Ian M. Brereton

Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro. Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA in vivo. The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development.

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has K_i values of 8 and 7 nM, respectively, for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 Å resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl l-phenylalanine) tetraamide prodrug of this compound was synthesized, and it has an IC₅₀ of 11.7 ± 3.2 μM against Pf lines grown in culture and a CC₅₀ in human A549 cell lines of 102 ± 11 μM, thus giving it a ∼10-fold selectivity index.

Purpose: To compare mono‐ and bi‐exponential relaxation model equations to discriminate between normal and fatty liver disease.Materials and Methods: Six rats on a choline deficient amino acid modified (CDAA) diet and six on normal chow were studied. Multiple spin echo images with increasing echo times (TEs) were collected at 9.4T. Pixel‐wise T₂ maps were generated using mono‐exponential decay function to calculate T_2M, and a bi‐exponential to calculate, short T₂ component (T_2S), long T₂ component (T_2L), and fractions of these components (ρ_S, ρ_L), respectively. Statistical F‐tests and Akaike's information criterion (AIC) were used to assess the relative performance of the two models.Results: F‐test and AIC showed that in the CDAA group, T₂ bi‐exponential model described the signal of T₂ weighted imaging of the liver better than the mono‐exponential model. Controls were best described by the mono‐exponential model. Mean values for T_2M, T_2L, T_2S, ρ_S, ρ_L were 31.2 ± 0.7 ms, 72.8 ± 3.3 ms, 8.2 ± 0.6 ms,71.2 ± 2.1%, 30.4 ± 1.3%, respectively, in CDAA rats, compared with 18.8 ± 0.5 ms, 32.3 ± 0.7 ms, 9.2 ± 1.8 ms, 79 ± 2%, 21.0 ± 1.1% in controls.Conclusion: In the fatty liver of CDAA rats we have shown that T₂ weighted images fit the bi‐exponential model better than mono‐exponential decays thus providing a better description of the data.Level of Evidence: 1Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2018;47:468–476.

Non-invasive quantification of the amyloid-load is of paramount interest in the field of Alzheimerŕs disease research. We present a combined PET/7 T-MRI and 16.4 T-microscopic-MRI (µMRI) dual-modality imaging approach enabling the quantification of the amyloid-load at high-sensitivity and high-resolution, and of regional cerebral blood flow (rCBF) in the brain of transgenic APP23 mice. Moreover, we demonstrate a novel, voxel-based correlative data analysis method which we used for in-depth evaluation of amyloid-PET and rCBF-data. Methods: We injected [¹¹C]PIB intravenously in transgenic (tg) and control (co) APP23 mice and performed dynamic PET measurements. rCBF-data were recorded with a flow sensitive alternating inversion recovery (FAIR) approach at 7 T. Subsequently, animals were sacrificed and brains harvested for ex vivo µMRI at 16.4 T with a T2*-weighted gradient-echo sequence at 30 µm spatial resolution. Additionally, correlative amyloid histology was performed. The [¹¹C]PIB-PET data were quantified to non-displaceable binding potentials (BPnd) using the Logan graphical analysis; FAIR-data were quantified with a simplified version of the Bloch equation. Results: Amyloid-load assessed by both, [¹¹C]PIB-PET and amyloid histology (AβH) was highest in the frontal cortex of tg mice ([¹¹C]PIB-BPnd: 0.93±0.08, AβH: 15.1±1.5%), followed by the temporoparietal cortex ([¹¹C]PIB-BPnd: 0.75±0.08, AβH: 13.9±0.7%) and the hippocampus ([¹¹C]PIB-BPnd: 0.71±0.09, AβH: 9.2±0.9%), and was lowest in the thalamus ([¹¹C]PIB-BPnd: 0.40±0.07, AβH: 6.6±0.6%). However, [¹¹C]PIB-BPnd and AβH linearly correlated (R²=0.82, p11C]PIB-BPnd and rCBF-MRI in the voxel-based analysis that was absent in control mice (slope_tg: -0.11±0.03; slope_co: 0.004±0.005, P = 0.014). Conclusion: Our dual-modality imaging approach employing [¹¹C]PIB-PET/7 T-MRI and 16.4 T-µMRI allowed amyloid-load quantification with high-sensitivity and high-resolution, the identification of substructures within single amyloid plaques, and the quantification of rCBF. Future applications of this multiparametrical imaging and data-analysis approach could simplify the identification of treatment impact on amyloid-load status and brain physiology and aid the development of new treatment strategies, enabling both a morphological and functional classification of underlying mechanisms-of-action.

Magnetic Resonance Imaging (MRI) of the rodent brain at ultra-high magnetic fields (> 9.4 Tesla) offers a higher signal-to-noise ratio that can be exploited to reduce image acquisition time or provide higher spatial resolution. However, significant challenges are presented due to a combination of longer T₁ and shorter T₂/T₂* relaxation times and increased sensitivity to magnetic susceptibility resulting in severe local-field inhomogeneity artefacts from air pockets and bone/brain interfaces. The Stejskal-Tanner spin echo diffusion-weighted imaging (DWI) sequence is often used in high-field rodent brain MRI due to its immunity to these artefacts. To accurately determine diffusion-tensor or fibre-orientation distribution, high angular resolution diffusion imaging (HARDI) with strong diffusion weighting (b >3000 s/mm²) and at least 30 diffusion-encoding directions are required. However, this results in long image acquisition times unsuitable for live animal imaging. In this study, we describe the optimization of HARDI acquisition parameters at 16.4T using a Stejskal-Tanner sequence with echo-planar imaging (EPI) readout. EPI segmentation and partial Fourier encoding acceleration were applied to reduce the echo time (TE), thereby minimizing signal decay and distortion artefacts while maintaining a reasonably short acquisition time. The final HARDI acquisition protocol was achieved with the following parameters: 4 shot EPI, b = 3000 s/mm², 64 diffusion-encoding directions, 125×150 μm² in-plane resolution, 0.6 mm slice thickness, and 2h acquisition time. This protocol was used to image a cohort of adult C57BL/6 male mice, whereby the quality of the acquired data was assessed and diffusion tensor imaging (DTI) derived parameters were measured. High-quality images with high spatial and angular resolution, low distortion and low variability in DTI-derived parameters were obtained, indicating that EPI-DWI is feasible at 16.4T to study animal models of white matter (WM) diseases.

Non-uniformly sampled HYSCORE data combined with maximum entropy reconstruction can shorten experimental times by approximately an order of magnitude.

Medical image registration is generally faced with the confounding effect of spatially dependent intensity variations. This can be the case when images have been acquired using the same imaging modality, for example, in magnetic resonance imaging and also when using various histology and staining processes. We propose the application of an intensity-invariant dense feature extraction method through the use of phase Fourier transforms. The approach allows medical images containing intensity in homogeneities to be aligned and warped as part of a feature-based registration technique. Registration performance was evaluated on mono-modality and multi-modality data, namely magnetic resonance and histology images. Qualitative and quantitative validation was conducted with respect to two established image intensity correction methods.

Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31–157) and an N-terminal signal peptide (residues 1–27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping.