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Renal vasculature, which is highly innervated by sympathetic fibers, contributes to cardiovascular homeostasis. This renal sympathetic outflow is inhibited by 5-HT in normoglycaemic rats. Considering that diabetes induces cardiovascular complications, we aimed to determine whether diabetic state modifies noradrenergic input at renal level and its serotonergic modulation in rats. Alloxan diabetic rats were anaesthetized (pentobarbital; 60 mg/kg i.p.) and prepared for in situ autoperfusion of the left kidney to continuously measure systemic blood pressure (SBP), heart rate (HR), and renal perfusion pressure (RPP). Electrical stimulation of renal sympathetic outflow induces frequency-dependent increases (Δ) in RPP (23.9 ± 2.1, 59.5 ± 1.9, and 80.5 ± 3.5 mm Hg at 2, 4, and 6 Hz, respectively), which were higher than in normoglycaemic rats, without modifying HR or SBP. Intraarterial bolus of 5-HT and 5-CT (5-HT 1/5/7 agonist) reduced electrically induced ΔRPP. Only L-694,247 (5-HT 1D agonist) reproduced 5-CT inhibition on sympathetic-induced vasoconstrictions, whereas it did not modify exogenous noradrenaline-induced ΔRPP. 5-CT inhibition was exclusively abolished by i.v. bolus of LY310762 (5-HT 1D antagonist). An inhibitor of guanylyl cyclase, ODQ (i.v.), completely reversed the L-694,247 inhibitory effect. In conclusion, diabetes induces an enhancement in sympathetic-induced vasopressor responses at the renal level. Prejunctional 5-HT 1D receptors, via the nitric oxide pathway, inhibit noradrenergic-induced vasoconstrictions in diabetic rats.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation., (© 2021. The Author(s).)

This study aims to determine the changes in, and bioaccessibility of, polyphenols and organosulfur compounds (OSCs) during the simulated gastrointestinal digestion of black onion, a novel product derived from fresh onion by a combination of heat and humidity treatment, and to compare it with its fresh counterpart. Fresh and black onions were subjected to in-vitro gastrointestinal digestion, and their polyphenol and OSC profiles were determined by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although to a lesser extent than in the fresh onion, the phenolic compounds in the black variety remained stable during the digestion process, presenting a higher bioaccessibility index (BI) with recovery corresponding to 41.1%, compared with that of fresh onion (23.5%). As for OSCs, apart from being more stable after the digestion process, with a BI of 83%, significantly higher quantities (21 times higher) were found in black onion than in fresh onion, suggesting that the black onion production process has a positive effect on the OSC content. Gallic acid, quercetin, isorhamnetin, and ɣ-glutamyl-S-(1-propenyl)-L-cysteine sulfoxide were the most bioaccessible compounds in fresh onion, while isorhamnetin, quercetin-diglucoside, ɣ-glutamyl-S-methyl-L-cysteine sulfoxide and methionine sulfoxide were found in black onion. These results indicate that OSCs and polyphenols are more bioaccessible in black onion than in fresh onion, indicating a positive effect of the processing treatment.

Background: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established., Methods: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response., Results: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition., Conclusions: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

The application of adaptive comfort models is among the determinant factors to reduce greenhouse gas emissions in the building sector. This research studies the region of Andalusia (south of Spain). A cluster analysis is applied to 786 Andalusian municipalities, and 4 groups are established according to the potential of adaptive strategies. A town is chosen from each group, and an hourly specific study is conducted for the last 20 years, as well as a daily study of the old time series by using an artificial neural network based on the existing climate data. The possibility of application of the EN 16798-1:2019 standard during the days of the year is analysed, as well as the possibilities of using natural ventilation and the possibility of using adaptive setpoint temperatures in comparison with both 3 fixed heating temperatures and 3 fixed cooling temperatures by considering the energy saving. The results to apply the standard ranged 69.0 and 100% of the days of each year. The possibilities of natural ventilation considered were greater than 10% of the hours of the year in all the assumptions. The energy saving of cooling degrees reveals a greater potential in the area studied than that of heating degrees; this tendency is supported by the study of old temporary series which are part of the climate variation predicted throughout the 21st century., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Elsevier Ltd. All rights reserved.)

