Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.     

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC₅₀) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC₅₀ shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.     

Depassivation and repassivation of stainless steels by stepwise pH change

Immersion tests with different stainless steels have been performed, while the pH was stepwise decreased and then increased again. During 8.5-day exposure, the depassivation and repassivation pH values as a function of pitting resistance equivalent number were determined. There is always a gap between both pH values (depassivation and repassivation), indicating that for every steel, there are conditions where an existing passive layer can be maintained but cannot be rebuilt after depassivation. In such environments, the passive layer is thicker, consisting mainly of molybdenum and iron rich oxides, while chromium is dissolved. Usually, depending on conditions, the passive layer is more chromium-rich, especially the inner layer. This is relevant, for example, for acidizing jobs in oil and gas industry, proving that repassivation after acidizing will happen promptly, when the pH is increased again.     

Life-cycle assessment of hydrogen technologies with the focus on EU critical raw materials and end-of-life strategies

We present the results of a life-cycle assessment (LCA) for the manufacturing and end-of-life (EoL) phases of the following fuel-cell and hydrogen (FCH) technologies: alkaline water electrolyser (AWE), polymer-electrolyte-membrane water electrolyser (PEMWE), high-temperature (HT) and low-temperature (LT) polymer-electrolyte-membrane fuel cells (PEMFCs), together with the balance-of-plant components. New life-cycle inventories (LCIs), i.e., material inputs for the AWE, PEMWE and HT PEMFC are developed, whereas the existing LCI for the LT PEMFC is adopted from a previous EU-funded project. The LCA models for all four FCH technologies are created by modelling the manufacturing phase, followed by defining the EoL strategies and processes used and finally by assessing the effects of the EoL approach using environmental indicators. The effects are analysed with a stepwise approach, where the CML2001 assessment method is used to evaluate the environmental impacts. The results show that the environmental impacts of the manufacturing phase can be substantially reduced by using the proposed EoL strategies (i.e., recycled materials being used in the manufacturing phase and replacing some of the virgin materials). To point out the importance of critical materials (in this case, the platinum-group metals or PGMs) and their recycling strategies, further analyses were made. By comparing the EoL phase with and without the recycling of PGMs, an increase in the environmental impacts is observed, which is much greater in the case of both fuel-cell systems, because they contain a larger quantity of PGMs.     

Effect of expander shape on cooling characteristics by using a model pulse tube refrigerator

Pulse tube refrigerators do not have moving parts in the cold section, and they have low vibration, high reliability, and long life. The expander in refrigerators typically has an inverted U or coaxial shape because this attains a wider absorber area, lower height, and compactness. However, the performance of a Stirling-type pulse tube refrigerator is inferior to that of a Stirling refrigerator. Cooling characteristics of the pulse tube refrigerator greatly depend on the shape of the expander. In this study, an inertance-type refrigerator, which uses ambient air for the working gas, was developed to examine the effect of expander shape. This refrigerator model with changeable expander operated with a Stirling cycle, and it was composed of a reciprocating compressor, after-cooler, regenerator, absorber, pulse tube, hot-end, and inertance tube with reservoir. The following expander shapes were tested: in-line, L shape, L-L shape, and coaxial shape. The effect of expander shape on cooling capacity was examined experimentally and numerically using the model pulse tube refrigerator. The results of experiments showed that the L shape expander had the highest performance and the coaxial expander had the lowest performance. In addition, the characteristics of the gas flow in each expander were confirmed by fluid dynamics analysis.