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1.  Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens 
PLoS Biology  2015;13(7):e1002198.
Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.
A study using Marek's disease virus in poultry shows that by reducing natural selection against highly virulent strains, imperfect vaccination enables the spread of viral strains that would otherwise be too lethal to persist.
Author Summary
There is a theoretical expectation that some types of vaccines could prompt the evolution of more virulent (“hotter”) pathogens. This idea follows from the notion that natural selection removes pathogen strains that are so “hot” that they kill their hosts and, therefore, themselves. Vaccines that let the hosts survive but do not prevent the spread of the pathogen relax this selection, allowing the evolution of hotter pathogens to occur. This type of vaccine is often called a leaky vaccine. When vaccines prevent transmission, as is the case for nearly all vaccines used in humans, this type of evolution towards increased virulence is blocked. But when vaccines leak, allowing at least some pathogen transmission, they could create the ecological conditions that would allow hot strains to emerge and persist. This theory proved highly controversial when it was first proposed over a decade ago, but here we report experiments with Marek’s disease virus in poultry that show that modern commercial leaky vaccines can have precisely this effect: they allow the onward transmission of strains otherwise too lethal to persist. Thus, the use of leaky vaccines can facilitate the evolution of pathogen strains that put unvaccinated hosts at greater risk of severe disease. The future challenge is to identify whether there are other types of vaccines used in animals and humans that might also generate these evolutionary risks.
PMCID: PMC4516275  PMID: 26214839
2.  An International Comparison Study of Pharmacy Students’ Achievement Goals and their Relationship to Assessment Type and Scores 
Objective: To identify pharmacy students’ preferred achievement goals in a multi-national undergraduate population, to investigate achievement goal preferences across comparable degree programs, and to identify relationships between achievement goals, academic performance, and assessment type.
Methods: The Achievement Goal Questionnaire was administered to second year students in 4 universities in Australia, New Zealand, England, and Wales. Academic performance was measured using total scores, multiple-choice questions, and written answers (short essay).
Results: Four hundred eighty-six second year students participated. Students showed an overall preference for the mastery-approach goal orientation across all sites. The predicted relationships between goal orientation and multiple-choice questions, and written answers scores, were significant.
Conclusion: This study is the first of its kind to examine pharmacy students’ achievement goals at a multi-national level and to differentiate between assessment type and measures of achievement motivation. Students adopting a mastery-approach goal are more likely to gain high scores in assessments that measure understanding and depth of knowledge.
PMCID: PMC4428420  PMID: 25995510
Achievement goals; assessment type; academic achievement; comparison study; pharmacy education
3.  Barriers and facilitators to patient recruitment to a cluster randomized controlled trial in primary care: lessons for future trials 
Primary-care based randomized controlled trials (RCTs) build an important evidence base for general practice but little evidence exists about barriers to recruitment which often hamper such trials.
We investigated the issues that impeded and facilitated recruitment to a clinical trial in general practice.
GPs participating in a cluster RCT that tested interventions for improving medication adherence and asthma control completed a survey comprising quantitative and free text questions about their recruitment experiences. We used backward regression to analyze quantitative data and coded free text responses into themes.
40/55 of enrolled GPs recruited patients, but only one-third reached the planned recruitment target (5 patients/GP). In univariate analyses, poor patient recruitment by GPs was significantly associated with longer time to first patient enrolment, GP-perceived poor access to eligible patients and GP working in a practice training medical students. In regression analysis, only the first was significant (p = 0.001); the explained variance of the model was 48%. Themes from free text responses described recruitment barriers at the level of GP (e.g. GPs excluding patients for whom research appeared too challenging), practice (e.g. practice cultures disempowered GPs), patient (e.g. reluctance to change treatment for research) and study (e.g. protocol requirements complicating recruitment). Facilitators included GPs perceiving good support from the research team.
