BackgroundExosomes 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).ResultsQuantification 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.ConclusionsBased 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.

minal 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 Ͻ 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.proteomics; mRNA expression; protein markers; sheep RUMINANTS ARE OF MAJOR WORLDWIDE interest and economic importance because of increasing meat and milk production. A substantial improvement in the productivity and performance of ruminants can only be achieved with adequate dietary supplementation. When high-yielding cattle, milk sheep, or goats merely consume forage, the intake of energy and protein is too low for the desired rates of animal performance. Hence, supplementation of forage-fed ruminants with sufficient amounts of energy and protein is necessary and significantly improves milk production or daily weight gain (41), but, particularly, a surplus of easily fermented carbohydrates or low-fiber intake will place the animals at risk. Such a change in the diet is a challenge for rumen microbes (31) and especially for the ruminal epithelium with respect to adaptation to the new fermentation pattern (47,55). Insufficient adaptation to diet may result in disintegration of the epithelium (22), translocation of lipopolysaccharides (30), and disturbed transport mechanisms (22) and can further cause subacute ruminal acidosis (14, 49). This disease not only can lead to depressed feed intake and milk production, but can also be associated with laminitis, inflammation, and liver abscesses (14,42,49).Hence, one objective of attaining an adequate diet composition for ruminants is to maintain the physiological functions of the forestomach, such as optimal fe...

Obesity is a growing health problem in humans as well as companion animals. In the development and progression of obesity-associated diseases, the members of the renin-angiotensin system (RAS) are proposed to be involved. Particularly, the prevalence of type 2 diabetes mellitus in cats has increased enormously which is often been linked to obesity as well as to RAS. So far, reports about the expression of a local RAS in cat adipocytes are missing. Therefore, we investigated the mRNA expression of various RAS genes as well as the adipocyte marker genes adiponectin, leptin and PPAR-γ in feline adipocytes using quantitative PCR. To characterize the gene expression during adipogenesis, feline pre-adipocytes were differentiated into adipocytes in a primary cell culture and the expression of RAS key genes measured. All major RAS components were expressed in feline cells, but obvious differences in the expression between pre-adipocytes and the various differentiation stages were found. Interestingly, the two enzymes ACE and ACE2 showed an opposite expression course. In addition to the in vitro experiments, mature adipocytes were isolated from subcutaneous and visceral adipose tissue. Significant differences between both fat depots were found for ACE as well as AT1 receptor with greater expression in subcutaneous than in visceral adipocytes. Visceral adipocytes had significantly higher adiponectin and PPAR-γ mRNA level compared to the subcutaneous fat cells. Concerning the nutritional status, a significant lower expression of ACE2 was measured in subcutaneous adipocytes of overweight cats. In summary, the results show the existence of a potentially functional local RAS in feline adipose tissue which is differentially regulated during adipogenesis and dependent on the fat tissue depot and nutritional status. These findings are relevant for understanding the development of obesity-associated diseases in cats such as diabetes mellitus.