Application of the cellular oxidation biosensor to Toxicity Identification Evaluations for high-throughput toxicity assessment of river water

Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 μL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale

The mechanism of SO2 -induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells.

Plants closing stomata in the presence of harmful gases is believed to be a stress avoidance mechanism. SO2 , one of the major airborne pollutants, has long been reported to induce stomatal closure, yet the mechanism remains unknown. Little is known about the stomatal response to airborne pollutants besides O3 . SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) and OPEN STOMATA 1 (OST1) were identified as genes mediating O3 -induced closure. SLAC1 and OST1 are also known to mediate stomatal closure in response to CO2 , together with RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs). The overlaying roles of these genes in response to O3 and CO2 suggested that plants share their molecular regulators for airborne stimuli. Here, we investigated and compared stomatal closure event induced by a wide concentration range of SO2 in Arabidopsis through molecular genetic approaches. O3 - and CO2 -insensitive stomata mutants did not show significant differences from the wild type in stomatal sensitivity, guard cell viability, and chlorophyll content revealing that SO2 -induced closure is not regulated by the same molecular mechanisms as for O3 and CO2 . Nonapoptotic cell death is shown as the reason for SO2 -induced closure, which proposed the closure as a physicochemical process resulted from SO2 distress, instead of a biological protection mechanism

The gut microbiota and cardiovascular health benefits: a focus on wholegrain oats

Existing scientific data suggest that a high intake of wholegrain foods contributes to improved gut health and a reduced risk of cardiovascular disease. Wholegrain oats are rich in dietary fibre and an important source of many bioactive components, including minerals, vitamins and phenolic compounds. The oat β‐glucans have been reported to lower low‐density lipoprotein cholesterol through their ability to increase the viscosity of intestinal chime, change the gut microbiota composition and increase the production of short‐chain fatty acids, which may contribute to the inhibition of hepatic cholesterol synthesis. Oats are also a rich source of phenolic acids, which are predominantly bound to cell wall polysaccharides through ester bonds. This bound state within oats means that phenolic acid bioavailability will largely be determined by interactions with the colonic microbiota in the large intestine. However, results from in vitro, animal and human studies have been inconsistent in relation to the impact of oats on the gut microbiota, possibly due to differences in experimental techniques and because compounds in oats, other than β‐glucans, have not been considered. This review focuses on the interaction of oat β‐glucans and phenolic acids with gut microbiota, and the subsequent link to cardiovascular health

The potential of biosensor as an early warning tool for disaster risk reduction at regional level

Recently, there is an increasing rate of environmental pollution cases reported, and that is closely related to technological hazard. Environmental monitoring (EM) is an approach to detect environmental risk before it develops into a disaster. Disaster Risk Reduction (DRR) is an important concept in reducing the impacts of hazards and disasters to social, economy and environment, especially at a regional level. Biosensors have been developed to detect pollutants and hazardous chemicals that are frequently and potentially found in the environment as a result of anthropogenic activity and as part of the natural phenomena. Efforts are focus in developing biosensors that are applicable in EM, some suggested biosensors could replace the conventional chemical analytical methods, but not all of them are practical. To evaluate the feasibility of biosensors in assisting EM in DRR, an analysis of articles published on biosensors in related field was carried out. Based on the evaluation, we concluded five major aspects to be considered when biosensors are to be applied as an early warning system tool in DRR at regional level, namely complexity of real sample, need of continuous environmental monitoring data, reproducibility of data, on-site testing and roles in risk characterization. This paper will help in the assessment of the applicability of biosensor in EM and as part in the DRR, and also as a guide to designing biosensor for EM purposes

Toxicity Biosensor for Sodium Dodecyl Sulfate Using Immobilized Green Fluorescent Protein Expressing Escherichia coli

