Role of evolutionary and ecological factors in the reproductive success and the spatial genetic structure of the temperate gorgonian Paramuricea clavata

Dispersal and mating features strongly influence the evolutionary dynamics and the spatial genetic structure (SGS) of marine populations. For the first time in a marine invertebrate, we examined individual reproductive success, by conducting larval paternity assignments after a natural spawning event, combined with a small‐scale SGS analysis within a population of the gorgonian Paramuricea clavata. Thirty four percent of the larvae were sired by male colonies surrounding the brooding female colonies, revealing that the bulk of the mating was accomplished by males from outside the studied area. Male success increased with male height and decreased with increasing male to female distance. The parentage analyses, with a strong level of self‐recruitment (25%), unveiled the occurrence of a complex family structure at a small spatial scale, consistent with the limited larval dispersal of this species. However, no evidence of small scale SGS was revealed despite this family structure. Furthermore, temporal genetic structure was not observed, which appears to be related to the rather large effective population size. The low level of inbreeding found suggests a pattern of random mating in this species, which disagrees with expectations that limited larval dispersal should lead to biparental inbreeding. Surface brooding and investment in sexual reproduction in P. clavata contribute to multiple paternity (on average 6.4 fathers were assigned per brood), which enhance genetic diversity of the brood. Several factors may have contributed to the lack of biparental inbreeding in our study such as (i) the lack of sperm limitation at a small scale, (ii) multiple paternity, and (iii) the large effective population size. Thus, our results indicate that limited larval dispersal and complex family structure do not necessarily lead to biparental inbreeding and SGS. In the framework of conservation purposes, our results suggested that colony size, proximity among colonies and the population size should be taken into consideration for restoration projects.     

Rigorous Training of Dogs Leads to High Accuracy in Human Scent Matching-To-Sample Performance

Human scent identification is based on a matching-to-sample task in which trained dogs are required to compare a scent sample collected from an object found at a crime scene to that of a suspect. Based on dogs’ greater olfactory ability to detect and process odours, this method has been used in forensic investigations to identify the odour of a suspect at a crime scene. The excellent reliability and reproducibility of the method largely depend on rigor in dog training. The present study describes the various steps of training that lead to high sensitivity scores, with dogs matching samples with 90% efficiency when the complexity of the scents presented during the task in the sample is similar to that presented in the in lineups, and specificity reaching a ceiling, with no false alarms in human scent matching-to-sample tasks. This high level of accuracy ensures reliable results in judicial human scent identification tests. Also, our data should convince law enforcement authorities to use these results as official forensic evidence when dogs are trained appropriately.     

Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS

Background

Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.

Methodology/Principal Findings

Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M’sila where P. (Phlebotomus) papatasi was the only sand fly species detected.

Conclusion

The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.     

Synthesis and biological evaluation of 3-amino-, 3-alkoxy- and 3-aryloxy-6-(hetero)arylpyridazines as potent antitumor agents

Various 3-amino-, 3-aryloxy- and alkoxy-6-arylpyridazines have been synthesized by an electrochemical reductive cross-coupling between 3-amino-, 3-aryloxy- or 3-alkoxy-6-chloropyridazines and aryl or heteroaryl halides. In vitro antiproliferative activity of these products was evaluated against a representative panel of cancer cell lines (HuH7, CaCo-2, MDA-MB-231, HCT116, PC3, NCI-H727, HaCaT) and oncogenicity prevention of the more efficient derivatives was highlighted on human breast cancer cell line MDA-MB 468-Luc prior establishing their interaction with p44/42 and Akt-dependent signaling pathways.     

High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

Propagation of fluorescent viruses in growing plaques

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.     

Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.     

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.