A metagenômica trouxe novas perspectivas ao estudo de comunidades microbianas no ambiente, permitindo explorar tanto a diversidade taxonômica de microrganismos ainda não-cultivados, como o acesso direto a genes e vias metabólicas. Neste trabalho, foram construídas bibliotecas metagenômicas a partir de amostras de sedimentos de mangue da Baía de Guanabara (RJ), impactadas com hidrocarbonetos de petróleo e metais pesados. Proteobacteria (33,3%), bactérias afiliadas a redutoras-de-sulfato (29,7%) e Firmicutes (20%) representaram os grupos principais nas amostras ambientais, baseado em análises filogenéticas de rDNA 16S, ao passo que isolamentos seletivos utilizando diesel e naftaleno permitiram a recuperação preferencial de delta-Proteobacteria e actinomicetos. Bibliotecas metagenômicas dos sedimentos enriquecidos com óleo diesel, com insertos entre 25 e 35 Kb clonados em fosmídeos, foram triadas para detecção de genes catabólicos de monoxigenases (alkB1) e expressão de epóxido-hidrolases, esterases, lipases e monoxigenases em ensaios de alto desempenho (HTS, high throughput screening). Clones reativos a alkB1 foram detectados, porém não foram funcionais nas condições de HTS testadas. Nas bibliotecas de fosmídeos triadas...

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates. © 2013 Cruz et al.

A biocatálise é uma área multidisciplinar de pesquisa, na qual as novas tecnologias têm sido desenvolvidas rapidamente a fim de oferecer opções e soluções únicas aos diversos desafios encontrados em síntese orgânica, atuando em complemento com a química sintética tradicional. Frente à necessidade de acompanhar o progresso desta área, este trabalho visa à implementação de metodologias de triagens de alto desempenho (HTS) que permitam a detecção do potencial enzimático microbiano de uma forma simples e rápida. Este trabalho está dividido em dois tópicos principais: epóxido-hidrolases (EH) e monooxigenases (MO). Cada um dos tópicos relatará a síntese das sondas fluorogênicas que foram utilizadas nos testes de HTS e, em seguida, encontrar-se-á os resultados de avaliações preliminares destas sondas com diferentes concentrações celulares e de reagentes. As melhores condições para os testes com células microbianas foram encontradas e empregadas para avaliar diferentes microrganismos provenientes do bioma brasileiro ou de coleções de cultura brasileiras. Os resultados obtidos com as sondas fluorogênicas foram comparados com os relatados na literatura ou os obtidos no nosso laboratório. No tópico de EH...

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions. Lower coefficients of variance (around 15%) were achieved using crude cell lysates of BL21 and KRX host strains growing under microaerobic conditions. Reproducible high-throughput screenings for the decolorization of high redox potential azo and anthraquinonic dyes were developed and optimized for identification of variants with increased redox potential. The enzymatic assays developed were tested for the screening of one mutant library from CotA-laccase created by error-prone PCR.

In this work we demonstrate the potential of using an automated cell viability analyzer for developing high-throughput screening of orthopedic bioactive materials. We used a biomaterial of carbon nanotubes (CNTs)-based composite integrated with hydroxyapatite/ polymethyl methacrylate (HA/PMMA) with controlled physical and chemical properties to evaluate the usefulness of morphometric analysis in conjunction with trypan blue dye exclusion assays in MG63 cell cultures. The MG63 cell line, derived from human bone osteo - sarcoma, is often used as a model for studying osteoblast-like cellular response to bioactive materials for orthopedic surgery. The viability analyzer, Vi-CELLTM XR, Beckman Coulter, was used with trypan blue dye exclusion method in cell suspensions obtained after trypsinization along with determining the distribution plots of cell diameter and circularity, which are critical cellular characteristics. In addition, the activity of alkaline phosphatase (ALP), a typical representation of osteogenic activity of osteoblasts, was also measured spectrophotometrically using p-nitrophenol phosphate as the substrate. Comparative analysis of the frequency histogram of average cell diameter and circularity allowed for the analyses of significant alterations in cell morphology not only over time in control cultures (spherical vs. a flat morphology) but also with respect to PMMA and HA nanocomposites. After cell exposure to HA/PMMA/CNTs...

Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde; As doenças músculo-esqueléticas têm um grande impacto na sociedade e economia actuais e visto que a população mundial está a envelhecer e a popularidade dos desportos recreativos a aumentar, é provável que a frequência de doenças músculo-esqueléticas também aumente. Ao contrário do osso e fígado, os tecidos músculo-esqueléticos moles, tais como tendão e ligamento, têm reduzida capacidade de regeneração. Ao invés disso, cicatrizam, o que afecta as propriedades mecânicas e função do tecido. Para melhorar a capacidade de regeneração dos tendões e ligamentos é essencial aumentar a proliferação e secreção da matriz extracelular (ECM) das células do tendão/ligamento (T/L) assim como aumentar a circulação sanguínea no seu interior. No presente estudo, usou-se “high-throughput screening” (HTS) para identificar rapidamente compostos que poderão aumentar a proliferação de células de ligamento e células endoteliais. Dois compostos que aumentam a proliferação/actividade metabólica das células de ligamento e sete compostos que aumentam o número/actividade metabólica das células endoteliais (EC) foram identificados. Neste trabalho...

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

The zebrafish larva is an ideal candidate for in vivo high-throughput screening: it is a small vertebrate, it is optically transparent, possesses complex organs, and is easy to culture. In addition, genetic mutants and models of human diseases are widely available. Despite these attractive features there are no tools capable of screening at sufficient throughput and resolution to fully exploit the zebrafish. Here, I present a collection of technologies that enable high-throughput studies on zebrafish larvae at cellular resolution.; Engineering and Applied Sciences

In the field of stem cell technology, future advancements rely on the effective isolation, scale-up and maintenance of specific stem cell populations and robust procedures for their directed differentiation. The stem cell microenvironment - or niche - encompasses signal inputs from stem cells, supporting cells and from the extracellular matrix. In this context, the contribution of physicochemical surface variables is being increasingly recognised. This paradigm can be exploited to exert control over cellular behaviour. However, the number of parameters at play, and their complex interactions, presents a formidable challenge in delineating how the decisions of cell fate are orchestrated within the niche. Additionally, in the case of mesenchymal stem cells (MSC), more than one type of stem cell niche has been identified. By employing high throughput screening (HTS) strategies, common and specific attributes of each MSC niche can be probed. Here, we explore biological, chemical and physical parameters that are known to influence MSC self-renewal and differentiation. We then review techniques and strategies that allow the HTS of surface properties for conditions that direct stem cell fate, using MSC as a case study. Finally, challenges in recapturing the niche...

Background: A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency. Results: A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R2) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93...

Chemical and physiological properties are related to individual or bioactive compounds such as essential oils, terpenoids, flavonoids, volatile compounds and other chemicals which are present in natural products in low concentrations (e.g. ppm or ppb). For many years, classical separation, chromatographic and spectrometric techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography (LC) and mass spectrometry (MS) have been used for the elucidation of isolated compounds from different matrices. Hence, the use of standard separation, chromatographic and spectrometric methods was found useful in chemical and both plant and animal physiology studies, for fingerprinting and comparing natural and synthetic samples, as well as to identify single active compounds. It has been generally accepted that a single analytical technique will not provide sufficient visualization of the metabolome, hence holistic techniques are needed for comprehensive analysis. In the last 40 years near infrared (NIR) spectroscopy became one of the most attractive and used methods of analyzing agricultural related products and plant materials which provide simultaneous, rapid and non-destructive quantitation of major. This technique has been reported to determine other minor compounds in plant materials such as volatile compounds and elements. The aim of this short review is to describe some recent applications of NIR spectroscopy combined with multivariate data analysis for high throughput screening of metabolites with an emphasis on food and medical applications.; Daniel Cozzolino