Numerous studies have reported health benefits associated with the consumption of fresh and black garlic, which are characterized by the presence of polyphenols and organosulfur compounds (OS). This study aims to analyze the bioaccessibility of the bioactive compounds in fresh and black garlic after in vitro gastrointestinal digestion by monitoring the individual profile of these compounds by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Polyphenols decreased from the beginning of the digestive process, is mainly affected during intestinal digestion. Regarding the OS, the S-alk(en)yl-L-cysteine (SACs) derivatives were more influenced by the acidic conditions of the gastric digestion, while the γ-glutamyl-S-alk(en)yl-L-cysteine (GSAk) derivatives were more susceptible to intestinal digestion conditions in both the fresh and black garlic samples. In conclusion, after in vitro gastrointestinal digestion, the compounds with the highest bioaccessibility were vanillic acid (69%), caffeic acid (52%), γ-glutamyl-S-methyl-L-cysteine sulfoxide (GSMCS) (77%), and S-allylmercapto-L-cysteine (SAMC) (329%) in fresh garlic. Meanwhile, in black garlic, the main bioaccessible compounds were caffeic acid (65%), GSMCS (89%), methionine sulfoxide (262%), trans-S-(1-propenyl)-L-cysteine (151%), and SAMC (106%). The treatment (heating + humidity) to obtain black garlic exerted a positive effect on the bioaccessibility of OS compounds, 55.3% of them remaining available in black garlic, but only 15% in fresh garlic. Polyphenols showed different behavior regarding bioaccessibility.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

This study explored plasma levels and urinary and fecal excretion of metabolites and microbial-derived catabolites over a 24 h period following the ingestion of red wine (RWP) or grape seed (GSP) proanthocyanidin-rich extracts by rats. In total, 35 structurally-related (epi)catechin metabolites (SREMs) and 5-carbon side chain ring fission metabolites (5C-RFMs) (phenyl-γ-valerolactones and phenylvaleric acids), and 50 phenolic acid and aromatic catabolites were detected after intakes of both extracts. The consumption of the RWP extract, but not the GSP extract, led to the appearance of a ∼200 nmol L -1 peak plasma concentration of SREMs formed from flavan-3-ol monomers. In contrast, ingestion of the GSPs, but not the RWPs, resulted in a substantial increase in microbiota-derived 5-carbon side chain ring fission metabolites (5C-RFMs) in plasma. 5C-RFMs, along with low molecular weight phenolic catabolites were detected in urine after ingestion of both extracts. The GSP and RWP extracts had respective mean degrees of polymerisation 5.9 and 6.5 subunits, and the RWP extract had an upper polymer size of 21 subunits compared to 44 subunits for the GSP extract. The differences in plasma metabolite profiles might, therefore, be a consequence of this polydispersity impacting on the microbiota-mediated rates of cleavage of the proanthocyanidin subunits and their subsequent metabolism and absorption. Urinary excretion of phenolic catabolites indicated that 11% of RWPs and 7% for GSPs were subjected to microbial degradation. In all probability these figures, rather than representing the percentage of proanthocyanidins that are completely degraded, indicate partial cleavage of monomer subunits producing a much higher percentage of shortened proanthocyanidin chains. Obtaining more detailed information on the in vivo fate of proanthocyanidins is challenging because of the difficulties in analysing unabsorbed parent proanthocyanidins and their partially degraded flavan-3-ol subunit chains in feces. Further progress awaits the development of improved purification and analytical techniques for proanthocyanidins and their use in feeding studies, and in vitro fecal and bacterial incubations, with radio and/or stable isotope-labelled substrates.

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., (©2018 American Association for Cancer Research.)

Objective: To correlate the vitreous concentration of transforming growth factor β-1 (TGF β-1) with the degree of clinical severity of proliferative vitreoretinopathy (PVR)., Design: A prospective, observational, cross-sectional study carried out on cases and controls., Participants: The study included 40 patients with a diagnosis of PVR secondary to rhegmatogenous retinal detachment., Methods: Vitreous was obtained in patients undergoing pars plana vitrectomy by rhegmatogenous retinal detachment, who were treated during the period from August 2015 to June 2016, in a national reference centre for ophthalmological care in Mexico City, Mexico. The levels of TGFβ-1 were quantified by ELISA technique. An ANOVA test was performed for the comparison of the different groups, together with a post-hoc Dunns test. A statistically significant difference was considered when obtaining P <.05., Results: The levels of TGFβ-1 were quantified, and the following means were found for each group: In the group with PVR grade A, 1150.6 ± 452.08 pg / ml, PVR grade B: 1129.6 ± 365.54 pg / ml, and PVR grade C: 1146.4 ± 330.21 pg / ml. The statistical analysis did not find significant differences when comparing the different PVR groups. (P=.53). However, when performing the differential analysis for each level of severity, a statistically significant increase in the expression of TGFβ-1 was observed in the group of patients with PVR-A at a greater number of days of evolution of the detachment. (P=.03). There were no statistically significant differences for PVR-B and PVR-C (P=.16 and P=.16, respectively)., Conclusion: Although the levels of TGFβ-1 are not directly related to the clinical severity grade, suggesting that there must be other factors involved in the advanced stages of PVR, TGFβ-1 may have greater relevance during the initial stages of the clinical course by promoting the epithelial-mesenchymal transition due to its greater expression in PVR-A. Thus, it can be concluded that each isoform plays a very particular role in the complex process of PVR., (Copyright © 2018 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.)

