Co-immunoprecipitation (co-IP) studies have confirmed CaR-14-3-3 in vivo interaction for both 14-3-3 theta and zeta. Confocal microscopy experiments have demonstrated that both 14-3-3 isoforms co-localise with the CaR most likely in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, which overlaps the filamin interaction domain. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx_1-2Sx_2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction.

To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 phosphorylation by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR.

To determine the possible role of 14-3-3 theta or zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that both 14-3-3 theta and zeta activated Rho signalling.

The enhancement of CaR-mediated Rho signalling by 14-3-3 isoforms suggests a general role for 14-3-3 proteins as chaperones in the regulation of Rho signalling, and possibly other CaR-mediated signalling pathways.

Co-immunoprecipitation (co-IP) and pulldown assays have confirmed CaR-14-3-3 in vivo and in vitro interaction, respectively, for 14-3-3 theta. Confocal microscopy has demonstrated that both 14-3-3 isoforms co-localise with the CaR in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx_1-2Sx_2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction.

To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 activity by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR. In addition, the possible role of 14-3-3 theta and zeta in CaR-mediated modulation of Rho-dependent stimulation of serum-response element transcription was examined in HEK293 cells using a luciferase assay. Preliminary results suggest that both 14-3-3 theta and zeta inhibit Rho signalling to a similar extent.

Differential binding of the two 14-3-3 isoforms to the CaR tail initially suggested differences in the way these isoforms may influence CaR-mediated signalling but experiments to date have not exposed such differences. The functional significance of the CaR-14-3-3 interaction will be further examined with other CaR-mediated signalling pathways.

Co-immunoprecipitation and confocal microscopy experiments have confirmed CaR-14-3-3 zeta in vivo interaction and co-localisation. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, overlapping the filamin binding site. Therefore, to determine the possible role of 14-3-3 zeta in CaR-mediated Rho signalling involving filamin, HEK-293 cells stably expressing the CaR and endogenously expressing filamin were transfected with 14-3-3 zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that 14-3-3 zeta inhibited Rho signalling. It is possible that 14-3-3 zeta interferes with the filamin interaction domain on the CaR by competitive binding leading to inhibition of Rho activity. These results suggest a role for 14-3-3 proteins as adapters in the regulation of CaR signalling involving filamin.

Co-immunoprecipitation and confocal microscopy experiments have confirmed CaR-14-3-3 zeta in vivo interaction and co-localisation. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, overlapping the filamin binding site. Therefore, to determine the possible role of 14-3-3 zeta in CaR-mediated Rho signalling involving filamin, HEK-293 cells stably expressing the CaR and endogenously expressing filamin were transfected with 14-3-3 zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that 14-3-3 zeta inhibited Rho signalling. It is possible that 14-3-3 zeta interferes with the filamin interaction domain on the CaR by competitive binding leading to inhibition of Rho activity. These results suggest a role for 14-3-3 proteins as adapters in the regulation of CaR signalling involving filamin.

The CaR has been shown to be involved in Rho signalling leading to subsequent serum response element (SRE)-mediated gene transcription, and activation of Rho signalling by the CaR is enhanced by the structural protein, filamin. The region of the CaR involved in Rho signalling is localised to residues 906-980. Furthermore, filamin has been demonstrated to interact with the CaR tail at residues 962-981. Preliminary Y2H studies in our laboratory have established that both 14-3-3 theta and zeta interact with the CaR tail. These findings implicate a possible role for 14-3-3 proteins in CaR-mediated Rho signalling involving filamin.

Objectives:

(1) To confirm CaR and 14-3-3 protein interaction in the mammalian system

(2) To delineate the 14-3-3 binding region on the CaR

(3) To determine whether 14-3-3 proteins modulate CaR-mediated Rho signalling

Methods:

(1) Co-immunoprecipitation studies to confirm the interaction between 14-3-3 proteins and the CaR

(2) A yeast two-hybrid beta-galactosidase interaction assay to delineate 14-3-3 binding regions on the CaR

(3) A luciferase reporter assay (as a measure of Rho-dependent stimulation of SRE-mediated gene transcription) to determine whether 14-3-3 proteins modulate CaR-mediated Rho signalling in HEK-293 cells stably expressing the CaR (HEK-293/CaR), and in melanoma cells not expressing filamin (M2 cells) and stably expressing filamin (A7 cells).

