Giemsa versus acridine orange staining in the fish micronucleus assay and validation for use in water quality monitoring

This study concerns a comparative analysis of the acridineorange and Giemsastaining procedures for the fish erythrocyte micronucleusassay. The goal was to optimize the assay in the context of field watermonitoring. Fish (Carassius carassius) were exposed to a reference genotoxic agent, cyclophosphamide monohydrate 5 mg l−1 for 2, 4, and 6 days before testing. Slides from each individual were scored using the two procedures. The results show that the assay was more sensitive when acridineorange was used. When slides were Giemsa stained, the presence of ambiguous artefacts, leading to false positives and increasing random variance, reduced the contrast between exposed and control samples. AcridineOrangestaining was then applied in the context of waterqualitymonitoring. Fish were exposed for 4 days to water sampled in two hydrological contexts: basal flow and spring flood. The results show that exposure to spring flood water in an agricultural stream can induce mutagenicity

Mutagenic impact on fish of runoff events in agricultural areas in south-west France

When heavy rainfall follows herbicide application, the intense surface runoff causes stream water contamination. Aquatic organisms are then briefly exposed to a complex mixture of contaminants. The aim of the present study is to investigate the genotoxic impact of such events on fish. A model fish, the Crucian carp (Carassius carassius) was exposed in controlled conditions, for 4 days, to water sampled daily in the Save River (France). The watershed of this stream is representative of agricultural areas in southwest France. Three hydrological conditions were compared: basal flow, winter flood, and spring flood. Chemical analysis of the water samples confirmed the higher contamination of the spring flood water,mainly explained by a peak of metolachlor. Genotoxicity was evaluated by micronucleus (MN) test and comet assay in peripheral erythrocytes. A significant increase in DNA breakdowns compared to controls was detected by the comet assay for all conditions. Exposure to spring flood water resulted in the highest damage induction. Moreover, induced chromosomal damage was only detected in this condition. In addition, fish were exposed, for 4 days, to an experimental mixture of 5 herbicides representative of the spring flood water contamination. Fish exhibited moderate DNA damage induction and no significant chromosomal damage. The mutagenicity induced by field-collected water is then suspected to be the result of numerous interactions between contaminants themselves and environmental factors, stressing the use of realistic exposure conditions. The results revealed a mutagenic impact of water contamination during the spring flood, emphasizing the need to consider these transient events in water quality monitoring programs

Caractérisation des effets génotoxiques sur poissons de produits phytosanitaires en période de crue

Les recherches effectuées dans le cadre de cette thèse visent à évaluer l'impact biologique des flashs de contaminations d'origine agricole associés aux crues. Dans un premier temps, nous avons cherché à évaluer les effets génotoxiques de tels événements. Ces mesures renseignent sur des effets précoces et pertinents écologiquement. Après avoir optimisé et validé le test micronoyaux dans nos conditions expérimentales, nous l'avons utilisé conjointement avec l'essai comète pour tester le potentiel génotoxique de 3 conditions hydrologiques contrastées. Il a ainsi été mis en évidence une plus grande génotoxicité de l'eau de la Save prélevée lors d'une crue de printemps par rapport à une crue d'hiver ou un débit de base. Ces résultats ont ensuite été confrontés avec ceux observés lors d'expositions à des mélanges expérimentaux reconstituant les contaminations observées dans le milieu. Les effets génotoxiques des mélanges d'herbicides ont été confirmés, et la complexité des mécanismes induisant la toxicité lors des épisodes de crues a été mise en évidence. Dans un second temps, afin de caractériser la contamination des organismes lors des épisodes de crue, un protocole d'extraction et d'analyse des herbicides accumulés dans les tissus de poissons a été évalué.This study aims at evaluating the biological impact of transient agricultural contamination events associated with floods. First, we investigated the genotoxic impact of such contamination events. These measures provide early and ecologically relevant data. Then, after the optimization and validation of the micronucleus assay in our experimental conditions, this test has been used together with the comet assay in order to test the genotoxic potential of 3 contrasted hydrological conditions. Spring flood water has been found to be more genotoxic than winter flood water or water sampled during the basal flow. These results have been compared with those of exposure to experimental mixtures, mimicking field contamination. The genotoxic potential of herbicides mixture has been confirmed, and the complexity of the processes inducing the toxicity during flood events has been highlighted. Second, in order to investigate the contamination pattern during flood events, a protocol allowing the extraction and quantification of the herbicides accumulated in fish tissues has been evaluated

Oc031--Generic Substitution Of Antiepileptic Drug (Aed) And Loss Of Seizure Control: A Population-Based Case-Crossover Study

Open access CC-BYInternational audienceThere are still controversies over pill substitution among AEDs: some studies claimed that switching between brand and generic AED (generic substitution) can lead to breakthrough seizures; other studies have refuted these concerns. France and some US states recommend limiting substitution of generic AED. We aimed at further estimating the association between generic substitution and loss of seizure control

