Selective X‐ray contrast enhancement of the spleen of living mice mediated by gold nanorods

Gold nanomaterials (AuNPs) represent a promising new class of contrast agents for X‐ray computed tomographic (CT) imaging in both research and clinical settings. These materials exhibit superior X‐ray absorption properties compared with other iodinated agents, and thus require lower injection doses. Gold is nonimmunogenic and therefore contributes to safety profile in living specimens. Unfortunately, most reports on the use of AuNPs as X‐ray CT enhancers only demonstrate marginal enhancement of the intended anatomical structure. In this study, we demonstrate the dramatic properties of gold nanorods (GNR) to serve as robust X‐ray CT contrast‐enhancing agent for selective imaging of the spleen. These organ‐specific uptake properties were delineated by performing longitudinal CT imaging of living mice that were dosed with GNR at 2 day intervals. Rapid uptake in spleen was noted within 12 h of first systemic administration with a change in contrast enhancement of 90 Hounsfield units (ΔHU = 90) and with two subsequent injections a total contrast enhancement of over 200 HU was observed. The resulting images provide excellent contrast that will enable the detailed anatomical visualization and study of a range of pre‐clinical models of spleen disease including infection and cancer. Copyright © 2014 John Wiley & Sons, Ltd.     

Design of ProCAs (Protein‐Based Gd3+ MRI Contrast Agents) with High Dose Efficiency and Capability for Molecular Imaging of Cancer Biomarkers

Magnetic resonance imaging (MRI) is the leading imaging technique for disease diagnostics, providing high resolution, three‐dimensional images noninvasively. MRI contrast agents are designed to improve the contrast and sensitivity of MRI. However, current clinically used MRI contrast agents have relaxivities far below the theoretical upper limit, which largely prevent advancing molecular imaging of biomarkers with desired sensitivity and specificity. This review describes current progress in the development of a new class of protein‐based MRI contrast agents (ProCAs) with high relaxivity using protein design to optimize the parameters that govern relaxivity. Further, engineering with targeting moiety allows these contrast agents to be applicable for molecular imaging of prostate cancer biomarkers by MRI. The developed protein‐based contrast agents also exhibit additional in vitro and in vivo advantages for molecular imaging of disease biomarkers, such as high metal‐binding stability and selectivity, reduced toxicity, proper blood circulation time, and higher permeability in tumor tissue in addition to improved relaxivities.     

Influence of different iodinated contrast media on the induction of DNA double‐strand breaks after in vitro X‐ray irradiation

The objective of this work was to examine differences in DNA double‐strand break induction in peripheral blood lymphocytes after in vitro X‐ray irradiation between iodinated contrast agents. Four different iodinated X‐ray contrast agents – three of them with two different iodine concentrations – and mannitol (negative control; concentration of 150 mg mannitol per ml blood) were pipetted into blood samples so that there was a concentration of 0, 7.5 or 15 mg of iodine per ml blood in the samples. Negative controls without contrast medium (0 mg of iodine per ml blood) were also processed for every irradiation dose. The tubes were exposed to 0, 20 or 500 mGy in vitro X‐ray irradiation. After that, the lymphocytes were separated by using density‐gradient centrifugation. Fluorescence microscopy was applied to determine the average number of γH2AX‐foci per lymphocyte in the presence or absence of different contrast media or mannitol. Differences in the number of γH2AX‐foci were statistically analysed by one‐way ANOVA and post‐hoc Tukey's honestly significant difference test. Iodinated contrast agents led to a statistically significant increase in DNA double‐strand breaks after in vitro irradiation. This effect increased statistically significant with rising radiation dose and appeared independent of the contrast agent used (iopromid, iodixanol, iomeprol, iopamidol). A statistically significant difference in DNA damage between the different tested contrast agents was not found. Therefore, the increase in DNA double‐strand breaks depends solely on the amount of iodine applied. For evaluation of clinical consequences, our findings could be tested in further animal studies. Copyright © 2014 John Wiley & Sons, Ltd.     

