Centre for Digital Transformation of Health - Research Publications

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    Fostering the use of Learning Health Systems through a fellowship program for interprofessional clinicians
    Dushyanthen, S ; Perrier, M ; Chapman, W ; Layton, M ; Lyons, K (WILEY, 2022-10)
    INTRODUCTION: To address Australian workforce needs, we developed a Learning Healthcare System (LHS) Academy fellowship program for clinicians. In the Academy, fellows complete foundational coursework, an LHS project, and other professional development deliverables to foster their future as digital health champions within their organizations. In this paper, we describe the 11-month-long program, as well as our evaluation results from the first 2 months of the program. METHODS: In the first week of the program, we sent all fellows an open-ended survey asking fellows to describe their digital health professional identities and what they expected to achieve from the fellowship program. At 2 months, we sent a follow-up open-ended survey that captured identical measures, their perceived barriers to participation in the program, perceived use of topics in the workplace and to their projects, and recommendations for program improvement. We analyzed the open text responses using qualitative content analysis, to identify categories of responses. RESULTS: Overall, 2 months into the program, it was evident that participants were finding the teaching model engaging, useful, valuable, and applicable to their work and projects. Fellows perceived barriers to engagement in the program as balancing other commitments, lacking technical expertise, and having difficulty seeing themselves as leaders. Fellows expected that the program will allow them to implement new models of care, provide them with enough expertise to become leaders and champions in digital health, and become mentors for future generations. As far as changes in their professional identity, there was a notable increase in the number of fellows perceiving themselves as leaders. CONCLUSION: Fellowship programs are one promising means of developing the healthcare workforce in LHS capabilities. Future studies should describe and evaluate LHS programs, to provide insights and recommendations for other educators interested in implementing similar programs of work within their own institutions.
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    Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity
    Layton, MJ ; Saad, M ; Church, NL ; Pearson, RB ; Mitchell, CA ; Phillips, WA (BMC, 2012-12-27)
    BACKGROUND: The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. RESULTS: We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα. CONCLUSIONS: Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.
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    Independent Interactions of Phosphorylated β-Catenin with E-Cadherin at Cell-Cell Contacts and APC at Cell Protrusions
    Faux, MC ; Coates, JL ; Kershaw, NJ ; Layton, MJ ; Burgess, AW ; Xu, W (PUBLIC LIBRARY SCIENCE, 2010-11-30)
    BACKGROUND: The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration. FINDINGS: In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions. CONCLUSIONS: We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.
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    Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells
    Layton, MJ ; Rynkiewicz, NK ; Ivetac, I ; Horan, KA ; Mitchell, CA ; Phillips, WA (PORTLAND PRESS LTD, 2014)
    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein-protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.
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    Immunopurification of adenomatous polyposis coli (APC) proteins.
    Elliott, KL ; Catimel, B ; Church, NL ; Coates, JL ; Burgess, AW ; Layton, MJ ; Faux, MC (Springer Science and Business Media LLC, 2013-10-25)
    BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.
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    Wnt Signalling Pathway Parameters for Mammalian Cells
    Tan, CW ; Gardiner, BS ; Hirokawa, Y ; Layton, MJ ; Smith, DW ; Burgess, AW ; Xu, W (PUBLIC LIBRARY SCIENCE, 2012-02-21)
    Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.