The role of the N-acetylglucosamine phosphoenolpyruvate phosphotransferase system from Lactobacillus plantarum 8014 in the mechanism of action of glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand

The rise in antibiotic-resistant bacteria is becoming a severe public health problem because of the shortage of new antibiotics to combat existing resistant bacterial pathogens. Should this trend of increasing bacterial drug resistance continue, the previously treatable conditions may once again become fatal. Using broad-spectrum antibiotics causes collateral damage to the commensal microbiota of the host leading to complications and a greater susceptibility to opportunistic pathogenic infection. As a result, narrow spectrum antibacterials effective against specific pathogens, are becoming increasingly sought after. Among the many alternative classes of narrow-spectrum antibiotics, is a diverse group of ribosomally-synthesised antimicrobial peptides known as bacteriocins. Glycocin F (GccF), a rare and uniquely diglycosylated bacteriocin produced by Lactobacillus plantarum KW80, appears to target a specific N-acetylglucosamine (GlcNAc) phosphotransferase system (PTS) and causes almost instant bacteriostasis by an as yet unknown mechanism. This thesis demonstrates how the GlcNAc-PTS is involved in the GccF mechanism of action and that the gccH gene provides immunity to GccF. Using transgenic and gene editing techniques, regions of the GlcNAc-PTS were either removed or altered to prevent normal function before being tested in vivo. The results demonstrated that only the EIIC domain of the GlcNAc-PTS is required in the GccF mechanism of action and that it acts like a "lure" that attracts the bacteriocin to the main target that is as yet unknown. Furthermore, the immunity gene was discovered, and using PTS knockout cell lines the immunity mechanism was shown to act independently of the GlcNAc-PTS. This work will form the foundation for the work needed to unravel the bacteriostatic mechanism of action of GccF, which may lead to the development a novel antimicrobial agent

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.We demonstrate that prophylactic and therapeutic inhibition of phosphodiesterase 4 with several different antagonists reduces lung fibrosis induced by a targeted injury to the type II alveolar epithelium. In conjunction with the reduction in lung collagen content, phosphodiesterase inhibition also reduced serum surfactant protein C levels and the expression of profibrotic genes by lung fibroblasts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144679/1/phy213753.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144679/2/phy213753_am.pd

The “One‐Step” approach for QT analysis increases the sensitivity of nonclinical QTc analysis

Abstract Whether a compound prolongs cardiac repolarization independent of changes in beat rate is a critical question in drug research and development. Current practice is to resolve this in two steps. First, the QT interval is corrected for the influence of rate and then statistical significance is tested. There is renewed interest in improving the sensitivity of nonclinical corrected QT interval (QTc) assessment with modern studies having greater data density than previously utilized. The current analyses examine the effects of moxifloxacin or vehicle on the QT interval in nonhuman primates (NHPs) using a previously described one‐step method. The primary end point is the statistical sensitivity of the assessment. Publications suggest that for a four animal crossover (4 × 4) in NHPs the minimal detectable difference (MDD) is greater than or equal to 10 ms, whereas in an eight animal crossover the MDD is ~6.5 ms. Using the one‐step method, the MDD for the four animal NHP assessments was 3 ms. In addition, the one‐step model accounted for day‐to‐day differences in the heart rate and QT‐rate slope as well as drug‐induced changes in these parameters. This method provides an increase in the sensitivity and reduces the number of animals necessary for detecting potential QT change and represents “best practice” in nonclinical QTc assessment in safety pharmacology studies

ATR-101, a selective ACAT1 inhibitor, decreases ACTH-stimulated cortisol concentrations in dogs with naturally occurring Cushing’s syndrome

Abstract Background Cushing’s syndrome in humans shares many similarities with its counterpart in dogs in terms of etiology (pituitary versus adrenal causes), clinical signs, and pathophysiologic sequelae. In both species, treatment of pituitary- and adrenal-dependent disease is met with limitations. ATR-101, a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase 1), is a novel small molecule therapeutic currently in clinical development for the treatment of adrenocortical carcinoma, congenital adrenal hyperplasia, and Cushing’s syndrome in humans. Previous studies in healthy dogs have shown that ATR-101 treatment led to rapid, dose-dependent decreases in adrenocorticotropic hormone (ACTH) stimulated cortisol levels. The purpose of this clinical study was to investigate the effects of ATR-101 in dogs with Cushing’s syndrome. Methods ATR-101 pharmacokinetics and activity were assessed in 10 dogs with naturally-occurring Cushing’s syndrome, including 7 dogs with pituitary-dependent disease and 3 dogs with adrenal-dependent disease. ATR-101 was administered at 3 mg/kg PO once daily for one week, followed by 30 mg/kg PO once daily for one (n = 4) or three (n = 6) weeks. Clinical, biochemical, adrenal hormonal, and pharmacokinetic data were obtained weekly for study duration. Results ATR-101 exposure increased with increasing dose. ACTH-stimulated cortisol concentrations, the primary endpoint for the study, were significantly decreased with responders (9 of 10 dogs) experiencing a mean ± standard deviation reduction in cortisol levels of 50 ± 17% at study completion. Decreases in pre-ACTH-stimulated cortisol concentrations were observed in some dogs although overall changes in pre-ACTH cortisol concentrations were not significant. The compound was well-tolerated and no serious drug-related adverse effects were reported. Conclusions This study highlights the potential utility of naturally occurring canine Cushing’s syndrome as a model for human disease and provides proof of concept for ATR-101 as a novel agent for the treatment of endocrine disorders like Cushing’s syndrome in humans

Characterization of two novel lytic bacteriophages having lysis potential against MDR avian pathogenic Escherichia coli strains of zoonotic potential

Abstract Avian pathogenic E. coli (APEC) is associated with local and systemic infections in poultry, ducks, turkeys, and many other avian species, leading to heavy economical losses. These APEC strains are presumed to possess zoonotic potential due to common virulence markers that can cause urinary tract infections in humans. The prophylactic use of antibiotics in the poultry sector has led to the rapid emergence of Multiple Drug Resistant (MDR) APEC strains that act as reservoirs and put human populations at risk. This calls for consideration of alternative strategies to decrease the bacterial load. Here, we report isolation, preliminary characterization, and genome analysis of two novel lytic phage species (Escherichia phage SKA49 and Escherichia phage SKA64) against MDR strain of APEC, QZJM25. Both phages were able to keep QZJM25 growth significantly less than the untreated bacterial control for approximately 18 h. The host range was tested against Escherichia coli strains of poultry and human UTI infections. SKA49 had a broader host range in contrast to SKA64. Both phages were stable at 37 °C only. Their genome analysis indicated their safety as no recombination, integration and host virulence genes were identified. Both these phages can be good candidates for control of APEC strains based on their lysis potential