Green fluorescent protein (GFP) is suitable as a toxicity sensor due to its ability to work alone without cofactors or substrates. Its reaction with toxicants can be determined with fluorometric approaches. GFP mutant gene (C48S/S147C/Q204C/S65T/Q80R) is used because it has higher sensitivity compared to others GFP variants. A novel sodium dodecyl sulfate (SDS) toxicity detection biosensor was built by immobilizing GFP expressing Escherichia coli in k-Carrageenan matrix. Cytotoxicity effect took place in the toxicity biosensor which leads to the decrease in the fluorescence intensity. The fabricated E. coli GFP toxicity biosensor has a wide dynamic range of 4–100 ppm, with LOD of 1.7 ppm. Besides, it possesses short response time (<1 min), high reproducibility (0.76% RSD) and repeatability (0.72% RSD, R2>0.98), and long-term stability (46 days). E. coli GFP toxicity biosensor has been applied to detect toxicity induced by SDS in tap water, river water, and drinking water. High recovery levels of SDS indicated the applicability of E. coli GFP toxicity biosensor in real water samples toxicity evaluation

Toxicity Biosensor for Sodium Dodecyl Sulfat Using Immobilized Green Fluorescent Protein expressing Escherichia coli

Green fluorescent protein (GFP) is suitable as a toxicity sensor due to its ability to work alone without cofactors or substrates. Its reaction with toxicants can be determined with fluorometric approaches. GFP mutant gene (C48S/S147C/Q204C/S65T/Q80R) is used because it has higher sensitivity compared to others GFP variants. A novel sodium dodecyl sulfate (SDS) toxicity detection biosensor was built by immobilizing GFP expressing Escherichia coli in k-Carrageenan matrix. Cytotoxicity effect took place in the toxicity biosensor which leads to the decrease in the fluorescence intensity. The fabricated E. coli GFP toxicity biosensor has a wide dynamic range of 4-100 ppm, with LOD of 1.7 ppm. Besides, it possesses short response time (&lt;1 min), high reproducibility (0.76% RSD) and repeatability (0.72% RSD, 2 &gt; 0.98), and long-term stability (46 days). E. coli GFP toxicity biosensor has been applied to detect toxicity induced by SDS in tap water, river water, and drinking water. High recovery levels of SDS indicated the applicability of E. coli GFP toxicity biosensor in real water samples toxicity evaluation

A High-Throughput Oxidative Stress Biosensor Based on Escherichia coli roGFP2 Cells Immobilized in a k-Carrageenan Matrix

Biosensors fabricated with whole-cell bacteria appear to be suitable for detecting bioavailability and toxicity effects of the chemical(s) of concern, but they are usually reported to have drawbacks like long response times (ranging from hours to days), narrow dynamic range and instability during long term storage. Our aim is to fabricate a sensitive whole-cell oxidative stress biosensor which has improved properties that address the mentioned weaknesses. In this paper, we report a novel high-throughput whole-cell biosensor fabricated by immobilizing roGFP2 expressing Escherichia coli cells in a k-carrageenan matrix, for the detection of oxidative stress challenged by metalloid compounds. The E. coli roGFP2 oxidative stress biosensor shows high sensitivity towards arsenite and selenite, with wide linear range and low detection limit (arsenite: 1.0 × 10−3–1.0 × 101 mg·L−1, LOD: 2.0 × 10−4 mg·L−1; selenite: 1.0 × 10−5–1.0 × 102 mg·L−1, LOD: 5.8 × 10−6 mg·L−1), short response times (0–9 min), high stability and reproducibility. This research is expected to provide a new direction in performing high-throughput environmental toxicity screening with living bacterial cells which is capable of measuring the bioavailability and toxicity of environmental stressors in a friction of a second

Signalling through retinoic acid receptors is required for reprogramming of both mouse embryonic fibroblast cells and epiblast stem cells to induced pluripotent stem cells

© 2014 AlphaMed Press. We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog-1 (LRH1 or NR5A2) with OCT4, MYC, KL F4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration-free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose-sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant-negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. Stem Cells 2015;33:1390-1404Link_to_subscribed_fulltex