E1a gene expression blocks the ERK1/2 signaling pathway by promoting nuclear localization and MKP up-regulation: implication in v-H-Ras-induced senescence

In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation.     

Myocardium proteome remodelling after nutritional deprivation of methyl donors

Methyl donor (MD: folate, vitamin B12 and choline) deficiency causes hyperhomocysteinemia, a risk factor for cardiovascular diseases. However, the mechanisms of the association between MD deficiency, hyperhomocysteinemia, and cardiomyopathy remain unclear. Therefore, we performed a proteomic analysis of myocardium of pups from rat dams fed a MD-depleted diet to understand the impact of MD deficiency on heart at the protein level. Two-dimension gel electrophoresis and mass spectrometry-based analyses allowed us to identify 39 proteins with significantly altered abundance in MD-deficient myocardium. Ingenuity Pathway Analysis showed that 87% of them fitted to a single protein network associated with developmental disorder, cellular compromise and lipid metabolism. Concurrently increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins after MD deficiency. To decipher the effect of MD deficiency on the abundance of specific proteins identified in vivo, we developed an in vitro model using the cardiomyoblast cell line H9c2. After a 4-day exposure to a MD-deprived (vs. complete) medium, cells were deficient of folate and vitamin B12, and released abnormal amounts of homocysteine. Western blot analyses of pup myocardium and H9c2 cells yielded similar findings for several proteins. Of specific interest is the result showing increased and decreased abundances of prohibitin and α-crystallin B, respectively, which underlines mitochondrial injury and endoplasmic reticulum stress within MD deficiency. The in vitro findings validate the MD-deficient H9c2 cells as a relevant model for studying mechanisms of the early metabolic changes occurring in cardiac cells after MD deprivation.     

New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7^T has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7^T that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).     

Plasma catecholamines and their physiologic thresholds during the first ten days of life in sheep

We studied serial plasma catecholamine levels in healthy newborn sheep over the first ten days of life. The results show that plasma norepinephrine values in newborn sheep are 3-4 fold higher, and plasma epinephrine values are two-fold higher than values in term fetal sheep. These elevations are sustained over the first 10 days of life. Cardiovascular (heart rate and blood pressure) and metabolic parameters (glucose and free fatty acids) are also significantly elevated above fetal levels. We performed graded catecholamine infusions in newborn animals and adult ewes to determine the minimum plasma catecholamine concentrations necessary for discernible physiologic effects. In response to step-wise increases in epinephrine or norepinephrine infusion rates, there were immediate increases in blood pressure and other physiologic responses. This pattern was seen in both newborn and adult animals, and differed from previous observations in fetal sheep where log-linear, dose response curves characteristic of a threshold response were seen. These results suggest that during the first two weeks of life plasma catecholamine levels are elevated above the threshold value for physiologic responses. These sustained elevations in circulating catecholamines are important in the maintenance of physiologic homeostasis.     

Hybridization and speciation

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.     

Obesity and body fat classification in the metabolic syndrome: Impact on cardiometabolic risk metabotype

Objective:

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by BMI and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study).

Design and Methods:

Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules, and hemostatic factors were determined at baseline and after 12 weeks of four dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA), and two low fat high complex carbohydrate (LFHCC) diets, one supplemented with long chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFAs)).

Results:

About 39 and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (≥30 kg/m²) and BF% (≥25% (men) and ≥35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as nonobese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more proinflammatory (higher C reactive protein (CRP) and leptin), prothrombotic (higher plasminogen activator inhibitor‐1 (PAI‐1)), proatherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA‐IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumor necrosis factor‐α (TNF‐α) concentrations were lower post‐intervention in NOO individuals compared with OO subjects (P < 0.001).

Conclusions:

In conclusion, assessing BF% and BMI as part of a metabotype may help to identify individuals at greater cardiometabolic risk than BMI alone.     

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus–oocyte complexes (COCs; n = 4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44 h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24 h with 0 or 10 μM forskolin, achieving a 2 × 2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n = 469) or kept fresh (n = 546). Fresh and vitrified-warmed blastocysts were cultured for 24 h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P < 0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P < 0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10 μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.