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.     

Design and evaluation of a mobile smart home interactive system with elderly users in Brazil

Personal and Ubiquitous Computing - This article presents a study concerning the evaluation of a smart home control system for elderly people with a sample of 10 users in a city in the interior of...     

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.     

Thermoelectric Converters of Human Warmth for Self-Powered Wireless Sensor Nodes

Solar cells are the most commonly used devices in customer products to achieve power autonomy. This paper discusses a complementary approach to provide power autonomy to devices on a human body, i.e., thermoelectric conversion of human heat. In indoor applications, thermoelectric converters on the skin can provide more power per square centimeter than solar cells, particularly in adverse illumination conditions. Moreover, they work day and night. The first sensor nodes powered by human heat have been demonstrated and tested on people in 2004-2005. They used the state-of-the-art 100-muW watch-size thermoelectric wrist generators fabricated at IMEC and based on custom-design small-size BiTe thermopiles. The sensor node is completed with a power conditioning module, a microcontroller, and a wireless transceiver mounted on a watchstrap     

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI)

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.     

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.     

Fibroblast protein profile analysis highlights the role of oxidative stress and vitamin K recycling in the pathogenesis of pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a genetic disorder associated to mutations in the ABCC6 gene; however, the pathogenetic mechanisms leading to elastic fibre calcifications and to clinical manifestations are still unknown. Dermal fibroblasts, directly involved in the production of the extracellular milieu, have been isolated from healthy subjects and from patients affected by PXE, cultured in vitro and characterized for their ability to produce reactive oxygen species, for structural and functional properties of their cell membranes, for changes in their protein profile. Data demonstrate that oxidative stress has profound and endurable consequences on PXE fibroblast phenotype being responsible for: reduced levels of global DNA methylation, increased amount of carbonylated proteins and of lipid peroxidation products, altered structural properties of cell membranes, modified protein expression. Data shed new light on the pathogenetic pathways in PXE, by identifying a network of proteins affecting elastic fibre calcification through inefficient vitamin K recycling, and highlight the role of differentially expressed proteins as targets for validating the efficacy of future therapeutic strategies aiming to delay and/or revert the pathologic phenotype of PXE fibroblasts. Moreover, data open new perspectives for investigating PXE-like phenotypes in the absence of ABCC6 mutations.