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This study aims to investigate whether the heat treatment applied during the production of black onion, a novel derived product made from fresh onion, produces changes in the content of flavonoids, organosulfur compounds, organic acids, water soluble sugars and amino acids in three onion varieties (‘Shallot’, ‘Chata’ and ‘Echalion’). The total flavonoid content decreased up to 12-fold in black onions compared with fresh onions while the quantities of isoalliin, the main organosulfur compound in black onions, drastically increased during the process. Moreover, the levels of fructose and glucose significantly increased during the elaboration process, contributing to the sweetness of black onions. The influence of heating on their antioxidant capacity showed a decreasing trend of the ORAC antioxidant activity of onion, while ABTS and DPPH did not show a clear tendency. These results present a comprehensive phytochemical characterization of black onions, highlighting the significant influence of the heating process on their phytochemical composition.

In the town of Cuevas Bajas (Málaga) an autochthonous variety of purple carrot is grown, known as "Carrot Morá". This variety is characterized by having a deep purple color on the outside, an intermediate layer of orange color and a characteristic purple heart. The purple color of the interior seems to be influenced by the area of cultivation, since it has been observed that this variety cultivated in another place does not develop it. In this study, the functional compounds of this carrot are characterized during the collection stage, according to the harvest period, the carrot tammage and the color of its heart. En la población de Cuevas Bajas (Málaga) se cultiva una variedad autóctona de zanahoria morada, conocida como “Zanahoria Morá”. Esta variedad se caracteriza por tener una tonalidad púrpura intensa en el exterior, una capa intermedia de color naranja y un característico corazón morado. El color morado del interior parece estar influenciado por la zona de cultivo, ya que se ha observado que esta variedad cultivada en otro lugar no lo desarrolla. En este estudio se caracterizan los compuestos funcionales de esta zanahoria durante la etapa de recolección, según el periodo de cosecha, el tamaño de la zanahoria y el color de su corazón. .

Purpose: Endoglin (ENG; CD105) is a coreceptor of the TGFβ family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGFβ, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma, an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG targeting has not been fully explored in this disease. Experimental Design: We characterized the expression pattern of transmembrane ENG, sENG, and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody–drug conjugates (ADC), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of Ewing sarcoma. Results: Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro. Moreover, the ADCs significantly delayed tumor growth in Ewing sarcoma cell line–derived xenografts and patient-derived xenografts in a dose-dependent manner. Conclusions: Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate Ewing sarcoma.

Ewing sarcoma (ES) is an aggressive tumour affecting mainly children and young adults. When metastases are detected, the survival rate is compromised as a result of a lack of appropriated treatments. Balanced chromosomal translocations, present in all primary ES, originate gene fusions, such as EWSR1-FLI1, which act as aberrant transcription factors. Targeting EWSR1-FLI1 by using RNA interference approaches reduced tumour growth and tumourigenicity of ES cells in vitro and in vivo and is a good tool in order to get knowledge in the ES biology. However, possibility of taking RNA interference into clinic is limited. At present, some drugs affecting gene fusion activity have been reported to reduce tumour growth. In addition, several approaches targeting ES genetic alterations not related to gene fusions or their genetic targets have been recently reported. The use of more appropriated animal models and the study of ES tumour microenvironment and epigenetics will unveil new therapeutic targets. Key Concepts ES is an aggressive developmental tumour. Balanced chromosomal translocation and associated gene fusions are the primary molecular event in ES. Secondary molecular events in ES can define new therapeutic targets and new prognostic markers. The role played by epigenetics in ES and the regulation of chromatin remodelling is beginning to be known. Detection of ES circulating cells or identification of gene expression profiles in peripheral blood cells could become good prognostic markers. Identification of new markers of cancer stem cells could help improve ES therapy. Endoglin could be a relevant therapeutic target in ES. Osteolysis and bone remodelling are important mechanisms of ES tumour growth. ES cells secrete extracellular vesicles, containing aberrant EWSR1-FLI1 mRNA and other growth factors, which interact with surrounding tumour cells and extracellular matrix components. Combination therapy of a dual inhibitor of IGF1R and IR pathways and a DNA damage agent can avoid resistance to IGF1R inhibitors. At present, there is a lack of appropriate genetically engineered mice (GEM) of ES. Keywords: Ewing sarcoma; tumour microenvironment; exosome; endoglin; animal model; epigenetic

