Generation of a novel three-dimensional scaffold-based model of the bovine endometrium.

Bovine in vitro endometrial models that resemble tissue function in vivo are needed to study infertility, long-term uterine alterations induced by pathogens and impact of endocrine disruptor chemicals on reproductive function and other reproductive system complications that cause high economic losses in livestock species. The present study aimed to generate an innovative, reproducible, and functional 3D scaffold-based model of the bovine endometrium structurally robust for long term-culture. We developed a multicellular model containing both endometrial epithelial and stromal cells. Epithelial cells organized to form a luminal-like epithelial layer on the surface of the scaffold. Stromal cells produced their own extracellular matrix forming a stable subepithelial compartment that physiologically resembles the normal endometrium. Both cell types released prostaglandin E2 and prostaglandin F2α following a treatment with oxytocin and arachidonic acid. Additionally signal pathways mediating oxytocin and arachidonic acid stimulation of prostaglandin synthesis were analyzed by real time PCR (RT-PCR). Oxytocin receptor (OXTR), prostaglandin E2 receptor 2 (EP2), prostaglandin E2 receptor 4 (EP4), prostaglandin F receptor (PTGFR), prostaglandin E synthase (PTGES), PGF-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (COX-2) expression was detected in both control and treatment groups, however, only significant changes in abundance of OXTR mRNA transcripts were found. The results obtained by this study are a step forward in bovine in vitro culture technology. This 3D scaffold-based model provides a platform to study regulatory mechanisms involved in endometrial physiology and can set the basis for a broader tool for designing and testing novel therapeutic strategies for recurrent uterine pathologies.

Influence of nutrition education in paediatric coeliac disease: impact of the role of the registered dietitian: a prospective, single-arm intervention study.

BACKGROUND: The diagnosis of coeliac disease (CD) involves a change in the diet of the individual, which may influence their quality of life and nutritional status. The present study aimed to determine whether nutrition education by a registered dietitian is able to improve eating habits and body composition in children with CD. METHODS: Dietary, physical activity and body composition changes were analysed, comparing baseline assessments with those 1 year after receiving education on healthy eating. At both time points, a 3-day dietary survey, a food frequency consumption questionnaire, an adherence to the Mediterranean diet test (Kidmed), duration of activity and an electrical bioimpedance study were conducted. Student's paired t-test and the McNemar test were also employed. RESULTS: Seventy-two subjects (42 girls) with an mean (range) age of 10 (2-16) years were included. Before the intervention, an unbalanced diet was observed, rich in protein and fat, and deficient in complex carbohydrates. Only 14% consumed an adequate Mediterranean diet. After nutrition intervention, a significant increase in the consumption of plant-based foods and a concomitant decrease in meat, dairy and processed food intake (P < 0.001) were observed. Moreover, 92% of the patients (P < 0.001) managed to consume an adequate Mediterranean diet. Similarly, an increase was observed in the duration of physical activity undertaken [mean (SD) 1.02 (1.79) h, P < 0.001] and improvements in body composition were recorded, with a 17% decrease in fat mass percentage (P < 0.001). CONCLUSIONS: Nutrition intervention focused on healthy eating is effective with respect to improving the nutritional status and diet quality in CD patients.

Evaluation of the aqueous phototransformation routes of phenyl ethyl azolic fungicides by liquid chromatography accurate mass spectrometry.

Similarities and differences among the phototransformation routes of four azolic fungicides (diniconazole, DIN, imazalil, IMA; penconazole, PEN; and propiconazole, PRO) in surface water aliquots are investigated. Selected compounds share a common chemical structure consisting on dichlorophenyl and azolic rings connected through an ethylene bridge, which is substituted with different functionalities. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was employed as analytical technique to follow the time-course of precursor fungicides and to detect and to identify their photo-transformation products (TPs). Under solar light, the substituents linked to the ethylene chain controlled the stability of the fungicides. Whilst PEN and PRO remained stable, DIN and IMA showed moderate reactivities, with half-lives (t1/2) of 5.1 and 33.5h, respectively. When exposed to UV (254nm) radiation, all compounds were effectively degraded with t1/2 in the range from seconds to a few minutes. Dechlorination followed by intramolecular cyclization, between phenyl and azolic rings, was identified as a common phototransformation route under UV irradiation. Depending on the length and the kind of the functionalities attached to the ethylene bridge, additional cyclization reactions are also possible. In-silico toxicity predictions pointed out to dechlorinated tricyclic TPs as the most concerning ones, with predicted lethal concentrations (LC50) in the same range as the precursor fungicides.

