symposia

Monday - Wednesday

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 21 symposia M moNDAY

SYM-01-01 SYM-O2-01 ASSESSMENT, RESEARCH, AND PUBLICATION: COTTON SEED FIBRE PROVIDE INSIGHTS NAVIGATING THE STAGES OF EDUCATION INTO CRITICAL DETERMINANTS OF CELL WALL SCHOLARSHIP DEVELOPMENTAL PROCESSES

Dolan E.L. Pettolino F., Liu S., Moncuquet P., Wilson I., White R., Macmillan C., University of Georgia, Athens, GA, USA. Yulia D. and Llewellyn D. CSIRO Agriculture & Food, Canberra, ACT 2601, Australia. Are you just learning what assessment means and wondering how to do it? Have you collected data in your class and are thinking about how Plant cell wall composition and structure are important for fundamental to share it with colleagues who may be interested? Have you conducted processes and agronomic traits. The composition and structure of an education study and are trying to figure out how to publish the the developing cotton seed fibre cell wall, and that of mature fibre, results? This session will touch on key issues related to all three of are critical determinants of fibre properties including length, fineness, these education scholarship topics: classroom assessment, education strength and maturity that define commercial fibre quality. For example, study design, and publication, ending with discussion about challenges fibre cells can reach great lengths due to the controlled flexibility of the conferees have faced in designing, conducting, and reporting on their walls which allows them to elongate but not widen, and the deposition educational work. Bring your questions! and organisation of the cellulosic secondary cell wall is essential for strength. We are using multiple approaches to understand and control cell wall composition and its impact on cotton fibre properties. We have undertaken a comprehensive analysis of cell wall composition and expression throughout seed fibre development (MacMillan & Birke et al., 2017. BMC Genomics, 18:539); manipulated the expression of cell wall involved in fibre composition and quality; and generated fibre quality mutants to identify genes and pathways involved in determining fibre properties. I will present our current findings from the study of a short seed fibre including phenotyping, from the whole plant to the cell wall; transcript profiling; genome sequencing; and gene mapping to identify the mutated gene and understand pathways associated with fibre and cell wall development.

SYM-02-02 SYM-02-03 MARCHANTIA: A SIMPLE MODEL SYSTEM TO STUDY IDENTIFICATION OF A NOVEL POLYSACCHARIDE IN CELL WALL BIOSYNTHESIS LAND PLANTS AND SYNTHESIS BY MEMBERS OF THE CELLULOSE SYNTHASE-LIKE F GENE FAMILY Lampugnani E.R.1, Golz J.F.1, Flores-Sandoval E.2, Roberts E.3, Ullah M.O.4, Khan G.A.1, Bacic A.5, Bulone V.4, Bowman J.L.2and Persson S.1 Little A.1, Lahnstein J.1, Jeffery D.W.2, Khor S.F.1, Schwerdt J.G.1, 1School of BioSciences, University of Melbourne, Australia. 2School Shirley N.J.1, Hooi M.3, Xing X.1, 3, Burton R.A.1 and Bulone V.1, 3 of Biological Sciences, Monash University, Australia. 3Department 1ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, of Biology, Rhode Island College, USA. 4ARC Centre of Excellence Food and Wine, University of Adelaide, Waite Campus, Glen in Plant Cell Walls, and School of Agriculture, Food and Wine, The Osmond, SA 5064, Australia. 2School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Australia. 5La Trobe Institute University of Adelaide, Waite Campus, Glen Osmond, SA 5064, for Agriculture and Food, School of Life Sciences, La Trobe University, Australia. 3Adelaide Glycomics, School of Agriculture, Food and Wine, Australia. University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia. The liverwort Marchantia polymorpha is a basal land plant with many of the key innovations seen in angiosperms. With little genetic As a significant component of monocot cell walls, (1,3;1,4)-β-glucan has redundancy in its genome, Marchantia is an ideal system for studying conclusively been shown to be synthesised by the cellulose synthase- cell wall biosynthesis in plants. Its walls are mostly composed like F6 protein. In this study, we investigated the synthetic activity of of cellulose with the main non-cellulosic polysaccharides being other members of the barley CslF gene family using heterologous xyloglucan and heteromannan. To facilitate dissection of the genes expression. As expected, the majority of the genes encode proteins involved in cell wall biosynthesis, we have constructed a library that are capable of synthesising detectable levels of (1,3;1,4)-β-glucan, of Marchantia glycosyltransferases (GTs) and used it to unravel however, overexpression of HvCslF3 and HvCslF10 genes resulted processes involved in cellulose biosynthesis. In angiosperms, such in the synthesis of a novel polysaccharide. A simple diagnostic assay as Arabidopsis, c e l l u l o s e i s p r o d u c e d by l a r g e C E L LU LO S E SY N T H A S E has been developed and the polysaccharide has been biochemically A (CESA) complexes (CSCs) that are embedded in the plasma characterised. To demonstrate that this product was not an aberration membrane. Each CSC is heteromeric, being composed of the products of the heterologous system, the characteristic polysaccharide linkages of three or more CESA genes. By contrast, Marchantia has only two were confirmed to be present in wild type barley tissues known to CESA genes with non-overlapping expression profiles. Freeze-fracture contain HvCslF3 and HvCslF10 transcripts. The finding of this linkage in electron microscopy experiments show rosette-like structures in the land plants has significant implications for defining the cell wall content plasma membrane, suggesting that Marchantia CSCs are homomeric. of many crop species and challenges the concept that members of a Loss of Marchantia CESA function leads to plantlets with severe growth single Csl family possess the same carbohydrate synthetic activity. defects linked to the loss of cellulose from their cell walls. Together, these data indicate that cellulose synthesis in Marchantia requires only a single protein, indicating that the heterotrimeric CESA dogma may need revision.

Page 22 ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 symposia M moNDAY

SYM-02-04 SYM-O2-05 PHI THICKENINGS IN BRASSICA ROOTS - AN COLD, ANTIOXIDANT AND OSMOTIC PRE-TREATMENTS ADAPTION TO WATER STRESS? MAINTAIN THE STRUCTURAL INTEGRITY OF MERISTEMATIC CELLS AND IMPROVE PLANT Collings D.A., Aleamotu’a M. and McCurdy D.W. REGENERATION IN CRYOPRESERVED KIWIFRUIT The University of Newcastle, Callaghan NSW 2308 Australia. SHOOT TIPS

With the Earth’s population to pass 9 billion by 2050, and with climate Mathew L.1, 2, McLachlan A.1, Jibran R.1, Burritt D.J.2 and Pathirana R.1 change increasing rainfall variability, breeding crop varieties more 1The New Zealand Institute for Plant and Food Research efficient in water usage and resistant to water stress is essential. We have Limited, Private Bag 11600, Palmerston North 4442, New investigated phi thickenings, unusual secondary cell wall thickenings Zealand. 2Department of Botany, University of Otago, PO Box 56, found in the root cortex where only thin, primary cell walls normally Dunedin 9054, New Zealand. occur. These bands extend around radial cell walls, with synthesis coordinated between adjacent cells so that in transverse view they Cryopreservation is a reliable and cost-effective method for the long- look similar to the Greek letter phi. Surprisingly little research into phi term preservation of clonally propagated plants. The number of clonally thickenings has been conducted, despite the observations that they can propagated species conserved by cryopreservation is increasing be induced by abiotic stresses in a wide variety of evolutionarily-diverse as vitrification-based methods are developed; droplet vitrification is species. Currently, their function(s) remain unclear. We investigated becoming the preferred method for many species, as it ensures fast phi thickenings in Brassica roots grown on agar plates. Confocal freezing and thawing rates. We investigated whether cold, antioxidant microscopy demonstrated the lignified cell walls of phi thickenings and osmotic pre-treatments could maintain the structural integrity forming a complex network of thick reinforcements surrounding the of cells, thus aiding in developing a droplet vitrification protocol for inner cortical cells of the root, and a more delicate, reticulate network kiwifruit, using Actinidia chinensis var. chinensis ‘Hort16A’ as a model. on the inner face of these cells adjoining the endodermis. Quantification Cold acclimation of donor plantlets at 4°C for 2 weeks followed by of phi thickenings showed induction in response to water stress caused sucrose pre-culture of shoot tips and supplementing all media used by salt (40 mM or higher) or sucrose (1% or higher). However, induction throughout the procedure with ascorbic acid (0.4 mM) resulted in 40% strongly depended on cultivar: of more than 20 commercial Brassica regeneration after cryopreservation. Transmission electron microscopy oleracea cultivars, some such as “golden acre” lacked inducible phi was used to examine meristematic cell structure at every critical step of thickenings whereas others such as “marathon F1” induced strongly. droplet vitrification. After treatment in vitrification solution, meristematic Similar strong cultivar differences occur in B. napus roots where the cells from cold-acclimated plantlets pre-treated with sucrose and winter varieties (for example “edimax”) lacked phi thickening induction ascorbic acid exhibited severe plasmolysis and some disruption of whereas spring varieties such as “hyola474CL” induced strongly. membrane and vacuoles. In contrast, cells without pre-treatments These observations provide a platform to test phi thickening functions, exhibited minimal changes even after exposure to vitrification solution. and discover the genetic pathways leading to their formation. Such However, after cryopreservation and recovery, all shoot-tip cells not pre- information might be applicable to crop breeding strategies in Brassica, treated showed rupturing of the plasma membrane, loss of cytoplasmic and other crops where phi thickenings occur, to develop varieties with contents and organelle distortion. By comparison, most pre-treated improved resistance to water stress. shoot-tip cells from cold-acclimated plantlets retained their structural integrity after cryopreservation, suggesting that only dehydrated and plasmolysed cells can withstand cryopreservation by vitrification.

