1,720,979 research outputs found
CARATTERIZZAZIONE MOLECOLARE ATTRAVERSO ANALISI DI IMMUNOFLUORESCENZA DI CARDIOMIOCITI PACEMAKER DERIVATI DA CELLULE STAMINALI EMBRIONALI MURINE INGEGNERIZZATE
Full text linkIntroduction and Aim. Mouse embryonic stem cells (mESCs) are able to differentiate in vitro into many cell types derived from the three germ layers, including pacemaker cardiomyocytes. ESCs thus represent a potential cell source for generation of biological pacemakers repair. The applicability of ESCs in regenerative therapies requires the isolation of a pure population of differentiated cells in order to avoid the contamination with teratogenic undifferentiated ESCs.
To identify ESC-derived pacemaker cardiomyocytes, we established a mESC line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the promoter of the HCN4 gene (pHCN4-EGFP). Although EGFP-positive spontaneously beating cells can be detected, non-beating EGFP-positive cells can be detected as well. The transmembrane protein CD166 is specifically expressed in the developing murine heart tube at embryonic day 8.5. Previous works have demonstrated that CD166 can be used as surface antigen to select a differentiated cell population enriched in cardiomyocytes.
The aim of this work is to characterize the cardiac nature of CD166-positive and negative cell populations isolated from differentiating mESC cultures at day 7+1.
Materials and Methods. pHCN4-EGFP mESCs-D3 were differentiated into embryoid bodies (EBs) by the “hanging drops” method. EBs at differentiation day 7+1 were dissociated into single cell-suspensions and sorted by flow cytometry. CD166+ and CD166 cells were plated in very low adhesion dish for 24 hours to facilitate their re-aggregation and then plated on cell culture dishes. Immunofluorescence experiments were carried out both on CD166+ and CD166 cell cultures. Samples were fixed in paraformaldehyde and labelled with antibodies against sarcomeric α-actinin to identify cardiomyocytes and against HCN4 and caveolin 3 to specifically detect cells with a pacemaker-like phenotype.
Results. While CD166+ cells form spontaneously and synchronously beating aggregate, CD166 cells form EBs-like aggregates in which only very few cells show signs of automaticity. Immunofluorescence analysis of these aggregates demonstrate that 60,13 % of CD166+ cells expresses a contractile apparatus organized in sarcomeric-like structures, as shown by α-actinin staining. In contrast CD166 aggregates present a much lower degree of α-actinin staining (3,60 % of cells). To identify cardiac cells with pacemaker-like phenotype, double staining with anti-HCN4 and anti-α-actinin antibodies was carried out. In CD166+ cell population 42,77 % of α-actinin positive cell expresses HCN4 channels whereas in CD166 cell population the percentage is only 5,12 %. Moreover CD166+ cells also stained positive for caveolin 3, a structural protein of muscular caveolae, known to interact with pacemaker channels in both native SAN myocytes and ESC-derived pacemaker cells. Interestingly, most CD166+ cells were also EGFP-positive confirming a persistent activation of the HCN4 promoter in these cells and thus their pacemaker phenotype.
Conclusions. Our data demonstrate that CD166 can be used, in a specific stage of differentiation, as a surface antigen to select a cell population enriched in cardiomyocytes presenting some peculiar features of sinoatrial pacemaker cells. For this reason, CD166+ cell population has the characteristics that made them a suitable cellular substrate for the creation of biological pacemakers
Mutation of the Caveolin-Interacting Sequence of the rabbit HCN4 channel affects its cellular localization
No full textResveratrol promotes myogenesis and hypertrophy in murine myoblasts
Full text linkBackground: Nutrigenomics elucidate the ability of bioactive food components to influence gene expression, protein synthesis, degradation and post-translational modifications.Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio- and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, prevention of mitochondrial disorder, avoidance of obesity-related diseases. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets in myogenesis process are not completely known. Myogenesis is a dynamic multistep process regulated by Myogenic Regulator Factors (MRFs), responsible of the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from cell cycle, elongate and fuse to form multinucleated myotubes. Skeletal muscle hypertrophy can be defined as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by increased protein synthesis, mainly regulated by Insulin Like Growth Factor 1 (IGF-1), PI3-K/AKT signaling pathways.Aim of this work was the study of RSV effects on proliferation, differentiation process and hypertrophy in C2C12 murine cells.Methods: To study proliferative phase, cells were incubated in growth medium with/without RSV (0.1 or 25 μM) until reaching sub confluence condition (24, 48, 72 h). To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without RSV (0.1 or 25 μM) for 24, 48, 72, 96 hours. After 72 hours of differentiation, the genesis of hypertrophy in neo-formed myotubes was analyzed.Results: Data showed that RSV regulates cell cycle exit and induces C2C12 muscle differentiation. Furthermore, RSV might control MRFs and muscle-specific proteins synthesis. In late differentiation, RSV has positive effects on hypertrophy: RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression.Conclusions: RSV might control cell cycle promoting myogenesis and hypertrophy in vitro, opening a novel field of application of RSV in clinical conditions characterized by chronic functional and morphological muscle impairment
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
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
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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