Selective extraction and determination of neonicotinoid insecticides in wine by liquid chromatography-tandem mass spectrometry.

A simplified, high throughput procedure for the determination of five neonicotinoid insecticides in red and white wines, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS), is presented. The effects of different experimental parameters (extraction sorbent, solvent elution and clean-up conditions) in the efficiency and the selectivity of the sample preparation process were assessed through calculation of the extraction yields and the matrix effects (MEs). Wines (10mL) were concentrated using OASIS HLB cartridges, on-line connected to Florisil clean-up cartridges, with acetonitrile serving as the elution solvent. The extract (5mLvol) was concentrated to 1mL and injected in the LC-ESI-MS/MS system. The optimized procedure provided quantitative extraction yields at the same time that the efficiency of ESI ionization remained unchanged between standards and sample extracts. Overall recoveries, calculated against authentic standards in ACN, varied between 77 and 119% and the attained limits of quantification remained below 0.2ngmL(-1). Analysis of commercial wines revealed imidacloprid residues in more than 50% of processed samples, with a maximum level of 14ngmL(-1).

Multiclass semi-volatile compounds determination in wine by gas chromatography accurate time-of-flight mass spectrometry.

The performance of gas chromatography (GC) with accurate, high resolution mass spectrometry (HRMS) for the determination of a group of 39 semi-volatile compounds related to wine quality (pesticide residues, phenolic off-flavours, phenolic pollutants and bioactive stilbenes) is investigated. Solid-phase extraction (SPE) was used as extraction technique, previously to acetylation (phenolic compounds) and dispersive liquid-liquid microextraction (DLLME) concentration. Compounds were determined by GC coupled to a quadrupole time-of-flight (QTOF) MS system through an electron ionization (EI) source. The final method attained limits of quantification (LOQs) at the very low ng mL(-1) level, covering the range of expected concentrations for target compounds in red and white wines. For 38 out of 39 compounds, performance of sample preparation and determination steps were hardly affected by the wine matrix; thus, accurate recoveries were achieved by using pseudo-external calibration. Levels of target compounds in a set of 25 wine samples are reported. The capabilities of the described approach for the post-run identification of species not considered during method development, without retention time information, are illustrated and discussed with selected examples of compounds from different classes.

Liquid chromatography quadrupole time-of-flight mass spectrometry selective determination of ochratoxin A in wine.

The performance of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for ochratoxin A (OTA) determination in wine is evaluated for the first time. Sample preparation was optimized to obtain quantitative recoveries at the same time that the efficiency of electrospray ionization (ESI) remained unaltered between sample extracts and calibration standards. Under final conditions, samples (20 mL) were concentrated using a reversed-phase solid-phase extraction (SPE) cartridge, followed by OTA elution with 1 mL of ethyl acetate. The absolute recoveries of the method, established against calibration standards, were 91-121% and 90-113% (without and with internal standard correction, respectively), for wines fortified at 3 concentration levels. The attained LOQ (0.05 ng mL(-1)) remained below the maximum permitted OTA concentration (2 ng mL(-1)) in dry wines. The method was applied to different samples, with OTA being found in some dessert wines at concentrations below 1 ng mL(-1). The ethyl ester of OTA (OTC) could be identified in the same wine samples from its accurate full product ion spectra.