Results: Co-immunoprecipitation studies confirmed CaR-14-3-3 in vivo interaction for both 14-3-3 theta and zeta. Y2H mapping studies delineated the interaction site for 14-3-3 zeta to residues 965-980 on the CaR tail, which overlaps the filamin interaction domain. By contrast, the 14-3-3 theta interaction site is confined to an upstream domain (residues 865-923).

To determine the possible role of 14-3-3 theta or zeta in CaR-mediated Rho signalling, HEK-293/CaR cells were transfected with 14-3-3 theta or zeta and a SRE luciferase reporter which allowed for the measurement of luciferase activity. Results demonstrated that over-expression of both 14-3-3 theta or zeta inhibited CaR-mediated Rho signalling in HEK-293/CaR cells. This effect was not seen in cells unable to express filamin (M2 cells). Further examination of Rho signalling in M2 cells stably expressing filamin (A7 cells) demonstrated that re-introducing filamin did not allow modulation of CaR-mediated Rho signalling by over-expression of 14-3-3 protein as seen in HEK-293/CaR.

Conclusions: We have demonstrated that both 14-3-3 theta and zeta interact with the CaR in vivo and bind differentially to the CaR tail. The inhibition of CaR-mediated Rho signalling by over-expression of 14-3-3 proteins suggests a regulatory role for the proteins in Rho signalling through competitive binding with filamin on the CaR tail. The inability to restore this inhibition in A7 cells may have been due to low level expression of filamin in these cells compared to HEK-293 cells.

Yeast two-hybrid mapping studies delineated the interaction site for both 14-3-3 isoforms to residues 865-922 on the CaR tail, and co-immunoprecipitation studies confirmed CaR/14-3-3 theta and zeta in vivo interaction in mammalian cells. To investigate the possible role of 14-3-3 theta and zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR (HEK-293/CaR) were transfected with either 14-3-3 theta or zeta and an SRE luciferase reporter, which allowed for the measurement of SRE activation. Results demonstrated that over-expression of both 14-3-3 theta and zeta inhibited CaR-mediated SRE activation in these cells. This effect was not seen in CaR-transfected M2 cells unable to express filamin but was not restored in CaR-transfected M2 cells stably expressing filamin. We propose a mechanism whereby 14-3-3 theta and zeta either competitively bind with or sequester filamin leading to the inhibition of CaR-mediated SRE activation in HEK-293/CaR cells.

To provide further insight into mechanisms controlling CaR signalling, a yeast two-hybrid screen was performed using the CaR intracellular tail as bait. A number of interacting proteins were identified including 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins which are most often phosphorylated. The proteins influence a multitude of cellular processes, including cell signalling and have been shown to be involved in various aspects of the Rho signalling pathway.

Yeast two-hybrid mapping studies delineated the interaction site for both 14-3-3 isoforms to residues 865-922 on the CaR tail, and co-immunoprecipitation studies confirmed CaR/14-3-3 theta and 14-3-3 zeta in vivo interaction in mammalian cells. To investigate the possible role of 14-3-3 theta and 14-3-3 zeta in CaR-mediated SRE activation, HEK-293 cells stably expressing the CaR (HEK-293/CaR) were transfected with either 14-3-3 theta or 14-3-3 zeta and an SRE luciferase reporter, which allowed for the measurement of SRE activation. Results demonstrated that over-expression of both 14-3-3 theta and 14-3-3 zeta inhibited CaR-mediated SRE activation in these cells. This effect was not seen in CaR-transfected M2 cells unable to express filamin nor in CaR-transfected M2 cells stably expressing filamin. We propose a mechanism whereby 14-3-3 theta and/or 14-3-3 zeta either competitively bind with or sequester filamin leading to the inhibition of CaR-mediated SRE activation in HEK-293/CaR cells.

