Esta pesquisa foi apoiada pelo nacional institutos de saúde neutralizar programa interagências acordo # AOD13015-001. Gostaríamos de agradecer a Stephanie Froberg e Peter Hurst esforços e expertise na produção de vídeo.

<ol>
	<li>Randleman, B., Hampton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drugs, Diseases and Procedures. , (2011).</li><li>Fish, R., Davidson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+ocular+thermal+and+chemical+injuries,+including+amniotic+membrane+therapy.">Management of ocular thermal and chemical injuries, including amniotic membrane therapy.</a> Current Opinion in Ophthalmology. 21 (4), 317-321 (2010).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+treatment+strategies+for+hydrofluoric+acid+burns:+current+status+and+future+prospects.">A review of treatment strategies for hydrofluoric acid burns: current status and future prospects.</a> Burns. 40 (8), 1447-1457 (2014).</li><li>Gaytan, B. D., Vulpe, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+toxicology:+tools+to+advance+the+future+of+toxicity+testing.">Functional toxicology: tools to advance the future of toxicity testing.</a> Frontiers in Genetics. 5, 110 (2014).</li><li>North, M., Vulpe, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+toxicogenomics:+mechanism-centered+toxicology.">Functional toxicogenomics: mechanism-centered toxicology.</a> International Journal of Molecular Sciences. 11 (12), 4796-4813 (2010).</li><li>McManus, M. T., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+in+mammals+by+small+interfering+RNAs.">Gene silencing in mammals by small interfering RNAs.</a> Nature Reviews Genetics. 3 (10), 737-747 (2002).</li><li>Bassik, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+mammalian+genetic+interaction+map+reveals+pathways+underlying+ricin+susceptibility.">A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility.</a> Cell. 152 (4), 909-922 (2013).</li><li>Solaimani, P., Damoiseaux, R., Hankinson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+high-throughput+screen+identifies+proteins+necessary+for+the+AHR-dependent+induction+of+CYP1A1+by+2,3,7,8-tetrachlorodibenzo-p-dioxin.">Genome-wide RNAi high-throughput screen identifies proteins necessary for the AHR-dependent induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin.</a> Toxicological Sciences. 136 (1), 107-119 (2013).</li><li>Cox, J. A., Gooding, R., Kolakowski, J. E., Whitmire, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Terrorism Risk Assessment; CSAC 12-006. , (2012).</li><li>Zang, R., Li, D., Tang, I., Wang, J., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+in+High-Throughput+Screening+for+Drug+Discovery.">Cell-Based Assays in High-Throughput Screening for Drug Discovery.</a> International Journal of Biotechnology for Wellness Industries. 1, 31-51 (2012).</li><li>Ray, R., Hauck, S., Kramer, R., Benton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+fluorometric+method+to+study+sulfur+mustard-induced+apoptosis+in+human+epidermal+keratinocytes+monolayer+microplate+culture.">A convenient fluorometric method to study sulfur mustard-induced apoptosis in human epidermal keratinocytes monolayer microplate culture.</a> Drug and Chemical Toxicology. 28 (1), 105-116 (2005).</li><li>Ruff, A. L., Dillman, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur+mustard+induced+cytokine+production+and+cell+death:+investigating+the+potential+roles+of+the+p38,+p53,+and+NF-kappaB+signaling+pathways+with+RNA+interference.">Sulfur mustard induced cytokine production and cell death: investigating the potential roles of the p38, p53, and NF-kappaB signaling pathways with RNA interference.</a> Journal of Biochemical and Molecular Toxicology. 24 (3), 155-164 (2010).</li><li>Smith, W. J., Gross, C. L., Chan, P., Meier, H. 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+use+of+human+epidermal+keratinocytes+in+culture+as+a+model+for+studying+the+biochemical+mechanisms+of+sulfur+mustard+toxicity.">The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity.</a> Cell Biology and Toxicology. 6 (3), 285-291 (1990).</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> Toxnet. , (2016).</li><li>Bancroft, W. D. <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 Medical Department of the United States Army in the World War. 14, (1926).</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> Chloropicrin Risk Characterization Document. , (2012).</li><li>Aigueperse, J., 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=.">.</a> Fluorine Compounds, Inorganic. Ullmann's Encyclopedia of Industrial Chemistry. , (2000).</li><li>Sotozono, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+Injury+in+the+Cornea:+The+Role+of+Inflammatory+Cytokines+in+Corneal+Damage+and+Repair.">Second Injury in the Cornea: The Role of Inflammatory Cytokines in Corneal Damage and Repair.</a> Cornea. 19 (6), S155-S159 (2000).</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> Guidelines and Recommendations for Dharmacon siRNA Libraries. , (2015).</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> Lipofectamine RNAiMAX Reagent. , (2017).</li><li>Denizot, F., Lang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cell+growth+and+survival.+Modifications+to+the+tetrazolium+dye+procedure+giving+improved+sensitivity+and+reliability.">Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.</a> Journal of Immunological Methods. 89 (2), 271-277 (1986).</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> IL-8 (human) AlphaLISA Detection Kit, 5,000 Assay Points. , (2016).</li><li>Araki-Sasaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+SV40-immortalized+human+corneal+epithelial+cell+line+and+its+characterization.">An SV40-immortalized human corneal epithelial cell line and its characterization.