Nuevos biomarcadores de cáncer de pulmón basados en miRNA / [New biomarkers of lung cancer based on miRNA]

Objetivo: Determinar si existen diferencias de expresión entre los miRNA de tejido sano y tumoral de adenocarcinoma de pulmón y carcinoma epidermoide de pulmón, con lo que podrían ser usados como biomarcadores. Material y métodos: Se ha extraído y secuenciado el miRNA de tejido tumoral y sano adyacente de muestras de adenocarcinoma y carcinoma epidermoide de dieciséis pacientes intervenidos en el Hospital Regional de Málaga. Esas secuencias se han analizado con un flujo de trabajo bioinformático específico que conlleva varios pasos: 1o) preprocesar las lecturas, 2o) mapearlas sobre el genoma humano de referencia, 3o) determinar la expresión de los miRNA en cada una de las muestras, 4o) calcular su expresión diferencial entre el tejido sano y el tumoral de cada paciente, 5o) realizar un análisis funcional de los miRNA encontrados. Resultados: Hemos analizado los miRNA con expresión diferencial en cada uno de los tipos histológicos estudiados. El análisis del adenocarcinoma y carcinoma epidermoide de pulmón ha dado como resultado un total de 82 y 360 miRNA diferencialmente expresados (miDE), respectivamente. Hemos encontrado 50 miRNA comunes a los dos tipos histológicos, y su análisis funcional indica que están implicados en el crecimiento, desarrollo y movimiento celular, que se produce tanto en la célula normal como en el cáncer. Conclusiones: Los miDE encontrados son una fuente de biomarcadores, al tener una reprogramación específica en cáncer, y ser obtenibles de forma no invasiva. (AU)

Objective: Determine if there are differences in expression between the miRNAs of healthy and tumor tissue of lung adenocarcinoma and squamous cell carcinoma of the lung, with which they could be used as biomarkers. Material and methods: miRNA has been extracted and sequenced from tumor and adjacent healthy tissue from samples of adenocarcinoma and squamous cell carcinoma from sixteen patients operated at the Regional Hospital of Malaga. These sequences have been analyzed with a specific bioinformatic workflow that involves several steps: 1st) preprocessing the reads, 2nd) mapping them onto the reference human genome, 3rd) determining the expression of the miRNAs in each of the samples, 4th) calculate its differential expression between the healthy and tumor tissue of each patient, 5th) perform a functional analysis of the miRNAs found. Results: We have analyzed the miRNAs with differential expression in each of the histological types studied. Analysis of adenocarcinoma and squamous cell carcinoma of the lung has resulted in a total of 82 and 360 differentially expressed miRNAs (miDE), respectively. We have found 50 miRNAs common to the two histological types, and their functional analysis indicates that they are involved in cell growth, development and movement, which occurs both in normal cells and in cancer. Conclusions: The miDEs found are a source of biomarkers, as they have a specific reprogramming in cancer, and are obtainable non-invasively. (AU)

Estudio de la desregulación de los transposones en el carcinoma epidermoide, el adenocarcinoma y el carcinoma microcítico de pulmón: en busca de nuevos biomarcadores / Study on the deregulation of transposons in squamous cell carcinoma, adenocarcinoma and small cell carcinoma of the lung: in search of new biomarkers

OBJETIVO: Determinar si existen biomarcadores entre los transposones, tanto para diagnóstico como pronóstico de adenocarcinoma, carcinoma epidermoide y carcinoma microcítico de pulmón, así como valorar diferencias y similitudes entre estos tres tipos histológicos. MATERIAL Y MÉTODOS: Se ha secuenciado el RNA total del tejido tumoral y sano adyacente de muestras de adenocarcinoma y carcinoma epidermoide de dieciséis pacientes intervenidos en el Hospital Regional de Málaga. En el caso del carcinoma microcítico, se han utilizado pacientes externos cuyos datos proceden de los repositorios genómicos disponibles para la comunidad científica. Esas secuencias se han analizado con un flujo de trabajo bioinformático específico que conlleva varios pasos: 1º) Preprocesar las lecturas, 2º) Mapearlas sobre el genoma humano de referencia, 3º) Determinar la expresión de los transposones en cada una de las muestras, y 4º) Calcular su expresión diferencial entre el tejido sano y el tumoral de cada paciente. RESULTADOS: En un primer paso, hemos analizado los transposones con expresión diferencial en cada uno de los tipos histológicos estudiados por separado. El análisis del adenocarcinoma, carcinoma microcítico y carcinoma epidermoide de pulmón ha dado como resultado un total de 7, 72 y 12 transposones diferencialmente expresados (TDE), respectivamente. Hemos encontrado transposones comunes a los tres tipos histológicos y otros cuyo comportamiento es específico en cada uno de ellos. CONCLUSIONES: Los transposones se reprograman específicamente cuando una célula normal del pulmón se vuelve cancerosa. Esta reprogramación es una fuente de biomarcadores que podría ayudar al diagnóstico precoz del cáncer

