Construction of gateway binary vector for selection with bialaphos or carboxin and GFP expression in fungi.

Genomic data has created a growing demand for tools and methodologies for studying the genes function, which can be realized through loss of function experiments (gene knockout) or by RNA silencing (knockdown). The develop-ment of binary vectors for Agrobacterium tumefaciens mediated transformation (ATMT) has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types. The selection of transformants using bialaphos and carboxin has the advantages of low cost in the transformation and availability of different selectable markers, also allowing the analysis of several genes and combination of study by knockout or knockdown, using selectable markers in the same transformant. Thus, this study aimed to build two binary vectors containing reporter gene and selectable markers that confer resistance to carboxin and bialaphos. The cassettes were constructed using the Gateway system to two fragments. The gene encoding the GFP protein and PtoxA and PtrpCpromoters were cloned into pDONR P1-P5R plasmid. Genes that confer bialaphos and carboxin resistance, bar and cbxr respectively, were cloned into pDONR P5-P2 plasmid. The pPGW plasmid was used as des-tination vector. The gfp gene transcription is controlled by PtoxA promoter and the bar and cbxr genes transcriptions are controlled by PtrpC promoter. These binary vectors were named pGWGFP-BAR and pGWGFP-CBXR. The assembly of cassettes was confi rmed by sequencing, and the validation of vectors is being accomplished through transformation (ATMT) with the plant pathogens Mycosphaerella fi jiensis and Fusarium oxysporum f. sp. cubense

Evolution of Chagas’ disease in Brazil. Epidemiological perspective and challenges for the future: a critical review

Aims: This paper aimed to provide a critical review of the evolution of Chagas’ disease in Brazil, its magnitude, historical development and management, and challenges for the future. Methods: A literature search was performed using PubMed, SciELO and Google Scholar and throughout collected articles’ references. Narrative analysis was structured around five main themes identified: vector transmission, control program, and transfusion, oral and congenital transmission. Results: In Brazil, the Chagas’ disease Control Program was fully implemented in the 1980s, when it reached practically all the endemic areas, and in 1991, the Southern Cone Initiative was created, aiming to eliminate the disease transmission through eliminating the Triatoma infestans and controlling blood banks. As a result, the prevalence of chagasic donors in blood banks reduced from 4.4% in the 80s to 0.2% in 2005. In 2006, PAHO certified the interruption of transmission of Chagas’ disease through this vector in Brazil. However, there are still challenges, such as the domiciliation of new vector species, the need for medical care of the infected individuals, the prevention of alternative mechanisms of transmission, the loss of political concern regarding the disease and, the weakening of the control program. Conclusion: Despite the progress towards control, there are still many challenges ahead to maintain and expand such control and minimise the risk of re-emergence

