Substituindo idéias, reduzindo conceitos errados e refinando argumentos: Uma reinterpretação dos 3Rs da experimentação animal

Purpose/Background. In the present work, a reinterpretation of the 3Rs (3Rs) proposed by William Russell and Rex Burch (Replacement, Reduction and Refinement) will be carried out with the aim of broadening its scope and improving nonhuman animal experimentation practices. Methodology/Approach. The meaning given by Russell and Burch to the 3Rs will be reviewed and the way in which each of them could be redefined or complemented in the light of current scientific practices, technical possibilities and bioethical knowledge related to the use of animals in research will be evaluated. Results/Findings. The article will show that 1) not only animals should be replaced, but also the misconceptions we have, both about them and about the importance of bioethics education in scientific training, 2) that the reduction, in addition to the number of subjects used in each experiment, should serve to end unnecessary, repetitive and superfluous research, as well as some persistent misconceptions about the way science operates, and 3) that refinement should go beyond the experimental space to extend to the way we think about animal ethics in the research setting. Discussion/Conclusions/Contributions. The paper reports on the importance of incorporating contemporary bioethical knowledge into animal experimentation practices to enhance the reflexive and ethical character of science.Propósito/Contexto. En el presente trabajo se llevará a cabo una reinterpretación de las tres erres (3R) propuestas por William Russell y Rex Burch (reemplazo, reducción y refinamiento), con el objetivo de ampliar su alcance y mejorar las prácticas de experimentación con animales no humanos. Metodología/Enfoque. Se revisará el sentido que le dieron Russell y Burch a las 3R y se evaluará el modo en que cada una de ellas podría redefinirse o complementarse a la luz de las prácticas científicas, las posibilidades técnicas y los conocimientos bioéticos actuales vinculados al uso de animales en investigación. Resultados/Hallazgos. El artículo mostrará que 1) no solo habrían de reemplazarse animales, sino también las ideas equívocas que tenemos, tanto sobre ellos, como sobre la importancia de la educación bioética en la formación científica, 2) que la reducción, además de referirse al número de sujetos utilizados en cada experimento, debería servir para acabar con investigaciones innecesarias, repetitivas y superfluas, así como con algunos persistentes equívocos sobre el modo de operar de la ciencia y 3) que el refinamiento tendría que salir del espacio experimental para extenderse al modo en que pensamos sobre ética animal en el ámbito de la investigación. Discusión/Conclusiones/Contribuciones. El trabajo da cuenta de la importancia que tiene la incorporación del conocimiento bioético contemporáneo en las prácticas de experimentación con animales para mejorar el carácter reflexivo y ético de la ciencia.Objetivo/Contexto. Neste documento, uma reinterpretação dos 3Rs (3Rs) propostos por William Russell e Rex Burch (Substituição, Redução e Refinamento) será realizada com o objetivo de ampliar seu escopo e melhorar as práticas não-humanas de testes em animais. Metodologia/ Abordagem. Revisaremos o significado dado por Russell e Burch aos 3Rs e avaliaremos como cada um deles poderia ser redefinido ou complementado à luz das práticas científicas atuais, possibilidades técnicas e conhecimentos bioéticos relacionados ao uso de animais na pesquisa. Resultados/Descobertas. O artigo mostrará que 1) não somente os animais devem ser substituídos, mas também conceitos errôneos sobre eles e a importância da educação bioética no treinamento científico, 2) que a redução, além do número de sujeitos utilizados em cada experimento, deve servir para eliminar pesquisas desnecessárias, repetitivas e supérfluas, assim como alguns conceitos errôneos persistentes sobre a maneira como a ciência funciona, e 3) que o refinamento deve se estender além do espaço experimental para a maneira como pensamos sobre a ética animal na pesquisa. Discussão/Conclusões/Contribuições. O artigo explica a importância de incorporar o conhecimento bioético contemporâneo nas práticas de experimentação animal para realçar o caráter reflexivo e ético da ciência

Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis.

