Coupled-column liquid chromatography method with photochemically induced derivatization for the direct determination of benzoylureas in vegetables

The coupled-column (LC-LC) system, consisting of a first column packed with internal surface reversed phase (ISRP) (50x4.6 mm ID) and a Chrompack C18 (100x4.6 mm ID) as second column, allowed the simultaneous determination of five benzoylurea insecticides in dichloromethane (CH2Cl2) extracts of vegetable samples without any clean-up step. This system was combined with a photochemically induced fluorescence (PIF) post-column derivatization in order to provide strongly fluorescent photoproducts from the non-fluorescent benzoylureas. Limits of detection ranged from 0.21 to 0.98 ug L–1 of pesticide (equivalent to 0.14–0.65 ug kg–1 in vegetable samples) and limits of determination ranged from 4.0 to 10.0 ug L–1 (equivalent to 2.7–6.7 ug kg–1). Linearity of the method was established between 2 and 1800 ug L–1, depending upon the compound. Validation of the total method was performed by randomly analyzing recoveries of four vegetable samples (aubergine, cucumber, green bean, and tomato) spiked at two levels of concentration (10.0 and 33.3 ug kg–1). The combination of the LC-LC system with PIF detection provides a sensitive, selective, and rapid method for the determination of pesticides in vegetable samples at levels lower than the maximum residue levels (MRLs) established for these compounds by Spanish legislation

Documentación relativa a dos expedientes de adopción

La existencia de adopciones internacionales en el siglo XX hace necesaria la traducción de distintos documentos redactados principalmente en inglés (lingua franca) que intervienen en el proceso de adopción. Estos documentos muestran los distintos pasos necesarios para llegar a un fin, el traslado de un menor al país receptor y su subsiguiente adopción. En este trabajo fin de máster muestro el proceso de traducción de estos documentos, el cual se completa gracias a una reflexión exhaustiva acerca de su naturaleza (análisis intra y extratextual), de los problemas encontrados en el proceso de traducción y de las estrategias empleadas. Para estas dos últimas secciones he seguido la clasificación propuesta por Cristiane Nord (Text Analysis in Translation, 1998) y Hurtado (Traducción y Traductología, 2001) respectivamente. Al mismo tiempo, ahondo en la noción de interrelación de diferentes géneros como son los representados por el informe del menor, certificado de idoneidad, afidávit y certificado de designación de tutores, apoyada por la teoría de las “colonias de géneros” formulada por Bhatia (2004) y por la noción de género en su dimensión dinámica (Devitt, 2004)

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.Junta de Andalucía CTS 5884Ministerio de Educación y Ciencia AP2007-04201European Molecular Biology Organization ASTF45-201

Reelin expression is up-regulated in mice colon in response to acute colitis and provides resistance against colitis

Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-β1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitisEspaña, Junta de Andalucía CTS 588

Biomarkers in ocular chronic graft versus host disease: tear cytokine- and chemokine-based predictive model.

Producción CientíficaPurpose: To develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). Methods: Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. Results: Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. Conclusions: A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.Ministry of Economy and Competitiveness, Madrid, Spain, SAF-2010 15631 (AES)

Lack of reelin modifies the gene expression in the small intestine of mice

We recently demonstrated that the mucosa of the small intestine of the rat expresses reelin and some components of its signaling system. The current study evaluates whether reelin affects the intestinal gene expression profile using microarray analysis and reeler mice, a natural mutant in which reelin is not expressed. The effect of the mutation on body weight and intestinal morphology is also evaluated. The mutation reduces body and intestinal weight during the first 2 months of age and modifies the morphology of the crypts and villi. For the microarray assays, total RNA was obtained from either isolated epithelial cells or intact small intestine. Of the 45,101 genes present in the microarray the mutation significantly alters the expression of 62 genes in the isolated epithelial cell samples and of 84 in the intact small intestine. The expression of 83% of the genes tested for validation was substantiated by reverse transcriptase polymerase chain reaction. The mutation notably up-regulates genes involved in intestinal metabolism, while it down-regulates genes related with immune response, inflammation, and tumor development. Genes involved in cell proliferation, differentiation, apoptosis, membrane transport and cytoskeleton are also differently expressed in the reeler mice as compared with the control. This is the first report showing that the lack of reelin modifies intestinal morphology and gene expression profile and suggests a role for reelin in intestinal epithelium homeostasis.Junta de Andalucía CTS 588

