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A Sorting Strategy For Bovine Mammary Progenitors
No full textThe mammary gland is a bilayered system organized into a series of branching ducts that terminate in secretory alveoli. Their epithelium is composed of an inner layer of cytokeratin(CK)18+ luminal cells, which secrete milk, and an outer layer of contractile CK14+ myoepithelial cells, which contribute to milk ejection during lactation. The existence of mammary renewable stem cells (MaSCs) has been deduced from the profound regenerative ability of the mammary epithelium. It undergoes different morphogenetic changes during puberty and during the subsequent pregnancies. The knowledge of MaSCs properties and functions should represent an interesting opportunity in the field of animal production as the appropriate regulation of these cells should benefit milk yeld. The aim of our study is to characterize the MaSCs population of bovine species and set up a sorting strategy to purify different primitive populations. The Colony-Forming Cell (CFC) assay performed on bovine mammary samples obtained from virgin, two years old cows revealed the presence in the mammary gland of both myoepithelial and luminal-restricted progenitor cells. In order to select and isolate the different types of progenitors we have performed a flow cytometric analysis of cells dissociated from bovine mammary tissue. Cells were then stained with antibodies directed to CD49f, whose expression at high levels is associated with progenitor and stem cell activity, and p-cadherin, that is normally expressed in the myoepithelial/basal layer of the mammary epithelium. After depletion of hematopoietic cells (using an anti-CD45 antibody) we identified four different subpopulations. They were characterized by different levels of CD49f/P-cad expression: CD49f/P-cad- (15,6%), CD49f++/P-cad- (25,4%), CD49f+/P-cad+ (27,1%), CD49f+/P-cad++ (10,2%). After sorting, the CFC assay performed with the cells of the different subpopulations showed that CD49f++/P-cad- population was able to generate colonies with an higher frequency compared to the others, revealing an enrichment in stem cells. In particular, 35,7% of colonies showed a myoepithelial phenotype and 64,3% a luminal one. In order to analyze whether these cells also had an in vivo tissue regenerative activity, the four subpopulation were implanted in collagen gels under the kidney capsule of NOD/SCID immunodeficient mice in numbers proportional to their frequency in the CD45- population. Immunohistochemical analysis of gels recovered 4 weeks later revealed that only CD49f++/P-cad- subpopulation was able to regenerate in vivo mammary alveolar structures characterized by a lumen surrounded by two layers of cells: an inner K18+ layer and an outer K14+ layer. The CFC assay performed with single cell suspension obtained from recovered gels showed a higher colony-forming ability of CD49f++/P-cad- population compared to the others. Therefore the structures produced in these gels contained clonogenic progenitors, that were able to form the same types of colonies observed from freshly isolated cells (36,2% of myoepithelial colonies and 63,8% of luminal ones). By using a novel sorting strategy we show that the mammary myoepithelial cell layer can be divided in different subpopulations. Each one has a different progenitor content and only in one fraction (CD49f++/P-Cad-) we were able to detect a subset of adult stem cells able to regenerate a mammary epithelium. This sorting strategy might prove useful for understanding the stem cells population dynamics during the different phases that the mammary gland undergoes. Besides, given the similar organization of the mammary tissue in the bovine and the human species, the cow might represent a much better animal model than the mouse
Functional effect of mir-27b on myostatin expression: a relationship in Piedmontese cattle with double-muscled phenotype
No full textAbstract
Background: In Piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point
mutation in the myostatin (MSTN) gene. The Piedmontese MSTN missense mutation G938A is translated to C313Y myostatin protein. This mutation alters MSTN function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. MiRNAs could play a role in skeletal muscle hypertrophy modulation by down-regulating gene
expression.
Results: After identifying a 30-UTR consensus sequence of several negative and positive modulator genes involved
in the skeletal muscle hypertrophy pathway, such as IGF1, IGF1R, PPP3CA, NFATc1, MEF2C, GSK3b, TEAD1 and
MSTN, we screened miRNAs matching to it. This analysis led to the identification of miR-27b, miR-132, miR-186 and
miR-199b-5p as possible candidates. We collected samples of longissimus thoracis from twenty Piedmontese and
twenty Friesian male bovines. In Piedmontese group miR-27b was up-regulated 7.4-fold (p < 0.05). Further, we
report that the level of MSTN mRNA was about 5-fold lower in Piedmontese cattle vs Friesian cattle (p < 0.0001)
and that less mature MSTN protein was detected in the Piedmontese one (p < 0.0001). Cotransfection of miR-27b
and psi-check2 vector with the luciferase reporter gene linked to the bovine wild-type 30-UTR of MSTN strongly
inhibited the luciferase activity (79%, p < 0.0001).
Conclusions: These data demonstrate that bovine MSTN is a specific target of miR-27b and that miRNAs contribute
to explain additive phenotypic hypertrophy in Piedmontese cattle selected for the MSTN gene mutation, possibly
outlining a more precise genetic signature able to elucidate differences in muscle conformation
Purification of ruminant mammary stem cell population and uses thereof for production of transgenic proteins in vivo
No full textCharacterization of ipscs derived from bovine mammary epithelial cells and bovine skin fibroblasts
No full textCorrelation between estrogen plasma level and miRNAs in muscle of Piedmontese cattle
Full text linkA loss-of-function mutation of the myostatin gene has a very high prevalence in the Piedmontese cattle breed. The effect of such mutation is a double-muscle phenotype because of hypertrophy. However, differences in muscle mass development can still be detected in individuals of this breed. Such differences must be generated by other factors controlling skeletal muscle development. MicroRNAs are short noncoding RNA molecules that modulate gene expression at a post-transcriptional level. MicroRNAs have been demonstrated to be involved in skeletal muscle development, and some of them are controlled by steroid hormone signaling. Data on estrogen signaling are lacking, whereas more studies have been carried out on the effect of androgens. We aimed at identifying putative estrogen responsive miRNAs that might be involved in skeletal muscle development. At a slaughterhouse, we collected muscle samples from longissimus dorsi and blood samples. Blood 17beta-estradiol concentration was assessed, and RNA was extracted from muscle samples. The animals we sampled were divided into groups according to estrogen blood concentration, and through qPCR expression, levels of 7 muscle-related miRNAs were evaluated. We found that miR-26b (P < 0.01), miR-27a-5p (P < 0.05), miR-27b (P < 0.05), and miR-199a-3p (P < 0.01) were differentially expressed among experimental groups. Expression levels of miR-26b were reduced approximately 50% in samples with a low blood estrogen concentrations, and the other miRNAs showed a tendency to increase their expression levels when blood estrogen levels were higher. The variations of the circulating concentrations of estrogen in Piedmontese cattle might influence muscle mass development through miRNAs and thus contribute to individual variability in a breed with a high prevalence of a myostatin point mutation
Reprogramming of Bovine Mammary Epithelial Cells And Bovine Skin Fibroblasts With Pluripotency Factors, a New Perspective in Veterinary Science and Milk Production
No full text
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
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