Assessment of trace element contamination and effects on Paracentrotus lividus using several approaches: Pollution indices, accumulation factors and biochemical tools.

Among the most common contaminants in marine ecosystems, trace elements are recognized as serious pollutants. In Corsica (NW Mediterranean Sea), near the old asbestos mine at Canari, trace elements from the leaching of mine residues have been discharged into the sea for several decades. The aim of this study was to assess the levels of contamination in this area and the potential effects on Paracentrotus lividus (Lamarck, 1816) using pollution indices, accumulation factors and biochemical tools. For this purpose, the concentration of 24 trace elements was measured in sea urchins (gonads and gut content), macroalgae, seawater column and sediment collected at 12 stations nearby the old asbestos mine and at a reference site. The bioaccumulation of trace elements occurs as follows: macroalgae > gut > gonads. TEPI contribute to highlight contamination gradients which are mainly due to the dominant marine currents allowing the migration of mining waste along the coastline. This hypothesis was supported by TESVI, which identified characteristic trace elements in the southern area of the mine. High hydrogen peroxide content, associated with elevated catalase and glutathione-S-transferase enzyme activities, were also identified at these sites and at the reference site. Trace elements contamination as well as several abiotic factors could explain these results (e.g. microbiological contamination, hydrodynamic events, etc.). The results obtained in this study suggest that oxidative stress induced by contamination does not affect the health of Paracentrotus lividus. This work has provided a useful dataset allowing better use of sea urchins and various tools for assessing trace element contamination in coastal ecosystems.

Stress response to trace elements mixture of different embryo-larval stages of Paracentrotus lividus.

This study investigated for the first time the oxidative biomarkers responses in all larval stages of sea urchin. The contamination effects were reproduced by using contaminated seawater to concentrations measured in the area adjacent to an old asbestos mine at factors of 5 and 10. The results suggested that the concentrations were not sufficiently high to induce a major oxidative stress. The biometric differences make this method a more sensitive approach for assessing the effects on sea urchin larvae. Measurements of specific activities of antioxidant enzymes at each stage suggested a high capacity of the larvae to respond to oxidative stress. This normal activity of the organism must be considered in future research. This work also highlighted the importance of spawners provenance in ecotoxicological studies. These data are essential to better understand the stress responses of sea urchin larvae and provide baseline information for later environmental assessment research.

Effects of trace elements contaminations on the larval development of Paracentrotus lividus using an innovative experimental approach.

Several experiments were performed using larvae of Paracentrotus lividus (Lamarck, 1816) in order to determine the consequences of different chronic contamination with mixtures of (i) fifteen trace elements from concentrations measured in the world ocean seawater, and (ii) seven trace elements from contamination resulting from mining. To predict the impact of increased marine pollution, higher concentrations were also used. These bioassays were conducted using spawners collected from Calvi (reference site, Corsica), and Albo (mining area, Corsica). The effects of trace elements have been studied on the entire larval development. The results show wider arms and delayed development as the number and concentration of trace elements increases. Therefore, the synergy between the different trace elements is of paramount importance with regard to the impact on organisms. Probably due to a hormesis phenomenon, larvae contaminated with seven trace elements at average concentrations developed more quickly. This work also highlighted the importance of the origin of spawners in ecotoxicological studies. To our knowledge, this is the first study to investigate the effects of such a broad combination of trace elements for chronic contamination on the entire larval stage of Paracentrotus lividus.

New insights into the functioning and structure of the PE and PP plastispheres from the Mediterranean Sea.

Plastic debris are accumulating in the marine environment and aggregate microorganisms that form a new ecosystem called the plastisphere. Better understanding the plastisphere is crucial as it has self-sufficient organization and carries pathogens or organisms that may be involved in the pollutant adsorption and/or plastic degradation. To date, the plastisphere is mainly described at the taxonomic level and the functioning of its microbial communities still remains poorly documented. In this work, metagenomic and metaproteomic analyzes were performed on the plastisphere of polypropylene and polyethylene plastic debris sampled on a pebble beach from the Mediterranean Sea. Our results confirmed that the plastisphere was organized as self-sufficient ecosystems containing highly active primary producers, heterotrophs and predators such as nematode. Interestingly, the chemical composition of the polymer did not impact the structure of the microbial communities but rather influenced the functions expressed. Despite the fact that the presence of hydrocarbon-degrading bacteria was observed in the metagenomes, polymer degradation metabolisms were not detected at the protein level. Finally, hydrocarbon degrader (i.e., Alcanivorax) and pathogenic bacteria (i.e., Vibrionaceae) were observed in the plastispheres but were not very active as no proteins involved in polymer degradation or pathogeny were detected. This work brings new insights into the functioning of the microbial plastisphere developed on plastic marine debris.

