Using single-species and algal communities to determine long-term adverse effects of silver nanoparticles on freshwater phytoplankton.

The physical and chemical properties of silver nanoparticles (AgNPs) have led to their increasing use in various fields such as medicine, food, and industry. Evidence has proven that AgNPs cause adverse effects in aquatic ecosystems, especially when the release of Ag is prolonged in time. Several studies have shown short-term adverse effects of AgNPs on freshwater phytoplankton, but few studies have analysed the impact of long-term exposures on these populations. Our studies were carried out to assess the effects of AgNPs on growth rate, photosynthesis activity, and reactive oxygen species (ROS) generation on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa, and additionally on microcystin (MC-LR) generation from these cyanobacteria. The tests were conducted both in single-species cultures and in phytoplanktonic communities exposed to 1 ngL-1 AgNPs for 28 days. The results showed that cell growth rate of both single-species cultures decreased significantly at the beginning and progressively reached control-like values at 28 days post-exposure. This effect was similar for the community-cultured cyanobacteria, but not for the green algae, which maintained a sustained decrease in growth rate. While gross photosynthesis (Pg) increased in both strains exposed in single cultures, dark respiration (R) and net photosynthesis (Pn) decreased in S. armatus and M. aeruginosa, respectively. These effects were mitigated when both strains were exposed under community culture conditions. Similarly, the ROS generation shown by both strains exposed in single-species cultures was mitigated when exposure occurred in community cultures. MC-LR production and release were significantly decreased in both single-species and community exposures. These results can supply helpful information to further investigate the potential risks of AgNPs and ultimately help policymakers make better-informed decisions about their utilization for environmental restoration.

Adverse effects of iron-based nanoparticles on freshwater phytoplankton Scenedesmus armatus and Microcystis aeruginosa strains.

Zero-valent nano-iron particles (nZVI) are increasingly present in freshwater aquatic environments due to their numerous applications in environmental remediation. However, despite the broad benefits associated with the use and development of nZVI nanoparticles, the potential risks of introducing them into the aquatic environment need to be considered. Special attention should be focused on primary producer organisms, the basal trophic level, whose impact affects the rest of the food web. Although there are numerous acute studies on the acute effects of these nanoparticles on photosynthetic primary producers, few studies focus on long-term exposures. The present study aimed at assessing the effects of nZVI on growth rate, photosynthesis activity, and reactive oxygen activity (ROS) on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa. Moreover, microcystin production was also evaluated. These parameters were assessed on both organisms singly exposed to 72 h-effective nZVI concentration for 10% maximal response for 28 days. The results showed that the cell growth rate of S. armatus was initially significantly altered and progressively reached control-like values at 28 days post-exposure, while M. aeruginosa did not show any significant difference concerning control values at any time. In both strains dark respiration (R) increased, unlike net photosynthesis (Pn), while gross photosynthesis (Pg) only slightly increased at 7 days of exposure and then became equal to control values at 28 days of exposure. The nZVI nanoparticles generated ROS progressively during the 28 days of exposure in both strains, although their formation was significantly higher on green algae than on cyanobacteria. These data can provide additional information to further investigate the potential risks of nZVI and ultimately help decision-makers make better informed decisions regarding the use of nZVI for environmental remediation.

Direct Tunneling Delay Time Measurement in an Optical Lattice.

We report on the measurement of the time required for a wave packet to tunnel through the potential barriers of an optical lattice. The experiment is carried out by loading adiabatically a Bose-Einstein condensate into a 1D optical lattice. A sudden displacement of the lattice by a few tens of nanometers excites the micromotion of the dipole mode. We then directly observe in momentum space the splitting of the wave packet at the turning points and measure the delay between the reflected and the tunneled packets for various initial displacements. Using this atomic beam splitter twice, we realize a chain of coherent micron-size Mach-Zehnder interferometers at the exit of which we get essentially a wave packet with a negative momentum, a result opposite to the prediction of classical physics.

Morphological and physiological changes exhibited by a Cd-resistant Dictyosphaerium chlorelloides strain and its cadmium removal capacity.