This document presents the results of the study of the microencapsulation of three types of probiotic bacteria (Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium longum) by means of the spray drying technique, both in free form and using different encapsulating agents (alginate, maltodextrin, chitosan-alginate and chitosan-maltodextrin) and evaluation of its morphology and viability in different storage conditions. En este documento se presentan los resultados del estudio de la obtención de microencapsulados de tres tipos de bacterias probióticas (Lactobacillus plantarum, Lactobacillus rhamnosus y Bifidobacterium longum) mediante la técnica de secado por pulverización tanto en forma libre como empleando diferentes agentes encapsulantes, (alginato, maltodextrina, quitosano-alginato y quitosano-maltodextrina) y evaluación de su morfología y viabilidad en diferentes condiciones de almacenamiento.

This study developed, optimized and validated an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method to identify and quantify metabolites and microbial-derived catabolites in urine, plasma and feces of rats following ingestion of 50 mg of a red wine proanthocyanidin-rich extract. The method was validated for specificity, linearity, limit of detection (LD) and quantification (LQ), intra-day and inter-day precision, recovery and matrix effects, which were determined for 34 compounds in the three biological matrices. After method validation, three parent flavan-3-ols, four 5-carbon side chain ring fission metabolites, and 27 phenolic acid and aromatic catabolites were quantified in plasma, urine and feces after red wine proanthocyanidin intake. These results establish the value of the UHPLC-HRMS protocol in obtaining a detailed picture of proanthocyanidin metabolites and their microbial-derived catabolites, along with their phase II metabolites, in biological fluids of rat, and potentially in human clinical studies designed to evaluate the bioavailability of dietary flavan-3-ols.

This document presents the results of the study of the changes produced in the profile of biosaludable compounds present in fresh onion when it is transformed into black onion. The profile is studied in bioactive compounds such as phenolic compounds, organosulfur compounds, sugars, organic acids as well as the antioxidant capacity of three onion varieties: Chalota, Chata and Echalion En este documento se presentan los resultados del estudio de los cambios producidos en el perfil de compuestos biosaludables presentes en la cebolla fresca cuando esta se transforma en cebolla negra. Se estudia el perfil en compuestos bioactivos tales como compuestos fenólicos, compuestos organosulfurados, azúcares, ácidos orgánicos así como de la capacidad antioxidante de tres variedades de cebolla: Chalota, Chata y Echalion .

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line. CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients., This work was supported in part by a grant from the Consejería de Educación, Junta de Castilla y León, Fondos FEDER (SA085U16 to JMHR and JCYL-EDU/346/2013 PhD scholarship to MHS), the ISCIII-FEDER Spanish Cancer Network (RD12/0036/0069) and the Fondo de Investigaciones Sanitarias (FIS) of the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) “Una manera de hacer Europa” (grant PI15/01471).

Biogenic amines (BA) are generally considered as a food hazard, even though there is not a threshold for these biomolecules in the European legislation, except for histamine in fishery products. These compounds are formed during the storage and processing of certain foods through microbiological activity, and when present in high concentrations, could have toxicological effects, causing health problems in consumers, especially to sensitive persons. This fact, in addition to the economical concern involved, makes it necessary to control the amounts of biogenic amines in foods. For all these reasons, literature on biogenic amines in different food products, especially in fermented beverages, is extensive. This review provides an overview of the most recent trends in the determination of biogenic amines in fermented beverages focusing on novelty, improvement and optimization of analytical methods. Hence, the different sample treatment procedures (including derivatization), the most important analytical techniques and the most frequent applications are described and discussed. Although biogenic amines have been determined in wine and other fermented beverages for decades, new advancements and technical possibilities have allowed to increase the accuracy and sensitivity of analytical methods, in order to overcome the challenges posed by the complex matrices and their high intrinsic variability. Thus, the different purposes of BA determination (food safety, production process or food microbiology research) and the most widely employed analytical techniques have been reviewed.