Discourse samples from the Cinderella story were selected from the AphasiaBank repository (McWhinney et al., 2011) if they met an empirically defined Signal: Noise ratio. To date a total of 28 cases have been analysed: 18 with a diagnosis of Broca’s aphasia and 10 controls. Dependent measures of long pauses, short pauses and speech segment durations (ln) for each aphasic were converted to z scores by comparison with the control group. All of the Broca’s aphasics showed shorter mean speech segment durations than the control group. Nine cases produced significantly longer mean short pause durations.

The results demonstrate that the FPS is a sensitive tool for characterisation of cognitive and motor processes associated with the impact of brain impairment on spontaneous speaking. It provides inferential statistics that quantify function across cognitive and motor domains beyond those provided by traditional categorical or model based diagnostic tools.

Methods: The School of Medicine, Sydney, University of Notre Dame examined a cohorts of 113 graduate-entry Year 3 medical students using 20 SCT questions in the formative midyear examination. Their answers were compared with the panel of 15 general practitioners (clinical tutors). This pilot study also correlates the medical students’ scores with their Year 2 summative performance.

Results: The mean and standard deviation (SD) of the students’ and panel’s scores were 15/20 (SD 2) and 17/20 (SD 2) respectively. The correlation with Year 2 summative written and total scores were 0.44 and 0.38 respectively (p

Conclusions: SCT is a new modality of assessment looking at the concordance of clinical thinking and decision making between the students and the panel. The pilot study showed a moderate correlation with other assessment scores. Further studies to investigate the correlation between SCT and clinical reasoning components of the clinical OSCE examination would be very useful.

The aim of the study was to evaluate whether targeted workshops will improve the confidence and competency of clinicians in writing assessment questions and therefore improve the overall quality of assessment items.

Methods: The School of Medicine, Sydney, University of Notre Dame is a new medical school having the first graduating cohort in 2011. Clinical year written assessment items are written by affiliate or adjunct clinicians of the School. Targeted question development workshops were conducted in one subschool site (Melbourne) to facilitate and train the clinicians in written question development. At the beginning of each workshop, a pre-workshop questionnaire was distributed to each participant collecting anonymous data on their self-perceived level of competency in developing quality MCQ and short answer questions (SAQ) on a 1-5 Likert scale. A 3-hour workshop then followed consisting of the 1^st part: training session on format and characteristics of high quality assessment items, pitfalls in writing questions, ways to improve an item to test higher order thinking and methods to avoid test-wise student’s strategies. 2^nd part: draft items from participants were shown to the multi-disciplinary panel for the purpose of further discussion and critique with respect to question quality. This provided the added advantage that each item was able to be modified and improved on at the time, thus providing immediate and ongoing feedback to the question writers. At the conclusion of the workshop, the same questionnaire was re-administered and participants rated their perceived competencies after the exercise. The Wilcoxon rank sum test with paired data was used to determine if there was any improvement in the post workshop competencies. The quality improvement of assessment items was analysed by determining the percentage of questions which needed further modifications at the subsequent review committee.

Results: There were a total of 11 clinicians participating in the workshop and a 100% response rate for the questionnaires. There was a highly significant (p=0.005) improvement in the perceived competency in writing both MCQ and SAQ items after the workshop (Table 1). The items developed post workshop also required significantly less modifications when compared to question items developed by non-workshop participants.

Conclusions: By delivering training and facilitating multi-disciplinary review and feedback on draft assessment items, the targeted question development workshops could improve the quality of assessment items as well as increasing the level of competency of the clinicians as part of the faculty development program.

Summary of work: Modified Angoff (MAM), Borderline Group (BGM), and Borderline Regression (BRM) standard setting methods were applied to nine OSCE stations for 103 first year and 106 second year student. The same set of five examiners standard set each OSCE station using each of the three methods.

