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.     

Test Case Generation as an AI Planning Problem

While Artificial Intelligence techniques have been applied to a variety of software engineering applications, the area of automated software testing remains largely unexplored. Yet, test cases for certain types of systems (e.g., those with command language interfaces and transaction based systems) are similar to plans. We have exploited this similarity by constructing an automated test case generator with an AI planning system at its core. We compared the functionality and output of two systems, one based on Software Engineering techniques and the other on planning, for a real application: the StorageTek robot tape library command language. From this, we showed that AI planning is a viable technique for test case generation and that the two approaches are complementary in their capabilities.     

Analysis of optical surfaces by means of surface plasmonspectroscopy

The sensitivity to material parameters of surface plasmon oscillations is explored for the design of a prototype system operating at a wavelength of 670 nm, for the simultaneous determination of thickness, complex permittivity and surface roughness of metal films. The apparatus was employed for characterization of gold and silver films with thicknesses within the 10-100 nm range. Thickness data measured with the prototype system were well correlated with those measured on a conventional surface profilometer. The Fourier spectrum of surface irregularities extracted from scattered light, enhanced by surface plasmon oscillations, was used to determine surface roughness parameters for the distinct samples. Those parameters were shown to be representative of typical surface structures observed with the atomic force microscope     

TGF-beta-induced apoptosis of cerebellar granule neurons is prevented by depolarization

The regulation of programmed cell death in the developing nervous system involves target-derived survival factors, afferent synaptic activity, and hormone- and cytokine-dependent signaling. Cultured immature cerebellar granule neurons die by apoptosis within several days in vitro unless maintained in depolarizing (high) concentrations of potassium (25 mM K+). Here we report that transforming growth factors (TGF)-beta1, -beta2, and -beta3 accelerate apoptosis of these neurons when maintained in physiological (low) K+ medium (5mM K+) as assessed by measures of viability, quantitative DNA fragmentation, and nuclear morphology. TGF-beta-induced apoptosis of these neurons is not blocked by CNTF and LIF, cytokines that enhance neuronal survival when applied alone, or by IGF-I, which prevents apoptosis upon potassium withdrawal. In contrast, neurons that differentiate in high K+ medium for several days in vitro acquire resistance to TGF-beta-mediated cell death. Granule neurons maintained in either low or high K+ medium produce latent, but not bioactive, TGF-beta1 and -beta2. Because neutralizing TGF-beta antibodies fail to augment survival of low K+ neurons, the cerebellar neurons are apparently unable to activate latent TGF-beta. Thus, apoptosis of low K+ neurons is not attributable to endogenous production of TGF-beta. Taken together, our data suggest that TGF-beta may limit the expansion of postmitotic neuronal precursor populations by promoting their apoptosis but may support survival of those neurons that have maturated, differentiated, and established supportive synaptic connectivity.     

Defensive allomones in three species ofHypselodoris (gastropoda: Nudibranchia) from the Cantabrian sea

ThreeHypselodoris species,H. villafranca, H. cantabrica, and H. tricolor, have been studied in order to further investigate the defensive strategies of Chromodorididae mollusks from Iberian coasts. All animals possess large amounts of furanosesquiterpenoids, probably derived from their diet of sponges. The products are transferred and stored in specific sites of the mantle (mantle dermal formations, MDFs) to be used as chemical deterrents against predators. Chemical analysis of CantabrianHypselodoris species has led to the characterization of eight sesquiterpenoids. Among these, six have been found previously in both sponges and nudibranchs, while two are new natural products.Hypselodoris species cooccurring in the same area contained comparable mixtures of sesquiterpenoids. However, two populations ofH. cantabrica from different sites of the Cantabrian sea showed different metabolic patterns. All sesquiterpenoids induce feeding deterrence inCarassius auratus and two are also toxic toGambusia affinis.     

