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Poster Abstracts POSTER ABSTRACTS SECTION TOPIC AWARD SPONSOR A Biochemistry & Molecular Biology ASBMB B Cell & Developmental Biology ANZSCDB C Genetics HGSA D Immunology ASI E Microbiology ASM F Neuroscience Perron Institute G Environmental and Plant Science CCDM Curtin University H One Health CBSM I ~Omics Royal Society WA J Senior Research Presentations CBSM Biochemistry & Molecular Biology A1 Development of Novel Aptamers Specific to Mesothelioma Cells for Targeted Drug Delivery Applications Milena Fratini1, Rakesh N. Veedu 1,2 1Centre for Comparative Genomics, Murdoch University, 2Perron Institute Neurological and Translational Science Introduction. Nucleic acid aptamer technology has attracted considerable attention in recent years in life sciences. Aptamer can bind to their target with very high affinity and specificity because of their ability to adopt three-dimensional structures, and this characteristic make them attractive molecules for disease specific drug delivery. Malignant pleural mesothelioma is a pulmonary cancer, caused by asbestos exposure. It is very aggressive and can lay dormant for 20- 40 years, though once detected is deadly within a year. Problem Statement. Nowadays there are many different anticancer therapies. However, there are various limitations such as such as poor delivery efficacy and side effects from chemotherapy, which drive us to look into other alternatives approaches. Although there are antibodies-based methods currently used for targeted cancer therapy, there are some limitations associated with this approach such as the requirement of living system for production, batch-to-batch variation, high immunogenicity and toxicity and shorter shelf-life. Alternatively, development of nucleic acid aptamers specific to mesothelioma cells could be one approach to achieve tissue specific cancer drug delivery and therapy. Aptamers possess some advantages such as easy laboratory production in large scale, low cost, high stability, no immunogenicity and no or low toxicity. Procedures. Cell-Selex methodology was used to develop aptamers specifically to AE17 mouse mesothelioma cells. Therefore, a 81mer DNA library was incubated with AE17 cells to isolate internalised aptamer candidates with high affinity. Seven rounds were performed. Negative selection steps were also performed using other cells to remove any non-specific binders.Results. Flow cytometry analysis data suggested that the selected aptamers candidates after seven rounds of selection showed a slight shift compared to the library control. Conclusions. We have performed seven rounds of Cell-Selex procedures and the isolated DNA aptamer products are currently being sequenced to identify the individual internalised aptamers specific to AE17 cells. A2 Uncovering the Mechanisms of Ciclopirox Olamine: an Anti-fungal Drug Repurposed for the Treatment of Metastatic Melanoma Conrad Hogg1,2, Rikki Brown2, Andrew Woo2, Peter Leedman2 1School of Biomedical Sciences, University of Western Australia, 2Laboratory for Cancer Medicine, Harry Perkins Institute of Medical Research Metastatic melanoma is an area of unmet clinical need, with less than 20% of patients surviving five years from their diagnosis. The standard of care is limited by the development of resistance to therapies, and the inability of many patients to respond to targeted or immunotherapies. Consequently, new treatments that can act alone or in combination with current therapies are urgently needed. Ciclopirox olamine (CPX) is an antifungal agent that has recently shown promising anti-cancer effects in phase I clinical trials of blood cancers, thus it can potentially be rapidly repurposed to treat metastatic melanoma. This study aims to uncover the underlying mechanisms of the action of CPX in melanoma by investigating the cytotoxicity of the drug in vitro and examining RNA and protein expression after treatment with the drug, or knockdown of pathways usually altered by CPX. While the mechanism of action of this drug remains unknown in melanoma, this study establishes that the action of CPX is strongly iron-dependent. It does not mediate its cytotoxic effect through reactive oxygen species or the Wnt/β-catenin pathway, which have previously been proposed as putative mechanisms in the literature. Furthermore, in a cell line with a BRAF mutation, CPX induces autophagy, which may contribute to the drug’s mechanism of action. CPX also induces the expression of the immune cell attracting chemokines including CCL3, suggesting that CPX has the potential to be immunogenic. Thus, CPX shows promise as both a monotherapy, and in combination with immunotherapy. Future work should focus on effect of chelating iron from essential enzymes in the cancer cells such as eIF5A and ribonucleotide reductase, as