SYM-03-01 SYM-03-02 yap and taz in tissue regeneration and REQUIREMENT OF DDX6 MEDIATED P-BODY cancer FORMATION FOR THE FUNCTION OF NANOS2 IN MALE GERM CELL DIFFERENTIATION Thompson, B. Saga Y. National Institute of Genetics, Mishima, Japan.

ABSTRACT NOT AVAILABLE In embryonic male gonads, germ cells produce abundant mRNA- AT TIME OF PUBLICATION protein complex called P-body. The formation of P-bodies is one of sex specific characteristics and is associated with male germ cell differentiation in the embryonic stage. We reported previously that an RNA binding protein NANOS2 is recruited to P-body and plays an essential role for germ cell differentiation. However, it is not clear whether P-body formation is necessary for NANOS2 to control the target and whether all NANOS2 functions are dependent on P-bodies. To understand relationship between P-body formation and NANOS2 function, we deleted P-bodies by knocking out DDX6 gene, essential for P-body formation in a germ cell-specific manner. For this purpose, we produced an XY-ES cell line which contains a germ- cell specific inducible Cre recombinase together with Cre reporter and introduced floxed DDX6 via Cas9-mediated homologous recombination. The results show that NANOS2-target Dazl was not repressed and male differentiation was not promoted similar to the case of NANOS2-null. On the other hand, some NANOS2-null properties, meiosis initiation and germ cell escape from seminiferous tubules are not observed in DDX6-KO germ cells, indicating that NANOS2 also works in a P-body-independent manner. This study also demonstrates that ES-mediated chimera method offers a powerful method as a standard genetic tool.

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 23 symposia moNDAY

SYM-03-03 SYM-O3-04 UNRAVELING COMMON PATHOGENIC PATHWAY A HIGHLY SELECTIVE MECHANISM TO SUPPRESS UNDERLYING THE FORMATION OF HYPOPLASTIC RETROTRANSPOSON EXPRESSION IN DROSOPHILA LEFT HEART SYNDROME Hayashi R.1, Handler D.2, Brandstaetter S.2, Helmrath S.2, Felder A.K.2 and Fonoudi H.1, 2, Bosman A.1, Humphreys D.1, Patrick R.1, Blue G.3, Hill A.1, Brennecke J.2 Ho J.1, Winlaw D.3 and Harvey R.1, 2 1The John Curtin School of Medical Research (JCSMR), The ,s Australian National University, 131 Garran Road, Acton ACT 2601׳1Victor Chang Cardiac Research Institute, Sydney. 2St. Vincent Clinical School, University of New South Wales, Sydney. 33. The Heart Australia. 2Institute of Molecular Biotechnology of Austrian Academy of Centre for Children, The Children’s Hospital at Westmead, Sydney. Sciences (IMBA), Dr Bohr-Gasse 3, 1030 Vienna, Austria.

Hypoplastic left heart syndrome (HLHS) is a genetically complex A selective and plastic mechanism to silence parasitic genetic elements disease, characterized by hypoplasia of the left side of the heart. is pivotal to maintain the integrity of eukaryotic genomes. In the somatic Although being one of the most severe forms of congenital heart compartment of fruit fly ovaries, gypsy class LTR-retrotransposons defects, our knowledge of the molecular underpinnings is very limited. are suppressed by a class of small RNA called Piwi-interacting RNAs Here, we have generated an in vitro model of HLHS using (piRNAs). piRNAs in this tissue are predominantly derived from a single induced pluripotent stem cells (hiPSCs) to uncover disease-causing precursor RNA called flamenco (flam), which is transcribed from the factors. hiPSCs were generated from 10 HLHS patients and their ~300 kb genomic that is enriched of gypsy-family transposon parents (3 clones per individual; 87 hiPSC lines in total). To investigate sequences. Although piRNAs are also derived from other cytoplasmic differences during early cardiovascular development, hiPSCs were transcripts, flam RNA produces piRNAs far more efficiently (10 ~ differentiated using both embryoid body and small moleculte cardiac- 10^3 fold). The mechanism that underlies the selectivity is not known. directed differentiation methods, and their cellular populations In the present work, we show that the piRNA biogenesis factor Fs(1) and were studied. Gene expression analysis of Yb selects flam RNA against other RNAs. Upon depletion of Fs(1) spontaneously differentiated cells showed lower expression of both Yb, flam-derived piRNAs are largely lost while other cellular RNAs cardiac and vascular smooth muscle markers in patients compared to including mRNAs become preferentially processed into piRNAs. Based controls. Flow cytometry analysis performed on hiPSC cultures after on a computational analysis, we were able to uncover the underlying directed cardiac differentiation at 5-day intervals (day 0-30) showed features in piRNA-precursor transcripts to recruit Fs(1)Yb. Our work cardiomyocyte differentiation in HLHS-hiPCSs was perturbed. Time- therefore sheds light on the fundamental question of how the cell course RNA-seq of 5 HLHS families revealed down-regulation of distinguishes self from non-self transcripts. cell cycle. This was confirmed using another 5 indipendent HLHS families. Cell phenotyping also indicated that beating cardiomyocytes derived from patients were immature and their calcium flux properties were significantly different. In summary, our findings suggest that the progression of cardiogenesis and vasculogenesis in HLHS-hiPSCs is perturbed, which may include problems in the cell cycle. Furthermore, the functionality of cardiomyocytes derived from HLHS-hiPSCs with respect to calcium flux properties was altered, suggestive of cardiomyocyte immaturity.

SYM-03-05 SYM-04-01 ELEVATED WNT SIGNALLING DISRUPTS HEART STRUCTURE OF A CHOLINERGIC POSTSYNAPTIC DEVELOPMENT AND MAY UNDERLIE SOME CASES MEMBRANE OF HUMAN HETEROTAXY Unwin P.N.T. Diamand K.E.M., Barratt K.S., Alzahrani A.S., Walsh K., Ahmed J.N. MRC Laboratory of Molecular Biology, Cambridge UK. and Arkell R.M. John Curtin School of Medical Research, The Australian National Cholinergic postsynaptic membranes are specialized membranes Univeristy, Canberra, ACT, 2601, Australia. of the nerve-muscle synapse, designed to depolarise rapidly when activated by the transmitter acetylcholine (ACh) released from a nerve Congenital heart disease (CHD) is the most common type of birth terminal into the synaptic cleft. The resident postsynaptic ion channels, defect and can occur in isolation or as part of a syndrome such as nicotinic ACh receptors, mediate the electrical response by opening Heterotaxy, in which the laterality of internal organs is disrupted. Many cation-selective pathways across the muscle cell membrane, signaling cardiovascular abnormalities are associated with low heritability, the muscle to contract. Membrane lipids are known to play a vital role hindering investigations into the genetic causes of CHD. Heterotaxy is in this process, which we are currently investigating by cryo-EM, using the most highly heritable cardiovascular abnormality and is frequently postsynaptic membranes from the Torpedo electric ray. We find that shown to arise from mutation of the ciliome. Mutation of the X-linked cholesterol segregates away from the phospholipids in the vicinity of factor ZIC3 is associated with both isolated CHD and the ACh receptors, associating robustly with specific transmembrane Heterotaxy but the cellular and molecular cause of ZIC3-associated sites and forming microdomain bridges between neighbouring protein Heterotaxy remains unknown. A genetic screen for mutations that affect molecules. The cholesterol-interacting parts of the receptor are those murine embryogenesis identified a novel null of Zic3, called katun most directly implicated in controlling cation conductance and gating (Ka). The mutant embryos exhibit Heterotaxy and also incompletely of the channel. Since cholesterol harbors a sterol group it would penetrant, partial (posterior) axis duplications and anterior truncation. confer local rigidity to the membrane, particularly when packed side- These latter two are redolent of elevated canonical by-side as in a microdomain. Our results suggest that such structural Wnt signalling. ZIC3 is a member of the Zic family of transcriptional support is needed to stabilize the transmembrane architecture and to regulators and previous work has shown that the ZICs can interact restrict mobility to relevant regions of the protein so that a productive with TCF proteins to inhibit Wnt/β-catenin mediated transcription when conformational change occurs upon acetylcholine release, ensuring a overexpressed in cell lines. This raises the possibility that dysregulated maximal depolarizing response. WNT signalling may underlie some cases of Heterotaxy and CHD. We have investigated this notion using mouse genetics and find that ZIC3 loss-of-function leads to elevated WNT signalling, that elevated WNT signalling is consistently associated with heart defects and Heterotaxy during embryogenesis in the absence of pronounced cilia defects. Precisely how elevated WNT signalling interferes with the laterality of internal organs is being investigated.