The present study compared and validated two analytical methods, HPLC-HRMS, and GC-MS using MSTFA as derivatization agent, for the analysis of microbiota-derived phenolic acids and aromatic compounds accumulating in urine, collected over a 24 h period after the consumption of 500 mL of orange juice. In addition, purification procedures using SDB-L and HLB solid phase cartridges were compared when HPLC-HRMS technique was used. Both HPLC-HRMS and GC-MS methodologies were successfully validated in terms of specificity, sensitivity, limit of detection and quantification, recovery and matrix effects. HPLC-HRMS, unlike GC-MS, does not require sample derivatization prior to analysis. GC-MS was not suitable for the analysis of phenolic sulfate and glucuronide metabolites because of their lack of volatility. These phase II metabolites could, however, be analysed by HPLC-HRMS which, as a consequence, provided more detailed and complete information on the phenolic compounds derived from microbiota-mediated degradation of orange juice (poly)phenols. Furthermore, the use of SDB-L and HLB cartridges for sample purification prior to HPLC-HRMS analysis is suitable for free phenolics and glucuronide metabolites but not sulfate derivatives. These findings highlight that the use of an inappropriate analytical protocol can adversely affect studies on the bioavailability of dietary (poly)phenols in which microbiota-derived phenolic catabolites play an important role., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Five free software packages were compared to assess their utility for the non-targeted study of changes in the volatile profile during the storage of a novel strawberry beverage. AMDIS coupled to Gavin software turned out to be easy to use, required the minimum handling for subsequent data treatment and its results were the most similar to those obtained by manual integration. However, AMDIS coupled to SpectConnect software provided more information for the study of volatile profile changes during the storage of strawberry beverage. During storage, volatile profile changed producing the differentiation among the strawberry beverage stored at different temperatures, and this difference increases as time passes; these results were also supported by PCA. As expected, it seems that cold temperature is the best way of preservation for this product during long time storage. Variable Importance in the Projection (VIP) and correlation scores pointed out four volatile compounds as potential markers for shelf-life of our strawberry beverage: 2-phenylethyl acetate, decanoic acid, γ-decalactone and furfural., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The treatment of infections in critically ill obese and morbidly obese patients is challenging because of the combined physiological changes that result from obesity and critical illness. The aim of this study was to describe the population pharmacokinetics of piperacillin in a cohort of critically ill patients, including obese and morbidly obese patients. Critically ill patients who received piperacillin-tazobactam were classified according to their body mass index (BMI) as nonobese, obese, and morbidly obese. Plasma samples were collected, and piperacillin concentrations were determined by a validated chromatographic method. Population pharmacokinetic analysis and Monte Carlo dosing simulations were performed using Pmetrics software. Thirty-seven critically ill patients (including 12 obese patients and 12 morbidly obese patients) were enrolled. The patients' mean ± standard deviation age, weight, and BMI were 50 ± 15 years, 104 ± 35 kg, and 38.0 ± 15.0 kg/m 2 , respectively. The concentration-time data were best described by a two-compartment linear model. The mean ± SD parameter estimates for the final covariate model were a clearance of 14.0 ± 7.1 liters/h, a volume of distribution of the central compartment of 49.0 ± 19.0 liters, an intercompartmental clearance from the central compartment to the peripheral compartment of 0.9 ± 0.6 liters · h -1 , and an intercompartmental clearance from the peripheral compartment to the central compartment of 2.3 ± 2.8 liters · h -1 A higher measured creatinine clearance and shorter-duration infusions were associated with a lower likelihood of achieving therapeutic piperacillin exposures in patients in all BMI categories. Piperacillin pharmacokinetics are altered in the presence of obesity and critical illness. As with nonobese patients, prolonged infusions increase the likelihood of achieving therapeutic concentrations., (Copyright © 2017 American Society for Microbiology.)

