RESUMEN

Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.

Asunto(s)

RESUMEN

The treatment of acute lymphoblastic leukemia is challenging due to side effects, efficacy of the available drugs, and costs. Utilization of L-asparaginase as a therapeutic agent is essential to increase survival of patients. However, costs are elevated and the bacterial forms of the enzyme cause reactions that result in its inhibition by the immune system. Therapeutics alternatives may be searched among eukaryote producers, like fungi. Twelve strains of filamentous fungi were evaluated regarding expression of L-asparaginase activity. The profile of nitrogen assimilation and radial growth were determined for strains which showed higher production ratios. Three media were formulated after selection of the carbon source and carbon/nitrogen ratio that better induced L-asparaginase expression by Penicillium sp. and Fusarium sp. The enzyme activity produced in liquid media reached 8.3 U min.-1 mL-1 (Penicillium sp. T6.2) and 11.4 U min.-1 mL-1 (Fusarium sp.) after 72 hours of cultivation in Bacelar-1 medium. These data show that good producers can be found among fungi, and adjustment of productive processes may offer an alternative to implement eukaryote L-asparaginase production.(AU)

O tratamento da leucemia linfoblástica aguda é desafiador devido aos efeitos adversos, à eficácia dos fármacos disponíveis e aos custos envolvidos. A utilização de L-asparaginase como agente terapêutico é fundamental para aumentar a sobrevida do paciente. Porém, seu custo é elevado e as formas bacterianas da enzima causam reações que resultam na sua inibição pelo sistema imune. Alternativas terapêuticas podem ser buscadas entre produtores eucariontes, como os fungos. Doze linhagens de fungos filamentosos foram avaliadas quanto à expressão de atividade de L-asparaginase. O perfil de assimilação de nitrogênio e o crescimento radial foram determinados para as linhagens com maior razão de produção. Três meios foram formulados após a seleção da fonte de carbono e da razão carbono/nitrogênio que melhor induziram a expressão de L-asparaginase por Penicillium sp. e Fusarium sp. A atividade enzimática produzida em meio líquido alcançou 8,32 U min.-1 mL-1 (Penicillium sp. T6.2) e 11,45 U min.-1 mL-1 (Fusarium sp.) após 72 horas de cultivo no meio Bacelar-1. Esses dados mostram que bons produtores podem ser encontrados entre os fungos e que ajustes nos processos produtivos podem oferecer uma alternativa para a implementação da produção de L-asparaginase eucarionte.(AU)

Asunto(s)

RESUMEN

Arthrospira (Spirulina) platensis, cianobactéria fotoautotrófica, é importante comercialmente devido ao seu valor nutricional (elevado teor de proteínas, lipídios, vitaminas e minerais), além da presença do ácido graxo γ-linolênico e pigmentos, os quais agregam valor a esta biomassa. Este micro-organismo foi cultivado em fotobiorreator tubular, e os seguintes parâmetros foram avaliados: aplicação de diferentes sistemas de circulação de células, intensidades luminosas, adição simultânea de diferentes proporções de sulfato de amônio e nitrato de sódio como fontes de nitrogênio, adição de dióxido de carbono proveniente de fermentação alcoólica e, finalmente, a aplicação desta biomassa para remoção de metais pesados. Para a avaliação do sistema de circulação de células e da intensidade luminosa foram utilizadas as variáveis dependentes: concentração celular máxima (Xm), produtividade em células (Px), fator de conversão de nitrogênio em células (YX/N), eficiência fotossintética (EF) e teores de proteínas e lipídios da biomassa. Os protocolos de alimentação de nitrogênio e o uso de CO2 proveniente de cilindro ou de fermentação alcoólica foram avaliados com uso das mesmas variáveis já citadas, exceto EF; e, para remoção de Ni2+, Zn2+ e Pb2+ em solução aquosa foram estudados o tempo de adsorção e a influência da concentração inicial de metal em solução; com posterior cálculo da eficiência e capacidade de adsorção, e apresentação de modelos cinéticos e isotermas de equilíbrio. A melhor combinação de resultados em valores médios (Xm = 4055 mg L-1, Px = 406 mg L-1 d-1, YX/N = 5,07 mg mg-1, lipídios totais = 8,94%, proteínas totais = 30,3%, EF = 2,04%) foi obtida nos cultivos realizados com intensidade luminosa = 120 µ mol fótons m-2 s-1. O tipo de sistema de circulação de células utilizado não influenciou estatisticamente quase nenhuma das respostas estudadas e isto indica a possibilidade de substituição do sistema tradicional airlift pelos outros testados...

