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RATIONALE: A full understanding of the biological impact of nanomaterials demands analytical procedures suitable for the detection/quantification of epigenetic changes that occur in the exposed organisms. Here, the effect of CuO nanoparticles (NPs) on global methylation of nucleic acids in Lepidium sativum was evaluated by liquid chromatography/ion trap mass spectrometry. Enhanced selectivity toward cytosine-containing nucleosides was achieved by using their proton-bound dimers formed in positive electrospray ionization (ESI(+)) as precursor ions for multiple reaction monitoring (MRM) quantification based on one or two ion transitions. METHODS: Plants were exposed to CuO NPs (0-1000 mg L(-1)); nucleic acid extracts were washed with bathocuproine disulfate; nucleosides were separated on a Luna C18 column coupled via ESI(+) to an AmaZon SL mass spectrometer (Bruker Daltonics). Cytidine, 2´-deoxycytidine, 5-methylcytidine, 5-methyl-2´-deoxycytidine and 5-hydroxymethyl-2´-deoxycytidine were quantified by MRM based on MS(3) ([2M+H](+)/[M+H](+)/[M+H-132](+) or [M+H-116](+)) and MS(2) ([2M+H](+)/[M+H](+) ). RESULTS: Bathocuproine disulfate, added as Cu(I) complexing agent, allowed for elimination of [2M+Cu](+) adducts from the mass spectra. Poorer instrumental detection limits were obtained for MS(3) (20-120 fmol) as compared to MS(2) (9.0-41 fmol); however, two ion transitions helped to eliminate matrix effects in plant extracts. The procedure was tested by analyzing salmon sperm DNA (Sigma) and applied for the evaluation of DNA and RNA methylation in plants; in the absence of NPs, 13.03% and 0.92% methylated cytosines were found in DNA and RNA, respectively; for NPs concentration >50 mg L(-1), DNA hypomethylation was observed with respect to unexposed plants. RNA methylation did not present significant changes upon plant exposure; 5-hydroxymethyl-2´-deoxycytidine was not detected in any sample. CONCLUSIONS: The MRM quantification proposed here of cytosine-containing nucleosides using their proton-bound homo-dimers as precursor ions proved its utility for the assessment of global methylation of DNA and RNA in plants under stress imposed by CuO NPs. Detection of copper adducts with cytosine-containing ions, and their elimination by washing extracts with Cu(I) chelator, calls for further investigation.

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The article describes the development of a new approach to teach molecular biology to undergraduate biology students. The 34 students who participated in this research belonged to the first period of the Biological Sciences teaching course of the Instituto Federal Goiano at Urutaí Campus, Brazil. They were registered in Cell Biology in the first semester of 2013. They received four 55 min-long expository/dialogued lectures that covered the content of "structure and functions of nucleic acids". Later the students were invited to attend four meetings (in a computer laboratory) in which some concepts of Bioinformatics were presented and some problems of the Rosalind platform were solved. The observations we report here are very useful as a broad groundwork to development new research. An interesting possibility is research into the effects of bioinformatics interventions that improve molecular biology learning.

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Haemophilus influenzae type b (Hib) is a human pathogen that causes meningitis in infants worldwide. Capsular polysaccharide linked to a protein has been used as an efficient vaccine, and this approach has reduced the incidence of Hib disease since its inclusion in national immunisation campaigns. The traditional polysaccharide downstream process is based on several ethanol precipitations, treatment with detergents and centrifugation. The aim of this study was to introduce tangential microfiltration (TMF) in the place of centrifugation to simplify handling and to scale up the process. The purity of the polysaccharide was RPNA=1747.2 and RPPrt=196.1 for nucleic acid and protein, respectively, meeting the quality requirements for this polysaccharide. Moreover, the polysaccharide was recognised by at specific antibody, and the ribose and phosphate contents were within the expected limits. Thus, we established a process for the purification of capsular polysaccharide produced by H. influenzae type b that is effective, robust and feasible to be scaling up.

