Portable Device for Quick Detection of Viable Bacteria in Water

(1) Background: Access to clean water is a very important factor for human life. However, pathogenic microorganisms in drinking water often cause diseases, and convenient/inexpensive testing methods are urgently needed. (2) Methods: The reagent contains 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phenazine methosulfate (PMS) and can react with succinate dehydrogenase within bacterial cell membranes to produce visible purple crystals. The colorimetric change of the reagent after reaction can be measured by a sensor (AS7262). (3) Results: Compared with traditional methods, our device is simple to operate and can provide rapid (i.e., 5 min) semi-quantitative results regarding the concentration of bacteria within a test sample. (4) Conclusions: This easy-to-use device, which employs MTT-PMS reagents, can be regarded as a potential and portable tool for rapid water quality determination.

Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

Repurposing the Electron Transfer Reactant Phenazine Methosulfate (PMS) for the Apoptotic Elimination of Malignant Melanoma Cells through Induction of Lethal Oxidative and Mitochondriotoxic Stress

Redox-directed pharmacophores have shown potential for the apoptotic elimination of cancer cells through chemotherapeutic induction of oxidative stress. Phenazine methosulfate (PMS), a N-alkylphenazinium cation-based redox cycler, is used widely as an electron transfer reactant coupling NAD(P)H generation to the reduction of tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a superoxide generating chemotherapeutic for the pro-oxidant induction of cancer cell apoptosis. In a panel of malignant human melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1–10 μM, 24 h) displayed pronounced apoptogenicity as detected by annexin V-ITC/propidium iodide flow cytometry, and PMS-induced cell death was suppressed by antioxidant (NAC) or pan-caspase inhibitor (zVAD-fmk) cotreatment. Gene expression array analysis in A375 melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced superoxide production was substantiated by MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating NADPH-driven (SOD-suppressible) superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375 melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of malignant melanoma cells.

Biodegradation of Picolinic Acid by Rhodococcus sp. PA18

Picolinic acid (PA), a C2-carboxylated pyridine derivative, is a significant intermediate used in industrial production. PA is considered hazardous for the environment and human health. In this study, a Gram-positive bacterium, Rhodococcus sp. PA18, which aerobically utilizes PA as a source of carbon and energy, was isolated. The strain completely degraded 100 mg/L PA within 24 h after induction and formed 6-hydroxypicolinic acid (6HPA), a major PA metabolite, which was identified using ultraviolet-visible spectroscopy, high performance liquid chromatography, and liquid chromatography/time of flight-mass spectrometry analyses. The cell-free extracts converted the PA into 6HPA when phenazine methosulfate was used as an electron acceptor. To our knowledge, this is the first report showing that PA can be metabolized by Rhodococcus. In conclusion, Rhodococcus sp. PA18 may be potentially used for the bioremediation of environments polluted with PA.

Eugenol Toxicity in Human Dental Pulp Fibroblasts of Primary Teeth

Objective: The aim of the study was to determine the eugenol concentrations at which toxicity occurs in human dental pulp fibroblasts of primary teeth. Study design: Samples of primary dental pulp tissue were taken. Tissue samples were seeded by means of explant technique and used in the 4th–5th pass. Single Cell Gel Electrophoresis (Comet), phenazine MeThoSulfate (MTS), LIVE/DEAD® Cell Viability/Toxicity and trypan blue assays for evaluation of the cytotoxicity of increasing concentrations of eugenol (0.06 to 810 μM) were performed. Results: The results of toxicity tests showed toxic effects on dental pulp fibroblasts, even at very low concentrations of eugenol (0.06 μM). Very low concentrations of eugenol produce high toxicity in human dental pulp fibroblasts. Conclusions: All of the concentrations of eugenol that we evaluated produced high toxicity in human dental pulp fibroblasts of primary teeth.