The combination of deep breathing and mental imagery promotes cardiovascular recovery in firefighters.

Firefighters' rescue operations involve intense physical activity associated with a high level of cardiovascular stress. To sustain such intense physical performance whilst maintaining a healthy heart, it is crucial that they benefit from rapid recovery between each intervention. This study aimed at investigating the impacts of a recovery protocol combining deep breathing and mental imagery. Forty firefighters were divided into two experimental groups which undertook two maximum fitness tests separated by either the control recovery protocol (30 min reading time; n = 20) or the experimental recovery protocol (30 min of deep breathing and mental imagery; n = 20). When compared to the pre-tests, the percentage evolution ratios in the post-tests for the Cooper performance, the heart rate recovery and the parasympathetic reactivation were promoted by the experimental protocol, compared to simple reading. In light of these results, we propose the use of practices of deep-breathing combined with mental imagery to improve firefighters' recovery. Practitioner summary: Firefighters' activities involve intense physical activities associated with a high level of psychological stress. Enhancing their recovery after each rescue intervention appears crucial. The results of this study showed that a recovery protocol combining deep breathing and mental imagery promotes heart rate recovery and better maintenance of physical fitness.

Fibrin-based scaffolds for dental pulp regeneration: from biology to nanotherapeutics.

Tissue engineering-based endodontic therapies, designed to regenerate the dental pulp (DP) in the devitalised endodontic space, have been proposed to improve tooth longevity compared to conventional root-filling therapies. Their aim is to restore tooth vitality and major DP functions necessary to maintain tooth health such as immunosurveillance, sensitivity and healing/repair/regenerative capacities. Several formulations based on the use of fibrin, the main component of the blood clot matrix, recently gave valuable results in the regeneration of the human DP. This review describes recent fibrin-based scaffolds designed for that purpose. After having presented the various strategies for DP regeneration, the main fibrin-based scaffolds reported so far for clinical use in endodontics were reviewed. Particular emphasis was given to hydrogel devices that may be improved by incorporation of bioactive molecules that stimulate vascularisation and tissue neoformation or provide antibacterial properties. Data indicate that fibrin-based scaffolds constitute a highly favourable environment for mesenchymal stem cells, which is maintained upon functionalisation. Additional knowledge is needed to understand how fibrin and functionalising agents affect adhesion, survival, proliferation, migration and differentiation of cells incorporated in the scaffold or which will colonise it from neighbouring host tissues. This knowledge is needed to adapt the hydrogel formulation for various clinical conditions.

A new agarose-based microsystem to investigate cell response to prolonged confinement.

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.

Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Spatial variations in trace element concentrations of the sea urchin, Paracentrotus lividus, a first reference study in the Mediterranean Sea.

A study on Trace Elements (TE) from sea urchin gonads has been conducted in the western Mediterranean Sea. Contamination data were used to determine a Trace Method Pollution Index (TEPI). TE concentrations varied considerably depending on the location of the sampling stations. The results showed that five trace elements (Zn, Fe, As, Al, Cu) are ubiquitous. The geographical area considered (Corsica) represents an important range of environmental conditions and types of pressure that can be found in the western Mediterranean Sea. TEPI was used to classify the studied sites according to their degree of contamination and allowed reliable comparison of TE contamination between local and international sites. TE contamination of the western Mediterranean Sea displayed a north-to-south gradient, from the Italian coasts down through the insular Corsican coasts to the north African littoral. Due to the increasing environmental pressure on the Mediterranean Sea, a regular monitoring of TE levels in marine organisms is necessary to prevent any further environmental deterioration.

Trace element concentrations in the apex predator swordfish (Xiphias gladius) from a Mediterranean fishery and risk assessment for consumers.

Swordfish (Xiphias gladius L., 1758) is an apex predator, highly migratory meso-pelagic fish widely distributed in the Atlantic Ocean and Mediterranean. As top predators, this fish may be the end reservoir of the bioaccumulation of trace elements in a food chain because they occupy higher trophic levels and are an important food source, causing them to be potentially hazardous to consume. This study aims to investigate the concentration of 18 trace elements of Swordfish, caught in the Mediterranean Sea and to discuss human exposure risks. The mean element levels in the fish muscles were clearly below the maximum allowable concentrations established by International food safety regulations. The data suggested that the risk is minor and acceptable for human health. The findings of this study amplify the scarce database on contaminants available, especially new data on "emerging elements", for this species from the Mediterranean Sea.

