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.     

Discovery of Affinity-Based Probes for Btk Occupancy Assays

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.     

Influence of device engineering on the analog and RF performances of SOI MOSFETs

This work presents a systematic comparative study of the influence of various process options on the analog and RF properties of fully depleted (FD) silicon-on-insulator (SOI), partially depleted (PD) SOI, and bulk MOSFET's with gate lengths down to 0.08 /spl mu/m. We introduce the transconductance-over-drain current ratio and Early voltage as key figures of merits for the analog MOS performance and the gain and the transition and maximum frequencies for RF performances and link them to device engineering. Specifically, we investigate the effects of HALO implantation in FD, PD, and bulk devices, of film thickness in FD, of substrate doping in SOI, and of nonstandard channel engineering (i.e., asymmetric Graded-channel MOSFETs and gate-body contacted DTMOS).     

An integrated system to remote monitor and control anaerobic wastewater treatment plants through the internet.

The TELEMAC project brings new methodologies from the Information and Science Technologies field to the world of water treatment. TELEMAC offers an advanced remote management system which adapts to most of the anaerobic wastewater treatment plants that do not benefit from a local expert in wastewater treatment. The TELEMAC system takes advantage of new sensors to better monitor the process dynamics and to run automatic controllers that stabilise the treatment plant, meet the depollution requirements and provide a biogas quality suitable for cogeneration. If the automatic system detects a failure which cannot be solved automatically or locally by a technician, then an expert from the TELEMAC Control Centre is contacted via the internet and manages the problem.     

Carbon mineralization from composts and food industry wastes added to soil

We have studied the short term C mineralization of six wastes from important food industries, one sludge from a biogas plant and three composts. All the wastes were characterized chemically and fractionated according to the Van Soest method. The fresh wastes were incubated under controlled environment conditions to determine the C mineralization rate. Based on first order mineralization kinetics, we calculated the hypothetical amount of stable C in the wastes as the amount of C that would not be mineralized within one year under field conditions. The percentage of stable organic C in the organic matter was in general much larger in the composts than in the other wastes, but when expressed on dry matter, the non-composted wastes had comparable or larger amounts of stable organic C than the composts and have a considerable potential for supplying organic matter to soils, and hence for C sequestration. The amount of stable organic C could best be predicted by the total N content of the wastes (R _a ² = 0.855), whereas the results of the fractionation had very little predictive power, probably due to problems related to the high ash content of some of the wastes. An index that combined stable organic C and N and P content in the wastes was calculated to assess possible limitations for applying these wastes in agriculture. Under current nutrient legislation in Western Europe, a number of these wastes will only be usable in small amounts, but these and other food industry wastes could still prove to be valuable soil amendments in nutrient poor situations, for increasing soil organic C content and supplying nutrients.     

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.     

Controlled pore size, thickness and surface free energy of super-hydrophobic PVDF® and Hyflon®AD membranes

Super-hydrophobic membranes were manufactured by using two per-fluorinated polymers such as PVDF and Hyflon AD. The combination of controlled structure and supra-molecular chemistry made these membranes ideal interfaces to be used in membrane contactors.     

Implementation of a real option in a sustainable supply chain: an empirical study of alkaline battery recycling

Green supply chain management (GSCM) has emerged as a key approach for enterprises seeking to become environmentally sustainable. This paper aims to evaluate and describe the advantages of a GSCM approach by analysing practices and performance consequences in the battery recycling sector. It seeks to integrate works in supply chain management (SCM), environmental management, performance management and real option (RO) theory into one framework. In particular, life cycle assessment (LCA) is applied to evaluate the environmental impact of a battery recycling plant project, and life cycle costing (LCC) is applied to evaluate its economic impact. Firms, also understanding the relevance of GSCM, have often avoided applying the green principles because of the elevated costs that such management involved. Such costs could also seem superior to the potential advantages since standard performance measurement systems are internally and business focused; for these reasons, we consider all the possible value deriving also by uncertainty associated to a green project using the RO theory. This work is one of the few and pioneering efforts to investigate GSCM practices in the battery recycling sector.