Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip

Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours – such as bone tumours – is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a DEP-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics

Caractérisation histologique et anatomique de la gamétogenèse de Radix balthica (linnaeus, 1758) en comparaison avec Lymnaea stagnalis (linnaeus, 1758)

International audienceFreshwater gastropods are frequently used as model organisms to assess the effects of certain chemical substances. Among them Radix balthica and Lymnaea stagnalis are commonly used in the laboratory, mesocosm and fields tests. In order to determine the effects of pollutants and more particularly endocrine disrupting substances on the reproduction of these organisms, histopathological analyses can be used. Because data are still scarce in the literature, knowledge development on anatomy of reproductive tissues and gametogenesis is a preliminary step before any studies on the impact of contaminants on the reproduction of these gastropods. The characterization of the anatomy and gametogenesis of Radix balthica and Lymnaea stagnalis was thus performed in this study. Important morphological differences exist between the two species. Despite this, the gonads of the two gastropods species have similar histological structure. In both species, spermatogonia are clustered; spermatozoids are anchored in the Sertoli cells and the male cells alternate with the female cells that rest on the epithelium of the hermaphroditic gland. This study is a prerequisite for any further histopathological studies on contaminated individuals

Etude protéomique de la fonction mâle chez Gammarus fossarum en réponse de pertubateurs endocriniens

International audienceWhile the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress

SILAR deposition of Ni(bpy)3X: {X = (NCS)2, (Fe(CN)5NO), and (Ag(CN)2)2} thin films on glass substrates

The authors focused on the preparation of thin layers based on hybrid materials (organometallic complexes) deposited onto glass substrates. The deposition experiments of [Ni(bpy)3](NCS)2, [Ni(bpy)3](Ag(CN)2)2, and [Ni(bpy)3](Fe(CN)5NO) were performed on glass slides (18 mm × 18 mm) by successive ionic layer adsorption and reaction (SILAR) method. The influence of some parameters, such as dipping cycle numbers (30, 60, and 120 dipping cycles), temperature (20°C, 30°C, 40°C, and 50°C), precursors concentration (10−3, 10−2, and 10−1 M), and the nature of the counteranions (NCS−, [Fe(CN)5NO]−2, [Ag(CN)2]−) were studied. Different methods (UV-Vis, SEM, FTIR, and XRD) were used to characterize the deposited layers to determine the absorption coefficient (α) and gap energy (Eg) of the materials

Proteome analysis of male gonads in Gammarus fossarum exposed to Pyriproxyfen: mining for endocrine disruption biomarkers

International audienceProteomics is becoming popular in ecotoxicology as an approach for the discovery of new candidate biomarkers of environmental contamination. Studies are currently based on the classical proteomic approach, in which proteomes from a control and a contaminated situation are resolved by 2D-PAGE, and proteins differentially expressed are then identified by mass spectrometry. Most of these detected proteins are abundant, usually housekeeping proteins, such as those involved in ATP metabolism and maintenance of the cytoskeletal structure, but rarely specific to a contamination exposure. This drawback is due to the few sequenced genomes in aquatic invertebrates that limit the interpretation of the mass spectra and the identification of proteins, and the low dynamic range of the 2D-PAGE approach. Recent technological advances in highthroughput “omics” methodologies, in which thousands of genes, proteins or metabolites can be assessed simultaneously, provide invaluable molecular information. Shotgun proteomics allows in-depth analysis of whole proteomes if performed with the most recent mass spectrometers. In order to unveil the novel perspectives offered by the latest generation of mass analyzer, we analyzed the performances of the Q-Exactive HF tandem mass spectrometer equipped with an ultra-high field Orbitrap analyzer. The aim of this study was to reconsider the discovery of new candidate biomarkers of exposure to Pyriproxyfen in male gammarids by shotgun proteomics, in the light of the latest generation of tandem mass spectrometer. Males were exposed to two concentrations of the xenobiotic Pyriproxyfen (0.5 and 50 μg/L) during two consecutive spermatogenesis cycles. The number of proteins identified, their abundance and the number of proteins that were modulated by the contamination were used to document the performance of the new

Utilisation de l’essai des comètes pour la surveillance des milieux aquatiques – problématique de la conservation des échantillons

National audienceLes milieux aquatiques constituent le réceptacle d’un grand nombre de contaminants, anthropiques et naturels, pouvant être dangereux pour les organismes qui y vivent

Chemical composition, antioxidant and insecticidal activities of a new essential oil chemotype of Pinus nigra ssp. mauritanica (Pinaceae), northern Algeria

