Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle.

Satellite cells are adult stem cells located between the basal lamina and sarcolemma of muscle fibers. Under physiological conditions, satellite cells are quiescent, but they maintain a strong proliferative potential and propensity to differentiate, which underlies their critical role in muscle preservation and growth. MicroRNAs (miRNAs) play essential roles during animal development as well as in stem cell self-renewal and differentiation regulation. MiRNA-1, miRNA-133a and miRNA-206 are closely related muscle-specific miRNAs, and are thus defined myomiRNAs. MyomiRNAs are integrated into myogenic regulatory networks. Their expression is under the transcriptional and post-transcriptional control of myogenic factors and, in turn, they exhibit widespread control of muscle gene expression. Very little information is available about the regulation and behavior of satellite cells in large farm animals, in particular during satellite cell differentiation. Here, we study bovine satellite cells (BoSCs) undergoing a differentiation process and report the expression pattern of selected genes and miRNAs involved. Muscle samples of longissimus thoracis from Holstein adult male animals were selected for the collection of satellite cells. All satellite cell preparations demonstrated myotube differentiation. To characterize the dynamics of several transcription factors expressed in BoSCs, we performed real-time PCR on complementary DNA generated from the total RNA extracted from BoSCs cultivated in growth medium (GM) or in differentiation medium (DM) for 4 days. In the GM condition, BoSCs expressed the satellite cell lineage markers as well as transcripts for the myogenic regulatory factors. At the time of isolation from muscle, PAX7 was expressed in nearly 100% of BoSCs; however, its messenger RNA (mRNA) levels dramatically decreased between 3 and 6 days post isolation (P<0.01). MyoD mRNA levels increased during the 1st day of cultivation in DM (day 7; P<0.02), showing a gradual activation of the myogenic gene program. During the subsequent 4 days of culture in DM, several tested genes, including MRF4, MYOG, MEF2C, TMEM8C, DES and MYH1, showed increased expression (P<0.05), and these levels remained high throughout the culture period investigated. Meanwhile, the expression of genes involved in the differentiation process also miRNA-1, miRNA-133a and miRNA-206 were strongly up-regulated on the 1st day in DM (day 7; P<0.05). Analysis revealed highly significant correlations between myomiRNAs expression and MEF2C, MRF4, TMEM8C, DES and MYH1 gene expression (P<0.001). Knowledge about the transcriptional changes correlating with the growth and differentiation of skeletal muscle fibers could be helpful for developing strategies to improve production performance in livestock.

Differential expression of living mammary epithelial cell subpopulations in milk during lactation in dairy cows.

Epithelial cells are shed into milk during lactation, and although they generally reflect the cellular characteristics of terminally differentiated luminal cells, previously the detection of more primitive cells was described in human milk where a cell population of epithelial lineage was detected expressing markers typical of progenitor cells. In this investigation, we report the development of flow cytometry analysis to allow multiparametric assessment of mammary epithelial cells observed in milk. Cells collected from milk samples of 10 healthy dairy cows were directly analyzed for 6 different markers: CD45, CD49f, cytokeratin 14, cytokeratin 18, presence of nucleus, and cell viability. Milk samples were collected in 3 different periods of lactation: early lactation (EL=d 0-30), mid-lactation (ML=d 90-120), and late lactation (LL=210-250). Here we identify the differential expression of precursor or differentiated cell markers (or both) in mammary epithelial cells present in bovine milk. Myoepithelial cells, as indicated by cells staining positively for cytokeratin 14(+)/cytokeratin 18(-), were observed to increase from EL to LL with a high correlation with nuclear staining inferring potential proliferative activity. Furthermore, a significant increase in CD49f(+) and cytokeratin 14(+)/cytokeratin 18(+) positive cells was observed in LL. This assay is a sensitive approach for evaluating the variations in the frequency and features of living epithelial cells, whose reciprocal balance may be significant in understanding mammary gland cellular function throughout lactation. These observations suggest that mammary epithelial cell immunophenotypes could be investigated as biomarkers for mammary gland function in dairy cows.

Characterization of volatile fraction of typical Irpinian wines fermented with a new starter yeast.

Non-Saccharomyces yeasts are microorganisms that play an important role in the fermentation dynamics, compositions and flavour of wine. The aromatic compounds responsible for varietal aroma in wine are mainly terpenes, of which the most important group are the monoterpenes because of their volatility and odour if present in a free form. In fact, some terpenyl-glycosides do not contribute to the aroma unless they are hydrolysed. The glycosylated form of terpenes can be converted by hydrolysis with ß-glycosidases produced by yeasts during the winemaking process, into aromatic compounds. In this study we utilized a non-Saccharomyces yeast, with a high extra-cellular glycosidase activity, isolated from grapes of cultivars typical of Irpinia region. This strain, identified as a Rhodotorula mucillaginosa (strain WLR12), was used to carry out an experimental winemaking process and the results were compared with those obtained with a commercial yeast starter. Chemical and sensorial analysis demonstrated that the wines produced with WLR12 strain had a more floral aroma and some sweet and ripened fruit notes compared to those obtained with commercial yeast. The data also showed an increasing of the free terpenes fraction that, however, did not significatively modify the bouquet of the wines.

