Can a virtual microbiology simulation be as effective as the traditional Wetlab for pharmacy student education?

BACKGROUND: Pharmacy practice education requires the development of proficiencies and an understanding of clinical microbiology. Learning in this area could be delivered using practical laboratory exercises, or potentially, simulation-based education. Simulation has previously successfully enhanced learning in health professional education. The current global climate due to COVID-19 has further highlighted the important role of technology-enhanced learning in delivering outcomes that meet the requisite learning objectives of a course. The aim of the present study was to compare the impact of a commercially available virtual microbiology simulation (VUMIE™) with a traditional wet laboratory (wetlab) on learner knowledge, skills and confidence in a second-year integrated pharmacotherapeutics course for Bachelor of Pharmacy students. METHODS: A randomised, crossover study was employed to determine whether the simulation intervention (VUMIE™) improves learning outcomes (knowledge, skills and confidence) of pharmacy students, when compared to a traditional wetlab intervention. Each student completed three 1-2 h length sessions, for both the wetlab and VUMIE™ interventions (6 sessions total). Data was collected using surveys deployed at baseline (pre-interventions), post-intervention 1 or 2 (VUMIE™ or wetlab) and endpoint (post-interventions 1 and 2). Statistical analysis was conducted using SPSS Statistics 25 and Instat™ software. RESULTS: Response rates were approximately 50% at initial survey and approximately 25% at endpoint survey. VUMIE™ produced higher post-intervention knowledge scores for the multiple-choice questions compared to the wetlab, however, the highest score was achieved at endpoint. Both interventions produced statistically significant differences for mean scores compared to baseline (pre-VUMIE™ and wetlab) across the domains of knowledge, skills and confidence. VUMIE™ produced higher post-intervention mean scores for knowledge, skills and confidence compared to post-intervention mean scores for the wetlab, however there was no statistical significance between the mean score for the two interventions, thus the VUMIE™ activity produced learning outcomes comparable to the wetlab activity. CONCLUSION: These findings suggest VUMIE™ provides similar effects on students' knowledge, skills, and confidence as a wetlab. The simulation's implementation was not cost-prohibitive, provided students with a physically and psychologically safe learning environment, and the benefit of being able to repeat activities, supporting deliberate practice.

Differences in statin associated neuroprotection corresponds with either decreased production of IL-1ß or TNF-α in an in vitro model of neuroinflammation-induced neurodegeneration.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) have been associated with conflicting effects within the central nervous system (CNS), with underlying mechanisms remaining unclear. Although differences between individual statins' CNS effects have been reported clinically, few studies to date have compared multiple statins' neuroprotective effects. This study aimed to compare six statins (atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin; 0-100 µM) using an in vitro model of lipopolysaccharide (LPS)-induced neuroinflammation and subsequent neurodegeneration. To achieve this, HAPI microglia were treated with LPS (0.1 µg/mL; 24 h), resulting in increased reactive oxygen species (ROS), nitric oxide, and IL-1ß, TNF-α and PGE2 release. Conditioned media ("HAPI-CM") was then transferred to SH-SY5Y neuroblastoma cells, and effects on cellular viability, mitochondrial membrane permeability, apoptosis, autophagy and ROS production assessed. Of the statins investigated, only atorvastatin, pravastatin and rosuvastatin protected SH-SY5Y cells from LPS-induced decreases in cellular viability; this appeared mediated through reduced caspase 3/7 activation and was associated with decreased IL-1ß (atorvastatin, pravastatin) and/or TNF-α (atorvastatin, pravastatin, rosuvastatin). Only pravastatin conferred protection at all tested concentrations. ROS production and autophagic vacuole formation was decreased by all statins, suggesting these two mechanisms are unlikely to be sole mediators of neuroprotection seen with selected statins. Ultimately, our model suggests that despite all statins reducing microglial inflammation, subsequent effects on neuronal viability and cell death signalling pathways varies between statins. Our findings highlight the need to consider individual statins as inducing discrete pharmacological effects within the CNS in future in vitro/in vivo studies and in clinical practice.

Statins Reduce Lipopolysaccharide-Induced Cytokine and Inflammatory Mediator Release in an In Vitro Model of Microglial-Like Cells.

