Hrvoje Lusic

X-Ray Computed Tomography Contrast Agents

X-ray computed tomography (CT) is a well-established tissue imaging technique employed in a variety of research and clinical settings.1 Specifically, CT is a non-invasive clinical diagnostic tool that allows for 3D visual reconstruction and segmentation of tissues of interest. High resolution CT systems can be used to perform non-destructive 3D imaging of a variety of tissue types and organ systems, such as: the gastrointestinal tract, cardiovascular system, renal tract, liver, lungs, bone, cartilage, tumorous tissue, etc. CT is one of the most prevalent diagnostic tools in terms of frequency-of-use and hospital availability.2 The use of CT is on the rise and the number of clinical CT scanners in operation worldwide is estimated at over 45,000.1b Today, over 70 million clinical CT scans are performed yearly in the U.S. alone. For a recent detailed analysis of the use of clinical CT imaging and data regarding the number of regular and contrast-enhanced CT scans performed annually in the U.S. we refer the reader to the “Nationwide Evaluation of X-ray Trends” survey published by the Conference for Radiation Control Program Directors (CRCPD).3 The idea of using tomography (Greek: tomos = slice, graphein = draw) as a diagnostic tool in medicine was adopted soon after the discovery of X-rays by W. C. Roentgen in 1895. However, several more decades passed before the technology advanced sufficiently to bring those ideas to fruition. The first successful CT imaging device was built in 1972 by G. N. Hounsfield, at Electric and Musical Industries Ltd. In 1979, G. N. Hounsfield and South African physicist A. M. Cormack shared a Nobel Prize in medicine for their contributions to the field of X-ray CT imaging and diagnostics.4 X-rays are a form of electromagnetic radiation with wavelengths roughly between 0.01 nm and 10 nm. Traditionally, X-rays are generated by a vacuum tube using high voltage to accelerate electrons from a cathode to a (usually) tungsten-alloy anode. In the process, the accelerated electrons release electromagnetic radiation in the form of X-rays and the maximum energy of the radiation is limited by the energy of the incident electron. Operating voltages of modern clinical CT scanners differ among instrument models and manufacturers, but generally fall between 80 kVp to 150 kVp. As a rule, materials possessing higher density (ρ) or high atomic number (Z) tend to better absorb X-rays. The relationship is best expressed in the formula for X-ray absorption coefficient (μ): μ≈ρZ4AE3 (1) where “A” is the atomic mass and “E” is the X-ray energy. The strong relationship between absorption and atomic number is of significant importance in clinical applications. The Z4 factor allows for contrast levels of several orders of magnitude between different tissues and types of contrast agents. When an incident X-ray has energy equal or slightly greater than the binding energy of the K-shell electron of the atom, a large sudden increase in absorption coefficient is observed. This energy value is known as absorption edge (k), and the k value increases with atomic number of the element. Consequently, X-ray attenuating contrast media containing atoms of high atomic number (most commonly iodine or barium), are frequently used in clinical settings to obtain images of soft tissues. To generate images with the highest contrast to the surrounding tissue, the energy of the X-ray source can be adjusted to closely match the absorption edge value (k) of the relevant imaging-agent atoms (i.e., iodine, barium, gold, etc.). Thus, it is also possible to do selective X-ray imaging and to differentiate between attenuating materials by fine tuning the energy source to the appropriate absorption edge value. A CT image is obtained by rotating an X-ray source around an object, with a detector positioned directly opposite the radiation source. Alternatively, in many preclinical CT scanners the object sometimes is rotated around its own axis. Such preclinical scanners are often being used for small animal in vivo imaging. Generally, X-ray scans are taken at small angular increments during rotation around the object over 360°. A series of attenuation profiles or projections is thus obtained. The projections are then processed mathematically to create a 3D rendition of the scanned object. An in depth description of the engineering principles underlying modern CT imaging instruments is beyond the scope of this manuscript, and the reader is referred to other published works.1c,5 A diagnostic imaging method related to CT is X-ray fluoroscopy. Fluoroscopy allows for the acquisition of real-time, continuous images of the internal organs. Like in CT, imaging agents are often used in fluoroscopy for better contrast resolution. Small iodinated agents are commonly injected into blood vessels for use in fluoroscopic angiography, allowing for the evaluation of blood flow and visualization of the vasculature system, while barium contrast media are introduced orally or with an enema to investigate the anatomy (and pathology) of the gastrointestinal tract. The introduction of magnetic resonance imaging (MRI) resulted in a loss of interest and reduction in CT contrast agent development throughout the 1980s. However, advances in computer technology, and the introduction and widespread adoption of spiral-CT in the mid-1990s have sparked a revival of interest in CT imaging and CT contrast media. Current clinical CT scanners are capable of acquiring high resolution 3D isotropic images of the body within several minutes. CT imaging today is less time consuming, less expensive, and more readily available than other medical imaging technologies such as MRI and positron emission tomography (PET). In the last several years, the emergence of novel technologies such as dual-source CT, and multi-detector CT has advanced the field of CT imaging even further. As a comparison to X-ray imaging diagnostic methods, PET imaging employs gamma-ray emitting radioactive nuclei “tracers” as contrast agents while MRI takes advantage of nuclear magnetic resonance principles by applying high magnetic fields to align magnetization of certain atomic nuclei. In contrast to CT and PET imaging, MRI uses no ionizing radiation and it is therefore often deemed safer than the other two.

Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry

We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNA(CUA) pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, containing a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddition reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.

Genetically encoded photocontrol of protein localization in mammalian cells.

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.

Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and xenopus embryos

Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts.

Gene Silencing in Mammalian Cells with Light-Activated Antisense Agents

Detailed knowledge of the external regulation of gene func-tion is a fundamental necessity in order to annotate sequencedgenomes and to understand biological processes in single cellsand multicellular organisms. One of the most widely used ap-proaches for the down-regulation of specific genes is the ap-plication of antisense agents. Antisense agents are oligomersthat have the ability to hybridize sequence specificslly tomRNAs, inhibiting translation and potentially leading to mRNAdegradation through RNAse H recruitment.

Generating Permissive Site-Specific Unnatural Aminoacyl-tRNA Synthetases

Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. Using these techniques, researchers can precisely control the position and number of novel chemical moieties in a protein, via introducing the novel R group of UAAs, that are genetically encoded in the proteins primary structure. The substrate recognition properties of a natural aminoacyl-tRNA synthetase (aaRS) must be modified in order to incorporate UAAs into proteins. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibility of this important technology. At present, engineered unnatural aminoacyl-tRNA synthetases (UaaRS) are evaluated on their translational efficiency (the extent to which they allow for incorporation of UAAs into protein) and fidelity (the extent to which they prevent incorporation of natural amino acids). We propose that a third parameter of substrate recognition, permissivity, is equally important. Permissive UaaRSs, whose relaxed substrate recognition properties allow them to incorporate multiple unnatural amino acids (but not natural amino acids), would eliminate the need to generate new UaaRSs for many new UAAs. Here, we outline methods for quickly and easily assessing the permissivity of existing UaaRSs and for generating permissive UaaRSs. In proof of principle experiments, we determined the degree of permissivity of two UaaRSs for a family of structurally related fluorinated UAAs ((19)F-UAAs). We then increased the permissivity of the initial UaaRSs to allow for incorporation of the family of (19)F-UAAs. Finally, we validated the utility of these new (19)F-UAAs as probes for fluorine NMR studies of protein structure and dynamics. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins.

Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery.

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapys appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individuals genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in vitro successes that we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have used in our research to provide readers with the tools to characterize and engineer new vectors.

The Human Mitochondrial tRNAMet: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA(Met)(CAU), to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA(Met)(CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f(5)C(34)), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL(Met)(CAU)), determined by NMR restrained molecular modeling, revealed how the f(5)C(34) modification facilitates the decoding of AUA at the P- and the A-sites. The f(5)C(34) defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL(Met)(CAU). The hmtASL(Met)(CAU) exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f(5)C(34) facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f(5)C(34) enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f(5)C(34), expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

Synthesis and investigation of the 5-formylcytidine modified, anticodon stem and loop of the human mitochondrial tRNAMet

Human mitochondrial methionine transfer RNA (hmtRNAMetCAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f5C34). The role of this modification in (hmtRNAMetCAU) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNAs anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNAs anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

PURPOSE To quantify the affinity of a cationic computed tomography (CT) contrast agent (CA(4+)) and that of an anionic contrast agent (ioxaglate) to glycosaminoglycans (GAGs) in ex vivo cartilage tissue explants and to characterize the in vivo diffusion kinetics of CA(4+) and ioxaglate in a rabbit model. MATERIALS AND METHODS All in vivo procedures were approved by the institutional animal care and use committee. The affinities of ioxaglate and CA(4+) to GAGs in cartilage (six bovine osteochondral plugs) were quantified by means of a modified binding assay using micro-CT after plug equilibration in serial dilutions of each agent. The contrast agents were administered intraarticularly to the knee joints of five New Zealand white rabbits to determine the in vivo diffusion kinetics and cartilage tissue imaging capabilities. Kinetics of diffusion into the femoral groove cartilage and relative contrast agent uptake into bovine plugs were characterized by means of nonlinear mixed-effects models. Diffusion time constants (τ) were compared by using a Student t test. RESULTS The uptake of CA(4+) in cartilage was consistently over 100% of the reservoir concentration, whereas it was only 59% for ioxaglate. In vivo, the contrast material-enhanced cartilage reached a steady CT attenuation for both CA(4+) and ioxaglate, with τ values of 13.8 and 6.5 minutes, respectively (P = .04). The cartilage was easily distinguishable from the surrounding tissues for CA(4+) (12 mg of iodine per milliliter); comparatively, the anionic contrast agent provided less favorable imaging results, even when a higher concentration was used (80 mg of iodine per milliliter). CONCLUSION The affinity of the cationic contrast agent CA(4+) to GAGs enables high-quality imaging and segmentation of ex vivo bovine and rabbit cartilage, as well as in vivo rabbit cartilage. SUPPLEMENTAL MATERIAL http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112246/-/DC1.