Regulation of the pyrimidine biosynthetic pathway by lysine acetylation of E. coli OPRTase.

The de novo pyrimidine biosynthesis pathway is an important route due to the relevance of its products, its implications in health and its conservation among organisms. Here, we investigated the regulation by lysine acetylation of this pathway. To this aim, intracellular and extracellular metabolites of the route were quantified, revealing a possible blockage of the pathway by acetylation of the OPRTase enzyme (orotate phosphoribosyltransferase). Chemical acetylation of OPRTase by acetyl-P involved a decrease in enzymatic activity. To test the effect of acetylation in this enzyme, K26 and K103 residues were selected to generate site-specific acetylated proteins. Several differences were observed in kinetic parameters, emphasizing that the kcat of these mutants showed a strong decrease of 300 and 150-fold for OPRTase-103AcK and 19 and 6.3-fold for OPRTase-26AcK, for forward and reverse reactions. In vivo studies suggested acetylation of this enzyme by a nonenzymatic acetyl-P-dependent mechanism and a reversion of this process by the CobB deacetylase. A complementation assay of a deficient strain in the pyrE gene with OPRTase-26AcK and OPRTase-103AcK was performed, and curli formation, stoichiometric parameters and orotate excretion were measured. Complementation with acetylated enzymes entailed a profile very similar to that of the ∆pyrE strain, especially in the case of complementation with OPRTase-103AcK. These results suggest regulation of the de novo pyrimidine biosynthesis pathway by lysine acetylation of OPRTase in Escherichia coli. This finding is of great relevance due to the essential role of this route and the OPRTase enzyme as a target for antimicrobial, antiviral and cancer treatments.

The Immunobiogram, a novel in vitro diagnostic test to measure the pharmacodynamic response to immunosuppressive therapy in kidney transplant patients.

BACKGROUND: Diagnostic tools to measure the response to individual immunosuppressive drugs for transplant patients are currently lacking. We previously developed the blood-based Immunobiogram bioassay for in-vitro characterization of the pharmacodynamic response of patients' own immune cells to a range of immunosuppressants. We used Immunobiogram to examine the association between patients' sensitivity to their prescribed immunosuppressants and clinical outcome. METHODS: We conducted an international, multicenter, observational study in a kidney transplant population undergoing maintenance immunosuppressive therapy. Patients were selected by clinical course poor [PCC] N = 53 (with renal dysfunction, and rejection signs in biopsy or/and an increase in DSA strength in last 12 months) versus good [GCC] N = 50 (with stable renal function and treatment, no rejection and no DSA titers). Immunobiogram dose-response curve parameters were compared between both subgroups in patients treated with mycophenolate, tacrolimus, corticosteroids, cyclosporine A or everolimus. Parameters for which significant inter-group differences were observed were further analyzed by univariate and subsequent multivariate logistic regression. RESULTS: Clinical outcome was associated with following parameters: area over the curve (AOC) and 25% (ID25) and 50% (ID50) inhibitory response in mycophenolate, tacrolimus, and corticosteroid-treated subgroups, respectively. These statistically significant associations persisted in mycophenolate (OR 0.003, CI95% <0.001-0.258; p = 0.01) and tacrolimus (OR < 0.0001, CI95% <0.00001-0.202; p = 0.016) subgroups after adjusting for concomitant corticosteroid treatment, and in corticosteroid subgroup after adjusting for concomitant mycophenolate or tacrolimus treatment (OR 0.003; CI95% <0.0001-0.499; p = 0.026). CONCLUSIONS: Our results highlight the potential of Immunobiogram as a tool to test the pharmacodynamic response to individual immunosuppressive drugs.

Ocular surface toxicity of depatuxizumab mafoditin (ABT-414): case reports / Toxicidade do depatuxizumabe mafodotina (ABT-414) na superfície ocular: relatos de casos

ABSTRACT The purpose of this study is to report the clinical features and outcomes of ocular surface toxicity following depatuxizumab mafoditin (ABT-414) therapy for unresectable glioblastoma. Ocular signs and symptoms of three patients treated with ABT-414 during a phase III trial for glioblastoma multiforme were evaluated. Both eyes of all patients were damaged during the week after the first infusion of the ABT-414 molecule. In all patients, mild-to-moderate keratitis could be ascertained, along with decreased visual acuity and blurred vision, as well as foreign-body sensation and redness. Symptoms and visual acuity improved 4 weeks. In conclusion, ABT-414 therapy may cause transient ocular surface toxicity. The initiation of artificial tears and lubricant ointment was enough to control the ocular surface signs and symptoms. A multidisciplinary approach, complete ophthalmologic monitorization, and elaboration of protocols are required to adequately manage these patients.

