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CELL-BASED BIOASSAYS FOR TESTING BIOACTIVE COMPOUNDS IN FARM ANIMALS
Full text linkCell-based assays can be adopted as in vitro method to evaluate the bioavailability and functionality of different nutraceutical and bioactive compounds, particularly in view of the need to use alternatives to animal studies. The interest in these bioactive compounds in animal sciences is not only related to medical research. It also represents an enormous benefit for health food companies and the animal produce sector in general.
The general aim of my PhD study was to study nutraceutical effects at a cellular level in response to different stress challenges.
In the first section of my thesis, the protective role of α-tocopherol in counteracting the cytotoxicity and DNA damage induced by Ochratoxin A (OTA) in primary porcine fibroblast cell cultures (ear and embryo), was determined by using the MTT assay, LDH release, DNA fragmentation, and TUNEL stain.
The aim of the second section was to evaluate the protective role of bovine Lactoferrin (bLf), added to the culture medium, against lipopolysaccharide (LPS) cytotoxicity using the established bovine mammary epithelial cell line BME-UV1 as an in vitro model of the bovine mammary epithelium. In addition, we assessed whether BME-UV1 cells were able to express endogenous bLf after in vitro exposure to LPS.
A further objective of my thesis work was to use cell-based bioassays to investigate the plasmin-plasminogen system. This system plays a key role in cellular responses, and is involved both in physiological and in pathological conditions in the mammary gland. The aim of the third section was to determine the effect of growth factors (IGF-1 and EGF) and three hormones (insulin, dexamethasone, and prolactin) on the expression of plasminogen activator (PA)-related genes (u-PA, u-PAR, PAI-1, PAI-2) and BME-UV1 cell proliferation. In addition we investigated the effects of E. coli LPS on cell viability, the modulation of cell-associated u-PA activity and the regulation of u-PA and u-PAR RNA expression in BME-UV1 cells.
Below are more details on what each section covers:
The first section reports how the role of α-tocopherol in counteracting OTA toxicity was evaluated in various experimental conditions using primary porcine fibroblasts. Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P < 0.01) reduced OTA cytotoxicity, lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α-tocopherol administration may counteract short-term OTA toxicity, thus supporting its defensive role at a cell membrane level.
The second section describes how BME-UV1 was used as an in vitro model to evaluate the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS). Exogenous bLf showed a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure did not involve endogenous bLf mRNA expression, suggesting that this cell line lacks functional LPS-responsive elements.
The third section details how cell proliferation was measured using the MTT assay and direct cell enumeration on BME-UV1 treated with physiological stimuli. Results showed that both IGF-1 and EGF increased cell proliferation. Neither of the growth factors had any effect on the expression of PAI-1 and PAI-2. In line with changes in gene expression, EGF and IGF-1 up regulated total cell-associated, membrane-bound and secreted u-PA activity. Dexamethasone alone and when combined with insulin or prolactin up regulated the gene expression of both PAI- and PAI-2, but not that of u-PA and u-PAR without affecting cell proliferation. Total decreased cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. This thus suggests that when dexamethasone acts synergistically with prolactin or insulin it inhibits the activation of the plasmin-plasminogen system, but this inhibition is not correlated with any changes in cell proliferation. In addition, the plasmin-plasminogen system was examined, using the BME-UV1 cell model, in order to evaluate the effects of Escherichia coli LPS on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression. LPS did not affect cell viability, but led to an increase in u-PA activity, with the maximum response after 6 h of incubation. In addition, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS increased u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes.
In conclusion, the application of appropriate in vitro models represents a fundamental requirement for the study of cellular responses to different stimuli as in the case covered in my thesis regarding nutraceutical compounds.
