Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils.

DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.

Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: preliminary investigations.

The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO(2) NP). For this purpose, freshwater mussels were exposed to P25 TiO(2) NP at the concentrations of 0.1, 1, 5 and 25mg/L during 24h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO(2) NP at 0.1 and 1mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO(2) NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment.

Ecotoxicological assessment of TiO2 byproducts on the earthworm Eisenia fetida.

The increasing production of nanomaterials will in turn increase the release of nanosized byproducts to the environment. The aim of this study was to evaluate the behaviour, uptake and ecotoxicity of TiO(2) byproducts in the earthworm Eisenia fetida. Worms were exposed to suspensions containing 0.1, 1 and 10 mg/L of byproducts for 24 h. Size of TiO(2) byproducts showed aggregation of particles up to 700 µm with laser diffraction. Only worms exposed at 10 mg/L showed bioaccumulation of titanium (ICP-AES), increasing expression of metallothionein and superoxide dismutase mRNA (Real-time PCR) and induction of apoptotic activity (Apostain and TUNEL). TiO(2) byproducts did not induce cytotoxicity on cœlomocytes, but a significant decrease of phagocytosis was observed starting from 0.1 mg/L. In conclusion, bioaccumulation of byproducts and their production of reactive oxygen species could be responsible for the alteration of the antioxidant system in worms.

Contribution of Miscanthus x giganteus root exudates to the biostimulation of PAH degradation: an in vitro study.

Phytoremediation is considered as a promising and cost-effective method to enhance bioremediation of polluted soils. Exudation of plant root secondary metabolites similar to organic pollutants may induce the expression of microbial degradative enzymes and favour cometabolism of xenobiotics. We investigated the contribution of Miscanthus x giganteus root exudates in the biostimulation of PAH-degradation. This perennial grass was chosen because of its capability to grow on polluted soils and its high biomass production for non-food purposes. First, the impact of cometabolism phenomena was evaluated on the selective enrichment of pyrene-degrading bacterial consortia. The identification of each isolated strains following incubation with pyrene only, "pyrene+phenanthrene", "pyrene+salycilate" or "pyrene+diesel fuel" showed a varying bacterial diversity and pyrene-degrading ability, depending on the co-substrate used. Then, a microplate assay was designed, based on the simultaneous measurement of bacterial consortia growth and degradation activity, in the presence of PAH and total root exudates. Results showed that i) the addition of root exudates was efficient for promoting bacterial growth, ii) but a selective enrichment of PAH-degraders compared to aliphatic ones could be clearly demonstrated, thereby conducing to an enhanced PAH catabolism. The identification of plant secondary metabolites showed the presence of a broad range of flavonoid-derived compounds that could play a role in cometabolic processes. Microplate assays with the two major molecules, quercetin and rutin, suggested a partial involvement of these compounds in biostimulation processes. Further investigations with the other identified secondary metabolites (apigenin, isovitexin, catechin, gallic and caffeic acid) should provide more information on the exudate-PAH cometabolic degradation phenomenon.

The impact of high anxiety level on cellular and humoral immunity in mice.

BACKGROUND/AIMS: In the present study, we aimed to examine whether a high anxiety level affects various parameters of immunity in mice. METHODS: We used the behavioral light/dark choice test to evaluate whether high anxiety has an impact on various parameters of cellular (granulocytes, monocytes, total lymphocytes, TCD4(+), TCD8(+) and NK numbers) and humoral (IgA, E and G concentrations) immunity. Secondly, we investigated whether the cellular and humoral immune systems of mice with contrasting levels of anxiety responded differently to stressors (such as physical restraint) by monitoring blood markers of the both types of immunity. RESULTS: High levels of anxiety inhibited part of the cellular and humoral immune systems by significantly decreasing total lymphocytes numbers (including TCD4(+) and TCD8(+)) and immunoglobulin (A and E) concentrations. However, no significant changes in the number of granulocytes, monocytes or NK cells were observed. As a consequence, overall, our results suggest that high anxiety led to a decrease in the efficiency of the immune system of anxious mice. On the other hand, our findings also showed that restraint stress (acute and subacute) produced the same immunological profile as high anxiety in mice. This was independent of the animals' anxiety status. At the same time, we observed that restraint stress produced significant increases in the levels of granulocytes and monocytes. CONCLUSIONS: High anxiety and restraint stress exerted adverse effects on cellular and humoral immunity in mice. While the effect of restraint stress was independent of the anxiety levels in mice, this stress led to an aggravation of the immune response from the high degree of anxiety. Therefore, anxious subjects could be more vulnerable to infections and inflammation, particularly when they are exposed to stressful situations.

Influences of ozone exposure upon macrophage responsivity to N-formyl-methionyl-leucyl-phenylalanine: mobility and metabolic changes.

Alveolar macrophages represent one of the first lines of cell defence in the lungs. They employ several mechanisms, including phagocytosis and secretion of reactive oxygen and nitrogen species. fMLP, a formylated peptide of bacterial origin, is a potent inducer of phagocyte chemotaxis and is also involved in generating antimicrobial agents such as nitric oxide (NO) and hydrogen peroxide (H2O2). In this study we analysed the in vitro effects of fMLP on the mobility of the THP-1 cell line, which served as a model for alveolar macrophages. Cell mobility and cytotoxicity were also analysed after pre-exposures to an atmosphere polluted with ozone (0.03-0.5 ppm) followed by a fMLP treatment. Finally, the secreted molecules (H2O2 and NO) were measured after ozone exposures ranging from 5 to 30 min and fMLP action. Activation by fMLP alone induced cell movement, whereas pre-exposure to the ozone concentrations decreased it. Addition of fMLP had different effects on cytotoxicity, mobility and metabolite secretion by the cells: (1) cytotoxicity increased depending on ozone concentrations and exposure times; (2) during the first 5 min and for all ozone concentrations, an average decrease of 50% of activated cell mobility was observed; (3) H2O2 was increased, even in combination with ozone; (4) NO was detected at 731 nM, a result that was not affected by ozone pre-exposure.

Precipitation of silver-thiosulfate complex and immobilization of silver by Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pMOL28 and pMOL30) that confer resistance to eleven metals. The ability of Cupriavidus metallidurans CH34 to resist silver is described here. Electronic microscopy, energy-dispersive X-ray (EDX) and X-ray diffractometry (DRX) observations revealed that C. metallidurans CH34 strongly associated silver with the outer membrane, under chloride chemical form. Using derivate strains of C. metallidurans CH34, which carried only one or no megaplasmid, we show that this resistance seems to be carried by pMOL30.

One-step, non-denaturing purification method of carp (Cyprinus carpio) vitellogenin.

A new single-step purification procedure was developed to purify carp (Cyprinus carpio) vitellogenin (VTG), from estradiol-treated carp plasma. This method was performed by high performance liquid weak anion-exchange chromatography, using a discontinuous elution gradient of NaCl (0-0.5 M, steps of 12.5 mM/4 min). SDS and native-PAGE analysis, of treated-fish plasma and purified solution, showed the appearance of a 370 kDa phospholipoprotein, composed of two 130 kDa monomers, with all VTG characteristics. The sequencing of a 130 kDa monomer confirmed that it was carp VTG. Consequently, this procedure is a rapid method, permitting high quantities of non-denatured carp VTG to be obtained.