RESUMO Nosso objetivo é relatar as características clínicas e os resultados da toxicidade na superfície ocular após a terapia com depatuxizumabe mafodotina (ABT-414) para glioblastoma irressecável. Os sinais e sintomas oculares de três pacientes que foram tratados com ABT-414 durante um estudo de fase III para glioblastoma multiforme foram avaliados. Ambos os olhos de todos os pacientes foram danificados durante a semana após a primeira infusão da molécula ABT-414. Em todos os pacientes, uma ceratite de leve a moderada pode ser verificada, juntamente com uma diminuição da acuidade visual e visão turva, bem como sensação de corpo estranho e vermelhidão. Os sintomas e a acuidade visual melhoraram em um período de 4 semanas. Em conclusão, a terapia com ABT-414 pode causar toxicidade transitória na superfície ocular. A iniciação com lágrimas artificiais e pomada lubrificante foi suficiente para controlar os sinais e sintomas na superfície ocular. Uma abordagem multidisciplinar, com acompanhamento oftalmológico completo e a elaboração de protocolos são necessários para o manejo adequado desses pacientes.

Ocular surface toxicity of depatuxizumab mafoditin (ABT-414): case reports.

The purpose of this study is to report the clinical features and outcomes of ocular surface toxicity following depatuxizumab mafoditin (ABT-414) therapy for unresectable glioblastoma. Ocular signs and symptoms of three patients treated with ABT-414 during a phase III trial for glioblastoma multiforme were evaluated. Both eyes of all patients were damaged during the week after the first infusion of the ABT-414 molecule. In all patients, mild-to-moderate keratitis could be ascertained, along with decreased visual acuity and blurred vision, as well as foreign-body sensation and redness. Symptoms and visual acuity improved 4 weeks. In conclusion, ABT-414 therapy may cause transient ocular surface toxicity. The initiation of artificial tears and lubricant ointment was enough to control the ocular surface signs and symptoms. A multidisciplinary approach, complete ophthalmologic monitorization, and elaboration of protocols are required to adequately manage these patients.

Orally Active Peptide Vector Allows Using Cannabis to Fight Pain While Avoiding Side Effects.

The activation of cannabinoid CB1 receptors (CB1R) by Δ9-tetrahydrocannabinol (THC), the main component of Cannabis sativa, induces analgesia. CB1R activation, however, also causes cognitive impairment via the serotonin 5HT2A receptor (5HT2AR), a component of a CB1R-5HT2AR heteromer, posing a serious drawback for cannabinoid therapeutic use. We have shown that peptides reproducing CB1R transmembrane (TM) helices 5 and 6, fused to a cell-penetrating sequence (CPP), can alter the structure of the CB1R-5HT2AR heteromer and avert THC cognitive impairment while preserving analgesia. Here, we report the optimization of these prototypes into drug-like leads by (i) shortening the TM5, TM6, and CPP sequences, without losing the ability to disturb the CB1R-5HT2AR heteromer, and (ii) extensive sequence remodeling to achieve protease resistance and blood-brain barrier penetration. Our efforts have culminated in the identification of an ideal candidate for cannabis-based pain management, an orally active 16-residue peptide preserving THC-induced analgesia.

