Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice

(2015). Cancer and the microbiome: potential applications as new tumor biomarker. Expert Review of Anticancer Therapy: Vol. 15, No. 3, pp. 317-330.

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA. Chromatin structure and function are regulated by the concerted activity of covalent modifiers of chromatin, nucleosome remodeling factors, and several emerging classes of non-coding RNAs. ISWI is an evolutionarily conserved ATP-dependent chromatin remodeler playing essential roles in chromosome condensation, gene expression, and DNA replication. ISWI activity has been involved in a variety of nuclear functions including telomere silencing, stem cell renewal, neural morphogenesis, and epigenetic reprogramming. Using an in vivo assay to identify factors regulating ISWI activity in the model system Drosophila melanogaster, we recovered a genetic interaction between ISWI and hsrω. The hsrω gene encodes multiple non-coding RNAs (ncRNAs), of which the >10 kb nuclear hsrω-n RNA, with functional homolog in mammals, is essential for the assembly and organization of hnRNP-containing nucleoplasmic omega speckles. These special nuclear compartments play essential roles in the storage/sequestration of hnRNP family and other proteins, thus playing important roles in mRNA maturation and other regulatory processes. Here we show that the hsrω-n ncRNA interacts in vivo and in vitro with ISWI to directly regulate its ATPase activity. We also provide in vivo data showing that omega speckle nuclear organization depends on ISWI function, highlighting a novel role for chromatin remodelers in nuclear speckles organization.

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton’s jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.