</a> Investigative Ophthalmology &amp; Visual Science. 36 (3), 614-621 (1995).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Ruff, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+for+sulfur+mustard-induced+ocular+injury+and+long-term+clinical+analysis+of+injury+progression.">Development of a mouse model for sulfur mustard-induced ocular injury and long-term clinical analysis of injury progression.</a> Cutaneous and Ocular Toxicology. 32 (2), 140-149 (2013).</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> GeneTitan MC Instrument North America/Japan (110V). , (2018).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pair+of+new+statistical+parameters+for+quality+control+in+RNA+interference+high-throughput+screening+assays.">A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays.</a> Genomics. 89 (4), 552-561 (2007).</li><li>He, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Sodium+Fluoride+on+the+Proliferation+and+Gene+Differential+Expression+in+Human+RPMI8226+Cells.">Effect of Sodium Fluoride on the Proliferation and Gene Differential Expression in Human RPMI8226 Cells.</a> Biological Trace Element Research. 167 (1), 11-17 (2015).</li><li>Tabuchi, Y., Yunoki, T., Hoshi, N., Suzuki, N., Kondo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+gene+networks+involved+in+sodium+fluoride-elicited+cell+death+accompanying+endoplasmic+reticulum+stress+in+oral+epithelial+cells.">Genes and gene networks involved in sodium fluoride-elicited cell death accompanying endoplasmic reticulum stress in oral epithelial cells.</a> International Journal of Molecular Sciences. 15 (5), 8959-8978 (2014).</li><li>Pesonen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloropicrin-induced+toxic+responses+in+human+lung+epithelial+cells.">Chloropicrin-induced toxic responses in human lung epithelial cells.</a> Toxicology Letters. 226 (2), 236-244 (2014).</li><li>Wilhelm, S. N., Shepler, K., Lawrence, L. J., Lee, H., Seiber, J. N., 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=.">.</a> Fumigants: Environmental Fate, Exposure, and Analysis. 652, (1997).</li><li>Adamek, E., Pawlowska-Goral, K., Bober, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+effects+of+fluoride+ions+on+enzyme+activity.">In vitro and in vivo effects of fluoride ions on enzyme activity.</a> Annales Academiae Medicae Stetinensis. 51 (2), 69-85 (2005).</li><li>Heard, K., Hill, R. E., Cairns, C. B., Dart, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+neutralizes+fluoride+bioavailability+in+a+lethal+model+of+fluoride+poisoning.">Calcium neutralizes fluoride bioavailability in a lethal model of fluoride poisoning.</a> Journal of Toxicology: Clinical Toxicology. 39 (4), 349-353 (2001).</li><li>Sparks, S. E., Quistad, G. B., Casida, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloropicrin:+reactions+with+biological+thiols+and+metabolism+in+mice.">Chloropicrin: reactions with biological thiols and metabolism in mice.</a> Chemical Research in Toxicology. 10 (9), 1001-1007 (1997).</li><li>Sparks, S. E., Quistad, G. B., Li, W., Casida, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloropicrin+dechlorination+in+relation+to+toxic+action.">Chloropicrin dechlorination in relation to toxic action.</a> Journal of Biochemical and Molecular Toxicology. 14 (1), 26-32 (2000).</li><li>Byron, K. A., Varigos, G., Wootton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrocortisone+inhibition+of+human+interleukin-4.">Hydrocortisone inhibition of human interleukin-4.</a> Immunology. 77 (4), 624-626 (1992).</li><li>van der Poll, T., Lowry, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+interleukin+8+production+by+human+whole+blood+is+enhanced+by+epinephrine+and+inhibited+by+hydrocortisone.">Lipopolysaccharide-induced interleukin 8 production by human whole blood is enhanced by epinephrine and inhibited by hydrocortisone.</a> Infection and Immunity. 65 (6), 2378-2381 (1997).</li><li>Solomon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxycycline+inhibition+of+interleukin-1+in+the+corneal+epithelium.">Doxycycline inhibition of interleukin-1 in the corneal epithelium.</a> Investigative Ophthalmology &amp; Visual Science. 41 (9), 2544-2557 (2000).</li><li>Mishra, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+oxidative+stress-induced+epigenetic+modifications+in+pancreatic+epithelial+cells.">Mitochondrial oxidative stress-induced epigenetic modifications in pancreatic epithelial cells.</a> International Journal of Toxicology. 33 (2), 116-129 (2014).</li><li>Stockholm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+phenotypic+heterogeneity+in+a+clonal+cell+population+in+vitro.">The origin of phenotypic heterogeneity in a clonal cell population in vitro.</a> PLoS One. 2 (4), e394 (2007).</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> ATCC Animal Cell Culture Guide. , (2014).</li><li>Lundholt, B. K., Scudder, K. M., Pagliaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+reducing+edge+effect+in+cell-based+assays.">A simple technique for reducing edge effect in cell-based assays.</a> Journal of Biomolecular Screening. 8 (5), 566-570 (2003).</li><li>Mpindi, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+normalization+methods+on+high-throughput+screening+data+with+high+hit+rates+and+drug+testing+with+dose-response+data.">Impact of normalization methods on high-throughput screening data with high hit rates and drug testing with dose-response data.</a> Bioinformatics. 31 (23), 3815-3821 (2015).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+score,+SSMD*,+z*+score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.</a> Journal of Biomolecular Screening. 16 (7), 775-785 (2011).</li></ol>