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SYM-04-02 SYM-O4-03 STRUCTURE OF HUMAN TELOMERASE STRUCTURAL BASIS FOR IMPORTIN ALPHA 3 BINDING SPECIFICITY OF W PROTEINS IN HENDRA Rhodes D. AND NIPAH VIRUSES NTU Institute of Structural Biology, Nanyang Technological University, Experimental Medicine Building, 6-03, 59 Nanyang Drive, Singapore Forwood J.K.1, Smith K.1, Tsimbulyak S.1, Cross E.1, Soares Dacosta T.2, 636921. Aragao D.3, Edwards M.4 and Basler C.4 1Charles Sturt University. 2La Trobe University. 3Australian Telomeres, the protein-DNA complexes that cap the ends of eukaryotic Synchrotron. 4Georgia State University. , are essential for genome stability and cell viability. In eukaryotes, telomere length is maintained by telomerase, a specialized Nucleocytoplasmic transport of proteins is an essential cellular process reverse transcriptase capable of de novo DNA synthesis. Telomere that mediates gene expression, cell differentiation, and a wide range length maintenance in stem cells and the majority of cancers is of disease pathways, including cancer and virus infection. Nuclear carried out by the specialized reverse transcriptase, telomerase. I translocation of proteins via the classical import pathway is initiated will describe our recent 3D-structural analysis of human telomerase upon recognition of a nuclear localization signal (NLS) by a member of using single-particle cryo-EM as well as the biochemical and structural the importin α subfamily. There are seven human isoforms of importin characterization of the telomerase recruitment complex. α that mediate nuclear import of cargo in a tissue- and isoform- specific manner. Our understanding as to how nuclear import adaptors differentially interact with cargo harbouring the same NLS remains poorly understood as all importin α isoforms are conserved in the domain responsible for NLS recognition. Here, we provide a structural basis for the nuclear import specificity of the W proteins of Hendra virus (HeV) and Nipah virus (NiV). Using structural approaches, we identify the interaction interfaces between importin α1 and α3 for the W proteins of both HeV and NiV, highlighting marked differences including a 2.4- fold more extensive interface and >50-fold binding affinity for importin α3. Using structure-guided design of importin α1 and α3 chimeric and mutant proteins, together with structures of importin α1 and α3 isoforms without cargo in the , we establish that the molecular basis of specificity resides in the differential positioning of the armadillo (ARM)-repeats 7 and 8. Overall, our study provides new mechanistic insights into a range of important cellular and disease processes that are reliant on isoform specificity of nuclear import adaptors.

SYM-04-04 SYM-04-05 REGULATION OF FAT MASS AND - STRUCTURAL BASIS OF NAD+ CLEAVAGE ACTIVITY ASSOCIATED (FTO) FUNCTION BY PROTEIN BY MAMMALIAN AND PLANT TIR DOMAINS UBIQUITINATION Horsefield S.1, Burdett H.1, Zhang X.2, 1, Shi Y.3, Manik M.K.1, Gu W.1, Widagdo J., Zhu T. and Anggono V. Chen J.1, Ve T.3, Dodds P.N.2 and Kobe B.1 Clem Jones Centre for Ageing Dementia Research, Queensland Brain 1University of Queensland, Brisbane, Qld 4072, Australia. 2Agriculture Institute, The University of Queensland, Brisbane, Queensland, 4072, and Food, Commonwealth Scientific and Industrial Research Australia. Organisation, Canberra, ACT 2601, Australia. 3Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia. The fat mass and obesity-associated (FTO) protein is an RNA demethylase that dynamically reverses the methylation of adenosine TIR (Toll/interleukin-1 receptor, resistance protein) domains are key base at the N6 position (m6A). Genetic polymorphisms in FTO gene components of innate immunity and cell-death signaling pathways have been strongly associated with obesity in . The cellular in animals and plants. Signaling depends on self-association and level of FTO is tightly regulated, with alterations in its expression homotypic association of TIR domains. We have recently reconstituted influencing energy metabolism, food intake and body weight. More large assemblies of the TLR (Toll-like receptor) adaptor TIR domains recently, I have discovered a role for FTO in regulating m6A dynamics and determined the structure of the filamentous assembly of TLR and memory consolidation in the mouse prefrontal cortex (Widagdo et adaptor MAL by cryo-electron microscopy. As an unexpected twist, we al., J. Neurosci., 2016). Accumulating evidence has demonstrated the found that the TIR domains involved in cell-death pathways, including physiological importance of FTO proteostasis, however, the cellular those from the mammalian TLR adaptor SARM, involved in axon mechanism underlying FTO protein turnover remains unknown. degeneration, and those from plant immune receptors (NLRs), possess Here, I will present evidence that FTO undergoes post-translational self-association-dependent NAD+-cleavage activity. Crystal structures ubiquitination on the evolutionary conserved Lys-216. CRISPR/Cas- of human SARM TIR domain and grapevine NLR Run1 TIR domain in 9-mediated knock-in cells harboring the ubiquitin-deficient K216R complex with small-molecule ligands shed light on the structural basis mutation displayed a slower rate of FTO turnover, resulting in an of this enzymatic activity. Our studies unify the mechanism of function increase in the level of FTO as well as enhanced phosphorylation of the of TIR domains as “signaling by cooperative assembly formation ribosomal S6 kinase. Surprisingly, K216R mutation reduced the level of (SCAF)” with prion-like features that leads to the activation of effector nuclear FTO and completely abolished the nuclear translocation of FTO , and show that some TIR domains can themselves function in response to starvation. Collectively, my results reveal the as effector enzymes. functional importance of ubiquitination in controlling FTO expression and localization, which may be crucial for determining body mass and composition, as well as synaptic plasticity, learning and memory.

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 25 symposia M moNDAY

SYM-05-01 SYM-O5-02 MATHEMATICAL MODELLING OF AMINO ACID CHARACTERISATION OF IMPAIRMENTS IN GLUCOSE TRANSPORTER FLUXES IN ASTROCYTES METABOLISM IN MODELS OF NEUROLOGICAL DISEASE AND TREATMENTS THEREOF Todd A.C., Hulme S.R., Broer S. and Billups B. The Australian National University, Canberra, ACT 2600. Borges K. University of QLD. Astrocytes in the brain are closely associated with synapses. They support neurotransmission by sequestering the released glutamate Energy is needed to prevent brain damage and seizures. In the CNS and converting it to glutamine for recycling back to presynaptic most ATP is generated by oxidative metabolism of glucose. In this talk I terminals. We have previously demonstrated that astrocytic glutamate will highlight biochemical techniques to study CNS glucose metabolism sequestration via excitatory amino acid transporters (EAATs) in rodent models of neurological disease. Data using 13C-glucose stimulates the release of glutamine via the System N neutral amino injection followed by subsequent quantification of 13C-glucose derived acid transporter SNAT3. Since the released glutamine plays a vital role metabolites in CNS tissue will be shown. This can be coupled with in resupplying the presynaptic neurotransmitter pool, it is important studying maximal activities of enzymes involved in glycolysis and the to know how this process is regulated to ensure a continuous supply Krebs cycle. Applying these techniques in a chronic epilepsy model, my of neurotransmitters. Therefore, the aim of our current work is to laboratory found that entry of glucose-derived carbons into the Krebs understand how the different transporters function cooperatively to cycle is impaired along with reductions in pyruvate dehydrogenase control amino acid fluxes. To investigate this, we have established (PDH) activity in the epileptogenic hippocampus. This indicates that a mathematical model of EAAT and SNAT3 fluxes based on kinetic ATP generation in the epileptogenic hippocampus will be reduced, rate equations. Parameters for the model were determined using which likely contributes to seizure generation, as ATP is vital to electrophysioloical recording and fluorescence imaging of astrocytes in keep neuronal membrane potentials stable and to regulate neuronal rat brain slices. Using this model, we show that EAAT-mediated fluxes signalling. To improve ATP generation, treatments should be aimed at of amino acids and sodium stimulate SNAT3-mediated glutamine either increasing PDH activity or circumventing the need of PDH for release and that modulation of the intracellular sodium concentration is ATP generation. Thus, fuels that can enter the CNS, such as ketones or the main regulator of astrocytic glutamine efflux. Our results provide a medium chain fats are likely to decrease seizure generation. Promising clear demonstration of how these two transporters interact to regulate data from a clinical trial with medium chain fats in people with epilepsy the release of amino acids from astrocytes. The model we have will be shown. employed can be extended to include multiple simultaneous transport processes, and we will develop it further to understand how different plasma membrane transporters coordinate the homeostasis of amino acids in brain cells. This will provide significant benefit in enabling the prediction of the role of individual transporters in essential functions such as neurotransmission, cell growth and survival.

SYM-05-03 SYM-05-04 METABOLIC DYSFUNCTION IN MOTOR NEURONE SLEEP AND CANCER: A CELL CULTURE MODEL OF DISEASE (MND): INSIGHTS GAINED FROM STUDIES IN SLEEP APNOEA ALTERS GENE EXPRESSION IN THE THE LAB AND THE CLINIC HYPOXIC, INFLAMMATORY AND CIRCADIAN RHYTHM PATHWAYS Ngo S.T.1, 2, 3 1Australian Institute for Bioengineering and Nanotechnology, Martinez C., Kerr B., Kataria N., Cistulli P. and Cook K.M. University of Queensland. 2Queensland Brain Institute, University University of Sydney, Charles Perkins Centre, Sydney, NSW. of Queensland. 3UQ Centre for Clinical Research, University of Queensland. Obstructive sleep apnoea (OSA) affects a significant proportion of the population and is characterised by episodic upper airway obstruction Characterised by the irreversible death of upper and lower motor resulting in systemic intermittent hypoxia. Recent epidemiological neurones, MND is a disease for which there is no effective treatment, studies have shown OSA is associated with higher rates of cancer and no cure. In recent years, there have been an increasing number development and cancer mortality. Animal models of OSA have found of reports that describe altered metabolic regulation in MND patients. increased metastasis and tumour growth. However, the molecular While it is proposed that changes in energy homeostasis is a modifier of mechanisms associated with the altered tumour behaviour seen in OSA disease outcome, the clinical impact of metabolic changes on disease is poorly understood. Hypoxia inducible factor (HIF) is a transcription progression and survival has remained mostly undefined. In this talk, I factor that can influence cancer growth and metabolism. In OSA, will present data from mouse studies where we seek to characterise the increased activity of HIF and NF-κB transcriptional activity is proposed impact of metabolic changes on disease course, while also interrogating to occur systemically and hence may affect tumours. However, we the origin of metabolic dysregulation. I will then present data from our have yet to understand how HIF or NF-κB may be activated by rapid prospective, multi-centre study that highlights, for the first time, the oxygen fluctuations, as compared to chronic tumour hypoxia, and how negative prognostic impact of altered energy expenditure in people this may lead to altered cancer outcomes. We have established a cell- living with MND. Finally, I will touch on some of our most recent work, in based model of OSA tissue oxygenation in order to study the effects of which we are investigating the relationship between whole body energy rapid, intermittent cycles of hypoxia in HCT116 colorectal cancer cells. expenditure and cellular bioenergetics using MND patient derived cells. Using quantitative rtPCR and western blotting, we found that HIF-1α increases during cycles of intermittent hypoxia and that expression of HIF target genes increase in response. We have also seen changes in inflammatory gene expression and changes in the pathways that regulate circadian rhythm. Inflammation and disruption of circadian rhythm have both been linked to cancer. Finally, we identified that the HIF-mediated response appears to be different in rapid intermittent hypoxia when compared to chronic tumour hypoxia. These differences may be the key to understanding how rapid intermittent hypoxia, as occurs in OSA, may influence tumour growth and metastasis.