Cinética ruminal de espécies forrageiras nativas da caatinga

This study aimed at evaluating the kinetics of in situ ruminal degradation of the dry matter (DM), crude protein (CP) and neutral detergent fiber (NDF) of five local forage species from the semi-arid region of Paraíba (Brazil): “Orelha de Onça” (Macroptilium Martii Benth), “Amor de Vaqueiro” (Desmodium canum), “Feijão Bravo” (Capparis Flexuosa), “Maniçoba” (Manihot Epruinosa) and “Imburana de Cambão” (Camniphora leptophloes). Four two-year-old male sheep, castrated, belonging to the breed Santa Inês, with an average weight of 40 kg, previously rumen-fistulated, were distributed in an entirely randomized statistical design, with subdivided portion. The animals were housed in individual stalls and fed ad libitum with diets containing hays of the studied forages and a mineral supplementation. The foods were ground, put into nylon bags and incubated during 0, 6, 12, 24, 48 and 96 hours. Chemical-bromatological analyses of the forages were carried out to determine  in situ ruminal degradation. The solubility of DM was larger for “Feijão Bravo” hay. For the potentially degradable fraction of DM, "Maniçoba" hay presented the highest average value. Compared to the other forages, "Maniçoba" and “Feijão Bravo” hays showed the lowest percentage of the undegradable fraction (P <0,01), but the highest PD and ED (2-5%/h) of DM  (P <0,01). “Feijão Bravo” hay presented the highest rates of PD and ED (2-5%/h) of CP. Among the  forages examined, "Maniçoba" and “Feijão Bravo” hays presented the best potential for feeding. The degradability in the DM, CP and NDF indicated the best potential of use for “Feijão Bravo” and "Maniçoba", since the nutritional quality and efficiency of utilization in the rumen was higher for these forages.Este trabalho tem como objetivo avaliar a cinética da degradação ruminal in situ da matéria seca (MS), proteína bruta (PB) e fibra em detergente neutro (FDN) de cinco espécies forrageiras nativas: orelha de onça (Macroptilium Martii Benth),  amor de vaqueiro (Desmodium canum), feijão bravo (Capparis Flexuosa), maniçoba (Manihot Epruinosa) e imburana de cambão (Camniphora  leptophloes) do semi-árido paraibano. Quatro ovinos machos castrados, de dois anos de idade, da  raça Santa Inês, com peso médio de 40 kg previamente canulados no rúmen, foram distribuídos em um delineamento experimental inteiramente casualizado, com parcela subdividida. Os animais foram alojados em baias individuais e alimentados à vontade com dietas contendo os fenos das forrageiras estudadas e suplementação mineral. Os alimentos foram moídos, colocados em sacos de náilon e incubados nos tempos 0, 6, 12, 24, 48 e 96 horas. Foram realizadas análises químico-bromatológicas (MS, PB e FDN) das forrageiras para determinação da degradação ruminal  in situ. A solubilidade da MS foi maior (P>0,01) para o feno de feijão bravo. Quanto à fração potencialmente degradável da MS, o feno de maniçoba apresentou o maior valor médio. Os fenos de maniçoba e de feijão bravo apresentaram um menor percentual da fração não degradável entre as forrageiras avaliadas (P<0,01). O feno de maniçoba e feijão bravo apresentaram DP e DE (2-5%/h) da MS superior (P<0,01) às demais forrageiras. O feijão bravo apresentou as maiores taxas de DP e DE (2-5%/h) da PB. Os fenos das forrageiras, maniçoba e feijão-bravo, apresentaram o melhor potencial para arraçoamento dentre as forragens estudadas. A degradabilidade da MS, PB e FDN indicaram para os fenos de maniçoba e feijão bravo melhor potencial de utilização, visto que a qualidade nutritiva e a eficiência de aproveitamento no rúmen foram superiores para estas forragens

Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome.

Guarana (Paullinia cupana var. sorbilis) is a plant native to the central Amazon basin. Roasted seed extracts have been used as medicinal beverages since pre-Colombian times, due to their reputation as stimulants, aphrodisiacs, tonics, as well as protectors of the gastrointestinal tract. Guarana plants are commercially cultivated exclusively in Brazil to supply the national carbonated soft-drink industry and natural product stores around the world. In this report, we describe and discuss the annotation of 15,387 ESTs from guarana seeded-fruits, highlighting sequences from the flavonoid and purine alkaloid pathways, and those related to biotic stress avoidance. This is the largest set of sequences registered for the Sapindaceae family

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

<p>Abstract</p> <p>Background</p> <p>The sequencing of the <it>D.melanogaster </it>genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of <it>Drosophila </it>protein-coding genes contain one or more alternative exons. A recent transcription map of the <it>Drosophila </it>embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of <it>Drosophila </it>transcriptome.</p> <p>Results</p> <p>Bioinformatic analysis of 1,303 <it>Drosophila </it>ORESTES clusters identified 68 sequences derived from unannotated regions in the current <it>Drosophila </it>genome version (4.3). Of these, a set of 38 was analysed by polyA⁺northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The <it>SP212 </it>gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this <it>locus </it>is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.</p> <p>Conclusion</p> <p>Using the ORESTES methodology we identified 17 novel exons from low abundance <it>Drosophila </it>transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.</p