Cervical cancer and its precursors low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) are associated with infection by human papillomavirus (HPV), in particular HPV 16 and 18. The distribution of the HPV genotype varies with the severity of cervical disease, age and the geographic location of the patients. We report the results of a population study carried out in a region of north-western (NW) Spain aimed at determining the prevalence of single and multiple infections by 35 types of HPV using low-density microarrays for 113 cases with negative for intraepithelial lesions or malignancies; 588 with atypical squamous cells of undetermined significance (ASCUS)/LSIL; 183 with HSIL; and seven cases of squamous cell carcinomas. Of the 891 patients analysed, 50.2% had single infections and 49.8% had multiple HPV infections. In women aged below 30 years, there was a predominance of multiple infections (p = 0.027). ASCUS/LSIL was associated with multiple and HSIL with single infections (p = 0.025). We observed significant increases in the percentage of infections due to a high-risk (HR) type of HPV when the severity of the cytological lesion increased (p = 0.001). No relationship was found between greater aggressiveness in the cytological diagnosis and a higher number of HPV types involved in multiple infections. The five most frequent genotypes were HPV 16 (26.3%), 53 (18.2%), 51 (17.3%), 6 (14.8%) and 66 (13.1%). The prevalence of HPV 16, 33 and 58 increased significantly from ACUS/LSIL to HSIL and the prevalence of HPV 51, 53 and 66 decreased. HPV 16 was the only genotype that showed a significant increase in prevalence when the severity of the cytological disease increased in single infections (p = 0.0001). The implementation of bivalent prophylactic vaccination could potentially lead to prevention in 32% of the population included in the study - in at least a quarter of patients with ACUS/LSIL (26.7%), and in half of HSIL (50.2%).

Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies. To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition). Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients. PI15/01471 SA085U16 JCyL-MQ FEHH-MH Disclosures García-Tuñón: Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Carcinoma in situ (CIS) is a non-papillary high-grade, potentially aggressive, and unpredictable manifestation of bladder urothelial carcinoma. The aim of this study was to assess patterns of Cyclin D3 gene amplification in Bacillus Calmette-Guerin (BCG)-treated CIS and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 28 primary (isolated) or secondary (concomitant) bladder CIS samples in which there was enough tissue material to assess Cyclin D3 gene status by fluorescent in situ hybridization was the study group. Cyclin D3 gene amplification was present in 29% of secondary CIS; none of primary CIS samples had Cyclin D3 gene amplification. Cyclin D3 amplification was related to recurrence- (p=0.046) and progression-free survival (p=0.002). Type of bladder CIS (primary vs. secondary) was unrelated to recurrence- or progression-free survival in the current series. Cox's regression analysis selected Cyclin D3 as an independent predictor of progression-free survival (p=0.041, relative risk=61.503, 95% confidence interval=1.1-274.710). None of primary CIS cases recurred on follow-up; nine secondary CIS recurred and four of them progressed to invasive bladder carcinoma HG T1 (n=1), T2b N0M0 (n=1), T3b N1M0 (n=1) and T4aN1M1 (n=1). Mean recurrence±SD (months) occurred at 19.5±2.06 (95% (confidence interval (CI)), 15.5-23.6); mean progression (months) occurred at 23.8±1.46 (95% (CI), 20.9-26.7). Our study suggests that Cyclin D3 gene amplification might be a predictor of aggressiveness in BCG-treated CIS. © 2010 Springer-Verlag., Supported by the grants SAF2007-64942 (Ministry of Education and Research, Madrid, Spain), P07-CVI-02974 and PI0003/2007 (Junta de Andalucia, Seville, Spain). Work at CIC is also funded by Instituto de Salud Carlos III (PI081828, RD06/020/0059). JLO is supported by ISCIII (Contratos Postdoctorales Sara Borrell CD6/00001).