Eficacia del mini mental y PFEIFFER (SPMSQ) para detectar deterioro cognitivo en mayores de 65 años / Eficácia do mini mental e PFEIFFER (SPMSQ) para detectar deterioração cognitiva em mais de 65 anos / Efficacy of the mini mental and PFEIFFER (SPMSQ) to detect cognitive impairment in those over 65 years of age

INTRODUCCIÓN: el deterioro cognitivo (DC) es un problema cada vez más frecuente, y a menudo no es diagnosticado, porque impide una terapéutica temprana, dificultando posteriormente la calidad de vida de quien lo padece. OBJETIVO: determinar la eficacia de los cuestionarios Mini Mental y PFEIFFER (SPMSQ) para detectar la existencia de deterioro cognitivo en personas mayores de 65 años. MÉTODO: estudio analítico de corte transversal. La muestra estuvo constituida por los adultos mayores del centro geriátrico de Macas. La utilidad diagnóstica se evaluó mediante el cálculo del área bajo la curva ROC (AUC), para el cálculo de los valores de sensibilidad (S), especificidad (E) se tomaron los puntos de corte más significativos y se comparó ambos test para determinar DC. RESULTADOS: el 53% de los pacientes fueron del género masculino y la media de 82 años. El 50% presentaron síntomas de DC con al menos un cuestionario positivo; el 58% fueron positivos con Pfeiffer y el 91% con Mini mental. Finalmente, los valores de sensibilidad y especificidad del mini mental fueron de 91% y 100% respectivamente y el AUC fue de 1 mientras Pfeiffer obtuvo una S y Ede 100% y AUC de 0.9, demostrando este último ser más efectivo para el diagnóstico de DC. CONCLUSIÓN: se determinó que el test de Pfeiffer fue más efectivo que el Mini mental para el diagnóstico de DC; además, la edad y el nivel de escolaridad son factores más asociados a esta patología.

INTRODUCTION: cognitive impairment (CD) is an increasingly frequent problem, and it is often not diagnosed, because it prevents early therapy, later hindering the quality of life of those who suffer from it. OBJECTIVE: to determine the effectiveness of the Mini Mental and PFEIFFER questionnaires (SPMSQ) to detect the existence of cognitive impairment in people over 65 years of age. METHOD: analytical cross-sectional study. The sample consisted of the elderly from the Macas geriatric center. The diagnostic utility was evaluated by calculating the area under the ROC curve (AUC), for the calculation of the sensitivity (S), specificity (E) values, the most significant cut-off points were taken and both tests were compared to determine DC. RESULTS: 53% of the patients were male and the average was 82 years old. 50% presented symptoms of CD with at least one positive questionnaire; 58% were positive with Pfeiffer and 91% with Mini mental. Finally, the sensitivity and specificity values of the mini mental were 91% and 100% respectively and the AUC was 1 while Pfeiffer obtained an S and E of 100% and AUC of 0.9, proving the latter to be more effective for the diagnosis of DC. CONCLUSION: it was determined that the Pfeiffer test was more effective than the Mini mental for the diagnosis of CD; Furthermore, age and level of education are factors most associated with this pathology.

INTRODUÇÃO: o déficit cognitivo (DC) é um problema cada vez mais frequente, muitas vezes não diagnosticado, pois impede a terapia precoce, prejudicando posteriormente a qualidade de vida de quem a sofre. OBJETIVO: determinar a eficácia dos questionários Mini Mental e PFEIFFER (SPMSQ) para detectar a existência de déficit cognitivo em pessoas com mais de 65 anos. MÉTODO: estudo transversal analítico. A amostra foi composta por idosos do centro geriátrico de Macas. A utilidade diagnóstica foi avaliada pelo cálculo da área sob a curva ROC (AUC), para o cálculo dos valores de sensibilidade (S), especificidade (E), os pontos de corte mais significativos foram tomados e ambos os testes foram comparados para determinar DC. RESULTADOS: 53% dos pacientes eram do sexo masculino e a média de idade foi de 82 anos. 50% apresentaram sintomas de DC com pelo menos um questionário positivo; 58% foram positivos com Pfeiffer e 91% com Mini mental. Finalmente, os valores de sensibilidade e especificidade do mini mental foram 91% e 100% respectivamente e a AUC foi 1 enquanto Pfeiffer obteve S e E de 100% e AUC de 0,9, provando que esta última é mais eficaz para o diagnóstico de DC. CONCLUSÃO: determinouse que o teste de Pfeiffer foi mais eficaz do que o Mini mental para o diagnóstico de DC; além disso, a idade e o nível de escolaridade são os fatores mais associados a essa patologia.

Music Therapy for Pain and Anxiety Management in Nasal Bone Fracture Reduction: Randomized Controlled Clinical Trial.

