29 research outputs found
Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions
More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-β-cyclodextrins (β-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with β - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + β - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells. Keywords: Taxus baccata; cell cultures; coronatine; gene expression; lipid droplets; paclitaxel; taxane accumulation
Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance
Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds - commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity
Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines
The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studied against three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chromatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer
Equidad en el cálculo de los expedientes universitarios. Expedientes cualitativos vs cuantitativos
Introducción: El expediente final de un graduado puede tener una importante repercusión en su futura carrera profesional, es por ello que también debería ser establecido de la forma más justa posible. Hasta hace unos años los expedientes en la universidad española se calculaban a partir de criterios cualitativos de 4 categorías (1-4). En el año 2003 se propone una calificación decimal (0-10) para calcular los expedientes, hecho que garantizaría más la equidad. El objetivo del estudio consiste en determinar la relación entre los 2 tipos de calificaciones y en observar las posibles diferencias y posibles repercusiones. Material y métodos: Para el estudio se registraron los datos de 6 promociones de la extinta licenciatura de biología, 5 promociones del grado de biología humana y de 3 promociones de medicina de la Facultad de Ciencias de la Salud y de la Vida de la Universidad Pompeu Fabra. Resultados: Se observaron correlaciones muy elevadas (superiores a 0,9) entre las 2 opciones de evaluación, pero se puso de manifiesto que con el sistema decimal existiría un mayor número de egresados con expedientes brillantes. Discusión: Dado que el sistema decimal de evaluación era más equitativo se propone que las autoridades responsables utilicen el sistema decimal de evaluación en vez del cualitativo que todavía continúa en vigor.Introduction: As the final academic record of a graduate can have an important impact on their future professional career, this should be established as fairly as possible. Until a few years ago, the academic grades in Spanish universities were calculated based on qualitative criteria of four categories (1-4). In 2003, a decimal rating (0-10) was proposed to calculate the grades; a fact that would further guarantee fairness. The objective of the study is to determine the relationship between the two types of qualifications and to observe any differences and repercussions. Material and methods: For the study data was recorder from 6 courses of the former Biology degree, 5 courses of the Human Biology degree, and of 3 Medicine courses of the Faculty of Health and Life Sciences of the Pompeu Fabra University. Results: Very high correlations (higher than 0.9) were observed between the two assessment options, but it was observed that with the decimal system there would be a greater number of graduates with brilliant results. Discussion: Since the decimal evaluation system was more equitable, it is proposed that the responsible authorities use the decimal assessment system instead of the qualitative one that is still in force
La presencia de las humanidades en las carreras de odontología de las universidades de Chile y de España
Se ha argumentado que la presencia de materias humanísticas permite una formación integral de los alumnos, lo que fomentaría en última instancia una mejor relación con los futuros pacientes y, por tanto, contribuiría a mejorar la salud de la población. El objetivo del presente estudio consistió en investigar la presencia de contenidos humanísticos en los grados de Odontología de las universidades de Chile y de España. Se encontró que en todas las universidades existen materias humanísticas que complementan la formación disciplinar de los graduados. Si bien se encontraron diferencias entre las universidades chilenas y españolas, las materias relacionadas con contenidos éticos, históricos y con el profesionalismo son ofrecidas por la gran mayoría de las universidades de los dos países. Por otra parte, la presencia de materias relacionadas con la literatura o con las artes plásticas, visuales y escénicas fue escasa, muy minoritaria en España y nula en Chile. Se aboga por aumentar la presencia de la formación humanística en los graduados de odontología, no solamente a partir de asignaturas específicas, sino introduciendo contenidos humanísticos en las materias troncales de formación técnica odontológica
Cultivos celulares de Taxus spp., una eficaz herramienta biotecnológica para la producción de taxanos y para el desarrollo de estudios básicos sobre su biosíntesis (Seminaris de Recerca 2017)
Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance
Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds – commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity
Exploring the Metabolic Stability of Engineered Hairy Roots after 16 Years Maintenance
Plants remain a major source of new drugs, leads and fine chemicals. Cell cultures deriving from plants offer a fascinating tool to study plant metabolic pathways and offer large scale production systems for valuable compounds - commercial examples include compounds such as paclitaxel. The major constraint with undifferentiated cell cultures is that they are generally considered to be genetically unstable and cultured cells tend to produce low yields of secondary metabolites especially over time. Hairy roots, a tumor tissue caused by infection of Agrobacterium rhizogenes is a relevant alternative for plant secondary metabolite production for being fast growing, able to grow without phytohormones, and displaying higher stability than undifferentiated cells. Although genetic and metabolic stability has often been connected to transgenic hairy roots, there are only few reports on how a very long-term subculturing effects on the production capacity of hairy roots. In this study, hairy roots producing high tropane alkaloid levels were subjected to 16-year follow-up in relation to genetic and metabolic stability. Cryopreservation method for hairy roots of Hyoscyamus muticus was developed to replace laborious subculturing, and although the post-thaw recovery rates remained low, the expression of transgene remained unaltered in cryopreserved roots. It was shown that although displaying some fluctuation in the metabolite yields, even an exceedingly long-term subculturing was successfully applied without significant loss of metabolic activity
Cultivos celulares de Taxus spp., una eficaz herramienta biotecnológica para la producción de taxanos y para el desarrollo de estudios básicos sobre su biosíntesis (Seminaris de Recerca 2017)
Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel
Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.This research was funded by Agencia Estatal de Investigación. REF: PID2020-113438RBI00/AEI/10.13039/501100011033 and by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) del Departament de Recerca i Universitats de la Generalitat de Catalunya 2021 SGR00693. D.H.-M. is a Postdoctoral researcher at María Zambrano at the University of Barcelona. His contract is financed by the Ministry of Universities, the European Union Next GenerationEU/PRTR.i and the Recovery, Transformation and Resilience Plan