Genome-wide footprints in the carob tree (Ceratonia siliqua) unveil a new domestication pattern of a fruit tree in the Mediterranean

Intense research efforts over the last two decades have renewed our understanding of plant phylogeography and domestication in the Mediterranean basin. Here we aim to investigate the evolutionary history and the origin of domestication of the carob tree (Ceratonia siliqua), which has been cultivated for millennia for food and fodder. We used >1000 microsatellite genotypes to delimit seven carob evolutionary units (CEUs). We investigated genome-wide diversity and evolutionary patterns of the CEUs with 3557 single nucleotide polymorphisms generated by restriction-site associated DNA sequencing (RADseq). To address the complex wild vs. cultivated status of sampled trees, we classified 56 sampled populations across the Mediterranean basin as wild, seminatural or cultivated. Nuclear and cytoplasmic loci were identified from RADseq data and separated for analyses. Phylogenetic analyses of these genomic-wide data allowed us to resolve west-to-east expansions from a single long-term refugium probably located in the foothills of the High Atlas Mountains near the Atlantic coast. Our findings support multiple origins of domestication with a low impact on the genetic diversity at range-wide level. The carob was mostly domesticated from locally selected wild genotypes and scattered long-distance westward dispersals of domesticated varieties by humans, concomitant with major historical migrations by Romans, Greeks and Arabs. Ex situ efforts to preserve carob genetic resources should prioritize accessions from both western and eastern populations, with emphasis on the most differentiated CEUs situated in southwest Morocco, south Spain and eastern Mediterranean. Our study highlights the relevance of wild and seminatural habitats in the conservation of genetic resources for cultivated trees

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-29-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5- HmdC, 5-formyl-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5- HmdU), and the (59S) diastereomer of 8,59-cyclo-29-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tetfamily dioxygenase enzymes in Arabidopsis

Adverse outcome pathways (AOPs) for radiation-induced reproductive effects in environmental species: state of science and identification of a consensus AOP network

Background Reproductive effects of ionizing radiation in organisms have been observed under laboratory and field conditions. Such assessments often rely on associations between exposure and effects, and thus lacking a detailed mechanistic understanding of causality between effects occurring at different levels of biological organization. The Adverse Outcome Pathway (AOP), a conceptual knowledge framework to capture, organize, evaluate and visualize the scientific knowledge of relevant toxicological effects, has the potential to evaluate the causal relationships between molecular, cellular, individual, and population effects. This paper presents the first development of a set of consensus AOPs for reproductive effects of ionizing radiation in wildlife. This work was performed by a group of experts formed during a workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. The work presents a series of taxon-specific case studies that were used to identify relevant empirical evidence, identify common AOP components and propose a set of consensus AOPs that could be organized into an AOP network with broader taxonomic applicability. Conclusion Expert consultation led to the identification of key biological events and description of causal linkages between ionizing radiation, reproductive impairment and reduction in population fitness. The study characterized the knowledge domain of taxon-specific AOPs, identified knowledge gaps pertinent to reproductive-relevant AOP development and reflected on how AOPs could assist applications in radiation (radioecological) research, environmental health assessment, and radiological protection. Future advancement and consolidation of the AOPs is planned to include structured weight of evidence considerations, formalized review and critical assessment of the empirical evidence prior to formal submission and review by the OECD sponsored AOP development program

Quantification of the 2-Deoxyribonolactone and Nucleoside 5 '-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of gamma-Radiation and Fe2+-EDTA

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC−MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1′-oxidation and the nucleoside 5′-aldehyde of 5′-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC−MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5′-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1I, was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5′-aldehyde per 106 nt per μM in accord with its established minor 1′- and major 5′-oxidation chemistry. Calicheamicin unexpectedly caused 1′-oxidation at a low level of 10 2-deoxyribonolactone per 106 nt per μM in addition to the expected predominance of 5′-oxidation at 560 nucleoside 5′-aldehyde per 106 nt per μM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe2+−EDTA, produced nucleoside 5′-aldehyde at a frequency of 57 per 106 nt per Gy (G-value 74 nmol/J) and 3.5 per 106 nt per μM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe2+−EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per μM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5′-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+−EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.National Institute of Environmental Health Sciences (ES002109)National Center for Research Resources (U.S.) (RR023783-01)National Center for Research Resources (U.S.) (RR017905-01)National Cancer Institute (U.S.) (CA103146

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

International audienceOxidative 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. © 2016 Elsevier Inc

Spéciation et dosimétrie interne: Des espèces chimiques aux modèles dosimétriques

International audienceSpeciation studies refer to the distribution of species in a particular sample or matrix. These studies are necessary to improve the description, understanding and prediction of trace element kinetics and toxicity. In case of internal contamination with radionuclides, speciation studies could help to improve both the biokinetic and dosimetric models for radionuclides. There are different methods to approach the speciation of radionuclide in a biological system, depending on the degree of accuracy needed and the level of uncertainties accepted. Among them, computer modelling and experimental determination are complementary approaches. This paper describes what is known about speciation of actinides in blood, GI-tract, liver and skeleton and of their consequences in terms of internal dosimetry. The conclusion is that such studies provide very valuable data and should be targeted in the future on some specific tissues and biomolecules. © 2004 EDP Sciences.Les études de spéciation correspondent à la détermination des espèces chimiques d'un élément et à l'analyse de leur distribution dans un échantillon ou un milieu donné. Ces études sont nécessaires pour mieux décrire, comprendre et prédire les biocinétiques des éléments traces. Dans le cas d'une contamination interne par des radionucléides, les études de spéciation permettent de mieux comprendre certains mécanismes d'absorption et de transfert. Elles contribuent de fait à améliorer les modèles biocinétiques et dosimétriques des radionucléides. Différentes approches peuvent être utilisées pour l'étude de la spéciation des radionucléides, en fonction du niveau d'étude demandé et du degré d'incertitude toléré. Parmi celles-ci, on distingue une approche théorique par modélisation et une approche expérimentale qui sont toutes deux complémentaires. Cet article résume et décrit ce qui est actuellement connu sur la spéciation des actinides dans le sang, le tractus gastro- intestinal (TGI), le foie et les os mais aussi sur les conséquences induites en radiotoxicologie. Dans le futur, les recherches devront être focalisées sur certains tissus spécifiques afin de répondre aux principales questions de la dosimétrie interne