Nonenzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in Arabidopsis.

In higher plants such as Arabidopsis thaliana, omega-3 trienoic fatty acids (TFAs), represented mainly by alpha-linolenic acid, serve as precursors of jasmonic acid (JA), a potent lipid signal molecule essential for defense. The JA-independent roles of TFAs were investigated by comparing the TFA- and JA-deficient fatty acid desaturase triple mutant (fad3-2 fad7-2 fad8 (fad3 fad7 fad8)) with the aos (allene oxide synthase) mutant that contains TFAs but is JA-deficient. When challenged with the fungus Botrytis, resistance of the fad3 fad7 fad8 mutant was reduced when compared with the aos mutant, suggesting that TFAs play a role in cell survival independently of being the precursors of JA. An independent genetic approach using the lesion mimic mutant accelerated cell death2 (acd2-2) confirmed the importance of TFAs in containing lesion spread, which was increased in the lines in which the fad3 fad7 fad8 and acd2-2 mutations were combined when compared with the aos acd2-2 lines. Malondialdehyde, found to result from oxidative TFA fragmentation during lesion formation, was measured by gas chromatography-mass spectrometry. Its levels correlated with the survival of the tissue. Furthermore, plants lacking TFAs overproduced salicylic acid (SA), hydrogen peroxide, and transcripts encoding several SA-regulated and SA biosynthetic proteins. The data suggest a physiological role for TFAs as sinks for reactive oxygen species.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N′-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Mètode de gestió de projectes informàtics d'adequació de programari

L'objectiu és triple: establir el cicle de vida d'un projecte i les seves diverses fases; proporcionar la metodologia, la documentació i les eines bàsiques per a gestionar el cicle de vida d'un projecte, i mirar d'extreure el màxim rendiment aprofitant la gestió d'un projecte actualment en execució.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight.

Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (approximately 40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

Software libre y Web 2.0 en la Educación Secundaria

Este proyecto ha sido pensado con un triple objetivo: Desarrollar una guía para el profesorado que desee introducirse en el uso de metodología didácticas basadas en Software Libre y la Web 2.0.Escribir un documento de consulta que sirva de base para desarrollar y utilizar diferentes herramientas de Software Libre y Web 2.0 dirigidas a cualquier etapa de la Educación Secundaria (ESO, F.P y los PCPI). Realizar un estudio del uso y conocimiento del Software Libre y la Web 2.0 entre los alumnos de los Programas de Cualificación Profesional Inicial.

Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

The hope for an HIV vaccine based on induction of CD8+ T lymphocytes: a review

The only long-term and cost-effective solution to the human immunodeficiency virus (HIV) epidemic in the developing world is a vaccine that prevents individuals from becoming infected or, once infected, from passing the virus on to others. There is currently little hope for an AIDS vaccine. Conventional attempts to induce protective antibody and CD8+ lymphocyte responses against HIV and simian immunodeficiency virus (SIV) have failed. The enormous diversity of the virus has only recently been appreciated by vaccinologists, and our assays to determine CD8+ lymphocyte antiviral efficacy are inadequate. The central hypothesis of a CTL-based vaccine is that particularly effective CD8+ lymphocytes directed against at least five epitopes that are derived from regions under functional and structural constraints will control replication of pathogenic SIV. This would be somewhat analogous to control of virus replication by triple drug therapy or neutralizing antibodies.

Colegio Nuestra Señora del Carmen-Ikastetxea

Este trabajo se centra en la atención a la diversidad, enfocado en aquellos alumnos con necesidades educativas especiales por dificultades de aprendizaje, por trastorno de déficit de atención, dislexia, inteligencia límite (bordeline) y síndrome de triple X. Con él también se pretende la adaptación de niños de nacionalidad china a nuestros hábitos y se les ilustra en el método silábico para que aprendan el idioma del castellano con prontitud para evitar la dislexia, entre otros problemas.

The Management of Patients on Oral Anticoagulation Undergoing Coronary Stent Implantation: A Survey among Interventional Cardiologists from Eight European Countries.

Purpose:  To evaluate the current management, and adherence to recommendations, of patients on oral anticoagulation (OAC) undergoing coronary stent implantation (PCI-S). Methods:  By means of a contact person who had been previously identified in 8 European countries, a questionnaire was electronically forwarded between April and July 2010 to the national institutions where PCI-S is performed. Results:  A total of  202 questionnaires (median response rate: 50%, range 33-78%) was received. The prevalence of OAC patients among those undergoing PCI-S is mostly reported 5-10% (97%). The peri-procedural pharmacological management mostly encompasses: preprocedural OAC interruption and bridging with low-molecular-weight heparin (59%), intraprocedural administration of an unfractionated heparin bolus (81%), and use of glycoprotein IIb/IIIa inhibitors on an individual basis (79%). The radial approach is reported as the preferred option (58%), as well as the implantation of bare metal stents (76%). Triple therapy (warfarin, aspirin, clopidogrel) is the most frequently prescribed (80%), generally for 1 month after bare metal stent (77%) and for at least 12 months after drug-eluting stent (60%). Throughout triple therapy, the International Normalized Ratio is mostly targeted to the lower end of the therapeutic range (77%), and gastric protection is routinely prescribed (69%), mostly by giving proton-pump inhibitors (70%). Conclusions:  Among the 202 interventional cardiologists from the 8 European countries interviewed, the management of patients on OAC undergoing PCI-S appears variable and only partially adherent to currently available recommendations. (J Interven Cardiol 2012;25:163-169).

Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

Present situation and new strategies for Chagas disease chemotherapy: a proposal

Treatments for Chagas disease have been administered since the first attempts by Mayer & Rocha Lima (1912, 1914) and up to the drugs currently in use (nifurtimox and benznidazole), along with potential drugs such as allopurinol and first, second and third-generation antifungal agents (imidazoles and triazoles), in separate form. Several diseases such as tuberculosis, leprosy and AIDS only came under control after they were treated with associations of drugs with different mechanisms of action. This not only boosts the action of the different compounds, but also may avoid the development of parasite resistance .To this end, over the short term, we propose experimental studies on laboratory animals and clinical trials with the following associations: (i) nifurtimox (8 mg/kg/day) + benznidazole (5 mg/kg/day) x 60 consecutive days; (ii) nifurtimox (8 mg/kg/day) or benznidazole (5 mg/kg/day) + allopurinol (8-10 mg/kg/day) x 60 days and (iii) nifurtimox (8 mg/kg/day) or benznidazole (5 mg/kg/day) + ketoconazole, fluconazole or itraconazole (5-6 mg/kg/day) x 60 consecutive days. The doses of the drugs and the treatment schedules for the clinical trials must be adapted according to the side effects. From these, other double or triple associations could be made, using drugs with different mechanisms of action. This proposal does not exclude investigations on new drugs over the median and long terms, targeting other aspects of the metabolism of Trypanosoma cruzi. Until such time as the ideal drug for specific treatment of Chagas disease might be discovered, we need to develop new strategies for achieving greater efficacy with the old drugs in associations and to develop rational experimentation with new drugs.

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock.

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed.