Population Structure in Naegleria fowleri as Revealed by Microsatellite Markers

Naegleria sp. is a free living amoeba belonging to the Heterolobosea class. Over 40 species of Naegleria were identified and recovered worldwide in different habitats such as swimming pools, freshwater lakes, soil or dust. Among them, N. fowleri, is a human pathogen responsible for primary amoeboic meningoencephalitis (PAM). Around 300 cases were reported in 40 years worldwide but PAM is a fatal disease of the central nervous system with only 5% survival of infected patients. Since both pathogenic and non pathogenic species were encountered in the environment, detection and dispersal mode are crucial points in the fight against this pathogenic agent. Previous studies on identification and genotyping of N. fowleri strains were focused on RAPD analysis and on ITS sequencing and identified 5 variants: euro-american, south pacific, widespread, cattenom and chooz. Microsatellites are powerful markers in population genetics with broad spectrum of applications (such as paternity test, fingerprinting, genetic mapping or genetic structure analysis). They are characterized by a high degree of length polymorphism. The aim of this study was to genotype N. fowleri strains using microsatellites markers in order to track this population and to better understand its evolution. Six microsatellite loci and 47 strains from different geographical origins were used for this analysis. The microsatellite markers revealed a level of discrimination higher than any other marker used until now, enabling the identification of seven genetic groups, included in the five main genetic groups based on the previous RAPD and ITS analyses. This analysis also allowed us to go further in identifying private alleles highlighting intra-group variability. A better identification of the N. fowleri isolates could be done with this type of analysis and could allow a better tracking of the clinical and environmental N. fowleri strains.     

Release of LL-37 by activated human Vgamma9Vdelta2 T cells: a microbicidal weapon against Brucella suis

Human Vgamma9Vdelta2 T cells play a crucial role in early immune response to intracellular pathogens. Moreover, in brucellosis, these cells are drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vgamma9Vdelta2 T cells are capable of inhibiting Brucella growth and development through a combination of mechanisms: 1) cytotoxicity, 2) macrophage activation and bactericidal activity through cytokine and chemokine secretion, and 3) antibacterial effects. We previously described that antibacterial factors were found in supernatants from activated Vgamma9Vdelta2 T cells. In this study, we show that Vgamma9Vdelta2 T cells express the human cathelicidin hCAP18 and its mature form, known as LL-37, is released upon activation of Vgamma9Vdelta2 T cells. We also show that LL-37 has an antibacterial effect on Brucella suis. Overall, our results demonstrate that LL-37 is a soluble factor responsible for a part of the bactericidal activity of Vgamma9Vdelta2 T cells.     

Sex-biased and body condition dependent dispersal capacity in the endangered saproxylic beetle <Emphasis Type="Italic">Osmoderma eremita</Emphasis> (Coleoptera: Cetoniidae)

The dispersal capacity of rare and endangered insect species has rarely been estimated even though it is essential for their management. For these species, laboratory based experiments are considered more appropriate for determining dispersal capacity as well as the factors influencing it. We aimed to characterize dispersal capacity of the endangered saproxylic beetle Osmoderma eremita (Scopoli, 1763) (Coleoptera: Cetoniidae). We studied the influence of sex and body condition on several parameters of dispersal (seven parameters of flight capacity measured in laboratory and pre-flight behaviour observed in the wild). Tethered flight experiments, conducted on 30 individuals collected in several regions of France, revealed: (1) maximal single flight distance of 1,454 m and maximal total flight distance of 2,361 m; (2) higher flight capacity in females than males; (3) flight speed and take-off completion decreasing with increasing body condition only for females. Additionally, 32 individuals displaying pre-flight behaviour in the wild showed similar interacting influences of sex and body condition: females initiating pre-flight behaviour had lower body condition than males. Thus, males and females have different dispersal strategies. We propose that body condition influences on dispersal capacity should be considered for species conservation by, for instance, managing adult food resources at the landscape scale and need to be taken into account in introduction programmes.     

A modular open platform for systematic functional studies under physiological conditions

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.     

Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance

AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem.     

Maturation of cytosolic iron-sulfur proteins requires glutathione

Glutathione is the major protective agent against oxidative stress in Saccharomyces cerevisiae. Deletion of the GSH1 gene (strain Deltagsh1) encoding the enzyme that catalyzes the first step of glutathione biosynthesis leads to growth arrest, which can be relieved by either glutathione or reducing agents such as dithiothreitol. Because defects in the biosynthesis of cellular iron-sulfur (Fe/S) proteins are associated with increases in glutathione levels, we examined the consequences of glutathione depletion on this essential process. No significant defects were detected in the amounts, activities, and maturation of mitochondrial Fe/S proteins in glutathione-depleted Deltagsh1 cells. On the contrary, the maturation of extra-mitochondrial Fe/S proteins was decreased substantially. The defect was rectified neither by addition of dithiothreitol nor under anaerobic conditions excluding oxidative damage of Fe/S clusters. A double mutant in GSH1 and ATM1 encoding a mitochondrial ATP binding cassette (ABC) transporter involved in cytosolic Fe/S protein maturation is nonviable even in the presence of dithiothreitol. Similar to atm1 and other mutants defective in cytosolic Fe/S protein maturation, mitochondria from glutathione-depleted Deltagsh1 cells accumulated high amounts of iron. Together, our data demonstrate that glutathione, in addition to its protective role against oxidative damage, performs a novel and specific function in the maturation of cytosolic Fe/S proteins.     

Control of Hydrogen Photoproduction by the Proton Gradient Generated by Cyclic Electron Flow in Chlamydomonas reinhardtii

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H₂ production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H₂ photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H₂ production.     

Coping with Pleistocene climatic fluctuations: Demographic responses in remote endemic reef fishes

Elucidating demographic history during the settlement of ecological communities is crucial for properly inferring the mechanisms that shape patterns of species diversity and their persistence through time. Here, we used genomic data and coalescent‐based approaches to elucidate for the first time the demographic dynamics associated with the settlement by endemic reef fish fauna of one of the most remote peripheral islands of the Pacific Ocean, Rapa Nui (Easter Island). We compared the demographic history of nine endemic species in order to explore their demographic responses to Pleistocene climatic fluctuations. We found that species endemic to Rapa Nui share a common demographic history, as signatures of population expansions were retrieved for almost all of the species studied here, and synchronous demographic expansions initiated during the last glacial period were recovered for more than half of the studied species. These results suggest that eustatic fluctuations associated with Milankovitch cycles have played a central role in species demographic histories and in the final stage of the community assembly of many Rapa Nui reef fishes. Specifically, sea level lowstands resulted in the maximum reef habitat extension for Rapa Nui endemic species; we discuss the potential role of seamounts in allowing endemic species to cope with Pleistocene climatic fluctuations, and we highlight the importance of local historical processes over regional ones. Overall, our results shed light on the mechanisms by which endemism arises and is maintained in peripheral reef fish fauna.