The branch-site test of positive selection is surprisingly robust but lacks power under synonymous substitution saturation and variation in GC

Positive selection is widely estimated from protein coding sequence alignments by the nonsynonymous-to-synonymous ratio omega. Increasingly elaborate codon models are used in a likelihood framework for this estimation. Although there is widespread concern about the robustness of the estimation of the omega ratio, more efforts are needed to estimate this robustness, especially in the context of complex models. Here, we focused on the branch-site codon model. We investigated its robustness on a large set of simulated data. First, we investigated the impact of sequence divergence. We found evidence of underestimation of the synonymous substitution rate for values as small as 0.5, with a slight increase in false positives for the branch-site test. When dS increases further, underestimation of dS is worse, but false positives decrease. Interestingly, the detection of true positives follows a similar distribution, with a maximum for intermediary values of dS. Thus, high dS is more of a concern for a loss of power (false negatives) than for false positives of the test. Second, we investigated the impact of GC content. We showed that there is no significant difference of false positives between high GC (up to similar to 80%) and low GC (similar to 30%) genes. Moreover, neither shifts of GC content on a specific branch nor major shifts in GC along the gene sequence generate many false positives. Our results confirm that the branch-site is a very conservative test.

Earthworm communities in organic and conventional coffee cultivation

The objective of this work was to evaluate the effect of organic and conventional coffee crops on biomass, population density and diversity of earthworms, in Lerroville, district of Londrina County, Paraná state, Brazil. Earthworm communities were sampled in three areas with organic coffee cultivation (CO1, CO2 and CO3), two with conventional coffee (CC1 and CC2), and a native forest fragment (MT). The soil of the areas CO1, CC1, and MT was classified as Nitossolo Vermelho (Rhodic Kandiudox), while CO2, CO3, and CC2 were on Latossolo Vermelho (Rhodic Hapludox). Eight samples were taken in each area on two occasions, winter and summer, using the Tropical Soil Biology and Fertility (TSBF) method in the 0-20 cm soil layer. The earthworms were handsorted and preserved in 4% formaldehyde, and were later weighed, counted and identified. The highest earthworm biomass, both in winter and summer, occurred in the CO3 area. For population density, the higher numbers of individuals were found in CO1 and CO3. The highest number of species was identified in the organic cultivation. The adoption of organic practices in coffee cultivation favored the diversity, density and biomass of earthworm communities.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Anti-angiogenic therapies in cancer: achievements and open questions.

The approval in 2004 of bevacizumab (Avastin), a neutralizing monoclonal antibody directed against vascular endothelial growth factor (VEGF) as the first anti-angiogenic systemic drug to treat cancer patients validated the notion introduced 33 years earlier by Dr. Judah Folkman, that inhibition of tumor angiogenesis might be a valid approach to control tumor growth. Anti-angiogenic therapy was greeted in the clinic a major step forward in cancer treatment. At the same time this success recently boosted the field to the quest for new anti-angiogenic targets and drugs. In spite of this success, however, some old questions in the field have remained unanswered and new ones have emerged. They include the identification for surrogate markers of angiogenesis and anti-angiogenesis, the understanding about how anti-angiogenic therapy and chemotherapy synergize, the characterization of the biological consequences of sustained suppression of angiogenesis on tumor biology and normal tissue homeostasis, and the mechanisms of tumor escape from anti-angiogenesis. In this review we summarize some of these outstanding questions, and highlight future challenges in clinical, translational and experimental research in anti-angiogenic therapy that need to be addressed in order to improve current treatments and to design new drugs.

Proliferative and Osteogenic Differentiation Potentials of Human Fetal Bone Cells

BACKGROUND. Human primary fetal bone cells (hFBC) are being characterized for use in bone tissue regeneration. Unlike human mesenchymal stem cells (hMSC), hFBC are partially differentiated with high expansion and regeneration potential. To date, proliferative and osteoblastic differentiation capacities of fetal bone cells remain poorly examined. The goal of this study was to define an environmental culture conditions for optimal proliferation and production of extracellular bone matrix leading to efficient bone repair. METHODS. Human primary FBC derived from our dedicated, consistent banks of bone cells comprising several fetal donors. For proliferation study, monolayer cultures of both cell types were expanded in DMEM or α- MEM media. Osteoblastic differentiation potentials of both hFBC and hMSC were evaluated through RT-PCR. Regulation of osteogenic differentiation by protein ligands Wnt3a and Wnt5a was studied by ALP enzymatic activity measurement. RESULTS. Evaluation of the proliferation rate demonstrated that hFBC proliferated more rapidly in α-MEM medium. Regarding growth factors that could stimulate cell proliferation rate, we observed that PDGF, FGF2 and Wnt3a had positive effects on proliferation of hFBC. Gene expression analysis demonstrated a higher expression of runx2 in hFBC cultured in basal conditions, which was was similar than that was observed in hMSC in osteoinductive culture conditions. Expression of sox9 was very low in hBFC and hMSC, compared to expression observed in fetal cartilage cells. Looking at osteogenic differentiation capacity, ALP activity was positively regulated byWnt5awhen hFBCwere cultured inα-MEM, but not in DMEM. Conversely, Wnt3a was shown to block the effect of osteogenic inductors on differentiation of both cell types. CONCLUSION. Data presented in this study indicate that the proliferation and differentiation of fetal and mesenchymal stem cells is optimal in α- MEM. Evidence for a pre-differentiated state of hBFC was given by extracellular matrix spontaneous mineralization as well as by higher ALP activity levels observed for these cells in baseline culture conditions, in comparison with hMSC. As we showed that, in vitro, hFBC express a higher capacity to differentiate in osteoblasts, they represent an attractive and promising prospect for fundamental research, and specifically for a new generation of skeletal tissue engineering.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

