Claire Sergeant

Microbial Community Analysis of Opalinus Clay Drill Core Samples from the Mont Terri Underground Research Laboratory, Switzerland

Opalinus Clay is a candidate host rock for a high-level radioactive waste repository in Switzerland. Microbial metabolism and its by-products could affect the physical and (geo)chemical conditions in such a repository. This study investigated the occurrence of indigenous microbes, their community size and-structure in an Opalinus Clay core from the Mont Terri Underground Research Laboratory, Switzerland, drilled with aseptic techniques. Core sub-samples were distributed to five laboratories, where they were analysed with microscopy, culture-and molecular biology techniques. Evidence supporting a viable microbial community in Opalinus Clay included: five positive culture results (including for sulphate-reducing bacteria, SRB) out of 20 culture attempts; extraction of 64 ng phospholipid fatty acids (PLFA) per g clay, suggesting the presence of viable cells; detection of PLFA biomarkers for anaerobic Gram-negative bacteria and SRB; and the presence of sufficient nutrients to support growth of indigenous and non-indigenous microorganisms for two months. Evidence against a thriving microbial community in Opalinus Clay included: 15 negative culture results out of 20 attempts; lack of cells by application of microscope techniques (phase contrast, AODC, CARD-FISH); consistent failure to extract PCR-amplifiable DNA from the core; the presence of 14 times higher amounts of lipids indicative of cell debris than those indicative of viable cells (PFLA); very small pore sizes; and very low water content. The combined results of this study (and evidence from other studies in comparable environments) suggest that unperturbed Opalinus Clay appears to contain only a small viable microbial community, which is probably metabolically almost inactive (dormant), due to space and water restrictions. However, any disturbances that would provide space, water and nutrients, as would be the case during repository excavation and construction, could revive the dormant organisms, unless the host rock was kept intact.

Fatty acids and plasma antioxidants in HIV-positive patients: Correlation with nutritional and immunological status

OBJECTIVE To investigate red blood cell (RBC) and plasma fatty acids (FA) in HIV-positive patients in relation to oxidative stress and nutritional or immunological status. DESIGN AND METHODS FA, plasma selenium, vitamins A and E were measured in 95 patients divided into four groups according to CD4 cells. RESULTS Poly- and di-unsaturated FA (PUFA, DUFA) decreased and saturated FA (SFA) increased in RBC in the patients below 400/mm3 and in plasma in the patients below 50/mm3. RBC SFA correlated to CD4 cells, PUFA to MDA. Unlike vitamin E, plasma vitamin A and selenium decreased in most groups. Plasma SFA and MUFA correlated negatively to selenium and PUFA and DUFA to vitamin E. No correlation was found between PUFA and nutritional markers. CONCLUSION FA seem to be modified during HIV infection by oxidative stress and disease evolution, but not by denutrition.

Microbial Investigations in Opalinus Clay, an Argillaceous Formation under Evaluation as a Potential Host Rock for a Radioactive Waste Repository

Various deep, compact, sedimentary formations have been studied in recent years as potential host rock for a repository for high-level, long-lived radioactive waste. Considering that microbial activities may influence radionuclide chemistry and migration in such environments, we investigated the potential presence of microorganisms in the Opalinus Clay formation, from unperturbed sediment samples (i.e., not affected by gallery excavation and borehole drilling) recovered under aseptic conditions in the Mont Terri Underground Rock Laboratory (Switzerland). A combination of molecular biology techniques and a cultivation-based approach suggested the presence of a few sparse autochthonous microbial cells in the Opalinus Clay. For the first time, ribosomal RNA (rRNA) genes were sequenced from enrichment cultures from such samples. The results suggested that at least two of the bacterial strains isolated were likely unknown species of the Sphingomonas and Alicyclobacillus genera, as their fully-sequenced 16S-rRNA genes shared less than 97% similarity with validly published sequences. Early genetic divergence occurring after physical isolation of bacterial ancestors in the geosphere by the sedimentation process or following later geological events may have resulted in the generation of particular taxa in the subsurface.

Use of combined microscopic and spectroscopic techniques to reveal interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions.

Iron in hereditary retinal degeneration: PIXE microanalysis: Preliminary results

Abstract Several types of hereditary retinal degeneration with progressive alteration of photoreceptors exist in men and animals. Recent immunohistochemical results have shown strong degradation of transferrin, the protein responsible for iron transport, in retinas of rats with hereditary retinal degeneration. Freeze-dried thin sections of rat retinas from different stages of the disease, and respective coeval control sections, have been analyzed using nuclear microprobe. In this first part of the study, the rat retinas at post-natal stages of 35 and 45 days have been analyzed. The sample preparation and the post-irradiation staining to determine precisely the retinal layers involved are described. Preliminary results of element distributions (K, Ca, Fe) in the rat retina layers are discussed. A very high content of calcium in the choriocapillaris of dystrophic rat retinas was observed. Preliminary results on iron distribution in the rat retina layers are presented.

Characterization of technetium(vII) reduction by cell suspensions of thermophilic bacteria and archaea

Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [99Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota.

Iron and other elements (Cu, Zn, Ca) contents in retina of rats during development and hereditary retinal degeneration

Abstract The retina as well as other tissues needs iron to survive, but modifications in iron metabolism have also been suggested to contribute to cerebral neurodegenerative diseases. Our study was intended to investigate iron distribution in the retina of normal rats and Royal College of Surgeons (RCS) rats affected by hereditary degeneration of the photoreceptors at different developmental stages (35, 45 and 55 days after birth). Iron (Fe) distribution was determined by proton induced X-ray emission (PIXE) microanalysis on retinal sections and compared to other tissues (cornea, liver, spleen) and to other elements (Cu, Zn, Ca). Elemental concentrations were determined in different retinal layers especially the photoreceptors, which are progressively altered and disappear in the RCS rats. Iron is unevenly distributed throughout the rat retina. The highest concentration is observed in the choroid and the retinal pigmented epithelium and in the inner segments of photoreceptors. Iron content is lower in the outer segments but still significant. It increases during both the development and the disease at the level of the segments. This last localised iron increase can result in an overproduction of free radicals and be correlated with the photoreceptor cell loss. The distributions of other elements (Cu, Zn, Ca) revealed interesting temporal progressions.

N-myc oncogene amplification is correlated to trace metal concentrations in neuroblastoma cultured cells

Abstract N- myc oncogene amplification is a powerful predictor of aggressive behavior of neuroblastoma (NB), the most common solid tumor of the early childhood. Since N- myc overexpression – subsequent to amplification – determines a phenotype of invasiveness and metastatic spreading, it is assumed that N- myc amplified neuroblasts synthesize zinc metalloenzymes leading to tumor invasion and formation of metastases. In order to test a possible relation between N- myc oncogene amplification and trace metal contents in human NB cells, Fe, Cu and Zn concentrations have been measured by nuclear microprobe analysis in three human neuroblastoma cell lines with various degrees of N- myc amplification. Elemental determinations show uniform distribution of trace metals within the cells, but variations of intracellular trace metal concentrations with respect to the degree of N- myc amplification are highly dependent on the nature of the element. Zinc concentration is higher in both N- myc amplified cell lines (IMR-32 and IGR-N-91) than in the non-amplified cells (SK–N–SH). In contrast, intracellular iron content is particularly low in N- myc amplified cell lines. Moreover, copper concentrations showed an increase with the degree of N- myc amplification. These results indicate that a relationship exists between intracellular trace metals and N- myc oncogene amplification. They further suggest that trace metals very probably play a determinant role in mechanisms of the neuroblastoma invasiveness.

Acides gras membranaires et anti-oxydants sériques chez 77 patients infectés par le VIH

We have measured the fatty acid (FA) composition of erythrocyte membranes and plasma anti-oxidants in HIV+ patients. Saturated FA are higher and poly-unsaturated FA lower than in controls (P = 0.02). Selenium (Se) is lower in patients less than 400 CD4 cells/mm3 (P = 0.002). Vitamin A is lower in the HIV+ regardless of the CD4 cell count. Se and vitamin A are correlated to nutritional markers (body mass index and albumin).

Trace metals and cancer: The case of neuroblastoma

Abstract N-myc oncogene amplification is one of the most established prognostic factors in neuroblastoma (NB), a young children solid tumor. Amounts of ferritin, an iron storage protein, are abnormally increased in serum of patients with advanced stage disease. N-myc amplified NB cells can synthesize zinc metalloenzymes allowing tumor invasion and metastases formation. The aim of this study was to find a relationship between N-myc amplification and trace metals in human neuroblasts. Coupling PIXE and RBS techniques, nuclear microprobe allowed to analyze elemental distributions and to determine trace metal concentrations within cultured neuroblasts characterized by various degrees of N-myc amplification. They were compared to trace metal distributions and concentrations in tumor xenograft models of human NB, after injection of cells from the same lines in athymic nude mice. Our data allowed to establish a relation between trace metal contents and mechanisms of NB oncogenesis, amplified cell lines representing more aggressive phenotypes of the disease. They should be confirmed by analysis of cultured neuroblasts and tumors issued from a non-amplified cell line transfected with the N-myc oncogene.