Benchmarking of protein carbonylation analysis in Caenorhabditis elegans: specific considerations and general advice.

Oxidative stress has been extensively studied due to its correlation with cellular disorders and aging. In proteins, one biomarker of oxidative stress is the presence of carbonyl groups, such as aldehyde and ketone, in specific amino acid side chains such as lysine, proline, arginine and threonine, so-called protein carbonylation (PC). PC study is now a growing field in general and medical science since PC accumulation is associated with various pathologies and disorders. At present, enzyme-linked immunosorbent assays (ELISA) seem to be the most robust method of quantifying the presence of carbonyl groups in proteins, despite having some recognised caveats. In parallel, gel-based approaches present cross-comparison difficulties, along with other technical problems. As generic PC analyses still suffer from poor homogeneity, leading to cross-data analysis difficulties and poor results overlap, the need for harmonisation in the field of carbonyl detection is now widely accepted. This study aims to highlight some of the technical challenges in proteomic gel-based multiplexing experiments when dealing with PC in difficult samples like those from Caenorhabditis elegans, from protein extraction to carbonyl detection. We demonstrate that some critical technical parameters, such as labelling time, probe concentration, and total and carbonylated protein recovery rates, should be re-addressed in a sample-specific way. We also defined a procedure to cost-effectively adapt CyDye™-hydrazide-based protocols to specific samples, especially when the experimental interest is focused on studying differences between stimulating conditions with a maximised signal-to-noise ratio. Moreover, we have improved an already-existing powerful solubilisation buffer, making it potentially useful for hard-to-solubilise protein pellets. Lastly, the depicted methodology exemplifies a simple way of normalising carbonyl-related signal to total protein in SDS-PAGE multiplexing experiments. Within that scope, we also proposed a simple way to quantify carbonyl groups by on-gel spotting diluted dye-containing labelling buffer. Proof of the robustness of the procedure was also highlighted by the high linear correlation between the level of carbonyls and the ultraviolet exposure duration of whole worms (R2=0.993). Altogether, these results will help to standardise existing protocols in the growing field of proteomic carbonylation studies.

Soil nematode assemblages as bioindicators of radiation impact in the Chernobyl Exclusion Zone.

In radioecology, the need to understand the long-term ecological effects of radioactive contamination has been emphasised. This requires that the health of field populations is evaluated and linked to an accurate estimate of received radiological dose. The aim of the present study was to assess the effects of current radioactive contamination on nematode assemblages at sites affected by the fallout from the Chernobyl accident. First, we estimated the total dose rates (TDRs) absorbed by nematodes, from measured current soil activity concentrations, Dose Conversion Coefficients (DCCs, calculated using EDEN software) and soil-to-biota concentration ratios (from the ERICA tool database). The impact of current TDRs on nematode assemblages was then evaluated. Nematodes were collected in spring 2011 from 18 forest sites in the Chernobyl Exclusion Zone (CEZ) with external gamma dose rates, measured using radiophotoluminescent dosimeters, varying from 0.2 to 22 µGy h(-1). These values were one order of magnitude below the TDRs. A majority of bacterial-, plant-, and fungal-feeding nematodes and very few of the disturbance sensitive families were identified. No statistically significant association was observed between TDR values and nematode total abundance or the Shannon diversity index (H'). The Nematode Channel Ratio (which defines the relative abundance of bacterial- versus fungal-feeding nematodes) decreased significantly with increasing TDR, suggesting that radioactive contamination may influence nematode assemblages either directly or indirectly by modifying their food resources. A greater Maturity Index (MI), usually characterising better soil quality, was associated with higher pH and TDR values. These results suggest that in the CEZ, nematode assemblages from the forest sites were slightly impacted by chronic exposure at a predicted TDR of 200 µGy h(-1). This may be imputable to a dominant proportion of pollutant resistant nematodes in all sites. This might result from a selection at the expense of sensitive species after the accident.

