MIDcor, an R-program for deciphering mass interferences in mass spectra of metabolites enriched in stable isotopes

Background: Tracing stable isotopes, such as 13 C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study. Results: Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D)between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2)D was found to decrease when the tested substance was 13 C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13 C labeling is known a priori. Conclusion: Using the developed program improves the reliability of stable isotope tracer data analysis

Metabolic reprogramming and vulnerabilities of prostate cancer stem cells independent or epithelial-mesenchymal transition

[spa] El proceso de la metástasis es la principal causa de mortalidad en pacientes de cáncer. En los últimos años se ha desvelado la importancia de la cooperación entre distintas subpoblaciones celulares que coexisten en el tumor. Entre estas subpoblaciones, encontramos las células denominadas cancer stem cells (CSCs), con un elevado potencial de autorenovación, pluripotencia y capacidad de iniciar tumores. Por otro lado, ciertas subpoblaciones celulares del tumor son capaces de incrementar sus capacidades migratorias e invasivas, mediante el proceso de epithelial-mesenchymal transition (EMT). Diversos estudios han demostrado que la cooperación entre CSCs y células que han activado el programa EMT facilita la colonización metastásica. Dado que la reprogramación metabólica es responsable de proveer a las células tumorales aquellos recursos bioenergéticos y de biosíntesis necesarios para el mantenimiento de su fenotipo tumoral, en este trabajo se ha caracterizado el metabolismo y las vulnerabilidades metabólicas de dos subpoblaciones celulares derivadas de la línea celular PC-3, con características diferenciadas de CSCs por un lado (PC-3M) y de EMT por otro (PC-3S). El estudio metabólico de estas subpoblaciones celulares desveló que las células PC-3M presentan una mayor preferencia para el uso de la glucólisis (efecto Warburg más marcado), mientras que las PC-3S son más dependientes del metabolismo energético mitocondrial. Estas subpoblaciones también difieren en el uso de las ramas oxidativa y no oxidativa de la vía de las pentosas fosfato y en las reacciones de biosíntesis y degradación de ácidos grasos, con el fin de satisfacer las distintas necesidades metabólicas que caracterizan estos fenotipos. Por otro lado, las PC-3M muestran una elevada flexibilidad y adaptación metabólica, siendo capaces de metabolizar numerosos substratos, entre ellos diferentes tipos de amino ácidos. Particularmente, el metabolismo de la glutamina en las PC-3M es más esencial que en las PC-3S, no sólo por su papel anaplerótico, si no por su función de tamponamiento de los excesos de ácido. El conjunto de estos resultados han desvelado las particularidades metabólicas y vulnerabilidades asociadas a los fenotipos, no solapados, de CSCs y EMT. El conocimiento adquirido podrá contribuir en el diseño de nuevas estrategias terapéuticas para el tratamiento de la metástasis.[eng] Metastasis represents the most life-threatening aspect of tumorigenesis and is the leading cause of death by cancer. Intensive research in this field has shed light on some of the molecular strategies employed by the heterogeneous cancer cell populations to leave the primary tumor, disseminate and grow new colonies in distant organs. In any given tumor, one important functional category of cancer cells is represented by cancer stem cells (CSCs), endowed with self-renewal and tumor-initiating potentials. Moreover, the epithelial-mesenchymal transition (EMT) program represents a process of fundamental importance conducive to tumor dissemination and metastatic spread of cancer cells. Some studies have pointed out that the EMT is responsible for the acquisition of the CSC-like state whereas others have shown that both cell entities can exist separately and cooperate to accelerate the process of metastasis. Here, we propose the combined use of metabolomics and fluxomics strategies to shed light on the metabolic reprogramming and vulnerabilities accompanying specific cancer cell phenotypes that differs in their metastatic and invasive capacities. The main objective of this thesis is focused on the characterization of the metabolic reprogramming and vulnerabilities of uncoupled CSC and EMT phenotypes present in a dual-cell prostate cancer cell model and represented by the highly related cell subpopulations PC-3M and PC-3S cells, respectively. Our results indicated that epithelial PC-3M cells, displaying CSC features and a high metastatic potential, preferentially rely on aerobic glycolysis (Warburg effect) for bioenergetics. Although these cells show low coupling between glycolysis and oxidative phosphorylation (OXPHOS) because of low pyruvate dehydrogenase activity, they display an increased metabolic flexibility to utilize different carbon sources, such as fatty acids, glutamine and other amino acids, that offset the decreased diversion of glucose-derived carbons into the tricarboxylic acid cycle and OXPHOS. The characterization of the non-CSC mesenchymal PC-3S cells expressing the EMT program and endowed with a high invasive capacity, showed a strong coupling between aerobic glycolysis and OXPHOS and a strong dependence on the mitochondrial metabolism for bioenergetics, which leads to higher levels of ROS that require increased levels of glutathione to provide an adequate antioxidant defense system. PC-3M and PC-3S cells differentially reprogram the use of the oxidative and non-oxidative branches of the pentose phosphate pathway to sustain their distinct metabolic needs. Glycolytic intermediates are preferentially directed to ribose synthesis in PC-3M cells to build up nucleotides whereas the generation of NADPH is more crucial for PC-3S cells to counteract their higher oxidative stress and sustain their increased fatty acid synthesis. Glutamine metabolism substantially contributes to TCA reactions in PC-3. For PC-3S cells, both glucose and glutamine are necessary to display a proper mitochondrial function. PC-3M cells are more dependent than PC-3S cells on the glutaminase reaction for proliferation and survival and this reliance lies mainly on the increased need for PC-3M cells to neutralize the excessive levels of protons (lactic acid) that result from their marked Warburg effect, which is achieved by the ammonia molecules released from glutamine metabolism. The high metabolic flexibility displayed by the CSC subpopulation including the participation of serine, glycine and one-carbon metabolism, the uptake of ketogenic amino acids, proline metabolism, among others, provide PC-3M cells with an extensive metabolic dynamics to obtain not only precursors but also to balance their redox status (NAD+/NADH and NADP+/NADPH) for metabolic processes to continue (e.g. glycolysis) and protect them from excessive acidity derived from a high glycolytic rate. Collectively, these results strengthen the notion that specific metabolic signatures are associated to CSC and EMT programs and highlight the importance of studying uncoupled cell phenotypes in order to univocally associate their characteristic metabolic reprogramming