Measurements of Dihadron Correlations Relative to the Event Plane in Au+Au Collisions at sNN=200\sqrt{s_{NN}}=200 GeV

Dihadron azimuthal correlations containing a high transverse momentum (\pt) trigger particle are sensitive to the properties of the nuclear medium created at RHIC through the strong interactions occurring between the traversing parton and the medium, i.e. jet-quenching. Previous measurements revealed a strong modification to dihadron azimuthal correlations in Au+Au collisions with respect to \pp\ and \dAu\ collisions. The modification increases with the collision centrality, suggesting a path-length dependence to the jet-quenching effect. This paper reports STAR measurements of dihadron azimuthal correlations in mid-central (20-60\%) Au+Au collisions at \snn=200~GeV as a function of the trigger particle's azimuthal angle relative to the event plane, \phis=|\phit-\psiEP|. The azimuthal correlation is studied as a function of both the trigger and associated particle \pt. The subtractions of the combinatorial background and anisotropic flow, assuming Zero Yield At Minimum (\zyam), are described. The away-side correlation is strongly modified, and the modification varies with \phis, which is expected to be related to the path-length that the away-side parton traverses. The pseudo-rapidity (\deta) dependence of the near-side correlation, sensitive to long range \deta correlations (the ridge), is also investigated. The ridge and jet-like components of the near-side correlation are studied as a function of \phis. The ridge appears to drop with increasing \phis while the jet-like component remains approximately constant. ...Comment: 50 pages, 39 figures, 6 table

Studies of di-jet survival and surface emission bias in Au+Au collisions via angular correlations with respect to back-to-back leading hadrons

We report first results from an analysis based on a new multi-hadron correlation technique, exploring jet-medium interactions and di-jet surface emission bias at RHIC. Pairs of back-to-back high transverse momentum hadrons are used for triggers to study associated hadron distributions. In contrast with two- and three-particle correlations with a single trigger with similar kinematic selections, the associated hadron distribution of both trigger sides reveals no modification in either relative pseudo-rapidity or relative azimuthal angle from d+Au to central Au+Au collisions. We determine associated hadron yields and spectra as well as production rates for such correlated back-to-back triggers to gain additional insights on medium properties.Comment: By the STAR Collaboration. 6 pages, 2 figure

Observation of the antimatter helium-4 nucleus

High-energy nuclear collisions create an energy density similar to that of the universe microseconds after the Big Bang, and in both cases, matter and antimatter are formed with comparable abundance. However, the relatively short-lived expansion in nuclear collisions allows antimatter to decouple quickly from matter, and avoid annihilation. Thus, a high energy accelerator of heavy nuclei is an efficient means of producing and studying antimatter. The antimatter helium-4 nucleus ($^4\bar{He}$), also known as the anti-{\alpha} ($\bar{\alpha}$), consists of two antiprotons and two antineutrons (baryon number B=-4). It has not been observed previously, although the {\alpha} particle was identified a century ago by Rutherford and is present in cosmic radiation at the 10% level. Antimatter nuclei with B < -1 have been observed only as rare products of interactions at particle accelerators, where the rate of antinucleus production in high-energy collisions decreases by about 1000 with each additional antinucleon. We present the observation of the antimatter helium-4 nucleus, the heaviest observed antinucleus. In total 18 $^4\bar{He}$ counts were detected at the STAR experiment at RHIC in 10$^9$ recorded Au+Au collisions at center-of-mass energies of 200 GeV and 62 GeV per nucleon-nucleon pair. The yield is consistent with expectations from thermodynamic and coalescent nucleosynthesis models, which has implications beyond nuclear physics.Comment: 19 pages, 4 figures. Submitted to Nature. Under media embarg