Purpose: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. Experimental Design: By chromatin immunoprecipitation, we analyzed EWS–FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. Results: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS–FLI1 (both type I and type II) binding to two representative target genes (TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS–FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. Conclusions: We showed that trabectedin may not only inhibit but also enhance the binding of EWS–FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors. Clin Cancer Res; 21(6); 1373–82. ©2015 AACR.

Producción Científica, Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting., Ministerio de Economía y Competitividad (PI081828), Ministerio de Economía y Competitividad (RD06/0020/0059 ), Ministerio de Economía y Competitividad (RD12/0036/0017), Ministerio de Economía y Competitividad (PT13/0010/0056)

Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.

This paper studies the amino acid profile of beverages obtained through the fermentation of strawberry puree by a surface culture using three strains belonging to different acetic acid bacteria species (one of Gluconobacter japonicus, one of Gluconobacter oxydans and one of Acetobacter malorum). An HPLC-UV method involving diethyl ethoxymethylenemalonate (DEEMM) was adapted and validated. From the entire set of 21 amino acids, multiple linear regressions showed that glutamine, alanine, arginine, tryptophan, GABA and proline were significantly related to the fermentation process. Furthermore, linear discriminant analysis classified 100% of the samples correctly in accordance with the microorganism involved. G. japonicus consumed glucose most quickly and achieved the greatest decrease in amino acid concentration. None of the 8 biogenic amines were detected in the final products, which could serve as a safety guarantee for these strawberry gluconic fermentation beverages, in this regard.

Ewing Sarcoma is a developmental tumor characterized by balanced chromosomal translocations and formation of new fusion genes, which are the main hallmark of this rare entity. Despite the vast knowledge regarding the molecular aspects of this rare malignancy obtained in the last few years, including the discovery of new therapeutic targets, many questions still remain open. In this review we focus on the research on targeted therapies in this malignancy, and discussed some bottlenecks related to this such as the possible role of pathologists, the availability of samples, the lack of appropriate animal models, and the resources needed to carry out preclinical and clinical research.

The European Food Safety Authority (EFSA) has accepted health claims for the food constituent melatonin because scientific evidence shows that it is effective at reducing sleep onset latency, and that it alleviates subjective feelings of jet lag. According to risk assessment data published by EFSA in 2011, histamine and tyramine are the most toxic biogenic amines and the ones that most affect food safety. The potential formation of biogenic amines is a concern in fermented foods because of the intense microbial activity. Conversely, Saccharomyces cerevisiase produces melatonin during fermentation in the winemaking process. This study aims to evaluate the production of potentially healthy melatonin and toxic biogenic amines during the winemaking process. To this end, 11 biogenic amines (agmatine, cadaverine, histamine, methylamine, 2-phenylethylamine, putrescine, spermidine, spermine, tyramine, tryptamine and melatonin) have been monitored during the making of 5 monovarietal wines (Merlot, Palomino Fino, Syrah, Tempranillo and Tintilla de Rota). This paper shows that alcoholic and malolactic fermentation plays a crucial role in the formation of these compounds. Bioactive melatonin is formed at safe levels of the other biogenic amines.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (∼30-300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent., Daniel J García-Domínguez is supported by a grant from the María García Estrada Foundation. Research in Enrique de Alava’s lab is also supported by the Ministry of Economy and Competitiveness of Spain-FEDER (PI081828, RD06/0020/0059, PI1100018, ISCIII_postdoc grant CD06/00001) and the European Commission (FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC).