The presence of iodinated contrast agents amplifies DNA radiation damage in computed tomography

The purpose of this study was to determine the influence of iodinated contrast agents on the formation of DNA double‐strand breaks in vitro in lymphocytes and to verify these results in patients undergoing diagnostic computed tomography examinations. Blood samples were irradiated in vitro in the presence of iodinated X‐ray contrast agent. Controls were irradiated without contrast agent. Fourteen patients were investigated using contrast‐enhanced computed tomography (CT), and 14 other patients with unenhanced CT. Blood samples were taken prior to and 5 min and 1, 2 and 24 h after the CT examination. In these blood samples the average number of γH2Ax‐foci per lymphocyte was enumerated by fluorescence microscopy. Statistical differences between foci numbers developed in the presence and absence of contrast agent were tested using an independent sample t‐test. In vitro foci numbers after irradiation were significantly higher when contrast agent was present during irradiation. In vivo, γH2Ax‐foci levels were 58% higher in patients undergoing contrast‐enhanced CT compared with those undergoing unenhanced CT. In the presence of iodinated contrast agents DNA, damage is increased and the radiation dose is not the only factor affecting the amount of DNA damage. Individual patient characteristics and biological dosimetry applications, e.g. the analysis of γH2Ax‐foci, have to be considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Imaging efficiency of an X‐ray contrast agent‐incorporated polymeric microparticle

Biocompatible polymeric encapsulants have been widely used as a delivery vehicle for a variety of drugs and imaging agents. In this study, X‐ray contrast agent (iopamidol) is encapsulated into a polymeric microparticle (polyvinyl alcohol) as a particulate flow tracer in synchrotron X‐ray imaging system. The physical properties of the designed microparticles are investigated and correlated with enhancement in the imaging efficiency by experimental observation and theoretical interpretation. The X‐ray absorption ability of the designed microparticle is assessed by Beer–Lambert–Bouguer law. Particle size, either in dried state or in solvent, primarily dominates the X‐ray absorption ability under the given condition, thus affecting imaging efficiency of the designed X‐ray contrast flow tracers. Copyright © 2011 John Wiley & Sons, Ltd.     

Stem cell labeling for magnetic resonance imaging

In vivo applications of cells for the monitoring of their cell dynamics increasingly use non‐invasive magnetic resonance imaging. This imaging modality allows in particular to follow the migrational activity of stem cells intended for cell therapy strategies. All these approaches require the prior labeling of the cells under investigation for excellent contrast against the host tissue background in the imaging modality. The present review discusses the various routes of cell labeling and describes the potential to observe both cell localization and their cell‐specific function in vivo. Possibilities for labeling strategies, pros and cons of various contrast agents are pointed out while potential ambiguities or problems of labeling strategies are emphasized.     

Myelin‐targeted,texaphyrin‐based multimodal imaging agent for magnetic resonance and optical imaging

Reliable methods of imaging myelin are essential to investigate the causes of demyelination and to study drugs that promote remyelination. Myelin‐specific compounds can be developed into imaging probes to detect myelin with various imaging techniques. The development of multimodal myelin‐specific imaging probes enables the use of orthogonal imaging techniques to accurately visualize myelin content and validate experimental results. Here, we describe the synthesis and application of multimodal myelin‐specific imaging agents for light microscopy and magnetic resonance imaging. The imaging agents were synthesized by incorporating the structural features of luxol fast blue MBS, a myelin‐specific histological stain, into texaphyrins coordinated to Gd^III. These new complexes demonstrated absorption of visible light, emission of near‐IR light, and relaxivity values greater than clinically approved contrast agents for magnetic resonance imaging. These properties enable the use of optical imaging and magnetic resonance imaging for visualization of myelin. We performed section‐ and en block‐staining of ex vivo mouse brains to investigate the specificity for myelin of the new compounds. Images obtained from light microscopy and magnetic resonance imaging demonstrate that our complexes are retained in white matter structures and enable detection of myelin. Copyright © 2016 John Wiley & Sons, Ltd.     

