Sensores y biosensores para evaluar la presencia de disruptores endócrinos

La disposición de sustancias nocivas al medio ambiente es un hecho cotidiano, pero, ¿cómo impactan estas sustancias en la salud de los seres vivos? Es difícil contestar esta pregunta, ya que es complejo conocer la real dimensión del riesgo que ellas generan al integrarse a diferentes ecosistemas. Dentro de estos compuestos encontramos aquellos que afectan al sistema endócrino, conocidos como “disruptores endócrinos”. Poder detectarlos y evaluar su concentración es un primer acercamiento al estudio del impacto del problema. Para ello es conveniente disponer de técnicas analíticas sensibles, sencillas, rápidas y de aplicación in situ, que permitan tener un mayor alcance y estudio de la situación en zonas de riesgo. El objetivo de esta Tesis fue el estudio y puesta a punto de diferentes técnicas analíticas para determinar in situ la presencia de dos disruptores endócrinos emergentes: el plomo y las nanopartículas de plata. Mediante análisis electroquímicos, espectroscopía UV-visible, dispersión de luz dinámica, laser, microscopía electrónica de transmisión y análisis de color, se estudiaron nanopartículas de plata (AgNPs) presentes en soluciones coloidales comerciales; mientras que se analizaron pinturas en paredes de hogares de familia y de edificios públicos buscando plomo, mediante un análisis colorimétrico in situ y posterior confirmación por análisis electroquímico y espectroscopia infrarroja por transformada de Fourier (FTIR). En el caso del plomo, se implementó una técnica electroquímica que puede ser fácilmente descentralizada, que permite la cuantificación de este elemento en pinturas. Esta técnica complementó un análisis colorimétrico que se implementó para su uso in situ como técnica de barrido previo.El uso de esta última técnica permitió proponer un protocolo general para la inspección de hogares en búsqueda de plomo que, en un ensayo exploratorio en dos barrios de Montevideo, resultó en un abatimiento muy importante de los costos. El estudio de las nanopartículas de plata en muestras comerciales mostró la necesidad de contar con una batería de técnicas para poder concluir acerca de la presencia y la concentración de las mismas. Se desarrolló una metodología electroquímica que permite discriminar iones plata de nanopartículas de plata, y se propone un protocolo analítico mayoritariamente descentralizable para la detección y cuantificación in situ de nanopartículas de plata. IX Las metodologías propuestas son una alternativa para el análisis de forma descentralizada, sencilla, económica y confiable, de dos disruptores endócrinos emergentes

Nanomolar CFTR Inhibition by Pore-Occluding Divalent Polyethylene Glycol-Malonic Acid Hydrazides

SummaryInhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2–100 kDa). IC50 values for CFTR inhibition were 10–15 μM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 μM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 ∼10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy

Intermolecular interactions in the TMEM16A dimer controlling channel activity

TMEM16A and TMEM16B are plasma membrane proteins with Ca2+ -dependent Cl- channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the "activating domain" to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl- transport

