Identification of the aquaporin gene family in Cannabis sativa and evidence for the accumulation of silicon in its tissues.

Cannabis sativa is an economically important crop providing bast fibres for the textile and biocomposite sector. Length is a fundamental characteristic determining the properties of bast fibres. Aquaporins, channel-forming proteins facilitating the passage of water, urea, as well as elements such as boron and silicon, are known to play a role in the control of fibre length in other species, like cotton. By mining the available genome, we here identify, for the first time, the aquaporin gene family of C. sativa. The analysis of published RNA-Seq data and targeted qPCR on a textile variety reveal an organ-specific expression of aquaporin genes. Computational analyses, including homology-based search, phylogeny and protein modelling, identify two NOD26-like intrinsic proteins harbouring the Gly-Ser-Gly-Arg (GSGR) aromatic/Arg selectivity filter and 108 amino acid NPA (Asn-Pro-Ala) spacing, features reported to be associated with silicon permeability. SIMS nano-analysis and silica extraction coupled to fluorescence microscopy performed on hemp plantlets reveal the presence of silicon in the bast fibres of the hypocotyl and in leaves. The accumulation of silica in the distal cell walls of bast fibres and in the basal cells of leaf trichomes is indicative of a mechanical role.

Do the pristine physico-chemical properties of silver and gold nanoparticles influence uptake and molecular effects on Gammarus fossarum (Crustacea Amphipoda)?

The specific and unique properties of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs), make them of high interest for different scientific and industrial applications. Their increasing use will inevitably lead to their release in the environment and aquatic ecosystems where they may represent a threat to aquatic organisms. Being a widespread and important component of the aquatic macroinvertebrate assemblage, amphipods and more specifically Gammarus fossarum will certainly be exposed to AgNPs and AuNPs. For these reasons, G. fossarum was selected as model organism for this study. The aim of the present work was the evaluation of the influence of both size (20, 40 and 80 nm) and surface coating (citrate CIT, polyethylene glycol PEG) on the acute toxicity of AgNPs and AuNPs on G. fossarum. We investigated the effects of AgNPs and AuNPs on the uptake by G. fossarum, NP tissue distribution and the expression of stress related genes by the use of ICP-MS, NanoSIMS50, Cytoviva®, and Rt-qPCR, respectively. Ag and Au bioaccumulation revealed a significant surface-coating dependence, with CIT-AgNPs and CIT-AuNPs showing the higher bio-accumulation potential in G. fossarum as compared to PEG-NPs. Opposite to that, no size-dependent effects on the bioaccumulation potential was observed. SIMS imaging and CytoViva® revealed an influence of the type of metal on the tissue distribution after uptake, with AgNPs detected in the cuticle and the gills of G. fossarum, while AuNPs were detected in the gut area. Furthermore, AgNPs were found to up-regulate CuZnSOD gene expression while AuNPs led to its down-regulation. Modulation of SOD may indicate generation of reactive species of oxygen and a possible activation of antioxidant defence in order to prevent and defend the organism from oxidative stress. However, further investigations are still needed to better define the mechanisms underlying the observed AgNPs and AuNPs effects.

Division-Based, Growth Rate Diversity in Bacteria.

To investigate the nature and origins of growth rate diversity in bacteria, we grew Escherichia coli and Bacillus subtilis in liquid minimal media and, after different periods of 15N-labeling, analyzed and imaged isotope distributions in individual cells with Secondary Ion Mass Spectrometry. We find a striking inter- and intra-cellular diversity, even in steady state growth. This is consistent with the strand-dependent, hyperstructure-based hypothesis that a major function of the cell cycle is to generate coherent, growth rate diversity via the semi-conservative pattern of inheritance of strands of DNA and associated macromolecular assemblies. We also propose quantitative, general, measures of growth rate diversity for studies of cell physiology that include antibiotic resistance.

Toxicity of Food-Grade TiO2 to Commensal Intestinal and Transient Food-Borne Bacteria: New Insights Using Nano-SIMS and Synchrotron UV Fluorescence Imaging.

Titanium dioxide (TiO2) is commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, a risk of intestinal barrier disruption, including dysbiosis of the gut microbiota, is increasingly suspected because of the presence of a nano-sized fraction in this additive. We hypothesized that food-grade E171 and Aeroxyde P25 (identical to the NM-105 OECD reference nanomaterial in the European Union Joint Research Centre) interact with both commensal intestinal bacteria and transient food-borne bacteria under non-UV-irradiated conditions. Based on differences in their physicochemical properties, we expect a difference in their respective effects. To test these hypotheses, we chose a panel of eight Gram-positive/Gram-negative bacterial strains, isolated from different biotopes and belonging to the species Escherichia coli, Lactobacillus rhamnosus, Lactococcus lactis (subsp. lactis and cremoris), Streptococcus thermophilus, and Lactobacillus sakei. Bacterial cells were exposed to food-grade E171 vs. P25 in vitro and the interactions were explored with innovative (nano)imaging methods. The ability of bacteria to trap TiO2 was demonstrated using synchrotron UV fluorescence imaging with single cell resolution. Subsequent alterations in the growth profiles were shown, notably for the transient food-borne L. lactis and the commensal intestinal E. coli in contact with food-grade TiO2. However, for both species, the reduction in cell cultivability remained moderate, and the morphological and ultrastructural damages, observed with electron microscopy, were restricted to a small number of cells. E. coli exposed to food-grade TiO2 showed some internalization of TiO2 (7% of cells), observed with high-resolution nano-secondary ion mass spectrometry (Nano-SIMS) chemical imaging. Taken together, these data show that E171 may be trapped by commensal and transient food-borne bacteria within the gut. In return, it may induce some physiological alterations in the most sensitive species, with a putative impact on gut microbiota composition and functioning, especially after chronic exposure.

Time-dependent effects of perfluorinated compounds on viability in cerebellar granule neurons: Dependence on carbon chain length and functional group attached.

The toxicity of long chained perfluoroalkyl acids (PFAAs) has previously been reported to be related to the length of the perfluorinated carbon chain and functional group attached. In the present study, we compared the cytotoxicity of six PFAAs, using primary cultures of rat cerebellar granule neurons (CGNs). Two perfluoroalkyl sulfonic acids (PFSAs, chain length C6 and C8) and four perfluoroalkyl carboxylic acids (PFCAs, chain length C8-C11) were studied. These PFAAs have been detected in human blood and the brain tissue of mammals. The cell viability trypan blue and MTT assays were used to determine toxicity potencies (based on LC50 values) after 24h exposure (in descending order): perfluoroundecanoic acid (PFUnDA)≥perfluorodecanoic acid (PFDA)>perfluorooctanesulfonic acid potassium salt (PFOS)>perfluorononanoic acid (PFNA)>perfluorooctanoic acid (PFOA)>perfluorohexanesulfonic acid potassium salt (PFHxS). Concentrations of the six PFAAs that produced equipotent effects after 24h exposure were used to further explore the dynamics of viability changes during this period. Therefore viability was assessed at 10, 30, 60, 90, 120 and 180min as well as 6, 12, 18 and 24h. A difference in the onset of reduction in viability was observed, occurring relatively quickly (30-60min) for PFOS, PFDA and PFUnDA, and much slower (12-24h) for PFHxS, PFOA and PFNA. A slight protective effect of vitamin E against PFOA, PFNA and PFOS-induced reduction in viability indicated a possible involvement of oxidative stress. PFOA and PFOS did not induce lipid peroxidation on their own, but significantly accelerated cumene hydroperoxide-induced lipid peroxidation. When distribution of the six PFAAs in the CGN-membrane was investigated using NanoSIMS50 imaging, two distinct patterns appeared. Whereas PFHxS, PFOS and PFUnDA aggregated in large hotspots, PFOA, PFNA and PFDA showed a more dispersed distribution pattern. In conclusion, the toxicity of the investigated PFAAs increased with increasing carbon chain length. For molecules with a similar chain length, a sulfonate functional group led to greater toxicity than a carboxyl group.

Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

BACKGROUND: The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. METHODS: The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. RESULTS: The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. CONCLUSIONS: The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were observed for uptake, pro-inflammatory response and changes at the proteome level. The low level of overlap between differentially abundant proteins observed in both Ag NPs and AgNO3 treated co-culture suggests size-dependent responses that cannot only be attributed to soluble Ag.

Analysis of dsDNA and RNA viromes in methanogenic digesters reveals novel viral genetic diversity.

Although viruses are not the key players of the anaerobic digestion process, they may affect the dynamics of bacterial and archaeal populations involved in biogas production. Until now viruses have received very little attention in this specific habitat; therefore, as a first step towards their characterization, we optimized a virus filtration protocol from anaerobic sludge. Afterwards, to assess dsDNA and RNA viral diversity in sludge samples from nine different reactors fed either with waste water, agricultural residues or solid municipal waste plus agro-food residues, we performed metagenomic analyses. As a result we showed that, while the dsDNA viromes (21 assigned families in total) were dominated by dsDNA phages of the order Caudovirales, RNA viruses (14 assigned families in total) were less diverse and were for the main part plant-infecting viruses. Interestingly, less than 2% of annotated contigs were assigned as putative human and animal pathogens. Our study greatly extends the existing view of viral genetic diversity in methanogenic reactors and shows that these viral assemblages are distinct not only among the reactor types but also from nearly 30 other environments already studied, including the human gut, fermented food, deep sea sediments and other aquatic habitats.

Determination of oral uptake and biodistribution of platinum and chromium by the garden snail (Helix aspersa) employing nano-secondary ion mass-spectrometry.

Environmental heavy metal contamination is a case of concern for both animal and human health. Studying the fate of metals in plant or animal tissues may provide information on pollution. In the present study, we investigated the possibility to follow the biological fate of chromium and platinum uptake in common garden snails (Helix aspersa), typically accumulating high concentrations of metals from their environment. Chromium and platinum were administered orally to snails in 5 groups (n=25/group): control, food contaminated by ca. 2.5 µg g(-1) and 19 µg g(-1) chromium and 2.5 µg g(-1) and 25 µg g(-1) platinum, for 8 weeks. Following exposure, surviving snails were sacrificed, shell and remaining tissue investigated by ICP-MS, and shell, midgut gland and mantle by nano-secondary ion mass-spectrometry (Nano-SIMS). (12)C(14)N-normalized platinum and (40)Ca-normalized chromium measurements indicated highest enrichments in cellular vesicles of the midgut gland, and lower concentrations in mantle and shell, with significantly higher platinum and chromium concentrations in the 2 exposure groups vs. control (P<0.05), with somewhat differing distribution patterns for chromium and platinum. Comparable results were obtained by ICP-MS, with both chromium and platinum fed snails showing drastically elevated concentrations of metals in shell (up to 78 and 122 µg g(-1) dw platinum and chromium, respectively) and in other tissues (up to 200 and 1125 µg g(-1) dw platinum and chromium, respectively). Nano-SIMS allowed for semi-quantitative comparison of metal fate in snail tissues, making this an interesting technique for future studies in the area of environmental pollution.