The anti-inflammatory effects of statins (HMG-CoA reductase inhibitors) within the cardiovascular system are well-established; however, their neuroinflammatory potential is unclear. It is currently unknown whether statins' neurological effects are lipid-dependent or due to pleiotropic mechanisms. Therefore, the assumption that all statin compounds will have the same effect within the central nervous system is potentially inappropriate, with no studies to date having compared all statins in a single model. Thus, the aim of this study was to compare the effects of the six statins (atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) within a single in vitro model of neuroinflammation. To achieve this, PMA-differentiated THP-1 cells were used as surrogate microglial cells, and LPS was used to induce inflammatory conditions. Here, we show that pretreatment with all statins was able to significantly reduce LPS-induced interleukin (IL)-1ß and tumour necrosis factor (TNF)-α release, as well as decrease LPS-induced prostaglandin E2 (PGE2). Similarly, global reactive oxygen species (ROS) and nitric oxide (NO) production were decreased following pretreatment with all statins. Based on these findings, it is suggested that more complex cellular models should be considered to further compare individual statin compounds, including translation into in vivo models of acute and/or chronic neuroinflammation.

Quality of warfarin control in atrial fibrillation patients in South East Queensland, Australia.

BACKGROUND: Warfarin is widely prescribed to decrease the risk of stroke in atrial fibrillation (AF) patients. Due to patient variability in response, regular monitoring is required, and time in therapeutic range (TTR) used to indicate quality of warfarin control with a TTR>60% is recommended. Recently, an Australian Government review of anticoagulants identified the need to establish current warfarin control and determine the potential place of the newer oral anticoagulants. AIM: To determine warfarin control by a pathology practice in Queensland, Australia and identify factors influencing TTR. METHODS: Retrospective data were collected from Sullivan Nicolaides Pathology, a major pathology practice offering a warfarin care programme in Australia. Patients enrolled in their programme as of September 2014 were included in the study. TTR was calculated using INR test results, and test dates using the Rosendaal method with mean patient TTR were used for analysis and comparison. Exclusions were target therapeutic range outside 2.0-3.0, less than two INR tests and programme treatment time of less than 30 days. RESULTS: The eligible 3692 AF patients had 73.6% of INR tests within the therapeutic range. The mean TTR was 81%, with 97% of patients above a TTR of 60%. TTR was not significantly influenced by age, gender or socioeconomic factors. CONCLUSIONS: The observed mean TTR of over 80% is superior to the minimum recommended threshold of 60%. The TTR achieved by the Queensland pathology practice demonstrates that dedicated warfarin programmes can produce high-quality warfarin care, ensuring the full benefit of warfarin for Australian patients.

Alterations in acetylcholine, PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide.

The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1-100 µM) or LPS (1-100 ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100 µM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100 ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100 µM), stretch-induced release for pseudomonal LPS (≥ 10 ng/mL) and both basal and stimulated release for E. coli LPS (≥ 10 ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100 µM) or LPS (100 ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.

Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

Resazurin assay of radiation response in cultured cells.

We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.

Further evidence for biological effects resulting from ionizing radiation doses in the diagnostic X-ray range.

In this study we assessed cell response to ionizing radiation doses in the diagnostic X-ray range, using metabolic performance as the biological end point. HeLa cells were irradiated between 3.5 mGy (standard deviation (SD=2.5%) and 55.1 mGy (SD=0.9%) with 110 kV X-rays, and after 7 days metabolic capacity of cultures was measured spectrophotometrically with resazurin. Cells receiving 7.5 mGy or greater showed impaired proliferation. At 7.5 mGy this effect was prevented by nitric oxide synthase inhibitor N G-monomethyl-L-arginine but not by ascorbic acid or p38 MAPK inhibitor SB203580. We conclude that HeLa cell proliferation is adversely affected by doses within the range of some radiological examinations, through a mechanism involving nitric oxide.

The effect of variations in pH and temperature on stability of melatonin in aqueous solution.