Targeted recruitment support early in the recruitment phase may enhance recruitment rates. Over time, interventions to enhance a general practice research culture are also likely to enhance skills to recruit patients, even for complex interventions. We recommend systematic evaluation of recruitment approaches and outcomes in future RCTs to optimize feasibility and success of these important trials.
Trial registration
Australian and New Zealand Clinical Trials Registry ACTRN12610000854033 (date registered 14/10/2010).
PMCID: PMC4369080  PMID: 25887970
Attitude of health personnel; Patient selection; General practitioners; Physician-patient relations; Randomized controlled trials as topic; Asthma/prevention & control
4.  An exploration of clinical interventions provided by pharmacists within a complex asthma service 
Pharmacy Practice  2015;13(1):529.
Pharmacists in Australia are accessible health care professionals, and their provision of clinical pharmacy interventions in a range of areas has been proven to improve patient outcomes. Individual clinical pharmacy interventions in the area of asthma management have been very successful. An understanding of the nature of these interventions will inform future pharmacy services. What we do not know is when pharmacists provide a complex asthma service, what elements of that service (interventions) they choose to deliver.
To explore the scope and frequency of asthma-related clinical interventions provided by pharmacists to patients in an evidence-based complex asthma service.
Pharmacists from 4 states/territories of Australia were trained in asthma management. People with asthma had 3 or 4 visits to the pharmacy. Guided by a structured patient file, the pharmacist assessed the patient’s asthma and management and provided interventions where and when considered appropriate, based on their clinical decision making skills. The interventions were recorded in a checklist in the patient file. They were then analysed descriptively and thematically.
Pharmacists provided 22,909 clinical pharmacy interventions over the service to 570 patients (398 of whom completed the service). The most frequently delivered interventions were in the themes ’Education on asthma’, ’Addressing trigger factors’, ’Medications - safe and effective use’ and ’Explore patient perspectives’. The patients had a high and ongoing need for interventions. Pharmacists selected interventions based on their assessment of perceived need then revisited and reinforced these interventions.
Pharmacists identified a number of areas in which patients required interventions to assist with their asthma management. Many of these were perceived to require continuing reinforcement over the duration of the service. Pharmacists were able to use their clinical judgement to assess patients and provide clinical pharmacy interventions across a range of asthma management needs.
PMCID: PMC4384270  PMID: 25883692
Asthma; Pharmaceutical Services; Pharmacists; Evidence-Based Practice; Australia
5.  Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus 
PLoS ONE  2014;9(12):e114466.
Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek’s disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of ‘closed loop’ structure of mRNA.
PMCID: PMC4263670  PMID: 25503397
6.  A B-cell targeting virus disrupts potentially protective genomic methylation patterns in lymphoid tissue by increasing global 5-hydroxymethylcytosine levels 
Veterinary Research  2014;45(1):108.
The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius. We discovered that embryonic B-cells have high 5mC content while 5hmC decreases during bursa development. We propose that a high 5mC level protects from the mutagenic activity of the B-cell antibody diversifying enzyme activation induced deaminase (AID). In support of this view, AID mRNA increases significantly within the developing bursa from embryonic to post hatch stages while mRNAs that encode Tet family members 1 and 2 reduce over the same period. Moreover, our data revealed that infectious bursal disease virus (IBDV) disrupts this genomic methylation pattern causing a global increase in 5hmC levels in a mechanism that may involve increased Tet 1 and 2 mRNAs. To our knowledge this is the first time that a viral infection has been observed to cause global increases in genomic 5hmC within infected host tissues, underlining a mechanism that may involve the induction of B-cell genomic instability and cell death to facilitate viral egress.
PMCID: PMC4258027  PMID: 25338704
7.  Glycemic Control and Blood Glucose Monitoring Over Time in a Sample of Young Australians With Type 1 Diabetes 
Diabetes Care  2013;36(10):2968-2973.
To determine whether personality traits (conscientiousness, agreeableness, emotional regulation, extraversion, and openness to experience) are associated with glycemic control and blood glucose monitoring behavior, and change or stability of these outcomes over time, in young people with type 1 diabetes.