The Effects Of Humic Substances On Pinus Callus Are Reversed By 2,4-Dichlorophenoxyacetic Acid

The reversal of humic matter-induced inhibition of callus growth and metabolism by 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in Pinus laricio. Two forest humic fractions (relative molecular mass (M_r) > 3500), derived from soil under Fagus sylvatica (Fs) and Abies alba (Aa) plantation, were used. Pinus laricio callus was grown for a subculture period (4 weeks) on Basal Murashige and Skoog (MS) medium plus forest humic matters (Fs or Aa), at a concentration of 1 mg C/l, and then was transferred, for an additional four weeks, to a MS medium culture without humic matter, but with different hormones: indole-3-acetic acid (IAA, 2 mg/1) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/1) and/or 6-benzylaminopurine (BAP, 0.25 mg/1). Growth of calluse, glucose, fructose, and sucrose contents, and activities of soluble and bound invertases, glucokinase, phosphoglucose isomerase, aldolase, and pyruvate kinase were monitored. The results show a negative effect of humic fractions on callus growth, due to decreased utilization of glucose and fructose, and decreased activities of glycolytic enzymes. The effects are reversible. Substitution of humic fractions with 2,4-D+BAP or 2,4-D is followed by an increase of glycolytic enzyme activities and, consequently, by the utilization of glucose and fructose that induces a restart of growth. In contrast, the inhibitory effects of humic fractions persist when they are substituted with BAP alone, indicating that only the auxin 2,4-D is capable of reversing the negative effects. A possible competitive action on the auxin-binding site between 2,4-D and the chemical structures in the forest humic fractions is suggested.     

Comparing and experimenting machine learning techniques for code smell detection

Several code smell detection tools have been developed providing different results, because smells can be subjectively interpreted, and hence detected, in different ways. In this paper, we perform the largest experiment of applying machine learning algorithms to code smells to the best of our knowledge. We experiment 16 different machine-learning algorithms on four code smells (Data Class, Large Class, Feature Envy, Long Method) and 74 software systems, with 1986 manually validated code smell samples. We found that all algorithms achieved high performances in the cross-validation data set, yet the highest performances were obtained by J48 and Random Forest, while the worst performance were achieved by support vector machines. However, the lower prevalence of code smells, i.e., imbalanced data, in the entire data set caused varying performances that need to be addressed in the future studies. We conclude that the application of machine learning to the detection of these code smells can provide high accuracy (>96 %), and only a hundred training examples are needed to reach at least 95 % accuracy.     

Engineered Multifunctional Albumin‐Decorated Porous Silicon Nanoparticles for FcRn Translocation of Insulin

The last decade has seen remarkable advances in the development of drug delivery systems as alternative to parenteral injection‐based delivery of insulin. Neonatal Fc receptor (FcRn)‐mediated transcytosis has been recently proposed as a strategy to increase the transport of drugs across the intestinal epithelium. FcRn‐targeted nanoparticles (NPs) could hijack the FcRn transcytotic pathway and cross the epithelial cell layer. In this study, a novel nanoparticulate system for insulin delivery based on porous silicon NPs is proposed. After surface conjugation with albumin and loading with insulin, the NPs are encapsulated into a pH‐responsive polymeric particle by nanoprecipitation. The developed NP formulation shows controlled size and homogeneous size distribution. Transmission electron microscopy (TEM) images show successful encapsulation of the NPs into pH‐sensitive polymeric particles. No insulin release is detected at acidic conditions, but a controlled release profile is observed at intestinal pH. Toxicity studies show high compatibility of the NPs with intestinal cells. In vitro insulin permeation across the intestinal epithelium shows approximately fivefold increase when insulin is loaded into FcRn‐targeted NPs. Overall, these FcRn‐targeted NPs offer a toolbox in the development of targeted therapies for oral delivery of insulin.     

Minimally invasive cardiac surgery

In a short period of time, tremendous progress has been made in the field of minimally invasive cardiac surgery. The goal of true "minimally invasive" cardiac surgery should be a totally thoracoscopic or transvascular procedure that allows a very short hospital stay and prompt patient recovery at acceptable costs. To accomplish this goal, efforts to miniaturize and refine instrumentation/cannulae/visualization and the development of innovative new techniques must be pursued. The concept of performing intracardiac repair of a variety of pathologies on the beating heart is under investigation and will require a new generation of technology, which will include through-blood imaging, such as blood displacement videoscopes, and task-specific instruments. Robotics may offer additional assistance in the performance of complex and fine maneuvers.