Uso de anticatarrales en menores de 14 años en consultas de Atención Primaria / Use of cold and cough medications prescribed in Primary Care clinics for children less than 14 years

OBJETIVO: Conocer el perfil de prescripción de anticatarrales en las consultas de Pediatría de Atención Primaria en el Área V del Servicio de Salud del Principado de Asturias y valorar su idoneidad. Material y métodos; Estudio transversal, descriptivo y retrospectivo. Se analizaron las prescripciones para el tratamiento de los procesos respiratorios en 6 consultas de Pediatría de Atención Primaria en el Área Sanitaria V del Servicio de Salud del Principado de Asturias en el año 2011. Se valoraron la idoneidad de los tratamientos prescritos mediante las fichas técnicas de los fármacos y las indicaciones clínicas según el diagnóstico, siguiendo las recomendaciones de guías clínicas, protocolos o en su defecto la bibliografía disponible más actual. RESULTADOS: Se analizaron 424 anticatarrales: 249 antitusígenos, 155 mucolíticos y 20 clasificados en otros. La media de edad de los pacientes es de 5 años. Un 85,1% de las prescripciones se consideran inadecuadas. El 11,6% de ellos fueron prescritos fuera de ficha técnica. Se consideraron inadecuados el 82,8% de los asociados al diagnóstico R74 y el 73% al R05. Todos los fármacos de los menores de 6 años se consideraron inadecuados. El 99,4% de los mucolíticos/otros y el 75,1% de los antitusígenos se consideraron inadecuados. CONCLUSIONES: Se observa un alto porcentaje de prescripción de fármacos anticatarrales en menores de 14 años en nuestro medio, encontrándose un 85% de las prescripciones inadecuadas. Los niños deberían recibir solo medicamentos con una relación beneficio-riesgo favorable; para ello es necesario mejorar la información sobre el uso pediátrico y promover acciones formativas dirigidas a los padres y a los profesionales sanitarios

OBJECTIVE:To evaluate cold and cough medications and their suitability in children in Primary Health Care in Area V of the Asturian Health Service. MATERIAL AND METHODS: A cross-sectional, descriptive and retrospective study was conducted in which an analysis was performed of the respiratory diseases and the prescriptions of 6 Primary Health Care paediatricians who worked in Area V of the Asturian Health Service in 2011. An evaluation was made on the suitability of these medications. An analysis was also made of the drug datasheet and clinical recommendations (clinical guidelines, protocols or reports). RESULTS: A total of 424 cold and cough drugs: 249 antitussives, 155 mucolytics, and 20 'others' were analyzed. The mean age was 5 years old. There was a total of 85.1% unsuitable prescriptions. Off-label drugs were used in 11.6%. The prescribing was considered unsuitable in 82.8% of prescriptions associated with R74, and 73% of R05. All of the prescription drugs in children under 6 years old were unsuitable. Mucolytics/'others' were not suitable in 99.4%, nor antitussives in 75.1%. CONCLUSIONS: There is a high level of cold and cough drugs being prescribed in children, with 85% of these being unsuitable. Children should only receive drugs with a good risk and benefit ratio. Pediatricians should try to improve the information about pediatric drug use and spread this information to parents, doctors and nurses

[Use of cold and cough medications prescribed in Primary Care clinics for children less than 14 years]. / Uso de anticatarrales en menores de 14 años en consultas de Atención Primaria.

OBJECTIVE: To evaluate cold and cough medications and their suitability in children in Primary Health Care in Area V of the Asturian Health Service. MATERIAL AND METHODS: A cross-sectional, descriptive and retrospective study was conducted in which an analysis was performed of the respiratory diseases and the prescriptions of 6 Primary Health Care paediatricians who worked in Area V of the Asturian Health Service in 2011. An evaluation was made on the suitability of these medications. An analysis was also made of the drug datasheet and clinical recommendations (clinical guidelines, protocols or reports). RESULTS: A total of 424 cold and cough drugs: 249 antitussives, 155 mucolytics, and 20 "others" were analyzed. The mean age was 5 years old. There was a total of 85.1% unsuitable prescriptions. Off-label drugs were used in 11.6%. The prescribing was considered unsuitable in 82.8% of prescriptions associated with R74, and 73% of R05. All of the prescription drugs in children under 6 years old were unsuitable. Mucolytics/"others" were not suitable in 99.4%, nor antitussives in 75.1%. CONCLUSIONS: There is a high level of cold and cough drugs being prescribed in children, with 85% of these being unsuitable. Children should only receive drugs with a good risk and benefit ratio. Pediatricians should try to improve the information about pediatric drug use and spread this information to parents, doctors and nurses.

