Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN7 promoter.

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline-N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

Metagenomic data of bacterial communities associated with Acropora species from Phu Quoc Islands, Vietnam.

Acropora is one of the most common coral genera found in Phu Quoc Islands, Vietnam. However, the presence of marine snails, such as the coralllivorous gastropod Drupella rugosa, was a potential threat to the survival of many scleractinian species, leading to changes in the health status and bacterial diversity of coral reefs in Phu Quoc Islands. Here, we describe the composition of bacterial communities associated with two species of Acropora (Acropora formosa and Acropora millepora) using the Illumina sequencing technology. This dataset includes 5 coral samples of each status (grazed or healthy), which were collected in Phu Quoc Islands (9°55'20.6″N 104°01'16.4″E) in May 2020. A total of 19 phyla, 34 classes, 98 orders, 216 families and 364 bacterial genera were detected from 10 coral samples. Overall, Proteobacteria and Firmicutes were the two most common bacterial phyla in all samples. Significant differences in the relative abundances of the genera Fusibacter, Halarcobacter, Malaciobacter, and Thalassotalea between grazed and healthy status were observed. However, there was no differences in alpha diversity indices between the two status. Furthermore, the dataset analysis also indicated that Vibrio and Fusibacter were core genera in the grazed samples, whereas Pseudomonas was the core genus in the healthy samples.

Flow cytometric enumeration of bacterial in the coral surface mucus layer.

The direct counts of bacteria inhabiting coral mucus were performed by flow cytometry testing four fluorescent dyes (SYBR®Green I, HCS, TOPRO®3, SYTO®62) with three different scleractinian species. Results obtained with SYTO62 were the most reliable based on the comparison with standardized epifluorescence counts and the resolution of cytograms.

Procarcinogens - Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes.

In Vietnam, a great number of toxic substances, including carcinogens and procarcinogens, from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. In the present study, we report the development of novel yeast-based biosensor systems to determine both genotoxic carcinogens and procarcinogens by cotransformation with two plasmids. One plasmid is carrying human CPR and CYP (CYP3A4, CYP2B6, or CYP2D6) genes, while the other contains the RAD54-GFP reporter construct. The three resulting coexpression systems bearing both CPR-CYP and RAD54-GFP expression cassettes were designated as CYP3A4/CYP2B6/CYP2D6 + RAD54 systems, respectively and used to detect and evaluate the genotoxic potential of carcinogens and procarcinogens by selective activation and induction of both CPR-CYP and RAD54-GFP expression cassettes in response to DNA damage. Procarcinogens were shown to be predominantly, moderately or not bioactivated by one of the CYP enzymes and thus selectively detected by the specific coexpression system. Aflatoxin B1 and benzo(a)pyrene were predominantly detected by the CYP3A4 + RAD54 system, while N-nitrosodimethylamine only moderately activated the CYP2B6 + RAD54 reporter system and none of them was identified by the CYP2D6 + RAD54 system. In contrast, the genotoxic carcinogen, methyl methanesulfonate, was detected by all systems. Our yeast-reporter system can be performed in 384-well microplates to provide efficient genotoxicity testing to identify various carcinogenic compounds and reduce chemical consumption to about 53% as compared with existing 96-well genotoxicity bioassays. In association with a liquid handling robot, this platform enables rapid, cost-effective, and high-throughput screening of numerous analytes in a fully automated and continuous manner without the need for user interaction.

Computer controlled automated assay for comprehensive studies of enzyme kinetic parameters.

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant") that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.