Gluconic fermentation of fruit substrates is an innovative strategy to elaborate nonalcoholic beverages which has not been studied so far. Futhermore, it prevents food waste of perishable products like strawberries. Amino acids, biogenic amines and ammonium ion were evaluated after the fermentation of strawberry puree using Gluconobacter japonicus in submerged culture. The process was monitored with the determination of pH, total acidity, glucose, fructose and gluconic acid concentrations. The determination of nitrogen compounds was accomplished using a HPLC–UV method involving diethyl ethoxymethylenemalonate (DEEMM) as the derivatization agent. Most amino acids did not present significant changes, except for lysine and histidine, which increased their concentration in the final product. However, the principal component analysis was able to group the initial and final samples in different sets using the amino acids concentration as variables. Biogenic amines were not detected in the final samples, thus ensuring the safety of this product for human consumption. Practical Applications Because of the high level of competitiveness, the success of a new product requires the use of a raw material different from the traditionally used ones, and the application of innovative techniques of production. Strawberry is a fruit prized for its aroma and flavor. In addition, Spain is the fifth strawberry producer in the world, and consequently there are surpluses of second quality strawberries. This second quality fruit is generally discarded, involving large economic losses. These facts make strawberry a good candidate to be used as raw material for the production of fruit derived products. Hence, gluconic acid fermentation is an innovative way of transforming surplus strawberries into beverages which do not present glucose, as it is transformed into gluconic acid whilst keeping fructose as sweetener. Hence, the final product could be the principal substract to elaborate a new beverage which could be consumed by diabetics. Gluconic acid submerged fermentations of strawberry puree were carried out with Gluconobacter japonicus. Additionally, the amount of biogenic amines was assessed for food safety purposes.

Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival., Fundació Privada Cellex (CdT); Instituto de Salud Carlos III (CD6/00001 to J.L.O).

This paper reports the determination of biogenic amines by high-performance liquid chromatography (HPLC) and fluorescence detection after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in balsamic, apple, and red, white, and Sherry wine vinegars. A solid-phase extraction (SPE) with mixed-mode resins method was used before analysis. The method was successfully validated obtaining adequate values of selectivity, response linearity, precision, accuracy, and low detection and quantification limits. The total content of biogenic amines in vinegars ranged from 23.35 to 1445.2 μg/L, being lower than those reported in wines. Putrescine was the amine that showed the highest concentrations in most samples. Methylamine and phenylethylamine were not determined in any vinegar. Balsamic and "Pedro Ximenez" Sherry vinegars reached the highest amounts of biogenic amines, while apple, white and Sherry wine vinegars had the lowest concentrations. Principal component analysis using the biogenic amines as variables, allowed to separate the different kind of vinegars, excepting red vinegars.

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling., Funding provided by EuroBoNet, the European research network on bone tumors (FP6-2004-Lifescihealth-5, proposal number 018814)-European Commission and the Instituto de Salud Carlos Tercero, FIS- project PI081828. J.L. Ordóñez is supported by the ISCIII (Contratos Perfeccionamiento CD6/00001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Funding provided by EuroBoNet, the European research network on bone tumors (FP6-2004-Lifescihealth-5, proposal number 018814)-European Commission and the Instituto de Salud Carlos Tercero, FIS- project PI081828. J.L. Ordo´n˜ ez is supported by the ISCIII (Contratos Perfeccionamiento CD6/00001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Cervical cancer and its precursors low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) are associated with infection by human papillomavirus (HPV), in particular HPV 16 and 18. The distribution of the HPV genotype varies with the severity of cervical disease, age and the geographic location of the patients. We report the results of a population study carried out in a region of north-western (NW) Spain aimed at determining the prevalence of single and multiple infections by 35 types of HPV using low-density microarrays for 113 cases with negative for intraepithelial lesions or malignancies; 588 with atypical squamous cells of undetermined significance (ASCUS)/LSIL; 183 with HSIL; and seven cases of squamous cell carcinomas. Of the 891 patients analysed, 50.2% had single infections and 49.8% had multiple HPV infections. In women aged below 30 years, there was a predominance of multiple infections (p = 0.027). ASCUS/LSIL was associated with multiple and HSIL with single infections (p = 0.025). We observed significant increases in the percentage of infections due to a high-risk (HR) type of HPV when the severity of the cytological lesion increased (p = 0.001). No relationship was found between greater aggressiveness in the cytological diagnosis and a higher number of HPV types involved in multiple infections. The five most frequent genotypes were HPV 16 (26.3%), 53 (18.2%), 51 (17.3%), 6 (14.8%) and 66 (13.1%). The prevalence of HPV 16, 33 and 58 increased significantly from ACUS/LSIL to HSIL and the prevalence of HPV 51, 53 and 66 decreased. HPV 16 was the only genotype that showed a significant increase in prevalence when the severity of the cytological disease increased in single infections (p = 0.0001). The implementation of bivalent prophylactic vaccination could potentially lead to prevention in 32% of the population included in the study - in at least a quarter of patients with ACUS/LSIL (26.7%), and in half of HSIL (50.2%).

Ewing sarcoma family tumors (ESFT) are a group of aggressive solid bone and soft tissue malignancies of children and young adults characterized by specific chromosomal translocations that give rise to EWS-ETS aberrant transcription factors. Identification of EWS-ETS target genes and their role in tumor signaling networks together with the unravelling of the cell of origin will facilitate the translation into new treatment modalities for these neoplasms, CIC-IBMCC belongs to NoE Eurobonet, FP6-2004- Lifescihealth-5, proposal number 018814, European Commission. Work at CIC is also funded by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation-FEDER (PI052524; RD06/0020/0059, CD06/00001). This work has been done with the support of the Fundación Memoria D. Samuel Solórzano Barruso.

Bone and soft tissue sarcomas are an infrequent and heterogeneous group of mesenchymal tumors including more than a hundred different entities attending to histologic patterns. Research into the molecular aspects of sarcomas has increased greatly in the last few years. This enormous amount of knowledge has allowed, for instance, to refine the classification of sarcomas, improve the diagnosis, and increase the number of therapeutical targets available, most of them under preclinical evaluation. However, other important key issues, such as sarcomagenesis and the cell of origin of sarcomas, remain unresolved. From a molecular point of view, these neoplasias are grouped into 2 main types: (a) sarcomas showing relatively simple karyotypes and translocations, which originate gene fusions (eg, EWS-FLI1 in Ewing sarcoma) or point mutations (eg, c-kit in the gastrointestinal tumors) and (b) sarcomas showing unspecific gene alterations, very complex karyotypes, and no translocations. The discovery of the early mechanisms involved in the genesis of sarcomas, the more relevant signaling pathways, and the development of genetically engineered mouse models could also provide a new individualized therapeutic strategy against these tumors. This review describes the clinical application of some of the molecular alterations found in sarcomas, some advances in the field of sarcomagenesis, and the development of animal models.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.-- et al., [BACKGROUND]: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. [METHODS]: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. [RESULTS]: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. [CONCLUSION]: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology. © 2009 Cancer Research., CIC-IBMCC and HHU belong to NoE Eurobonet, FP6-2004-Lifescihealth-5, proposal number 018814, European Commission. Work at CIC is also funded by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation-FEDER (PI052524; RD06/0020/0059, CD6/ 00001). Herrero-Martín was a recipient of a pre-doctoral fellowship from the Departamento de Educación, Junta de Castilla y León, Spain. This work has been done within the Acción Transversal en Cáncer program and the cooperative agreement between ISCIII and FICUS.

Ewing's sarcoma family tumors (EFT) are characterized by specific chromosomal translocations, which lead to EWS/ETS transcription factors.Elucidation of EWS/ETS target gene networks within the context of other signaling pathways, together with the identification of the initiating cell, and the development of genetically engineered mice will hopefully lead to biology-based therapeutic strategies for these tumors. 2009 American Association for Cancer Research., Grant support: The Centro de Investigación del Cáncer participates in EuroBoNeT, the European research network on bone tumors (FP6-2004-Lifescihealth-5, proposal number 018814)-European Commission. The Laboratory of Molecular Pathology of Sarcomas is also supported by the Spanish Ministry of Science and Innovation (FIS-FEDER, PI081828)and by the Junta de Castilla y León (SAN 673/SA 37/08). J. L. Ordóñez is supported by ISCIII (Contratos Perfeccionamiento CD6/00001); D. Osuna is supported by EuroBoNeT; J. Madoz-Gúrpide is supported by the Spanish Ministry of Science and Innovation, Ramón y Cajal Program.