Potential therapeutic applications of the genus Annona: Local and traditional uses and pharmacology

Ethno-pharmacological relevance Annona species (Annonaceae) have long been used as traditional herbal medicines by native peoples in tropical areas. In different countries they are used against a large variety of illnesses, such as parasitic and infectious diseases, cancer, diabetes, peptic ulcers, and mental disorders. Aim of the study This review aims to achieve a comprehensive understanding of the research conducted so far on the local and traditional uses, pharmacological activities, mechanism of actions of active compounds, toxicity, and possible interactions with other drugs of the Annona species. Through analysis of these findings, evidences supporting their applications in ethno-medicines are described. We discuss the possible research opportunities and stand out the weak points in our knowledge that deserves further investigation. Material and methods Information on ethno-medicinal uses and pharmacological activities of the Annona genus was collected. The main scientific biomedical literature databases (Cochrane, PubMed, Scopus, Lilacs, SeCiMed, Elsevier, SpringerLink, Google Scholar, SciFinder) were consulted. The search covered all the literature available until September 2017. National and regional databases of Herbal Medicine and Complementary and Alternative Medicine were also revised in order to explore further data. For a better understanding of the therapeutic importance of these species, we have classified the pharmacological activities within each group of disorders. The International Classification of Diseases (ICD), used from WHO Member States, was chosen as the reference classification. Results From among the 27 species revised, four species are highlighted for their important pharmacological activities in most of the groups of illnesses: A. muricata, A. squamosa, A. senegalensis, and A. cherimola. Many investigations have been performed with extracts from the leaves, bark, fruit and seeds and have shown a wide range of pharmacological activities, such as antiprotozoal, antitumoural, antidiabetic, hepato-protective, anti-inflammatory and anxiolytic activities. The chemistry on the annonaceous acetogenins (ACGs) has been extensively investigated due to their potent antitumoural activity. Many of the assays were carried out with the isolated acetogenins in different lines of tumour culture cells and were found effective at very low doses even in multidrug-resistant tumours, and hence constitute promising compounds in the treatment of different types of cancers. No studies were found with extracts rich in acetogenins in the clinical field. Conclusions The experimental results from the pharmacological research enable the validation of their traditional uses in several of the groups of diseases in the countries of origin and reveal these plants to be a valuable source for therapeutic molecules. However, more toxicity assays and clinical trials would be necessary to establish optimal and safe doses of consumption on the application of these medicinal plants

Reelin-Dab1 signaling system in human colorectal cancer

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-β1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers.Junta de Andalucía CTS 588

Reelin protects from colon pathology by maintaining the intestinal barrier integrity and repressing tumorigenic genes

We previously reported that reelin, an extracellular matrix protein first known for its key role in neuronal migration, reduces the susceptibility to dextran sulphate sodium (DSS)-colitis. The aim of the current study was to determine whether reelin protects from colorectal cancer and how reelin defends from colon pathology. In the colon of wild-type and of mice lacking reelin (reeler mice) we have analysed the: i) epithelium cell renewal processes, ii) morphology, iii) Sox9, Cdx2, Smad5, Cyclin D1, IL-6 and IFNγ mRNA abundance in DSS-treated and untreated mice, and iv) development of azoxymethane/DSS-induced colorectal cancer, using histological and real time-PCR methodologies. The reeler mutation increases colitis-associated tumorigenesis, with increased tumours number and size. It also impairs the intestinal barrier because it reduces cell proliferation, migration, differentiation and apoptosis; decreases the number and maturation of goblet cells, and expands the intercellular space of the desmosomes. The intestinal barrier impairment might explain the increased susceptibility to colon pathology exhibited by the reeler mice and is at least mediated by the down-regulation of Sox9 and Cdx2. In response to DSS-colitis, the reeler colon increases the mRNA abundance of IL-6, Smad5 and Cyclin D1 and decreases that of IFNγ, conditions that might result in the increased colitis-associated tumorigenesis found in the reeler mice. In conclusion, the results highlight a role for reelin in maintaining intestinal epithelial cell homeostasis and providing resistance against colon pathology.España, Junta de Andalucía CTS 588