Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

Notch signaling pathway is activated dynamically during evolution playing significant role in cell fate determination and differentiation. It has been known that alterations of this pathway may lead to human malignancies, including gastric cancer. Despite a decline in the overall incidence, this disease still remains an important global health problem. Therefore, a better understanding of the molecular alterations underlying gastric cancer may contribute to the development of rationally designed molecular targeted therapies. It has been reported that Notch1 receptor could become a prognostic marker of gastric cancer and novel target for gastric cancer therapy. Among the novel and targeted approaches for the treatment of gastric cancer is also the process of Notch receptors regulation by specific microRNA. γ-secretase inhibitors are also taken into consideration.

Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling.

Abstract:Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique

BackgroundDespite excellent results of bariatric surgery in the treatment of type 2 diabetes and weight loss in human subjects, some patients do not obtain desired results. One of the reasons for this is that not all patients follow caloric intake recommendations.AimThe aim of this study was to investigate the effect of duodenojejunal omega switch (DJOS) surgery on body weight, glucose tolerance, and incretins in rats.MethodsDJOS and SHAM surgery were performed on rats maintained for 8 weeks on high-fat diet (HF) and control diet (CD), respectively. After surgery, four groups were kept on the same diet as before the surgery, and four groups had a changed diet (CD vs. HF and HF vs. CD) for the next 8 weeks. Glucose tolerance, insulin tolerance, glucose-stimulated insulin, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) secretion, food intake, and body weight were measured.ResultsA change of diet after surgery resulted in reduced glucose tolerance. Plasma insulin levels were lowered between DJOS and SHAM surgeries for the HF/HF and CD/HF groups. DJOS surgery did not reduce body weight in the studied groups, irrespective of diet. In the HF/HF group, ΔGLP-1 was lower for DJOS surgery in comparison with other groups. Differences of weight changes were observed for groups HF/HF and HF/CD. After DJOS surgery, ΔGIP was lower in the CD/HF group compared with HF/HF.ConclusionsOur results show that applications of different types of diets, before and after surgery, is a sensitive method for studies of mechanism of glucose intolerance after DJOS surgery.