Arthrospira (Spirulina) platensis, photoautotrophic cyanobacterium, is commercially important due its high nutritional value (high content of proteins, lipids, vitamins, and minerals), besides of the presence of γ-linolenic fatty acids and pigments, which add value to this biomass. This microorganism was cultivated in tubular photobioreactor and the following parameters were evaluated: application of different cell circulation systems, light intensities, different protocols of simultaneous ammonium sulfate and sodium nitrate feeding, addition of CO2 from ethanol fermentation and, finally, the application of this biomass to the heavy metal removal. The dependent variables [maximum cell concentration (Xm), cell productivity (Px), nitrogen-to-cell conversion factor (YX/N), photosynthetic efficiency (EF) and proteins and lipids contents] were used to evaluate the influence of cell circulation systems and the light intensities. The nitrogen feeding protocols and the use of CO2 from cylinder or ethanol fermentation were evaluated using the same parameters aforementioned, except EF; and to analyze Ni2+, Zn2+ and Pb2+ removal, the adsorption time and the influence of initial metal concentration were evaluated with subsequent calculation of the efficiency and capacity adsorption. The kinetic models and equilibrium isotherms were also presented. The best combination of responses' mean values (Xm = 4055 mg L-1, Px = 406 mg L-1 d-1, YX/N = 5.07 mg mg-1, total lipids = 8.94%, total proteins = 30.3%, EF = 2.04%) was obtained at light intensity = 120 µ mol photons m-2 s-1. The cell circulation system did not exert statistical significant influence on almost all the responses, which suggests that the traditional airlift system could successfully be substituted by the others tested in this work. The mixture of the lower amount of nitrate in relation to ammonium (protocol I) (Xm = 4,543 g L-1; Px = 0,460 g L-1 d-1; YX/N = 15,6 g g-1; total lipids = 8,39% and total...

Asunto(s)

RESUMEN

A renina é um complexo natural de enzimas (endopeptidases aspárticas) utilizada na produção de queijos, extraída principalmente de estômagos de bezerros jovens. A maior demanda por queijos e o alto custo da renina têm incentivado a procura de fontes alternativas de proteases. Desse modo, o objetivo deste trabalho foi verificar as condições de tempo e temperatura para a produção de biomassa, exopolissacarídeos (EPS) e proteases por Pleurotus ostreatus em cultivo líquido. O micélio foi crescido em meio Pontecorvo, enriquecido com proteína isolada a 22, 25 ou 28oC, com amostragens aos 3, 6, 9, 14 e 17 dias de cultivo. A biomassa foi separada por centrifugação; EPS foram determinados por precipitação com etanol e a atividade proteolítica determinada pela ação do extrato enzimático sobre caseína solúvel alcalina 1% (m/v). Verificou-se que tanto o tempo quanto à temperatura afetaram (p?0,05) a produção de biomassa, EPS e proteases. Concluiu-se que a maior produção de biomassa de P. ostreatus ocorre após nove dias de cultivo a 25ºC; a produção de EPS aumenta com a ampliação do tempo de cultivo, com maior produção a 22ºC; e a máxima produção de proteases ocorre aos 14 dias de cultivo a 28ºC.(AU)

Rennet is a natural complex of enzymes (aspartic endopeptidases) and is often used in the production of cheese. Those enzymes (proteases) are extracted from abomasums of young ruminant animals. The high cheese demand associated with the high curdle cost have been stimulated the searching for alternative protease sources. The objective of this study was to verify the time and temperature conditions for biomass, exopolysaccharide (EPS) and protease production by Pleurotus ostreatus in liquid cultivation. The mycelium was grown in Pontecorvo medium added with isolated soy protein (5%) and maintained at 22, 25 or 28oC, with samplings at 3, 6, 9, 14 or 17 days of cultivation. The biomass was separated by centrifugation, EPS were separated by ethanol precipitation and the proteolytic activity was determined by enzymatic extract action on alkaline soluble casein 1% (m/v). It was verified that time and temperature affect (p?0.05) biomass, EPS and protease production. Most of P. ostreatus biomass production occurs after nine days of cultivation at 25ºC. EPS production rise with increasing of cultivation time, with higher production at 22ºC. Higher protease activity occurs at 28ºC on 14 days of cultivation.(AU)