Caracterização de méis de duas paisagens da região nordeste do Uruguai

The world honey market values ​​organic and natural honeys. Forest activity in the northeast of Uruguay, free of agricultural activities and with the presence of a protected area, is an opportunity for local beekeepers and transhumant people. The characterization of these honeys through melissopalynology and physical-chemical parameters could generate new valorization strategies. The objective of this study was to characterize honeys (n=27) from the protected area and from afforestation with Eucalyptus grandis under two production systems (transhumance and non-transhumance beekeepers). Botanical composition was analyzed by palynology, mineral profile, moisture, conductivity, pH, color (CIEL*a*b*), sugar profile and presence of glyphosate. The palynological analysis determined two types of honeys (monofloral from Eucalyptus sp. in the forestation and honeydew honeys with the presence of diverse pollens from native species in the protected area. No significant differences (p<0.05) were found between the honeys from eucalyptus for both beekeeping production systems in all the parameters evaluated. The honeydew honeys have an unknown origin and were statistically different from the previous ones. The average values ​​of pH and conductivity for these honeys were 6.37 ± 0.14 and 1113 ± 25.6 µS/cm respectively. The concentration of minerals K (2536.1 ± 382.1 mg/kg honey) and Fe (4.15 ± 0.27 mg/kg honey) was higher than those found in eucalyptus honey. The percentages of Isomaltulose (1.18 ± 0.62) and Trehalose (0.23 ± 0.05) sugars were higher in the honeys from the protected area while Maltose (0.46 ± 0.07) was lower. Glyphosate residues were not detected in all the samples analyzed. Should insist on good management practices carried out by beekeepers when they arrive from agricultural areas (transhumance beekeepers). Research should continue to determine the origin of the myelates in the protected area. Although the volumes produced are smaller, their valuation could increase due to the originality of these honeys. The northeast region of Uruguay has the potential to produce quality honey properly identified by geographic and botanical origin.El mercado mundial de miel valora las mieles orgánicas y naturales. La actividad forestal en el noreste del Uruguay, libre de actividades agrícolas y con la presencia de un área protegida, es una oportunidad para los apicultores locales y trashumantes. La caracterización de estas mieles a través de la melisopalinología y los parámetros fisicoquímicos podría generar nuevas estrategias de valorización. El objetivo de este trabajo fue caracterizar mieles (n=27) provenientes del área protegida y de la forestación con Eucalyptus grandis bajo dos sistemas de producción (apicultores trashumantes y no trashumantes). Se analizó la composición botánica mediante palinología, perfil de minerales, humedad, conductividad, pH, color (CIEL*a*b*), perfil de azúcares y presencia de glifosato. El análisis palinológico determinó dos tipos de mieles (monofloral de Eucalyptus sp.) en la forestación y mieles de mielada con presencia de pólenes diversos de especies nativas en el área protegida. No se encontraron diferencias significativas (p<0,05) entre las mieles de eucalipto para ambos sistemas de producción apícola en todos los parámetros evaluados. Las mieles de mieladas tienen origen desconocido y fueron estadísticamente diferentes respecto a las anteriores. Los valores promedios de pH y conductividad para estas mieles fueron 6,37± 0,14 y 1113 ± 25,6 µS/cm, respectivamente. Los minerales K (2536,1 ± 382,1 mg/kg miel) y Fe (4,15 ± 0,27 mg/kg miel) fueron mayores respecto a los encontrados en las mieles de eucaliptos. Los porcentajes de azúcares Isomaltulosa (1,18 ± 0,62) y Trehalosa (0,23 ± 0,05) fueron mayores en las mieles del área protegida, mientras que Maltosa (0,46 ± 0,07) fue menor. No se detectaron residuos de glifosato en todas las muestras analizadas. Sin embargo, se debe insistir en las buenas prácticas de manejo que realizan los apicultores cuando llegan desde áreas agrícolas (apicultores trashumantes). Se deberá continuar investigando para determinar el origen de los mielatos del área protegida. Si bien los volúmenes producidos son menores, podría aumentar su valoración debido a la originalidad de estas mieles. La región noreste de Uruguay tiene potencial para producir mieles de calidad debidamente identificadas por origen geográfico y botánico.O mercado mundial de mel valoriza os méis orgânicos e naturais. A atividade florestal no nordeste do Uruguai, livre de atividades agrícolas e a presença de uma área protegida, é uma oportunidade para apicultores locais e pessoas transumantes. A caracterização destes méis através da melissopalinologia e parâmetros físico-químicos poderá gerar novas estratégias de valorização. O objetivo deste trabalho foi caracterizar os méis (n=27) da área protegida e da arborização com Eucalyptus grandis sob dois sistemas de produção (apicultores com transumância e não transumância). A composição botânica foi analisada por palinologia, perfil mineral, umidade, condutividade, pH, cor (CIEL*a*b*), perfil de açúcares e presença de glyphosate. A análise palinológica determinou dois tipos de méis (monofloral de Eucalyptus sp. na florestação e méis de melada com presença de diversos pólens de espécies nativas da área protegida. Não foram encontradas diferenças significativas (p<0,05) entre os méis de eucalipto para ambos os sistemas de produção apícola em todos os parâmetros avaliados. Os méis de melada têm origem desconhecida e foram estatisticamente diferentes dos anteriores. Os valores médios de pH e condutividade para esses méis foram 6,37 ± 0,14 e 1113 ± 25,6 µS/cm respectivamente Os minerais K (2536,1 ± 382,1 mg/kg de mel) e Fe (4,15 ± 0,27 mg/kg de mel) foram maiores que os encontrados no mel de eucalipto. Nos méis da área protegida enquanto Maltose (0,46 ± 0,07) foi menor, resíduos de glifosato foram detectados em todas as amostras analisadas. Devem insistir nas boas práticas de gestão realizadas pelos apicultores quando chegam das zonas agrícolas (apicultores de transumância). A pesquisa deve continuar para determinar a origem dos mielatos na área protegida. Embora os volumes produzidos sejam menores, sua valorização pode aumentar devido à originalidade desses méis. A região nordeste do Uruguai tem potencial para produzir mel de qualidade devidamente identificado por origem geográfica e botânica

Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR

Ex vivo model predicted in vivo efficacy of CFTR modulator therapy in a child with rare genotype

Background: New drugs that target the basic defect in cystic fibrosis (CF) patients may now be used in a large number of patients carrying responsive mutations. Nevertheless, further research is needed to extend the benefit of these treatments to patients with rare mutations that are still uncharacterized in vitro and that are not included in clinical trials. For this purpose, ex vivo models are necessary to preliminary assessing the effect of CFTR modulators in these cases. Method: We report the clinical effectiveness of lumacaftor/ivacaftor therapy prescribed to a CF child with a rare genetic profile (p.Phe508del/p.Gly970Asp) after testing the drug on nasal epithelial cells. We observed a significant drop of the sweat chloride value, as of the lung clearance index. A longer follow-up period is needed to define the effects of therapy on pancreatic status, although an increase of the fecal elastase values was found. Conclusion: Drug response obtained on nasal epithelial cells correlates with changes in vivo therapeutic endpoints and can be a predictor of clinical efficacy of novel drugs especially in pediatric patients