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SYM-05-05 SYM-O6-01 COPPER(II) PREVENTS NEUROKININ B FUNCTIONAL TOOLS FOR THE DETECTION AND NETWORK AMYLOID FORMATION AND DISASSEMBLES CONTEXTUALISATION OF PROTEIN METHYLATION PREFORMED FIBRILS Tay A., Pang C.N.I., Winter D.L., Hart-Smith G., Hamey J. Jayawardena B.M. and Jones C.E. and Wilkins M.R. School of Science and Health, Western Sydney University, Locked School of Biotechnology and Biomolecular Sciences, University of bag 1797, Penrith, NSW, 2751. New South Wales.

Neurokinin B (NKB) belongs to a family of neuropeptides that have Protein methylation has recently emerged as one of the most diverse roles in cognition, neuroprotection and neuroinflammation. widespread post-translational modifications of proteins in the eukaryotic NKB has a key role in reproduction and consequently is found in high cell. Its detection by mass spectrometry, however, is affected by high concentrations in hypothalamic neurons that descend into the pituitary false discovery rates. Functionally, it is poorly understood yet it is gland. Some evidence suggests NKB is packed into secretory granules known to modulate many protein-protein interactions and, in some as an amyloid, termed ‘functional amyloid’, which is thought to allow cases, can interplay with other modifications. Here we describe two high peptide concentrations to be achieved in the granule. Several new tools that assist with the identification of methylated peptides from neuropeptides and hormones are known to form functional amyloids tandem mass spectrometry analyses (MS2 Deisotoper, MethylQuant). but interact with receptors in the monomeric form. The mechanisms The first increases peptide identification rates through the deisotoping underlying the disassembly of the amyloid are not clear, although of fragmentation spectra, whereas the second takes advantages dilution, pH and ionic strength are all thought to contribute. NKB of heavy isotopic labelling (methyl-SILAC) to unequivocally identify is known to bind copper with reasonably high affinity. Metal binding sites of protein methylation. We also describe a new Cytoscape app does not affect receptor activation and it has remained unclear as to (PTMoracle). This facilitates the contextualisation of methylation in why NKB binds copper. In this work we use a variety of biophysical protein interaction networks, with structural data associated with techniques to show that copper(II) has an important role in NKB protein-protein interactions and with all other known post-translational fibrillogenesis. At stoichiometric concentrations, copper will completely modifications. Case studies will be given to illustrate the utility of these inhibit formation of NKB fibrils and is the only biological metal that can tools. achieve this. Importantly, the metal can also promote disassembly of preformed fibrils. These results will be discussed in the context of NKB’s function and compared to the role of metals in amyloidogenic neurodegenerative diseases.

SYM-06-02 SYM-06-03 DIFFERENTIAL CORRELATION ACROSS RANKED MODELLING BREAST CANCER PROGRESSION USING SAMPLES FOR SINGLE CELL RNA-SEQUENCING SINGLE-CELL RNA-SEQ DATA Gloss B., Valdes-Mora F., Salomon R., Colino-Sanguino Y., Roden D., Ghazanfar S., Strbenac D., Ormerod J.T., Yang J.Y.H. and Patrick E. Ormandy C. and Gallego-Ortega D. The University of Sydney. Garvan Institute, 384 Victoria St Darlinghurst, NSW Australia.

Genes act as a system and not in isolation. Thus, it is important to Cancer cell diversity constitutes a challenge for cancer treatment consider coordinated changes of gene expression rather than single and deeply impact the outcome of cancer patients. A simultaneous genes when investigating biological phenomena. We have developed overview of cancer cells and associated stromal cells is critical for an approach for quantifying how changes in the association between the design of improved therapeutic regimes. Single-cell RNA-seq has pairs of genes may inform the outcome of interest called Differential emerged as a powerful method to unravel heterogeneity of complex Correlation across Ranked Samples (DCARS). Modelling gene biological systems; this has enabled in vivo characterization of cell correlation across a continuous sample ranking does not require the type compositions through unsupervised sampling and modelling dichotomisation of samples into two distinct classes and can identify of transcriptional states in single cells. Here we use the cell type differences in gene correlation across early, mid or late stages of agnostic, high-throughput microfluidic-based, single-cell RNA-seq the outcome of interest. We have recently demonstrated the utility method Drop-seq to elucidate the function and cellular composition of DCARS in the context of assessing differential correlation across of breast tumours. We use the MMTV-PyMT ± Elf5 mouse mammary survival ranking in TCGA, and further explore the use of DCARS in tumour model to provide high-resolution landscapes of the disease and the context of single cell RNA-Sequencing data. Furthermore, we highlight cellular events that result in the acquisition of the metastatic demonstrate that DCARS can be used in conjunction with network . We show breast cancer cell composition and tumour analysis techniques to extract biological meaning from multi-layered heterogeneity with unprecedented definition, elucidating the cellular and complex data. and molecular complexity of tumour progression within the context of a complex multicellular environment.

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SYM-06-04 SYM-O6-05 REMAPPING OUR RECENT EVOLUTIONARY HISTORY GENOMICS FOR COMMUNICABLE DISEASES SURVEILLANCE AND CONTROL IN AUSTRALIA Upton K.R. School of Chemistry and Molecular Biosciences, University of Howden B. Queensland. The Microbiological Diagnostic Unit Public Health Laboratory.The University of Melbourne at the Doherty Institute for Infection and Transposons are mobile DNA sequences that replicate within a host Immunity. genome and compose around half of the . Although they were originally described as ‘controlling elements’ that are able Microbial genomics is revolutionising the surveillance and response to to regulate gene expression, they are often derided as ‘Junk DNA’ communicable diseases in many countries around the world, however with minimal benefit to the host. This belief has been reinforced by the there are many challenges to ensure the full potential of genomics in technical limitation of uniquely identifying individual transposons in this area is realised. Key areas of need include platforms for rapid, short-read sequencing data. Most reads that identify these elements reproducible and accredited bioinformatics analyses; processes and align to multiple locations throughout the genome (multimapping regulatory changes to ensure effective data sharing at jurisdictional, reads). Traditional bioinformatic approaches have taken a conservative national and international levels; and effective engagement and training approach, only including reads that map to a unique location, resulting in of relevant public health professionals and epidemiologists. Through an underrepresentation of transposons in functional models of genome a coordinated approach, the implementation of genomics into routine regulation and reinforcing the Junk DNA hypothesis. Mappability communicable diseases surveillance and control in Australia will analysis indicates ~40% of the human genome is adversely affected significantly enhance the quality of this system. by the removal of multimapping reads, effectively masking the last 10 million years of our evolution from functional analysis. To address this gap in knowledge, my lab has developed and validated ReMapQ, a GPU-based machine learning algorithm incorporating a deep neural network to resolve the placement of multimapping reads. ReMapQ is able to incorporate all mappable reads, even those with 500 possible alignment locations. In-Silico validation with known-truth data sets has shown excellent performance, with ~90% precision in read placement. In a pilot analysis of Differentially Methylated Regions (DMRs) in triple negative breast cancer we have shown that biological signals identified by traditional analyses (including only high confidence single mapping reads) are highly conserved within ReMapQ processed data and have excellent correlation in log-fold-change values. Further, ReMapQ is able to identify around 50% more DMRs, not identified in single- mapping read analysis. We are working to refine this algorithm and apply it to integrated functional data sets to elucidate the functional impact of transposons and their role in disease and development in the human genome.