Comprehensive evaluation of the photo-transformation routes of trans-resveratrol.

Liquid chromatography (LC) combined with accurate mass spectrometry (MS), based on the use of a hybrid quadrupole time-of-flight (TOF) MS system, is employed to systematically investigate the photo-transformation routes of trans-resveratrol. Experiments were performed in quartz tubes, containing ethanolic solutions (12% v/v) of the precursor compound, exposed to different ultraviolet (UV) sources and to solar light. Time-courses of trans-resveratrol and transformation products (TPs) were investigated by direct injection of different reaction times aliquots in the LC-QTOF-MS system. Structural elucidation of detected TPs was derived from interpretation of their accurate product ion scan spectra. Trans-resveratrol labelled with (13)C6 in the mono-hydroxylated ring was also employed to further confirm the exact positions of some substituents in the generated TPs. In addition to the well-known trans-/cis-isomerization process, three different main reactions pathways were noticed under all the investigated conditions: (1) water addition to the exocycle double bond followed by oxidation to a ketone and cleavage of the molecule, (2) intramolecular cyclization to render a trihydroxylated phenanthrene, and (3) oxidation of the phenanthrene-like derivative to generate an orto-diquinone. Both, the trihydroxylated phenanthrene and the orto-diquinone underwent further aromatic hydroxylation reactions. The above transformation routes were also noticed for cis-resveratrol and the two analogue phytoalexins piceid and piceatannol. In addition to above transformation pathways, under solar light exposure, resveratrol underwent a molecular re-arrangement rendering the so-called resveratrone, whose structure consists of two fused aromatic rings bonded to a linear chain containing a carbonyl group conjugated with a double bond.

Assessment of dispersive liquid-liquid microextraction conditions for gas chromatography time-of-flight mass spectrometry identification of organic compounds in honey.

The suitability of the dispersive liquid-liquid microextraction (DLLME) technique for gas chromatography (GC) characterization of minor organic compounds in honey samples is evaluated. Under optimized conditions, samples were pre-treated by liquid-liquid extraction with acetonitrile followed by DLLME using carbon tetrachloride (CCl4, 0.075 mL) as extractant. The yielded settled phase was analyzed by GC using high resolution time-of-flight (TOF) mass spectrometry (MS). The whole sample preparation process is completed in approximately 10 min, with a total consumption of organic solvents below 4 mL, relative standard deviations lower than 12% and with more than 70 organic compounds, displaying linear retention index in the range from 990 to 2900, identified in the obtained extracts. In comparison with HS SPME extraction, higher peak intensities were attained for most volatile and semi-volatile compounds amenable to both extraction techniques. Furthermore, other species such as highly polar and water soluble benzene acids, long chain fatty acids, esters and flavonoids, which are difficult to concentrate by HS SPME, could be identified in DLLME extracts. Some of the compounds identified in DLLME extracts have been proposed as useful for samples classification and/or they are recognized as markers of honeys from certain geographic areas.

Investigation of liquid chromatography quadrupole time-of-flight mass spectrometry performance for identification and determination of hydroxylated stilbene antioxidants in wine.

The performance of liquid chromatography (LC) followed by quadrupole time-of-flight (QTOF) mass spectrometry (MS) for the determination of hydroxylated stilbene compounds in red and white wine samples is assessed. When combined with a solid-phase extraction step, LC-QTOF-MS allowed the selective determination of five target compounds (trans- and cis-resveratrol, trans-piceatannol, trans-piceid and trans-pterostilbene) attaining limits of quantification between 3 and 20ngmL(-1) and providing linear responses up to 4000ngmL(-1). Recoveries, established against standards prepared in methanol, varied between 93% and 115%. The distribution of the above species in wine is illustrated with the analysis of 15 samples. Trans-pterostilbene remained undetected in samples, whereas trans-piceid and trans-resveratrol maximum concentrations exceed the 6000ngmL(-1) level. Values for trans-piceatannol and cis-resveratrol ranged from non detected to 600ngmL(-1), and from 11 to more than 3200ngmL(-1), respectively. Accurate MS and MS/MS scan spectra were used to investigate the existence of reduced (dihydro) and oxidized (dehydro) forms of resveratrol and picetannol in the processed samples. Dihydro derivatives appeared, as free compounds, in 100% (dihydro-piceatannol) and 40% (dihydro-resveratrol) of the samples. On the other hand, dehydro derivatives were noticed as conjugated (glycosylated) species, with detection frequencies of 100% and 47% for dehydro-glucosyl-resveratrol and dehydro-glucosyl-piceatannol, respectively. Above findings confirm the suitability of LC-QTOF-MS for the comprehensive study of hydroxylated stilbene antioxidants in wine samples.

Liquid chromatography time-of-flight mass spectrometry evaluation of fungicides reactivity in free chlorine containing water samples.

Liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS), based on the use of a hybrid quadrupole-time-of-flight mass analyzer, was used to investigate the reactivity of nine fungicides in free chlorine-containing water samples. Three of the selected compounds (fenhexamid, FEN; pyrimethanil, PYR; and cyprodinil, CYP) displayed a poor stability in presence of moderate chlorine levels; thus, the effects of different parameters on their half-lives (t(1/2)) were evaluated. Sample pH, bromide traces, and the water matrix affected their relative stabilities. Despite such variations, the three fungicides are degraded at significant rates not only in ultrapure, but also in surface water spiked with chlorine levels up to 2 µg ml(-1), and when mixed with chlorinated tap water, generating several transformation products (TPs). The time-course of precursor species and their TPs was followed in the LC-MS mode, using the information contained in accurate, full scan mass spectra (MS) to propose the empirical formulae of TPs. Thereafter, their ion product scan (MS/MS) spectra were considered to set their chemical structures; allowing, in some cases, to distinguish between isomeric TPs. The reaction pathway of FEN, the less stable fungicide, involved just an electrophilic substitution of hydrogen per chlorine, or bromine, and cleavage of the molecule to render an amide. PYR and CYP shared common reaction routes consisting of halogenation, hydroxylation, and condensation processes leading to complex mixtures of TPs, which were relatively stable to further transformations.

Assessment of silicone as support to investigate the transformation routes of organic chemicals under environmental conditions and UV exposure. Application to selected fungicides.

The suitability of bulk silicone as support to follow the degradation of chemical compounds under environmental conditions and UV radiation is illustrated selecting three fungicides (fenhexamid, FEN; triadimenol, TRI and difenoconazole, DIF) as model compounds. These precursor species were first absorbed in silicone supports (10 mm length × 2 mm i.d. and 0.5 mm thickness) and then kept outdoors for several days (up to 2 months) or exposed to UV radiation (254 nm), from a low pressure mercury lamp, in the laboratory. Degradation of precursor fungicides and by-products formation was followed by liquid chromatography (LC) quadrupole time-of-flight (QTOF) mass spectrometry (MS), after desorption of silicone supports using 0.5 mL of acetonitrile. Half-lives (t(1/2)) measured under UV exposure varied from 5 to 100 min. As regards environmental conditions, the most stable fungicide was DIF, degraded by just 15 % after 2 months; whereas, t(1/2) values of 30 and 83 h were calculated for FEN during summer and autumn, respectively. Supports contained by-products arising from precursor species through de-chlorination, cleavage, hydroxylation, intra-molecular cyclation and oligomerization reactions. Most of them have been previously identified in soil surface, vegetable leaves and water after application of fungicides in agriculture fields. The low cost of silicone tubes (ca. 0.4 Euros), added to their excellent chemical stability and capability to retain precursor species and their by-products, make them ideal supports to follow the transformation routes of organic compounds under environmental and simulated conditions, even for relatively stable species with t(1/2) in the range of weeks or months.

Determination of hydroxylated stilbenes in wine by dispersive liquid-liquid microextraction followed by gas chromatography mass spectrometry.

Dispersive liquid-liquid microextraction (DLLME) has been proposed, for first time, to determine the concentrations of three hydroxylated stilbenes (trans-isomers of pterostilbene, resveratrol and piceatannol) in wine samples. DLLME was preceded by an acetylation step and followed by gas chromatography mass spectrometry (GC-MS) to achieve limits of quantification (LOQs) comprised between 0.6 and 5 ngmL(-1), linear responses up to 5000 ngmL(-1), excellent accuracy (overall recoveries ranged from 90 to 102% for samples spiked at different levels from 50 to 1000 ngmL(-1)) and standard deviations below 12%. Sample intake and organic solvents consumption remained at 1 and 0.6 mL, respectively, with a total sample preparation (derivatization and concentration) time around 15 min. The trans-isomers of piceatannol and resveratrol were measured in wine samples at concentrations up to 0.5 and 2.6 µgmL(-1), respectively. In addition to their cis-forms, the dihydrogenated analogues of both antioxidants were detected in red wines. To the best of our knowledge, this is the first report describing the presence of dihydropiceatannol in wine. On the other hand, pterostilbene remained below the detection limits of the method in all the processed samples. In addition to the relative low levels of this stilbene existing in grapes, its lipophilic character appears to limit the transfer from grapes skins to wine.

Dispersive liquid-liquid microextraction with non-halogenated extractants for trihalomethanes determination in tap and swimming pool water.

An effective, low solvent consumption, reduced cost and high throughput sample preparation method for the determination of four trihalomethanes (THMs) in tap and swimming pool water samples is presented. THMs extraction and concentration were performed by dispersive liquid-liquid microextraction (DLLME), based on the use of non-halogenated and low volatile solvents as extractants. Analytes were further determined by gas chromatography with micro-electron capture detection (GC-microECD). Under optimized conditions, the proposed method uses 18 mL volume samples, 0.7 mL of acetone (dispersant) and 0.05 mL of 1-undecanol (extractant). Achieved enrichment factors (EFs) varied from 67 to 104 times, the limits of quantification (LOQs) stayed between 0.05 and 1.3 ng mL(-1), and an excellent linearity was noticed up to 100 ng mL(-1). Relative recoveries, measured for spiked aliquots of tap and swimming pool water samples, remained between 79% and 113%, with associated standard deviations below 12%. The applicability of the developed methodology was demonstrated with chlorinated water samples analysis. As regards tap water samples, the sum of THMs concentrations remained under the limit fixed by the European Union (100 ng mL(-1)); however, some samples contained levels close to 80 ng mL(-1), the maximum allowable concentration established by the United States Environmental Protection Agency (EPA).

Dispersive liquid-liquid microextraction using non-chlorinated, lighter than water solvents for gas chromatography-mass spectrometry determination of fungicides in wine.

A novel and low solvent consumption method for the sensitive determination of fungicide residues in wine samples is proposed. Analytes were extracted by dispersive liquid-liquid microextraction (DLLME) and further determined by gas chromatography-mass spectrometry (GC-MS). Under optimized conditions, a binary mixture of acetone and 1-undecanol (0.5 and 0.05 mL, respectively) was used to extract target compounds from diluted (1:1) wine samples. After centrifugation, the floating phase of 1-undecanol was solidified and separated from the liquid hydro-alcoholic matrix. Thereafter, it was allowed to melt at room temperature and injected in the GC-MS system. The method showed relative standard deviations (RSDs, %) below 13%, limits of quantification (LOQs) between 0.2 and 3.2 ng mL(-1) and linear responses for concentrations up to 300 ng mL(-1). The efficiency of the liquid-phase microextraction process was scarcely affected by the characteristics of wine samples, consequently pseudo-external standard calibration (using matrix matched standards of red and white wine) sufficed to achieve acceptable accuracy values: relative recoveries from 81 to 120%. The applicability of the method was demonstrated with commercial wine samples.

Anemias nutricionales en el adolescente / Deficiency anemias in adolescents

Se revisan las recomendaciones para la prevención, diagnóstico y tratamiento de las anemias nutricionales en el adolescente, haciendo hincapié en las normas de alimentación; se define la población de adolescentes de mayor riesgo, y se exponen las pruebas biológicas para el diagnóstico de ferropenia y las deficiencias de folatos y vitamina B 12, el diagnóstico diferencial y el tratamiento con preparados orales y parenterales, así como sus indicaciones respectivas (AU)