Our objective was to describe the population pharmacokinetics of fluconazole in a cohort of critically ill nonobese, obese, and morbidly obese patients. Critically ill patients prescribed fluconazole were recruited into three body mass index (BMI) cohorts, nonobese (18.5 to 29.9 kg/m 2 ), obese (30.0 to 39.9 kg/m 2 ), and morbidly obese (≥40 kg/m 2 ). Serial fluconazole concentrations were determined using a validated chromatographic method. Population pharmacokinetic analysis and Monte Carlo dosing simulations were undertaken with Pmetrics. Twenty-one critically ill patients (11 male) were enrolled, including obese (n = 6) and morbidly obese (n = 4) patients. The patients mean ± standard deviation (SD) age, weight, and BMI were 54 ± 15 years, 90 ± 24 kg, and 31 ± 9 kg/m 2 , respectively. A two-compartment linear model described the data adequately. The mean ± SD population pharmacokinetic parameter estimates were clearance (CL) of 0.95 ± 0.48 liter/h, volume of distribution of the central compartment (V c ) of 15.10 ± 11.78 liter, intercompartmental clearance from the central to peripheral compartment of 5.41 ± 2.28 liter/h, and intercompartmental clearance from the peripheral to central compartment of 2.92 ± 4.95 liter/h. A fluconazole dose of 200 mg daily was insufficient to achieve an area under the concentration-time curve for the free, unbound drug fraction/MIC ratio of 100 for pathogens with MICs of ≥2 mg/liter in patients with BMI of >30 kg/m 2 A fluconazole loading dose of 12 mg/kg and maintenance dose of 6 mg/kg/day achieved pharmacodynamic targets for higher MICs. A weight-based loading dose of 12 mg/kg followed by a daily maintenance dose of 6 mg/kg, according to renal function, is required in critically ill patients for pathogens with a MIC of 2 mg/liter., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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 purée 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (©2015 American Association for Cancer Research.)

Fibromyalgia is a chronic pain syndrome associated with numerous somatic symptoms including gastrointestinal manifestations of nonspecific nature. Celiac disease and nongluten sensitivity frequently evolve in adults with gastrointestinal and extraintestinal symptoms similar to those found among patients with fibromyalgia. The objective of the present study was to evaluate the presence of celiac-type symptoms among patients with fibromyalgia in comparison with healthy subjects and with those experienced by adult celiac patients and subjects with gluten sensitivity. A list of typical celiac-type symptoms was developed, comparing the frequency of presentation of these symptoms between patients with fibromyalgia (N = 178) and healthy subjects (N = 131), in addition to those of celiac patients and gluten-sensitive patients reported in the literature. The frequency of presentation of every celiac-type symptom, excepting anemia, was significantly higher among patients with fibromyalgia compared to controls (p < 0.0001). Regarding the existing data in the literature, the prevalence of fatigue, depression, cognitive symptoms and cutaneous lesions predominated among patients with fibromyalgia, whereas the prevalence of gastrointestinal symptoms was higher among patients with fibromyalgia compared to gluten-sensitive patients and was similar among patients with fibromyalgia and celiac disease patient. The symptomatological similarity of both pathologies, especially gastrointestinal symptoms, suggests that at least a subgroup of patients with fibromyalgia could experience subclinical celiac disease or nonceliac gluten intolerance.

Background: Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin., Materials and Methods: In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples., Results: We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99., Conclusions: In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.

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.

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 Ximénez" 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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

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%)., (© 2011 The Authors. APMIS © 2011 APMIS.)

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.

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.

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.

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.

