Tricarbonyl rhenium(i) complexes with 8-hydroxyquinolines: structural, chemical, antibacterial, and anticancer characteristics.

Twelve tricarbonyl rhenium(i) complexes in the '2 + 1' system with the anionic bidentate N,O-donor ligand (deprotonated 8-hydroxyquinoline (HQ) or its 2-methyl (MeHQ) or 5-chloro (ClHQ) derivative) and neutral N-donor diazoles (imidazole (Him), 2-methylimidazole (MeHim), 3,5-dimethylpyrazole (Hdmpz), and 3-phenylpyrazole (HPhpz)) were synthesized: [Re(CO)3(LN,O)LN] (LN,O = Q-, MeQ-, ClQ-; LN = Him, MeHim, Hdmpz, HPhpz). Their crystal structures were determined by the scXRD method, compared with the DFT-calculated ones, and characterized by analytical (EA) and spectroscopic techniques (FT-IR, NMR, and UV-Vis) interpreted with DFT and TD-DFT calculations. Most of the Re(i) complexes did not show relevant antibacterial activity against Gram-negative and Gram-positive bacterial strains. Only [Re(CO)3(MeQ)Him] demonstrated significant action 4-fold better against Gram-negative Pseudomonas aeruginosa than the free MeHQ ligand. The cytotoxicity of the compounds was estimated using human acute promyelocytic leukemia (HL-60), ovarian (SKOV-3), prostate (PC-3), and breast (MCF-7) cancer, and breast non-cancerous (MCF-10A) cell lines. Only HQ and ClHQ ligands and [Re(CO)3(Q)Hdmpz] complex had good selectivity toward MCF-7 cell line. HL-60 cells were sensitive to all complexes (IC50 = 1.5-14 µM). Still, pure HQ and ClHQ ligands were slightly more active than the complexes.

New 5-Substituted SN38 Derivatives: A Stability Study and Interaction with Model Nicked DNA by NMR and Molecular Modeling Methods.

The new 5-substituted SN-38 derivatives, 5(R)-(N-pyrrolidinyl)methyl-7-ethyl-10-hydroxycamptothecin (1) and its diastereomer 5(S) (2), were investigated using a combination of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods. The chemical stability, configuration stability, and propensity to aggregate as a function of concentration were determined using 1H NMR. The calculated self-association constants (Ka) were found to be 6.4 mM-1 and 2.9 mM-1 for 1 and 2, respectively. The NMR experiments were performed to elucidate the interaction of each diastereomer with a nicked decamer duplex, referred to as 3. The calculated binding constants were determined to be 76 mM-1 and 150 mM-1 for the 1-3 and 2-3 complexes, respectively. NMR studies revealed that the interaction between 1 or 2 and the nicked decamer duplex occurred at the site of the DNA strand break. To complement these findings, molecular modeling methods and calculation protocols were employed to establish the interaction mode and binding constants and to generate molecular models of the DNA/ligand complexes.

Identification and structural characterization of three psychoactive substances, phenylpiperazines (pBPP and 3,4-CFPP) and a cocaine analogue (troparil), in collected samples.

PURPOSE: New psychoactive substances (NPSs) still appear on the market, mainly due to their legal status. This situation indicates and alarms that permanent recognition of the designer drug scene should be conducted. In this paper, we describe the detection of three psychoactive substances in samples collected from drug users. METHODS: Qualitative characterization was performed using liquid chromatography-high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyzer, gas chromatography with mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: In this study, we reported the detection and structural elucidation of three psychoactive substances: 1-(4-bromophenyl)piperazine (pBPP), 1-(3-chloro-4-fluorophenyl)piperazine (3,4-CFPP) and methyl 8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-4-carboxylate (troparil). CONCLUSIONS: To the best of our knowledge, this is the first report that presents an identification methodology for these substances found in illegal products. Comprehensive characterization of the NPSs presented in this paper facilitates their detection and identification by forensic and clinical laboratories.

Insight on the Interaction between the Camptothecin Derivative and DNA Oligomer Mimicking the Target of Topo I Inhibitors.

