Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc- finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1D the transcription of ADH2 in a ScAdr1-dependent fashion

Transmissão oral da doença de Chagas pelo consumo de açaí: um desafio para a Vigilância Sanitária

The occurrence of acute Chagas disease (ACD) by oral transmission related to food consumption, untill 2004, was a little known or investigated event. Currently becomes frequent in the Amazon region and is related to the occurrence of recent outbreaks in several states.Recent cases in Brazil of ACD are related to the consumption of acai juice, considered essencial food in the diet of the population in the Northern Region. The acai berry has been appreciated both for its nutritional value, as by its characteristic flavor, ceasing to be consumed in the producing regions, extending throughout the brazilian territory and even exported to other countries.The aim of this study was to highlight the new challenges faced by public health agencies in preventing the transmission of Chagas disease by consumption of açaí. Although there are important strategies being implemented by Brazil to combat Chagas disease transmitted via food, there is still a need for incentives to search for that knowledge generated assist in understanding the oral transmission of this parasite and its better interpretation of epidemiological, prevention and control. Besides the implementation of Good Hygiene Practices, Good Manufacturing Practices and the rapprochement between science institutions and producers of acai to contribute in solving this problem.Até o ano de 2004, a ocorrência de doença de Chagas aguda (DCA) por transmissão oral, relacionada ao consumo de alimentos, constituía um evento pouco conhecido ou investigado. Atualmente tornou-se frequente na região amazônica e está relacionada à ocorrência de surtos recentes em diversos estados brasileiros.Os casos recentes notificados no Brasil de DCA estão relacionados ao consumo do suco de açaí, considerado um alimento essencial na dieta da população da Região Norte e muito apreciado nos demais estados brasileiros e em outros países.O objetivo deste trabalho foi destacar os novos desafios a serem enfrentados pelos órgãos de saúde pública na prevenção da transmissão da doença de Chagas pelo consumo de açaí.Apesar de existirem importantes estratégias sendo implementadas pelo Brasil no combate à doença de Chagas transmitida via alimento, ainda há a necessidade de incentivos à pesquisa para que conhecimentos gerados auxiliem na compreensão da transmissão oral e sua melhor interpretação epidemiológica, de prevenção e controle. A implementação das Boas Práticas de Higiene, Boas Práticas de Manufatura e a aproximação entre instituições de ciência e os produtores de açaí também poderão contribuir na solução deste problema

Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5

Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively

Desenvolvimento de itens de ensaio de proficiência para pesquisa de Salmonella spp. em matriz chocolate

The aim of this study was to develop lyophilized test items (TI) containing Salmonella spp., in chocolate matrix to be used in proficiency testing programs (PTP). Microbial analysis was conducted on samples of granulated chocolate to verify that the sample was free of the target microorganisms. Homogeneity and stability studies in long and short term were carried out to monitor TI quality; the presence of vacuum in the samples was also verified, to ensure the efficiency of the lyophilization process. The results of the microbial testing indicated no contamination by Salmonella spp.; thus, the sample was appropriate to be used as matrix. The lyophilization technique, using trehalose as cryoprotectant, has proven to be effective for desiccation of TI produced. The Salmonella batch proved to be sufficiently homogeneous, because the microorganism was present in all analyzed flasks. The batch was held stable at -20°C (five weeks) and -70°C (26 weeks). As for the transportation stability, the batch was considered stable at 4°C (in four days). The TI produced batch in this study showed a quality level that makes it suitable to be used in PTP, to contribute to the increasing reliability of the test results from laboratories and to provide subsidies for identification of problems and troubleshooting.O objetivo desse estudo foi desenvolver itens de ensaio (IE) liofilizados contendo Salmonella spp., em matriz chocolate, para utilização em ensaio de proficiência (EP). Foi realizada a análise microbiológica de uma amostra de chocolate granulado para verificar se estava livre do micro-organismo alvo. Para monitoramento da qualidade dos IE, realizou-se estudos de homogeneidade e estabilidade em longo e curto prazo, bem como verificou-se a presença de vácuo nas amostras garantindo a eficiência do processo de liofilização. A análise microbiológica do chocolate indicou ausência de contaminação por Salmonella spp., estando apto para ser utilizado como matriz. A técnica de liofilização, com uso de trealose como crioprotetor, se mostrou eficaz para dessecação dos IE produzidos. O lote produzido se apresentou suficientemente homogêneo, pois o micro-organismo estava presente em todos os frascos analisados. O lote se apresentou estável à temperatura de -20ºC (em cinco semanas) e -70ºC (em 26 semanas); na estabilidade de transporte, foi considerado estável a 4ºC (em quatro dias). O lote de IE produzido nesse estudo apresentou qualidade que o torna apto para uso em EP, o que visou contribuir para o aumento da confiabilidade dos resultados das análises dos laboratórios e propiciar subsídios para a identificação e solução de problemas

From influence to impact: the multifunctional land-use in Mediterranean prehistory emerging from palynology of archaeological sites (8.0-2.8 ka BP)

Archaeobotany is used to discover details on local land uses in prehistoric settlements developed during the middle and beginning of late Holocene. Six archaeological sites from four countries (Spain, Italy, Greece, and Turkey) have pollen and charcoal records showing clear signs of the agrarian systems that had developed in the Mediterranean basin during different cultural phases, from pre-Neolithic to Recent Bronze Age. A selected list of pollen taxa and sums, including cultivated trees, other woody species, crops and annual or perennial synanthropic plants are analysed for land use reconstructions. In general, cultivation has a lower image in palynology than forestry, and past land uses became visible when oakwoods were affected by human activities. On-site palynology allows us to recognise the first influence of humans even before it can be recognised in off-site sequences, and off-site sequences can allow us to determine the area of influence of a site. Neolithic and Bronze Age archaeological sites show similar land use dynamics implying oak exploitation, causing local deforestation, and cultivation of cereal fields in the area or around the site. Although a substantial difference makes the Neolithic influence quite distant from the Bronze Age impact, mixed systems of land exploitation emerged everywhere. Multiple land use activities exist (multifunctional landscapes) at the same time within the area of influence of a site. Since the Neolithic, people have adopted a diffuse pattern of land use involving a combination of diverse activities, using trees\u2013crops\u2013domesticated animals. The most recurrent combination included wood exploitation, field cultivation and animal breeding. The lesson from the past is that the multifunctional land use, combining sylvo-pastoral and crop farming mixed systems, has been widely adopted for millennia, being more sustainable than the monoculture and a promising way to develop our economy

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements