Fluorescent Peptide–PNA Chimeras for Imaging Monoamine Oxidase A mRNA in Neuronal Cells

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 <i>in vitro</i> evaluation of novel fluorescent peptide–peptide nucleic acid (PNA) chimeras for <i>MAOA</i> mRNA imaging in live neuronal cells. The probes were designed to include <i>MAOA</i>-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 <i>K</i>_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

Safety and Effectiveness of Bone Marrow Cell Concentrate in the Treatment of Chronic Critical Limb Ischemia Utilizing a Rapid Point-of-Care System

Critical limb ischemia (CLI) is the end stage of lower extremity peripheral vascular disease (PVD) in which severe obstruction of blood flow results in ischemic rest pain, ulcers and/or gangrene, and a significant risk of limb loss. This open-label, single-arm feasibility study evaluated the safety and therapeutic effectiveness of autologous bone marrow cell (aBMC) concentrate in revascularization of CLI patients utilizing a rapid point-of-care device. Seventeen (17) no-option CLI patients with ischemic rest pain were enrolled in the study. Single dose of aBMC, prepared utilizing an intraoperative point-of-care device, the Res-Q™ 60 BMC system, was injected intramuscularly into the afflicted limb and patients were followed up at regular intervals for 12 months. A statistically significant improvement in Ankle Brachial Index (ABI), Transcutaneous Oxygen Pressure (TcPO2), mean rest pain and intermittent claudication pain scores, wound/ ulcer healing, and 6-minute walking distance was observed following aBMC treatment. Major amputation-free survival (mAFS) rate and amputation-free rates (AFR) at 12 months were 70.6% and 82.3%, respectively. In conclusion, aBMC injections were well tolerated with improved tissue perfusion, confirming the safety, feasibility, and preliminary effectiveness of aBMC treatment in CLI patients

Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting <i>KRAS2</i> Oncogenic mRNA Molecules: Theory and Experiment

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 <i>KRAS2</i> mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant <i>KRAS2</i> mRNA in tumors in mice by <i>in vivo</i> 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 <i>KRAS2</i> 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