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.     

Vertical ridge augmentation around dental implants using a membrane technique and autogenous bone or allografts in humans

This study investigated the effect on vertical bone regeneration of the addition of demineralized freeze-dried bone allograft or autogenous bone chips to a membrane technique. Twenty partially edentulous patients with vertical jawbone deficiencies were selected for this study. The patients were divided into two groups of 10 individuals. The 10 patients of Group A received 26 Br?nemark implants in 10 surgical sites. The 10 patients of Group B received 32 implants in 12 surgical sites. Fifty-two out of 58 implants (22 in Group A and 30 in Group B) extended 1.5 to 7.5 mm superior to the bone crest. Titanium-reinforced expanded polytetrafluoroethylene membranes were used to cover the implants and, before complete membrane fixation, demineralized freeze-dried bone allograft particles were condensed under the membrane in Group A, and autogenous bone chips were used in Group B. At the reentry after 7 to 11 months the membranes were removed and a small biopsy was collected from 11 sites comprehending the miniscrews. The clinical measurements from Group A demonstrated a mean vertical bone gain of 3.1 mm (SD = 0.9 mm, range 1 to 5 mm) with a mean percentage of bone gain of 124% (SD = 46.6%). The measurements from Group B showed a mean vertical bone gain of 5.02 mm (SD = 2.3 mm, range 1 to 8.5 mm) with a mean percentage of bone gain of 95% (SD = 26.8%). Histomorphometric analysis of the present study clearly demonstrated a direct correlation between the density of the pre-existing bone and the density of the regenerated bone. The mean percentage of new bone-titanium contact was from 39.1% to 63.2%, depending on the quality of the pre-existing bone. Both the clinical and histologic results indicate a beneficial effect of the addition of demineralized freeze-dried bone allograft or autogenous bone particles to vertical ridge augmentation procedures in humans.     

Detection of human papillomavirus DNA in laryngeal squamous cell carcinoma by polymerase chain reaction

Although human papillomaviruses (HPVs) have been found in many, but not all, tumours of the oral cavity, nose, pharynx and larynx, the true role of HPV in malignant tumours of the head and neck is still unclear. The presence of HPV DNA was investigated in 45 fresh squamous cell carcinoma (SCC) specimens and in 29 normal mucosa specimens collected from 45 primary laryngeal SCC patients. HPV DNA was detected using the polymerase chain reaction (PCR) with consensus primers that detect HPV types 6, 11, 16 and 18.9 of the 45 patients (20%) were HPV positive; the presence of HPV was also detected in the corresponding normal laryngeal mucosa of four of the 29 specimens (14%). No statistically significant differences were found between the presence of HPV DNA in normal specimens and in neoplastic mucosa specimens. No correlation was found between HPV DNA positive tumours and size, T classification, lymph node involvement and histological grading. This study adds further evidence suggesting a possible role of HPV DNA infection in laryngeal carcinogenesis.     

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.     

Structural characterization of Ti centres in Ti-silicalite and reaction mechanisms in cyclohexanone ammoximation

The main results obtained by means of many physical methods (IR, Raman, UV-Vis and XAFS spectroscopies) concerning the structure of the Ti centre in titanium silicalite and the reaction intermediates in the ammoximation of cyclohexanone are concisely reviewed.

The Ti is in tetrahedral coordination in vacuo and expands its coordination sphere upon interaction with adsorbates. In the presence of H₂O and H₂O/H₂O₂ solutions one of the SiOTi bridges is hydrolyzed with formation of (SiO)₃L₂TiOH (LH₂O) and (SiO)₃L₂TiOOH species, respectively. When NH₃ is dosed on (SiO)₃L₂TiOOH structures (SiO)₃L₂TiOO⁻NH₄⁺ species are formed. These species are thought to play an important role in the ammoximation reaction. The geometries of the peroxidic species (open or bridged) are discussed also on the basis of ab initio calculations.