The understanding of the mechanism of Topo I inhibition by organic ligands is a crucial source of information that has led to the design of more effective and safe pharmaceuticals in oncological chemotherapy. The vast number of inhibitors that have been studied in this respect over the last decades have enabled the creation of a concept of an 'interfacial inhibitor', thereby describing the machinery of Topo I inhibition. The central module of action of this machinery is the interface of a Topo I/DNA/inhibitor ternary complex. Most of the 'interfacial inhibitors' are primarily kinetic inhibitors that form molecular complexes with an "on-off" rate timing; therefore, all of the contacts between the inhibitor and both the enzyme and the DNA are essential to keep the complex stable and reduce the "off rate". To test this hypothesis, we designed the compound using a C-9-(N-(2'-hydroxyethyl)amino)methyl substituent in an SN38 core, with a view that a flexible substituent may bind inside the nick of a model of the DNA and stabilize the complex, leading to a reduction in the "off rate" of a ligand in a potential ternary complex in vivo. Using docking analysis and molecular dynamics, free energy calculations on the level of the MM-PBSA and MM-GBSA model, here we presented the in silico-calculated structure of a ternary complex involving the studied compound 1. This confirmed our suggestion that compound 1 is situated in a groove of the nicked DNA model in a few conformations. The number of hydrogen bonds between the components of a ternary complex was established, which strengthens the complex and supports our view. The docking analysis and free energy calculations for the receptor structures which were obtained in the MD simulations of the ternary complex 1/DNA/Topo I show that the binding constant is stronger than it was for similar complexes with TPT, CPT, and SN38, which are commonly considered as strong Topo I inhibitors. The binary complex structure 1/DNA was calculated and compared with the experimental results of a complex that was in a solution. The analysis of the cross-peaks in NOESY spectra allowed us to assign the dipolar interactions between the given protons in the calculated structures. A DOSY experiment in the solution confirmed the strong binding of a ligand in a binary complex, having a Ka of 746 mM-1, which was compared with a Ka of 3.78 mM-1 for TPT. The MALDI-ToF MS showed the presence of the biohybrid, thus evidencing the occurrence of DNA alkylation by compound 1. Because of it having a strong molecular complex, alkylation is the most efficient way to reduce the "on-off" timing as it acts as a tool that causes the cog to brake in a working gear, and this is this activity we want to highlight in our contribution. Finally, the Topo I inhibition test showed a lower IC50 of the studied compound than it did for CPT and SN38.

Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers.

Novel nontoxic derivatives of SN38 with favorable antineoplastic properties were characterized in water solution using NMR. The phenomena observed by NMR were linked to basic pharmacological properties, such as solubility, bioavailability, chemical and stereochemical stability, and binding to natural DNA oligomers through the terminal G-C base pair, which is commonly considered a biological target of Topo I inhibitors. Compound 1, with bulky substituents at both C5(R) and C20(S) on the same side of a camptothecin core, manifests self-association, whereas diastereomers 2, with bulky C5(S) and C20(S) substituents are mostly monomeric in solution. The stereogenic center at C5 is stable in water solution at pH 5-6. The compound with an (N-azetidinyl)methyl substituent at C9 can undergo the retro Mannich reaction after a prolonged time in water solution. Both diastereomers exhibit different abilities in terms of binding to DNA oligomers: compound 1 is strongly bound, whereas the binding of compound 2 is rather weak. Molecular modeling produced results consistent with NMR experiments. These complementary data allow linking of the observed phenomena in NMR experiments to basic preliminary information on the pharmacodynamic character of compounds and are essential for planning further development research.

Explanation of the Formation of Complexes between Representatives of Oxazolidinones and HDAS-ß-CD Using Molecular Modeling as a Complementary Technique to cEKC and NMR.

Molecular modeling (MM) results for tedizolid and radezolid with heptakis-(2,3-diacetyl-6-sulfo)-ß-cyclodextrin (HDAS-ß-CD) are presented and compared with the results previously obtained for linezolid and sutezolid. The mechanism of interaction of chiral oxazolidinone ligands belonging to a new class of antibacterial agents, such as linezolid, tedizolid, radezolid, and sutezolid, with HDAS-ß-CD based on capillary electrokinetic chromatography (cEKC), nuclear magnetic resonance (NMR) spectroscopy, and MM methods was described. Principles of chiral separation of oxazolidinone analogues using charged single isomer derivatives of cyclodextrin by the cEKC method were presented, including the selection of the optimal chiral selector and separation conditions, complex stoichiometry, and binding constants, which provided a comprehensive basis for MM studies. In turn, NMR provided, where possible, direct information on the geometry of the inclusion complexes and also provided the necessary structural information to validate the MM calculations. Consequently, MM contributed to the understanding of the structure of diastereomeric complexes, the thermodynamics of complexation, and the visualization of their structures. The most probable mean geometries of the studied supramolecular complexes and their dynamics (geometry changes over time) were determined by molecular dynamics methods. Oxazolidinone ligands have been shown to complex mainly the inner part of cyclodextrin, while the external binding is less privileged, which is consistent with the conclusions of the NMR studies. Enthalpy values of binding of complexes were calculated using long-term molecular dynamics in explicit water as well as using molecular mechanics, the Poisson-Boltzmann or generalized Born, and surface area continuum solvation (MM/PBSA and MM/GBSA) methods. Computational methods predicted the effect of changes in pH and composition of the solution on the strength and complexation process, and it adapted the conditions selected as optimal during the cEKC study. By changing the dielectric constant in the MM/PBSA and MM/GBSA calculations, the effect of changing the solution to methanol/acetonitrile was investigated. A fairly successful attempt was made to predict the chiral separation of the oxazolidinones using the modified cyclodextrin by computational methods.

Comprehensive Characterisation of the Ketoprofen-ß-Cyclodextrin Inclusion Complex Using X-ray Techniques and NMR Spectroscopy.

Racemic ketoprofen (KP) and ß-cyclodextrin (ß-CD) powder samples from co-precipitation (1), evaporation (2), and heating-under-reflux (3) were analysed using X-ray techniques and nuclear magnetic resonance (NMR) spectroscopy. On the basis of NMR studies carried out in an aqueous solution, it was found that in the samples obtained by methods 1 and 2, there were large excesses of ß-CD in relation to KP, 10 and 75 times, respectively, while the sample obtained by method 3 contained equimolar amounts of ß-CD and KP. NMR results indicated that KP/ß-CD inclusion complexes were formed and the estimated binding constants were approximately 2400 M-1, showing that KP is quite strongly associated with ß-CD. On the other hand, the X-ray single-crystal technique in the solid state revealed that the (S)-KP/ß-CD inclusion complex with a stoichiometry of 2:2 was obtained as a result of heating-under-reflux, for which the crystal and molecular structure were examined. Among the methods used for the preparation of the KP/ß-CD complex, only method 3 is suitable.

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

Hunt Trials as a Measure to Assess Level of Training in Boarhounds.

Boarhounds are hunting dogs bred for hunting wild boar, including terriers, dachshunds, and hounds. Hunt trials evaluate the individual hunting potential and trainability of the boarhounds in ten different competitions. The aim of this study was to determine the factors influencing the hunt trials for boarhounds in a large cohort of hunting dogs. The analysis was conducted based on the results of hunt trials for boarhounds conducted in 2005-2015. The database contained 1867 individuals belonging to 39 breeds. Effects of sex, age, breed group, and breed were estimated by non-parametric analysis of variance. Sex influenced (p < 0.01) the total score, and in almost all competitions dogs performed better than bitches. Age affected (p < 0.01 or p < 0.05) all competitions, indicating that the dogs perform better with age. The results analyzed by the breed group showed that the dachshunds performed better in courage (p < 0.01) and searching (p < 0.05). Breed influenced (p < 0.01) almost all scores except obedience and tracking on the lead. The best performing breed was Alpine Dachsbracke. In conclusion, all analyzed factors influenced the results of the hunt trials. The factors with the largest impact were breed and age, which reflect both the hunting potential and the level of training of the boarhounds.

New camptothecin derivatives for generalized oncological chemotherapy: Synthesis, stereochemistry and biology.

Derivatives of SN38 were synthesized that were either monosubstituted at C-5 or C-9 or disubstituted at both C-5 and C-9. Substitution to C-5 led to the generation of pairs of diastereomers (2c-2 h) in a one-pot reaction and was readily separable by HPLC. The absolute configurations of C-5 were established by electronic circular dichroism experiments. Compounds were tested in vitro against human cancer cell lines as well as a normal cell line. The impact of compounds 2a-2j on cancer cells is significant and the IC50 values against the normal cell line are several times higher than that of SN38. Using the Mannich reaction we obtained a new innovative group of derivatives with unique biological properties that preserves the high cytotoxicity in cancer cells and eliminates the acute toxicity to non-neoplastic cells, which can be considered a breakthrough in chemotherapy with the use of topoisomerase I inhibitors from the camptothecin family.

