Characterization In Vitro and In Vivo of a Pandemic H1N1 Influenza Virus from a Fatal Case

Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared in vitro and in vivo. The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).     

The Tomato Calcium Sensor Cbl10 and Its Interacting Protein Kinase Cipk6 Define a Signaling Pathway in Plant Immunity

Ca²⁺ signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca²⁺-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca²⁺, suggesting that tomato Cbl10 and Cipk6 constitute a Ca²⁺-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca²⁺ and ROS signaling in plant immunity.     

Alternative markers for the long-term detection of oral testosterone misuse

The screening of testosterone misuse in the doping control field is normally performed by the measurement of the ratio between the concentrations of testosterone and epitestosterone excreted as glucuronides (T/E). Despite the satisfactory results obtained with this approach, the measurement of T/E presents some limitations like the long-term detection of oral testosterone administration. Recently, several testosterone metabolites released after basic treatment of the urine have been reported (androsta-1,4-dien-3,17-dione, androsta-4,6-dien-3,17-dione, 17β-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione). In the present work, the usefulness of these metabolites for the detection of oral testosterone misuse has been evaluated and compared with the conventional T/E measurement. For this purpose, 173 urine samples collected from healthy volunteers were analysed in order to obtain reference concentrations for the four metabolites released after alkaline treatment. On the other hand, urine samples collected from five volunteers before and after testosterone undecanoate administration were also analysed. Concentrations of androsta-4,6-dien-3,17-dione and 17β-hydroxy-androsta-4,6-dien-3-one showed a similar behaviour as the T/E, allowing the detection of the misuse for several hours after administration. More promising results were obtained by quantifying androsta-1,4-dien-3,17-dione and 15-androsten-3,17-dione. The time in which the concentrations of these analytes could be differentiated from the basal level was between 3 and 6 times longer than the obtained with T/E, as a result, an improvement in the detection of testosterone abuse can be achieved. Moreover, several ratios between these compounds were evaluated. Some of them improved the detection of testosterone misuse when comparing with T/E. The best results were obtained with those ratios involving androsta-1,4-dien-3,17-dione.     

Ecological indicators of habitat and biodiversity in a Neotropical landscape: multitaxonomic perspective

Ecological indicators of habitat and biodiversity in a Neotropical landscape: multitaxonomic perspective. The use of indicator species to characterize specific ecological areas is of high importance in conservation/restoration biology. The objective of this study was to identify indicator species of diverse taxa that characterize different landscape units, and to better understand how management alters species composition. We identified two ecomosaics, tropical rain forest and the agricultural matrix, each one comprised of four landscape units. The taxonomic groups studied included birds (highly mobile), butterflies (moderately mobile), terrestrial gastropods (less mobile) and trees (sessile). Sampling efficiency for both ecomosaics was > or = 86%. We found 50 mollusks, 74 butterflies, 218 birds and 172 tree species, for a total of 514 species. Using ordination and cluster analysis, we distinguished three habitat types in the landscape: tropical rainforest, secondary vegetation and pastures with scattered trees and live fences. The InVal (> or = 50%) method identified 107 indicator species, including 45 tree species, 38 birds, 14 butterflies and 10 gastropods. Of these, 35 trees, 10 birds, four butterflies and eight gastropods were forest indicators. Additionally, 10, 28, 10 and two species, respectively per group, were characteristic of the agricultural matrix. Our results revealed a pattern of diversity decrease of indicator species along the rainforest-secondary forest-pasture gradient. In the forest, the gastropods Carychium exiguum, Coelocentrum turris, Glyphyalinia aff. indentata y Helicina oweniana were significantly correlated (p < 0.05) with 90% of the other groups of flora and fauna indicator species. These findings suggest that gastropods may be good indicators of forest habitat quality and biodiversity. The secondary vegetation is an intermediate disturbance phase that fosters high diversity in the agricultural matrix. We exemplify a multitaxa approach, including mesofauna, for ecological monitoring of agricultural landscapes.     

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1^−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1^−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1^−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1^−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1^+/+ cells, which was not the case in Cav1^−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.The transforming growth factor-beta (TGF-β) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. However, paradoxically, TGF-β also modulates processes such as cell invasion, immune regulation and microenvironment modification, which cancer cells may exploit to their advantage.¹ Indeed, a better knowledge about the mechanistic basis and clinical relevance of TGF-β is required for a better understanding of the complexity and therapeutic potential of this pathway. In hepatocytes, TGF-β induces both pro- and anti-apoptotic signals, whose balance decides cell fate.² Those hepatocytes that survive to TGF-β-mediated apoptosis induce an epithelial–mesenchymal transition (EMT) process, which confers migratory properties and apoptosis resistance.³ The anti-apoptotic signals are at least partially mediated by the epidermal growth factor receptor (EGFR) pathway, which is transactivated by TGF-β through a mechanism that involves upregulation of the EGFR ligands and activation of the metalloprotease TACE/ADAM17 responsible for their shedding.^{4, 5}Many efforts have been done in the recent years for a better understanding of spatial requirements on TGF-β signaling, including endocytic TGF-β receptors trafficking. Strong pieces of evidence support that TGF-β receptors can be located both in clathrin-coated pits and caveolin/cholesterol-enriched lipid rafts.^{6, 7} A pioneer study from Di Guglielmo et al.⁸ proposed that Smad2 phosphorylation would require clathrin-dependent endocytosis, whereas TGF-β receptors internalization via caveolae/lipid rafts would inhibit its signaling. From then, different studies have suggested that the endocytic pathways'' role on TGF-β signaling depend on the cell type and a general rule about the role of endocytosis in TGF-β signaling is not well understood yet.⁹ In hepatocytes, Smad activation is in a large extent accomplished on the hepatocyte plasma membrane in an AP-2 complex-dependent manner, being unnecessary the formation of clathrin-coated pits.¹⁰ In contrast, the non-Smad/AKT pathway activation requires caveolin-1 (Cav1)-dependent endocytosis,¹⁰ which is required for counteracting apoptosis.¹¹Cav1 is required for caveolae formation, which regulates not only endocytosis, but also lipid metabolism and energy homeostasis.¹² The localization of membrane receptors in the lipid raft pushed to investigate the role of Cav1 in regulating signaling events. In the case of epidermal growth factor (EGF) signaling, it was proposed that non-caveolae-coated pits are involved in the compartmentalization and internalization of the EGFR, although caveolin-rich domains may be required for signaling.¹³ In this line of evidence, different studies revealed an important role for Cav1 in EGFR-induced effects on cell proliferation and migration.^{14, 15}In this work, we have examined the role of Cav1 in the anti-apoptotic signals induced by TGF-β in hepatocytes, postulating that it may be required for TGF-β-mediated regulation of EGFR signaling. Using different experimental approaches and an immortalized hepatocyte cell line derived from Cav1^−/− mice, we demonstrate that transactivation of the EGFR pathway by TGF-β is impaired in Cav1^−/− hepatocytes. However, Cav1 is not required for the cellular response to EGFR ligands, but is necessary for TGF-β-mediated activation of the metalloprotease TACE/ADAM17, which is responsible for shedding of EGFR ligands and requires an intact lipid raft domain to be activated by TGF-β.     

Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower-living yeast

In addition to genetic differences between individuals as a result of nucleotide sequence variation, epigenetic changes that occur as a result of DNA methylation may also contribute to population niche width by enhancing phenotypic plasticity, although this intriguing possibility remains essentially untested. Using the nectar‐living yeast Metschnikowia reukaufii as study subject, we examine the hypothesis that changes in genome‐wide DNA methylation patterns underlie the ability of this fugitive species to exploit a broad resource range in its heterogeneous and patchy environment. Data on floral nectar characteristics and their use by M. reukaufii in the wild were combined with laboratory experiments and methylation‐sensitive amplified polymorphism (MSAP) analyses designed to detect epigenetic responses of single genotypes to variations in sugar environment that mimicked those occurring naturally in nectar. M. reukaufii exploited a broad range of resources, occurring in nectar of 48% of species and 52% of families surveyed, and its host plants exhibited broad intra‐ and interspecific variation in sugar‐related nectar features. Under experimental conditions, sugar composition, sugar concentration and their interaction significantly influenced the mean probability of MSAP markers experiencing a transition from unmethylated to methylated state. Alterations in methylation status were not random but predictably associated with certain markers. The methylation inhibitor 5‐azacytidine (5‐AzaC) had strong inhibitory effects on M. reukaufii proliferation in sugar‐containing media, and a direct relationship existed across sugar × concentration experimental levels linking inhibitory effect of 5‐AzaC and mean per‐marker probability of genome‐wide methylation. Environmentally induced DNA methylation polymorphisms allowed genotypes to grow successfully in extreme sugar environments, and the broad population niche width of M. reukaufii was largely made possible by epigenetic changes enabling genotype plasticity in resource use.