Development of an antibody technique for acetylcholinesterase expression detection in the gill of Nile tilapia (Oreochromis niloticus) as a glyphosate-based herbicide biomarker.

Widely used glyphosate-based herbicides can remain in agricultural fields or be spread into the surrounding environment. This study aimed to develop an antibody technique for assessing acetylcholinesterase (AChE) expression in Nile tilapia (Oreochromis niloticus) after glyphosate exposure in the assessed tissues consisting of plasma, muscle, gills, and liver. Results showed that the cumulative mortality of fish exposed to glyphosate increased with exposure time and glyphosate concentration. The LC50 was evaluated using probit analysis. A sub-lethal concentration of 2 µL L-1 glyphosate-based herbicide altered behavioural and physiological appearances. AChE expression decreased compared to that in the control group with increasing glyphosate exposure time. The 71 kDa AChE was consecutively expressed in plasma, muscle, gill, and liver under laboratory and field conditions, as detected by dot blot and Western blot analysis. Furthermore, laboratory and field studies of the gills showed positive immunohistochemical results. Although this study could detect AChE expression in many tissues, using gills to assess AChE exposure allowed the fish not to be sacrificed compared with other organ studies, and this technique can be used in both laboratory and field conditions. In conclusion, the antibody technique can be applied to measure AChE expression in the gill tissue to assess glyphosate-based herbicide exposure.

Distribution of Kazachstania Yeast in Thai Traditional Fermented Fish (Plaa-Som) in Northeastern Thailand.

Thai traditional fermented fish products (Plaa-som) from four provinces (Ubon Ratchathani, Surin, Sisaket, and Khon Kaen) in the northeast part of Thailand were collected and analyzed to determine their salt content, total acidity, and pH. Yeasts in all samples were isolated and identified to the genus and species level based on sequence analysis of the D1/D2 of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region. The results revealed that the salt content, total acidity, and pH values are in the range of 2.01-6.9%, 0.62-1.9%, and 4.4-6.57%, respectively. Moreover, 35 strains of yeast were isolated and identified as eight genera, namely Candida, Diutina, Filobasidium, Kazachstania, Pichia, Saccharomyces, Torulaspora, and Yarrowia with 17 species. The ascosporogenous yeast, Kazachstania, was the most dominant genus found and was widely distributed among the fermented food samples. In addition, a new strain of yeast, Kazachstania surinensis, was also discovered in Plaa-som samples. Thus, this study is the first to report the presence and wide distribution of these yeasts in fish fermentation products.

Diversity of fermentative yeasts with probiotic potential isolated from Thai fermented food products.

This research aimed to evaluate the diversity of yeasts recovered from fermented foods gathered from some areas of Northeastern Thailand. The fermented food items included Pla-som, Nham-pla, Kem-buknud, Isan-sausage, Pla-ra, Mhum-neu, Mhum-Khai-pla, Nham-neu, Nham-mu, Kung-joom, Som-pla-noi, and Poo-dong. Their probiotic characteristics were also investigated. A total of 103 yeast isolates of nine genera were identified using 28S rDNA sequencing. The yeast genera were Candida (20.3%), Diutina (2.9%), Filobasidium (1.0%), Kazachstania (33.0%), Pichia (3.9%), Saccharomyces (1.0%), Starmerella (28.2%), Torulaspora (2.9%), and Yarrowia (6.8%). Based on probiotic characteristic analysis of ten selected yeast strains, Kazachstania bulderi KKKS4-1 showed the strongest probiotic characteristics in terms of hemolytic activity, antimicrobial activity against pathogenic bacteria, tolerance to low pH and bile salt and hydrophobicity. Isolated yeasts with probiotic characteristics may be useful in fermented food and animal feed production to improve their nutritional values.

Acetylcholinesterase (AChE) monoclonal antibody generation and validation for use as a biomarker of glyphosate-based herbicide exposure in commercial freshwater fish.

Monoclonal antibody specific to acetylcholinesterase (AChE) was extracted from the brain of hybrid catfish after exposure to glyphosate-based herbicide for 24 h. AChE was partially purified using hydroxyapatite and chromatography columns. The specific characteristics of AChE were studied by western blot using commercial polyclonal antibody (Rabbit anti-Fish AChE). It was found that the protein band had a molecular weight of 71 kDa. After mice were injected with AChE 4 times, the spleen showed a response to the induction. Polyclonal B cells from the mouse's spleen were taken and fused with myeloma cells to produce hybrid cells. After two fusions were performed, the clones specific to AChE were selected by dot blot, ELISA, immunohistochemistry and western blot techniques. Two clones, ACHE 33 and ACHE 99, which had the isotype of IgM were found. These two produced monoclonal antibodies specific to AChE in both denatured and native forms. The ACHE 33 monoclonal antibody clone from hybrid catfish could be cross-react with two commercial freshwater fishes, Nile tilapia and climbing perch, based on dot blot, immunohistochemistry, and western blot techniques. Moreover, AChE in Nile tilapia and climbing perch with glyphosate- based herbicide exposure gave a positive result with ACHE 33 as protein with molecular weight of 66 kDa. Based on our results, the produced monoclonal antibody showed specificity and could be applied to test AChE expression to assess glyphosate-based herbicide contamination in hybrid catfish, Nile tilapia and climbing perch. It could be also be a useful tool in indicating the quality of water resources.

Soy isoflavones improves endometrial barrier through tight junction gene expression.

Contamination with bacterial endotoxin causes the disruption of the tight junction (TJ) barrier. We investigated the ameliorative effect of dietary flavonoids genistein (Ge) and daidzein (Di) in normal or lipopolysaccharide (LPS)-induced disruption of epithelial barrier function of the endometrium. Using the immortalized porcine glandular endometrial epithelial cells (PEG), transepithelial electrical resistance (TER) and FITC-dextran flux (FD-4) across the monolayer were measured. The mRNA expression of TJ proteins, zona occludens-1 (ZO1), and claudin-1, -3, -4, -7 and -8 was evaluated by real-time RT-PCR for coinciding effect of Ge or Di occurred at the gene transcription level. The results revealed that Ge and Di altered the TER, depending on times and concentrations. Low concentration (10(-10) M) of both compounds decreased the TER, whereas higher concentrations (10(-8) and 10(-6) M) increased the TER which was not related to the FD-4 flux. The increased TER by Ge or Di was parallel to the induction of claudin-3 and -4 or -8 mRNA expression respectively. With LPS inoculation, all isoflavone treatments inhibited the decreased TER induced by LPS, but only Ge (10(-8) or 10(-6) M) or Di (10(-10) or 10(-6) M) was coincidence with the decreased FD-4 flux. Under this LPS-stimulated condition, some or all examined TJ gene expressions appeared to be promoted by specific concentration of Ge or Di respectively. Our findings suggest that the soy isoflavones treatment could promote and restore the impaired endometrial barrier function caused by LPS contamination.