Electrospray ionization mass spectrometry screening of piperidine alkaloids from Senna spectabilis (Fabaceae) extracts: Fast identification of new constituents and co-metabolites

The fragmentation pattern of a homologous series of piperidine alkaloids isolated from S. spectabilis was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS and ESI-MS/MS analyses of EtOH extracts and fractions from flowers and fruits of S. spectabilis allowed to elucidate the structures of four new compounds. The identification of these co-metabolites, based on the fragmentation patterns of previously isolated compounds, and further confirmed by accurate mass spectrometry defines this technique as a powerful tool to determine the metabolomic profile of species which has pharmacological importance. ©2005 Sociedade Brasileira de Química.

Use of Physiological Parameters in Screening Drought Tolerance in Sugarcane Genotypes

The physiological response of four commercial sugarcane genotypes to water stress was evaluated by measuring the photochemical efficiency of the photosystem II (chlorophyll a fluorescence ratio, F v/F m), estimated chlorophyll content (SPAD unit), leaf temperature (LT) and leaf relative water content (RWC). A field trial was established in the subtropical area with well-watered and water-stressed genotypes, in completely randomized blocks with four replicates in a 4 × 2 × 3 factorial design (genotype × irrigation × evaluation date). Physiological measurements were done during a 90 day-period of formative stage of plants. The analysis of variance showed that the interaction of genotype × irrigation × evaluation date had a significant effect for three physiological markers tested, F v/F m, SPAD unit and RWC. Under non-stressed conditions, all genotypes showed similar responses for the four markers. Under water deficiency stress, two drought-tolerant genotypes, HOCP01-523 and TCP89-3505 displayed higher values for F v/F m, SPAD unit and RWC, and lower values for LT, and could be classified as tolerant. It is therefore possible to use these physiological water stress associated traits as scorable marker traits for selecting drought-tolerant sugarcane genotypes in future breeding programs. © 2011 Society for Sugar Research & Promotion.

Multiple minima hypersurfaces procedures for biomimetic ligands screening

In this work the interaction of the pesticide carbaryl with two groups of biomimetic ligands, peptides and MIPs was screened by multiple minima hypersurfaces (MMH) procedures, through the AM1 semiempirical method. Data related to the properties of the molecular association of the complex biomimetic ligand-pesticide were obtained and compared with another molecular modeling algorithm named Leapfrog, as included in the Sybyl software package, and experimental results from the literature, remarking good correlation between them. All important MMH program parameters (cells number, box size, conformers) were studied and optimized with the aim of getting the minimum computation time without losing the correlation with experimental data. The data demonstrated that MMH approach can be used as a fast biomimetic ligand screening tool for MIPs. In the case of peptides the computation time was not comparable with the molecular dynamics methods conventionally used for this approach. © 2011 Springer Science+Business Media B.V.

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota-LS-(vaccine strain) and Sao Joao do Meriti-SJM-strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity. © 2012 Springer Science+Business Media B.V.

Frequency of multiple endocrine neoplasia type 1 in a group of patients with pituitary adenoma: genetic study and familial screening

The purpose of this study it was to evaluate the frequency of Multiple Endocrine Neoplasia type 1 (MEN1) in patients with pituitary adenoma and to perform genetic analysis and familial screening of those individuals afflicted with MEN1. 144 patients with pituitary adenoma at Botucatu Medical School, UNESP-Univ Estadual Paulista, were assessed retrospectively for MEN1 during the years of 2005-2011. The patients were evaluated for the presence of primary hyperparathyroidism (PHP) and enteropancreatic tumors. Genetic analysis was performed for the individuals with clinically diagnosed MEN1. Thirteen patients met the diagnostic criteria for MEN1, but three individuals belong to the same family and they were considered as a single MEN1 event, revealing 7.7 % frequency of MEN1 in this patient group. Genetic analysis showed MEN1 mutations in four index cases: IVS4+1 G>A, IVS3-6 C>T, c.1547insC and a new D180A mutation. One patient did not agree to participate in the genetic study and another one was referred for follow up in other hospital. Only polymorphisms were found in the other individuals, one of which was novel. We identified a high frequency of MEN1 in pituitary adenoma patients. Since PHP is one of the most common MEN1 tumor and patients are mostly asymptomatic, we suggest that all pituitary adenoma patients have their calcium profile analyzed. © 2013 Springer Science+Business Media New York.

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

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.

