Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2017 |
| Outros Autores: | , , , , , |
| Tipo de documento: | Artigo |
| Idioma: | eng |
| Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
| Texto Completo: | https://www.arca.fiocruz.br/handle/icict/20137 |
Resumo: | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil. |
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Profeta, Gerson S.Pereira, Jessica A. S.Costa, Samara G.Azambuja, PatriciaGarcia, Eloi S.Moraes, Caroline da SilvaGenta, Fernando A.2017-07-18T15:18:10Z2017-07-18T15:18:10Z2017PROFETA, Gerson S. et al. Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels. Frontiers in Physiology, v.8, Article 308, 12p, May 2017.1664-042Xhttps://www.arca.fiocruz.br/handle/icict/2013710.3389/fphys.2017.00308engFrontiers MediaRhodnius prolixusLutzomyia longipalpisEnsaio fluorescenteDigestão de insetosHidrolase de glicosídeoglycoside hydrolasefluorescent assayinsect digestionRhodnius prolixusStandardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levelsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, β-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, β-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. β-Glucosidase, β-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/20137/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALgerson_profeta_etal_IOC_2017.pdfgerson_profeta_etal_IOC_2017.pdfapplication/pdf2453803https://www.arca.fiocruz.br/bitstream/icict/20137/2/gerson_profeta_etal_IOC_2017.pdfc9b7d4eb436aeb57873c6b4d0daa4e82MD52TEXTgerson_profeta_etal_IOC_2017.pdf.txtgerson_profeta_etal_IOC_2017.pdf.txtExtracted 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| dc.title.pt_BR.fl_str_mv |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| title |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| spellingShingle |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels Profeta, Gerson S. Rhodnius prolixus Lutzomyia longipalpis Ensaio fluorescente Digestão de insetos Hidrolase de glicosídeo glycoside hydrolase fluorescent assay insect digestion Rhodnius prolixus |
| title_short |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| title_full |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| title_fullStr |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| title_full_unstemmed |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| title_sort |
Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels |
| author |
Profeta, Gerson S. |
| author_facet |
Profeta, Gerson S. Pereira, Jessica A. S. Costa, Samara G. Azambuja, Patricia Garcia, Eloi S. Moraes, Caroline da Silva Genta, Fernando A. |
| author_role |
author |
| author2 |
Pereira, Jessica A. S. Costa, Samara G. Azambuja, Patricia Garcia, Eloi S. Moraes, Caroline da Silva Genta, Fernando A. |
| author2_role |
author author author author author author |
| dc.contributor.author.fl_str_mv |
Profeta, Gerson S. Pereira, Jessica A. S. Costa, Samara G. Azambuja, Patricia Garcia, Eloi S. Moraes, Caroline da Silva Genta, Fernando A. |
| dc.subject.other.pt_BR.fl_str_mv |
Rhodnius prolixus Lutzomyia longipalpis Ensaio fluorescente Digestão de insetos Hidrolase de glicosídeo |
| topic |
Rhodnius prolixus Lutzomyia longipalpis Ensaio fluorescente Digestão de insetos Hidrolase de glicosídeo glycoside hydrolase fluorescent assay insect digestion Rhodnius prolixus |
| dc.subject.en.pt_BR.fl_str_mv |
glycoside hydrolase fluorescent assay insect digestion Rhodnius prolixus |
| description |
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil. |
| publishDate |
2017 |
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2017-07-18T15:18:10Z |
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2017-07-18T15:18:10Z |
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2017 |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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PROFETA, Gerson S. et al. Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels. Frontiers in Physiology, v.8, Article 308, 12p, May 2017. |
| dc.identifier.uri.fl_str_mv |
https://www.arca.fiocruz.br/handle/icict/20137 |
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1664-042X |
| dc.identifier.doi.none.fl_str_mv |
10.3389/fphys.2017.00308 |
| identifier_str_mv |
PROFETA, Gerson S. et al. Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels. Frontiers in Physiology, v.8, Article 308, 12p, May 2017. 1664-042X 10.3389/fphys.2017.00308 |
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eng |
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Frontiers Media |
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