How the Intrinsically Disordered N-Terminus of Cancer/Testis Antigen MAGEA10 Is Responsible for Its Expression, Nuclear Localisation and Aberrant Migration.

Melanoma-associated antigen A (MAGEA) subfamily proteins are normally expressed in testis and/or placenta. However, aberrant expression is detected in the tumour cells of multiple types of human cancer. MAGEA expression is mainly observed in cancers that have acquired malignant phenotypes, invasiveness and metastasis, and the expression of MAGEA family proteins has been linked to poor prognosis in cancer patients. All MAGE proteins share the common MAGE homology domain (MHD) which encompasses up to 70% of the protein; however, the areas flanking the MHD region vary between family members and are poorly conserved. To investigate the molecular basis of MAGEA10 expression and anomalous mobility in gel, deletion and point-mutation, analyses of the MAGEA10 protein were performed. Our data show that the intrinsically disordered N-terminal domain and, specifically, the first seven amino acids containing a unique linear motif, PRAPKR, are responsible for its expression, aberrant migration in SDS-PAGE and nuclear localisation. The aberrant migration in gel and nuclear localisation are not related to each other. Hiding the N-terminus with an epitope tag strongly affected its mobility in gel and expression in cells. Our results suggest that the intrinsically disordered domains flanking the MHD determine the unique properties of individual MAGEA proteins.

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 AND 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'-dichlorodihydrofluorescein 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.

Antibody response against cancer-testis antigens MAGEA4 and MAGEA10 in patients with melanoma.

Melanoma-associated antigen A (MAGEA) represent a class of tumor antigens that are expressed in a variety of malignant tumors, however, their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. The restricted expression and immunogenicity of these antigens make them ideal targets for immunotherapy in human cancer. In the present study the presence of naturally occurring antibodies against two MAGEA subfamily proteins, MAGEA4 and MAGEA10, was analyzed in patients with melanoma at different stages of disease. Results indicated that the anti-MAGEA4/MAGEA10 immune response in melanoma patients was heterogeneous, with only ~8% of patients having a strong response. Comparing the number of strongly responding patients between different stages of disease revealed that the highest number of strong responses was detected among stage II melanoma patients. These findings support the model that the immune system is involved in the control of melanoma in the early stages of disease.