Discovery of selective dengue virus inhibitors using combination of molecular fingerprint-based virtual screening protocols,structure-based pharmacophore model development,molecular dynamics simulations and in vitro studies

Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1 log PFU/ml reduction of the virus infectious titre.     

Poly(2-hydroxyethylmethacrylate-co-2-folate ethylmethacrylate) and Folic acid/Poly(2-hydroxyethylmethacrylate) Solid Solution: Preparation and Drug Release Investigation

Poly(2-hydroxyethylmethacrylate-co-2-folatethylmethacrylate) was synthesized by free radical polymerization of 2-hydroxyethylmethacrylate with 2-folatethylmethacrylate and folic acid/poly(2-hydroxyethylmethacrylate) solid solutions was prepared by mixing folic acid with poly(2-hydroxyethylmethacrylate) using the solution casting method. The structure and the homogeneity distribution of folic acid in the polymer matrix are characterized by different methods. The diffusion behaviors of water and folic acid through poly(2-hydroxyethylmethacrylate-co-2-folatethylmethacrylate) and poly(2-hydroxyethylmethacrylate) matrixes were found obey to the Fick models. The in vitro cytotoxicity assessed by microculture tetrazolium test assay and the antioxidant activity of poly(2-hydroxyethylmethacrylate-co-2-folatethylmethacrylate) systems determined by 1,1-diphenyl-2-picryl-hydrazyl method revealed no significant toxicity of these systems and has excellent free radical scavenger property which can be as safe candidate in drug-carrier system. The solubility enhancement of folic acid in different pH media is also investigated and the results obtained reveal a maximum of 399–400?mg?L^?1. The release dynamic of folic acid from the poly(2-hydroxyethylmethacrylate-co-2-folatethylmethacrylate) containing 5?mol% of folic acid and that from folic acid/poly(2-hydroxyethylmethacrylate) system containing 10?mol% of folic acid have the best intestine/stomach ratio.     

Fabrication and characterization of hybrid coating on Mg–Zn–Ca Mg alloy for enhanced corrosion and degradation resistance as medical implant

Magnesium (Mg)-based alloys have appealing properties as promising implants for medical applications. However, their clinical applications are hindered due to the rapid corrosion and degradation rate in the physiological environment. In this investigation, we reported a novel interfacial engineering approach for the fabrication of polymer/ceramic hybrid coating on Mg–Zn–Ca Mg alloy. Firstly, hydroxyapatite (HA) coating was fabricated on the Mg–Zn–Ca sample followed by an alkali treatment that was performed in 1 M NaOH solution at 60 °C. Finally, polycaprolactone (PCL) coating was synthesized using a dip-coating approach on the top of the HA-coated Mg–Zn–Ca specimen. Microhardness test and adhesion test revealed that PCL/HA hybrid coating significantly improved mechanical properties and enhanced biointerface property between the substrate and coating. The immersion tests showed that the hybrid coating considerably slowed down the degradation in the simulated body fluid (SBF) solution. In addition, in vitro electrochemical investigations confirmed that PCL/HA coating significantly improved corrosion resistance and greatly reduced corrosion rate by about 10 times compared to HA coating and about 900 times to untreated Mg–Zn–Ca sample. Moreover, cytotoxicity assessment exhibited PCL/HA hybrid coating enhanced biocompatibility and bioactivity due to adopting a suitable interfacial engineering approach.     

Cytotoxicity of Ti–6Al–4V–5Cu Alloy to MC3T3-E1 Cells

The aim of this work was to study the cytotoxicity of Ti–6 Al–4 V–5 Cu(TC4–Cu) alloy for dental applications and evaluate the cell viability of diff erent fabricated TC4–Cu alloys in contact with MC3 T3-E1 cells in vitro. Ti–6 Al–4 V(TC4) alloy was used as a negative control to evaluate the cytotoxicity level of TC4–Cu alloy so as to provide basic support for the dental clinical application. Control group TC4 and experimental group TC4–Cu with diff erent fabrications were incubated in the cell culture medium. The absorbance value of mouse osteoblast MC3 T3-E1 cells was detected by CCK-8 assay after 1, 3 and 7 days, respectively. The relative proliferation rate of cells was calculated, and then the toxicity level was valued for each group. Cell morphology on the surface was also studied by observing the cytoskeleton through F-actin filament staining. The experimental results showed that the absorbance values for the experimental and the negative control groups were not same at diff erent time points. Compared with diff erent fabricated TC4–Cu alloys, the annealing-TC4–Cu alloy showed much better biocompatibility. The mouse osteoblast MC3 T3-E1 cells cultured with annealing-TC4–Cu, rolling-TC4–Cu and solution + aging-TC4–Cu have no toxic eff ects, and these alloys could promote the proliferation of mouse osteoblasts.     

