VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

Os procedimentos descritos neste protocolo foi aprovado pelo Comitê de Cuidado e Uso Institucional Animal da Universidade Indiana em Pensilvânia. 1. Os embriões de colheita <ol><li> Manter peixe-zebra a 28,5 ° C, pH 7, condutividade entre 500-1,500 uS, e um ciclo de luz / escuridão de 14 horas de luz e 10 horas escuro 24. Use estirpes do tipo selvagem, como a Turquia, AB ou TU / AB híbrido. Diferentes estirpes podem responder de forma diferente ao tratamento químico 25. </li><li> Configure o peixe para o acasalamento na noite anterior ovos de colheita por adição de água sistema de peixe em um tanque de acasalamento. Adicione uma peixes machos e fêmeas no tanque e separar os dois peixes com um divisor. Cada par peixe vai produzir uma gama de 50-300 ovos. Para garantir que os ovos suficientes será produzido, criou 30 pares de peixes. Normalmente, cerca de 50% de pares de peixes em idade de acasalamento prime (6-9 meses de idade) vai produzir ovos, resultando até 200 ovos por par e um máximo de até 3, 000 ovos para esta experiência. </li><li> Na manhã seguinte, após a luz acende, remova o divisor para iniciar o acasalamento. Verifique os tanques de acasalamento para os ovos a cada 15 minutos. </li><li> Uma vez que os peixes põem ovos, colher todos os embriões utilizando um coador de chá e combiná-los em um recipiente grande com tampão E3 (NaCl 5 mM, KCl 0,17 mM, CaCl 0,33 2, 0,33 mM MgSO 4). Em 1,5 hpf, remover e descartar ovos não fertilizados com uma pipeta de transferência de plástico sob um microscópio de dissecação. Ovos não fertilizados são opacas, enquanto ovos fertilizados são transparentes 34. </li><li> Transferir 50 embriões numa placa de Petri de 100 x 15 mm de vidro contendo 50 ml de tampão de E3 para cada condição de tratamento. Um total de 33 pratos são necessários 11 para as condições de tratamento e 3 repetições. </li></ol> 2. Os embriões Tratar <ol><li> Realizar tratamentos em 2 horas pós-fertilização (hpf) 35. Incubar os embriões / larvas a 28,5 ° C e examinar as larvas em 120 hpf. Realizar pelo least três repetições de cada condição de tratamento para análise estatística. </li><li> Para 3 placas de Petri (cada um contendo 50 embriões) em 2 HPF, remover o tampão de E3 com uma pipeta de transferência e adicionar 50 ml de etanol a 300 mM em tampão diluída E3. Cobrir as placas de Petri com Parafilm para minimizar a evaporação do etanol. <ol><li> Continue expondo os embriões em etanol durante 22 h. Em seguida, remover o etanol com uma pipeta de transferência. Adicionar 50 ml de tampão E3 e agite o prato várias vezes para lavar o etanol. Remover este buffer E3 com uma pipeta de transferência e repita o passo de lavagem mais 2 vezes. </li></ol></li><li> Para 3 placas de Petri (cada um contendo 50 embriões) em 2 HPF, remover o tampão de E3 com uma pipeta de transferência e adicionar 50 ml de tampão de novo E3. </li><li> Para placas de Petri de 9 (cada um contendo 50 embriões) em 2 HPF, remover o tampão de E3 com uma pipeta de transferência e adicionar 50 ml de 1,000 mg / L de nitrito de sódio dissolvido em tampão de E3. Confirmar a concentração da solução de nitrito de antemão usando ummodificação da diazotação (USEPA Método 354.1) método