Similarities and differences in the protein composition of cutaneous melanoma cells and their exosomes identified by mass spectrometry

Intercellular transport of proteins mediated by extracellular vesicles (EVs)—exosomes and ectosomes—is one of the factors facilitating carcinogenesis. Therefore, the research on protein cargo of melanoma-derived EVs may provide a better understanding of the mechanisms involved in melanoma progression and contribute to the development of alternative biomarkers. Proteomic data on melanoma-derived EVs are very limited. The shotgun nanoLC-MS/MS approach was applied to analyze the protein composition of primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) cutaneous melanoma cells and exosomes released by them. All cells secreted homogeneous populations of exosomes that shared a characteristic set of proteins. In total, 3514 and 1234 unique proteins were identified in melanoma cells and exosomes, respectively. Gene ontology analysis showed enrichment in several cancer-related categories, including cell proliferation, migration, negative regulation of apoptosis, and angiogenesis. The obtained results broaden our knowledge on the role of selected proteins in exosome biology, as well as their functional role in the development and progression of cutaneous melanoma. The results may also inspire future studies on the clinical potential of exosomes

Diverse expression of N-acetylglucosaminyltransferase V and complex-type \beta1,6-1,6-branched N-glycans in uveal and cutaneous melanoma cells

Although both uveal (UM) and cutaneous (CM) melanoma cells derive from the transformed melanocytes, their biology varies significantly in several aspects. Malignant transformation is frequently associated with alternations in cell glycosylation, in particular those concerning branched complex-type N-glycans. These changes occur principally in β1,4-N-acetylglucosaminyltransferase III (GnT-III) that catalyzes the synthesis of glycans with bisected N-acetylglucosamine (GlcNAc) and β1,6-N-acetylglucosaminyltransferase V (GnT-V) that is involved in forming β1,6-branched antenna in complex-type glycans. We searched for the reasons of a different behavior of CM and UM cells in the expression of GnT-III and GnT-V and their oligosaccharide products. Our study showed that UM cells have more β1,6-branched glycans than CM cells, what results from a higher expression of MGAT5 gene encoding GnT-V. The higher β1,6-branching of glycans in UM may contribute to their higher potential to migrate on fibronectin and weaker binding to main extracellular matrix proteins, observed in our previous studies

Low-vacuum filtration as an alternative extracellular vesicle concentration method : a comparison with ultracentrifugation and differential centrifugation

Recent years have brought great focus on the development of drug delivery systems based on extracellular vesicles (EVs). Considering the possible applications of EVs as drug carriers, the isolation process is a crucial step. To solve the problems involved in EV isolation, we developed and validated a new EV isolation method—low-vacuum filtration (LVF)—and compared it with two commonly applied procedures—differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cell culture media were characterized by (a) Transmission Electron Microscopy (TEM), (b) Nanoparticle Tracking Analysis (NTA), (c) Western blot and (d) Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Additionally, the membrane surface was imaged with Environmental Scanning Electron Microscopy (ESEM). We found that LVF was a reproducible and efficient method for EV isolation from conditioned media. Additionally, we observed a correlation between ATR-FTIR spectra quality and EV and protein concentration. ESEM imaging confirmed that the actual pore diameter was close to the values calculated theoretically. LVF is an easy, fast and inexpensive EV isolation method that allows for the isolation of both ectosomes and exosomes from high-volume sources with good repeatability. We believe that it could be an efficient alternative to commonly applied methods

Large extracellular vesicles do not mitigate the harmful effect of hyperglycemia on endothelial cell mobility

Extracellular vesicles, especially the larger fraction (LEVs – large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs

social advertising related to domestic violence and its reception by the public

W poniższej pracy znajdują się badania na temat wpływu reklam społecznych o tematyce przemocy domowej na odbiorców. Badania zostały przeprowadzone za pomocą ankiety na grupie 70 osób. Celem badań było znalezienie odpowiedzi na pytanie w jaki sposób reklamy społeczne zwiększają świadomość odbiorców na temat zjawiska przemocy domowej. Wyniki badań zawierają zarówno odpowiedź w jaki sposób adresaci odbierają przekaz, jaki jest komentarz na temat formy tego przekazu. Celem pracy jest rozeznanie w jaki sposób reklamy społeczne zwiększają świadomość odbiorców na temat zjawiska przemocy domowej. Pierwszy rozdział to wprowadzenie do tematu, oraz zapoznanie z zagadnieniem reklamy społecznej. W rozdziale drugim autor przybliżył problematykę przemocy domowej. Znajduje się tu opis zjawiska, sprawcy, ofiary przemocy domowej oraz przemoc wobec dziecka. Z kolei trzeci jest rozdziałem badawczym. Autor ukazał wyniki badań własnych oraz wnioski z nich płynące. Głównymi wnioskami płynącymi z badania są, że reklamy społeczne są odbierane przez społeczeństwo głównie poprzez emocje.The following text includes research about influence of social commercials of home violence on vievers. Research was examined on the group of 70 people using questionnaire. The target was to find answers about the way social commercials influence society. The results includes answers about the way people react to the message and their comments about its form. The goal of the thesis is to find out how social commercials about domestic violence increase their knowledge. First part is a introduction to the topic and to the thesis of social commercials. In second part author explained the problem of domestic violence. It includes the descriptions of the problem, culprit and victim. The third part is about research. Author shows results of the research and hers conclusions. The main conclusion is that teh social commercials influence on people the most via emotions

May metallic biomaterials used for orthopaedic implants promote carcinogenesis? Preliminary transcriptomic research on human chondrocytes

The aim of this research was to assess the risk of carcinogenesis induced by the metallic materials intended for orthopaedic implants. The report is an analytical summary of changes in the expression of cancer-related genes in human chondrocytes of normal and neoplastic phenotype. Cq values (quantification cycle values) obtained from qRT-PCR reactions (quantitative real-time polymerase chain reactions) were used to count Fc values (fold change values) for each gene. Differences in Fc values obtained for primary and cancer cells grown on the surface of medical steel AISI316L and titanium-aluminum-vanadium alloy Ti6Al4V were then analyzed by t-Student test. The results indicate that for cancer cells grown on the surfaces of both examined materials the fold change greater than 2, usually considered essential, was found for LUM gene involved in sarcoma induction. For FOS gene, also involved in sarcoma induction, the Fc value was also very close to 2 in the primary cells exposed to Ti6Al4V alloy. The remaining observed changes were rather subtle, although they cannot be omitted from further studies because differences in gene expression in primary and tumor cells grown on the same biomaterial were statistically significant in several cases. The compilation of qRT-PCR experiments carried out on primary and cancer cells in parallel allowed to identify possible future contraindications for patients with a genetic predisposition to cancer or with cancer history