Ewing Sarcoma (ES) shows several deregulated autocrine loops mediating cell survival and proliferation. Therefore, their blockade is a promising therapeutic approach. We previously reported the in vitro effect of insulin-like growth factor 1 receptor (IGF1R)/KIT pathway blockade on ES cell lines, and we now extend our observations to changes induced by this treatment in interacting proteins/networks. A proteomic analysis revealed that Heat Shock Protein (HSP)90 was differentially expressed between ES cell lines sensitive and resistant to specific IGF1R/KIT inhibitors. We therefore inhibited HSP90 with 17-allylamino-17-demethoxygeldanamycin (17-AAG) and siRNA, and observed that ES cell line growth and survival were reduced, especially in the resistant cell lines. Conversely, HSP90 induced-expression conferred resistance to anti-IGF1R/KIT treatment in the sensitive cell lines. 17-AAG treatment induced HSP90 client protein degradation, including AKT, KIT, or IGF1R, by inhibiting their physical interaction with HSP90. Xenograft models developed with A673 ES cell line confirmed that HSP90 inhibition, alone or combined with IGF1R inhibition, significantly reduced tumor growth and expression of client proteins. Remarkably, using two independent clinical sample sets, we have found that nearly half of IGF1R-positive tumors also show HSP90 overexpression. This delineates a subset of patients that could benefit from combination of anti-HSP90 agents when considering IGF1R-targeting therapies. Importantly, sensitivity to drugs such as ADW/IMA depends not only on the levels of expression and basal activation of IGF1R/KIT, but also, and for the first time reported in ES, on the development of the stress response mechanism. Accordingly, HSP90 expression could be a predictive factor of response to IGF1R-targeting therapies. ©2008 American Association for Cancer Research., Support was given by Fundación Ramón Areces, and Spanish Ministry of Health (FIS-FEDER: PI052524;ISCIII: CD6/00001), Centro de Investigación del Cáncer and Heinrich-Heine University belong to EuroBoNet, the European research network on bone tumors (FP6-2004-Lifescihealth-5, proposal number 018814)-European Commission, and by III Quadro Comunitário de Apoio, from the European Social Fund and Portuguese National Funds of MCES (A.S. Martins).

Bone and soft tissue sarcomas are an infrequent and heterogeneous group of mesenchymal tumors, including more than a hundred different entities attending to histological patterns. Sarcomas are quite resistant to conventional chemotherapy (anthracycline and ifosfamide) with the exception of some subtypes, such as Ewing's sarcoma (ES). New drugs with proved efficacy against sarcomas include taxanes, gemcitabine, and ET-743. Preclinical studies have also identified key molecular events leading to the progression and development of sarcomas which are good candidates to targeted therapy. Inhibitors of the tyrosine kinase receptors, such as IGF-1R, c-kit, PDGFR, VEGFR, or the mTOR signaling pathway, proteasome, angiogenesis, and stress response proteins are under clinical evaluation against sarcomas. ES, a tumor characterized by chromosomal translocations that originate gene fusions (EWS-FLI1, EWS-ERG), is an example of a good chemotherapy responder tumor whose survival rate shows a plateau in recent years. Preclinical studies have identified that new targets such as HSP90 are of relevance to ES. On the other hand, recent studies showed the role of cancer stem cells (CSCs) in sarcomas and the relevance of the identification of reliable molecular markers and possible therapeutic targets. New therapeutic approaches could be directed against CSCs. This review describes more recent targeted therapy in sarcomas, with special emphasis on ES and the role of CSCs. We also emphasize the role of high throughput proteomic techniques in identifying new therapeutic targets. © 2008 Elsevier Inc. All rights reserved.