espectrofotométrico 28. <ol><li> Continue expondo 3 pratos para 46 hr, 3 pratos para 70 horas e 3 pratos para 94 horas. Substitua a solução de nitrito com uma solução de nitrito acabado de fazer diariamente. </li><li> Depois de cada tempo de exposição, remover o nitrito com uma pipeta de transferência. Adicionar 50 ml de tampão E3 e agite o prato várias vezes para lavar o nitrito. Remover este buffer E3 com uma pipeta de transferência e repita o passo de lavagem mais 2 vezes. </li></ol></li><li> Para 3 placas de Petri (cada um contendo 50 embriões) em 2 HPF, remover o tampão de E3 com uma pipeta de transferência e adicionar 50 ml de 200 mg / L de nitrito de sódio. Repetir este com 400, 600, 800, e 1000 mg / L de nitrito de sódio. <ol><li> Continue expondo os embriões de nitrito por 70 h. Substitua a solução de nitrito com uma solução de nitrito acabado de fazer diariamente. </li><li> Após o tempo de exposição, remover o nitrito com uma pipeta de transferência. Adicionar 50 ml de tampão E3 e agite o prato váriasvezes para lavar o nitrito. Remover este buffer E3 com uma pipeta de transferência e repita o passo de lavagem mais 2 vezes. </li></ol></li><li> Para 3 placas de Petri (cada um contendo 50 embriões) em 2 HPF, remover o tampão de E3 com uma pipeta de transferência e adicionar 50 ml de 1,000 mg / L de nitrato de sódio dissolvido em tampão de E3. Confirmar a concentração da solução de nitrato de estoque de antemão, utilizando uma modificação da redução de cádmio (Método USEPA 353.3) 28 método espectrofotométrico. <ol><li> Continue expondo os embriões em nitrato por 70 hr. Substitua a solução de nitrato com solução de nitrato acabado de fazer diariamente. </li><li> Após o tempo de exposição, remover o nitrato com uma pipeta de transferência. Adicionar 50 ml de tampão E3 e agite o prato várias vezes para lavar o nitrato. Remover este buffer E3 com uma pipeta de transferência e repita o passo de lavagem mais 2 vezes. </li></ol></li><li> Durante cada dia da exposição, contar o número de mortos embrião / larvas usando um microscópio estereoscópico. Sinais de morteincluem a falta de batimentos cardíacos e a circulação sanguínea, ou falta de motilidade após 1 min de observação. Remover mortos embriões / larvas com uma pipeta de transferência para reduzir a contaminação do tampão E3. </li><li> Quando experimentos acabar a 120 hpf, sacrificar as larvas. <ol><li> Remover o tampão de E3 com uma pipeta de transferência. Em seguida, adicione 50 ml de 0,2% MS-222 (tamponada a pH 7) e esperar por 10 min. </li><li> Remover o MS-222 com uma pipeta de transferência. Adicionar 50 ml de tampão E3 e agite para lavar a MS-222. </li><li> Remover o tampão de E3 com uma pipeta de transferência e adicionar 20 ml de 4% de paraformaldeído (PFA) para fixar as larvas. Agitar o prato várias vezes. Usar uma pipeta de transferência para transferir as larvas para um frasco de vidro, juntamente com PFA suficiente para encher o frasco. Armazenar os tubos no frigorífico durante a noite (O / N). </li></ol></li><li> Tirar fotos de larvas fixo usando um estereoscópio com uma câmera digital. Use 30X ampliação, ISO 200, e 200 tempo de exposição ms. Orientar as larvas de modo que a anterior à lepé e dorsal é para o início do campo. </li></ol>