Water buffalo mozzarella cheese stored in polysaccharide-based gels: correlation between prolongation of the shelf-life and physicochemical parameters.

An innovative packaging system has been developed, based on natural gels, that has shown the peculiar characteristic to strongly increase the shelf life of water buffalo Mozzarella cheese. To explain the mechanism of action of the gel, measurements of Ca and Na in the cheese and in the storage liquid were carried out, together with pH determination. A correlation has been found between the constant level of Ca and pH in the cheese and the prolongation of nutritional characteristics; in fact, both parameters diminish significantly in the absence of gel. At the same time, the weight of the cheese in gel remained constant for as long as 30 d. Confocal laser microscopy gave direct evidence of the persistent physical structure of proteins and lipids of Mozzarella when stored in gel.

Migratory crustaceans as biomonitors of metal pollution in their nursery areas. The Lesina lagoon (SE Italy) as a case study.

The Lesina lagoon is located on the southern Adriatic coast of Italy; many marine species, such as the shrimp M. kerathurus, use the Lesina lagoon as a nursery, spending their initial growth phase there. In order to assess the usefulness of migratory species as biomonitors of the environmental quality of this nursery area, we evaluated the metal content of the M. kerathurus juveniles at the end of their growth phase in the lagoon (October), when they are assumed to have bioaccumulated the maximum level of metals from the lagoon environment. The concentrations of Cr, Cd, Pb, Zn, Mn and Cu were measured in the muscle and exoskeleton of the shrimp, and in the sediments and waters of three areas of the Lesina Lagoon. Both the water and sediment levels of the investigated metals tended to fall within the ranges recorded for other lagoon environments characterized by similar anthropic impact and texturally similar sediment; the juveniles of the shrimp M. kerathurus proved to be strong bioaccumulators of heavy metals such as Zn and Cu (biota-sediment accumulation factors - BSAFs - 6.01 and 25.0 respectively), which derive from agricultural activities; therefore, at the end of their growing phase in the lagoon they can be considered useful biomonitors of metal contamination of agricultural origin in their nursery area.

Natural polysaccharide-based gels for dairy food preservation.

The innovative packaging systems described in the present work, based on natural gels, have been shown to increase the shelf life of the Mozzarella cheese, without adding any chemical substance and without thermal procedures. Physical, physicochemical, microbiological, analytical, and mechanical analyses were used to monitor the quality of the cheese as a function of storage type and storage time. In particular, microbiological analysis confirmed that the characteristics of the Mozzarella cheese stored at 4 degrees C in gel are maintained for more than 15 d, whereas samples stored in the mother solution lost important characteristics after 5 d. A penetration test (texture) confirmed that the Mozzarella cheese preserved in the gel maintained mechanical properties similar to those of the fresh product, even after storage for 30 d at 4 degrees C.

Biocompatibility studies on biodegradable polyester-based composites of human osteoblasts: a preliminary screening.

A series of biodegradable composites with natural hydroxyapatite, designed for possible use in orthopedics applications, were preliminarily screened for biocompatibility by employing primary cultures of human osteoblasts in a direct contact method. The cells were seeded at low density onto the materials under investigation and allowed to grow for 2 weeks. They then were analyzed for morphology, proliferation, viability, alkaline phosphatase activity (AP), osteocalcin (OC) production, and extracellular matrix mineralization. The results showed that all materials have good biocompatibility. Cell viability tests demonstrated that in all cases the values were comparable to the control, and the addition of hydroxyapatite always resulted in an enhancement of performance with respect to the plain polymer. AP and OC analysis confirmed that all composites allowed the expression of phenotypic markers. Scanning electron microscopy provided direct evidence of intense cell adhesion and proliferation on the tested materials.

Investigation of the optothermal properties of a new polymeric blend: polymethyl-methacrylate-poly (ethylene-co-vinylacetate).

A new polymeric blend, polymethyl methacrylate-poly (ethylene-co-vinylacetate) (PMMA-EVA) has been produced recently in our laboratories. The optothermal characterization of PMMA-EVA was carried out by measuring the ultraviolet photoacoustic spectra of PMMA, EVA, and PMMA-EVA (25 degrees C, 220-350 nm), of a PMMA-EVA blend (250-750 nm) at two temperatures (19 degrees C and 65 degrees C), and by measuring the scattering coefficient (at 514.5 nm) of PMMA-EVA versus the temperature in the range 38-86 degrees C. Our measurements show that, in the explored thermal range, the blend undergoes a phase transition when its optical properties change drastically. The peculiar optical and thermal properties observed appear attractive for possible applications as temperature-controlled optical devices.