Cell-based assays are a valuable tool for assessing fundamental regulatory mechanisms at a cellular level, such as the plasmin-plasminogen system. Cell-based assays allow both the functionality of nutraceutical and cellular response mechanisms to be evaluated reducing use animal models in the preliminary study phase. Obviously, data obtained in cell culture models must be interpreted carefully, since this system represents a simplification of the intricacies of the numerous reactions and interactions that occur in vivo
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Cytotoxicity, DNA integrity and methylation in mammary and kidney epithelial cell lines exposed to Ochratoxin A
Full text linkOchratoxin A (OTA) is a secondary metabolite of moulds that may contaminate food and feedstuffs. OTA is recognized as a nephrotoxic, hepatotoxic and teratogenic substance in different animal species. The kidney is the target organ of OTA, whereas lower OTA concentrations could be detected in other tissues, such as adipose tissue, liver and muscles. In addition, OTA transfer to milk has been demonstrated in several species, such as humans, rabbits, rats and ruminants, identifying the mammary gland as one of the potential target of this mycotoxin. This study aimed to investigate the in vitro damage induced by OTA using two well established epithelial cell lines. MDCK cells have been used as a model of the kidney epithelium, while BME-UV1 have been employed as a model of the mammary gland epithelium. The effect of OTA on cultured cell lines, with subsequent evaluation of cell viability (MTT test) and membrane stability (LDH test), was assessed. In all experiments performed, control consisted of MDCK and BME-UV1 cells exposed to basal medium alone. After 24h of OTA treatment (concentration range 0.15 up to 10μg/mL), MDCK and BME-UV1 cell viability was strongly reduced in a dose-dependent way and LC50 has been calculated. LC50 for MDCK cells was 1 μg/mL while, for BME-UV1 cells, LC50 was 0.8 μg/mL. In light of LC50, the range of concentrations for further experiments was determined (0.3 up to 1.25μg/mL). The percentage of LDH released by MDCK and BME-UV1 cells increased in a dose-dependent way. In particular, 1.25 μg/mL of OTA determined a 35% of LDH released in MDCK cells and a 46% of LDH released in BME-UV1 cell line. Subsequently, the effect of the addition of OTA to MDCK and BME-UV1 cells has been evaluated on DNA integrity, which was detected by gel electrophoresis, by the analysis of DNA oxidation biomarker 8-OHdG (8-OHdG adduct) and the global DNA methylation status (5-mC). The level of 8-OHdG adduct was significantly (P<0.05) increased in BME-UV1 cells treated with 1.25 μg/ml of OTA. The analysis of 5-mC revealed that in MDCK and BME-UV1 cells, OTA has not induced alterations in the global DNA methylation status. The results obtained herein could represent the basis for the development of future studies investigating the in vitro relationship between DNA damage and the global DNA methylation status, promoting new strategies to control OTA cytotoxicity at different tissue level
Tobacco seeds as edible vaccine in pig livestock
Full text linkPlants have been recognized as an expression system for the production of edible vaccine because of the possibility of introducing antigenic proteins into their genome. In livestock, transformed plants for the expression of immunogenic proteins could be administered, orally, in feed to induce mucosal immune response in the gastrointestinal tract. the oral delivery of plant-made vaccines is attractive since the low costs, the heat stability, the avoidance of the injections and for the production of specific antibodies in the mucosa, where the major pathogens gain access to the body. Furthermore seeds provide a stable environment for transporting edible vaccine into the gut. In pig industry, verocytotoxic Escherichia coli (VTEC) strains are responsible for severe enterotoxaemia in the weaning period and novel strategies are required to control E. coli infections.
Currently, no vaccines are available and an outbreak of the disease requires antibiotic medication, which have disadvantages like the increasing of antimicrobial resistance and the environment impact; accordingly, the development of an effective oral vaccination strategy is of interest.
Tobacco presents many advantages including high transformation efficiency and easy cell culture protocols. Seeds do not contain significant levels of nicotine (less than 2 μg/Kg) and, once included in diets for weaned piglets, they showed a good palatability (Rossi et al., 2007)
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Vitamin E bioavailability : past and present insights
Full text linkAnimal health and production in dairy cows. Cow milk is considered as a remarkable source of bioactive components promoting human health, which has renewed interest in the effects of vitamin E supplementation on its nutritional value, sensory quality and shelf life. Thus, defining relative bioavailability, utilisation and transfer into milk of different vita- min E formulations is particularly important to assess the adequate levels of supplementation for animal health and milk quality. In nature vitamin E is present under one isomeric form, RRR α-tocopherol; when α-tocopherol is synthe- sized chemically, a racemic mixture of 8 possible isomers of α-tocopherol in equimolar concentrations is produced (all-rac α-tocopherol). The different stereoisomers have different biopotencies in humans and livestock; the conversion factor between RRR and all-rac vitamin E was estimated by early studies on the basis of the rat foetal resorption bio- assay, and then extended to other species. Recent advances on the distribution of vitamin E stereoisomers in plasma and tissues have highlighted the need to formulate new conversion factors in dairy cows as well as in humans. On ac- count of this, the present article aims to consider past and recent data related to vitamin E in dairy cow nutrition
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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