<ol>
	<li>Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogenic+causes+of+malformations.">Teratogenic causes of malformations.</a> Ann Clin Lab Sci. 40 (2), 99-114 (2010).</li><li>Brent, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cause+and+prevention+of+human+birth+defects:+What+have+we+learned+in+the+past+50+years.">The cause and prevention of human birth defects: What have we learned in the past 50 years.</a> Con Anom. 41 (1), 3-21 (2001).</li><li>Lin, S., Zhao, Y., Nel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+in+vivo+model+for+nano+EHS+studies.">Zebrafish: an in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li><li>Pamanji, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+effects+of+profenofos+on+embryonic+and+larval+development+of+zebrafish+(Danio+rerio).">Toxicity effects of profenofos on embryonic and larval development of zebrafish (Danio rerio).</a> Environ Toxicol Pharmacol. 39 (2), 887-897 (2015).</li><li>Simmons, A. E., Karimi, I., Talwar, M., Simmons, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitrite+on+development+of+embryos+and+early+larval+stages+of+the+zebrafish+(Danio+rerio).">Effects of nitrite on development of embryos and early larval stages of the zebrafish (Danio rerio).</a> Zebrafish. 9 (4), 200-206 (2012).</li><li>Mantecca, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+Evaluation+of+a+New+Zn-Doped+CuO+Nanocomposite+With+Highly+Effective+Antibacterial+Properties.">Toxicity Evaluation of a New Zn-Doped CuO Nanocomposite With Highly Effective Antibacterial Properties.</a> Toxicol Sci. , (2015).</li><li>Jensen, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+formation+from+nitrite+in+zebrafish.">Nitric oxide formation from nitrite in zebrafish.</a> J Exp Biol. 210, 3387-3394 (2007).</li><li>Scholz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+embryo+model+in+environmental+risk+assessment--applications+beyond+acute+toxicity+testing).">The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing).</a> Environ Sci Pollut Res Int. 15 (5), 394-404 (2008).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>CDC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+abortions+possibly+related+to+ingestion+of+nitrate-contaminated+well+water--LaGrange+County,+Indiana,+1991-1994.">Spontaneous abortions possibly related to ingestion of nitrate-contaminated well water--LaGrange County, Indiana, 1991-1994.</a> MMWR Morb Mortal Wkly Rep. 45 (26), 569-572 (1996).</li><li>Brender, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+nitrate+intake+from+drinking+water+and+selected+birth+defects+in+offspring+of+participants+in+the+national+birth+defects+prevention+study.">Prenatal nitrate intake from drinking water and selected birth defects in offspring of participants in the national birth defects prevention study.</a> Environ Health Perspect. 121 (9), 1083-1089 (2013).</li><li>Huber, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+dietary+intake+of+nitrates,+nitrites+and+nitrosamines+and+selected+birth+defects+in+offspring:+a+case-control+study.">Maternal dietary intake of nitrates, nitrites and nitrosamines and selected birth defects in offspring: a case-control study.</a> Nutr J. 12, 34 (2013).</li><li>Phillips, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+nitrate+and+nitrite+in+foods+in+relation+to+infant+methaemoglobinaemia.">Naturally occurring nitrate and nitrite in foods in relation to infant methaemoglobinaemia.</a> Food Cosmet Toxicol. 9 (2), 219-228 (1971).</li><li>Moncada, S., Palmer, R. M., Higgs, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+physiology,+pathophysiology,+and+pharmacology.">Nitric oxide: physiology, pathophysiology, and pharmacology.</a> Pharmacol Rev. 43 (2), 109-142 (1991).</li><li>Gladwin, M. T., Crawford, J. H., Patel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+nitric+oxide,+nitrite,+and+hemoglobin:+role+in+blood+flow+regulation.">The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation.</a> Free Radic Biol Med. 36 (6), 707-717 (2004).</li><li>Gupta, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+acute+respiratory+tract+infections+in+areas+with+high+nitrate+concentrations+in+drinking+water.">Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water.