Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP). We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Technetium 99m (99mTc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. 99mTc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quinidine, a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc Sestamibi. Cardiac targeting peptide’s transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quinidine and changes in potassium levels.

Respiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

Dimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives in Pseudomonas aeruginosa PAO1. First, results from cell growth experiments showed that deletion of either sfnR2 or sfnG, a gene encoding a DMSO 2 -monooxygenase, significantly inhibits the ability of P. aeruginosa PAO1 to use DMSP, DMS, DMSO, and DMSO 2 as sulfur sources. Deletion of the sfnR1 or msuEDC genes, which encode a MSA desulfurization pathway, did not abolish the growth of P. aeruginosa PAO1 on any sulfur compound tested. Second, data collected from ␤-galactosidase assays revealed that the msuEDC-sfnR1 operon and the sfnG gene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression of sfnR2 is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of the msuEDC-sfnR1 operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression of msuEDC-sfnR1, sfnG, and other target genes involved in DMS-related metabolism in P. aeruginosa PAO1. IMPORTANCE Dimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, including Pseudomonas, Xanthomonas, and Azotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism in P. aeruginosa PAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.