Estudio del perfil de energia libre de iones de K+ en el canal de potasio task-2

52 p.TASK-2 es un canal de potasio perteneciente a la familia KCNK, la que se caracteriza por formar dímeros. Cada subunidad contiene cuatro segmentos transmembranales y dos dominios de poro (P). TASK-2 pertenece a la subfamilia de Canales alcalino-activados. Experimentalmente se han propuesto varios sensores de pH para TASK-2. Nuestro grupo demostró que la neutralización de una Arginina (ARG224) cercana al poro, es el sensor de pH de TASK-2.Estudiando la conductancia de los iones de potasio en TASK-2, se puede deducir que las perturbaciones estructurales y energéticas generadas por el cambio del estado de protonación de los residuos sensores regulan el nivel de ocupancia y/o estabilidad conformacional del filtro de selectividad, lo que además podría generar la inactivación tipo C del canal. Para este estudio se aplicó el método ABF (Adaptive Biasing Force) implementado en NAMD. En los cálculos de ABF realizados para obtener el perfil de energía libre, se implementó una seria de restricciones sobre los iones de potasio alternados con agua, estos fueron ubicados en sitios específicos en el filtro de selectividad. Se realizaron simulaciones para los distintos estados de protonación de la ARG224.Los resultados obtenidos sugieren que trasladar el ión desde el sitio S0 con ambas ARG224 neutras presenta una menor barrera energética que con ambas ARG224 cargadas, lo que se correlaciona con los resultados experimentales observados. Estos resultados permiten analizar a nivel atómico los eventos que gobiernan la activación de TASK-

Characterization of novel genic SSR markers in Linum usitatissimum (L.) and their transferability across eleven Linum species

Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3%) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1% to 92.7% in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies

Genome diversification within a clonal population of pandemic <i>Vibrio parahaemolyticus </i>seems to depend on the life circumstances of each individual bacteria

Artículo de publicación ISIBackground: New strains of Vibrio parahaemolyticus that cause diarrhea in humans by seafood ingestion periodically emerge through continuous evolution in the ocean. Influx and expansion in the Southern Chilean ocean of a highly clonal V. parahaemolyticus (serotype O3:K6) population from South East Asia caused one of the largest seafood-related diarrhea outbreaks in the world. Here, genomics analyses of isolates from this rapidly expanding clonal population offered an opportunity to observe the molecular evolutionary changes often obscured in more diverse populations. Results: Whole genome sequence comparison of eight independent isolates of this population from mussels or clinical cases (from different years) was performed. Differences of 1366 to 217,729 bp genome length and 13 to 164 bp single nucleotide variants (SNVs) were found. Most genomic differences corresponded to the presence of regions unique to only one or two isolates, and were probably acquired by horizontal gene transfer (HGT). Some DNA gain was chromosomal but most was in plasmids. One isolate had a large region (8,644 bp) missing, which was probably caused by excision of a prophage. Genome innovation by the presence of unique DNA, attributable to HGT from related bacteria, varied greatly among the isolates, with values of 1,366 (ten times the number of highest number of SNVs) to 217,729 (a thousand times more than the number of highest number of SNVs). Conclusions: The evolutionary forces (SNVs, HGT) acting on each isolate of the same population were found to differ to an extent that probably depended on the ecological scenario and life circumstances of each bacterium.FONDECYT 114073

Characterization of novel genic SSR markers in Linum usitatissimum (L.) and their transferability across eleven Linum species

Little is known about the evolutionary relationships among Linum species, basically because of the lack of transferable molecular markers. Currently, expressed sequence tags available in public databases provide an opportunity for the rapid and inexpensive development of simple sequence repeat (SSR) markers in wild flax species. In this regard, fifty expressed sequence tag-derived microsatellite markers (EST-SSRs) were evaluated for polymorphism and transferability in 50 Linum usitatissimum cultivars/accessions and 11 Linum species. Among them 23 EST-SSRs were polymorphic in L. usitatissimum, while 2-4 alleles were detected (average 2.26 per locus). The polymorphism information content value ranged from 0.08 to 0.55 (average 0.38). Forty one genic markers (95.3%) produced strong amplicons in at least two of the 11 Linum species. The percentage of cross amplification ranged from 34.1% to 92.7% in L. tauricum and L. bienne, respectively. Moreover, the rate of transferability was associated positively with the botanical section. Our results suggest that the high degree of EST-SSRs transferability to Linum species can be a useful enhancement of the current database of SSR markers for future genetic and evolutionary studies