</a> Environ Health Perspect. 108 (4), 363-366 (2000).</li><li>Kross, B. C., Ayebo, A. D., Fuortes, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia:+nitrate+toxicity+in+rural+America.">Methemoglobinemia: nitrate toxicity in rural America.</a> Am Fam Physician. 46 (1), 183-188 (1992).</li><li>Greer, F. R., Shannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infant+methemoglobinemia:+the+role+of+dietary+nitrate+in+food+and+water.">Infant methemoglobinemia: the role of dietary nitrate in food and water.</a> Pediatrics. 116 (3), 784-786 (2005).</li><li>Sanchez-Echaniz, J., Benito-Fernandez, J., Mintegui-Raso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia+and+consumption+of+vegetables+in+infants.">Methemoglobinemia and consumption of vegetables in infants.</a> Pediatrics. 107 (5), 1024-1028 (2001).</li><li>Virtanen, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrate+and+nitrite+intake+and+the+risk+for+type+1+diabetes+in+Finnish+children.+Childhood+Diabetes+in+Finland+Study+Group.">Nitrate and nitrite intake and the risk for type 1 diabetes in Finnish children. Childhood Diabetes in Finland Study Group.</a> Diabet Med. 11 (7), 656-662 (1994).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol-+and+acetaldehyde-mediated+developmental+toxicity+in+zebrafish.">Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 769-781 (2004).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Loucks, E., Carvan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+effects+of+developmental+ethanol+exposure+in+zebrafish.">Strain-dependent effects of developmental ethanol exposure in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 745-755 (2004).</li><li>Bilotta, J., Barnett, J. A., Hancock, L., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+exposure+alters+zebrafish+development:+a+novel+model+of+fetal+alcohol+syndrome.">Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome.</a> Neurotoxicol Teratol. 26 (2), 737-743 (2004).</li><li>Li, J., Jia, W., Zhao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+nitrite+affects+zebrafish+valvulogenesis+through+yielding+too+much+NO+signaling.">Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.</a> PLoS One. 9 (3), e92728 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for chemical analysis of water and wastes. , (1983).</li><li>Loucks, E., Ahlgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+teratogenic+changes+in+a+zebrafish+model+of+fetal+alcohol+exposure.">Assessing teratogenic changes in a zebrafish model of fetal alcohol exposure.</a> J Vis Exp. (61), (2012).</li><li>Addiscott, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilizers+and+nitrate+leaching.">Fertilizers and nitrate leaching.</a> Agricultural Chemicals and the Environment, Issues in Environmental Science and Technology. , 1-26 (1996).</li><li>Su, Y. F., Lu, L. H., Hsu, T. H., Chang, S. L., Lin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+treatment+of+methemoglobinemia+in+an+elderly+couple+with+severe+cyanosis:+two+case+reports.">Successful treatment of methemoglobinemia in an elderly couple with severe cyanosis: two case reports.</a> Journal of Medical Case Reports. 6 (290), (2012).</li><li>Harvey, M., Cave, G., Chanwai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatal+methaemoglobinaemia+induced+by+self-poisoning+with+sodium+nitrite.">Fatal methaemoglobinaemia induced by self-poisoning with sodium nitrite.</a> Emergency Medicine Australasia. 22, 463-465 (2010).</li><li>Nishiguchi, M., Nushida, H., Okudaira, N., Nishio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autopsy+case+of+fatal+methemoglobinemia+due+to+ingestion+of+sodium.">An autopsy case of fatal methemoglobinemia due to ingestion of sodium.</a> Forensic Research. 6, (2015).</li><li>Avdesh, A., Chen, M., Martin-Iverson, M. T., Mondal, A., Ong, D., Rainey-Smith, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+Care+and+Maintenance+of+a+Zebrafish+(Danio+rerio)+Laboratory:+An+Introduction.">Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction.</a> J. Vis. Exp. (69), (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+tool+for+chemical+screens.">Zebrafish: A tool for chemical screens.</a> Birth Defects Res C Embryo Today. 90 (3), 185-192 (2010).</li></ol>