Melatonin (N-acetyl-5-methoxytryptamine) has a diverse range of functions, including the control of neuroendocrine events. A number of studies have shown that melatonin may be of potential benefit for the treatment of insomnia, as well as neurodegenerative disorders. At present, there are numerous dosage forms of melatonin, with the oral route of administration being most popular. Presently, there is little information on the stability of melatonin over a pH range. With the changes in pH in the gastro-intestinal tract, as well as in different experimental conditions, information on the stability of melatonin would be important. We used a high-performance liquid chromatography method to determine the stability of melatonin solutions over a pH range (1.2-12) at room temperature and at 37 degrees C over a period of 21 days. The results show that no melatonin degradation occurred in the first 2 days. From days 3 to 21, there was a gradual decline in melatonin at all pHs, with the decline not exceeding 30%. No decline in melatonin levels occurred in the first 2 days at 37 degrees C. From days 3 to 21, melatonin levels declined gradually, with the decline not exceeding 29%.

A sensitive and reliable method for the detection of lipid peroxidation in biological tissues.

A simple, accurate and cost effective method has been designed for the determination of lipid peroxidation in biological tissue samples. The method was a modification and improvement on existing methods available for lipid peroxidation determination. Solid-phase extraction was used to separate the thiobarbituric acid-malondialdehyde complex from thiobarbituric acid-reactive substances and HPLC was performed using a C18 (Waters Spherisorb, 5 microm, 250 x 4.6 mm i.d.) column to achieve isolation of the complex. The procedure was validated with respect to linearity of calibration (0.998), precision, sensitivity and limits of quantitation (1 nmol mL(-1)) and detection (0.5 nmol mL(-1)). Resorcinol was used as an external standard. The method was tested by inducing free radical generation with a known free radical generator, quinolinic acid, in rat brain homogenate. The results showed that the method presented allowed detection of lipid peroxidation products at concentrations in the nanomolar (nM) range compared with the micromolar (microM) range detected by other methods, thus rendering it suitable for use with biological samples. In addition, the modified method allowed for detection of the purified lipid peroxidation products, thus eliminating the possibility of simultaneous detection of impurities that absorb at the same wavelength.

Acetaminophen inhibits liver trytophan-2,3-dioxygenase activity with a concomitant rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic acid.

The effect of the analgesic agent, acetaminophen was determined on rat forebrain serotonin levels as well as hepatic tryptophan-2,3-dioxygenase (TDO) activity and urinary 5-hydroxyindole acetic acid (5-HIAA). The results show that acetaminophen administration (100mg/kg) over three hours does not affect the holoenzyme of tryptophan-2,3-dioxygenase but significantly inhibits the apoenzyme. This inhibition is accompanied by a concomitant rise in forebrain serotonin levels. This phenomenon is also accompanied by a reduction in urinary 5-HIAA levels. These results suggest that acetaminophen use is accompanied by changes in brain serotonin levels due to inhibition of hepatic tryptophan-2,3-dioxygenase activity. This in turn could explain the possible abuse potential of acetaminophen and its effects on mood at high doses.

Cyanide-induced free radical production and lipid peroxidation in rat brain homogenate is reduced by aspirin.

The neuroprotective properties of aspirin were investigated using cyanide-induced neurotoxicity as model. Cyanide, a known neurotoxic agent significantly increased lipid peroxidation and superoxide anion levels in rat brain homogenate in a concentration-dependent manner (0.25-1.0 mM). When homogenate, containing 1.0 mM KCN was co-treated with aspirin (1.0 mM) there was a significant decrease in lipid peroxidation. Aspirin (0.5 mM and 1.0 mM) also significantly reduced KCN-induced superoxide anion generation. The results of the present report therefore indicate a neuroprotective role for aspirin.

An investigation into the neuroprotective properties of ibuprofen.

There is increasing evidence suggesting a protective role for anti-inflammatory medications in neurological disorders such as Alzheimer's disease (AD). While there has not been any direct evidence for this, a number of clinical studies indicate that those patients who have had a history of nonsteroidal anti-inflammatory use, have a lower incidence of AD. Since there is currently no evidence on the mechanism by which these agents offer possible neuroprotection, we investigated the potential neuroprotective properties of the nonsteroidal anti-inflammatory drug, ibuprofen, by examining whether this agent could reduce lipid peroxidation and superoxide radical generation. Quinolinic acid and cyanide, known neurotoxins, were used to induce lipid peroxidation and superoxide anion formation respectively, in rat brain homogenate. The results show that ibuprofen significantly (p<0.05) reduced quinolinic acid-induced lipid peroxidation and cyanide-induced superoxide production. The results of the present report therefore suggest a possible mechanism for the neuroprotective effect of ibuprofen.