One of the key aspects that must be taken into consideration within the framework of Sustainable Construction is the management of Construction and Demolition (C&D) Debris. As for other types of waste, specific handling procedures are required to manage C&D Debris; these include reduction, reuse, recycling, and if all other possibilities fail, recovery or disposal. For public planning strategies aimed at the management of C&D Debris to be effective, it is first necessary to have specific knowledge of the type of waste materials generated in a particular region. After verifying that the methods available to determine the production and composition of C&D Debris are limited, this paper presents a procedure to ascertain the production and composition of C&D Debris, in any region. The procedure utilizes data on the surface areas of newly constructed buildings, renovations and demolitions, which are estimated from available data for recent years, as well as information on the quantity of debris generated per surface area in any type of construction site, which is obtained from recently executed constructions or from the ground plans of older buildings. The method proposed here has been applied to Galicia, one of Spain's autonomous communities, for which the quantity and composition of C&D Debris have been estimated for the horizon year 2011., (Copyright 2009 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Purpose: Ewing tumor cell survival and proliferation depends on several autocrine loops. Targeting these loops is a promising therapeutic approach. We recently showed the cytostatic role of imatinib, an inhibitor of the SCF-KIT loop, on Ewing tumor cells, and in this study, we intend to analyze the inhibition of the insulin-like growth factor I receptor (IGF1R) loop., Experimental Design: We analyzed IGF1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, vincristine, and doxorubicin on Ewing tumor cell lines. We studied the effect on proliferation, apoptosis, cell cycle, pathway phosphorylation, soft-agar growth, motility, and vascular endothelial growth factor expression levels., Results: Treatment with ADW742 induced down-regulation of IGF1R/AKT/mammalian target of rapamycin (mTOR) phosphorylation, which was deeper in cell lines having higher IGF1R activation levels. Treatment also induced dose-dependent inhibition of cell proliferation (IC50 = 0.55-1.4 micromol/L), inducing a G1 phase blockage and apoptosis. Addition of imatinib to ADW742 synergistically augmented these effects and was especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF1R activation levels. Combination with usual chemotherapeutic agents vincristine and doxorubicin showed synergistic interactions., Conclusions: Inhibition of Ewing tumor cell proliferation by ADW742 is mediated through blockade of IGF1R signaling. Combination of ADW742 with imatinib, vincristine, and doxorubicin induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF1R activation levels. This study supports a potential role for ADW742 in the treatment of Ewing tumor and AKT/mTOR as a possible surrogate marker of response to therapy.

The objective of this article was to identify the effects of bromocriptine on the ultrastructure of the caudate nucleus in rats with a 6-hydroxidopamine (6-OHDA) unilateral lesion of the substantia nigra pars compacta. Eighteen Wistar male rats were stereotactically lesioned with 6-OHDA (n=12), or sham lesioned (n=6). Two days after rotational behavior was tested, and 2 days later, 6 rats were treated with 0.3 mg/Kg bromocriptine orally for a month and 6 rats were kept for the same time without treatment. The neuropile of the sham operated and bromocriptine-treated rats was well preserved contrary to the non-bromocriptine-treated rats. Also, it was found that there was a significant difference in the number of synaptic endings with edema in caudate of bromocriptine-treated rats compared with non-treated rats; however, the size of the synaptic endings were different to those found in the sham lesioned rats. Also, as in the sham lesioned group, the bromocriptines showed more synaptic contacts with dendritic spines contrasting to the non-treated group. The results suggest that bromocriptine possesses antioxidant properties because it decreased the ultrastructural alterations after 6-OHDA lesion.

This study was undertaken to compare the ability of five uveal melanocytic cell lines to produce primary and metastatic uveal melanomas in immunosuppressed rabbits and to determine whether animal survival was improved by antibiotic administration. One hundred albino rabbit eyes, five groups of 20, were implanted in the suprachoroidal space with four melanoma cell lines (MKT-BR, OCM-1, 92-1 and SP 6.5) and one melanocytic line (UW-1). Rabbits were immunosuppressed with cyclosporin A (CsA) at a dosage of 15 mg/kg/day, decreased to 10 mg/kg/day after the fourth week. Prophylactic penicillin G, 10 to 2 x 10 IU, was administered intramuscularly at 5-day intervals. Animals were followed for 12 weeks and the ophthalmoscopic findings, weight and general well-being were recorded weekly. Autopsies were performed to study the eyes, liver and lungs under light microscopy. The mean global survival time in the groups was 43+/-4 days. Ophthalmoscopic intraocular tumours developed in 37% of the MKT-BR group, 50% of the OCM-1 group, 100% of the 92-1 group, 23% of the UW-1 group and 75% of the SP 6.5 group; histologically, tumours appeared in 36.8%, 45%, 100%, 58.8% and 100%, respectively. The 92-1 and SP 6.5 cell lines were associated with the most aggressive local behaviour. Lung metastases developed in the OCM-1 group (5%), 92-1 group (61.1%), UW-1 group (7.1%) and SP 6.5 group (42.1%), but were not present in the MKT-BR group. The 92-1 and SP 6.5 cell lines were the most efficient in local and metastatic tumour production. Prophylactic antibiotic administration did not improve animal survival.