A 3-year longitudinal study was conducted using data from 142 individuals with type 1 diabetes, 8–19 years of age. Personality was assessed at baseline using the Five-Factor Personality Inventory for Children. Data relating to glycemic control (HbA1c) and frequency of blood glucose monitoring (based on meter memory) were collected annually. Relationships between personality traits and HbA1c and monitoring frequency were examined using regression models and mixed-design ANOVA.
Three of the Five-Factor domains were independently associated with glycemic control. Individuals high in conscientiousness and agreeableness had a lower and more stable HbA1c across the 3-year study period. In contrast, the HbA1c of individuals scoring low on these traits was either consistently worse or deteriorated over time. Low or high emotional regulation scores were also associated with worse glycemic control. By the third year, these domains, together with initial HbA1c, accounted for 39% of HbA1c variance. Conscientiousness was the only personality factor associated with blood glucose monitoring behavior.
Results of this study underline the importance of personality in contributing to diabetes outcomes. Attention to a young person’s personality, and appropriate tailoring of diabetes management to ensure an individualized approach, may help to optimize diabetes outcomes.
PMCID: PMC3781533  PMID: 23835696
8.  An international validation study of two achievement goal measures in a pharmacy education context 
Achievement goal theory helps us understand what motivates students to participate in educational activities. However, measuring achievement goals in a precise manner is problematic. Elliot and McGregor’s Achievement Goal Questionnaire (AGQ) and Elliot and Murayama’s revised Achievement Goal Questionnaire (AGQ-R) are widely used to assess students’ achievement goals. Both instruments were developed and validated using undergraduate psychology students in the USA.
In this study, our aims were to first of all, assess the construct validity of both questionnaires using a cohort of Australian pharmacy students and, subsequently, to test the generalizability and replicability of these tools more widely in schools of pharmacy in other English-speaking countries. The AGQ and the AGQ-R were administered during tutorial class time. Confirmatory factor analysis procedures, using AMOS 19 software, were performed to determine model fit.
In contrast to the scale developers’ findings, confirmatory factor analysis supported a superior model fit for the AGQ compared with the AGQ-R, in all countries under study.
Validating measures of achievement goal motivation for use in pharmacy education is necessary and has implications for future research. Based on these results, the AGQ will be used to conduct future cross-sectional and longitudinal analyses of the achievement goals of undergraduate pharmacy students from these countries.
PMCID: PMC4186489  PMID: 25298743
confirmatory factor analysis; achievement goal theory; pharmacy education
9.  Identifying Achievement Goals and Their Relationship to Academic Achievement in Undergraduate Pharmacy Students 
Objectives. To compare the achievement goal orientations of first-year with those of third-year undergraduate Australian pharmacy students and to examine the relationship of goal orientations to academic achievement.
Methods. The Achievement Goal Questionnaire was administered to first-year and third-year students during class time. Students’ grades were obtained from course coordinators.
Results. More first-year students adopted performance-approach and mastery-approach goals than did third-year students. Performance-approach goals were positively correlated with academic achievement in the first year. Chinese Australian students scored the highest in adopting performance-approach goals. Vietnamese Australian students adopted mastery-avoidance goals more than other ethnicities. First-year students were more strongly performance approach goal-oriented than third-year students.
Conclusion. Adopting performance-approach goals was positively correlated with academic achievement, while adopting avoidance goals was not. Ethnicity has an effect on the adoption of achievement goals and academic achievement.
PMCID: PMC4174375  PMID: 25258438
achievement goals; achievement goal questionnaire; academic achievement; ethnicity
10.  Investigating the Relationship Between Pharmacy Students’ Achievement Goal Orientations and Preferred Teacher Qualities 
Objective. To investigate the relationships between pharmacy students’ preferred teacher qualities and their academic achievement goal orientations.