In Anbetracht der aktuellen Situation bei der Untersuchung und Entdeckung von pharmazeutisch wirksamen Substanzen war das Ziel der Arbeit eine Untersuchung der Möglichkeiten, die markierungsfreie Detektion für den Einsatz im Hochdurchsatzscreening zu nutzen und durch ein breites Angebot an Sensoroberflächen und Assay-Testformaten ein möglichst breites Anwendungsgebiet zu eröffnen. Als Detektionsmethode wurde die Reflektometrische Interferenzspektroskopie (RIfS) verwendet. Das Augenmerk lag zum einen auf der Bereitstellung und experimentellen Charakterisierung von unterschiedlichen Transduceroberflächen, Zum anderen wurde ein für High-Throughput Screening-Anwendungen vorgesehener Prototyp auf seine Eignung hin grundlegend charakterisiert und an unterschiedlichen Beispielassays seine Anwendungsbreite sowie die Grenzen aufgezeigt. Effektive Immobilisierungstechniken, zur kovalenten Immobilisierung von Biotin, (Strept?)Avidin und NTA wurden entwickelt und es wurde gezeigt, dass sich mit diesen Systemen Biotin-Konjugate bzw. His-Tag-Fusionsproteine in unterschiedlicher Dichte auf der Sensoroberfläche immobilisieren lassen, so dass kinetische und thermodynamische Untersuchungen möglich waren. Am Beispiel von drei Assays, die in die Kategorien indirekter Assay...

Microfluidic devices fabricated by mass production offer an immense potential of applications such as high-throughput drug screening, clinical diagnostics and gene analysis [1]. The low unit production costs of plastic substrates make it possible to produce single-use devices, eliminating the need for cleaning and reuse [2]. Fabrication of microfluidic devices can be applied by microtechnical fabrication processes in combination with plastic molding techniques [3]. Basically, replication in plastics requires a hot embossing or injection molding tool. Various microfabrication technologies for the masterfabrication are established, such as the LIGA technique, mechanical micromachining and the micro electrical discharge machining technique (µEDM). Depending on the specific requirements, the most suitable process can be selected. The availability of these technologies allows to generate robust metal molding tools which exhibit the inverse shapes of the intended microstructures. In close collaboration, Greiner Labortechnik and Forschungszentrum Karlsruhe have fabricated prototype single-use plastic microfluidic devices in a standard microplate format by hot embossing with a mechanical micromachined molding tool and subsequent sealing of the microchannels. The microfluidic lab-on-chip structures are compatible with existing plate and liquid handling robotics. Sub-microliter sample volumes can be applied in the 96-channel multiplexed microstructures. Additionally...

The GPCRs are the target of a significant portion of all approved drugs and also represent an important parcel of potential drug targets for new indications. Activation of GPCRs can be detected by the utilization of functional cell-based assays like measuring transient intracellular calcium mobilization. The emergence of the fluorescent imaging plate reader and Ca2+-sensitive fluorescent dyes has made the high-content screening of GPCR possible. Here was applied a dual automated FLIPR platform to testing a large collection of compounds to identification of both agonist and antagonists form a single screen of GPCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study...

7 pages, 4 figures, 1 scheme, 2 tables.-- PMID: 15762752 [PubMed].-- Printed version published Mar 2005.; Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose−response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.; This work was supported by a grant from Fundació La Caixa (4th Program for Research on Neurodegenerative Diseases...

Plantas são uma importante fonte de novos protótipos há pelo menos quatro décadas. A descoberta e introdução no mercado de paclitaxel, dos alcalóides da vinca, etoposídeo e muitos agentes antibacterianos têm servido de apoio ao desenvolvimento de programas de descobrimento de novos fármacos baseados em produtos naturais. Produtos naturais foram recentemente redescobertos como importante ferramenta na descoberta de novos fármacos devido às estruturas moleculares complexas capazes de interagir com alvos em células de mamíferos. A flora brasileira, a mais rica do mundo, tornou-se um importante sítio para prospecção de novos protótipos em decorrência da riqueza de espécies vegetais relacionada a uma possível diversidade química. Programas de triagem têm sido introduzidos no Brasil contrapondo a devastação de florestas relacionadas a atividades rurais, extrativismo madeireiro e mineral. Ensaios em larga escala permitem a análise de um grande número de amostras em um tempo relativamente pequeno, e pode ser considerado como o método mais eficaz para a descoberta de novos fármacos a partir de plantas. Dados atualizados relativos a programas de triagem que objetivam a identificação de novos protótipos antitumorais e antibacterianos são comentados.; Plants have played a significant role in the treatment of cancer and infectious diseases for the last four decades. The discovery and introduction to market of paclitaxel...

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337