Cell- and Tissue-Containing Biosensors

International audienceWhole cell biosensors allow detection of modifications of a cell or a microorganism used as an integrated system. Cells and tissues from prokaryotic to higher eukaryotic origins are used as standard biosensors with two main outputs, fluorescence and bioluminescence. These living systems can be used as biosensors per se. Alternatively, their properties can be transposed by molecular engineering to any desired cell model with many applications and benefits especially in terms of access to sophisticated and accurate detection modes. Looking ahead, new developments mainly concern transposition of whole cell biosensors to the human body for monitoring and diagnosis purposes

Conception d'une protéine kinase A modifiée (une nouvelle méthode d'analyse de la voie AMPc)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

In this work, both direct and indirect cell-based antioxidant profiles were established for 27 plant extracts and 1 algal extract. To evaluate the direct antioxidant effects, fluorescent AOP1 cell assay was utilized, which measures the ability of different samples to neutralize intracellular free radicals produced by a cell-based photo-induction process. As the intestinal barrier is the first cell line crossed by the product, dose response curves obtained from Caco-2 cells were used to establish EC50 values for 26 out of the 28 natural extracts. Among them, 11 extracts from Vitis, Hamamelis, Syzygium, Helichrysum, Ilex and Ribes genera showed remarkable EC50s in the range of 10 µg/mL. In addition to this, a synergistic effect was found when combinations of the most potent extracts (S. aromaticum, H. italicum, H. virginiana, V. vinifera) were utilized compared to extracts alone. Indirect antioxidant activities (i.e., the ability of cells to trigger antioxidant defenses) were studied using the ARE/Nrf2 luminescence reporter-gene assay in HepG2 cells, as liver is the first organ crossed by an edible ingredient once it enters in the bloodstream. Twelve extracts were subjected to an intestinal epithelial barrier passage in order to partially mimic intestinal absorption and show whether basolateral compartments could maintain direct or indirect antioxidant properties. Using postepithelial barrier samples and HepG2 cells as a target model, we demonstrate that indirect antioxidant activities are maintained for three extracts, S. aromaticum, H. virginiana and H. italicum. Our experimental work also confirms the synergistic effects of combinations of post-intestinal barrier compartments issued from apical treatment with these three extracts. By combining cell-based assays together with an intestinal absorption process, this study demonstrates the power of cell systems to address the issue of antioxidant effects in humans

Dosimetry Performances of a MultiWell-Plate-Based Near-Field RF Applicator for the Investigation of EM Impact on Biological Cells

International audienceWhile electromagnetic fields are now quasiubiquitous on earth and are constantly growing in use, RF radiation impact on living beings still continues to question. Within this context, a near-field RF applicator compatible with routine in vitro microplates for cellular investigations is proposed. This paper specifically focuses on its dosimetry performances, which are evaluated through simulation and measurements at 225 MHz, and its facility in biological postanalysis enabled by the use of standard bio-microplates

From the Yeast cell wall to new strategies in the fight against Ciguatera

Séminaire Scientifique au Centre de Recherches Insulaires et Observatoire de l’Environnement (CRIOBE, USR 3278)I will attempt to explain my scientific journey, from studying the cell wall and stress responsemechanisms of yeasts, up to the development of innovative approaches to fight Ciguatera, a majorcause of food poisonings by seafood worldwide.Yeasts are eukaryotic microorganisms traditionally used by man to make bread, beer and wineand thus very well studied. Their cells are surrounded by a thick cell wall composed of glycans andproteins organized in a complex multilayered architecture. Essential for the survival of yeasts andfungi, this highly dynamic organelle gets constantly remodeled during the cell cycle to allowmorphogenetic events as well as upon environmental changes. It is a key player in cellular adhesionand resistance to antifungal compounds. While studying the mechanisms of cell wall adaptations tovarious stresses, I came across the different cellular signaling pathways involved notably MAP kinasecascades and the Calcineurin pathway which are conserved with higher eukaryotes.Ciguatoxins (CTXs) are lipid-soluble, highly neurotoxic, polyether compounds produced bydinoflagellates from the genus Gambierdiscus spp.. mostly found in tropical and subtropical zones.CTXs bind to Voltage Gated Sodium Channels at the surface of human sensory neurons where theyremain, causing Ciguatera Fish Poisoning or CFP. This severe disease is characterized by with avariety of gastrointestinal, cardiovascular and neurological symptoms (paresthesia, ataxia, coldallodynia), including persistent neurological effects. Despite the importance and prevalence of CFP,there is so far no simple and quick way of detecting these toxins in contaminated samples. Currently,only heavy and expensive laboratory methods are available to detect them: LC-MS/MS, receptorbinding assays by competition with labeled compounds, and neuroblastoma cell-based assaysperformed on mammalian neurons. We have started to engineer biosensors based on the detection ofa transcriptional signal in the model yeast Saccharomyces cerevisiae, using the very goodconservation of its signaling pathways with higher eukaryotes.Finally, I will also present a smaller project (NeuroSens) based on a different strategy, to studyand characterize the binding of Ciguatoxins on mammalian neuronal cells by Single Molecule ForceSpectroscopy using Atomic Force Microscopy

New strategies in the fight against Ciguatera

International audienceCiguatera is the major cause of food poisonings by seafood worldwide, with an estimated 50 000 to 500 000 victims per year. It is caused by very potent neurotoxins, Ciguatoxins which are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp.. Ciguatoxins are mostly found in tropical and subtropical zones, including notably the Caribbean’s, French Polynesia, New Caledonia, Indian Ocean and Southern Asia. Within the last decade, they have been also identified in fishes caught in European waters (ex.: in Madeira and Canary Islands), while Gambierdiscus spp. have also been found both in the NE Atlantic Ocean and in the Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels at the surface of human sensory neurons where they remain, causing Ciguatera Fish Poisoning or CFP. This severe disease is characterized by with a variety of gastrointestinal, cardiovascular and neurological symptoms (paresthesia, ataxia, cold allodynia), including persistent neurological effects. Despite the importance and prevalence of CFP, there is so far no simple and quick way of detecting these toxins in contaminated samples. Currently, only heavy and expensive laboratory methods are available to detect them: LC-MS/MS, receptor-binding assays by competition with radiolabeled compounds, and neuroblastoma cell-based assays performed on mammalian neurons. We have started to engineer biosensors based on the detection of a transcriptional signal in the yeast model Saccharomyces cerevisiae. This unicellular eukaryotic model is well-known and easy to genetically modify, grows fast and presents a very good conservation of signaling pathways with higher eukaryotes. Moreover, in an attempt to use developing Nanobiotechnology approaches, we will present our current strategy to study and characterize the binding of Ciguatoxins on mammalian neuronal cells by Single Molecule Force Spectroscopy using AFM

Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

International audienceCiguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1μM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products

Development of new biosensors to detect ciguatoxins

Le poster a été présenté à l'oralInternational audienceCiguatoxins are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp.. Ciguatoxins are mostly found in tropical and subtropical zones; however, within the last decade, they have been identified in fishes caught in European waters, notably in Madeira (1) and Canary Islands (2), while Gambierdiscus spp. have also been found both in the NE Atlantic Ocean (3) and in the Mediterranean Sea (4). These toxins bind to Voltage Gated Sodium Channels at the surface of human sensory neurons where they remain, causing Ciguatera Fish Poisoning with a variety of gastrointestinal, cardiovascular and neurological symptoms (paresthesia, ataxia, cold allodynia), including persistent neurological effects. Ciguatera is the major cause of food poisonings by seafood worldwide, with an estimated 50 000 to 500 000 victims per year. However, there is so far no simple and quick way of detecting these toxins in contaminated samples. Currently, only heavy and expensive laboratory methods are available to detect them: LC-MS/MS, receptor-binding assays by competition with radiolabeled compounds, and neuroblastoma cell-based assays performed on mammalian neurons (5). We have started to engineer biosensors based on the detection of a transcriptional signal in the yeast model Saccharomyces cerevisiae. This unicellular eukaryotic model is well-known and easy to genetically modify, grows fast and presents a very good conservation of signaling pathways with higher eukaryotes. We present a series of genetically modified yeast strains which allow us to follow the activation of specific signaling pathways responding linearly to ciguatoxin exposure

Cellular Antioxidant Effect of an Aronia Extract and Its Polyphenolic Fractions Enriched in Proanthocyanidins, Phenolic Acids, and Anthocyanins

Oxidative stress and chronic inflammation contribute to some chronic diseases. Aronia berries are rich in polyphenols. The aim of the present study was to characterize the cellular antioxidant effect of an aronia extract to reflect the potential physiological in vivo effect. Cellular in vitro assays in three cell lines (Caco-2, HepG2, and SH-SY5Y) were used to measure the antioxidant effect of AE, in three enriched polyphenolic fractions (A1: anthocyanins and phenolic acids; A2: oligomeric proanthocyanidins; A3: polymeric proanthocyanidins), pure polyphenols and microbial metabolites. Both direct (intracellular and membrane radical scavenging, catalase-like effect) and indirect (NRF2/ARE) antioxidant effects were assessed. AE exerted an intracellular free radical scavenging activity in the three cell lines, and A2 and A3 fractions showed a higher effect in HepG2 and Caco-2 cells. AE also exhibited a catalase-like activity, with the A3 fraction having a significant higher activity. Only A1 fraction activated the NRF2/ARE pathway. Quercetin and caffeic acid are the most potent antioxidant polyphenols, whereas cyanidin and 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone showed the highest antioxidant effect among polyphenol metabolites. AE rich in polyphenols possesses broad cellular antioxidant effects, and proanthocyanidins are major contributors. Polyphenol metabolites may contribute to the overall antioxidant effect of such extract in vivo