OBJECTIVE: To evaluate whether listening to music through binaural headphones contributes to the perception of pain and anxiety in patients undergoing closed nasal bone fracture reductions. STUDY DESIGN: Randomized controlled trial. SUBJECTS AND METHODS: We recruited patients from San Juan de Dios Hospital with displaced nasal fractures who required a reduction and assigned them to a control group or a music group. For both groups, a protocolized closed reduction of the nasal fracture with local anesthesia was performed. The music group heard music through headphones during the pre-, intra-, and postprocedural periods of the intervention. Physiological variables (blood pressure and heart rate) were measured. An anxiety survey (State-Trait Anxiety Inventory) and the visual analog scale for measuring pain were also applied. RESULTS: The music group exhibited significantly lower levels of systolic blood pressure (P = .0001), anxiety (P < .0001), and pain (P = .0004) than the control group. CONCLUSION: Listening to music through headphones-a safe and low-cost intervention-appears to aid in pain and anxiety management associated with procedures that are usually uncomfortable, such as the reduction of nasal bone fractures with local anesthesia. We believe that this effect is achieved by the modulation of pain and anxiety on an emotional-affective dimension at a central level. Given its safety, feasibility, and low cost, music therapy should be considered a complementary treatment for pain and anxiety management for nasal fracture reduction performed with local anesthesia, as well as for other medical procedures of similar pain levels conducted without general anesthesia.

Characterization of X-ray gas attenuator plasmas by optical emission and tunable laser absorption spectroscopies.

X-ray gas attenuators are used in high-energy synchrotron beamlines as high-pass filters to reduce the incident power on downstream optical elements. The absorption of the X-ray beam ionizes and heats up the gas, creating plasma around the beam path and hence temperature and density gradients between the center and the walls of the attenuator vessel. The objective of this work is to demonstrate experimentally the generation of plasma by the X-ray beam and to investigate its spatial distribution by measuring some of its parameters, simultaneously with the X-ray power absorption. The gases used in this study were argon and krypton between 13 and 530 mbar. The distribution of the 2p excited states of both gases was measured using optical emission spectroscopy, and the density of argon metastable atoms in the 1s5 state was deduced using tunable laser absorption spectroscopy. The amount of power absorbed was measured using calorimetry and X-ray transmission. The results showed a plasma confined around the X-ray beam path, its size determined mainly by the spatial dimensions of the X-ray beam and not by the absorbed power or the gas pressure. In addition, the X-ray absorption showed a hot central region at a temperature varying between 400 and 1100 K, depending on the incident beam power and on the gas used. The results show that the plasma generated by the X-ray beam plays an essential role in the X-ray absorption. Therefore, plasma processes must be taken into account in the design and modeling of gas attenuators.

Multiple signals modulate the activity of the complex sensor kinase TodS.

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

FAK dimerization controls its kinase-dependent functions at focal adhesions.

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

Validación preliminar del cuestionario del clima motivacional iniciado por los padres-2 (PIMCQ-2) / Preliminary validation of the Parent-Initiated Motivational Climate Questionnaire-2 (PIMCQ-2)

El propósito de este trabajo fue validar al contexto español el Cuestionario del Clima Motivacional Iniciado por los Padres-2 (PIMCQ-2). Para ello, se utilizó una muestra de 108 jugadores de tenis, con edades comprendidas entre los 12 y 17 años. Se analizaron sus propiedades psicométricas realizando un análisis factorial exploratorio, un análisis factorial confirmatorio y un análisis de la consistencia interna a través del alfa de Cronbach. Además, se examinó la validez convergente analizando las correlaciones entre el clima motivacional iniciado por padres y madres, y el clima motivacional del entrenador. Los resultados reflejaron la necesidad de eliminar seis ítems del instrumento original para obtener unos índices de ajuste aceptables en el análisis factorial confirmatorio, aunque se mantuvo la misma estructura factorial. Se obtuvieron valores de consistencia interna aceptables para los tres factores. Además, se hallaron evidencias externas de validez, puesto que las dimensiones del clima motivacional de padres y madres se relacionaron con las dimensiones del clima motivacional del entrenador. En conclusión, el estudio demostró de forma preliminar que la versión española del PIMCQ-2 revelaba unas adecuadas propiedades psicométricas.

The purpose of this study was to validate the Parent-Initiated Motivational Climate Questionnaire-2 (PIMCQ-2) in the Spanish context. To achieve this goal, a sample of 108 tennis players aged between 12 and 17 was used. The psychometric properties of the PIMCQ-2 were analyzed using exploratory factor analysis, confirmatory factor analysis and an analysis of the internal consistency through Cronbach alpha. Convergent validity was also examined analyzing the correlations among parent-initiated motivational climate and coach motivational climate. The results showed the necessity to eliminate six items from the original instrument to obtain acceptable fit indices in confirmatory factor analysis, although the factor structure remained unchanged. Acceptable values of internal consistency were obtained for the three factors. There was also external evidence of validity, since the dimensions of parent-initiated motivational climate were related to the dimensions of coach motivational climate. In conclusion, this preliminary study showed that the Spanish version of the PIMCQ-2 revealed appropriate psychometric properties.

Genome expression analysis of nonproliferating intracellular Salmonella enterica serovar Typhimurium unravels an acid pH-dependent PhoP-PhoQ response essential for dormancy.

Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

Human G3BP1 interacts with beta-F1-ATPase mRNA and inhibits its translation.

The post-transcriptional regulation of nuclear mRNAs that encode core components of mitochondria has relevant implications in cell physiology. The mRNA that encodes the catalytic subunit of the mitochondrial H(+)-ATP synthase subunit beta (ATP5B, beta-F1-ATPase) is localized in a large ribonucleoprotein (RNP) complex (beta-F1-RNP), which is subjected to stringent translational control during development and the cell cycle, and in carcinogenesis. Because downregulation of beta-F1-ATPase is a conserved feature of most prevalent human carcinomas, we have investigated the molecular composition of the human beta-F1-RNP. By means of an improved affinity-chromatography procedure and protein sequencing we have identified nine RNA-binding proteins (RNABPs) of the beta-F1-RNP. Immunoprecipitation assays of Ras-GAP SH3 binding protein 1 (G3BP1) and fluorescent in-situ hybridization of mRNA indicate a direct interaction of the endogenous G3BP1 with mRNA of beta-F1-ATPase (beta-F1 mRNA). RNA-bridged trimolecular fluorescence complementation (TriFC) assays confirm the interaction of G3BP1 with the 3'-UTR of beta-F1 mRNA in cytoplasmic RNA-granules. Confocal and high-resolution immunoelectron-microscopy experiments suggest that the beta-F1-RNP is sorted to the periphery of mitochondria. Molecular and functional studies indicate that the interaction of G3BP1 with beta-F1 mRNA inhibits its translation at the initiation level, supporting a role for G3BP1 in the glycolytic switch that occurs in cancer.

Up-regulation of the ATPase inhibitory factor 1 (IF1) of the mitochondrial H+-ATP synthase in human tumors mediates the metabolic shift of cancer cells to a Warburg phenotype.

The H(+)-ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1). The expression of IF1 in human tissues and its participation in the development of human pathology are unknown. Here, we have developed monoclonal antibodies against human IF1 and determined its expression in paired normal and tumor biopsies of human carcinomas. We show that the relative mitochondrial content of IF1 increases significantly in carcinomas, suggesting the participation of IF1 in oncogenesis. The expression of IF1 varies significantly in cancer cell lines. To investigate the functional activity of IF1 in cancer, we have manipulated its cellular content. Overexpression of IF1 or of its pH-insensitive H49K mutant in cells that express low levels of IF1 triggers the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation with concurrent mitochondrial hyperpolarization. Treatment of the cells with the H(+)-ATP synthase inhibitor oligomycin mimicked the effects of IF1 overexpression. Conversely, small interfering RNA-mediated silencing of IF1 in cells that express high levels of IF1 promotes the down-regulation of aerobic glycolysis and the increase in oxidative phosphorylation. Overall, these findings support that the mitochondrial content of IF1 controls the activity of oxidative phosphorylation mediating the shift of cancer cells to an enhanced aerobic glycolysis, thus supporting an oncogenic role for the de-regulated expression of IF1 in cancer.

Selective inhibition of beta-F1-ATPase mRNA translation in human tumours.

