1,321 research outputs found

    DoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatography

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    P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O phospho-L-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chro matographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-en coding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was ap plied. The dynamic binding capacity of the O-phospho-L-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/ mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of en dotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agenciesinfo:eu-repo/semantics/publishedVersio

    Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

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    Funding: This work was supported by FEDER funds through the POCI—COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi /00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.Acknowledgments: The authors would like to thank Thomas Roberts for providing the pcDNA3–FLAG–p53 construct through Addgene, ref: 10838.The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.info:eu-repo/semantics/publishedVersio

    Actividade antioxidante de azeitonas verdes descaroçadas produzidas em Trás-os-Montes: estudo do efeito da cultivar.

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    A azeitona de mesa e parte integrante da dieta Mediterrânica, sendo considerada uma fonte importante de antioxidantes. Em Trás-os-Montes, no inicio do Outono, são processadas de forma artesanal um tipo de azeitona de mesa denominada "alcaparras". Para 0 seu processamento, os frutos são colhidos verdes, partidos, retirado o caroço e introduzidos em agua, mudada diariamente, durante aproximadamente uma semana ate que possam ser consumidos. De uma maneira geral, o factor cultivar não e tido em conta no processamento, apesar de apresentarem composição química e propriedades tecnológicas distintas. Assim, com o presente trabalho pretendeu-se estudar 0 efeito da cultivar na actividade antioxidante das "alcaparras". Neste sentido, foram processados lotes de azeitonas verdes descaroçadas monovarietais, à escala laboratorial das cultivares Cobrançosa, Madura!, Negrinha de Freixo, Santulhana e Verdeal Transmontana

    Characterization of several olive (Oleae europaea L.) cultivars based on fatty acids, triacilglycerol and tocopherol compositions.

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    Olive oil is the principal lipid source of the Mediterranean countries being largely consumed in Portugal. In the last years, the olive oil sector showed a great dynamics and olive oils with Protected Designation of Origin (PDO) and varietal olive oils (olive oils from just one cultivar) are introduced in the market In this context, this work intends to contribute for a better knowledge of the most representative cultivars of Olea europaea L grown in Tnis-os-Montes and Beira Interior, regions with importance in the production of these products. 34 samples of olives belonging to 14 olive cultivars were analyzed

    Estudo preliminar sobre as formigas (Hymenoptera: Formicidae) associadas ao olival da Terra Quente Transmontana (Nordeste de Portugal).

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    Com o presente estudo pretendeu-se contribuir para identificar as principais espécies de formigas associadas ao olival da Terra Quente Transmontana (Nordeste de Portugal) e por outro lado, conhecer diferentes aspectos do seu comportamento, como os que se referem à época de maior ocorrência das diferentes espécies, ao período do dia em que apresentam maior actividade nas árvores e à sua importância na protecção contra espécies nocivas. As observações decorreram entre Abril e Junho de 1999, num olival de cerca de 50 anos de idade, isento de tratamentos fitossanitários há vários anos e conduzido em regime de sequeiro. Com periodicidade semanal seleccionaram-se ao acaso três árvores, cujo tronco se observou durante 10 minutos em quatro períodos distintos do dia - 9, 11, 15 e 17 horas -, para recolha de todas as formigas presentes. Estas identificaram-se como pertencentes a dez espécies: Crematogaster scutellaris Oliv., Tapinoma nigerrimwn Nyl., Tetramorium semilaeve André, Cataglyphis hispânica Emery, C. ibérica Emery, Camponotus piceus Leach, C. lateralis Oliv., C.foreli Emery, Leptothorax angustulus Nyl. e Plagiolepis pygmaea (Latr.). T. nigerrimum foi a espécie mais abundante, quer no conjunto dos exemplares identificados, com 89,4% destes exemplares, quer em cada uma das datas de amostragem e períodos do dia. A maior actividade registou-se nas amostragens correspondentes às 9 e 17 horas, quando se obtiveram 30,7% e 30,2% do total de exemplares. Nove dos indivíduos recolhidos transportavam presas visíveis, que eram, em seis casos, lagartas de Prays oleae (Bern.), num caso um adulto de Euphyllura olivina Costa e noutro, uma ninfa de Psocoptera e um indivíduo não identificado. Das formigas que transportavam presas, três pertenciam à espécie T. nigerrimum, três a C. piceus, duas a C. hispânica e uma a C. scutellaris

    Spiders actively choose and feed on nutritious non-prey food resources

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    Spiders are generalist predators adapted to consume a wide range of prey although their ability to exploit non-prey foods such as pollen, nectar, and honeydew has been referred but less studied. In this work, we investigated the effect of different non-prey food items (Glucose at 0.5 M; aphid honeydew; black scale honeydew; a mixture of glucose 0.5 M, phenylalanine 0.1 mM, proline 0.1 mM, and tryptophan 0.1 mM; honey at 10%, and pollen at 10%) on the survival of immature spiders of two functional groups represented by Haplodrassus rufipes (ground hunters) and Synema globosum (ambushers), and their feeding choices, in laboratory experiments. The overall survival of both species fed on non-prey foods significantly increased compared to individuals fed on water. The black-scale honeydew was the best food for H. rufipes increasing longevity up to 117 days. The highest survival reached by S. globosum was observed when fed on a mixture of glucose 0.5 M and three amino acids. When different non-prey food items were offered together, the exploring rate was significantly higher for H. rufipes than for S. globosum. H. rufipes chose to feed on honey whereas S. globosum chose the mixture treatment. The most chosen food items corresponded with those that provided the highest longevities in both species. Our results suggest that spiders could search, recognize and actively select the most beneficial non-prey food. Habitat management practices such as maintaining weed strips in the crop may provide these valuable supplementary food resources within agroecosystems contributing for biological pest control.info:eu-repo/semantics/publishedVersio