Essential oil (EO) of aerial parts (twigs and needles) of an Algerian-Moroccan endemic pine Pinus nigra Arn. ssp. mauritanica (Mair. & Pey.) (Pinaceae) collected in Northern Algeria were extracted by hydrodistillation (HD) and solid phase micro-extraction (SPME). The compounds were analysed using Gas Chromatography (GC) and GC-Mass Spectrometry (GC-MS). The mean oil yield was 0.23%, for twigs and 0.47% for needles. 83 and 91 compounds have been identified for needles and twigs respectively. Chemical divergences from all other studied Black pines worldwide were observed in needles where the major components identified by HD are β-caryophyllene (26.2%), germacrene D (17.2%), α-pinene (9.4%) unveiling a new chemotype. Significative qualitative and quantitative differences were observed in chemical composition, depending on both technics HD and SPME. Twigs showed a higher potential for the antioxidant activities DPPH, ABTS and β-carotene system than EO needles. In other hand, needles revealed a higher activity for all concentrations than twigs with significant differences (p < 0.05). For phosphomolybden tests, both EOs exhibited more significant antioxidant potential compared to the DPPH, ABTS, β-carotene and reducing power systems. Noticeable insecticidal effects with variable rate depending on used plant parts, concentration, duration and frequency of exposure were also observed

Effets transgénérationnels d'une exposition parentale chez l'espèce sentinelle Gammarus fossarum

International audienceSince the 80s, the development of molecular biomarkers is an important component of ecotoxicology. Unfortunately, field studies that univocally link biomarker responses to fitness impacts and finally to population level are scarce. This is mainly due to the discordance in time scales between toxicological and ecological responses. In previous studies and under laboratory conditions with high contamination levels, a direct link has been established between genotoxic impacts in gametes of the sentinel species Gammarus fossarum, and impairment in embryos production. However, in a previous field study, abnormal genotoxic impacts levels observed in gametes, lower than ones obtained under laboratory exposure, failed to demonstrate rapid consequence on the production of embryos by exposed genitors. Taking advantage of the availability of multiple scale marker measurement in this species, from molecular (Comet assay, global DNA methylation) to physiological (feeding rate, molting success, vitellogenesis) and life history traits (growth, fertility, embryonic survival), along with the possibility to conduct rearing culture in the lab (time to puberty about 4 months), the objective of this study was to assess whether biomarker responses recorded in adult gammarids exposed to chemical stress may be predictive of long term effects on the fitness of their future progeny (i.e. transgenerational effects). For this, exposure of genitors to cadmium as model compound in the lab at environmentally relevant concentrations (0.3 and 3 µg/L) was followed by a post-exposure population culture of at least F1 and F2 individuals in uncontaminated conditions. Feeding rates (leaf disc surface measurements) of exposed organisms did not differ significantly from control individuals (Mann Whitney test p>0.05). The number of oocyte per female and the indicator of investment in oocyte quality (oocyte volume) of exposed females were also not significantly different compared to control organisms. In contrast, the first results analyzed on the F1 generation demonstrated a sharp significant effect of F0 parental cadmium exposure on the embryo number produced by F1 females after 6 months of lab maintenance in uncontaminated conditions (figure 3). Hence, exposure at environmentally relevant level of cadmium which only induces weak biomarker response (here sperm DNA damage) could generate an important delayed decrease in the population recruitment for future generations of this sentinel invertebrate species. To further assess population consequence of such effect, we plan to integrate these effects in the demographic population model available in Gammarus fossarum (5). Establishing a causal link between sperm DNA damage and drop in future generations is too speculative at this stage because other pathways of toxicity could be in cause in this transgenerational effects. Nevertheless, regarding our results on reproduction of these individuals (feeding rate and oocyte areas), We can discard the hypothesis of a limitation of investment by F0 females in F1 generation during Cd exposure. In addition to these results, other markers are being further analyzed (epigenetic marker and growth rate). Similar measurements will be done in the F2 generation in the next few weeks.This study enhances the importance to scrutinize transgenerational and delayed impacts of contaminant exposure, and the relevance of invertebrate species such as Gammarus to address this issue. Ongoing experiments considering field exposure of caged gammarids are implemented in order to generalize the outcome of this first laboratory case study

Utilisation de Gammarus sp. en écotoxioclogie. Etude de cas avec l'insecticide régulateur de croissance fénoxycarbe

International audienceGammarus sp. (Amphipoda) are widely distributed across European freshwater systems. In the present study, we evaluated the effect of a fenoxycarb exposure on Gammarus sp.. More specifically, i) we assessed the sensitivity of the embryo stage, ii) we identified embryogenesis' sensitive period, iii) we evaluated the sensitivity of the reproductive period and iv) we compared the response to fenoxycarb exposure among three European gammarid species. Fenoxycarb is a growth regulator insecticide, analog of the insect juvenile hormone, used for pest management and for veterinary purpose. This study demonstrated that 5 and 50 µg

Utilisation de l’essai des comètes pour la surveillance des milieux aquatiques – problématique de la conservation des échantillons

Les milieux aquatiques constituent le réceptacle d’un grand nombre de contaminants, anthropiques et naturels, pouvant être dangereux pour les organismes qui y vivent