Bioassessment of trace element contamination of Mediterranean coastal waters using the seagrass Posidonia oceanica.

A large scale survey of the trace element (TE) contamination of Mediterranean coastal waters was performed from the analysis of Ag, As, Cd, Cu, Hg, Ni and Pb in the bioindicator Posidonia oceanica, sampled at 110 sites differing by their levels of exposure to contaminants. The holistic approach developed in this study, based on the combined utilization of several complementary monitoring tools, i.e. water quality scale, pollution index and spatial analysis, accurately assessed the TE contamination rate of Mediterranean coastal waters. In particular, the mapping of the TE contamination according to a new proposed 5-level water quality scale precisely outlined the contamination severity along Mediterranean coasts and facilitated interregional comparisons. Finally, the reliability of the use of P. oceanica as bioindicator species was again demonstrated through several global, regional and local detailed case studies. NB: The designations employed and the presentation of the information in this document do not imply the expression of any opinion whatsoever on the part of the authors concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips.

The present study gives a summary using state-of-the-art technology to monitor Posidonia oceanica and Mytilus galloprovincialis as bioindicators of the pollution of the Mediterranean littoral with trace elements (TEs), and discusses their complementarity and specificities in terms of TE bioaccumulation. Furthermore, this study presents two complementary indices, the Trace Element Spatial Variation Index (TESVI) and the Trace Element Pollution Index (TEPI): these indices were shown to be relevant monitoring tools since they led to the ordering of TEs according to the overall spatial variability of their environmental levels (TESVI) and to the relevant comparison of the global TE pollution between monitored sites (TEPI). In addition, this study also discusses some underestimated aspects of P. oceanica and M. galloprovincialis bioaccumulation behaviour, with regard to their life style and ecophysiology. It finally points out the necessity of developing consensual protocols between monitoring surveys in order to publish reliable and comparable results.

Experimental in situ exposure of the seagrass Posidonia oceanica (L.) Delile to 15 trace elements.

The Mediterranean seagrass Posidonia oceanica (L.) Delile has been used for trace element (TE) biomonitoring since decades ago. However, present informations for this bioindicator are limited mainly to plant TE levels, while virtually nothing is known about their fluxes through P. oceanica meadows. We therefore contaminated seagrass bed portions in situ at two experimental TE levels with a mix of 15 TEs (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Pb and Bi) to study their uptake and loss kinetics in P. oceanica. Shoots immediately accumulated pollutants from the beginning of exposures. Once contaminations ended, TE concentrations came back to their original levels within two weeks, or at least showed a clear decrease. P. oceanica leaves exhibited different uptake kinetics depending on elements and leaf age: the younger growing leaves forming new tissues incorporated TEs more rapidly than the older senescent leaves. Leaf epiphytes also exhibited a net uptake of most TEs, partly similar to that of P. oceanica shoots. The principal route of TE uptake was through the water column, as no contamination of superficial sediments was observed. However, rhizomes indirectly accumulated many TEs during the overall experiments through leaf to rhizome translocation processes. This study thus experimentally confirmed that P. oceanica shoots are undoubtedly an excellent short-term bioindicator and that long-term accumulations could be recorded in P. oceanica rhizomes.

Establishing research strategies, methodologies and technologies to link genomics and proteomics to seagrass productivity, community metabolism, and ecosystem carbon fluxes.

A complete understanding of the mechanistic basis of marine ecosystem functioning is only possible through integrative and interdisciplinary research. This enables the prediction of change and possibly the mitigation of the consequences of anthropogenic impacts. One major aim of the European Cooperation in Science and Technology (COST) Action ES0609 "Seagrasses productivity. From genes to ecosystem management," is the calibration and synthesis of various methods and the development of innovative techniques and protocols for studying seagrass ecosystems. During 10 days, 20 researchers representing a range of disciplines (molecular biology, physiology, botany, ecology, oceanography, and underwater acoustics) gathered at The Station de Recherches Sous-marines et Océanographiques (STARESO, Corsica) to study together the nearby Posidonia oceanica meadow. STARESO is located in an oligotrophic area classified as "pristine site" where environmental disturbances caused by anthropogenic pressure are exceptionally low. The healthy P. oceanica meadow, which grows in front of the research station, colonizes the sea bottom from the surface to 37 m depth. During the study, genomic and proteomic approaches were integrated with ecophysiological and physical approaches with the aim of understanding changes in seagrass productivity and metabolism at different depths and along daily cycles. In this paper we report details on the approaches utilized and we forecast the potential of the data that will come from this synergistic approach not only for P. oceanica but for seagrasses in general.