Assessment of DNA double‐strand breaks induced by intravascular iodinated contrast media following in vitro irradiation and in vivo,during paediatric cardiac catheterization

Paediatric cardiac catheterizations may result in the administration of substantial amounts of iodinated contrast media and ionizing radiation. The aim of this work was to investigate the effect of iodinated contrast media in combination with in vitro and in vivo X‐ray radiation on lymphocyte DNA. Six concentrations of iodine (15, 17.5, 30, 35, 45, and 52.5 mg of iodine per mL blood) represented volumes of iodinated contrast media used in the clinical setting. Blood obtained from healthy volunteers was mixed with iodinated contrast media and exposed to radiation doses commonly used in paediatric cardiac catheterizations (0 mGy, 70 mGy, 140 mGy, 250 mGy and 450 mGy). Control samples contained no iodine. For in vivo experimentation, pre and post blood samples were collected from children undergoing cardiac catheterization, receiving iodine concentrations of up to 51 mg of iodine per mL blood and radiation doses of up to 400 mGy. Fluorescence microscopy was performed to assess γH2AX‐foci induction, which corresponded to the number of DNA double‐strand breaks. The presence of iodine in vitro resulted in significant increases of DNA double‐strand breaks beyond that induced by radiation for ≥17.5 mg/mL iodine to blood. The in vivo effects of contrast media on children undergoing cardiac catheterization resulted in a 19% increase in DNA double‐strand breaks in children receiving an average concentration of 19 mg/mL iodine to blood. A larger investigation is required to provide further information of the potential benefit of lowering the amount of iodinated contrast media received during X‐ray radiation investigations. Copyright © 2015 John Wiley & Sons, Ltd.     

多模态超声造影剂的现状及研究进展

近年来,随着多种医学成像技术的相互融合和分子影像技术的迅速发展,以超声分子显像为基础,同时具有多种影像学对比显影能力的多模态超声造影剂已成为当前超声影像学领域的研究热点之一。多模态超声造影剂不仅可以提高超声成像的分辨力、敏感性、对比度,还能在一定程度改善传统单一影像诊断的局限性,实现多种方式成像的优势互补,具有广阔的应用前景。本文就多模态超声造影剂的现状及进展进行综述。     

An HPLC/mass spectrometry platform for the development of multimodality contrast agents and targeted therapeutics: prostate‐specific membrane antigen small molecule derivatives

The production of disease‐targeted agents requires the covalent conjugation of a targeting molecule with a contrast agent or therapeutic, followed by purification of the product to homogeneity. Typical targeting molecules, such as small molecules and peptides, often have high charge‐to‐mass ratios and/or hydrophobicity. Contrast agents and therapeutics themselves are also diverse, and include lanthanide chelates for MRI, ^99mTc chelates for SPECT, ⁹⁰Y chelates for radiotherapy, ¹⁸F derivatives for PET, and heptamethine indocyanines for near‐infrared fluorescent optical imaging. We have constructed a general‐purpose HPLC/mass spectrometry platform capable of purifying virtually any targeted agent for any modality. The analytical sub‐system is composed of a single dual‐head pump that directs mobile phase to either a hot cell for the purification of radioactive agents or to an ES‐TOF MS for the purification of nonradioactive agents. Nonradioactive agents are also monitored during purification by ELSD, absorbance and fluorescence. The preparative sub‐system is composed of columns and procedures that permit rapid scaling from the analytical system. To demonstrate the platform's utility, we describe the preparation of five small molecule derivatives specific for prostate‐specific membrane antigen (PSMA): a gadolinium derivative for MRI, indium, rhenium and technetium derivatives for SPECT, and an yttrium derivative for radiotherapy. All five compounds are derived from a highly anionic targeting ligand engineered to have a single nucleophile for N‐hydroxysuccinimide‐based conjugation. We also describe optimized column/mobile phase combinations and mass spectrometry settings for each class of agent, and discuss strategies for purifying molecules with extreme charge and/or hydrophobicity. Taken together, our study should expedite the development of disease‐targeted, multimodality diagnostic and therapeutic agents. Copyright © 2006 John Wiley & Sons, Ltd.     

