Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells.The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF)

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistanc

Las agrupaciones corales en España: espacios para la convivencia y la educación musical.

This paper presents the most relevant data obtained from a study conducted with the participation of 1255 choirs, most of them registered in the database of the Music and Dance Documentation Center from the National Institute of Performing Arts and Music of Spain. The main aim of the study of such an extensive array of choirs is to get sufficient information to describe the basic profile of choral activity in Spain. In order to conduct this study a questionnaire was designed including some specific questions for each one of the three types of choirs in which the sample was classified: children/youth choirs, mixed choirs and equal voices choirs. The questionnaire includes variables referring to the basic profile of the choirs, institutional linkage, repertoires, financing, data about the activities of the choirs and the conductors’ profile. The results show, among other things, that the choirs establish long lasting associative experiences and that, though many conductors are trained as musicians, they are not specialized in choral conducting. Finally, the qualitative and quantitative importance of the choral activity to enhance cultural heritage, musical education and social relevance of the choral movement in Spain stands out.Este trabajo presenta los datos más relevantes obtenidos en un estudio en el que han participado 1.225 agrupaciones corales, muchas de ellas registradas en la base de datos del Centro de Documentación de Música y Danza del Instituto Nacional de las Artes Escénicas y de la Música (INAEM). Este estudio, realizado sobre una extensa muestra de coros, tiene como objetivo central la recogida de información descriptiva del perfil básico de la actividad coral en España. Para desarrollar este trabajo se ha diseñado un cuestionario con algunas preguntas específicas formuladas a los tres tipos de agrupaciones en las que se clasifica esta muestra: coros infantiles-juveniles, mixtos y de voces iguales. El cuestionario incluye variables referidas a los datos de identificación de los coros, la vinculación institucional, el repertorio básico, la financiación, información sobre las actividades realizadas en las agrupaciones corales e información sobre el perfil de la dirección coral. Los resultados muestran, entre otros aspectos, que las agrupaciones corales constituyen experiencias asociativas duraderas y que la mayor parte de las personas que asumen la dirección tienen formación musical pero no cuentan con especialización en dirección coral. Finalmente, se destaca la importancia cualitativa y cuantitativa de la actividad coral para la mejora del patrimonio cultural, la enseñanza musical así como la relevancia social que tiene el coralismo en España

Las agrupaciones corales en España: espacios para la convivencia y la educación musical

This paper presents the most relevant data obtained from a study conducted with the participation of 1255 choirs, most of them registered in the database of the Music and Dance Documentation Center from the National Institute of Performing Arts and Music of Spain. The main aim of the study of such an extensive array of choirs is to get sufficient information to describe the basic profile of choral activity in Spain. In order to conduct this study a questionnaire was designed including some specific questions for each one of the three types of choirs in which the sample was classified: children/youth choirs, mixed choirs and equal voices choirs. The questionnaire includes variables referring to the basic profile of the choirs, institutional linkage, repertoires, financing, data about the activities of the choirs and the conductors’ profile. The results show, among other things, that the choirs establish long lasting associative experiences and that, though many conductors are trained as musicians, they are not specialized in choral conducting. Finally, the qualitative and quantitative importance of the choral activity to enhance cultural heritage, musical education and social relevance of the choral movement in Spain stands out

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Abstract Background Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.</p

Intoxicaciones graves con medicamentos. Administración de carbón activado

Las intoxicaciones agudas constituyen un motivo frecuente de consulta en los Servicios de Urgencias; aproximadamente el 80%, acusan una sintomatología leve pero un 5% pueden presentar un compromiso multiorgánico requiriendo su ingreso en una Unidad de Cuidados Intensivos (U.C.I). Muchas de estas intoxicaciones son intentos de autólisis o accidentes domésticos y uno de los tratamientos de elección para este tipo de pacientes es el carbón activado (C.A), administrado conjuntamente con un catártico. En este estudio se identifican los efectos deseables e indeseables de la administración terapéutica de carbón activado y catárticos, en pacientes con intoxicación medicamentosa aguda grave (I.M.A.G.). Se estudiaron 30 pacientes con criterios para el uso de carbón activado, observando que un 23% presentaron vómitos, un 7% se broncoaspiraron, y un 90% sufrieron diarreas en las primeras 24 horas. Este estudio demuestra que la administración de carbón activado y catárticos resulta efectiva pero no inocua, y que presenta algunos problemas que requieren estrecha vigilancia, lo cual nos ha llevado a elaborar un plan de cuidados y una guía clínica para un mejor seguimiento de estos pacientes

Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance. Methods: The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for β-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway. Results: S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. β-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells. Conclusions: S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistanc

Intoxicaciones graves con medicamentos. Administración de carbón activado

Las intoxicaciones agudas constituyen un motivo frecuente de consulta en los Servicios de Urgencias; aproximadamente el 80%, acusan una sintomatología leve pero un 5% pueden presentar un compromiso multiorgánico requiriendo su ingreso en una Unidad de Cuidados Intensivos (U.C.I). Muchas de estas intoxicaciones son intentos de autólisis o accidentes domésticos y uno de los tratamientos de elección para este tipo de pacientes es el carbón activado (C.A), administrado conjuntamente con un catártico. En este estudio se identifican los efectos deseables e indeseables de la administración terapéutica de carbón activado y catárticos, en pacientes con intoxicación medicamentosa aguda grave (I.M.A.G.). Se estudiaron 30 pacientes con criterios para el uso de carbón activado, observando que un 23% presentaron vómitos, un 7% se broncoaspiraron, y un 90% sufrieron diarreas en las primeras 24 horas. Este estudio demuestra que la administración de carbón activado y catárticos resulta efectiva pero no inocua, y que presenta algunos problemas que requieren estrecha vigilancia, lo cual nos ha llevado a elaborar un plan de cuidados y una guía clínica para un mejor seguimiento de estos pacientes