OBJECTIVE: To determine whether biomarkers exist among transposons for both the diagnosis and prognosis of adenocarcinoma, squamous cell carcinoma and small cell carcinoma of the lung, as well as to evaluate differences and similarities between these three histological types. MATERIAL AND METHODS: The total RNA was sequenced for the tumor tissue and adjacent healthy tissue in adenocarcinoma and squamous cell carcinoma samples from sixteen patients being treated at the Hospital Regional de Málaga. In the case of small cell carcinoma, external patients were used whose data came from genomic repositories available to the scientific community. These sequences were analyzed with a specific bioinformatic workflow which includes several steps: 1) Pre-process readings, 2) Map them on the reference human genome, 3) Determine transposon expression in each of the samples, and 4) Calculate the differential expression between the healthy and tumor tissue in each patient. RESULTS: In the first step, we analyzed the transposons with differential expression in each of the histological types studied separately. The analysis of adenocarcinoma, small cell carcinoma and squamous cell carcinoma of the lung has resulted in a total of 7, 72 and 12 differentially expressed transposons, respectively. We found transposons common to all three histological types and others whose behavior is specific to each type. CONCLUSIONS: Transposons are specifically reprogrammed when a normal lung cell becomes cancerous. This reprogramming is a source of biomarkers that could help with the early diagnosis of cancer

Expression-based, consistent biomarkers for prognosis and diagnosis in lung cancer.

OVERVIEW: Lung cancer is one of the deadliest cancers in the world. Its histological classification depends on early diagnosis and successful treatment. Therefore, having specific biomarkers for a quick sorting widens the successful output of lung cancer treatment. MATERIAL AND METHODS: High-throughput sequencing (RNA-seq) was performed of small cohorts of BioBanco samples from healthy and tumour cells from lung adenocarcinoma (LUAD) and squamous cell carcinoma of the lung (lSCC). RNA-seq samples from small cell lung cancer (SCLC) were downloaded from databases. A bioinformatic workflow has been programmed for the identification of differentially expressed genes (DEGs). RESULTS: A total of 4777 DEGs were differentially expressed in SCLC, 3676 DEGs were in lSCC, while the lowest number of DEGs, 2819, appeared in LUAD. Among them, 945 DEGs were common to the three histological types. Once validated their expression profile and their survival predictive capacity in large, public cohorts, three DEGs can be exclusively considered as diagnostic biomarkers, three as prognosis biomarkers, and other three exhibit both diagnosis and prognosis capabilities. CONCLUSIONS: This prospective study presents evidences for the diagnostic and prognostic capabilities of expression changes in CAPN8-2, TMC5 and MUC1 in LUAD, while they are non-significant in SCLC and lSCC. Their translation to clinical practice is proposed.

Estabilidad de pantoprazol en unidades para nutricion parenteral / Stability of pantoprazole in parenteral nutrition units

Introduccion: El pantoprazol es una base debil (pka * 4) y su estabilidad en solucion acuosa es dependiente del pH. Teniendo en cuenta que el pH de las unidades de nutricion parenteral (UNP) puede oscilar entre 6,0 y 6,5, y puesto que pantoprazol parece ser el inhibidor de la bomba de protones mas estable a pH acido, se evaluo la posibilidad de adicionarlo a las UNP con objeto de facilitar su administracion a los pacientes. Metodos: Se determina por cromatografia liquida de alta resolucion (HPLC, del ingles high performance liquid chromatographic) la riqueza de pantoprazol en una UNP a diferentes intervalos de tiempo. Resultados: La determinacion cromatografica de las concentraciones de pantoprazol reflejo un rapido y progresivo envejecimiento de la muestra. Pasadas 24 h la cantidad de farmaco detectado en la UNP es inferior al 50% del total adicionado. Conclusiones: A la vista de estos resultados se desaconseja este vehiculo para la administracion de pantoprazol, ya que puede poner en riesgo la seguridad de los pacientes al infradosificar la medicacion que requieren y exponerlos a los posibles efectos desconocidos de los diferentes productos de degradacion del farmaco (AU)

Introduction: Pantoprazole is a weak base (pka ¡Ö 4) with its stability in aqueous solution dependent on pH. Keeping in mind that the pH of the parenteral nutrition units (PNU) can range between 6.0 and6.5 and since pantoprazole seems to be the most stable protonpump inhibitor (PPI) for pH acid, we want to assess the possibility of adding it to PNU with the aim of simplifying its administration to patients. Methods: Using high performance liquid chromatographic (HPLC) to measure pantoprazole content in PNU at different time intervals. Results: The chromatographic determination of pantoprazole concentration reflected a rapid and progressive aging of the sample. After 24 h the quantity of drug detected in the PNU was below 50% of the total added. Conclusions: In view of these results, we therefore do not suggest this as a suitable vehicle for pantoprazole administration as it could put patients at risk of being under-dosed and therefore exposing them to potential unknown side effects of the different drug degradation products (AU)

[Stability of pantoprazole in parenteral nutrition units]. / Estabilidad de pantoprazol en unidades para nutrición parenteral.