Methods. Participants completed an achievement goal questionnaire and a build-a-teacher task. For the latter, students were given a $20 hypothetical budget to purchase amounts of 9 widely valued teachers’ qualities.
Results. Three hundred sixty-six students participated. Students spent most of their budget on the traits of enthusiasm, expertise, and clear presentation style, and the least amount of money on interactive teaching, reasonable workload, warm personality, and intellectually challenging. In relation to achievement goals, negative associations were found between avoidance goals and preferences for teachers who encourage rigorous thinking and self-direction.
Conclusion. These novel findings provide a richer profile of the ways students respond to their learning environment. Understanding the relationships between teachers’ characteristics and pharmacy students’ achievement goal orientations will contribute to improving the quality of pharmacy learning and teaching environments.
PMCID: PMC4174377  PMID: 25258440
achievement goals; motivation; pharmacy education; teacher qualities; student preferences
11.  Reflective Practice and Its Implications for Pharmacy Education 
Pharmacy students require critical-thinking and problem-solving skills to integrate theory learned in the classroom with the complexities of practice, yet many pharmacy students fall short of acquiring these skills.1-2 Reflective practice activities encourage learning from the student’s own experiences and those of others, and offer a possible solution for the integration of knowledge-based curricula with the ambiguities of practice, as well as enhance communication and collaboration within a multidisciplinary team. Although reflective practices have been embraced elsewhere in health professions education, their strengths and shortcomings need to be considered when implementing such practices into pharmacy curricula. This review provides an overview of the evolution of theories related to reflective practice, critically examines the use of reflective tools (such as portfolios and blogs), and discusses the implications of implementing reflective practices in pharmacy education.
PMCID: PMC3930242  PMID: 24558286
reflective practice; reflection; reflective learning; self-directed learning; pharmacy education
12.  An Avian Retrovirus Uses Canonical Expression and Processing Mechanisms To Generate Viral MicroRNA 
Journal of Virology  2014;88(1):2-9.
To date, the vast majority of known virus-encoded microRNAs (miRNAs) are derived from polymerase II transcripts encoded by DNA viruses. A recent demonstration that the bovine leukemia virus, a retrovirus, uses RNA polymerase III to directly transcribe the pre-miRNA hairpins to generate viral miRNAs further supports the common notion that the canonical pathway of miRNA biogenesis does not exist commonly among RNA viruses. Here, we show that an exogenous virus-specific region, termed the E element or XSR, of avian leukosis virus subgroup J (ALV-J), a member of avian retrovirus, encodes a novel miRNA, designated E (XSR) miRNA, using the canonical miRNA biogenesis pathway. Detection of novel microRNA species derived from the E (XSR) element, a 148-nucleotide noncoding RNA with hairpin structure, showed that the E (XSR) element has the potential to function as a microRNA primary transcript, demonstrating a hitherto unknown function with possible roles in myeloid leukosis associated with ALV-J.
PMCID: PMC3911700  PMID: 24155381
13.  ‘Help for Hay Fever’, a goal-focused intervention for people with intermittent allergic rhinitis, delivered in Scottish community pharmacies: study protocol for a pilot cluster randomized controlled trial 
Trials  2013;14:217.
Despite the availability of evidence-based guidelines for managing allergic rhinitis in primary care, management of the condition in the United Kingdom (UK) remains sub-optimal. Its high prevalence and negative effects on quality of life, school performance, productivity and co-morbid respiratory conditions (in particular, asthma), and high health and societal costs, make this a priority for developing novel models of care. Recent Australian research demonstrated the potential of a community pharmacy-based ‘goal-focused’ intervention to help people with intermittent allergic rhinitis to self-manage their condition better, reduce symptom severity and improve quality of life. In this pilot study we will assess the transferability of the goal-focused intervention to a UK context, the suitability of the intervention materials, procedures and outcome measures and collect data to inform a future definitive UK randomized controlled trial (RCT).