Research and characterization of pathogenic vibrios from bathing water along the Conero Riviera (Central Italy)

The occurrence and pathogenicity of vibrios in bathing water were investigated along the Conero Riviera (Adriatic Sea, Central Italy). Vibrio spp. enumeration was performed on thiosulfate-citrate-bile-salts-sucrose-agar by the membrane filter method, and identification was done through a biochemical protocol. All isolates were tested for the presence of cytotoxicity, protease, lipase, elastase, gelatinase, urease, haemolytic activity, ctx, tdh and trh genes by conventional methods. In all, 200 vibrios were isolated from 132 samples that were analysed. Vibrio harveyi and Vibrio alginolyticus were the species most frequently recovered. All strains were cytotoxic and some of them showed protease, gelatinase, lipase, elastase, urease and haemolytic activity. One isolate of V. alginolyticus and one of V. harveyi had the trh gene, while another strain of V. harveyi and one of Vibrio parahaemolyticus had the ctx gene. These results demonstrate the presence of potentially pathogenic vibrios in the Conero Riviera and the risk of infection due to bathing water exposure.     

In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles

The recent advances in nanotechnology and the corresponding popular usage of nanomaterials have resulted in uncertainties regarding their environmental impacts. In this study, we used a systematic approach to study and compare the in vitro cytotoxicity of selected engineered metal oxide nanoparticles to the test organisms — E. coli. Among the seven test nano-sized metal oxides, ZnO, CuO, Al₂O_3, La₂O_3, Fe₂O_3, SnO₂ and TiO_2, ZnO showed the lowest LD₅₀ of 21.1 mg/L and TiO₂ had the highest LD₅₀ of 1104.8 mg/L. Data of ¹⁴C-glucose mineralization test paralleled the results of bacteria viability test. After regression calculation, the cytotoxicity was found to be correlated with cation charges (R² = 0.9785). The higher the cation charge is, the lower the cytotoxicity of the nano-sized metal oxide becomes. To the best of our knowledge, this finding is the first report in nanotoxicology.     

Lycopene-rich fractions derived from pink guava by-product and their potential activity towards hydrogen peroxide-induced cellular and DNA damage

Effects of solvent and supercritical carbon dioxide (SC-CO₂) extraction on antioxidant and cytotoxic activities of lycopene-rich fractions of decanted pink guava by-product (decanter) were determined with lycopene-equivalent antioxidant capacity, β-carotene bleaching and MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assays. Extraction with SC-CO₂ gave a higher yield than solvent extraction (3.15 vs. 0.68 mg/100 g dried decanter, corresponding to 42.99 and 33.63 mg of lycopene). No cytotoxicity was found in Chang liver cells supplemented with either extracts (6.25–200 μg/ml). Solvent extract at 25 μg/ml (2.32 μM lycopene) and SC-CO₂ extract at 200 μg/ml (5.09 μM lycopene) had protective effect against hydrogen peroxide-induced cytotoxicity. However, only high concentrations of solvent extract (200 μg/ml; lycopene = 18.65 μM) or lycopene standard (10 μM) protected cells against DNA damage. Supercritical fluid extraction demonstrated a higher yield in lycopene-rich fraction from decanter. These fractions have the potential to be developed as a functional ingredient to prevent oxidative stress and other related diseases.     

In Vitro Inactivation of Herpes Virus by Ozone

Viruses represent a major threat to human health and might be transmitted by direct and indirect contact. Reducing the viral load, either in the host or in the environment greatly reduces virus spreading. In this work we aimed to evaluate the virucidal activity of ozone against herpes virus of human (Herpes Simplex Virus 1 – HSV-1) and bovine (Bovine Herpes Virus 1 – BoHV-1) origin. The virucidal activity was measured by tittering aliquots of HSV-1 and BoHV-1 exposed for 1, 2, and 3 h to ozone generated by a domestic device. In addition, the possible cytotoxic effect of ozone to cultured MDBK cells was also assessed using the MTT method. MDBK cells exposed to ozone for 3 h and tested immediately after exposure, or after culturing for 24 h, had viability similar to non-exposed cells, indicating that ozone per se was not cytotoxic to the cells. Furthermore, a significant reduction in BoHV-1 (99.62%) and HSV-1 (90.0%) titer was observed after 3 h exposure to ozone. Our results indicate that ozone might be safely used to reduce environmental load of herpes virus.