Anivellament de coneixements en el trànsit batxillerat/universitat: anàlisi dels continguts en microcòpia al grau de Farmàcia

Podeu consultar la Setena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/43352Durante el curso 2010-2011, en la asignatura Iniciació al Treball de Laboratori (ITL) del grado de Farmacia, se recopilaron datos de encuestas incluídas en las libretas de laboratorio de los alumnos, para comparar el nivel de conocimientos previos adquiridos durante el Bachillerato sobre el contenido de la práctica “Microscopía y estereomicroscopía ópticas” con el nivel de esta unidad de enseñanza universitaria. Se procesaron datos de 200 estudiantes. A partir de las respuestas se ha definido un total de 20 ítems: 19 desglosan los contenidos adquiridos en la sesión (no adquiridos previamente en Bachillerato) y 1 es para los estudiantes con todos los contenidos adquiridos antes de ingresar en la UB

Anivellament de coneixements en el trànsit batxillerat/universitat: anàlisi dels continguts en microcòpia al grau de Farmàcia

Podeu consultar la Setena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/43352Durante el curso 2010-2011, en la asignatura Iniciació al Treball de Laboratori (ITL) del grado de Farmacia, se recopilaron datos de encuestas incluídas en las libretas de laboratorio de los alumnos, para comparar el nivel de conocimientos previos adquiridos durante el Bachillerato sobre el contenido de la práctica “Microscopía y estereomicroscopía ópticas” con el nivel de esta unidad de enseñanza universitaria. Se procesaron datos de 200 estudiantes. A partir de las respuestas se ha definido un total de 20 ítems: 19 desglosan los contenidos adquiridos en la sesión (no adquiridos previamente en Bachillerato) y 1 es para los estudiantes con todos los contenidos adquiridos antes de ingresar en la UB

Model-driven discovery of long-chain fatty acid metabolic reprogramming in heterogeneous prostate cancer cells.

Epithelial-mesenchymal-transition promotes intra-tumoral heterogeneity, by enhancing tumor cell invasiveness and promoting drug resistance. We integrated transcriptomic data for two clonal subpopulations from a prostate cancer cell line (PC-3) into a genome-scale metabolic network model to explore their metabolic differences and potential vulnerabilities. In this dual cell model, PC-3/S cells express Epithelial-mesenchymal-transition markers and display high invasiveness and low metastatic potential, while PC-3/M cells present the opposite phenotype and higher proliferative rate. Model-driven analysis and experimental validations unveiled a marked metabolic reprogramming in long-chain fatty acids metabolism. While PC-3/M cells showed an enhanced entry of long-chain fatty acids into the mitochondria, PC-3/S cells used long-chain fatty acids as precursors of eicosanoid metabolism. We suggest that this metabolic reprogramming endows PC-3/M cells with augmented energy metabolism for fast proliferation and PC-3/S cells with increased eicosanoid production impacting angiogenesis, cell adhesion and invasion. PC-3/S metabolism also promotes the accumulation of docosahexaenoic acid, a long-chain fatty acid with antiproliferative effects. The potential therapeutic significance of our model was supported by a differential sensitivity of PC-3/M cells to etomoxir, an inhibitor of long-chain fatty acid transport to the mitochondria

MIDcor, an R-program for deciphering mass interferences in mass spectra of metabolites enriched in stable isotopes

Background: Tracing stable isotopes, such as 13 C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study. Results: Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D)between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2)D was found to decrease when the tested substance was 13 C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13 C labeling is known a priori. Conclusion: Using the developed program improves the reliability of stable isotope tracer data analysis

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors