A second-generation diagnostic single nucleotide polymorphism (SNP)-based assay, optimized to distinguish among eight poplar (Populus L.) species and their early hybrids

Rapid identification of Populus L. species and hybrids can be achieved with relatively little effort through the use of primer extension-based single nucleotide polymorphism (SNP) genotyping assays. We present an optimized set of 36 SNP markers from 28 gene regions that diagnose eight poplar species (Populus angustifolia James, Populus balsamifera L., Populus deltoides Bartram, Populus fremontii Watson, Populus laurifolia Ledeb., Populus maximowiczii Henry, Populus nigra L., and Populus trichocarpa Torr. & Gray). A total of 700 DNA sequences from six Populus species (1–15 individuals per species) were used to construct the array. A set of flanking and probe oligonucleotides was developed and tested. The accuracy of the SNP assay was validated by genotyping 448 putatively “pure” individuals from 14 species of Populus. Overall, the SNP assay had a high success rate (97.6 %) and will prove useful for the identification of all Aigeiros Duby and Tacamahaca Spach. species and their early-generation hybrids within natural populations and breeding programs. Null alleles and intraspecific polymorphisms were detected for a few locus/species combinations in the Aigeiros and Tacamahaca sections. When we attempted to genotype aspens of the section Populus (Populus alba L., Populus grandidentata Michx., Populus tremula L., and Populus tremuloides Michx.), the success rate of the SNP array decreased by 13 %, demonstrating moderate cross-sectional transferability.     

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.     

Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.     

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

Gut microflora is a key player in host energy homeostasis

Gut microflora is now considered as a key organ involved in host energy homeostasis. Recent data suggest that the alterations of the gut bacteria ecosystem could contribute to the development of metabolic disorders such as type 2 diabetes and obesity. First, gut microflora may increase energy efficiency of non digested food via the fermentation, thus providing more energy to the host. Secondly, fatty acids flux and storage in the adipose tissue is under the control of the fasting-induced adipocyte factor FIAF, which expression depends on gut microflora. Third, high-fat diet feeding changes gut bacteria profile, leading to a drop in bifidobacteria content, which correlates with a higher LPS plasma levels, thereby participating to the onset of inflammation, insulin resistance and type 2 diabetes associated with obesity. Changing gut microflora composition could be a useful tool to prevent or to treat high-fat/low fibres diet-induced metabolic syndrome. double dagger.     

Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Medicago truncatula is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant’s efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N₂ fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for M. truncatula was identification of two epistatic interactions in controlling plant N₂ fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions.     

Socioeconomic Inequalities in Smoking and Smoking Cessation Due to a Smoking Ban: General Population-Based Cross-Sectional Study in Luxembourg

This study aimed to measure changes in socioeconomic inequalities in smoking and smoking cessation due to the 2006 smoking ban in Luxembourg. Data were derived from the PSELL3/EU-SILC (Panel Socio-Economique Liewen Zu Letzebuerg/European Union—Statistic on Income and Living Conditions) survey, which was a representative survey of the general population aged ≥16 years conducted in Luxembourg in 2005, 2007, and 2008. Smoking prevalence and smoking cessation due to the 2006 smoking ban were used as the main smoking outcomes. Two inequality measures were calculated to assess the magnitude and temporal trends of socioeconomic inequalities in smoking: the prevalence ratio and the disparity index. Smoking cessation due to the smoking ban was considered as a positive outcome. Three multiple logistic regression models were used to assess social inequalities in smoking cessation due to the 2006 smoking ban. Education level, income, and employment status served as proxies for socioeconomic status. The prevalence of smoking decreased by 22.5% between 2005 and 2008 (from 23.1% in 2005 to 17.9% in 2008), but socioeconomic inequalities in smoking persisted. Smoking prevalence decreased by 24.2% and 20.2% in men and women, respectively; this difference was not statistically significant. Smoking cessation in daily smokers due to the 2006 smoking ban was associated with education level, employment status, and income, with higher percentages of quitters among those with a lower socioeconomic status. The decrease in smoking prevalence after the 2006 law was also associated with a reduction in socioeconomic inequalities, including differences in education level, income, and employment status. Although the smoking ban contributed to a reduction of such inequalities, they still persist, indicating the need for a more targeted approach of smoke-free policies directed toward lower socioeconomic groups.     

Magnesium supplementation and deoxycorticosterone acetate--salt hypertension: effect on arterial mechanical properties and on activity of endothelin-1

The aim of this study was to show whether the decrease in blood pressure induced by Mg supplementation in deoxycorticosterone acetate - salt (DOCA-salt) hypertensive rats is associated with mechanical modifications of blood vessels and (or) changes in tissular production and (or) vasoconstrictor activity to endothelin-1. DOCA-salt treatment increased blood pressure, media thickness, cross-sectional area, and lumen diameter of carotid arteries. Distensibility and incremental elastic modulus versus stress were not altered in carotid arteries, suggesting that the DOCA-salt vessel wall adapts structurally to preserve its blood pressure buffering capacity. Magnesium supplementation attenuated DOCA-salt hypertension. In comparison with normotensive rats, systolic, mean, and pulse pressures were higher whereas diastolic pressure was not different in Mg-supplemented DOCA-salt rats. Magnesium supplementation did not significantly modify the elastic parameters of carotid arteries. In resistance mesenteric arteries, DOCA-salt hypertension induces an inward hypertrophic remodeling. Magnesium supplementation attenuates wall hypertrophy and increases lumen diameter to the normotensive diameter, suggesting a decrease in peripheral resistance. Magnesium supplementation normalizes the altered vasoconstrictor activity of endothelin-1 in mesenteric arteries and attenuates endothelin-1 overproduction in kidney, left ventricle, and aorta of DOCA-salt rats. These findings suggest that Mg supplementation prevents blood pressure elevation by attenuating peripheral resistance and by decreasing hypertrophic effect of endothelin-1 via inhibition of endothelin-1 production.     

Effects of deletion of the prolactin receptor on ovarian gene expression

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.