Downregulation of Gap Junctions in Astrocytes by Monoclonal Antibodies against the Second Extracellular Loop of Connexin-43

Downregulation of gap junctions by monoclonal antibodies against the second extracellular loop of connexin-43 (E2Cx43) was studied in a passaged culture of astrocytes. The results of confocal laser scanning microscopy demonstrated that, after two hours of coincubation of cells loaded with Calcein AM and Dil according to Goldberg et al. and unlabelled cells, the cytoplasmic dye Calcein AM was actively transferred to unlabelled cells through newly formed gap junctions. This transfer could be almost completely blocked by addition of 60 μg/ml of anti-E2Cx43 antibodies. Flow cytometric analysis showed that, in experiments carried out according to Goldberg et al., with approximately 2% of labeled cells added to unlabelled ones, about 2.5% of the total cell population took up Calcein AM through gap junctions, thus forming a cell pool characterized by low-intensity green fluorescence. In the presence of antibodies, the proportion of these cells was no more than 0.6%, which indicates an at least fourfold suppression of the gap junction function by E2Cx43 antibodies. The data obtained were reproduced in several independent series. Thus, we obtained monoclonal antibodies capable of modulating the gap junction function in cultures of Cx43-positive cells

Current and Future Trends on Diagnosis and Prognosis of Glioblastoma: From Molecular Biology to Proteomics

Glioblastoma multiforme is the most aggressive malignant tumor of the central nervous system. Due to the absence of effective pharmacological and surgical treatments, the identification of early diagnostic and prognostic biomarkers is of key importance to improve the survival rate of patients and to develop new personalized treatments. On these bases, the aim of this review article is to summarize the current knowledge regarding the application of molecular biology and proteomics techniques for the identification of novel biomarkers through the analysis of different biological samples obtained from glioblastoma patients, including DNA, microRNAs, proteins, small molecules, circulating tumor cells, extracellular vesicles, etc. Both benefits and pitfalls of molecular biology and proteomics analyses are discussed, including the different mass spectrometry-based analytical techniques, highlighting how these investigation strategies are powerful tools to study the biology of glioblastoma, as well as to develop advanced methods for the management of this pathology