Changes induced on freshwater microalga Dictyosphaerium chlorelloides (Dc(wt)) acclimated in the laboratory until their survival in culture media enriched with cadmium 100 µM have been studied. Cadmium removal by living cells of this Cd-resistant (Dc(CdR100)) strain was tested in cultures exposed to 100 µM Cd during 30 days. Cell dimensions were measured under light microscopy, and cell growth was studied. Photosynthetic yield (ΦPSII) was analyzed and the photosynthetic oxygen development and respiration response was obtained. Results show that Dc(CdR100) strain exhibited significant cell morphology changes in comparison to Dc(wt) cells, which affected both surface area and cell biovolume. Malthusian fitness analysis showed that Dc(CdR100) strain living in Cd-enriched culture had developed a lower capacity of nearly 50% growth, and its photosynthetic oxygen development and respiration response were significantly reduced in both light and dark photosynthetic phases. Dc(CdR100) strain showed a very high capacity to remove cadmium from the aquatic environment (over 90%), although most of the removed heavy metal (≈70%) is adhered to the cell wall. These specific characteristics of Dc(CdR100) cells suggest the possibility of using this strain in conjunction with Dc(wt) strain as bioelements into a dual-head biosensor, and in bioremediation processes on freshwater polluted with Cd.

Interference of heavy metals on the photosynthetic response from a Cr(VI)-resistant Dictyosphaerium chlorelloides strain.

The successful selection of a particular type of bioelement and its association to the appropriate transducer determines the specificity of a biosensor. Therefore, from a strain of chloroficea Dictyosphaerium chlorelloides, modified in laboratory to tolerate high Cr(VI) concentrations, the possible interferences of other heavy metals on photosynthetic activity were studied. After exposing wild type and Cr(VI)-resistant cells to increasing Ag(+1), Co(+2), Hg(+2), Cr(+3), Cu(+2), Zn(+2), Fe(+3) and Cd(+2) concentrations, both photosynthetic quantum yields was compared. Photosynthetic electron transport rates were measured with a TOXY-PAM chlorophyll fluorometer, non-linear regression analysis of each of the toxicity tests was done, and means of both groups were compared using unpaired t test. The results show no significant differences between both cell types when they were exposed to Ag(+1), Co(+2), Hg(+2), Cr(+3), Cu(+2), Fe(+3) and Cd(+2) metal ions, and extremely significant differences (p < 0.0001) to Zn(+2) exposures. These results demonstrate the suitability of this Cr(VI)-resistant type D. chlorelloides strain as a suitable bioelement to be coupled to a biosensor based on dual-head microalgae strategy to detect and quantify Cr(VI) in water courses and waste water treatment plants. However, some disturbance may be expected, especially when certain analyte species such as zinc are present in water samples tested. The analysis of binary mixtures between Zn(+2) and other heavy metals showed a slight antagonistic phenomenon in all cases, which should not alter the potential Zn(+2) interference in the Cr(+6) detection process.

A full GMP process to select and amplify epitope-specific T lymphocytes for adoptive immunotherapy of metastatic melanoma.

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

A simple competitive assay to determine peptide affinity for HLA class II molecules: a useful tool for epitope prediction.

We have designed a cytometry-based competition assay to evaluate peptide binding to empty recombinant HLA class II molecules. The efficiency of this assay was evaluated using recombinant HLA-DP0401 molecules (HLA-DP) produced in insect cells and 13 peptides from human telomerase reverse transcriptase (hTERT). We demonstrate that our method allowed accurate measurements of peptide Ki values and can thus discriminate strong, moderate and poor HLA-DP binders. In parallel, we showed that among hTERT peptides, the most immunodominant in healthy individuals were those with moderate affinity for HLA-DP while no T cell response could be evidenced against peptides with very strong or very low affinities for HLA-DP. This strongly suggests that the precise determination of peptide affinity with our method can improve HLA class II epitope prediction.

4-Hydroxybenzoate uptake in Klebsiella planticola strain DSZ1 is driven by DeltapH.

Klebsiella planticola strain DSZ1 has the ability to degrade different aromatic compounds such as benzoate and organochlorinated as propachlor and alachlor. DSZ1 strain cells mineralised 4-hydroxybenzoate (4HBA) through a meta-cleavage pathway, yielding protocatechuate as dihydroxylated intermediate, with a specific rate of CO2 formation 0.12 x 10-6 (cpm/OD) h-1, and a rate of 4-HBA utilisation of 0.75 mmol h-1. Aerobically the 4HBA transport system is driven by gradient of protons (DeltapH), but is not ATP-driven. Under anaerobic conditions, the system can use the nitrate reduction as a final electron acceptor in respiration. A kinetic analysis of the 4HBA transport system revealed a Kt value of 16 microM with a Vmax value of 25 nmol/min.mg at pH 7.