Contrast enhancement by lipid‐based MRI contrast agents in mouse atherosclerotic plaques; a longitudinal study

The use of contrast‐enhanced MRI to enable in vivo specific characterization of atherosclerotic plaques is increasing. In this study the intrinsic ability of two differently sized gadolinium‐based contrast agents to enhance atherosclerotic plaques in ApoE^?/? mice was evaluated with MRI. We obtained a kinetic profile for contrast enhancement, as the literature data on optimal imaging time points is scarce, and assessed the longer‐term kinetics. Signal enhancement in the wall of the aortic arch, following intravenous injection of paramagnetic micelles and liposomes, was followed for 1 week. In vivo T₁‐weighted MRI plaque enhancement characteristics were complemented by fluorescence microscopy of NIR₆₆₄ incorporated in the contrast agents and quantification of tissue and blood Gd–DTPA. Both micelles and liposomes enhanced contrast in T₁‐weighted MR images of plaques in the aortic arch. The average contrast‐to‐noise ratio increased after liposome or micelle injection to 260 or 280% respectively, at 24 h after injection, compared with a pre‐scan. A second wave of maximum contrast enhancement was observed around 60–72 h after injection, which only slowly decreased towards the 1 week end‐point. Confocal fluorescence microscopy and whole body fluorescence imaging confirmed MRI‐findings of accumulation of micelles and liposomes. Plaque permeation of contrast agents was not strongly dependent on the contrast agent size in this mouse model. Our results show that intraplaque accumulation over time of both contrast agents leads to good plaque visualization for a long period. This inherent intraplaque accumulation might make it difficult to discriminate passive from targeted accumulation. This implies that, in the development of targeted contrast agents on a lipid‐based backbone, extensive timing studies are required. Copyright © 2012 John Wiley & Sons, Ltd.     

Design of functionalized gold nanoparticle probes for computed tomography imaging

The development of new molecules able to efficiently act as long‐circulating computed tomography (CT) contrast agents is one of the most crucial topics in the biomedical field. In the last years, the chance to manipulate materials at the nano‐size level gave new boost to this research, with the specific aim to design innovative nanoprobes. Gold nanoparticles (AuNPs) have showed unique X‐rays attenuation properties which, combined with their easy surface functionalization, makes them ideal candidates for the next generation of contrast agents. In this paper, we present a rational and facile approach to synthesize engineered and water‐stable AuNPs, achieving concentrated colloidal solution with high Hounsfield Units (HU). An accurate control of reagents ratio allowed us to design AuNPs with different shapes, from symmetrical to anisotropic morphology, in a convenient ‘one‐pot’ fashion. Their activity as efficient and reliable CT contrast agents has been evaluated and compared. Moreover, glucosamine‐functionalized gold nanoparticles have been developed ([Au] = 31.20 mg/mL; HU = 2453), in order to obtain a CT contrast agent able to combine spatial resolution with metabolic information. Copyright © 2016 John Wiley & Sons, Ltd.     

Modified lipoproteins as contrast agents for imaging of atherosclerosis

The ability to detect and characterize atherosclerosis with targeted contrast agents may enable initiation of therapy for atherosclerotic lesions prior to becoming symptomatic. Since lipoproteins such as high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) play a critical role in the regulation of plaque biology through the transport of lipids into and out of atherosclerotic lesions, modifying HDL and LDL with radioisotopes for nuclear imaging, chelates for magnetic resonance imaging (MRI) or other possible contrast agents for computed tomography imaging techniques may aid in the detection and characterization of atherosclerosis. This review focuses on the literature employing lipoproteins as contrast agents for imaging atherosclerosis and the feasibility of this approach. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and characterization of new low‐molecular‐weight lysine‐conjugated Gd‐DTPA contrast agents

Various blood pool contrast agents (CAs), characterized by intravascular distribution, have been developed to assist contrast enhanced magnetic resonance angiography (MRA). Among these CAs, the DTPA derivatives conjugated to synthetic polypeptides, such as polylysine, represent attractive candidates for blood pool imaging. However, due to the presence of charged residues located on their backbone, these agents are retained in the kidneys and this compromises their long blood half‐life. In order to overcome this major drawback of the polylysine compounds, two new low‐molecular‐weight CAs were synthesized in the present work by conjugating four or six 1‐p‐isothiocyanatobenzyl‐DTPA moieties to tri‐ or penta‐Lys peptides [(Gd‐DTPA)₄Lys₃ and (Gd‐DTPA)₆Lys₅], respectively. All the –NH₂ groups of Lys were thus blocked by covalent conjugation to DTPA. The stability and relaxometric properties of these compounds, as well as their pharmacokinetic and biodistribution characteristics, were then evaluated. The half‐life in blood of these new polylysine derivatives, as determined in rats, is twofold longer than that of Gd‐DTPA. The compounds could thus be optimal blood pool markers for MRA, which typically uses fast acquisition times. The absence of positive molecular charge did not limit their retention in kidneys 2 h after administration. On the other hand, (Gd‐DTPA)₄Lys₃ is retained in kidneys to a lesser extent than (Gd‐DTPA)₆Lys₅. Their moderate retention in blood and their higher stability and relaxivity in comparison with Gd‐DTPA highlight these polylysine derivatives as optimal compared with previously developed polylysine compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Thermodynamic stability and kinetic inertness of a Gd–DTPA bisamide complex grafted onto gold nanoparticles