SYM-07-01 SYM-07-02 USING MACHINE LEARNING TO PERSONALIZE DEVELOPMENT OF AN OPTIMIZED NICOTIANA CRISPR-CAS9 APPLICATIONS BENTHAMIANA HOST LINE FOR TRANSIENT EXPRESSION OF HUMANIZED IGG Wilson L.1, O’Brien A.1, Reti D.1, Horlbacher M.1, Dunne R.2 and Bauer D.1 1CSIRO, Sydney, NSW, Australia. 2CSIRO, Data61, NSW, Australia. Leblanc Z., Mortimer C., Naim F. and Waterhouse P. Centre for Tropical Crops and Biocommodities, Queensland University Numerous studies have sought to build machine learning models of Technology, Brisbane, Australia. that predict general CRISPR-Cas9 activity and while great progress has been made, these approaches are still limited. Small sequence Production of biologics in plants is forecast to be a disruptive variations can have a dramatic effect on the CRISPR-Cas9 system, technology in the pharmaceutical industry. An ideal plant system for leading to changes in on-target activity or increases the number of this application is N. benthamiana, which is a relative of the tobacco off-targets. Despite this risk, current tools are not accounting for plant and native to Australia. A diminished viral defence response in genetic variation among a population. To address this, we developed N. benthamiana facilitates its transient transformation by the plant VARiant-aware detection and SCoring of Off-Targets (VARSCOT), pathogen Agrobacterium tumefaciens. In commercial biologics which allows researchers to design personalized CRISPR-Cas9 production systems this pathogen/host interaction is exploited to applications for specific individuals or populations. VARSCOT is able produce large amounts of a desired recombinant protein. Genome to use variant information to identify CRISPR-Cas9 target sites unique editing technologies such as CRISPR/Cas9 provide a means to to a specific individual or population. We find our tool to be the most further improve N. benthamiana as a pharmaceutical and general sensitive detection method for off-targets, finding 40% to 70% more biologics production host by increasing yield and human compatibility experimentally verified off-targets compared to other popular software of recombinant proteins synthesized in this system. Leveraging the tools. VARSCOT uses a machine learning model to score off-target increased editing efficiencies seen in plant protoplast transformation activity, leading to a 98% reduction in false positives when predicting as well as the granularity provided by this transformation medium, which off-targets are active. As off-target activity varies with CRISPR- this study aims to generate ideal plant host lines for the production of Cas9 concentration, VARSCOT’s model provides a probabilistic scores biologics through multiplex editing of genes involved in glycosylation that accounts for different conditions. and pathogen defence.

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SYM-07-03 SYM-O7-04 MODELLING CANCER MUTATIONS USING CRIPSR/ MOUSE KNOCKOUT OF NUCLEAR FACTOR I GENES CAS9 GENOME EDITING CAUSE CORTICAL MALFORMATIONS THAT PERSIST Lannagan T.R.M.1, Lee Y.K.1, Wang T.1, Roper J.2, Bettington M.3, 4, INTO ADULTHOOD 4 1 5 1 1 Fennell L. , Vrbanac L. , Jonavicius L. , Somashekar R. Gieniec K. , 1 1 1 1 1 1 1 1 1 1 Bunt J.1, Boogert S. , Lim J.W.C. , Huth S.F. , Dean R.J. , Bridges C. , Yang M. , Ng J.Q. , Suzuki N. , Ichinose M. , Wright J.A. , Kobayashi 2 1 1 6 7 8 5 9 Gronostajski R.M. and Richards L.J. H. , Putoczki T.L. , Abud H.E. , Marker J. , Kleve S. , Wirapati A. , 1 10 4 4 1 The University of Queensland, Queensland Brain Institute, Brisbane, Tejpar S. , Leggett B.A. , Whitehall V.L. , Worthley, D.L. * and Woods 2 S.L.1* Australia. State University of New York at Buffalo, Department of 1School of Medicine, University of Adelaide and South Australian Biochemistry- Program in Genetics- Genomics and Bioinformatics- Health and Medical Research Institute, Adelaide, SA Australia. 2Tufts Center of Excellence in Bioinformatics and Life Sciences, Buffalo, Medical Center & Tufts University, School of Medicine, Boston, MA USA. USA. 3Envoi Specialist Pathologists, Brisbane, QLD Australia. 4QIMR Berghofer Medical Research Institute, Brisbane, QLD The Nuclear factor I (NFI) family of transcription factors are required Australia. 5Department of Anatomical Pathology, Flinders Medical for the development of multiple organ systems. Using mouse Centre, Bedford Park, SA Australia. 6Department of Medical Biology, knockout models, we previously demonstrated that family members University of Melbourne and the Walter and Eliza Hall Institute of NFIA, NFIB and NFIX are important for normal brain development. Medical Research, Melbourne, VIC Australia 7Cancer Program, These proteins have overlapping biological functions in regulating Monash Biomedicine Discovery Institute and the Department of the transition of progenitor cells from proliferation to differentiation Anatomy and Developmental Biology, Monash University, Clayton, and therefore result in overlapping neurodevelopmental defects in Victoria, Australia embryos. Nfia and Nfib knockout mice die at birth due to kidney and lung 8Cancer Voices SA, Adelaide, South Australia, Australia 9Swiss defects, respectively. To investigate whether the phenotypes observed Institute of Bioinformatics, Bioinformatics Core Facility, Lausanne, in embryonic development persist into adulthood, we generated cortex- Switzerland. 10Digestive Oncology Unit, Department of Oncology, specific conditional models of Nfia and Nfib. These University Hospitals Leuven, Leuven, Belgium. *=contributed equally Nfiaflox or Nfibflox; Emx1-Cre mice are viable and fertile. Their postnatal cortical development is delayed, but no major defects are observed. In this age of next generation sequencing we are fast accruing more In adulthood, these mutants have enlarged brains due to increased information on cancer associated genetic alterations than ever before. volume of the cerebral cortex and, in particular, the cingulate cortex. How do we translate this new knowledge into better outcomes for Preliminary assessment revealed only a minor behavioural phenotype. cancer patients? Clearly we must prioritise genetic alterations for study These observed phenotypes are comparable to those in humans with from this wealth of data. Here we utilise the organoid culture technique, a deletion or mutation of NFI. Hence, we now have a model to further combined with CRISPR/Cas9 genome engineering, to sequentially study the aetiology and the functional defects in the human disorders. introduce genetic alterations associated with the serrated pathway to colorectal cancer (CRC). Our novel preclinical models enable therapeutic evaluation in known, complex genetic landscapes. These models can also be readily personalised to investigate the many leads generated by next generation sequencing of our patients.

SYM-07-05 SYM-O8-01 KEEP CALM AND CRISPR: JOINING THE GENOME APPLYING FOR A TEACHING AWARD - INTERACTIVE EDITING REVOLUTION WORKSHOP

Adikusuma A.1, Piltz S.1, 2, White M.1, 2, Robertson L.1, Dawson R.1, Costabile M.1, 2 Hughes J.1 and Thomas P.Q.1, 2 1School of Pharmacy and Medical Sciences, University of South 1University of Adelaide, SA, Australia 5005. 2South Australian Health Australia. 2Molecular Signalling Laboratory, University of South & Medical Research Institute, Adelaide, SA, Australia 5000. Australia Cancer Research Institute.

CRISPR genome editing technology enables targeted genetic This session is aimed at academic staff interested in applying for a modification of virtually any species with unprecedented efficiency. Teaching Award in the near future. The session will be run by Dr. Maurizio For biomedical research, CRISPR technology offers unparalleled Costabile, who has been awarded teaching awards at University (UniSA opportunities to develop accurate and sophisticated cell and animal Citation, 2013 and UniSA Digital Citation, 2015), National (Office disease models using virtually any species or cell type. Importantly, of Learning and Teaching Citation, 2014) and International (Online CRISPR can also be used to modify the human genome in vivo, Learning Consortium, Effective practice award, 2016, New Orleans) enabling functional correction of disease-causing mutations for as well as being a finalist in the SA Science Excellence Awards for precision medicine applications. Prof Paul Thomas is Director of the SA STEM Educator of the Year: Tertiary Teaching in 2017. The session will Genome Editing (SAGE) facility and the Genome Editing Laboratory cover how to identify “excellence” in teaching, the forms of evidence (GEL) at SAHMRI. He was an early adopter of CRISPR technology required in an application and how it can be presented. How to craft and his lab has generated over 60 mutant mouse lines using CRISPR your application, including creating and developing your narrative will editing. Prof Thomas will provide an overview CRISPR editing and be covered. The session will be run in an interactive workshop style describe novel applications and unexpected outcomes of this relatively with ample opportunity for questions and feedback during the session. new technology. Attendees that are at the start of their journey in applying for an award through to staff that have won local awards and are now seeking broader recognition are encouraged to attend.