Placement automatique de sondes d’irradiance

Nous proposons une méthode pour placer automatiquement des sondes dans une scène par minimisation d’une fonction d’erreur. Nous guidons les sondes vers les sites d’échantillonnage optimaux en appliquant la descente de gradient à une fonction d’erreur qui représente la similarité entre la structure en construction et un ensemble de référence. En utilisant la pondération inverse à la distance comme fonction interpolante, nous avons construit avec fiabilité des ensembles de sondes dans trois scènes. En comparant nos résultats avec ceux produits par un ensemble de sondes de référence placées sur une grille régulière, nous atteignons théoriquement notre objectif dans une des trois scènes, où nous obtenons des valeurs d’erreur inférieures à la référence avec beaucoup moins de sondes. Nous avons eu des succès partiels dans les autres scènes, selon le nombre d’échantillons utilisés.Diffuse global illumination within a 3D scene can be approximated in real time using irradiance probes. Probe placement typically relies on significant human input, and final quality of the approximation is often left to the subjectivity of a lighting artist. As demand for realism in rendering increases, the need to enhance the quality of such approximations is greater. We propose a method to automatically place probes in a scene by minimizing an error function. We guide probes to optimal sampling locations by applying gradient descent to an error function that represents similarity between our interpolated results and reference irradiance values. Using weighted nearest neighbour interpolation, we were able to reliably construct probe sets with minimal input in three scenes. Comparing our results to those produced by a set of probes placed on a 3D grid, we were theoretically successful in one scene, in which we could obtain lower error values with fewer probes. We obtained partial success in the other scenes, depending on the number of samples used

The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome

Exposure to phosphine in maritime transport: a real and important occupational risk: a report of three cases

In maritime transport, to assess the risks of insect pests spreading, fumigation is recommended by the Food and Agriculture Organisation. Fumigant mostly used for foodstuffs is the phosphine gas generated by the reaction of aluminium phosphide and moisture in the atmosphere. In this article, we first discuss phosphine toxicity to humans and then we describe three cases of occupational exposure in maritime transport of cereals. We found phosphine level higher than 20 ppm in tank atmosphere of bulk carriers and levels from 2 to 3.5 ppm in port silos and port warehouses where cereals were unloaded. Two weeks later, atmospheric measurements in a silo were still at 0.8 ppm. In this case, 3 workers described symptoms which could be linked with phosphine. Exposures to phosphine and cases in maritime transport are surely underestimated. Exposure could occur at sea, in harbour but also in port warehouses, trucks and silos or warehouses along logistic chain. All workers in the chain could be exposed. We can recommend research aiming at the development of alternative techniques using a less harmful gas for humans. At individual level, we propose that, along with the training for employees, workers potentially exposed should wear a test strip (phosphine detector strips) or a personal gas badge with appropriate maintenance

The role of network bridging organisations in compensation payments for agri-environmental services under the EU Common Agricultural Policy

Compensation payments to farmers for the provision of agri-environmental services are a well-established policy scheme under the EU Common Agricultural Policy. However, in spite of the success in most EU countries in the uptake of the programme by farmers, the impact of the scheme on the long term commitment of farmers to change their practices remains poorly documented. To explore this issue, this paper presents the results of structured field interviews and a quantitative survey in the Walloon Region of Belgium. The main finding of this study is that farmers who have periodic contacts with network bridging organisations that foster cooperation and social learning in the agri-environmental landscapes show a higher commitment to change. This effect is observed both for farmers with high and low concern for biodiversity depletion. Support for network bridging organisations is foreseen under the EU Leader programme and the EU regulation 1306/2013, which could open-up interesting opportunities for enhancing the effectiveness of the current payment scheme for agri-environmental services

The lactococcal abortive infection protein AbiP is membrane-anchored and binds nucleic acids

AbstractAbiP, a lactococcal abortive phage infection system, has previously been shown to arrest phage bIL66M1 DNA replication around 10 min after infection and to inhibit the switch off of phage early transcripts. We report here the functional characterization and implication in the abortive infection phenotype of two domains identified in the AbiP sequence. We show that AbiP is a protein anchored to the membrane by an N-terminal membrane-spanning domain. Our results further suggest that membrane localization may be required for the anti-phage activity of AbiP. The remainder of the protein, which contains a putative nucleic acid binding domain, is shown to be located on the cytosolic side. Purified AbiP is shown to bind nucleic acids with an approximately 10-fold preference for RNA relative to ssDNA. AbiP interaction with both ssDNA and RNA molecules occurs in a sequence-independent manner. We have analyzed the effect of substitutions of aromatic and basic residues on the surface of the putative binding fold. In vitro and in vivo studies of these AbiP derivatives indicate that the previously reported effects on phage development might be dependent on the nucleic acid binding activity displayed by the membrane-bound protein

IS911 transpososome assembly as analysed by tethered particle motion

Initiation of transposition requires formation of a synaptic complex between both transposon ends and the transposase (Tpase), the enzyme which catalyses DNA cleavage and strand transfer and which ensures transposon mobility. We have used a single-molecule approach, tethered particle motion (TPM), to observe binding of a Tpase derivative, OrfAB[149], amputated for its C-terminal catalytic domain, to DNA molecules carrying one or two IS911 ends. Binding of OrfAB[149] to a single IS911 end provoked a small shortening of the DNA. This is consistent with a DNA bend introduced by protein binding to a single end. This was confirmed using a classic gel retardation assay with circularly permuted DNA substrates. When two ends were present on the tethered DNA in their natural, inverted, configuration, Tpase not only provoked the short reduction in length but also generated species with greatly reduce effective length consistent with DNA looping between the ends. Once formed, this ‘looped’ species was very stable. Kinetic analysis in real-time suggested that passage from the bound unlooped to the looped state could involve another species of intermediate length in which both transposon ends are bound. DNA carrying directly repeated ends also gave rise to the looped species but the level of the intermediate species was significantly enhanced. Its accumulation could reflect a less favourable synapse formation from this configuration than for the inverted ends. This is compatible with a model in which Tpase binds separately to and bends each end (the intermediate species) and protein–protein interactions then lead to synapsis (the looped species)