Resting heart rate: Its correlations and potential for screening metabolic dysfunctions in adolescents

Background: In pediatric populations, the use of resting heart rate as a health index remains unclear, mainly in epidemiological settings. The aims of this study were to analyze the impact of resting heart rate on screening dyslipidemia and high blood glucose and also to identify its significance in pediatric populations.Methods: The sample was composed of 971 randomly selected adolescents aged 11 to 17 years (410 boys and 561 girls). Resting heart rate was measured with oscillometric devices using two types of cuffs according to the arm circumference. Biochemical parameters triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and glucose were measured. Body fatness, sleep, smoking, alcohol consumption and cardiorespiratory fitness were analyzed.Results: Resting heart rate was positively related to higher sleep quality (β = 0.005, p = 0.039) and negatively related to cardiorespiratory fitness (β = -0.207, p = 0.001). The receiver operating characteristic curve indicated significant potential for resting heart rate in the screening of adolescents at increased values of fasting glucose (area under curve = 0.611 ± 0.039 [0.534 - 0.688]) and triglycerides (area under curve = 0.618 ± 0.044 [0.531 - 0.705]).Conclusion: High resting heart rate constitutes a significant and independent risk related to dyslipidemia and high blood glucose in pediatric populations. Sleep and cardiorespiratory fitness are two important determinants of the resting heart rate. © 2013 Fernandes et al.; licensee BioMed Central Ltd.

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShotTM (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. © 2013 Elsevier B.V.

Screening of genomic libraries

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs. © Springer Science+Business Media, LLC 2013.

Cost-effective composite electrode for the fast voltammetric screening and determination of riboflavin (B2) and pyridoxine (B6) in pharmaceuticals

Different solid composites made by mechanical dispersions of graphite particles into heated paraffin (from 65 to 80% graphite, in mass) were prepared and assessed in order to optimize their use in electrochemical and electroanalytical procedures for bioanalysis. Besides these, composites were also evaluated by thermoanalytical techniques aiming to study their conservation and long-term stability (over eight months without special care), among others. Best results were found at 80% m/m graphite in paraffin. Such electrode combines low-cost, stability, sensitivity, ease of maintenance and clearance, besides the possibilities of manufacture in many different forms and shapes (with or without modifications) and applicability in a wide range of pH. Electrochemical studies by different voltammetric techniques involving vitamins from complex B (riboflavin and pyridoxine) leaded to a better understanding about their electrooxidative processes onto carbon-composite electrodes, specially regarding reversibility and pH-dependence. Data were also acquired and optimized with analytical purposes, being square-wave voltammetry in pH 4.2 chosen by its many advantages. Good linearity between peak responses as function of concentration were reached from 5 to 43 μmol L-1 for riboflavin (peak at -0.257 V) and up to 8.5 × 10-4 mol L -1 for pyridoxine (peak at +1.04 V), best studied conditions; limits of detection (at an S/N of 3) for both analites showed to be circa 1.0 mol L-1. Different commercial samples were analyzed for riboflavin (EMS® complex B syrup) and pyridoxine (Citoneurin 5000 Merck® ampoules) providing 96.6% and 98.7% recoveries, respectively.

In vivo analgesic activity, toxicity and phytochemical screening of the hydroalcoholic extract from the leaves of Psidium cattleianum Sabine

Ethnopharmacological relevance: Psidium cattleianum Sabine is extensively used in Brazilian traditional medicine to treat several diseases including painful disorders. Aim of the study to investigate the toxicity and the possible analgesic activities of the hydroalcoholic extract from the leaves of Psidium cattleianum Sabine (ELPCS), to support its use in folk medicine. To screen the major phytochemical constituents of this extract and evaluate their antioxidant activity. Materials and methods: ELPCS was assessed for its antioxidant activity using the DPPH model. Its analgesic activity was examined using mouse models of acetic acid-induced writhing and hot plate paw licking models. The major phytochemical constituents of the extract were screened; their toxicity on LLC-MK2 mammalian cells was evaluated. Results: ELPCS exhibited significant peripheral analgesic activity at doses of 60, 80, 100, 200 and 400 mg/kg in mice, but it did not display central analgesic activity and not was toxic to LLC-MK2 cell (LD 50>400 μg/mL). The extract exhibited free radical scavenging activity as evidenced by IC 50 values (15.9 μg/mL) obtained by the DPPH method. Phytochemical screening detected flavonoids, saponins, cardiac glycosides, anthraquinones, and tannins. Conclusions: The results of the experimental studies proved the analgesic activity of ELPCS and supported the traditional use of this plant. © 2013 Elsevier B.V. All rights reserved.

Desenvolvimento de sistemas de screening utilizando procedimentos em fluxo para monitoramento de sulfonamidas em urina e produtos de origem animal

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento de sistemas de screening automatizados para determinação de sulfonamidas em mel e procedimento limpo para a quantificação de sulfaquinoxalina em medicamentos veterinários

Pós-graduação em Química - IQ

Desenvolvimento de métodos limpos para screening e determinação de sulfonamidas em matrizes diversas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)