Proteomic and Biochemical Analysis of Extracellular Vesicles Isolated from Blood Serum of Patients with Melanoma

Background: Malignant melanoma is the most serious type of skin cancer with the highest mortality rate. Extracellular vesicles (EVs) have potential as new tumor markers that could be used as diagnostic and prognostic markers for early detection of melanoma. Methods: EVs were purified from the blood serum of melanoma patients using two methods—ultracentrifugation and PEG precipitation—and analyzed by mass spectrometry and immunoblot. Results: We identified a total of 585 unique proteins; 334 proteins were detected in PEG-precipitated samples and 515 in UC-purified EVs. EVs purified from patients varied in their size and concentration in different individuals. EVs obtained from stage II and III patients were, on average, smaller and more abundant than others. Detailed analysis of three potential biomarkers—SERPINA3, LGALS3BP, and gelsolin—revealed that the expression of SERPINA3 and LGALS3BP was higher in melanoma patients than healthy controls, while gelsolin exhibited higher expression in healthy controls. Conclusion: We suggest that all three proteins might have potential to be used as biomarkers, but a number of issues, such as purification of EVs, standardization, and validation of methods suitable for everyday clinical settings, still need to be addressed

Oxidative properties of blood-derived extracellular vesicles in 15 patients after myocardial infarction

Background: In this study, we investigated the yield and composition of extracellular vesicles (EVs) derived from 40- to 60-year-old healthy male controls and post-myocardial infarction (post-MI) patients' blood samples and assessed their pro-inflammatory and oxidative-related properties. Our study aimed to determine the EV yield and composition differences between both groups and to find out if there were differences between EV-mediated oxidative stress reactions. Material/Methods: Fifteen post-MI patients and 25 healthy individuals were included. EVs were isolated by ultracentrifugation and analyzed using nanotracking analysis (NTA), western blotting and fluorescent flow cytometry (FFC). Oxidative stress (OS) in blood samples was identified by measuring malondialdehyde concentration from serum, while EVs-induced OS was measured in the human vein endothelium cells (HUVEC) using H2DCFDA (2',7'-dichloro-dihydrofluorescein diacetate) fluorescence as a marker. Results: We found higher EVs concentration in healthy controls than in the post-MI group (7.07±3.1 E+10 ml vs 3.1±1.9 E+10 ml, P<0.001) and a higher level of CD9-positive exosomes (MFI 275±39.5 vs 252±13, P<0.001). Post-MI patients' EVs carry pro-oxidative nicotinamide adenine dinucleotide phosphate (NADPH) oxidases isoforms NOX1 (NADPH oxidase 1), NOX5 (NADPH oxidase 5) and NOX2 (NADPH oxidase 2) and anti-oxidative thioredoxin, extracellular signal-regulated kinases 1/2 (ERK1/2), and protein kinase B (Akt B). In the post-MI EVs, there was a higher predominance of enzymes with anti-oxidative effects, leading to weaker OS-inducing properties in the HUVEC cells. Conclusions: We conclude that post-MI patient blood sample EVs have stronger anti- than pro-oxidative properties and these could help fight against post-MI consequences

Identification and Tracking of Antiviral Drug Combinations

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.Peer reviewe