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SYM-09-01 SYM-09-02 DYNAMIC CELLULAR INTERPLAY IN TISSUE ANALYSIS OF CARDIAC DIFFERENTIATION AT SINGLE REGENERATION CELL RESOLUTION REVEALS A REQUIREMENT OF HYPERTROPHIC SIGNALING FOR HOPX TRANSCRIPTION 1, 2 Rosenthal N. Friedman C.1, Nguyen Q.1, Lukowski S.1, Chiu H.1, Bar-Joseph Z.5, Tam P.4, 1The Jackson Laboratory, Bar Harbor Maine USA. 2Imperial College Murry C.E.3, Ruohola Baker H.3, Powell J.2 and Palpant N.J.1 London UK. 1University of Queensland. 2Garvan Institute. 3University of Washington. 4University of Sydney. 5Carnegie Mellon University. Regeneration, the restoration of original tissue structure and function in response to damage or disease, differs from tissue repair, in Differentiation into diverse cell lineages requires the orchestration of gene which collagen deposition and scar formation often lead to functional regulatory networks guiding diverse cell fate choices. Utilizing human impairment. In both scenarios, an early inflammatory response is pluripotent stem cells, we measured expression dynamics of 17,718 genes essential to clear damaged cells and initiate organ repair, but the f r o m 4 3 ,16 8 c e ll s ac r o s s fi ve t im e p o int s ove r a t hi r t y day t i m e - c o ur s e of i n v i t r o cardiac-directed differentiation. We used unsupervised clustering to identify quality and extent of immune engagement affects the outcome, with transcriptional networks underlying lineage derivation of 15 subpopulations distinct responses underlying the repair of heart, skeletal muscle and including mesoderm, definitive endoderm, vascular endothelium, cardiac skin. The diverse regenerative capacity of adult mammalian organs precursors, and definitive cardiac fates including contractile cardiomyocytes is also reflected in the tissue-specific composition and transcriptomic and non-contractile derivatives. Utilizing customized machine learning profiles of resident immune cell subsets and the stromal cells with algorithms, we analyzed scRNA-seq data to identify transcription factor which they interact. In the damaged heart, immune cells sense and regulatory networks linking the trajectory of subpopulations in vitro with cell modulate inflammation through a dynamic interplay with stromal cells types derived during cardiac development in vivo. We leveraged this data in the cardiac interstitium, which either leads to recapitulation of cardiac to study gene networks governing cardiomyocyte differentiation in vivo to morphology by rebuilding functional scaffolds to support muscle advance translational applications of stem cells in disease modelling and regrow th, or fails to resolve the inflammator y response and forms fibrotic therapies. Among a network of known genetic drivers of differentiation, we identified dysregulation of the non-DNA binding homeodomain protein, scar tissue. Investigation into the mechanistic basis of homeostasis and HOPX as a candidate cause for the immature state of in vitro derived restoration of cardiac function in an unbiased screen of recombinant cardiomyocytes. While HOPX is expressed in cardiac progenitor cells (CPC) inbred mouse panels has yielded dramatic strain-specific variation, in vivo, we show during in vitro differentiation that HOPX is expressed in offering unexpected insights into the functions of cardiac interstitial only 16% of hPSC-derived cardiomyocytes. Using genetic models we components and highlighting the potential for more genetically precise, determined the mechanisms underlying transcriptional regulation of HOPX. organ-specific treatments of degenerative diseases. We show that HOPX is situated downstream from hypertrophic signaling, HOPX directly drives hypertrophic growth, and is required for expression of myofibrillar genes involved in cardiomyocyte maturation. Through genetic dissection underlying the HOPX transcriptional landscape, we show that the distal HOPX transcriptional start site is the primary regulatory driver of HOPX expression underlying hypertrophic stimulation. Taken together, we utilized single cell analysis of cardiac in vitro differentiation to identify mechanisms for activating gene networks in cardiac differentiation as they occur during in vivo heart development that enhance the utility of hPSCs for cardiac translational applications.

SYM-09-03 SYM-09-04 THE MECHANOSENSOR YAP DRIVES CUTANEOUS REPROGRAMMING HUMAN IPSC INTO SENSORY TYPE 2 INFLAMMATION AND ECZEMA DEVELOPMENT NEURONS TO STUDY RETT SYNDROME

Mendoza-Reinoso V.1, Corley S.2, Lim J.Y.1, Goh L.F.3, Tong P.4, Sharmin S.1, 2, Lesperance L.S.1, Wei W.1, Piekna A.1, Prescott S.A.1 Wilkins M.2, Common J.E.3, Roediger B.4 and Beverdam A.1, 5 and Ellis J.1 1School of Medical Sciences, UNSW Sydney, NSW 2052, 1The Hospital for Sick Children, Toronto, ON, Canada. 2The University Australia. 2School of Biotechnology and Biomolecular Sciences, of Queensland, Brisbane, QLD, Australia. UNSW Sydney, NSW 2052, Australia. 3Institute of Medical Biology, A(⁎)STAR, 8A Biomedical Grove, Immunos 06-08, Singapore There is a large and growing population of individuals diagnosed with 138648, Singapore. 4Skin Inflammation Group, Centenary Institute, neurodevelopmental disorders. Rett syndrome is one of the major The University of Sydney, NSW 2050, Australia. 5School of Biomedical neurodevelopmental disorders. Symptoms include difficulties in social Sciences, The University of Queensland, QLD 4072, Australia. interaction, communication and repetitive behaviours as well as altered touch and pain sensitivity. Sensory neurons in the peripheral nervous One in five people in the Western world is affected by atopic dermatitis system respond to somatosensory input including touch and pain. (AD)/eczema. This allergic skin disease develops as result of intrinsic Our studies are focused on evaluating sensory neurons from control epidermal barrier defects. These drive a local and systemic type subjects for comparison neurons from subjects with Rett syndrome. 2 immune response, resulting in a pathological cycle of itching, To this end we adapted a previously published protocol (Chambers scratching and inflammation, and eventually in AD pathogenesis. et al., Nature Biotechnology, 2012) to produce populations of sensory The exact mechanism of how epidermal barrier dysfunction, itch and neurons from induced Pluripotent Stem Cell (iPSC) lines derived from type 2 inflammation connect at the molecular level remains poorly healthy control and Rett syndrome individuals (Cheung et al., Human understood. Yes-associated protein (YAP) is a mechanosensor that Molecular Genetics, 2011). Our modification of the growth factor and responds to mechanical stimuli to control tissue homeostasis. Our transcriptomics analyses demonstrated a strong activation of the type media composition in the Chambers et al. protocol resulted in 85% of 2 immune response in skin of transgenic mice expressing the dominant the cells exhibiting a repetitive spiking pattern at 5 weeks consistent active YAP2-5SA-ΔC protein the basal epidermis. Re-assessment of with nociceptor-like neurons. In contrast, preliminary lentivirus- the phenotype of YAP2-5SA-ΔC mice indeed demonstrated behavioral, mediated NGN2 infections and addition of neurotrophin-3 (NT-3) histological, immunological and genetic features of atopic dermatitis. may accelerate the formation of single spiking mechanoreceptor / Interestingly, we also found that YAP was activated in an independent proprioceptor cell populations with repetitive spiking nociceptor AD mouse model and in skin biopsies of human AD patients. population in sensory neuron culture. Western blots for TRK family Furthermore, we identified that YAP activity in keratinocytes drives IL- proteins and immunostaining for sensory neuron markers support these 33 and CTSS expression in vitro and in the murine skin in vivo. We conclusions and FACS analysis is underway. Preliminary screening of propose the YAP2-5SA-ΔC transgenic mouse line as a new mouse Rett syndrome patient lines using the adapted protocol suggests TRKA model for AD development. Furthermore, we revealed that YAP drives and glutamate receptor 1(GLUR1) are down regulated whereas gamma type 2 inflammation and itch, eventually leading to AD pathogenesis, amino-butyric acid (GABA) receptor seems unchanged in western blot through activation of IL33 and CTSS production in the epidermis. Our which indicates potential synaptic / morphological abnormalities. In studies identify YAP as a hitherto unrecognized upstream inducer of future, electrophysiology and morphological analysis will be performed cutaneous atopic inflammation. This provides fundamental insights into for more depth analysis. the mechanisms of allergic sensitization in vivo, thereby defining novel treatment strategies for individuals with established atopic dermatitis.

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SYM-09-05 SYM-10-01 THE INTEGRATED STRESS RESPONSE IN SKELETAL ABERRANT PROTEIN FOLDING AND AGGREGATION DEVELOPMENT AND DISEASE AND INCREASED VULNERABILITY IN MOTOR NEURONS IN ALS Cheah K.S.E. School of Biomedical Sciences, University of Hong Kong, 5 Sassoon Ooi L.1 Rd, Hong Kong. 1University of Wollongong, Wollongong, NSW 2522, Australia. 2Australia and Illawarra Health and Medical Research The integrated stress response (ISR) has a central role in maintaining Institute, Wollongong, NSW 2522, Australia. homeostasis in cells experiencing many forms of cellular stress such as biomechanical loading, oxidative stress, hypoxia, endoplasmic Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative reticulum (ER) stress. Induction of the ISR is important for maintaining disease that causes the degeneration of motor neurons in the brain and cells in normal development and is associated with diverse congenital spinal cord. The proteostasis network comprises pathways that regulate and common diseases. Human congenital skeletal dysplasia caused the biogenesis, folding, trafficking and degradation of proteins. The by disrupted development of the growth plate is often associated with role of protein aggregates in the pathogenesis of ALS remains unclear. mutations that trigger ER stress that induces the Unfolded Protein However misfolded and aggregated proteins contribute to a failure in Response (UPR). Here, we exploited a mouse model of Metaphyseal proteostasis, which is thought to underlie motor neuron vulnerability in Chondrodysplasia type Schmid (MCDS) to provide mechanistic insight ALS. The aim of this research was to identify mechanisms underlying into the impact of the ISR on cell fate. We show that the ISR core vulnerability of motor neurons in ALS. To address this we have used protein kinase RNA-like ER kinase (PERK) signaling pathway that biochemical and electrophysiological tools to analyse motor neurons modulates translation, dominates in causing dysplasia by reverting from induced pluripotent stem cells (iPSCs) from ALS patients, a chondrocyte differentiation via ATF4 directed transactivation of Sox9. SOD1G93A mouse model and post mortem tissue from ALS cases. Treatment of mutant mice with a chemical inhibitor of PERK signaling We have identified alterations in membrane proteins, alterations in prevents the differentiation defects and ameliorates chondrodysplasia. protein solubility and an increased vulnerability to cell stress in motor By preventing aberrant differentiation, titrated inhibition of the ISR may neurons in ALS. Further we have identified mechanisms that contribute be a therapeutic strategy for stress-induced skeletal disorders. to increased vulnerability and aberrant folding and aggregation of proteins in motor neurons in ALS.

