotide deletions ranging between -3 bp and -25 bp in size in both two genes of interest. This study laid an important basis for future gene function studies and targeted gene editing investigations on soybean plants in Vietnam.

Production of in vitro strawberry (Fragaria × ananassa) plantlets in large-scale system supplemented with silver nanoparticles

The growth of strawberry plantlets in the rooting stage on culture medium supplemented with silver nanoparticles (AgNPs) and the ethylene gas accumulation in plantlet culture bottles were investigated. In addition, different culture systems were first used to produce large-scale Strawberry plantlets. The results showed that shoots (3 cm) were cultured on MS medium supplemented with 0.02 mg/L NAA, 1 g/L activated charcoal, 30 g/L sucrose, 8 g/L agar and 0.5 mg/L AgNPs showed about 4 days earlier rooting formation and the plantlet growth such as plantlet height (5.60 cm), fresh weight (242.67 mg), dry weight (34,67 mg), number of roots/plantlet (6.67), root length (3.40 cm), SPAD (39.30 nmol/cm2) were higher than those in the control after 15 days of culture. Besides, the ethylene gas content in the culture bottle (0.06 ppm) in the 0.5 mg/L AgNPs treatment was lower than as compared to that in the control (0.15 ppm) after 15 days of culture. A shoot density (10 shoots) in 250 mL culture bottle with 40 mL of medium gave optimal growth than those in other treatments after 15 days of culture. Square plastic box culture system (length × width × height: 19 cm × 19 cm × 7 cm; 2.5 L in volume) containing 250 mL MS medium added to 0.5 mg/L AgNPs produced 100 vigorous plantlets; meanwhile, rectangular plastic box system (34 cm × 23 cm × 13 cm; 10 L in volume; 10 L in volume) produced 200 vigorous plantlets. Plantlets derived from 0.5 mg/L AgNPs treatment in the plastic box systems exhibited well acclimatization after 30 and 60 days of culture in the greenhouse.

Isolation, screening and identification of microorganisms having antimicrobial activity and causing cytotoxicity isolated from sediment samples of the Vung Ang bay in Ha Tinh

Actinomycetes has been extensively studied due to its ability to produce secondary compounds with high application value. Especially their antibiotic ability, more than 40% of antibiotics are derived from actinomycetes of which genus Streptomyces predominates. Moreover, marine actinomycetes have been being of great interest in recent years due to their ability to produce bioactive substances capable of providing diverse and novel chemical structures. In this study, from 8 samples of marine sediments collected in Vung Ang bay in Ha Tinh provinces, we have isolated 20 strains of actinomycetes. The strains were fermented in A1+ medium, the fermentation fluid was extracted 5 times with ethyl acetate, recovered sediment and determined antibacterial and cytotoxic activity. From the screening results, two strains with the highest antibacterial activity and highest cytotoxicity were selected ie., HT03 and HT06. Both strains had antagonistic activity of Enterococcus faecalis ATCC29212 with MICHT03= 32 μg/mL, MICHT06= 16μg/mL, with Stapphylococus aureus ATCC25923 with MICHT03= 64 μg/mL, MICHT06= 32 μg/mL and with Bacillus cereus ATCC 13245 has the same MIC = 16 μg / mL. In addition, the two strains HT03 and HT06 were able to strongly inhibit the yeast Candida albicans ATCC10231 with MICHT03= 16 μg/mL, MICHT06= 8 μg/mL. Especially, two strains, HT03 and HT06, exhibited very good toxicity on all 5 cancer cell lines (MCF-7 breast cancer cell; MDA-MB-231 breast cancer cell; lung cancer cell). NCI-H1975; HeLa cervical cancer cell; AGS gastric cancer cell) at both test concentrations of 30 µg/mL and 100µg/mL. By the analysis of 16S rRNA sequence, the results showed that the HT03 strain had the highest similarity (99.93%) to that of Streptomyces fradiaes and Streptomyces fradiae ATCC. The HT06 strain was defined to belong to Nocardiopsis synnemataformans with the 16S sequence identity of 99.89% to the Japanese standard Nocardiopsis synnemataformans DSM 44143 strain NBRC-102581.

Parkinson's disease: Certain features of pathology, genetics, and pathogenesis

Parkinson disease (PD) is the second-most common and complex neurodegenerative disorders in humans, characterized by motor symptoms such as tremor, rigidity, bradykinesia, and non-motor symptoms such as insomnia, constipation, anxiety, depression and fatigue. Up to now, the diagnosis of PD has been mainly based on clinical symptoms with motor features being the mainstay and this limits the possibility of early detection. PD is usually diagnosis after the sixth decade of life, however about 5–10% of patients who develop the disease before the age of 50 are early-onset PD. The rapid development of genetic studies and their application may induce the early diagnosis of PD in the near future, especially for the early-onset PD. A few mechanisms have been implicated in PD pathogenesis, with α-synuclein aggregation central to the development of the disease. Multiple other processes are thought to be involved, with several studies suggesting that abnormal protein clearance, mitochondrial dysfunction, and neuroinflammation play a role in the onset and progression of PD. There are many PD patients in Vietnam, however, the studies are mainly based on clinical symtom descriptions. Given the aging of the population, the prevalence of PD is to increase dramatically, which would lead to increased urgency for the need to identify improved methods in diagnosis and treatment this disease.

Development of an in-house Lepto-LAT kit for screening and detection of Leptospira infection

Leptospirosis is a common zoonotic disease in the tropics and subtropics. Leptospira infected human without prompt detection and treatment will face serious consequences such as acute hepatitis-kidneys, pulmonary hemorrhage which can lead to death. Besides the MAT gold standard method, Leptosipra antigens developed by DNA recombination technology have been widely studying and applying in diagnosis of Leptospira infection in human and animal. Overcoming the disadvantages of MAT and ELISA such as complicated protocol, which requires highly qualified staff and specialized equipment, the latex agglutination method has been studied and widely used in detecting pathogens such as Salmonella, E. coli, Leptospira in the world. The advantages of this method are simple operation, fast and cheap. In the previous article, we expressed Leptospira LigB antigen in E. coli cells and successfully purified it by affinity chromatography with 98% purity. In this paper, we present the process for establishment of a Lepto-LAT kit to detect Leptospira infection in dogs. This kit had the sensitivity and specificity of 91.75% and 91.57%, respectively.

Construction of DNA ladder for determination of small size DNA fragments

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.