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PROCEDIMENTO: O Teste de Amplificação de Ácidos Nucleicos (NAT) é uma tecnologia desenvolvida para a detecção do ácido nucleico de agentes infecciosos. Os testes NAT desenvolvidos no Brasil foram implantados na rede de estabelecimentos de hematologia e hemoterapia (Hemorrede) para detecção do Vírus da Imunodeficiência Humana (HIV) e do Vírus da Hepatite tipo C (HCV), em bolsas de sangue destinadas à transfusão. A implantação do NAT, neste caso, tem o propósito de reduzir o risco de transmissão de agentes virais transmissíveis por transfusão de sangue, como HIV e HCV, uma vez que é possível a detecção mais precoce destes agentes infecciosos em doações realizadas durante o período próximo à contaminação, porém, ainda em janela imunológica (antes da soro-conversão). Em conjunto com os testes sorológicos a implementação do NAT em estabelecimentos de hemoterapia é um consenso mundial visando garantir maior segurança transfusional e auxiliar na atualização dos dados epidemiológicos destas doenças. CRITÉRIOS PARA VALIDAÇÃO DOS ESTABELECIMENTOS DE HEMOTERAPIA: Os critérios para validação dos estabelecimentos de hemoterapia que receberiam a plataforma de testes, conjuntos diagnósticos (kits) registrados na ANVISA, o ressarcimento ao serviço público, no caso de realização do NAT para estabelecimentos de hemoterapia exclusivamente privados, a formalização dos contratos ou convênios a fim de definir as responsabilidades entre as partes foi estabelecida na Portaria GM/MS nº 79 de 31 de janeiro de 2003, que revogou as portarias anteriores de 2002. Essa última Portaria estabelecia ainda, que a Secretaria Executiva e a Secretaria de Atenção à Saúde (SAS), ambas do Ministério da Saúde (MS), ficavam autorizadas a emitir, conjuntamente, normas complementares e a adotar as providências necessárias para o custeio do NAT. Porém seu cumprimento foi inviabilizado devido à falta de recursos para aquisição e implantação dos testes. Em 2002, na Portaria GM/MS nº 112/04, foram definidas as efetivas ações para implantação gradativa do teste na Hemorrede brasileira. Assim, foi criado um consórcio público formado pelo Instituto de Tecnologia em Imunobiológicos ­ Bio-Manguinhos/FIOCRUZ, pela Universidade Federal do Rio de Janeiro ­ UFRJ e Instituto de Biologia Molecular do Paraná ­ IBMP, sob demanda e direção nacional da SAS/MS, por meio da sua Coordenação Geral de Sangue e Hemoderivados (CGSH) para garantir o desenvolvimento do projeto produção de insumos com tecnologia nacional. IMPLANTAÇÃO E ADEQUAÇÃO DO CONJUNTO DIAGNÓSTICO: A implantação e adequação do conjunto diagnóstico desenvolvido por Bio-Manguinhos na rotina dos estabelecimentos de hemoterapia foi possível após a realização de etapas importantes como os estudos piloto e multicêntrico: a) Estudo Piloto: Projeto realizado no HEMOSC em 2008, para avaliação da plataforma brasileira de testes NAT multiplex HIV/HCV e seus processos na Hemorrede Pública, sendo possível identificar adaptações técnicas necessárias ao conjunto diagnóstico, aquisição de equipamentos para compor a plataforma NAT nos estabelecimentos de hemoterapia componentes do estudo multicêntrico, reformas nas estruturas físicas do HEMORIO/RJ; HEMOPE/PE e Fundação Pró-Sangue/SP e desenvolvimento do aplicativo informatizado para controle do envio e recebimento de amostras e liberação de resultados dos testes, o Gerenciador do Sistema Multicêntrico ­ GSM NAT. Ao final do estudo piloto, foram realizados cerca de 5.000 testes nas amostras coletadas e processadas no HEMOSC/SC; b) Estudo Multicêntrico: Realizado entre 2009 a 2010, com a participação de oito estabelecimentos de hemoterapia, definidos pelo Ministério da Saúde, sendo eles: HEMORIO ­ Rio de Janeiro, HEMOES ­ Espírito Santo, Fundação Pró-Sangue ­ São Paulo, Santos ­ SP, HEMOPE ­ Pernambuco, HEMONORTE ­ Rio Grande do Norte, HEMOÍBA ­ Paraíba e HEMOSC ­ Santa Catarina, este teve como principal ação avaliar a implantação na rotina, logística de transporte de amostras e liberação de resultados "on line". Ao final, o estudo multicêntrico testou cerca de 220 mil amostras. RECOMENDAÇÃO DA CONITEC: Diante do exposto, os membros da CONITEC presentes na 20ª reunião do plenário, realizada nos dias 06/11 e 07/11/2013, recomendaram a incorporação do procedimento: Teste do Ácido Nucleico (NAT) em amostras de sangue de doador. DECISÃO: PORTARIA Nº 13, de 15 de maio de 2014 - Torna pública a decisão de incorporar o procedimento do teste do ácido nucleico (NAT) em amostras de sangue de doador no Sistema Único de Saúde - SUS.

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Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.

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The proven ability of gel electrophoresis to simultaneously resolve, in a single experiment, many components from complex biological samples, has determined its preference over a variety of well-established chromatographic methods. Therefore, procedures placed at the interface between gel separation and microanalysis have earned increasing significance with respect to the overall success of the microanalytical strategy. The first of these procedures is the detection technique. The most important requirement for compatibility with further analysis or bioapplications is that the staining method does not compromise the chemical integrity and the biological properties of micropurified biomolecules. Procedures for negative detection of proteins with metal salts that have been proven to comply with this condition have been known for about 15 years. Only recently have these procedures been extended to the field of nucleic acids and lipopolysaccharides. The focus of this review is to chronicle the development and current status of the negative or reverse stain procedure based on the in-gel reaction of imidazole with zinc salts and its applications forthe micropurification and analysis of unmodified proteins, nucleic acids and bacterial lipopolysaccharides. We highlight the common aspects in the detection of the three types of biomolecules, and their applications to structural and biological analyses. Emphasis is given on the mechanism underlying imidazole-zinc staining, as it contributes to a deeper understanding of a general detection mechanism with metal salts. Finally, we discuss the latest applications of the techniques in proteomics and their possible impact on the characterization of gel-separated single components from complex lipopolysaccharides.

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New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but less toxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH(4) (compound II), by reducing the cyclohexenone and lactone moieties with LiAlH(4) (compound III) and by transforming the lactone moiety into an amide (compound IV) using dimethylamine. The cytotoxicity of these derivatives from DHC was assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). IC(50) values of 540 and 350 microM were obtained for compound II in the NRU and NAC tests, respectively. Compound III was less toxic than the other DHC derivatives (IC(50)=1800 microM) on V79 cells based on NAC assay. Compound IV showed an IC(50) ranging from 350 to 600 microM based on the three endpoints evaluated. The three compounds were less toxic on V79 cells than DHC. DHC, compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay.

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Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

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Os isolados de Giardia duodenalis de individuos assintomaticos, de pacientes e de animais apresentam diferencas e semelhancas em sua composicao proteica, expressao de antigenos, proteases e isoenzimas, assim como na constituicao dos acidos nucleicos. A presente revisao analisa estas diferencas com enfase na interacao parasita e hospedeiro, e nos possiveis mecanismos de virulencia do protozoario

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