Prospective study of insulin-like growth factor-I, insulin-like growth factor-binding protein 3, genetic variants in the IGF1 and IGFBP3 genes and risk of coronary artery disease.

Although experimental studies have suggested that insulin-like growth factor I (IGF-I) and its binding protein IGFBP-3 might have a role in the aetiology of coronary artery disease (CAD), the relevance of circulating IGFs and their binding proteins in the development of CAD in human populations is unclear. We conducted a nested case-control study, with a mean follow-up of six years, within the EPIC-Norfolk cohort to assess the association between circulating levels of IGF-I and IGFBP-3 and risk of CAD in up to 1,013 cases and 2,055 controls matched for age, sex and study enrolment date. After adjustment for cardiovascular risk factors, we found no association between circulating levels of IGF-I or IGFBP-3 and risk of CAD (odds ratio: 0.98 (95% Cl 0.90-1.06) per 1 SD increase in circulating IGF-I; odds ratio: 1.02 (95% Cl 0.94-1.12) for IGFBP-3). We examined associations between tagging single nucleotide polymorphisms (tSNPs) at the IGF1 and IGFBP3 loci and circulating IGF-I and IGFBP-3 levels in up to 1,133 cases and 2,223 controls and identified three tSNPs (rs1520220, rs3730204, rs2132571) that showed independent association with either circulating IGF-I or IGFBP-3 levels. In an assessment of 31 SNPs spanning the IGF1 or IGFBP3 loci, none were associated with risk of CAD in a meta-analysis that included EPIC-Norfolk and eight additional studies comprising up to 9,319 cases and 19,964 controls. Our results indicate that IGF-I and IGFBP-3 are unlikely to be importantly involved in the aetiology of CAD in human populations.

The origins, evolution, and functional potential of alternative splicing in vertebrates.

Alternative splicing (AS) has the potential to greatly expand the functional repertoire of mammalian transcriptomes. However, few variant transcripts have been characterized functionally, making it difficult to assess the contribution of AS to the generation of phenotypic complexity and to study the evolution of splicing patterns. We have compared the AS of 309 protein-coding genes in the human ENCODE pilot regions against their mouse orthologs in unprecedented detail, utilizing traditional transcriptomic and RNAseq data. The conservation status of every transcript has been investigated, and each functionally categorized as coding (separated into coding sequence [CDS] or nonsense-mediated decay [NMD] linked) or noncoding. In total, 36.7% of human and 19.3% of mouse coding transcripts are species specific, and we observe a 3.6 times excess of human NMD transcripts compared with mouse; in contrast to previous studies, the majority of species-specific AS is unlinked to transposable elements. We observe one conserved CDS variant and one conserved NMD variant per 2.3 and 11.4 genes, respectively. Subsequently, we identify and characterize equivalent AS patterns for 22.9% of these CDS or NMD-linked events in nonmammalian vertebrate genomes, and our data indicate that functional NMD-linked AS is more widespread and ancient than previously thought. Furthermore, although we observe an association between conserved AS and elevated sequence conservation, as previously reported, we emphasize that 30% of conserved AS exons display sequence conservation below the average score for constitutive exons. In conclusion, we demonstrate the value of detailed comparative annotation in generating a comprehensive set of AS transcripts, increasing our understanding of AS evolution in vertebrates. Our data supports a model whereby the acquisition of functional AS has occurred throughout vertebrate evolution and is considered alongside amino acid change as a key mechanism in gene evolution.

Body image perception and weight-related behaviour among adolescents of the Seychelles

Background: We examined one's own body image perception and its association with reported weight-related behavior among adolescents of a rapidly developing country in the African region. Methods: We conducted a school-based survey of 1432 students aged 11-17 years in the Seychelles. Weight and height were measured, and thinness, normal weight and overweight were assessed along standard criteria. A self-administered and anonymous questionnaire was administered. Perception of body image was assessed using both a closed-ended question (CEQ) and the Stunkard's pictorial silhouettes (SPS). Finally, a question assessed voluntary attempts to change weight. Results: Overall, 14.1% of the students were thin, 63.9% were normal-weight, and 22.0% were overweight or obese. There was fair agreement between actual weight status and self-perceived body image based on either CEQ or SPS. However, a substantial proportion of the overweight students did not consider themselves as overweight (SPS: 24%, CEQ: 34%) and, inversely, a substantial proportion of the normal-weight students considered themselves as too thin (SPS: 29%, CEQ: 15%). Among the overweight students, an adequate attempt to lose weight was reported more often by boys and girls who perceived themselves as overweight vs. not overweight (72-88% vs. 40-71%, p <0.05 for most comparisons). Among the normal-weight students, an inadequate attempt to gain weight was reported more often by boys and girls who perceived themselves as thin vs. not thin (27-68% vs. 11-19%, p <0.05). Girls had leaner own body ideals than boys. Conclusions: We found that substantial proportions of overweight students did not perceive themselves as overweight and/or did not want to lose weight and, inversely, that many normalweight students perceived themselves as too thin and/or wanted to gain weight: this points to forces that can drive the upwards overweight trends. Appropriate perception of one's weight was associated with adequate weight-control behavior, although not strongly, emphasizing that appropriate weight perception is only one of several factors driving adequate weight-related behavior. These findings emphasize the need to address appropriate perception of one's own weight and adequate weight-related behavior in adolescents for both individual and community weight-related interventions.

Pollination requirements of seeded and seedless mini watermelon varieties cultivated under protected environment

The objective of this work was to evaluate the floral biology and pollination requirements of seeded and seedless mini watermelon varieties, and to determine the best varieties to cultivate under protected environment. Three seedless (HA-5106, HA-5158, and HA-5161) and two seeded (Minipol and Polimore) genotypes were tested. Flowers were monitored from the pre-anthesis stage to senescence, and fruit quality was also evaluated. The evaluated treatments were hand-geitonogamous pollination (MG), cross-pollination with pollen from the Polimore variety (MCP), cross-pollination with pollen from the Minipol variety (MCM), and restricted pollination. All varieties had monoecious plants with diclinous flowers, and the stigmas remained receptive throughout anthesis. Fruit set rates of 84.62% (MG), 61.54% (MCP), 48% (MCM), and 0% (restricted) were obtained for seeded varieties, but of 0% (MG), 76.36% (MCP), 82.69% (MCM), and 0% (restricted) for seedless varieties. Fruits did not differ in quality among treatments within each genotype. Therefore, all the studied varieties require a pollination agent and diploid pollen for fruit set to occur, regardless of the donor variety; and Minipol or Polimore with HA-5106 or HA-5158 are the varieties recommended for cultivation in protected environment.

Evidence of recombination in putative ancient asexuals.

Ancient asexuals have been considered to be a contradiction of the basic tenets of evolutionary theory. Barred from rearranging genetic variation by recombination, their reduced number of gene arrangements is thought to hamper their response to changing environments. For the same reason, it should be difficult for them to avoid the build-up of deleterious mutations. Several groups of taxonomically diverse organisms are thought to be ancient asexuals, although clear evidence for or against the existence of recombination events is scarce. Several methods have recently been developed for predicting recombination events by analyzing aligned sequences of a given region of DNA that all originate from one species. The methods are based on phylogenetic, substitution, and compatibility analyses. Here we present the results of analyses of sequence data from different loci studied in several groups of evolutionarily distant species that are considered to be ancient asexuals, using seven different types of analysis. The groups of organisms were the arbuscular mycorrhizal fungi (Glomales), Darwinula stevensoni (Darwinuloidea crustacean ostracods) and the bdelloid rotifers (Bdelloidea), which are thought to have been asexual for the last 400, 25-100, and 35-40 Myr, respectively. The seven different analytical methods evaluated the evolutionary relationships among haplotypes, and these methods had previously been shown to be reliable for predicting the occurrence of recombination events. Despite the different degree of genetic variation among the different groups of organisms, at least some evidence for recombination was found in all species groups. In particular, predictions of recombination events in the arbuscular mycorrhizal fungi were frequent. Predictions of recombination were also found for sequence data that have previously been used to infer the absence of recombination in bdelloid rotifers. Although our results have to be taken with some caution because they could signal very ancient recombination events or possibly other genetic variation of nonrecombinant origin, they suggest that some cryptic recombination events may exist in these organisms.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.