The Role of Extracellular Vesicles in the Progression of Human Neuroblastoma

The long-underestimated role of extracellular vesicles in cancer is now reconsidered worldwide by basic and clinical scientists, who recently highlighted novel and crucial activities of these moieties. Extracellular vesicles are now considered as king transporters of specific cargoes, including molecular components of parent cells, thus mediating a wide variety of cellular activities both in normal and neoplastic tissues. Here, we discuss the multifunctional activities and underlying mechanisms of extracellular vesicles in neuroblastoma, the most frequent common extra-cranial tumor in childhood. The ability of extracellular vesicles to cross-talk with different cells in the tumor microenvironment and to modulate an anti-tumor immune response, tumorigenesis, tumor growth, metastasis and drug resistance will be pinpointed in detail. The results obtained on the role of extracellular vesicles may represent a panel of suggestions potentially useful in practice, due to their involvement in the response to chemotherapy, and, moreover, their ability to predict resistance to standard therapies—all issues of clinical relevance.     

Conjugation of amino-bioactive glasses with 5-aminofluorescein as probe molecule for the development of pH sensitive stimuli-responsive biomaterials

Bioceramics, such as silica-based glasses, are widely used in bone and teeth restoration. Nowadays, the association between nanotechnology and pharmacology is one of the most promising research fields in cancer therapy. The advanced processing methods and new chemical strategies allow the incorporation of drugs within them or on their functionalized surfaces. Bioceramics can act as local drug delivery systems to treat bone and teeth diseases. The present paper reports data related to the development of a pH-stimuli responsive bioactive glass. The glass conjugation with 5-aminofluorescein (5-AF), through a pH-sensitive organic spacer, allows to produce a pH-responsive bioactive biomaterial: when it is exposed to specific pH changes, it can favour the release of 5-AF directly at the target site. 5-AF has been chosen as a simple, low cost, non toxic model to simulate doxorubicin, an anticancer drug. As doxorubicin, 5-AF contains an amino group in its structure in order to form an amide bond with the carboxylic functionalities of the glass. Raman spectroscopy and thermal analysis confirm the glass conjugation of 5-AF by means of an amide bond; the amount of 5-AF loaded was very high (≈65 and 44 wt%). The release tests at two different pH (4.2 and 7.4) show that the amount of released 5-AF is higher at acid pH with respect to physiological one. This preliminary datum evidenced that a pH-sensitive drug delivery system has been developed. The low amount of 5-AF released (<1 wt% of the total 5-AF) is due to the very low solubility of 5-AF in aqueous medium. This disadvantage, may be overcome in a dynamic environment (physiological conditions), where it is possible to obtain a drug release system ensuring an effective therapeutic dose for long times and, at the same time, avoiding the drug toxicity.     

gp 120s derived from four syncytium-inducing HIV-1 strains induce different patterns of CD4 association with lymphocyte surface molecules

This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.     

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.     

Performances of Pressure Reducing Valves in Variable Demand Conditions: Experimental Analysis and New Performance Parameters

Pressure Reducing Valves (PRV) play a critical role in Water Distribution Networks (WDN): they regulate pressure ensuring an efficient service to users and preventing damage to pipelines. In recent years, the attention of water utilities towards pressure management and leakage control led to the necessity of more flexible and responsive technologies that can guarantee a higher level of pressure control accuracy. Because of this the common performance parameters based on steady state conditions are no longer satisfactory to evaluate the effective behaviour of the devices when used in situations where demand can change. In the present paper the pressure control effectiveness of different types of PRV (electric actuated, pilot operated and direct acting) in variable demand conditions is discussed. The data used are from experimental tests, literature and field application. To assess valves’ pressure control performance, the use of new parameters, which consider the peak of pressure reached during control operations and the accuracy of target pressure regulation, has been proposed. The use of these parameters allows the comparison between different type of valves giving to WDN managers a direct overview on the valves ability to regulate pressure under variable demand conditions.

     

Upgrading the data acquisition and control systems of the European Breeding Blanket Test Facility

The EBBTF (European Breeding Blanket Test Facility) experimental plant is a key component for the development of the breeding blankets (TBMs test blanket modules, HCLL helium cooled lithium lead and HCPB helium cooled pebble bed types) used by ITER. EBBTF is an experimental plant which provides the double breeding/cooling loops (liquid metal and gas) required for HCLL testing. EBBTF is composed of four subsystems (TBM, IELLLO integrated European lead lithium loop, HE-FUS3 helium fusion loop, version 3 and helium compressor build by ATEKO) with dedicated control systems realized with hardware/software combinations covering 15 years (1995–2010) time span. At the end of 2010 we began to upgrade the HE-FUS3 data acquisition control systems (DACS) replacing the obsolete PLC Siemens S5 with National Instruments Compact FieldPoint and LabVIEW. The control room has been completely reorganized using high resolution monitors and workstations linked with standard Ethernet interfaces. The data acquisition, control, safety and SCADA software has been completely developed in ENEA using LabVIEW. In this paper we are going to discuss the technical difficulties and the solutions that we have used to accomplish the upgrade.     

Improving Operation of Pressurized Irrigation Systems by an Off-grid Control Devices Network

Water demand in irrigation is expected to increase in the near future, and it will be seriously impacted by climate change, specifically in semi-arid areas. The increase of water demand, along with the reduction of water availability, can seriously intensify the frequency and magnitude of pressure deficits in pressurized irrigation networks, with dangerous consequences on the healthy crop growth and on the crop production. In this framework, the present paper investigates the contribution of a network of smart control valves, named GreenValve System (GVS, patented by the Politecnico di Milano), to improve the management of a pressurized irrigation system. The GVS is able to recover energy for its operation from the flow, to be remotely commanded and to introduce management logics based on real-time data in order to create a stand-alone real-time monitoring and control network. In the paper, specifically, a three-step general and replicable methodological approach for the definition of installation and operating conditions for these valves is proposed. The effectiveness of a specific management logic, allowed by the use of the GVS, to limit pressure deficit and failure occurrence in the network is discussed. Reference is made to a case study on a critical on-demand irrigation network. The results showed that the introduction of simple management rules can reduce and even avoid the occurrence of hydrant failure, creating the conditions for more effective use of the resources.