Amelia J. McFarland

Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa

Pyocyanin has recently emerged as an important virulence factor produced by Pseudomonas aeruginosa. The redox-active tricyclic zwitterion has been shown to have a number of potential effects on various organ systems in vitro, including the respiratory, cardiovascular, urological, and central nervous systems. It has been shown that a large number of the effects to these systems are via the formation of reactive oxygen species. The limitations of studies are, to date, focused on the localized effect of the release of pyocyanin (PCN). It has been postulated that, given its chemical properties, PCN is able to readily cross biological membranes, however studies have yet to be undertaken to evaluate this effect. This review highlights the possible manifestations of PCN exposure; however, most studies to date are in vitro. Further high quality in vivo studies are needed to fully assess the physiological manifestations of PCN exposure on the various body systems.

Molecular Mechanisms Underlying the Effects of Statins in the Central Nervous System

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly referred to as statins, are widely used in the treatment of dyslipidaemia, in addition to providing primary and secondary prevention against cardiovascular disease and stroke. Statins’ effects on the central nervous system (CNS), particularly on cognition and neurological disorders such as stroke and multiple sclerosis, have received increasing attention in recent years, both within the scientific community and in the media. Current understanding of statins’ effects is limited by a lack of mechanism-based studies, as well as the assumption that all statins have the same pharmacological effect in the central nervous system. This review aims to provide an updated discussion on the molecular mechanisms contributing to statins’ possible effects on cognitive function, neurodegenerative disease, and various neurological disorders such as stroke, epilepsy, depression and CNS cancers. Additionally, the pharmacokinetic differences between statins and how these may result in statin-specific neurological effects are also discussed.

Effects of Pseudomonas Aeruginosa Virulence Factor Pyocyanin on Human Urothelial Cell Function and Viability

PURPOSE We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. MATERIALS AND METHODS RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. RESULTS The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. CONCLUSIONS Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection.

Paradoxical Role of 3-Methyladenine in Pyocyanin-Induced Toxicity in 1321N1 Astrocytoma and SH-SY5Y Neuroblastoma Cells

The role of autophagy in pyocyanin (PCN)-induced toxicity in the central nervous system (CNS) remains unclear, with only evidence from our group identifying it as a mechanism underlying toxicity in 1321N1 astrocytoma cells. Therefore, the aim of this study was to further examine the role of autophagy in PCN-induced toxicity in the CNS. To achieve this, we exposed 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells to PCN (0-100 μmol/L) and tested the contribution of autophagy by measuring the impact of the autophagy inhibitor 3-methyladenine (3-MA) using a series of biochemical and molecular markers. Pretreatment of 1321N1 astrocytoma cells with 3-MA (5 mmol/L) decreased the PCN-induced acidic vesicular organelle and autophagosome formation as measured using acridine orange and green fluorescent protein-LC3 -LC3 fluorescence, respectively. Furthermore, 3-MA (5 mmol/L) significantly protected 1321N1 astrocytoma cells against PCN-induced toxicity. In contrast pretreatment with 3-MA (5 mmol/L) increased PCN-induced toxicity in SH-SY5Y neuroblastoma cells. Given the influence of autophagy in inflammatory responses, we investigated whether the observed effects in this study involved inflammatory mediators. The PCN (100 μmol/L) significantly increased the production of interleukin-8 (IL-8), prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) in both cell lines. Consistent with its paradoxical role in modulating PCN-induced toxicity, 3-MA (5 mmol/L) significantly reduced the PCN-induced production of IL-8, PGE2, and LTB4 in 1321N1 astrocytoma cells but augmented their production in SH-SY5Y neuroblastoma cells. In conclusion, we show here for the first time the paradoxical role of autophagy in mediating PCN-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells and provide novel evidence that these actions may be mediated by effects on IL-8, PGE2, and LTB4 production.

ERK1/2 activation modulates pyocyanin-induced toxicity in A549 respiratory epithelial cells

Pyocyanin (PCN), a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate oxidative stress. However mechanisms underlying PCN-induced oxidative injury remain unclear. Although oxidative stress and subsequent MAPK signaling has been shown to modulate cell death in other models, its role in PCN-induced cytotoxicity remains unknown. Therefore the aim of this study was to investigate the role of redox-sensitive MAPK in PCN-induced toxicity in A549 cells. Here we show that PCN (50μM) rapidly increased ERK1/2 phosphorylation after 5min. Pre-treatment of A549 cells with the MEK1/2 inhibitor U0126 (10μM) decreased PCN-induced ERK1/2 phosphorylation and protected cells against apoptosis and cell injury suggesting a role for ERK signalling. In contrast, JNK and p38 MAPK phosphorylation remained unchanged following exposure to PCN and pretreatment with either the JNK or p38 MAPK inhibitors (10μM SP600125 and 10μM SB203580, respectively) did not afford protection against PCN toxicity. This would suggest that PCN-induced cytotoxicity appears to occur independently of JNK and p38 MAPK signaling pathways. Finally, although we confirm that oxidative stress contributes to PCN-induced toxicity, our data suggest the contribution of oxidative stress is independent of ERK1/2 signaling. These findings may provide insight for novel targeted therapies to reduce PCN-mediated lung injury in patients with chronic P. aeruginosa respiratory infections.