Gold nanoparticles coated by gadolinium (III) chelates (Au@DTDTPA) where DTDTPA is a dithiolated bisamide derivative of diethylenetriamine‐N,N,N′,N′′,N′′‐pentaacetic acid (DTPA), constituted contrast agents for both X‐ray computed tomography and magnetic resonance imaging. In an MRI context, highly stable Gd³⁺ complexes are needed for in vivo applications. Thus, knowledge of the thermodynamic stability and kinetic inertness of these chelates, when grafted onto gold nanoparticles, is crucial since bisamide DTPA chelates are usually less suited for Gd³⁺ coordination than DTPA. Therefore, these parameters were evaluated by means of potentiometric titrations and relaxivity measurements. The results showed that, when the chelates were grafted onto the nanoparticle, not only their thermodynamic stability but also their kinetic inertness were improved. These positive effects were correlated to the chelate packing at the nanoparticle surface that stabilized the corresponding Gd³⁺ complexes and greatly enhanced their kinetic inertness. Copyright © 2014 John Wiley & Sons, Ltd.     

In‐vivo high‐resolution X‐ray microtomography for liver and spleen tumor assessment in mice

The present study sought to validate the use of glycery1‐2‐oley‐1,3‐bis‐[7‐(3‐amino‐2,4,6‐triiodophenyl)‐ heptanoate] (DHOG) contrast agent for mouse spleen tumor and liver metastasis imaging by high‐resolution X‐ray microtomography. Three groups of female nude mice were compared: controls (n = 5), and mice injected with 2.5 × 10⁶ STC1 tumor cells in the spleen, imaged at 15 days (group G15, n = 5) and at 30 days (group G30, n = 5, of which one died before imaging). Micro‐CT scans (X‐ray voltage, 50 kVp; anode current, 200 µA; exposure time, 632 ms; 180 rotational steps resulting in 35 µm isotropic spatial resolution) were acquired at 0, 0.75, 2 and 4 h after i.v. injection of DHOG. CT number (Hounsfield units: HU) and contrast‐to‐noise ratios (CNR) were determined in three organs. Statistical analysis was performed by Mann–Whitney U‐test. Contrast enhancement in normal spleen and liver increased, respectively to 1020 ± 159 and 351 ± 27 HU over baseline at 4 h, and 482 ± 3 and 203 ± 14 HU on day 6 after a single contrast injection. Automated three‐dimensional reconstruction and modeling of the spleen provided accurate and quantifiable images. Spleen tumor and liver metastases did not take up DHOG, making them detectable in contrast to the increased signal in normal tissue. The smallest liver metastasis detected measured 0.3 mm in diameter. High‐resolution X‐ray micro‐CT in living mice using DHOG contrast agent allowed visualization and volume quantification of normal spleen and of spleen tumor and its liver metastases. Copyright © 2007 John Wiley & Sons, Ltd.     

Serine‐ and mannose‐modified liposomal contrast agent for computed tomography: evaluation of the enhancement in rabbit liver VX‐2 tumor model