La renina es un complejo natural de enzimas (endopeptidasas aspárticas) utilizada en la producción de quesos, extraída principalmente de estómagos de becerros jóvenes. La mayor demanda por quesos y el alto costo de la renina ha incentivado la búsqueda de fuentes alternativas de proteasas. Así que, el objetivo de esta investigación fue verificar las condiciones de tiempo y temperatura para la producción de biomasa, exopolisacáridos (EPS) y proteasas por Pleurotus ostreatus en cultivo líquido. El micelio creció en medio a Ponte corvo, enriquecido con proteína aislada a 22, 25 o 28ºC, con muestras a los 3, 6, 9, 14 y 17 días de cultivo. Se separó la biomasa por centrifugación; EPS fueron determinados por precipitación con etanol y la actividad proteolítica determinada por la acción del estrato enzimático sobre caseína soluble alcalina 1% (m/v). Se verificó que tanto el tiempo como la temperatura afectaron (p?0,05) a la producción de biomasa, EPS y proteasas. Se concluye que la mayor producción de biomasa de P. ostreatus ocurre tras nueve días de cultivo a 25ºC; la producción de EPS aumenta con la ampliación del tiempo de cultivo, con mayor producción a 22ºC; y la máxima producción de proteasas ocurre a los 14 días de cultivo a 28ºC.(AU)

Asunto(s)

RESUMEN

Este estudo objetivou quantificar as perdas de sementes (jovens mexilhões) no cultivo e discutir as principais causas, especialmente as ocasionadas por predadores e parasitas. O experimento ocorreu na Praia da Ponta do Sambaqui, no período de dezembro de 2004 a agosto de 2005. Foram confeccionadas 54 cordas de mexilhões, contendo 600 sementes cada. Metade das cordas foi disposta em um espinhel de cultivo e protegidas com malha de traineira, sendo mensalmente retirada essa proteção de três delas. As outras 27 cordas permaneceram em um tanque-rede. Por mês, eram coletadas três cordas de cada tratamento e feitas as contagens. Foram fixadas amostras de 30 animais de cada tratamento, sendo realizados procedimentos clássicos de histologia, com coloração HE. A salinidade e temperatura da água do mar foram medidas, respectivamente, de forma semanal e diária. As sementes das cordas no espinhel sofreram intensa predação no verão. Nos meses seguintes não houve diferença significativa na perda de animais entre os 2 tratamentos. Os mexilhões Perna perna jovens são mais vulneráveis ao ataque dos predadores que indivíduos adultos. Além da predação, podem ser causas de perdas questões relacionadas às técnicas de manejo e excesso de sementes por corda. A prevalência do trematódeo Bucephalus sp. foi baixa (5,11%), não havendo relação direta com as perdas observadas/

This study had the objective of quantifying seed loss in the culture of young mussels as well as discussing the main causes of this loss, especially that derived from predators and parasites. The experiment was carried out in Ponta do Sambaqui beach, between December of 2004 and August of 2005. Fifty four mussel ropes were prepared, containing 600 seeds/m each. Half the ropes were disposed in a long line and protected with fishing net. This protection was removed from three of them every month. The other 27 ropes remained in a net-tank. Every month three ropes from each treatment were collected and the mussels counted. Thirty animals from each treatment were fixed and classical histology procedures were carried out, with stain HE. The salinity and the water temperature of the sea were measured weekly and diary, respectively. The seeds from the ropes of the long line suffered intense predation in the summer. In the following months there was no significant difference in the loss of animals between both treatments. The young Perna perna mussels are more vulnerable to predators than adult individuals. Besides predation, questions related with handling techniques and the excess of seeds in each rope can be causes for loss of animals. The prevalence of the trematod Bucephalus sp. was low (5.11%), there was no directly relation with the observed losses

Asunto(s)