A NMR study of binding the metabolite of SN38 derivatives to a model nicked DNA decamer mimicking target of Topo I inhibitors.

In this account we present NMR based results of the interaction of 7-ethyl-9-hydroxymethyl-10-hydroxycamptothecin (1), a derivative of SN38, with a model nicked DNA decamer mimicking the wild type DNA target of Topoisomerase I inhibitors from the camptothecin family. The title compound 1 can be considered a main metabolite of phase I in the metabolic pathway of camptothecin derivatives bearing the alkylamino substituent. Therefore, its pharmacodynamic properties are of interest. It was established by DOSY (Diffusion Ordered Spectroscopy) that compound 1 forms a fairly stable molecular complex with a model nicked DNA decamer with affinity constant Ka 3.02 mM-1. The analysis of NOESY experiments revealed intermolecular cross peaks and mutual induced shifts on both interacting components allowing the conclusion that guest molecule 1 is stacking the nitrogen bases inside the nick. MD (Molecular Dynamics) analysis of four possible inclusions of 1 inside the nick allows establishing the detailed geometry of a complex. Two conformations are suggested as the ones best representing the results of molecular modeling reconciled with experimental NOESY results. The aromatic core of both structures is stacking the nitrogen bases in a nick facing the unbroken strand with ring A. The protons in ring E interact with ribose protons of edge bases of a nick. In conclusion, it can be asserted that SN38 derivative 1 can effectively bind the molecular target of Topo I enzyme and play a role as a Topo I inhibitor.

Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli.

PURPOSE: Identification of human insulin analogs' impurity with a mass shift +14 Da in comparison to a parent protein. METHODS: The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing. RESULTS: The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs. CONCLUSIONS: Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.

Enantioselective recognition of sutezolid by cyclodextrin modified non-aqueous capillary electrophoresis and explanation of complex formation by means of infrared spectroscopy, NMR and molecular modelling.

A method for the enantioseparation of sutezolid, the next analogue after linezolid and tedizolid, belonging to the truly new class of antibacterial agents, the oxazolidinones, was developed based on non-aqueous capillary electrophoresis (NACE), using a single isomer of cyclodextrins as a chiral pseudophase. During the experiment, the enantioseparation of sutezolid together with its predecessor, linezolid, both weak base antibacterial agents, was evaluated using anionic single-isomers of cyclodextrins from hydrophilic, up to hydrophobic: heptakis-(2,3-dihydroxy-6-sulfo)-ß-cyclodextrin, heptakis-(2,3-diacetyl-6-sulfo)-ß-cyclodextrin (HDAS-ß-CD), as well as heptakis-(2,3-dimethyl-6-sulfo)-ß-cyclodextrin (HDMS-ß-CD), respectively. Based on the observed results, the cyclodextrins, HDAS-ß-CD and HDMS-ß-CD which carry the acetyl and methyl groups at the C2 and C3 positions, respectively, provided the baseline separation of sutezolid enantiomers. However, HDMS-ß-CD led to a reversal of enantiomer migration order (EMO) in comparison to HDAS-ß-CD. Instead, enantiomers of linezolid were separated only by HDMS-ß-CD. During the experiments, different organic solvents and their mixtures in various ratios were tested. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the type of organic solvent, and the concentrations of trifluoroacetic acid (TFA) in the NACE buffer system. Focusing on the desired EMO in which the eutomers (S)-sutezolid and (S)-linezolid migrated last, the highest enantioresolution using the NACE method was achieved at normal polarity mode with 45 mM HDMS-ß-CD dissolved in MeOH/ACN (85:15, v/v) containing 200 mM TFA/20 mM ammonium formate. Moreover, infrared spectroscopy, NMR and molecular modelling were investigated to provide information about complex formation.

Nuclear magnetic resonance spectroscopic study of the inclusion complex of (R)-tedizolid with HDAS-ß-CD, ß-CD, and γ-cyclodextrin in aqueous solution.