INTRODUCTION: Pantoprazole is a weak base (pka approximately equal to 4) with its stability in aqueous solution dependent on pH. Keeping in mind that the pH of the parenteral nutrition units (PNU) can range between 6.0 and 6.5 and since pantoprazole seems to be the most stable proton pump inhibitor (PPI) for pH acid, we want to assess the possibility of adding it to PNU with the aim of simplifying its administration to patients. METHODS: Using high performance liquid chromatographic (HPLC) to measure pantoprazole content in PNU at different time intervals. RESULTS: The chromatographic determination of pantoprazole concentration reflected a rapid and progressive aging of the sample. After 24 h the quantity of drug detected in the PNU was below 50% of the total added. CONCLUSIONS: In view of these results, we therefore do not suggest this as a suitable vehicle for pantoprazole administration as it could put patients at risk of being under-dosed and therefore exposing them to potential unknown side effects of the different drug degradation products.

[Comparison of high-resolution liquid chromatography versus microparticle enzyme immunoassay for the measurement of sirolimus levels in renal transplantation]. / Comparación de la cromatografía líquida de alta resolución frente al enzimoinmunoensayo de micropartículas para la determinación de sirolimus en trasplante renal.

OBJECTIVE: To compare sirolimus levels measured in whole blood using two analytical techniques: high-resolution liquid chromatography and microparticle enzyme immunoassay, and to evaluate whether hemoglobin, hematocrit, and time from kidney transplantation influence results obtained using the immune-enzymatic technique. METHOD: A retrospective, observational study in which all transplanted patients with at least one measurement of sirolimus levels using high-resolution liquid chromatography or microparticle enzyme immunoassay from October 2004 to May 2005 were consecutively included. For statistical comparisons simple linear regression, ANCOVA, intra-class correlation coefficient, and the method of agreement limits were all used. RESULTS: Ninety-one patients were assessed for a total of 307 measurements (median: 2, inter-quartile range: 1-4, range: 1-15) of sirolimus levels. The straight-line equation using the linear regression analysis was as follows: MEIA = 0.70 (95% CI: 0.39-1.01) + 1.14 (95% CI: 1.10-1.17) x HPLC/UV. The intra-class correlation coefficient between both measurements was 0.955 (95% CI 0.944-0.964). Mean overestimation using enzyme immunoassay was 24.8% +/- 19.4%. Difference in means between both measurements was 1.9 +/- 1.3 ng/mL. Agreement limits were established between -0.8 ng/mL (95% CI: -1.05; -0.55) and +4.6 ng/mL (95% CI: 4.35; 4.85). Factors such as post-transplant time, hemoglobin, and hematocrit did not influence overestimates obtained using enzyme immunoassays. These results were not influenced by non-independence in measurements. CONCLUSIONS: Despite enzyme immunoassay overestimates in establishing sirolimus levels in whole blood, its correlation with chromatography is acceptable. Added to its benefits versus chromatographic techniques, this renders enzyme immunoassay a good alternative for the measurement of sirolimus levels in whole blood.

Comparación de la cromatografía líquida de alta resolución frente al enzimoinmunoensayo de micropartículas para la determinación de sirolimus en trasplante renal / No disponible

