Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity

Autores
Garcia Bustos, Maria Fernanda; Moya Alvarez, Agustin; Pérez Brandan, Cecilia María; Parodi Ramoneda, Cecilia María; Sosa, Andrea Mabel; Buttazzoni Zuñiga, Valeria Carolina; Pastrana, Oscar Marcelo; Manghera, Paula Mariana; Peñalva, Pablo Alejandro; Marco, Jorge Diego; Barroso, Paola Andrea
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis
Fil: Garcia Bustos, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Moya Alvarez, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Parodi Ramoneda, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Sosa, Andrea Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Buttazzoni Zuñiga, Valeria Carolina. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Pastrana, Oscar Marcelo. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Manghera, Paula Mariana. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Peñalva, Pablo Alejandro. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Materia
FLUORESCENT
DRUGS
LEISHMANIA
GALANGIN
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/154227
Acceder
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network_name_str CONICET Digital (CONICET)
spelling Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial ActivityGarcia Bustos, Maria FernandaMoya Alvarez, AgustinPérez Brandan, Cecilia MaríaParodi Ramoneda, Cecilia MaríaSosa, Andrea MabelButtazzoni Zuñiga, Valeria CarolinaPastrana, Oscar MarceloManghera, Paula MarianaPeñalva, Pablo AlejandroMarco, Jorge DiegoBarroso, Paola AndreaFLUORESCENTDRUGSLEISHMANIAGALANGINhttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasisFil: Garcia Bustos, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Moya Alvarez, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Parodi Ramoneda, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Sosa, Andrea Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Buttazzoni Zuñiga, Valeria Carolina. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;Fil: Pastrana, Oscar Marcelo. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;Fil: Manghera, Paula Mariana. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;Fil: Peñalva, Pablo Alejandro. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFrontiers Media2021-06-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/154227Garcia Bustos, Maria Fernanda; Moya Alvarez, Agustin; Pérez Brandan, Cecilia María; Parodi Ramoneda, Cecilia María; Sosa, Andrea Mabel; et al.; Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity; Frontiers Media; Frontiers in Cellular and Infection Microbiology; 11; 4-6-2021; 1-132235-2988CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fcimb.2021.666746/fullinfo:eu-repo/semantics/altIdentifier/doi/10.3389/fcimb.2021.666746info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:21:10Zoai:ri.conicet.gov.ar:11336/154227instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:21:11.06CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
title Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
spellingShingle Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
Garcia Bustos, Maria Fernanda
FLUORESCENT
DRUGS
LEISHMANIA
GALANGIN
title_short Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
title_full Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
title_fullStr Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
title_full_unstemmed Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
title_sort Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity
dc.creator.none.fl_str_mv Garcia Bustos, Maria Fernanda
Moya Alvarez, Agustin
Pérez Brandan, Cecilia María
Parodi Ramoneda, Cecilia María
Sosa, Andrea Mabel
Buttazzoni Zuñiga, Valeria Carolina
Pastrana, Oscar Marcelo
Manghera, Paula Mariana
Peñalva, Pablo Alejandro
Marco, Jorge Diego
Barroso, Paola Andrea
author Garcia Bustos, Maria Fernanda
author_facet Garcia Bustos, Maria Fernanda
Moya Alvarez, Agustin
Pérez Brandan, Cecilia María
Parodi Ramoneda, Cecilia María
Sosa, Andrea Mabel
Buttazzoni Zuñiga, Valeria Carolina
Pastrana, Oscar Marcelo
Manghera, Paula Mariana
Peñalva, Pablo Alejandro
Marco, Jorge Diego
Barroso, Paola Andrea
author_role author
author2 Moya Alvarez, Agustin
Pérez Brandan, Cecilia María
Parodi Ramoneda, Cecilia María
Sosa, Andrea Mabel
Buttazzoni Zuñiga, Valeria Carolina
Pastrana, Oscar Marcelo
Manghera, Paula Mariana
Peñalva, Pablo Alejandro
Marco, Jorge Diego
Barroso, Paola Andrea
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv FLUORESCENT
DRUGS
LEISHMANIA
GALANGIN
topic FLUORESCENT
DRUGS
LEISHMANIA
GALANGIN
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis
Fil: Garcia Bustos, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Moya Alvarez, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Parodi Ramoneda, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Sosa, Andrea Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Buttazzoni Zuñiga, Valeria Carolina. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Pastrana, Oscar Marcelo. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Manghera, Paula Mariana. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Peñalva, Pablo Alejandro. Facultad de Ciencias Agrarias y Veterinarias ; Universidad Catolica de Salta;
Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina
description Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis
publishDate 2021
dc.date.none.fl_str_mv 2021-06-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/154227
Garcia Bustos, Maria Fernanda; Moya Alvarez, Agustin; Pérez Brandan, Cecilia María; Parodi Ramoneda, Cecilia María; Sosa, Andrea Mabel; et al.; Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity; Frontiers Media; Frontiers in Cellular and Infection Microbiology; 11; 4-6-2021; 1-13
2235-2988
CONICET Digital
CONICET
url http://hdl.handle.net/11336/154227
identifier_str_mv Garcia Bustos, Maria Fernanda; Moya Alvarez, Agustin; Pérez Brandan, Cecilia María; Parodi Ramoneda, Cecilia María; Sosa, Andrea Mabel; et al.; Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity; Frontiers Media; Frontiers in Cellular and Infection Microbiology; 11; 4-6-2021; 1-13
2235-2988
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
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info:eu-repo/semantics/altIdentifier/doi/10.3389/fcimb.2021.666746
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eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
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dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
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reponame_str CONICET Digital (CONICET)
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repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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