NMR spectroscopy is used to investigate the host-guest complexation of (R)-tedizolid, such as tedizolid with the hydroxymethyl substituent at the C5 position of the oxazolidinone ring ((R)-TED) or tedizolid with 5-methyl dihydrogen phosphate ((R)-TED-PO4) with heptakis-(2,3-diacetyl-6-sulfo)-ß-cyclodextrin (HDAS-ß-CD), ß-CD and γ-CD, in particular to obtain information about the mode and strength of the guest complexation into the hydrophobic cavity of the host. The complex stoichiometries of 1:1 (host:guest) and 1:2 were detected in millimolar concentrations for HDAS-ß-CD and γ-CD with TED-PO4 complexes, respectively. In the meantime, the mixed of complexes with stoichiometries of 1:1 and 2:1 were found for ß-CD with both TED and TED-PO4, however the 1:1 complex had a significant advantage.The binding mode was proposed. The estimated binding constants Ka of the complexes of TED or TED-PO4 with CDs differ significantly in the order HDAS-ß-CD<<ß-CD<<γ-CD.

Insight into human insulin aggregation revisited using NMR derived translational diffusion parameters.

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

Comprehensive spectral identification of key intermediates to the final product of the chiral pool synthesis of radezolid.

Radezolid (RAD, 12), biaryl oxazolidinone, was synthesised with small modifications according to the methods described in the literature. The pharmacological activity is observed only for (S)-enantiomer, therefore its synthesis is oriented towards obtaining a single isomer of required purity and desired optical configuration. The intermediate products of RAD synthesis were characterised using 1H- and 13C-NMR, as well as the 2D correlation HSQC and HMBC (2, 5, 9, 10), furthermore studied using infrared radiation (FT-IR), Raman scattering (3, 5, 9), and electronic circular dichroism (ECD) (5, 12) spectroscopy. Each technique provides a unique and specific set of information. Hence, the full spectral characteristics of key intermediates obtained from the chiral pool synthesis to the finished product of RAD were summarised and compared. For a more accurate analysis, and due to the lack of reliable and reproducible reference standards for intermediate products, their vibrational analysis was supported by quantum chemical calculations based on the density functional theory (DFT) utilising the B3LYP hybrid functional and the 6-311G(d,p) basis set. Good agreement was observed between the empirical and theoretical spectra. Graphical abstract Comprehensive spectral identification (ECD, NMR, FT-IR, Raman) of key intermediates of the chiral pool synthesis of radezolid.

Application of spectroscopic methods (FT-IR, Raman, ECD and NMR) in studies of identification and optical purity of radezolid.

In the presented study, N-{[(5S)-3-(2-fluoro-4'-{[(1H-1,2,3-triazol-5-ylmethyl)amino]methyl}biphenyl-4-yl)-2-oxo-1,3-oxazolidin-5-yl]methyl}acetamide (radezolid) was synthesized and characterized using FT-IR, Raman, ECD and NMR. The aim of this work was to assess the possibility of applying classical spectral methods such as FT-IR, Raman, ECD and NMR spectroscopy for studies on the identification and optical purity of radezolid. The experimental interpretation of FT-IR and Raman spectra of radezolid was conducted in combination with theoretical studies. Density functional theory (DFT) with the B3LYP hybrid functional was used for obtaining radezolid spectra. Full identification was carried out by COSY, 1H {13C} HSQC and 1H {13C} HMBC experiments. The experimental NMR chemical shifts and spin-spin coupling constants were compared with theoretical calculations using the DFT method and B3LYP functional employing the 6-311++G(d,p) basis set and the solvent polarizable continuum model (PCM). The experimental ECD spectra of synthesized radezolid were compared with experimental spectra of the reference standard of radezolid. Theoretical calculations enabled us to conduct HOMO and LUMO analysis and molecular electrostatic potential maps were used to determine the active sites of microbiologically active form of radezolid enantiomer. The relationship between results of ab initio calculations and knowledge about chemical-biological properties of S-radezolid and other oxazolidinone derivatives are also discussed.

Preliminary study of mechanism of action of SN38 derivatives. Physicochemical data, evidence of interaction and alkylation of DNA octamer d(GCGATCGC)2.

The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC)2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd.