Implementation of a hepatitis-themed virtual escape room in pharmacy education: A pilot study.

As we enter a world of blended learning in higher education, an increased need for adaptation of teaching strategies to enhance engagement has been recognised to amplify learning outcomes online. Gamification has been identified as a creative tool to engage the current cohort of learners who are also characteristically tech-savvy. To this end, escape room games have gained considerable traction in medical and pharmacy education to promote learning, critical thinking and teamwork. In this pilot study we describe the implementation of a 60-minute, web-based hepatitis-themed escape room game within a Year 3 Pharmacotherapy unit at Monash University. A total of 418 students participated in this activity. Students' knowledge gain on the topic was assessed through a pre- and post-intervention assessment, whereby a statistically significant improvement was seen in the knowledge score following implementation of the gaming activity (58.66% pre-intervention vs. 72.05% post-intervention, p < 0.05). The innovative learning activity was also well perceived by the students. Virtual escape room game is a viable pedagogical approach to teach and reinforce clinical concepts among pharmacy students. With the evolving landscape of education and learner demographics, investment in technology- enhanced game-based learning is a promising trajectory to support students' growth in a learner-centered environment. A comparison between virtual escape room game and traditional teaching will further inform effectiveness of the gamification on long term knowledge retention.

Students' and Examiners' Experiences of Their First Virtual Pharmacy Objective Structured Clinical Examination (OSCE) in Australia during the COVID-19 Pandemic.

Objective Structured Clinical Examinations (OSCEs) are routinely used in healthcare education programs. Traditionally, students undertake OSCEs as face-to-face interactions to assess competency in soft skills. Due to physical distancing restrictions during COVID-19, alternative methods were required. This study utilized a mixed-method design (online survey and interviews) to evaluate second-year pharmacy students' and examiners' experiences of their first virtual OSCEs in Australia. A total of 196 students completed their first virtual OSCE in June 2020 of which 190 students completed the online survey. However, out of the 190 students, only 88% (n = 167) consented to the use of the data from their online survey. A further 10 students and 12 examiners were interviewed. Fifty-five students (33%) who participated in the online survey strongly agreed or agreed that they preferred the virtual experience to face-to-face OSCEs while 44% (n = 73) neither agreed nor disagreed. Only 20% (n = 33) felt more anxious with the virtual OSCEs. Additionally, thematic analysis found non-verbal communication as a barrier during the OSCE. Positive aspects about virtual OSCEs included flexibility, decreased levels of anxiety and relevance with emerging telehealth practice. The need for remote online delivery of assessments saw innovative ways of undertaking OSCEs and an opportunity to mimic telehealth. While students and examiners embraced the virtual OSCE process, face-to-face OSCEs were still considered important and irreplaceable. Future opportunities for OSCEs to be delivered both face-to-face and virtually should be considered.

Incorporation of MyDispense, a Virtual Pharmacy Simulation, into Extemporaneous Formulation Laboratories.

A core competency of Australian Pharmacy graduates is to prepare and compound extemporaneous formulations. Students in our pharmacy course would traditionally formulate extemporaneous products in laboratory classes while simultaneously preparing a handwritten label, with students divorcing this laboratory activity from the entire dispensing process. As a way to incorporate the dispensing process into the preparation of extemporaneous products without adding excessive time to the laboratory, we integrated MyDispense, a virtual pharmacy simulation, in pre-laboratory activities. This meant that students could complete all the dispensing activities for prescribed extemporaneous formulations prior to attending the laboratory. Prescriptions for solutions, suspensions, creams and ointments were developed in MyDispense, including essential components for dispensing an extemporaneous formulation (e.g., formulation name, dosing instructions). These prescriptions were provided to students at least 1 week prior to their laboratory classes, whereas for the laboratory assessments, the prescription was provided at the commencement of the extemporaneous exam. Due to the implementation of dispensing via MyDispense, we found that students demonstrated pre-laboratory engagement as all students presented their printed labels upon entering the laboratory. We also observed an increase in interaction between students and laboratory facilitators, mainly focused on the principles of formulation integrated around patient outcomes. Virtual simulations such as MyDispense can therefore provide a guided realistic learning experience, whilst overcoming time pressures associated with laboratory timetabling. This approach also encourages students to engage in the dispensing process prior to extemporaneous laboratories providing more opportunity to discuss higher-level formulation principles and patient-centred outcomes during laboratory classes.

Analysis of Dispensing Errors Made by First-Year Pharmacy Students in a Virtual Dispensing Assessment.

Pharmacists have a crucial role in the supply of medications and ensuring optimal patient outcomes. However, with the increased use of prescription medications, there is a potential for dispensing errors to occur. Some dispensing errors can result in patient harm, with some leading to death. The development of safe and accurate dispensing skills in pharmacy students is an essential part of the pharmacy curriculum to prevent such dispensing errors from occurring. A retrospective study was conducted on a virtual dispensing assessment completed by first-year pharmacy students using MyDispense at Monash University. Students were assessed on their ability to safely and accurately dispense four prescriptions. The students' answers in the assessment were then analyzed using qualitative and quantitative methods. Errors in drug quantity, number of repeats, product, patient and prescriber selection were quantitatively analyzed. Through the development of a codebook, frequency of errors was determined for label directions and appropriate use of ancillary labels. In this study, the dispensing errors that were identified depended on the class of medication. Errors in label directions were most common, with the majority of errors displaying incorrect route of administration, drug formulation and/or frequency of dosing. Identified errors were then further categorized into potential severity of harm, ranging from "no harm" to "severe harm". The findings from this study show the types of errors made by students that are preventable and the potential for first-year pharmacy students to benefit from more comprehensive introductions to dispensing guides and safe environments to practice.

Effect of Changing From Closed-Book to Formulary-Allowed Examinations.

Objective. To determine whether allowing final-year Bachelor of Pharmacy students to use a medicines formulary during examinations modified their learning behaviors and performance, and to investigate students' perceptions about having this resource available during examinations.Methods. Student performance and examination difficulty (as measured by classification of examination questions as high or low according to Bloom's taxonomy of learning) in second semester examinations (formulary allowed) was compared with first semester examinations (closed book) in successive years. Students completed a survey regarding their study and examination approaches and experiences after both semesters.Results. Examinations in semester two had more questions rated higher on Bloom's taxonomy than did examinations in semester one. Data were collected from student surveys for closed book examinations (response rate of 25% and 19% in 2015 and 2016, respectively) and open book examinations (response rate of 22% and 15% in 2015 and 2016, respectively). Students' study approaches, hours studied per week, and anxiety (all self-reported) did not differ between semesters one and two, but students in semester two spent more time studying with a formulary compared with students in semester one. Qualitative analysis of student comments revealed students preferred the formulary-allowed examinations over the closed-book examinations. The majority of students (68%) agreed with the statement: "Knowing that I will have access to the AMH [Australian Medicines Handbook] during the exams allowed me to pay more attention to higher level skills such as analysis and evaluation."Conclusion. When students were allowed to use a formulary for examinations, they studied more using their formulary prior to the examination. Students performed similarly on examinations with a greater proportion of questions addressing higher levels of Bloom's taxonomy and on closed-book examinations that were comparatively less cognitively challenging.

Surface association sensitizes Pseudomonas aeruginosa to quorum sensing.

In the pathogen Pseudomonas aeruginosa, LasR is a quorum sensing (QS) master regulator that senses the concentration of secreted autoinducers as a proxy for bacterial cell density. Counterintuitively, previous studies showed that saturating amounts of the LasR ligand, 3OC12-HSL, fail to induce the full LasR regulon in low-density liquid cultures. Here we demonstrate that surface association, which is necessary for many of the same group behaviors as QS, promotes stronger QS responses. We show that lasR is upregulated upon surface association, and that surface-associated bacteria induce LasR targets more strongly in response to autoinducer than planktonic cultures. This increased sensitivity may be due to surface-dependent lasR induction initiating a positive feedback loop through the small RNA, Lrs1. The increased sensitivity of surface-associated cells to QS is affected by the type IV pilus (TFP) retraction motors and the minor pilins. The coupling of physical surface responses and chemical QS responses could enable these bacteria to trigger community behaviors more robustly when they are more beneficial.

