Helicobacter pylori persistence is associated to its capability of developing biofilms as a response to environmental stress and changes. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of bOMVs and pOMVs. The presence of eDNA in bOMVs and pOMVs was carried out using a DNase I-gold complex and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterized using the dynamic light scattering (DLS) analysis. The eDNA associated to OMVs was detected and quantified by using PicoGreen assay and spectrophotometer, while its extraction was performed through a DNA Kit. The TEM images showed that the eDNA was mainly isolated and identified on OMVs-membrane surface; while the PicoGreen staining showed a 4-fold increase of dsDNA in bOMVs compared to pOMVs. The eDNA extracted from OMVs was visualized by using gel electrophoresis. The DLS analysis demonstrated that H. pylori generate vesicles, both in its planktonic and biofilm phenotypes, with sizes in the nanometer scales and a broad size distribution. The DLS aggregation study of H. pylori OMVs demonstrated that eDNA may play a role in the OMVs aggregation, particularly for biofilm phenotype. The eDNA associated with vesicle membrane can affect the DNase I activity on H. pylori biofilms. OMVs derived from H. pylori ATCC 43629/NCTC 11639, particularly its biofilm phenotype, may play a structural role by preventing eDNA degradation by nucleases and provide a bridging function between eDNA strands.

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (e_MVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the e_MVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of death by cancer in the world. What makes this pathological condition particularly lethal is a combination of clinical and molecular heterogeneity, lack of early diagnostic indexes, and underwhelming results from current therapeutic protocols. A major cause of PDAC chemoresistance seems to lie in the ability of cancer cells to spread out and fill the pancreatic parenchyma, exchanging nutrients, substrates, and even genetic material with cells from the surrounding tumor microenvironment (TME). Several components can be found in the TME ultrastructure, including collagen fibers, cancer-associated fibroblasts, macrophages, neutrophils, mast cells, and lymphocytes. Cross-talk between PDAC and TME cells results in the latter being converted into cancer-favoring phenotypes; this behavior could be compared to an influencer guiding followers into supporting his activity. Moreover, TME could be a potential target for some of the newest therapeutic strategies; these include the use of pegvorhyaluronidase-α and CAR-T lymphocytes against HER2, FAP, CEA, MLSN, PSCA, and CD133. Other experimental therapy options are being currently studied, aiming to interfere with the KRAS pathway, DNA-repairing proteins, and apoptosis resistance in PDAC cells. Hopefully these new approaches will grant better clinical outcomes in future patients.

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Normal epithelial thyroid cells in culture are inhibited by TGF-1. Instead, transformed thyroid cell lines are frequently resistant to its growth inhibitory effect. Loss of TGF- responsiveness could be due to a reduced expression of TGF- receptors, as shown in transformed rat thyroid cell lines and in human thyroid tumors, or to alterations of other genes controlling TGF- signal transduction pathway. However, in thyroid neoplasia, a complex pattern of alterations occurring during transformation and progression has been identified. Functionally, TGF-1 acts as a tumor suppressor in the early stage of transformation or as a tumor promoter in advanced cancer. This peculiar pleiotropic behaviour of TGF- may result from cross-talk with signalling pathways mediated by other growth factors, among which EGF-like ligands play an important role. This paper reports evidences on TGF-1 and EGF systems in thyroid tumors and on the cross-talk between these growth factors in thyroid cancer.

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.