SYM-10-02 SYM-10-03 DYNAMIC STRUCTURAL PROPERTIES OF 14-3-3 ZETA STRUCTURAL CAPACITANCE IN PROTEIN EVOLUTION PROTEIN UNDERPIN ITS MOLECULAR CHAPERONE AND HUMAN DISEASES ACTION AGAINST AMORPHOUS PROTEIN AGGREGATION Buckle A.M. Department of Biochemistry and Molecular Biology, Monash Woodcock J.M.1, Goodwin K.L.2, Sandow J.3, Coolen C.1, Rekas A.4 and University. Carver J.A.2, 5 1Centre for Cancer Biology, SA Pathology and University of South Canonical mechanisms of protein evolution include the duplication Australia, SA. 2School of Physical Sciences, University of Adelaide, and diversification of pre-existing folds through genetic alterations SA. 3Walter and Eliza Hall Institute of Medical Research, Parkville, that include point mutations, insertions, deletions, and copy number VIC. 4Australian Nuclear and Science Technology Organisation, amplifications, as well as post-translational modifications that modify NSW. 5Research School of Chemistry, Australian National University, processes such as folding efficiency and cellular localization. Following Canberra, ACT. a survey of the human mutation database, we have identified an additional mechanism, that we term ‘structural capacitance’, which The family of 14-3-3 proteins are dimeric phospho-serine binding results in the de novo generation of microstructure in previously proteins that function as adaptors with important roles in the regulation disordered regions. We suggest that the potential for structural of many signaling responses in eukaryotic cells. Less well described, capacitance confers select proteins with the capacity to evolve 14-3-3 proteins also exhibit molecular chaperone activity that over rapid timescales, facilitating saltatory evolution as opposed to attenuates the amorphous aggregation of proteins. This property may exclusively canonical Darwinian mechanisms. Our results implicate explain the occurrence of the 14-3-3 zeta isoform in the pathological the elements of protein microstructure generated by this distinct protein aggregation associated with neurodegenerative conditions mechanism in the pathogenesis of a wide variety of human diseases. including Alzheimer’s and Parkinson’s diseases. To better understand The benefits of rapidly furnishing the potential for evolutionary change this aspect of 14-3-3 proteins’ function, we have examined the regions conferred by structural capacitance are consequently counterbalanced of 14-3-3 zeta that play a role in its molecular chaperone action. by this accompanying risk. The phenomenon of structural capacitance We determined that neither the flexible C-terminus region nor the has implications ranging from the ancestral diversification of protein amphipathic phospho-serine binding groove contribute to molecular folds to the engineering of synthetic proteins with enhanced evolvability. chaperone action. Published studies using mutant forms of 14-3-3 zeta that are engineered to disrupt the dimeric state of the protein suggest that monomeric 14-3-3 zeta represents the chaperone-competent form of the protein. However, our recent results suggest that this is a simplistic view and that the dimer interface of 14-3-3 zeta represents a structurally dynamic region that is involved simultaneously in both 14-3- 3 protein dimer formation and molecular chaperone function.

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SYM-10-04 SYM-10-05 A BIOSENSOR BASED FLIM-FRET PHASOR CONTRIBUTION OF THE RESIDUE AT POSITION APPROACH TO MEASURE PROTEOSTASIS CAPACITY 4 WITHIN CLASSICAL NUCLEAR LOCALIZATION IN CELLS SIGNALS TO MODULATING INTERACTION WITH IMPORTINS AND NUCLEAR TARGETING Moily N.S., Hinde E. and Hatters D. Smith K.M.1, Di Antonio V.2, Bellucci L.3, Thomas D.R.4, Caporuscio F.5, Department of Biochemistry and Molecular Biology, University of 4 1 4 2 2 Melbourne, Australia. Wagstaff K.M. , Forwood J.K. , Jans D.A. , Palu G. and Alvisi G. 1School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia. 2Department of Molecular The pool of quality control proteins that maintain protein-folding Medicine, University of Padua, Via Gabelli 63, 35121 Padua,Italy. 3NEST, (proteostasis) is dynamic but can become quickly depleted in cellular Istituto Nanoscienze del CNR and Scuola Normale Superiore, P.zza stress and disease. The ability of these quality control chaperones S. Silvestro 12, 56127 Pisa, Italy. 4Department of Biochemistry and to maintain the proteome in a folded state in health and response to Molecular Biology, Monash University, Wellington Road, Clayton, stressors is not yet defined quantitatively. We have developed a family Victoria 3800, Australia. 5Department of Life Sciences, University of of barnase FRET-based biosensors with differing folding stabilities that Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy. engage primarily with HSP70 and HSP90 family proteins and modify Nuclear import involves the recognition by importin (IMP) superfamily its foldedness and aggregation[1]. Here we quantify the ability of these members of nuclear localization signals (NLSs) within protein cargoes critical cellular chaperones to bind to the barnase biosensor using the destined for the nucleus, the best understood being recognition of phasor approach to FLIM analysis of FRET in a living cell. The phasor classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS method is a fit free approach to fluorescence lifetime analysis that has consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, the capacity to quantify the FRET efficiency of the barnase sensor recent studies indicated that the contribution made by different residues in each pixel of a FLIM image and therefore spatially map protein at the P4 position can vary. Here, we apply a combination of microscopy, foldedness in a living cell[2]. Using phasor FLIM-FRET analysis we are molecular dynamics, crystallography, in vitro binding, and bioinformatics able to calculate the chaperone occupancy rates in folded and unfolded approaches to show that the nature of residues at P4 indeed modulates barnase biosensors fractions, thereby quantifying their holdase cNLS function in the context of a prototypical Simian Virus 40 large abilities in baseline conditions and when challenged through stress. tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic By multiplexing this technology with image correlation spectroscopy, substitutions in place of R impaired binding to IMPα and nuclear targeting, we also hope to understand the stickiness of unfolded proteins in cells with the largest effect exerted by a G residue at P4. Substitution of R with by measuring barnase diffusion rates and predict how cells react to neutral hydrophobic residues caused the loss of electrostatic and van der excess unfolded protein load and aggregate formation. References: Waals interactions between the P4 residue side chains and IMPα. Detailed 1. Wood, R.J., et al., A biosensor-based framework to measure latent bioinformatics analysis confirmed the importance of the P4 residue for proteostasis capacity. Nature Communications, 2018. 9(1): p. 287. 2. cNLS function across the human proteome, with specific residues such as Hinde, E., et al., Biosensor FRET detection by the phasor approach to G being associated with low activity. Furthermore, we validate our findings fluorescence lifetime imaging microscopy (FLIM). Microsc Res Tech, for two additional cNLSs from human cytomegalovirus (HCMV) DNA 2012. 75(3): p. 271-81. polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

SYM-11-01 SYM-11-02 METABOLOMIC ANALYSIS OF INSULIN RESISTANCE INCREASED NUCLEAR NAD+ BIOSYNTHESIS ALTERS ACROSS DIFFERENT MOUSE STRAINS AND DIETS SKELETAL MUSCLE PHYSIOLOGY

Stoeckli J.1, Fisher-Wellman K.H.2, Muoio D.M.2 and James D.E.1 Samsudeen A.F.1, Fiveash C.E.1, Brandon A.E.2, Das A.3, Kiriaev L.4, 1Charles Perkins Centre, School of Life and Environmental Sciences, Araki T. 5, Head S.I.4, Cooney G.J.2, Osborne B.1 and Turner N.1 The University of Sydney, Australia. 2Duke Molecular Physiology 1Mitochondrial Bioenergetics Laboratory, UNSW Sydney. 2The Institute, Duke University, Durham, NC, USA. Charles Perkins Centre, University of Sydney. 3Molecular Biology of Ageing Laboratory, UNSW Sydney. 4Department of Physiology, UNSW Insulin resistance is a major risk factor for many diseases. However, Sydney. 5National Center of Neurology and Psychiatry, Tokyo, Japan. its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors including Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous co-substrate genes and the environment. Here we used metabolomics combined used in a multitude of cellular reactions. Recent recognition of the with computational methods to identify factors that classified insulin role of NAD+ in obesity and ageing has sparked a surge in interest resistance across individual mice derived from three different mouse in NAD+ biology. Nicotinamide mononucleotide adenylyltransferase strains fed two different diets. Three inbred ILSXISS strains were (NMNAT) is a key regulating NAD+ levels, however the fed high fat or chow diets and subjected to metabolic phenotyping metabolic consequences of NMNAT manipulation has not been and metabolomics analysis of skeletal muscle. There was significant explored. Our studies investigated transgenic mice overexpressing metabolic heterogeneity between strains, diet and individual animals. NMNAT1 (the nuclear NMNAT isoform) where NMNAT1Tg mice had a Distinct metabolites were changed with insulin resistance, diet and reduced lean mass compared to wild-type (WT) littermates, primarily between strains. Computational analysis revealed 113 metabolites driven by a marked reduction (~30-40%) in skeletal muscle mass. that were correlated with metabolic phenotypes. Using these 113 Functionally, NMNAT1Tg mice showed reduced forelimb grip strength in metabolites, combined with machine learning to segregate mice comparison to WT littermates, but surprisingly no difference in exercise based on insulin sensitivity we identified C22:1-CoA, C2-carnitine endurance. Immunohistochemical analysis showed a decrease in the and C16-ceramide as the best classifiers. Strikingly, when these three average cross-sectional area of muscle fibres underpinned the reduced metabolites were combined into one signature, they classified mice muscle mass. NMNAT1Tg muscle was characterised by an increase in based on insulin sensitivity more accurately than each metabolite on its more oxidative myosin heavy chain (MHC) isoforms (MHC1, MHC2a) own or other published metabolic signatures. Furthermore, C22:1-CoA, and decreased fast-twitch MHC2b expression. A potential shift to a was 2.3-fold higher in insulin resistant mice and correlated significantly more oxidative phenotype in NMNAT1Tg vs. WT mice was confirmed with insulin resistance. We have identified a metabolomic signature by twitch characteristics and force/fatigue experiments in isolated comprised of three functionally unrelated metabolites that accurately extensor digitorum longus and soleus muscles. At a whole-body level, predicts whole body insulin sensitivity across three mouse strains. NMNAT1Tg mice showed higher energy expenditure, improved glucose These data indicate the power of simultaneous analysis of individual, tolerance and greater clearance of glucose into skeletal muscle in genetic and environmental variance in mice for identifying novel factors hyperinsulinaemic-euglycaemic clamp experiments. Overall, our that accurately predict metabolic phenotypes like whole body insulin findings indicate that enhancing nuclear NAD+ biosynthesis invokes sensitivity. widespread changes in skeletal muscle physiology.