Os autores não têm nada para divulgar.
DISCLAIMER: as opiniões expressadas neste artigo são dos autores e não refletem a política oficial do departamento do exército, departamento de defesa ou o governo dos EUA. Esta pesquisa foi apoiada por um acordo interinstitucional entre NIAID/NIH e o USAMRICD e em parte por uma entrevista para o programa de participação de pesquisa pós-graduação em defesa dos Estados Unidos exército médico pesquisa Instituto de química administrada pelo carvalho Cume Instituto de ciência e educação através de um acordo interinstitucional entre o departamento de energia dos EUA e USAMRMC.

Neste documento descrevemos nossos métodos e resultados no desenvolvimento de uma célula epitelial de alto throughput córnea triagem modelo para o estudo das lesões HF e CP. Também apresentamos os resultados a partir da tela de siRNA primário para lesão de HF. Havia muitos desafios para o desenvolvimento de modelos HTS para o estudo das lesões TIC. Métodos que encontramos na literatura relacionada ao estudo de HF, HFA ou CP em modelos de cultura de células foram de pouca ajuda. A maioria dos estudos em vitr...

Lesão ocular induzida a toxicidade é uma verdadeira emergência ocular porque os produtos químicos têm o potencial para rapidamente infligir dano tecidual significativa. Infelizmente, tratamentos para toxicidade induzida por lesão da córnea só são geralmente favorável como nenhuma terapêutica específica existe para tratar essas lesões. A atual estratégia de tratamento é específico e principalmente inclui tratamentos terapêuticos tópicos tais como os lubrificantes, antibióticos, e cycloplegics seguido por anti-inflamatórios (por exemplo, esteroides) uma vez a córnea tem re-epithelialized1 ,2. Apesar das melhor tratamento terapêutico opções atuais disponíveis, prognóstico a longo prazo é geralmente pobre devido à turvação corneal progressiva e neovascularização2,3.
Modelos animais tem sido tradicionalmente usados para investigar a toxicidade química e compreender os mecanismos de lesão. No entanto, estudos em animais são demorado e caro. Existem também os esforços para reduzir a experimentação animal. Por exemplo, a legislação REACH (1907/2006 CE) na União Europeia tem disposições destinadas a reduzir a experimentação animal. As disposições incluem uma exigência que as empresas compartilham dados para evitar testes animais e obtenção de aprovação da Agência Europeia dos produtos químicos, antes de realizar os testes propostos em animais. Ao abrigo das disposições do REACH, a experimentação animal deve ser um último recurso. Há também o regulamento europeu de cosméticos (CE 1223/2009) que eliminadas a testes de cosméticos em animais. Quando são realizados estudos em animais, eles são guiados pelos princípios do 3Rs (refinamento, redução e substituição), que fornecem uma estrutura para a realização de pesquisas com animais mais humana, reduzindo o número de animais utilizados e usando alternativas não-animais sempre que possível. Por estas razões, o campo de toxicologia tem procurado adoptar em vitro ensaios que podem fornecer a introspecção do mecanismo molecular de toxicidade e podem ser feitos em maior taxa de transferência4. Esta é uma abordagem de toxicologia funcional onde substâncias tóxicas são definidas por sua função em vez de unicamente por sua química. Levou um passo adiante, funcional toxicogenomics procurar compreender as funções que desempenhar os efeitos de substâncias tóxicas5genes específicos. Com a aplicação da tecnologia siRNA, telas para investigar a função do gene nas respostas moleculares e celulares a substâncias tóxicas podem ser feitas com alto rendimento. siRNA são moléculas de RNA encalhadas dobro que são 19-25 nucleotides longos que aproveitar o post transcriptional gene silencioso caminho presente em todas as células de mamíferos6. Estes são sinteticamente feitos e projetados para atingir um gene específico. Quando introduzido em uma célula, o siRNA é processado e um fio, o fio guia, é carregado no complexo silencioso induzido por RNA (RISC). O siRNA direciona o RISC para uma região complementar em uma molécula de mRNA, e o RISC degrada o mRNA. Isso resulta na redução da expressão do gene específico. A redução resultante da expressão do gene específico em seguida pode ser estudada em células expostas tóxicas para verificar a função desse gene na resposta celular para a toxicidade. Esta abordagem tem sido usada para entender os mecanismos de susceptibilidade de ricina e a indução de AHR-dependente de CYP1A17,8.
A lista de avaliação de risco de terrorismo químico (CTRA) e as listas de produtos químicos industriais tóxicos (TIC) têm discriminados selecionados produtos químicos com base na sua toxicidade e potencial para ser liberado durante um terrorista, guerra ou acidente industrial evento9. Nós estamos aplicando um alto throughput de siRNA, abordagem de toxicogenomic (HTS) para o estudo de substâncias tóxicas de lista CTRA, que foram identificados com alto risco de utilização em um incidente terrorista de triagem. Toxicologia tradicional procura entender os efeitos adversos que produtos químicos em organismos vivos; no entanto, temos um desejo mais para entender os mecanismos de lesão com a finalidade de informar o desenvolvimento de abordagens terapêuticas e terapêutica e, possivelmente, descobrir moléculas que podem ser direcionadas para o desenvolvimento de terapêutico. Este esforço de certa forma pode ser considerado análogo ao uso de triagem de siRNA alto throughput e ensaios de célula com base na droga descoberta processo10. A principal diferença seria que essa descoberta de drogas normalmente procura um destino singular para descoberta terapêutica Considerando que em nossa abordagem é pouco provável que haja um destino singular com elevado valor terapêutico para o tratamento de exposição tóxica. Antecipamos que qualquer paradigma de tratamento eficaz para a exposição tóxica exigiria uma abordagem multi-facetada para alcançar alto valor terapêutico, e toxicogenomic dados vital podem informar uma paradigma de tratamento eficaz.
Automação de bancada traz metodologia alto throughput para laboratórios fora as indústrias farmacêuticas ou biotecnologia. Os estudos em vitro no nosso Instituto têm sido historicamente ensaios tradicionais que são de baixa taxa de transferência11,12,13. Nos últimos anos, nosso laboratório possui uma transição para o uso da robótica de bancada para realizar a triagem de siRNA alto throughput. Aqui, apresentamos o refinamento dos modelos de célula ocular e o desenvolvimento in vitro métodos de exposição para o fluoreto de hidrogênio (HF) e cloropicrina (CP) apropriada para triagem de siRNA alto throughput. Nosso objetivo é identificar as moléculas que regulam a lesão celular em resposta a essas substâncias tóxicas. Os alvos da biblioteca siRNA selecionados incluem receptores G proteína quinase de proteína, proteases, fosfatases, canais iônicos e outros alvos potencialmente druggable. HF e CP foram selecionados para o estudo por agentes de lista CTRA com os relatórios de ToxNet dos acidentes industriais a cruzar para encontrar aqueles que apresentam o maior risco de lesão ocular através do vapor exposição9,14. CP (fórmula química Cl3CNO2, número CAS 76-06-2) foi originalmente usado como um gás lacrimogêneo em WWI15. Atualmente é usado como um fumigante agrícola e funções como um inseticida, fungicida e nematicida16. Fluoreto de hidrogênio (HF) é usado em processos incluindo alquilação em refinarias de petróleo e fluoração eletroquímica de compostos orgânicos17. HF (fórmula química HF, número CAS 62778-11-4) é um gás, mas na sua forma aquosa é o ácido fluorídrico (HFA, CAS número 7664-39-3). Portanto, eleito para usar HFA em nosso celular em modelos de exposição. O antígeno de T grande SV40 imortalizado SV40-HCEC foi selecionado para o estudo de linha de célula epitelial da córnea humana. Viabilidade celular e o marcador inflamatório IL-8 foram selecionadas como pontos de extremidade alvos que estão envolvidos na lesão celular devem reflectir-se na morte celular e a resposta inflamatória. Especificamente, se o alvo fosse a desempenhar um papel protetor na exposição tóxica, morte celular e/ou produção de citocinas inflamatórias deve aumentar quando a expressão-alvo é inibida por siRNA. O contrário seria verdadeiro para alvos que desempenham um papel negativo. Além disso, inflamação crônica parece desempenhar um papel na patologia da córnea após exposição e intervenção em caminhos de morte celular podem melhorar os resultados clínicos2,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:879px;" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo liquid handing platform</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">G5409A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo plate shaker</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96LT disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 178</td>
