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.     

Highly efficient preparation of free all-trans-astaxanthin from Haematococcus pluvialis extract by a rapid biocatalytic method based on crude extracellular enzyme extract

A highly efficient, rapid, green and safe procedure for the preparation of free all-trans-astaxanthin from Haematococcus pluvialis algal extract, by a crude extracellular enzyme extract, was reported. The free all-trans-astaxanthin obtained by the biocatalysed method had fewer side products compared to the saponification procedure. Through single-factor experiments and a Box–Behnken design, it was possible to find the optimal biocatalytic conditions for the hydrolysis of 2 mg of H. pluvialis oil with 14.7 mg (protein content) of lyophilised crude extracellular enzyme extract obtained from Pseudomonas aeruginosa. The reaction was carried out in 30 min at pH 9.16 and 36 °C, in 5.5 mL total reaction volume, under nitrogen atmosphere and dark conditions. The hydrolysis ratio of the astaxanthin esters was 98.72%, and the production of free all-trans-astaxanthin was 82.83 μg per mg of H. pluvialis oil. The method herein reported was simpler than other enzymatic methods previously described and allowed saving of time and costs.     

Pseudospectral method for assessing stability robustness for linear time-periodic delayed dynamical systems

The article presents a pseudospectral approach to assess the stability robustness of linear time-periodic delay systems, where periodic functions potentially present discontinuities and the delays may also periodically vary in time. The considered systems are subject to linear real-valued time-periodic uncertainties affecting the coefficient matrices, and the presented method is able to fully exploit structure and potential interdependencies among the uncertainties. The assessment of robustness relies on the computation of the pseudospectral radius of the monodromy operator, namely, the largest Floquet multiplier that the system can attain within a given range of perturbations. Instrumental to the adopted novel approach, a solver for the computation of Floquet multipliers is introduced, which results into the solution of a generalized eigenvalue problem which is linear w.r.t. (samples of) the original system matrices. We provide numerical simulations for popular applications modeled by time-periodic delay systems, such as the inverted pendulum subject to an act-and-wait controller, a single-degree-of-freedom milling model and a turning operation with spindle speed variation.     

Subspace Clustering for Situation Assessment in Aquatic Drones: A Sensitivity Analysis for State-Model Improvement

Abstract

In this paper, we propose the use of subspace clustering to detect the states of dynamical systems from sequences of observations. In particular, we generate sparse and interpretable models that relate the states of aquatic drones involved in autonomous water monitoring to the properties (e.g., statistical distribution) of data collected by drone sensors. The subspace clustering algorithm used is called SubCMedians. A quantitative experimental analysis is performed to investigate the connections between i) learning parameters and performance, ii) noise in the data and performance. The clustering obtained with this analysis outperforms those generated by previous approaches.     

Designer portfolio archetypes in design-intensive industries

ABSTRACT

The paper analyzes the designer portfolio configurations employed by firms, in design-intensive industries, to implement different product design strategies. Using the fuzzy set qualitative comparative analysis methodology, the paper explores how decorative lamps manufacturers, that first adopted the new LED technology, assembled their designer portfolios. The study shows that, in the early phases of LED lamps, four different designer portfolio archetypes were adopted by firms, two of them related to a product language divergence strategy and two to a product language convergence strategy: international design-star archetype, crowd design-innovator archetype, local ambassador archetype, international bridge archetype. These four archetypes are discussed, contributing to a better understanding of the relationship between product design strategies and designers’ management in design-intensive industries.     

Distribution of POPs,pesticides and antibiotic residues in organic honeys from different production areas

Demand for honey is increasing, especially if it is organic and if its nutritional properties are linked to untreated environments in order to guarantee quality for health. Sources of contamination of honey can be divided into environmental and apicultural. Therefore, the distribution of persistent organic pollutants, pesticides and antibiotic residues from geographical areas with different contamination sources (high anthropic impact, intensive farming, husbandry and low anthropic impact) was investigated in order to confirm the potential transfer of xenobiotics into the supply chain and to give beekeepers tools for the selection of areas dedicated to organic production. The presence of polychlorinated biphenyls, polybrominated diphenyl ether and polycyclic aromatic hydrocarbons was confirmed, not only in proximity to highly urbanised centres, where the concentrations were higher, but in all environment contexts, confirming their ubiquity. No antibiotics or neonicotinoids were detected in 95 organic honeys, demonstrating the absence of apicultural treatments and consequently the good quality of honey of different areas. These results are important due to the undefined regulatory European situation on honey antibiotic limits.     

Assessment of intradialysis calcium mass balance by a single pool variable‐volume calcium kinetic model

Introduction: A reliable method of intradialysis calcium mass balance quantification is far from been established. We herein investigated the use of a single‐pool variable‐volume Calcium kinetic model to assess calcium mass balance in chronic and stable dialysis patients. Methods: Thirty‐four patients on thrice‐weekly HD were studied during 240 dialysis sessions. All patients were dialyzed with a nominal total calcium concentration of 1.50 mmol/L. The main assumption of the model is that the calcium distribution volume is equal to the extracellular volume during dialysis. This hypothesis is assumed valid if measured and predicted end dialysis plasma water ionized calcium concentrations are equal. A difference between predicted and measured end‐dialysis ionized plasma water calcium concentration is a deviation on our main hypothesis, meaning that a substantial amount of calcium is exchanged between the extracellular volume and a nonmodeled compartment. Findings: The difference between predicted and measured values was 0.02 mmol/L (range ?0.08:0.16 mmol/L). With a mean ionized dialysate calcium concentration of 1.25 mmol/L, calcium mass balance was on average negative (mean ± SD ?0.84 ± 1.33 mmol, range ?5.42:2.75). Predialysis ionized plasma water concentration and total ultrafiltrate were the most important predictors of calcium mass balance. A significant mobilization of calcium from the extracellular pool to a nonmodeled pool was calculated in a group of patients. Discussion: The proposed single pool variable‐volume Calcium kinetic model is adequate for prediction and quantification of intradialysis calcium mass balance, it can evaluate the eventual calcium transfer outside the extracellular pool in clinical practice.     

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.     

Unpatterned Bioactive Poly(Butylene 1,4-Cyclohexanedicarboxylate)-Based Film Fast Induced Neuronal-Like Differentiation of Human Bone Marrow-Mesenchymal Stem Cells

Herein, we present poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE) films characterized by an unpatterned microstructure and a specific hydrophobicity, capable of boosting a drastic cytoskeleton architecture remodeling, culminating with the neuronal-like differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). We have used two different filming procedures to prepare the films, solvent casting (PBCE) and compression-moulding (PBCE*). PBCE film had a rough and porous surface with spherulite-like aggregations (Ø = 10–20 μm) and was characterized by a water contact angle = 100°. PBCE* showed a smooth and continuous surface without voids and visible spherulite-like aggregations and was more hydrophobic (WCA = 110°). Both surface characteristics were modulated through the copolymerization of different amounts of ether-oxygen-containing co-units into PBCE chemical structure. We showed that only the surface characteristics of PBCE-solvent-casted films steered hBM-MSCs toward a neuronal-like differentiation. hBM-MSCs lost their canonical mesenchymal morphology, acquired a neuronal polarized shape with a long cell protrusion (≥150 μm), expressed neuron-specific class III β-tubulin and microtubule-associated protein 2 neuronal markers, while nestin, a marker of uncommitted stem cells, was drastically silenced. These events were observed as early as 2-days after cell seeding. Of note, the phenomenon was totally absent on PBCE* film, as hBM-MSCs maintained the mesenchymal shape and behavior and did not express neuronal/glial markers.