A pilot cluster RCT with associated preliminary economic analysis and embedded qualitative evaluation. The pilot trial will take place in two Scottish Health Board areas: Grampian and Greater Glasgow & Clyde. Twelve community pharmacies will be randomly assigned to intervention or usual care group. Each will recruit 12 customers seeking advice or treatment for intermittent allergic rhinitis. Pharmacy staff in intervention pharmacies will support recruited customers in developing strategies for setting and achieving goals that aim to avoid/minimize triggers for, and eliminate/minimize symptoms of allergic rhinitis. Customers recruited in non-intervention pharmacies will receive usual care. The co-primary outcome measures, selected to inform a sample size calculation for a future RCT, are: community pharmacy and customer recruitment and completion rates; and effect size of change in the validated mini-Rhinoconjunctivitis Quality of Life Questionnaire between baseline, one-week and six-weeks post-intervention. Secondary outcome measures relate to changes in symptom severity, productivity, medication adherence and self-efficacy. Quantitative data about accrual, retention and economic measures, and qualitative data about participants’ experiences during the trial will be collected to inform the future RCT.
This work will lay the foundations for a definitive RCT of a community pharmacy-based ‘goal-focused’ self-management intervention for people with intermittent allergic rhinitis. Results of the pilot trial are expected to be available in April 2013.
Trial registration
Current Controlled Trials ISRCTN43606442
PMCID: PMC3721993  PMID: 23856015
Respiratory; Allergy; Community pharmacy; Self-care
14.  A Learning and Teaching Resource on Patient Self-Management of Chronic Pain 
Objective. To develop, pilot test, and evaluate an instructional module on patient self-management for undergraduate pharmacy students in an Australian university.
Design. Learning outcomes and associated content and assessment tasks were developed, featuring lecture and readings, in-class discussions, and online delivery of in-depth interviews with patients who were living with chronic pain.
Assessment. Students completed a premodule and postmodule questionnaire and were further assessed by multiple-choice questions following completion of the module and again at the end of the semester. Positive changes were identified in the students’ discourse surrounding patient self-management of chronic pain. Responses to multiple-choice questions showed that knowledge was sustained over the course of the semester.
Conclusions. Completion of a comprehensive module on patient self-management increased undergraduate pharmacy students’ understanding and knowledge of patients experiencing chronic pain. The module could be implemented across other healthcare disciplines.
PMCID: PMC3602859  PMID: 23518902
online learning; curriculum patient interviews; patient self-management; student perceptions; assessment
15.  Experiences of community pharmacists involved in the delivery of a specialist asthma service in Australia 
The role of community pharmacists in disease state management has been mooted for some years. Despite a number of trials of disease state management services, there is scant literature into the engagement of, and with, pharmacists in such trials. This paper reports pharmacists’ feedback as providers of a Pharmacy Asthma Management Service (PAMS), a trial coordinated across four academic research centres in Australia in 2009. We also propose recommendations for optimal involvement of pharmacists in academic research.
Feedback about the pharmacists’ experiences was sought via their participation in either a focus group or telephone interview (for those unable to attend their scheduled focus group) at one of three time points. A semi-structured interview guide focused discussion on the pharmacists’ training to provide the asthma service, their interactions with health professionals and patients as per the service protocol, and the future for this type of service. Focus groups were facilitated by two researchers, and the individual interviews were shared between three researchers, with data transcribed verbatim and analysed manually.
Of 93 pharmacists who provided the PAMS, 25 were involved in a focus group and seven via telephone interview. All pharmacists approached agreed to provide feedback. In general, the pharmacists engaged with both the service and research components, and embraced their roles as innovators in the trial of a new service. Some experienced challenges in the recruitment of patients into the service and the amount of research-related documentation, and collaborative patient-centred relationships with GPs require further attention. Specific service components, such as the spirometry, were well received by the pharmacists and their patients. Professional rewards included satisfaction from their enhanced practice, and pharmacists largely envisaged a future for the service.