RESUMEN

Este estudo objetivou quantificar as perdas de sementes (jovens mexilhões) no cultivo e discutir as principais causas, especialmente as ocasionadas por predadores e parasitas. O experimento ocorreu na Praia da Ponta do Sambaqui, no período de dezembro de 2004 a agosto de 2005. Foram confeccionadas 54 cordas de mexilhões, contendo 600 sementes cada. Metade das cordas foi disposta em um espinhel de cultivo e protegidas com malha de traineira, sendo mensalmente retirada essa proteção de três delas. As outras 27 cordas permaneceram em um tanque-rede. Por mês, eram coletadas três cordas de cada tratamento e feitas as contagens. Foram fixadas amostras de 30 animais de cada tratamento, sendo realizados procedimentos clássicos de histologia, com coloração HE. A salinidade e temperatura da água do mar foram medidas, respectivamente, de forma semanal e diária. As sementes das cordas no espinhel sofreram intensa predação no verão. Nos meses seguintes não houve diferença significativa na perda de animais entre os 2 tratamentos. Os mexilhões Perna perna jovens são mais vulneráveis ao ataque dos predadores que indivíduos adultos. Além da predação, podem ser causas de perdas questões relacionadas às técnicas de manejo e excesso de sementes por corda. A prevalência do trematódeo Bucephalus sp. foi baixa (5,11%), não havendo relação direta com as perdas observadas(AU)/

This study had the objective of quantifying seed loss in the culture of young mussels as well as discussing the main causes of this loss, especially that derived from predators and parasites. The experiment was carried out in Ponta do Sambaqui beach, between December of 2004 and August of 2005. Fifty four mussel ropes were prepared, containing 600 seeds/m each. Half the ropes were disposed in a long line and protected with fishing net. This protection was removed from three of them every month. The other 27 ropes remained in a net-tank. Every month three ropes from each treatment were collected and the mussels counted. Thirty animals from each treatment were fixed and classical histology procedures were carried out, with stain HE. The salinity and the water temperature of the sea were measured weekly and diary, respectively. The seeds from the ropes of the long line suffered intense predation in the summer. In the following months there was no significant difference in the loss of animals between both treatments. The young Perna perna mussels are more vulnerable to predators than adult individuals. Besides predation, questions related with handling techniques and the excess of seeds in each rope can be causes for loss of animals. The prevalence of the trematod Bucephalus sp. was low (5.11%), there was no directly relation with the observed losses (AU)

Asunto(s)

RESUMEN

We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1beta), transforming growth factor beta 1 (TGF-beta1), fibronectin, and prostaglandin E2 (PGE2) by pure fibroblasts (fourth passage). A fibroblast colony-forming units (CFU-F) assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta, TGF-beta1, and fibronectin in conditioned mediums (CM) of fibroblast cultures were measured by enzyme-linked immunosorbent assay (ELISA) kit and PGE(2) by radioimmunoassay (RIA) kit. Confluence was achieved in the 60% of LCP and 78% of BCP primary cultures compared with 100% of NC, and only fibroblasts from seven LCP and six BCP cultures had the capacity to proliferate following four subcultures. Levels of IL-1beta were below 10 pg/ml in both patient groups, while NC had a mean value of 5882.57+/-221.61 pg/ml. Levels of TGF-beta1 in BCP were lower than NC values ( P<0.05). LCP and BCP had significantly decreased levels of fibronectin when compared to NC values ( P<0.05 and P<0.01, respectively). Levels of PGE2 in LCP were higher compared to NC ( P<0.01). In conclusion, BM fibroblasts from LCP and BCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta, TGF-beta1, fibronectin, and PGE2 production.

Asunto(s)

RESUMEN

One hundred sixty eight bacterial strains, isolated from soil and samples of vegetable in decomposition, were screened for the use of citrus pectin as the sole carbon source. 102 were positive for pectinase depolymerization in assay plates as evidenced by clear hydrolization halos. Among them, 30(per cent) presented considerable pectinolytic activity. The cultivation of these strains by submerged and semi-solid fermentation for polygalacturose production indicated that five strains of "Bacillus" sp. produced high quantities of the enzyme. The physico-chemical characteristics, such as optimum pH of 6.0-7.0, optimum temperatures between 45ºC and 55ºC, stability at temperatures above 40ºC and in neutral and alkaline pH, were determined

Asunto(s)