The authors have nothing to disclose.

O método descrito aqui demonstra a utilidade do peixe-zebra na avaliação do potencial teratogênico de nitrito e nitrato. Em comparação com outros vertebrados, zebrafish têm vantagens que incluem alta fecundidade, fecundação externa, transparência óptica e rápido desenvolvimento. Os mutantes que carecem de pigmentação disponíveis (tais como o peixe-zebra) Casper 36 também ajudam a aumentar a visibilidade dos órgãos internos. Também é fácil de gerar peixes-zebra transgénicos com genes rep?...

Teratogénese é um processo que interrompe o desenvolvimento normal de um embrião ou feto por determinação de alterações funcionais e estruturais permanentes, retardamento do crescimento, ou aborto em casos graves 1. Ela pode ser causada por certos agentes naturais (teratógenos), os quais interferem com o desenvolvimento embrionário de várias maneiras 2. Durante o desenvolvimento fetal humano, teratogens comum, tais como a radiação, agentes infecciosos, metais tóxicos e químicos orgânicos foram relatados para causar defeitos de dobras epicanthic (a dobra de pele na pálpebra superior) e clinodactilia (dedo curvo ou pés) através de erros morfogenéticos 1. Compreender o mecanismo molecular de teratogenesis é o primeiro passo para o desenvolvimento de tratamento e prevenção. Vários modelos de vertebrados, tais como o sapo com garras Africano (Xenopus laevis) e peixe-zebra (Danio rerio) têm sido utilizados para determinar as vias moleculares afectadas por TeratOgens. Estudos anteriores têm usado peixe-zebra como um modelo para a epidemiologia, toxicologia e teratogenesis 3-7. Scholz et ai. considerada peixe-zebra como um &quot;padrão ouro&quot; para a avaliação da toxicidade ambiental. Isto é devido, em parte, para a transparência do embrião do peixe-zebra, o que permite aos investigadores visualizar o defeito de desenvolvimento, uma vez que ocorre 8. Aproximadamente 70% dos genes humanos ortólogos tem no peixe-zebra, fazendo com que um modelo de peixe-zebra vertebrado desejável para o estudo de defeitos humanos 9. Alguns estudos epidemiológicos têm sugerido que o nitrato e nitrito, comumente presente em alimentos agrícolas e água, estão associados a defeitos de nascimento ou abortos espontâneos 10,11, enquanto outros estudos não suportam essa associação 12. Nitrato (NO3 -) e nitrito (NO 2 -) estão naturalmente presentes no solo e na água. Eles são uma fonte de nitrogênio para as plantas e são uma parte do nciclo itrogen 13. Alimentos como feijão verde, cenoura, abóbora, espinafre e beterraba de fazendas que usam fertilizantes ricos em nitrato de ter aumentado significativamente os níveis de nitrato e nitrito 7. Leite de vacas alimentadas com alimentos à base de nitrato altos e peixes em água de alta nitrato (principalmente de escoamento do solo 30) pode levar a seres humanos que consomem grandes quantidades de nitrato e nitrito 14. Nitratos e nitritos são também vulgarmente utilizados na conservação de alimentos, o que aumenta dramaticamente a quantidade ingerida pelo ser humano 12. Níveis óptimos de nitrato e nitrito desempenham papéis fundamentais em processos fisiológicos como a homeostase vascular e função, neurotransmissão e mecanismos de defesa do hospedeiro imunológicos 13-15. No entanto, a exposição a altos níveis de nitrato e nitrito pode levar a efeitos adversos, especialmente em lactentes e crianças de 16. nitrato ingerido é ainda convertido em nitrito na cavidade oral e pela microflora no the trato gastrointestinal pela microflora intestinal 17. Nitrato coloca crianças em um alto risco para a síndrome do bebê azul por oxidação da hemoglobina em metemoglobina, prejudicando a hemoglobina a partir do seu transporte de oxigénio capacidade 18. Isto resulta em que a cor azul da pele que se estende para os tecidos periféricos, em casos mais graves. Oxigenação inibido de tecidos resulta em outros sintomas, mais severamente levando a coma e morte 19,20. Sintomas semelhantes são observados em bebés e adultos em concentrações mais altas de nitrato de 21. Níveis elevados de metemoglobina em adultos devido a resultados de intoxicação de nitrito em cianose, dor de cabeça, distúrbios respiratórios 31, e morte se não for tratada devido a complicações relacionadas com a hipóxia tecidual vital 32,33. Nitrato de ingerido em níveis mais elevados também pode resultar em várias complicações de saúde. diabetes infância, diarreia recorrente e infecções recorrentes do trato respiratórioem crianças têm sido relacionados com alta nitrato 11,17,22 ingestão. A exposição crônica a um elevado nível de nitrato está associado com a micção e hemorragia do baço. Exposição aguda a doses elevadas de nitratos pode levar a um largo espectro de condições médicas tais como dor abdominal, fraqueza muscular, sangue nas fezes e urina, desmaios, e da morte 11. A exposição pré-natal ao nitrato em níveis elevados tem sido associada a defeitos do tubo neural e músculo-esquelético 11. Um relatório recente mostrou que o tratamento de embriões de peixe-zebra com nitrito levou a gema edema sac, malformações craniofaciais e axiais, e nadar bexiga noninflation 5. No presente estudo, nós demonstramos um método para o tratamento de embriões de peixes-zebra com nitrato e nitrito para determinar o seu potencial teratogénico. Os embriões foram expostos a nitrito em diferentes concentrações e em diferentes períodos de tempo. O etanol foi utilizado como um controlo positivo, uma vez que é um agente teratogénico estabelecido em 23. Our método mostraram que ambas as concentrações elevadas e longos tempos de exposição a nitrito eram prejudiciais à sobrevivência e resultou em vários fenótipos, variando de leve (edema) a graves defeitos de desenvolvimento (bruto). Portanto, o peixe-zebra é um modelo útil para explorar diretamente os potenciais efeitos teratogênicos de nitrato e nitrito em embriões para complementar os estudos epidemiológicos.

