Genetic structure and core collection of the World Olive Germplasm Bank of Marrakech: towards the optimised management and use of Mediterranean olive genetic resources

The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding.     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.     

Ultrasmall gold-doxorubicin conjugates rapidly kill apoptosis-resistant cancer cells

Ultrasmall (mean diameter, 2.7 nm) gold nanoparticles conjugated to doxorubicin (Au-Dox) are up to 20-fold more cytotoxic to B16 melanoma cells than the equivalent concentration of doxorubicin alone, and act up to six times more quickly. Ultrasmall Au-Dox enters the cell endocytic vesicles and is also seen free in the cytoplasm and nuclei. This is in distinct contrast to larger particles reported in previous studies, which are excluded from the nucleus and which show no increased toxicity over Dox alone. Cell death with Au-Dox is confirmed to be apoptotic by TUNEL staining and ultrastructural examination using transmission electron microscopy. To further explore the mechanism of action, two other cell lines were examined: HeLa cells which are highly sensitive to Dox, and HeLa cells overexpressing Bcl-2 which show impaired apoptosis and Dox resistance. Interestingly, the Dox-sensitive cells show a slightly decreased sensitivity to Au-Dox relative to Dox alone, whereas the Dox-resistant cells are not resistant to Au-Dox. These results have implications for the design of chemotherapeutic nanoparticles, suggesting that it is possible to selectively target apoptosis-resistant cancer cells while at the same time reducing cytotoxicity to normal cells.     

The anti-inflammatory effect of paraoxonase 1 against oxidized lipids depends on its association with high density lipoproteins

AimThe aims of this study were to investigate whether purified PON1 can reduce the pro-inflammatory effect of oxidized phospholipids and whether the effect depended on its association with HDL.Main methodsLipid peroxidation was induced by copper ions and was measured using the conjugated diene method. Lysophosphatidylcholine (lyso-PC) formation was measured by HPLC with evaporative light scattering detection (ELSD) and ICAM-1 expression on Ea.hy926 endothelial cells was analyzed by flow cytometry.Key findingsPurified PON1 significantly inhibited copper-induced oxidation of LDL and HDL, causing a 60.5% and 77.7% decrease in conjugated diene formation, respectively. Incubating PON1 with oxLDL caused a significant increase in lyso-PC levels, while oxHDL caused a significant decrease. PON1 (12.5 to 50 μg/mL) had a pro-inflammatory effect in the presence of oxLDL, increasing ICAM-1 levels in Ea.hy926 cells by 33.0% and 40.6% (p < 0.001) respectively, and had an anti-inflammatory effect in the presence of oxHDL, causing a 3-fold reduction in ICAM-1 levels. PON1 also caused a significant decrease in TNFα? and purified lyso-PC-induced ICAM-1 expression. The results obtained with reconstituted HDL as well as LCAT and PAF-AH inhibitors suggested that the anti-inflammatory effect of PON1 against oxidized lipids is dependent on its association with HDL.SignificanceOur results clearly showed that PON1 is involved in the anti-inflammatory effect of HDL and that the effect appears to depend on its association with HDL.     

Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis

Blastocystis hominis is an anaerobic parasite of the human intestinal tract belonging to the Stramenopile group. Using genome sequencing project data, we describe here the complete sequence of a 29,270-bp circular DNA molecule that presents mitochondrial features (such as oxidative phosphorylation complex I subunits) but lacks complexes III, IV and V. Transmission electron microscopy analyses reveal that this molecule, as well as mitochondrial (NADH:ubiquinone oxidoreductase subunit 7 (NAD7), beta-succinyl-CoA synthetase (beta-SCS)) and hydrogenosomal (pyruvate ferredoxin oxido-reductase (PFOR), iron-hydrogenase) proteins, are located within double-membrane surrounded-compartments known as mitochondria-like organelles (MLOs). As there is no evidence for hydrogen production by this organism, we suggest that MLOs are more likely anaerobic mitochondria.     

Critical and differential roles of NKp46- and NKp30-activating receptors expressed by uterine NK cells in early pregnancy

In early human pregnancy, uterine decidual NK cells (dNK) are abundant and considered as cytokine producers but poorly cytotoxic despite their cytolytic granule content, suggesting a negative control of this latter effector function. To investigate the basis of this control, we examined the relative contribution to the cytotoxic function of different activating receptors expressed by dNK. Using a multicolor flow cytometry analysis, we found that freshly isolated dNK exhibit a unique repertoire of activating and inhibitory receptors, identical among all the donors tested. We then demonstrated that in fresh dNK, mAb-specific engagement of NKp46-, and to a lesser extent NKG2C-, but not NKp30-activating receptors induced intracellular calcium mobilization, perforin polarization, granule exocytosis and efficient target cell lysis. NKp46-mediated cytotoxicity is coactivated by CD2 but dramatically blocked by NKG2A coengagement, indicating that the dNK cytotoxic potential could be tightly controlled in vivo. We finally found that in dNK, mAb-specific engagement of NKp30, but not NKp46, triggered the production of IFN-gamma, TNF-alpha, MIP-1alpha, MIP-1beta, and GM-CSF proinflammatory molecules. These data demonstrate a differential, controlled role of NKp46- and NKp30-activating receptors expressed by dNK that could be critical for the outcome of pregnancy and the killing of uterine cells infected by pathogens.     

Adsorptive features of acid-treated olive stones for drin pesticides: Equilibrium,kinetic and thermodynamic modeling studies

The adsorption behavior of drin pesticides from aqueous solution onto acid treated olive stones (ATOS) was investigated using stir bar sorptive extraction and gas chromatography coupled with mass spectroscopy. The effects of sorbent particle size, adsorbent dose, contact time, concentration of pesticide solution and temperature on the adsorption processes were systematically studied in batch shaking sorption experiments. Maximum removal efficiency (94.8%) was reached for aldrin (0.5 mg L⁻¹) using the fraction 63–100 μm of ATOS (solid/liquid ratio: 1 g L⁻¹). Experimental data were modeled by Langmuir, Freundlich and Dubinin–Radushkevich (D–R) isotherms. The Freundlich isotherm model (R² = 0.98–0.99) fitted the equilibrium data better than the Langmuir and D–R isotherm models, with low sum of error values (SE = 1.4–9.2%). The mean adsorption free energy derived from the D–R isotherm model (R² = 0.95–0.99) showed that the adsorption of drin pesticides was taken place by weak physical forces, such as van der Waals forces and hydrogen bonding. The calculated thermodynamic parameters, ΔH, ΔS and ΔG prove that drin pesticides adsorption on ATOS was feasible, spontaneous and exothermic under examined conditions. The pseudo first order, pseudo second order kinetic and the intra-particle diffusion models were used to describe the kinetic data and rate constants were evaluated.