RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus

Background  

In all jawed vertebrates, highly polymorphic genes of the major histocompatibility complex (MHC) encode antigen presenting molecules that play a key role in the adaptive immune response. Their polymorphism is composed of multiple copies of recently duplicated genes, each possessing many alleles within populations, as well as high nucleotide divergence between alleles of the same species. Experimental evidence is accumulating that MHC polymorphism is a result of balancing selection by parasites and pathogens. In order to describe MHC diversity and analyse the underlying mechanisms that maintain it, a reliable genotyping technique is required that is suitable for such highly variable genes.     

Genotoxic effects of bitumen fumes in Big Blue transgenic rat lung

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 degrees C, total particulate matter: 100mg/m3) and perform a nose-only exposure of Big Blue transgenic rodents 6h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.     

Viability and gene expression responses to polymeric nanoparticles in human and rat cells

Applications of polymeric nanoparticles (NP) in medical fields are rapidly expanding. However, the influence of polymeric NP on cell growth and functions is widely underestimated. Therefore, we have studied cell and polymeric NP interactions by addressing two cell types with two endpoints (viability and gene expressions). Rat NR8383 and human THP-1 monocytic cell lines were exposed to 6 to 200 μg/mL of Eudragit^® RL NP for 24 h, and cellular viability was estimated using MTT, WST-1, and trypan blue tests. A decrease of viability was observed with NR8383 cells (down to 70 % for 200 μg/mL), and on the contrary, an increase with THP-1 cells (up to 140 % for 200 μg/mL). Differential expression of genes involved in oxidative damage (NCF1), inflammation (NFKB, TNFA, IL6, IL1B), autophagy (ATG16L), and apoptotic balance (PDCD4, BCL2, CASP8) was analyzed. ATG16L, BCL2, and TNFA were up-regulated in NR8383 cells, which are consistent with an induction of autophagy and inflammation. On the other hand, NCF1, NFKB, and IL1B were down-regulated in THP-1 cells, which may contribute to explain the increase of cellular viability. Our results show that (1) the toxic potency of NP is dependent on the cellular model used and (2) mechanistic toxicology should be the corner stone for the evaluation of NP hazard.     

The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line.

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.     

Back to the sea twice: identifying candidate plant genes for molecular evolution to marine life

Background  

Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have adapted to a completely submerged lifestyle in marine waters. Here, we exploit two collections of expressed sequence tags (ESTs) of two wide-spread and ecologically important seagrass species, the Mediterranean seagrass Posidonia oceanica (L.) Delile and the eelgrass Zostera marina L., which have independently evolved from aquatic ancestors. This replicated, yet independent evolutionary history facilitates the identification of traits that may have evolved in parallel and are possible instrumental candidates for adaptation to a marine habitat.     

Temporal variation overshadows the response of leaf litter microbial communities to simulated global change

Bacteria and fungi drive the decomposition of dead plant biomass (litter), an important step in the terrestrial carbon cycle. Here we investigate the sensitivity of litter microbial communities to simulated global change (drought and nitrogen addition) in a California annual grassland. Using 16S and 28S rDNA amplicon pyrosequencing, we quantify the response of the bacterial and fungal communities to the treatments and compare these results to background, temporal (seasonal and interannual) variability of the communities. We found that the drought and nitrogen treatments both had significant effects on microbial community composition, explaining 2–6% of total compositional variation. However, microbial composition was even more strongly influenced by seasonal and annual variation (explaining 14–39%). The response of microbial composition to drought varied by season, while the effect of the nitrogen addition treatment was constant through time. These compositional responses were similar in magnitude to those seen in microbial enzyme activities and the surrounding plant community, but did not correspond to a consistent effect on leaf litter decomposition rate. Overall, these patterns indicate that, in this ecosystem, temporal variability in the composition of leaf litter microorganisms largely surpasses that expected in a short-term global change experiment. Thus, as for plant communities, future microbial communities will likely be determined by the interplay between rapid, local background variability and slower, global changes.     

Microdissection, mRNA amplification and microarray: a study of pleural mesothelial and malignant mesothelioma cells

The studies of molecular alterations in tumor cells with microarrays are often hampered by inherent tissue heterogeneity. The emergence of Laser Capture Microdissection (LCM) allowed us to overcome this challenge since it gives selective access to cancer cells that are isolated from their native tissue environment. In this report, we microdissected mesothelial cells and malignant mesothelioma cells of ex vivo resected specimens using LCM. Amplified RNA from mesothelial and mesothelioma microdissected cells allowed us to measure global gene expression with 10 K-microarrays in four independent experiments. We screened 9850 annotated human genes, 1275 of which have satisfied our data analysis requirements. They included 302 overexpressed genes and 160 downregulated genes in mesothelioma microdissected cells as compared to mesothelial microdissected cells. Among them, the expression levels of eight genes, namely BF, FTL, IGFBP7, RARRES1, RARRES2, RBP1, SAT, and TXN according to HUGO nomenclature, were increased, whereas six: ALOX5AP, CLNS1A, EIF4A2, ELK3, REQ and SYPL, were found to be underexpressed in mesothelioma microdissected cells. The ferritin light polypeptide (FTL) gene overexpression was confirmed by real time quantitative PCR. Our approach allowed a comprehensive in situ examination of mesothelioma and provided an accurate way to find new marker genes that may be useful for diagnosis and treatment of malignant pleural mesothelioma.     

Vivid molecular divergence over volcanic remnants: the phylogeography of Megadromus guerinii on Banks Peninsula,New Zealand

Megadromus guerinii, an endemic carabid beetle (Carabidae), is the most common carabid throughout its restricted range on Banks Peninsula, a formation of extinct volcanoes in Canterbury, New Zealand. This study characterises the small-scale phylogeographic patterns of M. guerinii across the formerly volcanically active Banks Peninsula using mitochondrial and ribosomal genes. Between the eastern and western areas of the peninsula, the mitochondrial, but not nuclear, DNA has a well-defined geographic distribution. Specifically, mitochondrial cytochrome c oxidase subunit I (CO1) identifies two distinct groups (> 6% divergence between eastern and western beetles) while ribosomal genes show no discernible pattern. Whether such a pattern represents male-biased dispersal, Wolbachia infection, a recent range expansion of a divergent lineage, or a deeper historic separation is explored. There is potential that male-biased dispersal could have occurred. Wolbachia infection was not detected. We conclude that historical processes have likely separated taxa in the eastern and western peninsula.     

Sequence variation in ZFX introns in human populations

DNA variation in human populations was studied by examining the last intron of the ZFX gene (about 1, 151 bp) with a worldwide sample of 29 individuals. Only one polymorphic site was found, which is located in an Alu sequence. This polymorphism is present at an intermediate frequency in all populations studied, and could be a shared polymorphism or due to migration among populations in Asia, Europe, and Africa. The nucleotide diversity is 0.04%, supporting the view that the level of nucleotide variation in nuclear DNA is very low in humans. From the sequence data, the age (T) of the most recent common ancestor of the sampled sequences is estimated: the mode of T is about 306,000 years, and the 95% confidence interval of T is 162,000-952,000 years. This mode estimate is considerably older than the estimates from Y- linked sequences.