Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations

Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.     

Tracing the progeny of the aortic hemangioblast in the avian embryo

A population of hematopoietic progenitors becomes committed within the embryo proper in the floor of the aorta (P-Sp/AGM in the mouse). In birds, this first aspect of intraembryonic hematopoiesis is prominent during embryonic day 3 (E3) as endothelium-associated "intra-aortic clusters." Between E6 and E8, diffuse hematopoiesis then occurs as "para-aortic foci" located in the dorsal mesentery ventral to the aorta. These foci are not associated with endothelium. Whether these two hematopoietic cell populations arise from distinct or common progenitors is not known. We could recently trace back the origin of intra-aortic clusters in the avian embryo by labeling aortic endothelial cells (EC) in vivo with acetylated low-density lipoproteins. This approach established the derivation of early intraembryonic hemopoietic cells from the endothelium, but did not indicate how long during ontogeny such a relationship may exist, since the progeny of EC labeled at E2 could be traced for 1-2 days at most. Here we report that, when E2 aortic ECs were infected prior to the formation of intra-aortic clusters with a nonreplicative LacZ-bearing retroviral vector, numerous cells were labeled in the para-aortic foci at E6. In contrast, when the retroviral vector was inoculated at E4 rather than E2, that is, after the disappearance of intra-aortic clusters, no cells in the para-aortic foci were labeled. Taken together, our results demonstrate that ECs from the aortic floor seed the two aspects of aorta-associated hemopoiesis and that these ECs with hemangioblastic potential are present only transiently in the aorta.     

Ecotourism disturbance to wildfowl in protected areas: historical, empirical and experimental approaches in the Camargue, Southern France

Ecotourism is becoming very popular, especially in protected areas where wildlife concentrate and is easier to observe, but the consequences of associated disturbance have seldom be quantified other than in the short-term, making the sustainability of this activity untested. We combined a historical, an empirical and an experimental approach to assess the long-, medium- and short-term consequences of disturbance to wintering wildfowl (Anatidae) in a wetland of international importance in the Camargue, Southern France. In the short-term, disturbance made teal (Anas crecca) move away temporarily from observation blinds without leaving the waterbody. Wildfowl fed more after disturbance, disrupting their normal resting activities. In the medium-term, waterbodies with more tourists did not host fewer birds: conversely the most heavily disturbed one hosted the highest wildfowl density. In the long term, wildfowl numbers were not related with the number of visitors. When practiced with appropriate guiding of people, and where appropriate facilities are provided to limit human disturbance as done here, ecotourism may not affect wintering wildfowl other than reversibly in the very short term. The legitimate demand of the public for access, even in fragile protected areas, may therefore be sustainable under some conditions.     

Two Down syndrome patients with rec(21),dupq,inv(21)(p11;q2109) from a familial pericentric inversion

Aneusomie de recombinaison arose from a familial pericentric inversion of a chromosome 21. Two female patients had a typical Down syndrome; one of them had slight psychomotor retardation. There was partial trisomy 21q2109----qter in these two patients but ZnCu SOD activity was normal.     

Genetic variation and drought response in two Populus × euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides × Populus nigra, cv. ‘Agathe_F’ and ‘Cima’, were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra.A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, ‘Cima’ displayed the highest abundance of antioxidant enzymes.In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while ‘Cima’ differed from ‘Agathe_F’ by increased abundance of enzymes involved in photorespiration and in oxidative stress, ‘Agathe_F’ was mainly differentiated by increased abundance of enzymes involved in photosynthesis.     

Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells

To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1β, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.     

Histone phosphorylation: how to proceed

Among all posttranslational modifications that occur on histone tails, phosphorylation is the one that establishes a direct link between chromatin remodeling and intracellular signaling pathways. Specific, conserved serine residues are present on the N-terminal tails of each histone. These are phosphoacceptor sites for a number of kinases, whose identification is essential to decipher the transduction routes leading to various physiological responses. In the case of histone H3, phosphorylation at the Ser10 residue may lead to either activated gene expression or chromatin condensation during mitosis. In addition, phosphorylation at specific sites may be coupled to other distinct modifications, such as acetylation and methylation, generating the so-called "histone code" which postulates that well defined combinatorial modifications at histone tails correspond to specific physiological responses. Here we describe a number of experimental methodologies that are essential for the study of histone phosphorylation. While chromatin immunoprecipitation is useful in recognizing gene targets, the in-gel kinase assay is a first, essential step in establishing the identity of the kinase(s) that operates in response to a specific signaling pathway. The subsequent use of in vitro kinase assays is helpful in validating the implication of a candidate kinase. These powerful approaches are important as identification of the signaling transduction routes leading to chromatin remodeling is critical to an understanding of all cellular processes.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

A Genetic Analysis of Mancozeb Resistance in Typhlodromus Pyri (Acari: Phytoseiidae)

A field population of Typhlodromus pyri (Acari: Phytoseiidae) tolerant to mancozeb was selected in the laboratory. After 10 mancozeb selections the LC₅₀ value for mancozeb was 73 times higher in the selected-10 strain compared to the standard susceptible strain. A genetic analysis using reciprocal crosses and backcrosses of female F₁ progeny found no maternal effect. Resistance in the selected-10 strain was codominant in expression, dominance value was about −0.1. Backcrosses between F₁ females and the susceptible strain indicate that the resistance to mancozeb could be principally conferred by a predominant gene, but additional factors would also be involved.