Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Aplicação de filmes de suberina na produção de materiais bio-funcionais

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Dietary patterns in pregnancy and birth weight

OBJECTIVE To analyze if dietary patterns during the third gestational trimester are associated with birth weight.METHODS Longitudinal study conducted in the cities of Petropolis and Queimados, Rio de Janeiro (RJ), Southeastern Brazil, between 2007 and 2008. We analyzed data from the first and second follow-up wave of a prospective cohort. Food consumption of 1,298 pregnant women was assessed using a semi-quantitative questionnaire about food frequency. Dietary patterns were obtained by exploratory factor analysis, using the Varimax rotation method. We also applied the multivariate linear regression model to estimate the association between food consumption patterns and birth weight.RESULTS Four patterns of consumption – which explain 36.4% of the variability – were identified and divided as follows: (1) prudent pattern (milk, yogurt, cheese, fruit and fresh-fruit juice, cracker, and chicken/beef/fish/liver), which explained 14.9% of the consumption; (2) traditional pattern, consisting of beans, rice, vegetables, breads, butter/margarine and sugar, which explained 8.8% of the variation in consumption; (3) Western pattern (potato/cassava/yams, macaroni, flour/farofa/grits, pizza/hamburger/deep fried pastries, soft drinks/cool drinks and pork/sausages/egg), which accounts for 6.9% of the variance; and (4) snack pattern (sandwich cookie, salty snacks, chocolate, and chocolate drink mix), which explains 5.7% of the consumption variability. The snack dietary pattern was positively associated with birth weight (β = 56.64; p = 0.04) in pregnant adolescents.CONCLUSIONS For pregnant adolescents, the greater the adherence to snack pattern during pregnancy, the greater the baby’s birth weight.

Chlamydospore formation by Paracoccidioides brasiliensis mycelial form

To investigate the role of some adverse environmental conditions in chlamy-dospore formation by the mycelial form of P. brasiliensis, we cultured four P. brasiliensis isolates (18, Bt4, 1183, Pb9) at 25°C within solid agar medium either rich or poor in nutrients. Isolates 18 and 1183 were also cultured under anaerobiosis in a nitrogen atmosphere. Isolate 18 produced great number of terminal and intercalary chlamydospore after 7-10 days of culture in a medium poor in nutrients (2% agar with 0.1% dextrose and polypepton). The three other isolates also produced chlamydospores under the same conditions, but in lower numbers. Chlamydospore production by isolate 18 was abolished when the fungus was cultured in two agar media rich in nutrients (brain heart infusion and potato dextrose agar). Anaerobic incubation of isolate 18 under an atmosphere of N2 showed small mycelial outgrowth with numerous chlamydospores. At the electron microscopical level, the chlamydospores showed one or various nuclei and numerous mitochondria, indicating great potential for further development. Accordingly, chlamydospores produced multiple budding after only 24 h incubation at 35°C. The results demonstrate that under adverse environmental conditions P. brasiliensis mycelial form produces chlamydospores within a short period of time.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Vitamin A Deficiency and Training to Farmers: Evidence from a Field Experiment in Mozambique

Vitamin A deficiency is a widespread public health problem in Sub-Saharan Africa. This paper analyzes the impact of a food-based intervention to fight vitamin A deficiency using orange-fleshed sweet potato (OFSP). We conducted a randomized evaluation of OFSP-related training to female farmers in Mozambique, in which the treatment group was taught basic concepts of nutrition, and OFSP-planting and cooking skills. We found encouraging evidence of changes in behavior and attitudes towards OFSP consumption and planting, and considerable increases in nutrition-related knowledge, as well as knowledge on cooking and planting OFSP.

Vitamin A deficiency and training to farmers: Evidence from a field experiment in Mozambique

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Economics from the NOVA – School of Business and Economics

Eco-epidemiologic study of emerging fungi related to the work of babaçu coconut breakers in the State of Maranhão, Brazil