The PAMS provided pharmacists an opportunity to become involved in an innovative service delivery model, supported by the researchers, yet trained and empowered to implement the clinical service throughout the trial period and beyond. The balance between support and independence appeared crucial in the pharmacists’ engagement with the trial. Their feedback was overwhelmingly positive, while useful suggestions were identified for future academic trials.
PMCID: PMC3439711  PMID: 22709371
Pharmacy; Asthma; Disease management service; Experiences; Feedback
16.  Clonal Structure of Rapid-Onset MDV-Driven CD4+ Lymphomas and Responding CD8+ T Cells 
PLoS Pathogens  2011;7(5):e1001337.
Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both “public” and “private” CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.
Author Summary
Many viral infections target the immune system, making use of the long lived, highly proliferative lymphocytes to propagate and survive within the host. This characteristic has led to an association between some viruses such as Epstein Barr Virus (EBV), Human T cell Lymphotrophic Virus-1 (HTLV-1) and Mareks Disease Virus (MDV) and lymphoid tumors. We employed methods for identifying the T cell receptor repertoire as a molecular bar-code to study the biology of MDV-induced tumors and the anti-tumor response. Each individual contained a small number of large (high frequency) tumor clones alongside some smaller (lower frequency) clones in the CD4+ T cell population. The tumor infiltrating CD8+ T cell response was highly focused with a small number of large clones, with one representing a public CDR3 sequence. This data is consistent with the recognition of a small number of dominant antigens and understanding the relationship between these and protective immunity is important to improve development of new vaccination strategies. Collectively, our results provide insights into the clonal structure of MDV driven tumors and in the responding CD8+ T cell compartment. These studies advance our understanding of MDV biology, an important poultry disease and a natural infection model of virus-induced tumor formation.
PMCID: PMC3088711  PMID: 21573129
17.  Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas 
PLoS Pathogens  2011;7(2):e1001305.
Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.
Author Summary
MicroRNAs (miRNAs), encoded in the genomes of a number of organisms including several viruses, belong to a class of small RNA molecules that can function as key regulators of gene expression influencing various biological processes and diseases including cancer. Among all the miRNAs, miR-155 has been well documented for its direct role of oncogenesis in a number of species including chickens. Remarkably, miR-K12-11 and miR-M4, the miRNAs encoded by the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV) respectively, have been shown to be functional orthologs of miR-155. There are no animal models of KSHV-induced tumors to examine the oncogenic potential of miR-K12-11. However, using recombinant mutant viruses in excellent models of MDV-induced lymphomas in their natural chicken hosts, we demonstrate that miR-M4 is critical for the induction of tumors. This is the first study that clearly demonstrates a direct role for a single miRNA in inducing cancer in an in vivo animal model. The ability of gga-miR-155 to rescue the oncogenic potential of miR-M4-deleted virus demonstrated the conservation of oncogenic functions the two miRNAs. Moreover, we show that virus attenuated by deleting the miRNAs can function as vaccines against virulent virus infection, enabling the prospects of generating novel molecularly-defined vaccines.
PMCID: PMC3044692  PMID: 21383974
18.  Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes 
Bacterial artificial chromosome (BAC) vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs) are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130) of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.
PMCID: PMC2992818  PMID: 21127705
19.  Pharmacy Students' Approaches to Learning in Undergraduate and Graduate Entry Programs 
To compare longitudinal data with previous cross-sectional data regarding Australian undergraduate pharmacy students' approaches to learning, and explore the differences in approaches to learning between undergraduate and postgraduate cohorts.
Longitudinal, repeated measures design using a validated self-report survey instrument were used to gather data.
Undergraduate students' preferences for meaning directed, undirected, and reproduction-directed approaches to learning displayed the same pattern across the 2 studies; however, application-directed scores increased significantly in the second half of the undergraduate degree program. Commencing postgraduate students' approaches to learning were similar to finishing undergraduate students, and this group was significantly more oriented towards meaning-directed learning compared to undergraduate students.