well as the assessing synergy of the drug in combination with immunotherapy in vivo. A3 Insights from Magnetofection Agent Development Kerr, R.H.1, Evans, C.W.1, Kretzmann, J.A.1, Clemons, T.D.1, Iyer, K.S.1 1The University of Western Australia Magnetofection describes transfection protocols that rely on magnetic materials associated with gene delivery agents to provide targeting and amplified action through interaction with an external magnetic field. Improvements in DNA delivery have relevance for future adjuvants in the treatment of cancer, the first world’s leading cause of mortality, in gene therapy, and preparation of transformed cells in research. It was expected that a magnetofection agent consisting of superparamagnetic iron oxide nanoparticles (SPION) and an existing high-performance transfection agent could utilise an external magnetic field to achieve effective transfection under challenging conditions such as co-transfection, or short transfection timeframes. Transfection agents were electrostatically associated with SPION and used to deliver a GFP encoding 5.3kb reporter plasmid to immortalised cell lines inside an oscillating magnet array. The success of DNA expression, and vehicle internalisation, were monitored by photometry, fluorescence microscopy, and flow cytometry. Effective magnetofection agents were similar to non-magnetic transfection over long transfection times, however for 1 hour or less of transfection time, magnetofection protocols resulted in approximately 2-fold improved (**) expression at 48 hours post-treatment. The ratio of the rate of vehicle internalisation to the rate of reporter gene expression was found to increase over time (~1 to ~1.2 over 4 hours) and was generally higher in magnetofection (~1.4 vs. ~1.2 for traditional transfection). This leads to the conclusion that magnetofection is not an avenue to universal improvement of transfection outcomes, but has advantages as a technique to shorten the exposure period necessary for effective gene delivery. A4 Functional and Structural Studies of a Lipid A Modifying Enzyme in Pathogenic Neisseria Katherine Lim1, Anandhi Anandan2, Alice Vrielink2 & Charlene Kahler 1 1School of Biomedical Sciences, UWA, 2School of Molecular Sciences, UWA Neisseria meningitidis (Nm) is usually an asymptomatic coloniser of the upper respiratory tract but can cause meningitis and septicaemia in susceptible individuals. Neisseria gonorrhoeae (Ng) is a sexually transmitted pathogen that usually infects the urogenital tract. If left untreated in females, it can lead to pelvic inflammatory disease, infertility and spontaneous abortions. Current treatment for gonorrhoea is with antibiotics but widespread global antibiotic resistance has led to the need for alternative treatment methods. The enzyme phosphoethanolamine transferase A (EptA) is conserved in both Ng and Nm, and is a pivotal virulence factor used to transfer phosphoethanolamine from phosphatidylethanolamine to the lipid A of the lipooligosaccharide. EptA is being developed as a therapeutic drug target because inactivation of EptA causes complete attenuation in mouse and human infection models due to its role in protecting the bacteria from cationic antimicrobial peptides, thus enabling the natural immune system to clear the infection. Comparisons between EptA and other members of the alkaline phosphatase superfamily identified several conserved residues in the enzyme active site involved with coordinating the Zn2+ ion and substrate binding. These conserved residues were mutated and confirmed to have caused a reduced resistance to polymyxin B. To further understand the impact of these mutations, the mutant proteins were purified and tested for activity and stability. All eight mutant proteins were found to be inactive compared to wild-type (WT) EptA. Biophysical analysis using circular dichroism showed that the secondary structures of all mutant proteins were similar to WT EptA. However, differential scanning fluorimetry results showed three mutant proteins had increased thermal stability compared to WT EptA. Two mutants that had the largest change in thermal stability are undergoing crystallisation trials to examine the effect of the mutations on the binding and active site pocket of the enzyme. A5 Investigating the Therapeutic Potential of an Axl Inhibitor, BGB324, in Overcoming Sorafenib Resistance in Hepatocellular Carcinoma Shelby Margolius1,2, Tasnuva Kabir1, Peter Leedman1 1Laboratory for Cancer Medicine, Centre for Medical Research, Harry Perkins Institute of Medical Research, 2School of Biomedical Sciences, University of Western Australia Introduction: Hepatocellular carcinoma (HCC) is the 2nd most lethal cancer worldwide, commonly diagnosed at an advanced stage. Currently, sorafenib
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