			<td>96-channel large tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 177</td>
			<td>96-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 179</td>
			<td>384-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96 250 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19477-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 30 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 70 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EnSpire multimode plate reader</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">2300-0000</td>
			<td>AlphaLISA assay detector with high power laser excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IL-8 (human) AlphaLISA Detection Kit&#160;</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">AL224F</td>
			<td>no-wash bead-based assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">ProxiPlate-384 Plus white 384-shallow well microplates</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td>6008359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Lipofectamine RNAiMAX</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">13778500</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Opti-MEM 1 Reduced Serum Medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">TrypLE Express</td>
			<td style="width:247px;">Gibco or equivalent</td>
			<td style="width:119px;">12605010</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IncuCyte Zoom</td>
			<td style="width:247px;">Essen Instruments or equivalent</td>
			<td style="width:119px;">ESSEN BIOSCI 4473</td>
			<td>Incubator-housed automated microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chloropicrin</td>
			<td style="width:247px;">Trinity Manufacturing or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Acute toxicity and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM-F12 cell culture medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>11330-057</td>
			<td>Contains HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal bovine serum</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>1891471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Human epidermal growth factor (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>E9644-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Recombinant human insulin (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin-Streptomycin solution (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Hydrocortisone (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td>H0888-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PBS&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">P5493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA</td>
			<td style="width:247px;">Dharmacon or equivalent</td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">M5655</td>
			<td>MTT assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA buffer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">B002000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96-well cell culture plates</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">T150 cell culture flasks</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">BSL-2 cell culture hood</td>
			<td style="width:247px;">Nuaire or equivalent</td>
			<td style="width:119px;">NU-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">300 mL robotic reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">12-565-572&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96 baffled automation reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">1064-15-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">500 mL sterile disposable storage bottles</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-1443A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealing foil</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-0475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cardamonin</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2509</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SKF 86002&#160;</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2008</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMSO</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">48% hydrofluoric acid</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">339261</td>
			<td>Corrosive and acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 1000 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 200 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 20 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemical fume hood</td>
			<td style="width:247px;">Jamestown Metal Products</td>
			<td style="width:119px;">MHCO_229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">384-well sample storage plates</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">262261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sodium chloride</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">50 mL conical tubes</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">14-959-49A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 50 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 25 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 10 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 5 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-573</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">SV40-HCEC immortalized human corneal epithelial cells</td>
			<td style="width:247px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sceptor Handheld Automated Cell Counter</td>
			<td style="width:247px;">Millipore or equivalent</td>
			<td style="width:119px;">PHCC20060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">GeneTitan Multi-Channel (MC) Instrument</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">00-0372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Affymetrix 24- and 96-array plates</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">901257; 901434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube HF</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube CP</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger pump</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">6400000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Clear Plate seals</td>
			<td style="width:247px;">Resesarch Products International or Equivalent</td>
			<td style="width:119px;">202502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Reagent reservoirs</td>
			<td style="width:247px;">VistaLab Technologies or equivalent</td>
			<td style="width:119px;">3054-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Xlfit</td>
			<td style="width:247px;">IDBS or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Excel add-in used for automated curve fitting</td>
		</tr>
	</tbody>
</table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