Status and origin of Haemonchinae (Nematoda: Trichostrongylidae) in deer: a survey conducted in France from 1985 to 1998.

During our investigations on helminthofauna in roe deer (Capreolus capreolus) and red deer (Cervus elaphus) in France (1985-1998) we isolated nematodes not only of the genus Haemonchus Cobb, 1898 but also of the genus Ashworthius Le Roux 1930, both of which belong to the same subfamily of Haemonchinae. The prevalence of Ashworthius was 22% (65/294) in roe deer and 40% (10/25) in red deer. H. contortus was not found in red deer, whereas its prevalence was only 3% (9/294) in roe deer. These data contrast with those observed in other European countries. The presence of A. sidemi in red and roe deers in France suggests a case of parasitism imported via sika deer. A hypothesis of their spread is proposed.

Molecular systematics of the phlebotomine sandflies of the subgenus Paraphlebotomus (diptera, psychodidae, phlebotomus) based on ITS2 rDNA sequences. Hypotheses Of dispersion and speciation.

Phylogenetic Paraphlebotomus relationships are inferred by a study based on the sequences of ITS2, which has been sequenced in nine Paraphlebotomus species: P. alexandri, P. andrejevi, P. jacusieli, P. kazeruni, P. mireillae, P. mongolensis, P. saevus, P. sergenti and P. similis and in two out-groups species of the subgenus Phlebotomus: P. papatasi and P. duboscqi. Paraphlebotomus alexandri appears as the sister group of all other Paraphlebotomus sandflies. Among the other species, three groupings are clearly highlighted: andrejevi and mongolensis; mireillae and saevus; jacusieli, kazeruni, sergenti and similis. These groupings are related to speculations about the migration of Paraphlebotomus from a centre of dispersion located in the Middle East sometime from the early Eocene to the late Miocene.

Downregulation of interleukin-12 (IL-12) responsiveness in human T cells by transforming growth factor-beta: relationship with IL-12 signaling.

Interleukin-12 (IL-12) is a cytokine that plays a central role in the control of cell-mediated immunity. We have previously shown that transforming growth factor-beta1 (TGF-beta) inhibitory effects on human primary allogeneic cytotoxicity and proliferative responses interfere with IL-12 pathway. The present study was undertaken to further elucidate the biochemical basis of the functional interaction between these two cytokines and to define the site of TGF-beta action on the signaling pathway activated by IL-12. Our data indicate that TGF-beta induced an inhibition of interferon-gamma (IFN-gamma) production without affecting the IL-12Rbeta1 and IL-12Rbeta2 subunits mRNA expression by activated T cells. We further show that TGF-beta has a significant inhibitory effect on the early signal transduction events following interaction of IL-12 with its receptor on activated T cells, resulting in the inhibition of both JAK2 and Tyk2 phosphorylation. In addition, TGF-beta was found to significantly inhibit IL-12-induced phosphorylation of the STAT4 transcription factor. Electrophoretic mobility shift assay indicated that TGF-beta induced a decrease in IL-12-induced STAT4 DNA binding activity in T lymphocytes. This study suggests that TGF-beta influences IL-12 responsiveness at least in part by inhibiting early signaling events essential to gene induction in IL-12-activated T cells.

Erythropoietin-induced erythroid differentiation of the human erythroleukemia cell line TF-1 correlates with impaired STAT5 activation.

The TF-1 cell line has been established from a patient with erythroleukemia. While various cytokines induce TF-1 cell proliferation, erythropoietin (Epo) only sustains the short-term growth of these cells and induces their differentiation along the erythroid lineage. A truncated Epo receptor (EpoR) is overexpressed in these cells. The truncation removed the 96 C-terminal amino acids, including seven tyrosine residues. An additional single mutation at position +3 of Tyr344 led to the replacement of leucine 347 by proline. Stimulation by Epo induced an impaired activation of the STAT5 transcription factor in these cells. The same defect in STAT5 activation was found in the murine FDCP-1 cell line transfected with a chimeric EpoR containing the abnormal TF-1 EpoR cytoplasmic domain. Infection of TF-1 cells with a retrovirus containing a normal murine EpoR was able to restore both Epo-induced STAT5 activity and cellular proliferation. In contrast, Epo-induced differentiation was reduced strongly in infected TF-1ER cells. These results suggest that Epo-induced differentiation correlates with impaired Epo-induced STAT5 activation.

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.