Pharmacy students' maturation in approach to their learning was evident and this bodes well for pharmacists' engaging in life-long learning and capacity to work in increasingly complex health settings.
PMCID: PMC2933015  PMID: 21045948
lifelong learning; learning; Vermunt's Inventory of Learning Styles; longitudinal study
20.  Pharmacy Asthma Care Program (PACP) improves outcomes for patients in the community 
Thorax  2007;62(6):496-592.
Despite national disease management plans, optimal asthma management remains a challenge in Australia. Community pharmacists are ideally placed to implement new strategies that aim to ensure asthma care meets current standards of best practice. The impact of the Pharmacy Asthma Care Program (PACP) on asthma control was assessed using a multi‐site randomised intervention versus control repeated measures study design.
Fifty Australian pharmacies were randomised into two groups: intervention pharmacies implemented the PACP (an ongoing cycle of assessment, goal setting, monitoring and review) to 191 patients over 6 months, while control pharmacies gave their usual care to 205 control patients. Both groups administered questionnaires and conducted spirometric testing at baseline and 6 months later. The main outcome measure was asthma severity/control status.
186 of 205 control patients (91%) and 165 of 191 intervention patients (86%) completed the study. The intervention resulted in improved asthma control: patients receiving the intervention were 2.7 times more likely to improve from “severe” to “not severe” than control patients (OR 2.68, 95% CI 1.64 to 4.37; p<0.001). The intervention also resulted in improved adherence to preventer medication (OR 1.89, 95% CI 1.08 to 3.30; p = 0.03), decreased mean daily dose of reliever medication (difference −149.11 μg, 95% CI −283.87 to −14.36; p = 0.03), a shift in medication profile from reliever only to a combination of preventer, reliever with or without long‐acting β agonist (OR 3.80, 95% CI 1.40 to 10.32; p = 0.01) and improved scores on risk of non‐adherence (difference −0.44, 95% CI −0.69 to −0.18; p = 0.04), quality of life (difference −0.23, 95% CI −0.46 to 0.00; p = 0.05), asthma knowledge (difference 1.18, 95% CI 0.73 to 1.63; p<0.01) and perceived control of asthma questionnaires (difference −1.39, 95% CI −2.44 to −0.35; p<0.01). No significant change in spirometric measures occurred in either group.
A pharmacist‐delivered asthma care programme based on national guidelines improves asthma control. The sustainability and implementation of the programme within the healthcare system remains to be investigated.
PMCID: PMC2117224  PMID: 17251316
21.  MicroRNA-26a-mediated regulation of interleukin-2 expression in transformed avian lymphocyte lines 
Micro(mi)RNAs are a class of small non-coding RNAs that play critical roles in the induction of various cancers, including lymphomas induced by oncogenic viruses. While some of the miRNAs are oncogenic, miRNAs such as miR-26a are consistently downregulated in a number of cancers, demonstrating their potential tumor suppressor functions. Global miRNA expression profiles of a number of virus-transformed avian lymphoma cell lines have shown downregulation of gga-miR-26a expression, irrespective of molecular mechanisms of transformation or the viral aetiology. The neoplastic transformation of lymphocytes by many viruses accompanies high levels of proliferative responses, mostly mediated through cytokines such as IL-2. Chicken IL-2 can modulate T-cell proliferation and cytotoxicity in vitro and in vivo and dysregulation of IL-2 expression is observed in diseases such as leukaemia.
The expression levels of gga-miR-26a in chicken lymphoma cells transformed by 3 distinct avian oncogenic viruses, viz Marek's disease virus (MDV), avian leukosis virus (ALV) and Reticuloendotheliosis virus (REV) were consistently downregulated compared to the levels in the normal lymphocytes. This downregulation of miR-26a regardless of the viral etiology and molecular mechanisms of transformation was consistent with the tumor suppressor role of this miRNA. Notwithstanding this well-established role in cancer, we demonstrate the additional role of this miRNA in directly targeting chicken IL-2 through reporter and biochemical assays. The downregulation of miR-26a can relieve the suppressive effect of this miRNA on IL-2 expression.