Objetivo: Comparar los niveles de sirolimus obtenidos en sangretotal, mediante dos técnicas analíticas: cromatografía líquidade alta resolución y enzimoinmunoensayo de micropartículas yevaluar si los valores de hemoglobina, hematocrito y tiempo transcurridodesde el trasplante renal influyen en los resultados obtenidoscon la técnica inmunoenzimática.Método: Estudio observacional, retrospectivo, en el que seincluyeron de manera consecutiva todos aquellos pacientes trasplantadosrenales con al menos una determinación de sirolimus,mediante cromatografía líquida de alta resolución y enzimoinmunoensayode micropartículas, entre los meses de octubre de2004 a mayo de 2005, ambos inclusive. Para los contrastesestadísticos se utilizaron la regresión lineal simple, el ANCOVA,el coeficiente de correlación intraclase y el método de los límitesdel acuerdo.Resultados: Se evaluaron 91 pacientes con un total de 307determinaciones (mediana: 2, rango intercuartílico: 1-4, rango:1-15) de sirolimus. La ecuación de la recta mediante análisis deregresión lineal fue la siguiente: MEIA = 0,70 (IC95%: 0,39-1,01)+ 1,14 (IC95%: 1,10-1,17) x HPLC/UV. El coeficiente de correlaciónintraclase entre ambas mediciones fue de 0,955 (IC95%0,944-0,964). La media de sobreestimación por enzimoinmunoensayofue de 24,8% ± 19,4%. La diferencia de medias entreambas mediciones fue de 1,9 ± 1,3 ng/mL. Los límites del acuerdoquedaron establecidos entre -0,8 ng/mL (IC95%: -1,05; -0,55)y +4,6 ng/mL (IC95%: 4,35; 4,85). Los factores tiempo postrasplante,hemoglobina y hematocrito no influyeron en la sobreestimaciónobtenida con el enzimoinmunoensayo. Estos resultadosno se vieron influidos por la no independencia de las mediciones.Conclusiones: A pesar de la sobreestimación del enzimoinmunoensayoen la determinación de sirolimus en sangre total, sucorrelación con la técnica cromatográfica es aceptable. Esto, unidoa las ventajas que presenta frente a las técnicas cromatográficas,hace del enzimoinmunoensayo una buena alternativa en ladeterminación de sirolimus en sangre total

Objective: To compare sirolimus levels measured in wholeblood using two analytical techniques: high-resolution liquid chromatographyand microparticle enzyme immunoassay, and to evaluatewhether hemoglobin, hematocrit, and tome from kidneytransplantation influence results obtained using the immune-enzymatictechnique.Method: A retrospective, observational study in which alltransplanted patients with at least one measurement of sirolimuslevels using high-resolution liquid chromatography or microparticleenzyme immunoassay from October 2004 to May 2005 wereconsecutively included. For statistical comparisons simple linearregression, ANCOVA, intra-class correlation coefficient, and themethod of agreement limits were all used.Results: Ninety-one patients were assessed for a total of 307measurements (median: 2, inter-quartile range: 1-4, range: 1-15)of sirolimus levels. The straight-line equation using the linearregression analysis was as follows: MEIA = 0.70 (95% CI: 0.39-1.01) + 1.14 (95% CI: 1.10-1.17) x HPLC/UV. The intra-classcorrelation coefficient between both measurements was 0.955(95% CI 0.944-0.964). Mean overestimation using enzyme immunoassay was 24.8% ± 19.4%. Difference in means betweenboth measurements was 1.9 ± 1.3 ng/mL. Agreement limits wereestablished between –0.8 ng/mL (95% CI: -1.05; -0.55) and +4.6ng/mL (95% CI: 4.35; 4.85). Factors such as post-transplanttime, hemoglobin, and hematocrit did not influence overestimatesobtained using enzyme immunoassays. These results were notinfluenced by non-independence in measurements.Conclusions: Despite enzyme immunoassay overestimates inestablishing sirolimus levels in whole blood, its correlation withchromatography is acceptable. Added to its benefits versus chromatographictechniques, this renders enzyme immunoassay agood alternative for the measurement of sirolimus levels in wholeblood

Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development.

Ammonium assimilation during the initial stages of Scots pine growth involves two cytosolic glutamine synthetase (GS, EC: 6.3.1.2) isoenzymes encoded by separate genes, GS1a and GS1b. GS1a was most exclusively expressed in photosynthetic tissues of the seedling whereas GS1b was expressed ubiquitously showing higher levels in non-photosynthetic tissues such as root and hypocotyl. Temporal expression analysis has shown that when germination starts GS1b is the predominant form in the embryo, however, its relative abundance in the tissue decreased in the postgerminative stages when green cotyledons are developed. In contrast GS1a was present at a low level in the embryo but its abundance increased markedly during germination and seedling growth. These data suggest that GS1a and GS1b genes display different and non-redundant roles in the nitrogen metabolism of conifers. The precise localization of individual transcripts by in situ hybridization strongly supports this possibility. GS1 gene products are mainly expressed in different cellular types: GS1a in chlorophylic parenchyma and GS1b in the vascular bundles of all tissues examined in the seedling. Our data support that glutamine biosynthesis in pine seedlings follows a different pattern related to angiosperms involving two cytosolic GS proteins: one of them a typical cytosolic GS which may be involved in the generation of glutamine for N transport and a second cytosolic GS generating amino donors for the biosynthesis of major N compounds in photosynthetic tissues, a closer role to angiosperm chloroplastic GS. The results are discussed with regard to recent studies on N mobilization and metabolism during the initial stages of conifer development.