Lesão ocular induzida a toxicidade é uma verdadeira emergência ocular porque os produtos químicos têm o potencial para rapidamente infligir dano tecidual significativa. Tratamentos para toxicidade induzida por lesão da córnea são geralmente favorável como nenhuma terapêutica específica existe para tratar essas lesões. Nos esforços para desenvolver tratamentos e terapias para cuidar de exposição, pode ser importante para compreender os mecanismos moleculares e celulares destes ferimentos. Propomos que a utilização dos rastreio pequena de RNA (siRNA) inibitório alto throughput pode ser uma importante ferramenta que pode ajudar mais rapidamente elucidar os mecanismos moleculares da lesão epitelial de córnea química. siRNA são dobro encalhadas moléculas de RNA que são 19-25 nucleotides longos e utilizam o silenciamento caminho para degradar o mRNA do gene pós-transcricional que tem homologia com o siRNA. A redução resultante da expressão do gene específico em seguida pode ser estudada em células expostas tóxicas para verificar a função desse gene na resposta celular para a toxicidade. O desenvolvimento e validação de modelos de exposição em vitro e métodos para o alto throughput screening (HTS) de fluoreto de hidrogênio (HF) e cloropicrina-(CP) induzido por lesão ocular são apresentados neste artigo. Embora nós selecionamos essas duas substâncias tóxicas, nossos métodos são aplicáveis ao estudo de outras substâncias tóxicas, com pequenas modificações para o protocolo de exposição tóxica. O antígeno de T grande SV40 imortalizado SV40-HCEC foi selecionado para o estudo de linha de célula epitelial da córnea humana. Viabilidade celular e produção de IL-8 foram selecionados como pontos de extremidade no protocolo de triagem. Diversos desafios associados ao desenvolvimento de exposição tóxica e métodos de cultura celular adequados para estudos HTS são apresentados. Permite a criação de modelos HTS para estas substâncias tóxicas mais estudos entender melhor o mecanismo da lesão e a tela para terapêutica potencial para lesão ocular química.