We show that miR-26a is globally downregulated in a number of avian lymphoma cells irrespective of the mechanisms of transformation, reiterating the highly conserved tumor suppressor function of this miRNA. However, with the potential for directly targeting chicken IL-2, the downregulation of miR-26a in these tumor cells could relieve the inhibitory effect on IL-2 expression assisting in the proliferative features of the transformed lymphocyte lines.
PMCID: PMC2873332  PMID: 20441582
22.  Homodimerization of the Meq Viral Oncoprotein Is Necessary for Induction of T-Cell Lymphoma by Marek's Disease Virus ▿  
Journal of Virology  2009;83(21):11142-11151.
Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B MeqBZIP/BZIP) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B MeqHom/Hom). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.
PMCID: PMC2772798  PMID: 19692466
23.  Novel MicroRNAs (miRNAs) Encoded by Herpesvirus of Turkeys: Evidence of miRNA Evolution by Duplication▿  
Journal of Virology  2009;83(13):6969-6973.
Herpesviruses account for 134 out of the 140 virus-encoded microRNAs (miRNAs) known today. Here we report the identification of 11 novel miRNAs encoded by herpesvirus of turkey (HVT), a virus used as a live vaccine in poultry against the highly oncogenic Marek's disease virus type 1. Ten of these miRNAs were clustered together within the repeat long region of the viral genome, demonstrating some degree of positional conservation with other mardiviruses. Close sequence and phylogenetic relationships of some miRNAs in this cluster indicate evolution by duplication. HVT miRNAs represent the first example of virus-encoded miRNAs that show evolution by duplication.
PMCID: PMC2698521  PMID: 19403687
24.  Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene▿ †  
Journal of Virology  2009;83(11):5846-5853.
In this study, we have identified an internal ribosome entry site (IRES) from the highly infectious herpesvirus Marek's disease virus (MDV). The IRES was mapped to the intercistronic region (ICR) of a bicistronic mRNA that we cloned from the MDV-transformed CD4+ T-cell line MSB-1. The transcript is a member of a family of mRNAs expressed as immediate-early genes with two open reading frames (ORF). The first ORF encodes a 14-kDa polypeptide with two N-terminal splice variants, whereas the second ORF is contained entirely within a single exon and encodes a 12-kDa protein also known as RLORF9. We have shown that the ICR that separates the two ORFs functions as an IRES that controls the translation of RLORF9 when cap-dependent translation is inhibited. Deletion analysis revealed that there are two potential IRES elements within the ICR. Reverse genetic experiments with the oncogenic strain of MDV type 1 indicated that deletion of IRES-controlled RLORF9 does not significantly affect viral replication or MDV-induced mortality.
PMCID: PMC2681985  PMID: 19297480
25.  Undergraduate and Postgraduate Pharmacy Students' Perceptions of Plagiarism and Academic Honesty 
To assess undergraduate and postgraduate pharmacy students' perceptions of plagiarism and academic honesty.
A questionnaire was administered to undergraduate and postgraduate pharmacy students to determine their levels of awareness of university policy concerning academic honesty; attitudes to plagiarism by rating the acceptability of a range of plagiarizing and cheating practices; and choice of appropriate penalties for a first and second occurrence. The choice of behaviors in response to a scenario about the preparation of a reading-based written assignment and the strategies that students would be prepared to use in order to submit the assignment on time were also assessed.
Findings indicated widespread deficiencies in student knowledge of, and attitudes towards, plagiarism. Students did not perceive plagiarism as a serious issue and the use of inappropriate strategies for sourcing and acknowledging material was common.
The study highlights the importance of achieving a balance among the 3 dimensions of plagiarism management: prevention, detection and penalty.
PMCID: PMC2769527  PMID: 19885074
academic honesty; plagiarism; cheating; Australia

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