Anesthesia and Monitoring of Animals During MRI Studies.

The use of imaging represents a major impact on the refinement and the reduction of in vivo studies in animal models, in particular for allowing longitudinal monitoring of the onset and the progression of disease within the same animal, and studying the biological effects of drug candidate and their therapeutic effectiveness. But the use of imaging procedures can affect animal physiology, and the need to anesthetize the animals for imaging entails potential health risks. During anesthesia, there is an inevitable autonomic nervous system depression which induces cardiovascular depression, respiratory depression, and hypothermia. Also other procedures associated with imaging such as animal preparation (e.g., fasting, premedication), blood sampling, and dosage/contrast agent injections can also affect physiology and animal welfare. All these factors are likely to have confounding effect on the outcome of the imaging studies and pose important concerns regarding the animal's well-being, particularly when imaging immune deprived animals or diseased animals. We will discuss these challenges and considerations during imaging to maximize efficacious data while promoting animal welfare

Flexible scintillator autoradiography for tumor margin inspection using 18F-FDG

Autoradiography potentially offers high molecular sensitivity and spatial resolution for tumor margin estimation. However, conventional autoradiography requires sectioning the sample which is destructive and labor-intensive. Here we describe a novel autoradiography technique that uses a flexible ultra-thin scintillator which conforms to the sample surface. Imaging with the flexible scintillator enables direct, high-resolution and high-sensitivity imaging of beta particle emissions from targeted radiotracers. The technique has the potential to identify positive tumor margins in fresh unsectioned samples during surgery, eliminating the processing time demands of conventional autoradiography. We demonstrate the feasibility of the flexible autoradiography approach to directly image the beta emissions from radiopharmaceuticals using lab experiments and GEANT-4 simulations to determine i) the specificity for 18 F compared to 99m Tc-labeled tracers ii) the sensitivity to detect signal from various depths within the tissue. We found that an image resolution of 1.5 mm was achievable with a scattering background and we estimate a minimum detectable activity concentration of 0.9 kBq/ml for 18 F. We show that the flexible autoradiography approach has high potential as a technique for molecular imaging of tumor margins using 18 F-FDG in a tumor xenograft mouse model imaged with a radiation-shielded EMCCD camera. Due to the advantage of conforming to the specimen, the flexible scintillator showed significantly better image quality in terms of tumor signal to whole-body background noise compared to rigid and optimally thick CaF 2 :Eu and BC400. The sensitivity of the technique means it is suitable for clinical translation

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

Purpose Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP® peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Procedures Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP® variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP® variants in multidimensional space of spectral parameters. Results The fluorescently labeled pHLIP® variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP® peptide. Conclusions Different cargo molecules conjugated with pHLIP® peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP®-based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions

Revealing the configuration and crystal packing of organic compounds by solid-state NMR spectroscopy: Methoxycarbonylurea, a case study

The molecular configuration and intermolecular arrangement of polycrystalline methoxycarbonylurea (MCU) has been studied by a combination of chemical editing, rotational echo double resonance (REDOR) spectroscopy and ab initio calculations. From the multispin ISn REDOR experiments several dipolar couplings were determined and converted into distance constraints. Intra- and intermolecular dipolar couplings were distinguished by isotope dilution. The configuration of the MCU molecule can be determined from three torsion angles Psi(1), Psi(2), and Psi(3). Ab initio calculations showed that these angles are either 0degrees or 180degrees (Z or E). From the REDOR experiments, the E configuration was found for Psi(1) and Psi(2) and the Z configuration for Psi(3). Thus the configuration of MCU in the solid state was determined to be EEZ. Distance constraints for the intermolecular arrangement of MCU were obtained by performing REDOR experiments on (CN2)-C-13-N-15 MCU with different degrees of isotope dilution and on a cocrystallized 1:1 mixture of C-13(urea) MCU and N-15(amide) MCU. By combining these distance constraints with molecular modeling, three different possible packing motifs for MCU molecules were found. The molecules in these motifs are arranged as linear chains with methoxy groups at the borders of the chains. All the intermolecular hydrogen bond donors and acceptors in the interior of the chain are saturated

NMR crystallography of amides, peptides and protein-ligand complexes

NMR crystallography, the combination of solid-state NMR techniques, chemical modelling, quantum chemical calculations and other characterization techniques, allows the determination of molecular and supramolecular structures which are not amenable to standard X-ray crystallography. The method is demonstrated on a set of application examples. First the principles and practical considerations of NMR crystallography based on dipolar NMR spectroscopy are outlined in conformational studies of polymorphs of N-octyl-gluconamide and of methoxycarbonyl urea. Then structural studies of two substrate-inhibitor complexes, human manganese superoxide dismutase with azide and nickel superoxide dismutase with cyanide, are reviewed. Finally an example of ongoing developments in the related field of EPR crystallography is reported

Revealing CSA tensors and hydrogen bonding in methoxycarbonyl urea: A combined 13C, 15N and 13C14N2 dipolar chemical shift NMR and DFT study

Methoxycarbonyl urea (MCU), a potential long-term nitrogen fertilizer, is studied by C-13 and N-15 dipolar chemical shift NMR spectroscopy and ab initio calculations. Employing a combination of dipolar chemical shift NMR, selective isotope labeling and ab initio gas phase calculations, possible molecular structures and chemical shielding tensors of all N-15 nuclei and of two out of the three C-13 nuclei were revealed. Four possible stable configurations of the molecule with different energies were found in the calculations. The CSA tensors were calculated for these configurations. While the calculated C-13(urea) CSA tensor orientation of the configuration with the lowest energy is in good agreement with the experimental tenser orientation, there are pronounced differences between calculated and experimental tensor eigenvalues. These differences are a clear indication of the presence of intermolecular hydrogen bonds in the experimental sample, which are neglected in the gas phase calculations. Four different possible orientations of the experimental C-13(urea) CSA tensor exist, due to symmetry. This ambiguity is solved by comparison with results from GIAO calculations of the C-13 CSA tensor, employing the minimum energy configuration (EEZ). It is found that the orientation, where delta(11) points approximately in direction of N(imide), delta(22) approximately in direction of the C=O bond, and delta(33) is oriented perpendicular to the molecular frame, is adopted in the molecule

Revealing CSA tensors and hydrogen bonding in methoxycarbonyl urea: A combined C-13, N-15 and (CN2)-C-13-N-14 dipolar chemical shift NMR and DFT study

Methoxycarbonyl urea (MCU), a potential long-term nitrogen fertilizer, is studied by C-13 and N-15 dipolar chemical shift NMR spectroscopy and ab initio calculations. Employing a combination of dipolar chemical shift NMR, selective isotope labeling and ab initio gas phase calculations, possible molecular structures and chemical shielding tensors of all N-15 nuclei and of two out of the three C-13 nuclei were revealed. Four possible stable configurations of the molecule with different energies were found in the calculations. The CSA tensors were calculated for these configurations. While the calculated C-13(urea) CSA tensor orientation of the configuration with the lowest energy is in good agreement with the experimental tenser orientation, there are pronounced differences between calculated and experimental tensor eigenvalues. These differences are a clear indication of the presence of intermolecular hydrogen bonds in the experimental sample, which are neglected in the gas phase calculations. Four different possible orientations of the experimental C-13(urea) CSA tensor exist, due to symmetry. This ambiguity is solved by comparison with results from GIAO calculations of the C-13 CSA tensor, employing the minimum energy configuration (EEZ). It is found that the orientation, where delta(11) points approximately in direction of N(imide), delta(22) approximately in direction of the C=O bond, and delta(33) is oriented perpendicular to the molecular frame, is adopted in the molecule

Revealing the configuration and crystal packing of organic compounds by solid-state NMR spectroscopy: Methoxycarbonylurea, a case study

The molecular configuration and intermolecular arrangement of polycrystalline methoxycarbonylurea (MCU) has been studied by a combination of chemical editing, rotational echo double resonance (REDOR) spectroscopy and ab initio calculations. From the multispin ISn REDOR experiments several dipolar couplings were determined and converted into distance constraints. Intra- and intermolecular dipolar couplings were distinguished by isotope dilution. The configuration of the MCU molecule can be determined from three torsion angles Psi(1), Psi(2), and Psi(3). Ab initio calculations showed that these angles are either 0degrees or 180degrees (Z or E). From the REDOR experiments, the E configuration was found for Psi(1) and Psi(2) and the Z configuration for Psi(3). Thus the configuration of MCU in the solid state was determined to be EEZ. Distance constraints for the intermolecular arrangement of MCU were obtained by performing REDOR experiments on (CN2)-C-13-N-15 MCU with different degrees of isotope dilution and on a cocrystallized 1:1 mixture of C-13(urea) MCU and N-15(amide) MCU. By combining these distance constraints with molecular modeling, three different possible packing motifs for MCU molecules were found. The molecules in these motifs are arranged as linear chains with methoxy groups at the borders of the chains. All the intermolecular hydrogen bond donors and acceptors in the interior of the chain are saturated