Modular exchange of substrate-binding loops alters both substrate and cofactor specificity in a member of the aldo-keto reductase superfamily.

Substrate specificity in the aldo-keto reductase (AKR) superfamily is determined by three mobile loops positioned at the top of the canonical (α/ß)(8)-barrel structure. These loops have previously been demonstrated to be modular in a well-studied class of AKRs, in that exchanging loops between two similar hydroxysteroid dehydrogenases resulted in a complete alteration of substrate specificity (Ma,H. and Penning,T.M. (1999) Proc. Natl Acad. Sci. USA, 96, 11161-11166). Here, we further examine the modularity of these loops by grafting those from human aldose reductase (hAR) into the hyperthermostable AKR, alcohol dehydrogenase D (AdhD), from Pyrococcus furiosus. Replacement of Loops A and B was sufficient to impart hAR activity into AdhD, and the resulting chimera retained the thermostability of the parent enzyme. However, no active chimeras were observed when the hAR loops were grafted into a previously engineered cofactor specificity mutant of AdhD, which displayed similar kinetics to hAR with the model substrate dl-glyceraldehyde. The non-additivity of these mutations suggests that efficient turnover is more dependent on the relative positioning of the cofactor and substrate in the active site than on binding of the individual species. The ability to impart the substrate specificities of mesostable AKRs into a thermostable scaffold will be useful in a variety of applications including immobilized enzyme systems for bioelectrocatalysis and fine chemical synthesis.

Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins.

The post-translational sulfation of tyrosine residues occurs in numerous secreted and integral membrane proteins and, in many cases, plays a crucial role in controlling the interactions of these proteins with physiological binding partners as well as invading pathogens. Recent advances in our understanding of protein tyrosine sulfation have come about owing to the cloning of two human tyrosylprotein sulfotransferases (TPST-1 and TPST-2), the development of novel analytical and synthetic methodologies and detailed studies of proteins and peptides containing sulfotyrosine residues. In this article, we describe the TPST enzymes, review the major techniques available for studying the presence, location and function of tyrosine sulfation in proteins and discuss the biological functions and biochemical interactions of several proteins (or protein families) in which tyrosine sulfation influences the protein function. In particular, we describe the detailed evidence supporting the importance of tyrosine sulfation in the cellular adhesion function of P-selectin glycoprotein ligand-1, the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the ligand binding and activation of other G-protein-coupled receptors by complement proteins, phospholipdis and glycoprotein hormones.

Probing the fibrate binding specificity of rat liver fatty acid binding protein.

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

Characterization of the drug binding specificity of rat liver fatty acid binding protein.

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli.

Rat liver fatty acid binding protein (L-FABP) was efficiently expressed in Escherichia coli and purified to homogeneity. The cDNA encoding L-FABP was ligated into the pTrc99A expression vector and expressed by induction with isopropyl-beta-d-thiogalactopyranoside under the control of the P(trc) promoter. Following an 18 h induction period, L-FABP constituted approximately 3% of the cytosolic protein. The protein could be purified to electrophoretic homogeneity (silver-stained polyacrylamide gel detection) by ammonium sulfate fractionation (65% saturation) of the soluble bacterial lysate followed by the chromatographic sequence of anion-exchange-->hydrophobic interaction-->anion-exchange chromatography. The recombinant protein displayed an isoelectric point of 7.0 and cross-reactivity with rabbit anti-(human L-FABP) polyclonal antibody. The ligand binding properties of the delipidated L-FABP were examined by titration with the fluorescent probe 1-anilino-8-naphthalene sulfonic acid and isothermal titration calorimetric analysis of oleic acid binding. The purified rat L-FABP displayed a binding stoichiometry of 2:1 (ANS:L-FABP) with dissociation constants (K(d)) of 1.7 and 15.5 microM for the high and low affinity binding sites, respectively. The K(d) values determined by ITC for oleic acid binding were 0.155 and 4.04 microM with a binding stoichiometry of approximately 2 mol of fatty acid/mol of protein. These physicochemical and binding properties are in agreement with those of L-FABP isolated from rat liver tissue.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.