An alternative method of particulate fluorescent tracer analysis in sediments using a microplate fluorimeter.

Conservative particulate fluorescent tracers (e.g. luminophores and microspheres) are commonly used in a wide range of sediment transport studies. Traditionally, their spatial redistribution is estimated by counting them in sediments under ultraviolet light (e.g. by epifluorescence microscopy), a time-consuming but effective method. While alternative methods have recently been developed (e.g. photodetection, digital image analyses), this 'classical' counting method remains the most commonly used. This article describes an alternative procedure for measuring the concentration of fluorescent tracers (here, microspheres) using a microplate fluorimeter. This technique enables simultaneous analysis of numerous samples while reducing the sediment preparation and quantification time. After a calibration step from sediment samples with known microsphere content, the method was validated by comparing results from the epifluorescence microscopy method. Different adjustments were also reported, as well as application examples. The different calibration tests showed high linear relationships between the microsphere concentration of sediment samples and the measured fluorimetric intensities (R2-0.99) with a detection limit of 6%. In comparison with the previously used method, very similar results were obtained, as illustrated in recent studies using both luminophores and microspheres. The rapid and reliable method proposed here will enable increasingly complex experiments to be performed with less time-consuming qualitative analyses.

Effects of uranium-contaminated sediments on the bioturbation activity of Chironomus riparius larvae (Insecta, Diptera) and Tubifex tubifex worms (Annelida, Tubificidae).

Freshwater sediments represent a compartment for accumulation of toxic substances, notably of metallic pollutants such as uranium. However, they also constitute a privileged habitat for many benthic macro-invertebrate species with important roles in the functioning of these ecosystems, particularly through their bioturbation activities. Uranium accumulation in sediments can thus have harmful effects on these organisms (e.g., developmental delay, malformations, mortality). The present study aimed to evaluate the consequences of these effects on the bioturbation activity of Chironomus riparius larvae and Tubifex tubifex worms. These two species, which are widespread in freshwater ecosystems, are characteristic of two different modes of bioturbation: bioirrigation and upward bioconveying, respectively. By quantifying the burial and redistribution of fluorescent particulate tracers (microspheres), sediment reworking induced by these macro-invertebrates was measured after 12d of exposure. Biodiffusion D(b) and bioadvection W rates, as well as several other parameters, were estimated to assess and compare the bioturbation activity of the two species, separately and in combination, between uncontaminated and uranium-spiked sediments. The results reveal that C. riparius larvae were more sensitive to uranium, but their bioturbation activity, even under uncontaminated conditions, had little effect on sediment reworking. Particle mixing was mainly induced by T. tubifex worms, which were only affected by uranium at high concentrations in the sediment. Finally, bioturbation by T. tubifex led to a high degree of uranium release from sediment to the overlying water, which highlights the crucial role of this mostly dominant species on uranium biogeochemical cycles at concentrations existing in naturally contaminated sites.

Influence of Chironomus riparius (Diptera, Chironomidae) and Tubifex tubifex (Annelida, Oligochaeta) on oxygen uptake by sediments. Consequences of uranium contamination.

The diffusive oxygen uptake (DOU) of sediments inhabited by Chironomus riparius and Tubifex tubifex was investigated using a planar oxygen optode device, and complemented by measurements of bioturbation activity. Additional experiments were performed within contaminated sediments to assess the impact of uranium on these processes. After 72h, the two invertebrate species significantly increased the DOU of sediments (13-14%), and no temporal variation occurred afterwards. Within contaminated sediments, it was already 24% higher before the introduction of the organisms, suggesting that uranium modified the sediment biogeochemistry. Although the two species firstly reacted by avoidance of contaminated sediment, they finally colonized it. Their bioturbation activity was reduced but, for T. tubifex, it remained sufficient to induce a release of uranium to the water column and an increase of the DOU (53%). These results highlight the necessity of further investigations to take into account the interactions between bioturbation, microbial metabolism and pollutants.