Down-regulation of beta-F1-ATPase (the catalytic subunit of the mitochondrial H+-ATP synthase) is a hallmark of many human tumours. The expression level of beta-F1-ATPase provides a marker of the prognosis of cancer patients, as well as of the tumour response to chemotherapy. However, the mechanisms that participate in down-regulating its expression in human tumours remain unknown. In the present study, we have investigated the expression of beta-F1-ATPase mRNA (termed beta-mRNA) in breast, colon and lung adenocarcinomas and squamous carcinomas of the lung. Despite the down-regulation of the protein, tumour beta-mRNA levels remained either unchanged (breast and lung adenocarcinomas) or significantly increased (colon and squamous lung carcinomas) when compared with paired normal tissues, suggesting a specific translation-masking event for beta-mRNA in human cancer. Consistently, we show using cell-free translation assays that a large fraction (approximately 70%) of protein extracts derived from breast and lung adenocarcinomas specifically repress the translation of beta-mRNA. We show that the 3'UTR (3' untranslated region) of human beta-mRNA is a relevant cis-acting element required for efficient translation of the transcript. However, an RNA chimaera bearing the 3'UTR of human beta-mRNA does not recapitulate the inhibitory effect of tumour extracts on beta-mRNA translation. Overall, the findings of the present study support the hypothesis that down-regulation of the bioenergetic activity of mitochondria in human tumours is exerted by translation silencing of beta-mRNA.

Cancer abolishes the tissue type-specific differences in the phenotype of energetic metabolism.

Nowadays, cellular bioenergetics has become a central issue of investigation in cancer biology. Recently, the metabolic activity of the cancer cell has been shown to correlate with a proteomic index that informs of the relative mitochondrial activity of the cell. Within this new field of investigation, we report herein the production and characterization of high-affinity monoclonal antibodies against proteins of the "bioenergetic signature" of the cell. The use of recombinant proteins and antibodies against the mitochondrial beta-F1-ATPase and Hsp60 proteins and the enzymes of the glycolytic pathway glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase M2 in quantitative assays provide, for the first time, the actual amount of these proteins in normal and tumor surgical specimens of breast, lung, and esophagus. The application of this methodology affords a straightforward proteomic signature that quantifies the variable energetic demand of human tissues. Furthermore, the results show an unanticipated finding: tumors from different tissues and/or histological types have the same proteomic signature of energetic metabolism. Therefore, the results indicate that cancer abolishes the tissue-specific differences in the bioenergetic phenotype of mitochondria. Overall, the results support that energetic metabolism represents an additional hallmark of the phenotype of the cancer cell and a promising target for the treatment of diverse neoplasias.

The tumor suppressor function of mitochondria: translation into the clinics.

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life-death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial beta-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.

Glucose avidity of carcinomas.

The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the "abnormal" aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg's formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg's hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the "selfish" pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.

HuR and the bioenergetic signature of breast cancer: a low tumor expression of the RNA-binding protein predicts a higher risk of disease recurrence.

Downregulation of the catalytic subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) is a hallmark of many types of cancer. The expression of beta-F1-ATPase is stringently controlled by posttranscriptional mechanisms. Herein, we pursue the identification of beta-F1-ATPase messenger RNA-binding proteins (beta-mRNABPs) that interact and could define the bioenergetic phenotype of the cancer cell in order to establish its relevance as markers of breast cancer progression. RNA immunoprecipitation and RNA affinity chromatography identify HuR as a beta-mRNABP that interacts with the 3'-untranslated region of the transcript. Subcellular fractionation and high-resolution immunoelectron microscopy revealed the cofractionation and presence of HuR in subcellular structures associated to liver mitochondria. Analysis of the expression level of HuR in a cohort of breast carcinomas shows its association with the degree of alteration of the bioenergetic phenotype of the tumor. Moreover, HuR expression is shown to be an independent marker of breast cancer prognosis. A low tumor expression of HuR predicts a higher risk of disease recurrence in early stage breast cancer patients as assessed by clinical and bioenergetic markers of prognosis, strongly supporting the incorporation of HuR as an additional marker for the follow-up of these patients. Mechanistically, overexpression experiments and short hairpin RNA-mediated silencing of HuR in human embryonic kidney and HeLa cells indicate that HuR is not regulating beta-F1-ATPase expression. Overall, the participation of additional RNA-binding proteins in controlling beta-F1-ATPase expression and therefore in defining the bioenergetic signature of the cancer cell is expected.