Introduction: There are more than 300,000 extractors using the babaçu coconut as a source of income in the States of Maranhão, Pará, Tocantins and Piauí, and this activity is associated with fungal infections. The objective of this study was to examine the occurrence of emergent fungi in the conjunctiva, nails and surface and subcutaneous injuries of female coconut breakers in Esperantinópolis, Maranhão. Additionally, soil samples and palm structures were collected. Methods: The obtained samples were cultured in Petri dishes containing potato-dextrose-agar and chloramphenicol. The etiological agent was confirmed by a direct mycological exam and growth in culture. Results: In total, 150 domiciles were visited, and samples were collected from 80 patients. From the ground, the most frequently isolated fungus was Aspergillus niger (53. 8%). the most frequently detected fungus in babaçu coconut was Aspergillus niger (66.7%). Conjunctival fungal growth occurred in 76.3% of the women. The ocular fungal microbiota consisted of filamentous fungi (80.6%), and yeasts were present in 19.4% of cases. Onychomycosis was diagnosed in 44% (11/25) of the women. Conclusions: The identification of the genera Neosartorya, Rhizopus and Curvularia in onychomycoses shows that emergent filamentous fungi can be isolated. Aspergillus sp., Penicillium sp. and Scedosporium sp. were the predominant genera found in the babaçu coconut. From ocular conjunctiva, Candida spp. were the most prevalent species isolated, and Fusarium sp. was present only in one woman. The nearly permanent exposure of coconut breakers to the external environment and to the soil is most likely the reason for the existence of a mycotic flora and fungal infections, varying according to the individual's practices and occupation.

Characterization of a foodborne outbreak caused by Salmonella Enteritidis in Aracaju, State of Sergipe, Brazil

INTRODUCTION: In December 2001, an outbreak of foodborne gastroenteritis infected 114 of 161 people who ate at a restaurant in Aracaju, State of Sergipe, Brazil. METHODS: The epidemiological and microbiological aspects of the outbreak were characterized. RESULTS: Potato salad made with homemade mayonnaise and stored at unsuitable temperatures was associated with increased risk of foodborne infection. Salmonella Enteritidis was isolated from the diarrheal stools of the hospitalized patients, and genotyping of the fecal samples generated identical randomly amplified polymorphic deoxyribonucleic acid (DNA) profiles. CONCLUSIONS : To the best of our knowledge, this is the first and the only record of a gastrointestinal outbreak in Sergipe.

Aspergilli and Penicillia related to the surface of ripening italian grana type cheese and its ripening environment

Information available on the mycoflora associated to ripening Italian “grana type” cheese is very poor. Recently, ochratoxin A (OTA) was detected in samples of packed grated cheese [1]; therefore, the need of information to perform a risk management was highlighted. Moreover, sterigmatocystin (STC) has been reported in cheese and it is considered an emerging problem. Despite the fact that both of them are mycotoxins included in group 2B by IARC [2,3], no European regulation exists. So, the main goal of this work is to give for the first time a general overview about Penicillia and Aspergilli growing on the surface of ripening “grana type” cheese, with particular attention on mycotoxigenic species. To perform this, in 2013 and 2014 crust samples were scratched from ripening grana cheese wheels and also Potato Dextrose Agar plates were exposed to monitor ripening house air. Then, 140 fungal isolates were randomly chosen, purified and monosporic colonies were obtained for their identification at specie level. A polyphasic approach is followed, based on morphological characterisation, toxic extrolites profiling and gene sequencing. The identification is still in progress, but the first results based on the morphological approach showed the presence of mycotoxigenic Aspergilli (Aspergillus flavus and A. versicolor) and various Penicillium species; among them Penicillium chrysogenum, P. implicatum and P. solitum were identified. Only P. chrysogenum was reported to produce the mycotoxins cyclopiazonic acid (CPA) and roquefortine-C (ROQ-C) [4]. These results will be presented and discussed. [1] A. Biancardi, R. Piro, G. Galaverna, C. Dall’Asta, "A simple and reliable liquid chromatography–tandem mass spectrometry method for determination of ochratoxin A in hard cheese" International Journal of Food Sciences and Nutrition 64 (5), 2013, 632 – 640. [2] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of Carcinogenic Risks to Humans” 31, 1983, 191 – 199. [3] International Agency for Research on Cancer (IARC) “IARC Monographs on the Evaluation of carcinogenic Risks to Humans”, suppl. 7, 1987, 72. [4] J. I. Pitt, D. A. Hocking, “Fungi and Food Spoilage” 1997, 291.

Daldinia eschscholtzii (Ascomycota, Xylariaceae) isolated from the Brazilian Amazon: taxonomic features and mycelial growth conditions

The Amazon has a high diversity of fungi, including species of the genus Daldinia (Ascomycota, Xylariaceae), which produce secondary metabolites with recognized nematicidal and antimicrobial activity. The ecological role of Daldinia is important, as stromata serve as refuges to many insects and arthropodes, and the fungi contribute to the degradation of vegetable organic matter. The aim of this study was to analyze the taxonomic features and mycelial growth conditions in vitro of a Daldinia specimen collected in the Brazilian Amazon. Morphological and molecular studies of the fungus identified it as D. eschscholtzii. To evaluate mycelial growth, we cultivated the fungus at 20, 25, 30, 35, and 40 °C in malt extract-peptone agar (MEPA), malt extract-peptone (MEP), potato dextrose (PD), and minimum medium (MM). The best mycelial growth occurred at 35 °C, although the greatest amount of biomass was obtained at 25 °C and 30 °C. PD proved to be the best medium for biomass production.

Água e alimentação no contexto das alterações climáticas: um caso de estudo - Região Norte

Dissertação de mestrado integrado em Engenharia Civil

Food consumption of adolescents. A simplified questionnaire for evaluating cardiovascular risk

OBJECTIVE: To develop a simplified questionnaire for self-evaluation by adolescents of foods associated with the risk of coronary diseases. METHODS: Frequency questionnaires about 80 foods were answered by representative samples of 256 adolescents aged 12 to 19 from Rio de Janeiro as part of the Nutrition and Health Research project. The dependent variable was the serum cholesterol predicting equation as influenced by diet, and the independent variables were the foods. The variables were normalized and, using Pearson's correlation coefficient, those with r>0.10 were selected for the regression model. The model was analyzed for sex, age, random sample, and total calories. Those food products that explained 85% of the cholesterol variation equation were present in the caloric model, and contained trans fatty acids were selected for the questionnaire. RESULTS: Sixty-five food products had a statistically significant correlation (P<0.001) with the dependent variable. The simplified questionnaire included 9 food products present in all tested models: steak or broiled meat, hamburger, full-fat cheese, French fries or potato chips, whole milk, pies or cakes, cookies, sausages, butter or margarine. The limit of the added food points for self-evaluation was 100, and over 120 points was considered excessive. CONCLUSION: The scores given to the food products and the criteria for the evaluation of the consumption limits enabled the adolescents to get to know and to balance their intake.

ETIOLOGÍA E IMPORTANCIA ECONÓMICA DE UNA NUEVA ENFERMEDAD VIRAL DE BATATA EN LA PROVINCIA DE CÓRDOBA

La batata se ubica en el séptimo lugar como cultivo destinado a la alimentación humana, y en el quinto luego de arroz, trigo, maíz y mandioca. Globalmente, existen 8 millones de hectáreas plantadas con batata y, aproximadamente el 95% de esa superficie se ubica en más de un centenar de países en desarrollo. En Argentina, la región pampeana (Buenos Aires, Córdoba y Santa Fe) y el NEA representan el 83% de la superficie plantada. Córdoba y Buenos Aires constituyen las principales provincias productoras. A pesar de su importancia potencial en la alimentación humana y animal, como producto exportable y para industrialización, se viene registrando una marcada reducción en el área cultivada con esta hortícola y, entre las causas más relevantes que determinan este fenómeno, se encuentran las enfermedades virales. Históricamente estas patologías han sido la principal limitante en la producción de este cultivo en Argentina y, especialmente en Córdoba. Recientemente y, tras brindar solución al grave problema ocasionado por el “enanismo clorótico” (Sweet potato chlorotic dwarf disease), virosis que afectó al cv Morada INTA en la década del 90, se observó, en nuestra provincia, la aparición de una severa sintomatología viral en lotes de producción implantados con el cv Arapey INIA, genotipo de creciente difusión en el cultivo por sus buenas características agronómicas. En virtud de dicha sintomatología, se sugiere que en la nueva patología viral se halla involucrado más de un agente etiológico y que la misma produce daños económicos en la producción de Arapey INIA. Por otra parte, la identificación de el/los virus presentes en la nueva patología es el primer eslabón para la búsqueda de resistencia a los mismos. Se supone, además, que, en germoplasma selecto de batata existen fuentes de resistencia a el/los virus involucrados y, que, al menos uno de los agentes patógenos de esta virosis de Arapey INIA, es transmitido por moscas blancas. Se propone, como paso inicial para el control de la nueva etiología: caracterizar biológica, serológica y molecularmente a el/los virus involucrados en ella; preparar reactivos de diagnóstico para los mismos y evaluar la gravedad de esta virosis a través de la estimación de su incidencia, prevalencia y severidad y de los daños que provoca sobre los componentes de rendimiento, en zonas productoras de la provincia de Córdoba. Por otra parte y, debido a que una de las principales formas de control de estas enfermedades es a través del empleo de germoplasma resistente y, considerando que la mayoría de los cultivares comerciales de batata, incluído Arapey INIA poseen escasa variabilidad genética por ser monoclonales, se pretende explorar molecularmente para genes de resistencia en aproximadamente 30 genotipos (clones) promisorios procedentes de la EEA INTA San Pedro ( Bs.As.), empleados como parentales en policruzamientos, además de hacerlo en el genotipo bajo estudio (Arapey INIA).