Eric Wickstrom

Two-dimensional (2D) positron emission tomography (PET) and computed tomography (CT) are used for diagnosis and evaluation of cancer patients, requiring surgeons to look through multiple planar images to comprehend the tumor and surrounding tissues. We hypothesized that experienced surgeons would consistently evaluate three-dimensional (3D) presentation of CT images overlaid with PET images when preparing for a procedure. We recruited six Jefferson surgeons to evaluate the accuracy, usefulness, and applicability of 3D renderings of the organs surrounding a malignant pancreas prior to surgery. PET/CT and contrast-enhanced CT abdominal scans of a patient with a ductal pancreatic mass were segmented into 3D surface renderings, followed by co-registration. Version A used only the PET/CT image, while version B used the contrast-enhanced CT scans co-registered with the PET images. The six surgeons answered 15 questions covering a) the ease of use and accuracy of models, b) how these models, with/without PET, changed their understanding of the tumor, and c) what are the best applications of the 3D visualization, on a scale of 1 to 5. The six evaluations revealed a statistically significant improvement from version A (score 3.6±0.5) to version B (score 4.4±0.4). A paired-samples t-test yielded t(14) = −8.964, p<0.001. Across the surgeon cohort, contrast-enhanced CT fused with PET provided a more lifelike presentation than standard CT, increasing the usefulness of the presentation. The experienced surgeons consistently reported positive reactions to 3D surface renderings of fused PET and contrast-enhanced CT scans of a pancreatic cancer and surrounding organs. Thus, the 3D presentation could be a useful preparative tool for surgeons prior to making the first incision. This result supports proceeding to a larger surgeon cohort, viewing prospective 3D images from multiple types of cancer.

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson–Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.

Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression, and addiction. Here we report the synthesis, characterization, and in vitro evaluation of novel fluorescent peptide–peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a μ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through μ-opioid receptor-mediated endocytosis. The K_d by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ∼5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack μ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near-infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.

We are developing agents for positron emission tomography (PET) imaging of cancer gene mRNA expression and software to fuse mRNA PET images with anatomical computerized tomography (CT) images to enable volumetric (3D) haptic (touch-and-feel) simulation of pancreatic cancer and surrounding organs prior to surgery in a particular patient. We have identified a novel ligand specific for epidermal growth factor receptor (EGFR) to direct PET agent uptake specifically into cancer cells, and created a volumetric haptic surgical simulation of human pancreatic cancer reconstructed from patient CT data. Young's modulus and the Poisson ratio for each tissue will be adjusted to fit the experience of participating surgeons.

Infections are a devastating complication of titanium alloy orthopedic implants. Current therapy includes antibiotic-impregnated bone cement and antibiotic-containing coatings. We hypothesized that daptomycin, a Gram-positive peptide antibiotic, could prevent bacterial colonization on titanium alloy surfaces if covalently bonded via a flexible, hydrophilic spacer. We designed and synthesized a series of daptomycin conjugates for bonding to the surface of 1.0 cm² Ti6Al4V foils through bisphosphonate groups, reaching a maximum yield of 180 pmol/cm². Daptomycin-bonded foils killed 53 ± 5% of a high challenge dose of 3 × 10⁵ cfu Staphylococcus aureus ATCC 29213.

95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, ⁶⁴Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([¹¹¹In]DOTA-AEEA)_n-PDAP^m-AEEA₂-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [¹¹¹In]_n-PDAP^m-KRAS2 G12D probes increased from 3.1 ± 0.2 at n = 2, m = 1, to 4.1 ± 0.3 at n = 8, m = 3, to 6.2 ± 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [¹¹¹In]_n-PDAP^m-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 ± 0.6 at n = 2, m = 1, 2.6 ± 0.9 at n = 8, m = 3, and 3.7 ± 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [¹¹¹In]DOTA control results. Simultaneous administration of nonradioactive Gd_n-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [¹¹¹In]₈KRAS2 G12 V probes 3−6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of ¹¹¹In uptake increased from 3.1 ± 0.5 to 6.5 ± 1.0, and the kidney/tumor ratio of ¹¹¹In uptake decreased by more than 5-fold from 174.8 ± 17.5 to 30.8 ± 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd_n-KRAS2 G12 V probes, enhanced tumor uptake of [¹¹¹In]_nKRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd_n-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.

We hypothesized that chelating Gd(III) to 1,4,7‐tris(carboxymethylaza)cyclododecane‐10‐azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would provide a magnetic resonance genetic imaging agent capable of hybridization to a specific mRNA. Because of the low sensitivity of Gd(III) as an magnetic resonance imaging (MRI) contrast agent, a single Gd‐DO3A complex per PNA hybridization agent could not provide enough contrast for detection of cancer gene mRNAs, even at thousands of mRNA copies per cell. To increase the Gd(III) shift intensity of MRI genetic imaging agents, we extended a novel DO3An‐polydiamidopropanoyl (PDAP^m) dendrimer, up to n = 16, from the N‐terminus of KRAS PNA hybridization agents by solid phase synthesis. A C‐terminal D(Cys‐Ser‐Lys‐Cys) cyclized peptide analog of insulin‐like growth factor 1 (IGF1) was included to enable receptor‐mediated cellular uptake. Molecular dynamic simulation of the (Gd‐DO3A‐AEEA)₁₆‐PDAP⁴‐AEEA₂‐KRAS PNA‐AEEA‐D(Cys‐Ser‐Lys‐Cys) genetic imaging nanoparticles in explicit water yielded a pair correlation function similar to that of PAMAM dendrimers, and a predicted structure in which the PDAP dendron did not sequester the PNA. Thermal melting measurements indicated that the size of the PDAP dendron included in the (DO3A‐AEEA)_n‐PDAP^m‐AEEA₂‐KRAS PNA‐AEEA‐D(Cys‐Ser‐Lys‐Cys) probes (up to 16 Gd(III) cations per PNA) did not depress the melting temperatures (T_m) of the complementary PNA/RNA hybrid duplexes. The Gd(III) dendrimer PNA genetic imaging agents in phantom solutions displayed significantly greater T₁ relaxivity per probe (r₁ = 30.64 ± 2.68 mM⁻¹ s⁻¹ for n = 2, r₁ = 153.84 ± 11.28 mM⁻¹ s⁻¹ for n = 8) than Gd‐DTPA (r₁ = 10.35 ± 0.37 mM⁻¹ s⁻¹), but less than that of (Gd‐DO3A)₃₂‐PAMAM dendrimer (r₁ = 771.84 ± 20.48 mM⁻¹ s⁻¹) (P < 0.05). Higher generations of PDAP dendrimers with 32 or more Gd‐DO3A residues attached to PNA‐D(Cys‐Ser‐Lys‐Cys) genetic imaging agents might provide greater contrast for more sensitive detection. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 1061–1076, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Guanylyl cyclase C (GC‐C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18‐residue heat‐stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12‐residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides3and6, were synthesized by orthogonal solid phase synthesis routes. Peptides3and6bound GC‐C with a rank order potency of STa > peptide3> peptide6. Peptides3and6behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 713–723, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com