Yellow lupin (<it>Lupinus luteus</it> L.) transcriptome sequencing: molecular marker development and comparative studies

Abstract Background Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species. Results Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession’s origin. Conclusion L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discovery, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparisons and putative gene candidates for QTL detection.</p

Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing

Abstract Microsatellites are valuable molecular markers for evolutionary and ecological studies. Next generation sequencing is responsible for the increasing number of microsatellites for non-model species. Penguins of the Pygoscelis genus are comprised of three species: Adélie (P. adeliae), Chinstrap (P. antarcticus) and Gentoo penguin (P. papua), all distributed around Antarctica and the sub-Antarctic. The species have been affected differently by climate change, and the use of microsatellite markers will be crucial to monitor population dynamics. We characterized a large set of genome-wide microsatellites and evaluated polymorphisms in all three species. SOLiD reads were generated from the libraries of each species, identifying a large amount of microsatellite loci: 33,677, 35,265 and 42,057 for P. adeliae, P. antarcticus and P. papua, respectively. A large number of dinucleotide (66,139), trinucleotide (29,490) and tetranucleotide (11,849) microsatellites are described. Microsatellite abundance, diversity and orthology were characterized in penguin genomes. We evaluated polymorphisms in 170 tetranucleotide loci, obtaining 34 polymorphic loci in at least one species and 15 polymorphic loci in all three species, which allow to perform comparative studies. Polymorphic markers presented here enable a number of ecological, population, individual identification, parentage and evolutionary studies of Pygoscelis, with potential use in other penguin species

Ezetimibe added to statin therapy after acute coronary syndromes

BACKGROUND: Statin therapy reduces low-density lipoprotein (LDL) cholesterol levels and the risk of cardiovascular events, but whether the addition of ezetimibe, a nonstatin drug that reduces intestinal cholesterol absorption, can reduce the rate of cardiovascular events further is not known. METHODS: We conducted a double-blind, randomized trial involving 18,144 patients who had been hospitalized for an acute coronary syndrome within the preceding 10 days and had LDL cholesterol levels of 50 to 100 mg per deciliter (1.3 to 2.6 mmol per liter) if they were receiving lipid-lowering therapy or 50 to 125 mg per deciliter (1.3 to 3.2 mmol per liter) if they were not receiving lipid-lowering therapy. The combination of simvastatin (40 mg) and ezetimibe (10 mg) (simvastatin-ezetimibe) was compared with simvastatin (40 mg) and placebo (simvastatin monotherapy). The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, unstable angina requiring rehospitalization, coronary revascularization ( 6530 days after randomization), or nonfatal stroke. The median follow-up was 6 years. RESULTS: The median time-weighted average LDL cholesterol level during the study was 53.7 mg per deciliter (1.4 mmol per liter) in the simvastatin-ezetimibe group, as compared with 69.5 mg per deciliter (1.8 mmol per liter) in the simvastatin-monotherapy group (P<0.001). The Kaplan-Meier event rate for the primary end point at 7 years was 32.7% in the simvastatin-ezetimibe group, as compared with 34.7% in the simvastatin-monotherapy group (absolute risk difference, 2.0 percentage points; hazard ratio, 0.936; 95% confidence interval, 0.89 to 0.99; P = 0.016). Rates of pre-specified muscle, gallbladder, and hepatic adverse effects and cancer were similar in the two groups. CONCLUSIONS: When added to statin therapy, ezetimibe resulted in incremental lowering of LDL cholesterol levels and improved cardiovascular outcomes. Moreover, lowering LDL cholesterol to levels below previous targets provided additional benefit