Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays

The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.     

Epigenetic regulation of the ELOVL6 gene is associated with a major QTL effect on fatty acid composition in pigs

Background

In previous studies on an Iberian x Landrace cross, we have provided evidence that supported the porcine ELOVL6 gene as the major causative gene of the QTL on pig chromosome 8 for palmitic and palmitoleic acid contents in muscle and backfat. The single nucleotide polymorphism (SNP) ELOVL6:c.-533C > T located in the promoter region of ELOVL6 was found to be highly associated with ELOVL6 expression and, accordingly, with the percentages of palmitic and palmitoleic acids in longissimus dorsi and adipose tissue. The main goal of the current work was to further study the role of ELOVL6 on these traits by analyzing the regulation of the expression of ELOVL6 and the implication of ELOVL6 polymorphisms on meat quality traits in pigs.

Results

High-throughput sequencing of BAC clones that contain the porcine ELOVL6 gene coupled to RNAseq data re-analysis showed that two isoforms of this gene are expressed in liver and adipose tissue and that they differ in number of exons and 3’UTR length. Although several SNPs in the 3’UTR of ELOVL6 were associated with palmitic and palmitoleic acid contents, this association was lower than that previously observed with SNP ELOVL6:c.-533C > T. This SNP is in full linkage disequilibrium with SNP ELOVL6:c.-394G > A that was identified in the binding site for estrogen receptor alpha (ERα). Interestingly, the ELOVL6:c.-394G allele is associated with an increase in methylation levels of the ELOVL6 promoter and with a decrease of ELOVL6 expression. Therefore, ERα is clearly a good candidate to explain the regulation of ELOVL6 expression through dynamic epigenetic changes in the binding site of known regulators of ELOVL6 gene, such as SREBF1 and SP1.

Conclusions

Our results strongly suggest the ELOVL6:c.-394G > A polymorphism as the causal mutation for the QTL on pig chromosome 8 that affects fatty acid composition in pigs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0111-y) contains supplementary material, which is available to authorized users.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

The Role of Microscopy in the Investigation of Paramyosin as a Vaccine Candidate against Schistosoma japonicum

There has been growing interest in paramyosin as a vaccine component to combat schistosomiasis. Immunological and molecular techniques have been used in the past to investigate the effectiveness of a paramyosin vaccine as an anti-schistosomal treatment. However, recent localization studies at ultrastructural and morphological levels have highlighted a number of questions concerning the role of paramyosin within schistosome parasites. Debates about how a non-surface protein such as paramyosin might provide protection against schistosome infections have recently been addressed by microscopy results. Immunolocalization studies have indicated multiple functions of paramyosin within the parasite and provided insights into how a vaccine may target the parasite, as discussed here by Geoffrey Gobert.     

FIELD MEASUREMENTS OF INORGANIC NITROGEN UPTAKE BY EPIFLORA COMPONENTS OF THE SEAGRASS POSIDONIA OCEANICA (MONOCOTYLEDONS,POSIDONIACEAE)1

Crustose corallines, crustose and erect brown algae, and sessile animals are major components of the epiphytic community of the Mediterranean seagrass Posidonia oceanica (L.) Delile. Production, biomass, and specific composition of this epiphyte–seagrass association are impacted by anthropogenic increase of nutrient load in this oligotrophic area. In this context, nitrogen uptake by P. oceanica and its epiflora was measured using the isotope ¹⁵N at a 10 m depth in the Revellata Bay (Corsica, Mediterranean Sea). Epiflora components showed various seasonal patterns of biomass and abundance. The epiphytic brown algae appeared at the end of spring, later than the crustose corallines, and after the nitrate peak in the bay. Because of their later development in the season, epiphytic brown algae mostly rely on ammonium for their N needs. We hypothesize that the temporal succession of epiphytic organisms plays a crucial role in the N dynamics of this community under natural conditions. The epiphytic brown algae, which have a growth rate one order of magnitude greater than that of crustose corallines, showed lower N‐uptake rates. The greater N‐uptake rates of crustose corallines probably reflect the greater N requirements (i.e., lower C/N ratios) of red algae. We determined that the epiflora incorporated ammonium and nitrate more rapidly than their host. Nevertheless, when biomass was taken into account, P. oceanica was the most important contributor to N uptake from the water column by benthic macrophytes in this seagrass bed.     

Giardia intestinalis: study of infestation procedures of young mice before weaning

Despite the prevalence of giardiasis little is known about the host-parasite specificity. With Giardia intestinalis-suckling mouse model, the success of infestation was depending on the parasite form, cyst or trophozoite, and, for the same parasite form, on the studied strain. Furthermore, the intestinal modification during the wean were unfavourable to Giardia intestinalis colonization.     

Genomics and proteomics approaches to the study of cancer-stroma interactions

Background

The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.

Methods

The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.

Results

We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.

Conclusions

A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

     

Food sources of two detritivore amphipods associated with the seagrass Posidonia oceanica leaf litter

This study focused on the ingestion and assimilation of Posidonia oceanica (L.) Delile litter by Gammarella fucicola Leach and Gammarus aequicauda Martynov, two dominant detritivore amphipods of the P. oceanica leaf litter. Scanning electron microscope observations indicated that leaf litter is highly colonized by diverse diatoms, bacteria and fungi, which may constitute a potential food source for the litter fauna. Gut content observations demonstrated that these species eat P. oceanica litter, and that this item is an important part of their ingested diet. Stable isotope analyses showed that the species do not experience the same gains from the ingested Posidonia. Gammarella fucicola displayed isotopic values, suggesting a major contribution of algal material (micro- and macro-epiphytes or drift macro-algae). On the other hand, the observed isotopic values of G. aequicauda indicated a more important contribution of P. oceanica carbon. The mixing model used agreed with this view, with a mean contribution of P. oceanica to approximately 50% (range 40-55%) of the assimilated biomass of G. aequicauda. This demonstrated that the two species, suspected to be detritus feeders, display in reality relatively different diets, showing that a certain degree of trophic diversity may exist among the detritivore community of the seagrass litter.     

Forskolin induction of late-LTP and up-regulation of 5' TOP mRNAs translation via mTOR, ERK, and PI3K in hippocampal pyramidal cells

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5' terminal oligopyrimidine (5' TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5' TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral – CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5' TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5' TOP-myr-dYFP reporter to selectively monitor 5' TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5' TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.