BackgroundAlthough much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.ResultsIn this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.ConclusionsThis study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae

Comparative Proteomic Analysis of Aedes aegypti Larval Midgut after Intoxication with Cry11Aa Toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes

The Teaching of Bioethics in Science

Tema del mesEl objetivo del presente trabajo es discutir la importancia que tiene la enseñanza de la bioética en la formación científica. La ciencia es una actividad que ha aportado innumerables avances y beneficios en términos sociales, sin embargo, su construcción a través de la historia no ha estado exenta de abusos, violaciones y prácticas desproporcionadas por parte del ser humano hacia su propia especie, los animales, las plantas, los ecosistemas y el planeta entero. Este trabajo es una reflexión sobre alternativas más juiciosas, sustentables, benéficas y libres de daño hacia los otros mediante la educación de estrategias alternativas para minimizar el daño antropogénico. Asimismo, plantea interrogantes respecto a los mitos sobre la ciencia y los científicos, así como la conveniencia de cambiar estos dogmas por nuevos paradigmas, y discute la idea de que un científico con formación bioética no tiene la opción de permanecer en un silencio informado respecto a los alcances, riesgos y consecuencias de su labor científica. Finalmente, respecto al gremio científico, se sugiere aplicar los principios de la triple RRR (Reemplazar, Reducir y Refinar) desde un punto de vista diferenteThe aim of this paper is to discuss the importance of bioethics teaching to both science students and teachers. Science has brought countless advances and benefits to human well-being, however, its historical development has occurred in parallel with abuse, slavery and disproportionate practices from human beings towards their own species, animals, plants, ecosystems and the entire planet. We discuss on alternatives in education considered more judicious, sustainable, benefic and damage free towards “the others” in order to minimize anthropogenic damage. Questions regarding the myths of science and scientists are raised along from the convenience of changing old to new paradigms. Scientific community with bioethical training no longer has the option to remain uninformed about the implications, risks and consequences of their scientific work. Finally, regarding the scientific guild, it is suggested to apply 3Rs principles (Replacement, Reduction, and Refinement) from a different point of vie

Aedes aegypti Mos20 Cells Internalizes Cry Toxins by Endocytosis, and Actin Has a Role in the Defense against Cry11Aa Toxin

Bacillus thuringiensis (Bt) Cry toxins are used to control Aedes aegypti, an important vector of dengue fever and yellow fever. Bt Cry toxin forms pores in the gut cells, provoking larvae death by osmotic shock. Little is known, however, about the endocytic and/or degradative cell processes that may counteract the toxin action at low doses. The purpose of this work is to describe the mechanisms of internalization and detoxification of Cry toxins, at low doses, into Mos20 cells from A. aegypti, following endocytotic and cytoskeletal markers or specific chemical inhibitors. Here, we show that both clathrin-dependent and clathrin-independent endocytosis are involved in the internalization into Mos20 cells of Cry11Aa, a toxin specific for Dipteran, and Cry1Ab, a toxin specific for Lepidoptera. Cry11Aa and Cry1Ab are not directed to secretory lysosomes. Instead, Mos20 cells use the Rab5 and Rab11 pathways as a common mechanism, most probably for the expulsion of Cry11Aa and Cry1Ab toxins. In conclusion, we propose that endocytosis is a mechanism induced by Cry toxins independently of specificity, probably as part of a basal immune response. We found, however, that actin is necessary for defense-specific response to Cry11Aa, because actin-silenced Mos20 cells become more sensitive to the toxic action of Cry11A toxin. Cry toxin internalization analysis in insect cell lines may contribute to a better understanding to Cry resistance in mosquitoes

Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis.

BackgroundAlthough much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.ResultsIn this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.ConclusionsThis study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae

Addendum: Bedoya-Pérez, L.P. et al. Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis. Int. J. Mol. Sci. 2013, 14, 8467–8478

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