Desenvolvimento do método de exposição
Nós refinado e avaliamos a adequação da linha de células epiteliais da córnea humana SV40-HCEC para uso em estudos HTS. SV40-HCEC foram imortalizados usando o antígeno de T grande SV-40 e foi um presente do amigo Dhanajay23. Havia muitas variáveis exploradas no desenvolvimento de metodologia de exposição para apresentar de forma concisa n...

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

A triagem de RNA inibitória pequena de alta taxa de transferência é uma importante ferramenta que pode ajudar mais rapidamente elucidar os mecanismos moleculares da lesão epitelial de córnea química. Aqui, apresentamos o desenvolvimento e validação de modelos de exposição e métodos para o rastreio de alto rendimento de fluoreto de hidrogênio e cloropicrina-induzido por lesão epitelial de córnea.

High Throughput Screening de SiRNA para lesão de células epiteliais de cloropicrina e córnea induzida pelo fluoreto do hidrogênio

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Research

JoVE Journal

Biology

5.8K visualizações.  US Army Medical Research Institute of Chemical Defense. A triagem de RNA inibitória pequena de alta taxa de transferência é uma importante ferramenta que pode ajudar mais rapidamente elucidar os mecanismos moleculares da lesão epitelial de córnea química. Aqui, apresentamos o desenvolvimento e validação de modelos de exposição e métodos para o rastreio de alto rendimento de fluoreto de hidrogênio e cloropicrina-induzido por lesão epitelial de córnea.

Vídeo: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Alto rendimento Triagem e Biossensoriais com Fluorescente C. elegans Cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biologia

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Alto rendimento Yeast Tela superexpressão Plasmid

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening para pequenas moléculas Moduladores de canais de potássio Interiores retificador

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificação de pares Kinase-substrato utilizando High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzima Triagem Usando um de alta capacidade Enzyme genética Sistema de Triagem

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.