Monocytes influence the fate of T cells challenged with oxidised low density lipoproteins towards apoptosis or MHC-restricted proliferation.

Atherosclerosis has been implicated in myocardial infarction, stroke and a host of cardiovascular diseases. The presence of activated T lymphocytes and macrophages, and the increased expression of HLA-DR antigen are consistent with the notion of immune activity in the atherosclerotic plaque. The nature of the causative antigen has not been established although oxidised low density lipoproteins (oxLDL) that accumulate in atherosclerotic plaques could fulfil this role. Here, we report that monocytes play a key role in influencing the fate of purified peripheral human T lymphocytes from healthy donors when the cells are exposed to LDL oxidised under the controlled conditions of water radiolysis. Our data showed that oxLDL generated under these conditions were chemoattractants for T cells. However, they induced a state of apoptosis in T lymphocytes cultured in the absence of monocytes. The extent of apoptosis was related to the degree of oxidation of LDL and the time of T cell exposure to oxLDL. OxLDL-dependent apoptosis did not involve a scavenger-like receptor. CD4(+) cells were more sensitive to the apoptotic effect of oxLDL than CD8(+) cells. OxLDL-primed (12 h) autologous monocytes triggered a robust proliferation of T lymphocytes cultured in the absence of oxLDL. The strength of T cell stimulation was related to the degree of oxidation of the LDL used in priming. Heterologous monocytes exposed to oxLDL under similar conditions induced a response that was not different than monocytes exposed to untreated LDL (natLDL) which did not induce T cell proliferation. Fucoidan did not modify the oxLDL-, monocyte-dependent T cell response to proliferation, suggesting that a scavenger-like receptor was not involved. The expression of the HLA-DR marker and the B7.2 protein were up-regulated in monocytes exposed to oxLDL but not to natLDL. The levels of B7.1 were unchanged. Our data are consistent with the notion that monocytes are critical for T cell survival in the presence of oxLDL and MHC-restricted T cell proliferative response to oxLDL.

4-Hydroxybenzoate uptake in an isolated soil Acinetobacter sp.

The isolated soil bacteria Acinetobacter strain BEM2 is able to utilize some xenobiotic aromatic compounds as a carbon source. In this study the metabolism of 4-hydroxybenzoate (4-HBA) by strain BEM2 was characterized. Degradation involved a meta-cleavage pathway yielding 3,4-dihydroxybenzoate (3,4-DHBA) as an intermediate and CO(2) as the principal product from the C atoms in the aromatic ring. 4-HBA uptake was studied, and the kinetic parameters were determined. The uptake was shown to be directly coupled to ATP hydrolysis and its synthesis, according to the Mitchell chemiosmotic hypothesis.

Novel 21-aminosteroid U-74389G inhibits low-density lipoprotein peroxidation induced by .OH and O2-. free radicals.

LDL peroxidation represents one of the first event in the atherogenesis process. Inhibiting LDL oxidation may impede this process and offers a new mechanism to retard atherogenesis. 21-Aminosteroids, derived from methylprednisolone, have recently excited much interest by virtue of their ability to inhibit lipid peroxidation. The aim of our work was to investigate the effect of a novel 21-aminosteroid, U-74389G, in the LDL peroxidation initiated in a metal- and cell-free system by oxygen free radicals, .OH and O2-., generated by water gamma-radiolysis. In a concentration dependent manner, U-74389G increased the resistance of LDL to oxidation measured by the length of the lag phase, reduced the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), and also reduced the alpha-tocopherol disappearance by about 47% at the concentration 20 microM. U-74389G was also able to reduce the chemotactic activity of oxidized LDL towards monocytes, as well as the cholesterol accumulation in macrophages. These observations suggest that the U-74389G is a potent antioxidant by decreasing LDL peroxidation and this should be evaluated in in vivo models as a potential therapy to retard atherogenesis.