Kupffer cell imaging is a powerful tool for the detection of liver cancer. This diagnostic procedure depends on the faculty of the reticuloendothelial system (RES) which takes up foreign bodies, including small particles. The current study aimed to develop a novel RES targeting liposomal contrast agent that functionalized with serine or mannose, the moiety specifically binding to a corresponding receptor on phagocytic cells. Liposomes loaded with non‐ionic X‐ray contrast media, Iohexol, were prepared by supercritical carbon dioxide reverse‐phase evaporation method and were intravenously injected to healthy rabbits in order to evaluate the liver parenchymal enhancement in X‐ray computed tomography (CT). From 10 to 40 min after injection, the mean enhancement value of the liver parenchyma approached 45 and 34 Hounsfield units (HU) when serine‐modified iodinated liposomal contrast agent (ILCA) and mannose‐modified ILCA were applied, respectively. The tumor‐to‐liver contrast values were also evaluated after the administration of the prepared ILCA to rabbits with VX‐2 carcinoma. For serine‐modified ILCA, tumor‐to‐liver contrast was 82 HU at 1 min and >24 HU at 10–40 min; for mannose‐modified ILCA, the values were 58 HU at 0.5 min and >21 HU at 10–40 min. These vales estimated from the region of intrest and the imaging figures of liver indicate the potential of ILCA for clinical use. Copyright © 2010 John Wiley & Sons, Ltd.     

Solid silica nanoparticles: applications in molecular imaging

Silica and silica‐based nanoparticles have been widely used for therapeutic and diagnostic applications in cancer mainly through delivery of drugs, genes and contrast agents. Development of synthesis methods has provided the possibility of fabricating silica nanoparticles with different sizes in nanometer ranges as well as silica‐based multimodal nanoparticles with many innovative properties and intriguing applications in biomedicine. The surface of silica particles facilitates different methods of surface modifications and allows conjugation of various biomolecules such as proteins and nucleic acids. In this review, different methods of fabrication of silica and silica‐based nanoparticles, their surface modification and the application of these nanoparticles in molecular imaging are discussed. Overall, the aim of this review is to address the development of silica and silica‐based multifunctional nanoparticles that are introduced mainly for molecular imaging applications using optical, magnetic (MRI), X‐ray (computed tomography) and multimodal imaging techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

A physiologically based pharmacokinetic model of vascular–extravascular exchanges during liver carcinogenesis: application to MRI contrast agents

The extraction of physiological parameters by non‐invasive imaging techniques such as dynamic magnetic resonance imaging (MRI) or positron emission tomography requires a knowledge of molecular distribution and exchange between microvascularization and extravascular tissues. These phenomena not only depend on the physicochemical characteristics of the injected molecules but also the pathophysiological state of the targeted organ. We developed a five‐compartment physiologically based pharmacokinetic model focused on hepatic carcinogenesis and MRI contrast agents. This model includes physical characteristics of the contrast agent, dual specific liver supply, microvessel wall properties and transport parameters that are compatible with hepatocarcinoma development. The evolution of concentrations in the five compartments showed significant differences in the distribution of three molecules (differentiated by their diameters and diffusion coefficients ranging, respectively, from 0.9 to 62 nm and from 68.10^?9 to 47.10^?7 cm² s^?1) in simulated regeneration nodules and dysplastic nodules, as well as in medium‐ and poorly differentiated hepatocarcinoma. These results are in agreement with known vascular modifications such as arterialization that occur during hepatocarcinogenesis. This model can be used to study the pharmacokinetics of contrast agents and consequently to extract parameters that are characteristic of the tumor development (like permeability), after fitting simulated to in vivo data. Copyright © 2007 John Wiley & Sons, Ltd.     

Prolonged in vivo circulation time by zwitterionic modification of magnetite nanoparticles for blood pool contrast agents

Long circulation time is critical for blood pool contrast agents used in high‐resolution magnetic resonance angiography. For iron oxide particle contrast agents, size and surface properties significantly influence their in vivo performance. We developed a novel long‐circulating blood pool contrast agent by introducing zwitterionic structure onto the particle surface. Zwitterionic structure was fabricated by 3‐(diethylamino)propylamine (DEAPA) grafted onto the surface of ployacrylic acid coated magnetite nanoparticles via EDC/NHS [N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbo‐diimide hydrochloride/N‐hydroxysuccinimide] coupling chemistry. Zwitterionic particles demonstrated five times lower macrophage cell uptake than the original particles and low cell toxicity. Magnetic resonance angiography indicated that zwitterionic nanoparticles had much longer in vivo circulation time than the original particles and were an ideal candidate for blood pool contrast agent. We suppose that zwitterionic modification by DEAPA and EDC/NHS can be used generally for coating nanoparticles with carboxyl surface and to prolong their circulating time. Copyright © 2012 John Wiley & Sons, Ltd.