A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators

In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000–2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers (e.g. forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended

Caracterização de méis de diferentes regiões do Uruguai, marcadores de origem

Uruguay produces and exports honey. Honey is appreciated worldwide and has been well studied in terms of its chemical composition. These studies help determine botanical origin and prevent fraud. However, Uruguay exports honey without differentiating; the diversity of soils and vegetation in the country allows different types of honey production. Therefore, the aim of this work was to characterize honey from four regions of the country including three protected areas. The samples were collected during one year in two stations and electrical conductivity, humidity, sugar profile, macrominerals (K, Ca, Na and Mg) and pollen content were analyzed. The quality of the studied environments was evaluated by determining the presence of glyphosate. Results from this study confirm significant differences among the analyzed honeys from the different regions (Tukey-Kramer, p 0.8 mS / cm) associated with high mineral content were found. 37 different pollen taxa (family, genus or species) were detected. Some samples are monofloral (main pollen> 45%) of cultivated species (Lotus sp and Trifolium repens) or native species (Parkinsonia aculeata, Lithraea brasiliensis, Myrcianthes sp and Tripodanthus acutifolius). Calcium and sodium are suggested as markers of geographic origin. Mannose is suggested as a marker of botanical origin. The detection of glyphosate appears associated with agricultural activities even in protected areas. Uruguay produces different honeys that can be marketed indicating origin. More regions should be studied and for longer periods.Uruguay produce y exporta miel, alimento mundialmente apreciado y estudiado en términos de su composición química. Estos estudios ayudan a determinar el origen geográfico y botánico y a prevenir fraudes. Sin embargo, Uruguay exporta miel sin diferenciar. La diversidad de suelos y vegetación del país permite diferentes tipos de producción de miel. El objetivo de este trabajo fue caracterizar la miel de cuatro regiones del país, de las que tres son áreas protegidas. Las muestras se recolectaron durante un año en dos estaciones. Se analizó conductividad eléctrica, humedad, perfil de azúcar, macrominerales (K, Ca, Na y Mg) y contenido de polen. Se evaluó la calidad de los ambientes estudiados determinando la presencia de glifosato. Se encontraron diferencias entre las mieles analizadas (Tukey-Kramer, p 0.8 mS / cm) asociadas a altos contenidos minerales. Se detectaron 37 taxas de pólenes diferentes (familia, género o especie). Algunas muestras son monoflorales (polen principal> 45%) de especies cultivadas (Lotus sp y Trifolium repens) o especies nativas (Parkinsonia aculeata, Lithraea brasiliensis, Myrcianthes sp y Tripodanthus acutifolius). Se sugieren calcio y sodio como marcadores de origen geográfico. Se sugiere la manosa como marcador de origen botánico. La detección de glifosato aparece asociada con las actividades agrícolas incluso en áreas protegidas. Uruguay produce diferentes mieles que se pueden comercializar indicando origen, se deberían estudiar más regiones y durante más tiempo.O Uruguai produz e exporta mel. Um alimento apreciado em todo o mundo e estudado quanto à sua composição química. Esses estudos ajudam a determinar a origem geográfica e botânica e a prevenir fraudes. No entanto, o Uruguai exporta mel sem se diferenciar. A diversidade de solos e vegetação do país permite diferentes tipos de produção de mel. O objetivo deste trabalho foi caracterizar mel de quatro regiões do país. Três são áreas protegidas. As amostras foram coletadas durante um ano em duas estações. Condutividade elétrica, umidade, perfil de açúcar, macrominerais (K, Ca, Na e Mg) e conteúdo de pólen foram analisados. A qualidade dos ambientes estudados foi avaliada pela determinação da presença de glifosato. Foram encontradas diferenças entre os méis analisados ​​(Tukey-Kramer, p 0,8 mS / cm) associadas a alto teor de minerais foram encontradas. 37 taxa de pólen diferentes (família, gênero ou espécie) foram detectados. Algumas amostras são monoflorais (pólen principal> 45%) de espécies cultivadas (Lotus sp e Trifolium repens) ou espécies nativas, Parkinsonia aculeata, Lithraea brasiliensis, Myrcianthes sp e Tripodanthus acutifolius). O cálcio e o sódio são sugeridos como marcadores de origem geográfica. A manose é sugerida como um marcador de origem botânica. A detecção do glifosato aparece associada às atividades agrícolas mesmo em áreas protegidas. O Uruguai produz diferentes méis que podem ser comercializados com indicação de origem. Mais regiões devem ser estudadas e por mais tempo

Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin–induced intestinal fluid secretion

n/