A pilot study assessing the value of 3D printed molecular modelling tools for pharmacy student education

BACKGROUND AND PURPOSE Medicinal chemistry and pharmacology are difficult topics to both teach and learn given the complex nature of drug mechanisms and drug-receptor interactions. This highlights the need for innovative teaching methods to deliver this information to students. One such method is through three-dimensional (3D) printing of enzymes and ligands in the teaching of molecular modelling concepts relating to drug-receptor and enzyme interactions be ligands. This type of printing has been shown to be beneficial in several educational settings; however, to our knowledge, its effectiveness in pharmacy, medicinal chemistry and pharmacology learning and teaching is largely unknown. Therefore, the aim of this study was to evaluate pharmacy student perceptions and the educational benefits of 3D printed molecules in molecular modelling with regards to engagement and learning outcomes when used in a drug-target interaction topic. EDUCATIONAL ACTIVITY AND SETTING This aim was achieved through administering students a short questionnaire designed to evaluate their engagement and learning outcomes with students also free to provide comments. FINDINGS This study found that nearly all (>90%) students found the activity was useful in improving both student engagement and learning outcomes. DISCUSSION AND SUMMARY In conclusion, 3D printing may provide an alternative learning activity to help pharmacy students understand the drug-target interaction.

Pyocyanin induces systemic oxidative stress, inflammation and behavioral changes in vivo

Abstract Pyocyanin (PCN) is a virulence factor secreted by Pseudomonas aeruginosa (P. aeruginosa) that has been shown to have numerous toxic effects in both in vitro and in vivo studies. Such toxicities include pro-inflammatory and pro-oxidant mediated responses. It is hypothesized that PCN can cross biological membranes and reach the systemic circulation, but no previous studies have investigated this. The aim of this study was, therefore, to quantify PCN in plasma and assess if systemic responses were occurring after localized intranasal administration in C57BL/6 J mice. This was achieved through the plasma quantification of PCN and assessment of changes to behavior using two commonly used tests, the forced swimming test and the open field test. Furthermore, evidence of systemic oxidative stress and inflammation was measured using malondialdehyde (MDA) and TNF-α post PCN exposure. PCN was found to cross into systemic circulation but in a variable manner. Furthermore, significant increases in plasma TNF-α and MDA (both p < 0.001) were observed along with changes in behavior indicative of systemic inflammatory responses.

Appraising the role of the virtual patient for therapeutics health education

BACKGROUND Face-to-face instruction, paper-based case-studies and clinical placements remain the most commonly used teaching methods for therapeutics curricula. Presenting clinical content in a didactic manner presents challenges in engaging learners and developing their clinical reasoning skills which may be overcome by inclusion of the virtual patient (VP). Currently there is limited literature examining the use of the VP in therapeutics teaching and learning. This review aimed to determine the role of VPs in therapeutics education, specifically the impact on student experiences, performance, and clinical skills. METHODS A search of primary literature was conducted with search terms including virtual patient, education, health, AND learning. Boolean operators were applied to include studies from health relevant fields with article titles and abstracts vetted. RESULTS Nine of the 21 included studies were control-matched, and all but one compared VPs to traditional teaching. VPs enhanced the learning experience in all 17 studies that measured this outcome. Fourteen studies measured performance and clinical skills and 12 found VPs were beneficial, while two did not. The VP was not superior to traditional teaching in all studies, but the VP appeared beneficial to the student learning experience. Discrepancy was found between the impact of VPs on short- and long-term knowledge. IMPLICATIONS The VP appears to enhance the student learning experience and has a role in therapeutics education, however a blended-learning (BL) approach may be required to account for individual learning styles. Additional investigation is required to clarify the efficacy of the VP, particularly as a component of BL, on longer-term knowledge retention.

Folic acid supplementation improves vascular endothelial function, yet not skin blood flow during exercise in the heat, in patients with heart failure

Heart failure (HF) patients are susceptible to heat strain during exercise, secondary to blunted skin blood flow (SkBF) responses, which may be explained by impaired nitric oxide (NO)-dependent vasodilation. Folic acid improves vascular endothelial function and SkBF through NO-dependent mechanisms in healthy older individuals and patients with cardiovascular disease. We examined the effect of folic acid supplementation (5 mg/day for 6 wk) on vascular function [brachial artery flow-mediated dilation (FMD)] and SkBF responses [cutaneous vascular conductance (CVC)] during 60 min of exercise at a fixed metabolic heat production (300 ẆHprod) in a 30°C environment in 10 patients with HF (New York Heart Association Class I-II) and 10 healthy controls (CON). Serum folic acid concentration increased in HF [preintervention (pre): 1.4 ± 0.2; postintervention (post): 8.9 ± 6.7 ng/ml, P = 0.01] and CON (pre: 1.3 ± 0.6; post: 5.2 ± 4.9 ng/ml, P = 0.03). FMD improved by 2.1 ± 1.3% in HF ( P < 0.01), but no change was observed in CON postintervention ( P = 0.20). During exercise, the external workload performed on the cycle ergometer to attain the fixed level of heat production for exercise was similar between groups (HF: 60 ± 13; CON: 65 ± 20 external workload, P = 0.52). Increases in CVC during exercise were similar in HF (pre: 0.89 ± 0.43; post: 0.83 ± 0.45 au/mmHg, P = 0.80) and CON (pre: 2.01 ± 0.79; post: 2.03 ± 0.72 au/mmHg, P = 0.73), although the values were consistently lower in HF for both pre- and postintervention measurement intervals ( P < 0.05). These findings demonstrate that folic acid improves vascular endothelial function in patients with HF but does not enhance SkBF during exercise at a fixed metabolic heat production in a warm environment.