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">DREL/2010 instrument</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">26700-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
			<td style="width:185px;">VWR</td>
			<td style="width:104px;">89001-244</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">03-338B</td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish as a model to assess the teratogenic potential of nitrite

Altos níveis de nitrato no ambiente pode resultar em defeitos congênitos ou abortos espontâneos em humanos. Presumivelmente, isto deve-se a conversão de nitrato em nitrito pelas bactérias intestinais e salivares. No entanto, em outros estudos de mamíferos, altos níveis de nitrito não causar defeitos de nascimento, embora possam levar a resultados reprodutivos pobres. Assim, o potencial teratogénico de nitrito não é clara. Seria útil dispor de um sistema de modelo animal vertebrado para avaliar facilmente efeitos teratogénicos de nitrito ou qualquer outro produto químico de interesse. Aqui, nós demonstrar a utilidade de peixe-zebra (Danio rerio) para rastrear compostos para toxicidade e defeitos embrionárias. embriões de peixe-zebra são fertilizados externamente e ter um desenvolvimento rápido, o que os torna um bom modelo para estudos teratogênicos. Nós mostramos que o aumento do tempo de exposição ao nitrito afecta negativamente a sobrevivência. O aumento da concentração de nitrito também afecta adversamente a sobrevivência, enquanto que o nitrato não. Para tha embriõest sobreviver à exposição nitrito, vários defeitos podem ocorrer, incluindo pericárdio e gema de edema sac, nadar noninflation bexiga, e malformação craniofacial. Os nossos resultados indicam que o peixe-zebra é um sistema conveniente para estudar o potencial teratogénico de nitrito. Esta abordagem pode ser facilmente adaptada para testar outros produtos químicos para os seus efeitos sobre o desenvolvimento dos vertebrados precoce.

A exposição ao etanol de 300 mM durante 22 horas não teve nenhum efeito sobre a sobrevivência (dados não apresentados), consistente com relatos anteriores 5,23,26. Isto é esperado, como o etanol é um agente teratogénico conhecido e serviu como um controlo positivo. Fenótipos observados incluíram edema pericárdico, noninflation bexiga natatória (Figura 1), defeitos craniofaciais, e atraso do desenvolvimento (dados não apresentados). <p class="j...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

A exposição a teratógenos podem causar defeitos de nascimento. Os peixes-zebra são úteis para a determinação do potencial teratogénico de produtos químicos. Nós demonstrar a utilidade de peixe-zebra, expondo embriões para vários teores de nitritos e também em diferentes tempos de exposição. Mostramos que os nitritos podem ser tóxicos e causar graves defeitos do desenvolvimento.

Peixe-zebra como um modelo para avaliar o potencial teratogênico de nitrito

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

zebrafish-as-a-model-to-assess-the-teratogenic-potential-of-nitrite

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Immunology and Infection

Unit 1

Adverse Drug Effects and Chemical Toxicity

SCIENTISTS IN ACTION

9.4K visualizações.  Indiana University of Pennsylvania.  A exposição a teratógenos podem causar defeitos de nascimento. Os peixes-zebra são úteis para a determinação do potencial teratogénico de produtos químicos. Nós demonstrar a utilidade de peixe-zebra, expondo embriões para vários teores de nitritos e também em diferentes tempos de exposição. Mostramos que os nitritos podem ser tóxicos e causar graves defeitos do desenvolvimento. 

Vídeo: Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Imunologia e Infecção

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Utilizando o rato como um BLT humanizado baseado Stem Modelo Gene Therapy célula tumoral

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

O inseto<em&gt; Galleria mellonella</em&gt; Como um modelo de infecção poderosa para investigar Patogênese Bacteriana

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantificação da Resposta explosão respiratória como um indicador da imunitário inatas Saúde em Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Utilização de<em&gt; Galleria mellonella</em&gt; Como um organismo modelo para estudar<em&gt; Legionella pneumophila</em&gt; Infecção

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cecal ligadura e punção induzida Sepsis como um modelo para estudar a autofagia em Ratos

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Transdução retroviral de células T helper como uma abordagem genética para estudar mecanismos que controlam a sua diferenciação e função

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpressão e purificação do ser humano<em&gt; Cis</em&gt; -preniltransferase em<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

O Madagascar silvando barata como um modelo de animais não-mamíferos alternativo para investigar a eficácia da droga, patogenia e virulência

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...