Exploration of serum- and cell culture-derived exosomes from dogs.

BACKGROUND: Exosomes are defined as extracellular membrane vesicles, 30-150 nm in diameter, derived from all types of cells. They originate via endocytosis and then they are released through exocytosis to the extracellular space, being found in various biological fluids as well as in cell culture medium. In the last few years, exosomes have gained considerable scientific interest due to their potential use as biomarkers, especially in the field of cancer research. This report describes a method to isolate, quantify and identify serum- and cell culture-derived exosomes from dog samples, using small volumes (100 µL and 1 mL, respectively). RESULTS: Quantification and sizing of exosomes contained in serum and cell culture samples were assessed by utilizing nanoparticle tracking analysis, transmission electron microscopy and immunoelectron microscopy. Detected particles showed the normal size (30-150 nm) and morphology described for exosomes, as well as presence of the transmembrane protein CD63 known as exosomal marker. CONCLUSIONS: Based on a validated rapid isolation procedure of nanoparticles from small volumes of different types of dog samples, a characterization and exploration of intact exosomes, as well as facilitation for their analysis in downstream applications was introduced.

Identification of differentially expressed proteins in ruminal epithelium in response to a concentrate-supplemented diet.

Ruminal epithelium adapts to dietary change with well-coordinated alterations in metabolism, proliferation, and permeability. To further understand the molecular events controlling diet effects, the aim of this study was to evaluate protein expression patterns of ruminal epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to 6 wk. The control group received hay only. Proteome analysis with differential in gel electrophoresis technology revealed that, after 2 days, 60 proteins were significantly modulated in ruminal epithelium in a comparison between hay-fed and concentrate-fed sheep (P < 0.05). Forty proteins were upregulated and 20 proteins were downregulated in response to concentrate diet. After 6 wk of this diet, only 14 proteins were differentially expressed. Among these, 11 proteins were upregulated and 3 downregulated. To identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionization mass spectrometry. The differential expression of selected proteins, such as esterase D, annexin 5, peroxiredoxin 6, carbonic anhydrase I, and actin-related protein 3, was verified by immunoblotting and/or mRNA analysis. The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. These results suggest new candidate proteins that may contribute to a better understanding of the signaling pathways and mechanisms that mediate rumen epithelial adaptation to high-concentrate diet.