    EcoPred: an educational individual based model to explain biological control, a case study within an arable land

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    Individual based models (IBMs) are up-to-date tools both in research and educational areas. Here we introduce an IBM built on NetLogo platform that simulates a top-down trophic cascade controlled by the pressure exerted by two model predators (web-building spiders and ground runner spiders) on a model pest (the olive fruit fly) within a hypothetical agricultural landscape (the olive crop). EcoPred is an IBM that aims to be an educational tool that can help teachers to explain concepts related to ecology in a modern, enjoyable and comprehensive way. EcoPred reflects the changes on a fly population within a simulated olive crop according to (1) the mortality rate caused by the predation of two spider species and energy loss, (2) the energy gain by feeding on flowers and (3) the reproduction rate in olive trees. The model was tested with 26 students achieving very good results in terms of acceptance and interest on the learning method. EcoPred can be used for educational purposes with 16 year old students and older to explain ecological concepts such as trophic level, species interactions, limiting factor and biological control in an interactive way simultaneously introducing students to biology oriented programming languages.This work was supported by the Portuguese Foundation of Science and Technology [PTDC/AGR-PRO/111123/200, SFRH/BD/97248/2013; Centro 2020 [Centro-01-0145-FEDER-000007].info:eu-repo/semantics/publishedVersio

    Functional responses of three guilds of spiders: Comparing single- and multiprey approaches

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    Spiders are successful natural enemies of pests occurring throughout the different strata of an agroecosystem. The study of their functional responses can provide information related to the potential effectiveness of different species and guilds on reducing a pest population. However, multiple prey availability may change the functional response of a predator. In this study, the functional responses of three species of spiders in single-prey and multiple-prey experiments were modelled. The spider species Haplodrassus rufipes, Araniella cucurbitina and Synema globosum were chosen as being representatives of ground runners, orb-weavers and ambushers, respectively. Three prey species were selected: a target prey, the Mediterranean fruit fly Ceratitis capitata and two alternate prey species, the flour moth Ephestia kuehniella and the house cricket Acheta domesticus. When the selected target prey C. capitata was supplied in the single-prey experiments, the most and the least efficient spider species were H. rufipes and S. globosum, respectively. However, opposite results were obtained when alternative preys were supplied. Also, A. cucurbitina significantly changed its functional response with the presence of alternative prey species. Considering the prey preference during the multiple-prey experiments, A. domesticus, used as representative of a heavy, long-sized and highly motile prey, was avoided whereas C. capitata, the target pest used as representative of light, small-sized and moderately motile prey was preferred by the three species of spiders. Ephestia kuehniella, used as representative of light, medium-sized and low motile prey was occasionally consumed. Each guild could include efficient predators against pests according to its hunting strategies and the ecological exploited niches. Orb-weavers could be efficient predators against flying pests; ambushers such as S. globosum could contribute to the reduction of the populations of flower-visiting pests, whereas active ground hunters may also play an important role preying on pests that develop a part or all of its life cycle in the ground. However, further research on feeding behaviour such as prey switching is needed for a better understanding of the effectiveness of spiders as natural enemies.This work was funded by Portuguese Foundation for Science and Technology (FCT) through the project EXCL/AGR-PRO/0591/2012 “Olive crop protection in sustainable production under global climatic changes: linking ecological infrastructures to ecosystem functions” and by the project ReNATURE – Valorization of the Natural Endogenous Resources of the Centro Region” funded by Centro 2020 (Centro-01-0145-FEDER-000007). Jacinto Benhadi-Marín is grateful to the Portuguese Foundation of Science and Technology for financial support through the Ph.D. grant SFRH/BD/97248/2013. This manuscript is part of Jacinto Benhadi-Marín's Ph.D. thesis.info:eu-repo/semantics/publishedVersio

    Distribution of the spider community in the olive grove agroecosystem (Portugal): potential bioindicators

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    Spiders are successful natural enemies in different crops. Research on the role of spiders as natural enemies within agroecosystems needs to address the distribution of their communities across different spatial scales, as well as the dominant species. We studied the spatial distribution of the community of spiders of the olive agroecosystem in Northeastern Portugal and investigated potential species that could be used as bioindicators of agricultural management in the olive grove. We found nine functional groups (ambushers, foliage runner hunters, ground hunters, orb-web builders, sheet web builders, sensing web-builders, space web builders, stalkers and wandering sheet/tangle weavers) encompassing a community that changed significantly across the horizontal and vertical gradient. We propose Thanatus vulgaris Simon, 1870 as potential bioindicator for the ground of the olive grove central area and Ozyptila pauxilla (Simon, 1870) for the ground of the olive grove peripheral area. Adjacent shrubland areas could play an important role in biological control of pests, allowing the exchange of species and individuals with the olive crop. The role of agrobiont species as indicators of agricultural managements deserves further investigation towards the enhancement of the effectiveness of spiders within low-impact crop management in arable landscapes.Thisworkwas funded byCentro 2020 (Centro-01-0145-FEDER- 000007) through the project ‘ReNATURE – Valorization of the Natural Endogenous Resources of the Centro Region’. The authors are indebted to all of the farmers who allowed access to their olive groves. Jacinto Benhadi-Marín is grateful to the Portuguese Foundation of Science and Technology (FCT) for financial support through the PhD grant SFRH/BD/97248/2013. This manuscript is part of Jacinto Benhadi-Marín’s PhD thesis. The authors declare that they have no conflicts of interest.info:eu-repo/semantics/publishedVersio
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