Summary of results: Pass marks and standard errors for MAM were higher than the other two methods in most stations. Pass / fail decisions agreement between MAM and BGM/BRM was statistically significant, but marginally convincing (Kappa = 0.488, p

Conclusions: Empirical evidence seems to indicate that BGM/BRM is more credible and defensible than MAM for standard setting pass mark in OSCE.

Take-home messages: Evidence-informed optimization of defensibility, credibility and feasibility of standard setting procedures for OSCE should be an on-going goal.

Summary of work: A new instrument with a wide spectrum of scales that allow objectivity and precision (i.e. checklist, process performance rating, and an overall global rating) was introduced, replacing the use of holistic numerical scoring. The new marking format was piloted and used in nine OSCE stations for first year students and 14 OSCE stations for second year students in 2010. It was also specifically designed for computerized scoring using Optical Mark Recognition software.

Summary of results: Findings suggest that the new instrument is well accepted by examiners for its ease of use and enhanced objectivity. Empirical evidence also indicates better discrimination for students’ clinical skills, stronger internal consistency, greater inter- and intra-station reliability, and improved individual examiner marking consistency.

Conclusions: The validity and reliability of OSCEs can be enhanced with the use of practical scoring scales that offer examiners greater objectivity and precision.

Take-home messages: The procedural nature of the OSCE should not preclude the incorporation of scales targeting various levels of objectivity and precision to enhance the validity and reliability of student results.

Methods: The School examined 110 graduate-entry medical students in Year 3 (2010) using OSCE across 2 sites. There were 8 stations examining: history taking, clinical examination skills on simulated patients, real patients or plastic models with a total examination time of 100 minutes. The content of each station had been standardized by a multi-disciplinary panel. A detailed criterion-based marking assessing various clinical competencies was employed. Participating examiners and standard setters were trained before the examination. An examiner and a standard setter were assigned to each station. The two markers did not confer or discussion in the process. The Intra-Class Correlation (ICC) co-efficient between the examiner and standard setter, the overall reliability score (Cronbach’s alpha) were calculated using the SPSS® statistical package.

Results: The ICC correlations between the examiner and standard setter were significant (p<0.001) (Table 1). There was a low correlation in Station 7 at Melbourne School. The overall Cronbach’s alpha reliability score is 0.7.

Conclusions: The significant correlations between examiners and standard setters revealed that pre-examination training and standardisation reduce inter-rater variability and improve overall reliability. The analysis could also pick up potential discrepancy in marking (eg Station 7) and appropriate score adjustment made. The standard setter also serves as a reserve/backup examiner.

Summary of work: The School examined 110 graduateentry Year 3 medical students. There were 10 stations (total examination time 120 minutes). Competency-based marking was developed to assess the students’ abilities to: demonstrate respect and compassion, elicit systematic history/physical examination, formulate working diagnosis and differential diagnoses, develop and interpret investigations and formulate management plans. A score of ‘0’ (failed), ‘1’ (achieved) or ‘2’ (achieved well) was given for each competency assessed. A separate independent global scoring was also given. The Pearson’s correlation coefficient between the competency based and global scoring marks and the reliability score (Cronbach’s alpha) was calculated.

Summary of results: The correlation between the competency based and global scoring marks was highly significant (p

Conclusions: The competency based method of assessment is reliable. The examiners gave highly valued feedback on this new marking scheme. Take-home messages: Competency based marking in OSCE is reliable and can be considered to be used in assessing the clinical year medical students.

The forum was addressed by Professor Bruce Armstrong, (Professor of Public Health, University of Sydney) who attended the inaugural meeting of ACOSH on November 17, 1971, and who was able to provide a background and history into the founding of ACOSH.

Professor Kingsley Faulkner (Director of Clinical Teaching, University of Notre Dame) reflected on the impact of smoking in his medical practice, outlining highlights of tobacco control over the past 40 years.

Maurice Swanson (Chief Executive Officer of the WA Heart Foundation) provided the background and story of tobacco control since 1971.