An in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption

There is a pressing need for high throughput methods to assess potential effects of endocrine disrupting chemicals (EDCs). released into the environment. Currently our ability to identify effects in vitro exceeds that for in vivo monitoring. However, only in vivo analysis provides the full spectrum of physiological impacts exerted by a given chemical. With the aim of finding a physiological system compatible with automatic plate reading we tested the capacity of early embryonic stage Xenopus laevis tadpoles to monitor thyroid hormone (TH) disruption. Fluorescent transgenic X. laevis embryos bearing a TH/bZIP-eGFP construct, placed in 96 well plates, were used for a physiological-based screen for potential TH signaling disruptors. Using stage NF-45 embryos (time of thyroid gland formation) allowed rapid detection of chemical interference with both peripheral TR signaling and production of endogenous TH. Nanomolar concentrations of TH receptor agonists could be detected within 72 h. Moreover, when testing against a 5nM T3 challenge, the effects of inhibitors of TH production were revealed, including inhibitors of TH synthesis, (methimazole: 1 mM or sodium perchlorate: 3.56 microM), as well as antagonists acting at the receptor level (NH3: 2 microM) and a deiodinase inhibitor (iopanoic acid: 10 microM). Finally, we show that the thyroid disrupting activities of BPA (10 microM) and TBBPA (1 microM) can also be detected in this rapid screening protocol. Finally, this noninvasive technology using an automatic reading system shows low variability (around 5%) and permits detection of subtle changes in signaling by EDCs that either inhibit or activate TH signaling in vivo.     

Technical note: High-throughput method for antifungal activity screening in a cheese-mimicking model

In this study, we developed a high-throughput antifungal activity screening method using a cheese-mimicking matrix distributed in 24-well plates. This method allowed rapid screening of a large variety of antifungal agent candidates: bacterial fermented ingredients, bacterial isolates, and preservatives. Using the proposed method, we characterized the antifungal activity of 44 lactic acid bacteria (LAB) fermented milk-based ingredients and 23 LAB isolates used as protective cultures against 4 fungal targets (Mucor racemosus, Penicillium commune, Galactomyces geotrichum, and Yarrowia lipolytica). We also used this method to determine the minimum inhibitory concentration of a preservative, natamycin, against 9 fungal targets. The results underlined the strain-dependency of LAB antifungal activity, the strong effect of fermentation substrate on this activity, and the effect of the screening medium on natamycin minimum inhibitory concentration. Our method could achieved a screening rate of 1,600 assays per week and can be implemented to evaluate antifungal activity of microorganisms, fermentation products, or purified compounds compatible with dairy technology.     

Activation of fibroblast metabolism in a dermal and skin equivalent model: a screening test for activity of peptides

Skin firmness, elasticity and tone are gradually lost with age. These changes originate in the dermis and correspond to a decrease in the ability of cells, particularly the fibroblasts, to regenerate the molecules which make up the extracellular matrix. Skin ageing is also characterized by a reduction of the epidermal thickness and by a flattening of the basal membrane. The recent development of two 3-dimensional culture systems, in which the cells develop within a porous structure reproducing the extracellular matrix of the human dermis, is a way of reproducing in vivo conditions and demonstrating the biological effects of anti-ageing compounds. The dermal equivalent model used in this study is composed of a dermal matrix made of collagen-chitosan-glycosaminoglycans populated by normal human fibroblasts which synthesized their own extracellular matrix. A skin equivalent model is obtained by the cell culture of normal human keratinocytes onto a dermal equivalent elevated at the air-liquid interface. Such models were used to prove anti-ageing activity of promising compounds. Cosmetic Science has used many protein hydrolysates in order to fight skin ageing, but up to now, these natural peptides were poorly studied, and their efficacy poorly demonstrated. Eight protein hydrolysates were screened in a proliferation study in monolayered cultures giving two selected polypeptides. A soya derived peptide was used for an efficiency study in 3-dimensional models. In the dermal equivalent model, this peptide increased fibroblast proliferation by 40% and led to a stimulation of collagen formation (165%) and elastin (116%) synthesis. The effect of this soya peptide on glycosaminoglycan synthesis was also significant, with increases of 36% for chondroitin-4-sulfate and 68% for hyaluronic acid. These results were confirmed using a skin equivalent model. In this model, the soya peptide increased the thickness of the epidermis.     

Exposure-effect model for calculating copper effect concentrations in sediments with varying copper binding properties: a synthesis

An exposure-effects model is described for calculating copper effect concentrations for benthic organisms in sediments with varying copper-binding properties. It is based on a bioenergetic-based kinetic model that describes the rate of assimilation of copper by benthic organisms from dissolved and particulate phases. During acute exposures, the total copper assimilated by the organisms was used as a measure of the organisms' exposure to bioavailable copper, and toxicity occurs when the copper exposure exceeds a threshold value. Exposure-effects models were developed for nine benthic organisms and were used to predict the effect of sediment-water partitioning (Kd) and copper assimilation from ingested solids on toxic effects and how these factors will influence derived sediment quality guideline (SQG) concentrations. Species sensitivity distributions were used to calculate SQG concentrations for copper for sediments of varying copper binding properties. The modeling indicated that "single value" SQG concentrations would be ineffective for predicting the toxicity of metals in sediment. It is proposed that, for all contaminants (not just metals), a better approach would be to have SQG concentrations, or ranges, that are applied to different sediment types. SQGs should account for contaminant exposure from both water-filtration and particulate-ingestion exposure routes, as can be achieved by models of sediment-water contaminant partitioning (Kd) and the contaminant assimilation efficiency (AE) of ingested particles. The study indicates that improved mechanistic models of contaminant exposure, as influenced by both organism physiology and sediment properties, are needed to predict toxic effects in sediments.     

Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms

Budding yeast can be considered to have two distinct lifespans: (a) a replicative (budding, non-chronological) lifespan, measured as the number of daughters produced by each actively dividing mother cell; and (ii) a chronological lifespan, measured as the ability of stationary cultures to maintain viability over time. In non-dividing cells, essential components that become damaged cannot be diluted out through cell division but must, of necessity, be turned over and renewed. By elevating stress resistances, many of the activities needed for such renewal should be elevated with commensurate reduction in the steady-state levels of damaged cell components. Therefore, chronological lifespan in particular might be expected to relate to stress resistance. For yeast to attain a full chronological lifespan requires the expression of the general stress response. It is more important, though, that the cells should be efficiently adapted to respiratory maintenance, since it is cultures grown to stationary phase on respiratory media that usually display the longest chronological lifespans. For this reason, respiration-adapted cells potentially provide a better model of chronological ageing than cultures pre-grown on glucose.