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SYM-11-03 SYM-11-04 METABOLISM REGULATES MUSCLE STEM MITOCHONDRIAL COQ DEFICIENCY DRIVES INSULIN CELL SELF RENEWAL BY CONNECTING THE RESISTANCE BY INCREASING MITOCHONDRIAL MICROENVIRONMENT AND HISTONE ACETYLATION OXIDANTS Fazakerley D.J.1, Chaudhuri R.1, Yang P.2, Maghzal G.J.3, Krycer Ly C.H.1, Su S.2, Tian L.2, Zalcenstein D.2, Naik S.2, Ritchie M.2, Lynch G.S.1 J.R.1, Minard A.Y.1, Samocha-Bonet D.4, Murphy M.P.5, Stocker R.3, 6 and Ryall J.G.1 and James D.E.1, 7 1Centre for Muscle Research, Department of Physiology, The University of 1Charles Perkins Centre, School of Life and Environmental Sciences, Melbourne. 2The Walter and Eliza Hall Institute of Medical Research. University of Sydney, Camperdown, NSW, Australia. 2School of Mathematics and Statistics, University of Sydney, Camperdown, NSW, Australia. 3Vascular Skeletal muscle contains a resident population of somatic stem cells Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, which are capable of both self-renewal and differentiation. The signals NSW, Australia. 4Garvan Institute of Medical Research, Darlinghurst, Sydney, 5 that regulate this important decision have yet to be fully elucidated. NSW, Australia. MRC Mitochondrial Biology Unit, Hills Road, University of Cambridge, Cambridge, UK. 6St Vincent’s Clinical School, University of New Here we use scRNAseq to identify the innate metabolic signature of 7 muscle stem cells. We show that committed muscle progenitor cells South Wales, Sydney, Australia. Charles Perkins Centre, Sydney Medical exhibit an enrichment of glycolytic and TCA cycle genes and that School, University of Sydney, Camperdown, NSW, Australia. extracellular carbohydrate availability regulates intracellular citrate Insulin resistance in muscle, adipose and liver tissue is a gateway to a number levels and global histone acetylation. Muscle stem cells exposed to a of metabolic diseases including . Alterations in several facets reduced (or altered) carbohydrate environment demonstrate reduced of mitochondrial biology are implicated in insulin resistance including impaired global histone acetylation and transcription of myogenic determination oxidative phosphorylation and increased mitochondrial reactive oxygen factors (including myod1). Importantly, reduced carbohydrate species (ROS). However, disentangling the respective roles of these processes in insulin resistance has been difficult since they often occur in tandem. We availability was directly linked to increased rates of asymmetric division have used a new small molecule (mitochondria-targeted paraquat) to acutely and muscle stem cell self-renewal. Our results reveal an important role generate superoxide within mitochondria, without disrupting the respiratory for the extracellular metabolic environment in the decision to undergo chain, to show that mitochondrial oxidants alone are sufficient to induce self-renewal or myogenic commitment, suggesting local metabolite insulin resistance. Increased ROS, specifically in mitochondria, are a common production may be a therapeutic target to improve muscle regeneration. feature of an array of in vitro and in vivo models of insulin resistance, yet the drivers of mitochondrial ROS under these conditions are not completely understood. To address this, we assessed the proteome of insulin resistant 3T3-L1 adipocytes and adipose tissue from mice and humans. We found lower expression of mevalonate/Coenzyme Q (CoQ) biosynthesis pathway proteins in insulin resistant samples. Analysis of subcellular CoQ content revealed selective depletion of CoQ from mitochondria in both insulin resistant adipose and muscle tissue. Given its role in electron transport, we investigated whether loss of CoQ caused insulin resistance via mitochondrial ROS. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered insulin resistance through increased mitochondrial ROS, while CoQ supplementation in either insulin resistant cell models or mice lowered ROS and restored insulin sensitivity. Our data place loss of mitochondrial CoQ upstream of mitochondrial ROS in the pathway to insulin resistance and suggest that interventions that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance.

SYM-11-05 SYM-12-01 MITOCHONDRIAL ENERGY GENERATION THE USE OF RECOMBINANT HONEYBEE SILK FOR DISORDERS: GENES AND MECHANISMS RATIONAL DESIGN OF ADVANCED MATERIALS Thorburn D.R.1, 2, 3 1 2 Sutherland T.D. and Rapson T.D. Murdoch Childrens Research Institute, Parkville, VIC 3052. Victorian CSIRO, Health and Biosecurity. Clinical Genetics Services, Parkville, VIC 3052. 3Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052. Precise control of a polymers composition is required for the Inherited disorders of oxidative phosphorylation (OXPHOS) cause a rational design of advanced materials such as stimuli responsive clinically and genetically heterogeneous range of mitochondrial diseases. or multifunctional materials. Recombinant protein systems provide Mutations causing these disorders have been identified in 35 of the 37 this control, and in contrast to synthetic equivalents are compatible genes encoded by mitochondrial DNA (mtDNA) and over 250 nuclear with large scale production. However, despite the promise of genes, with many still to be discovered. Apart from primary defects proteins for advanced material design, protein-based materials are affecting OXPHOS subunits, assembly factors, electron carriers, and underrepresented in materials science. Here we describe our efforts mtDNA maintenance or expression, many patients with mitochondrial to develop recombinant silk proteins that are tolerant of amino acid disease have secondary defects related to enzyme cofactors, metabolite modifications without compromising the ability to produce the proteins transport or other aspects of mitochondrial biogenesis, homeostasis at large scale in recombinant systems or fabricate them into material and quality control. Several examples of recent collaborative studies forms. We describe design of the silk proteins into materials with diverse illustrate the challenges of elucidating disease mechanisms. Firstly, we functional properties ranging from recoverable -silk sponges with identified four families with Leigh syndrome with bi-allelic mutations that peroxidase activity, stable nitric oxide-sensing protein films and in destabilized the MRPS34 protein, located in the foot of the small 28S electrodes capable of fully reducing oxygen to water. mitoribosome subunit. Ribosome profiling and quantitative proteomic analyses of patient fibroblasts showed this led to loss of most of the 30 proteins of the 28S subunit, relative sparing of the large subunit, failure to assemble actively translating mitoribosomes and loss of many subunits of OXPHOS complexes I and IV. Secondly, we identified large deletions and gene conversions in a highly repetitive chromosomal region refractory to conventional analyses. The resulting ATAD3B/ATAD3A gene fusions cause lethal pontocerebellar hypoplasia and lead to mtDNA aggregation in patient fibroblasts, with multiple indicators of altered cholesterol metabolism. ATAD3 appears to be a key protein linking formation of mitochondrial nucleoids in cholesterol-rich mitochondrial inner membrane domains to cellular cholesterol metabolism. A final example is the SURF1 gene, in which patients typically have bi-allelic knockout mutations and suffer severe neurological and other symptoms but mouse knockouts show no clear phenotype. We used gene editing to generate human embryonic stem cell SURF1 knockouts. These differentiate well to cardiomyocytes and show marked abnormalities of OXPHOS function and calcium signaling as well as decreased contractility in 3D organoids.

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SYM-12-02 SYM-12-03 TISSUE ENGINEERED MULTICELLULAR STRUCTURES MEASURING AND MODIFYING TRANSLATION IN FOR AQUATIC DETOXIFICATION ESCHERICHIA COLI THROUGH START CODON AND Pollak N.M.1, 2, 3, Glass N.R.2, Suzuki K.G.H.2, Cooper-White J.J.2, 4 ORTHOGONAL tRNA ENGINEERING 1, 5, 6 and Macdonald J. 1 1 2, 3, 4 2, 3, 4 2, 3, 4 1Genecology Research Centre, School of Science and Engineering, Vincent R.M. , Wright B.M. , Hecht A. , Glasgow J. , Bawazer L. , Munson M.S.2, 3, 4, Cochran J.R.2, 4, Endy D.2, 4, Salit M.2, 3, 4 and Jaschke University of the Sunshine Coast, Sippy Downs, Queensland, 1 2 P.R. Australia. Australian Institute for Bioengineering and Nanotechnology, 1 3 Department of Molecular Sciences, Macquarie University, The University of Queensland, Brisbane, Queensland, Australia. CSIRO 2 Synthetic Biology Future Science Platform. 4UQ Centre for Stem Cell Sydney, NSW. Joint Initiative for Metrology in Biology, Stanford, USA. 3Genome-scale Measurements Group, National Institute Ageing and Regenerative Engineering, The University of Queensland, 4 Brisbane, Queensland, Australia. 5Inflammation and Healing Research of Standards and Technology, Stanford, USA. Department of Cluster, School of Science and Engineering, University of the Sunshine Bioengineering, Stanford, USA. Coast, Sippy Downs, Queensland, Australia. 6Division of Experimental Therapeutics, Department of Medicine, Columbia University, New York, Efficient translation initiation in bacteria requires the specific interaction USA. between the start codon on the mRNA and the anticodon on the initiator tRNA. Recently, we measured the interaction strength of E. coli initiator The development of new systems to target environmental pollution is tRNA with all 64 possible start codons in vivo. We found a surprising critically important for improved bioremediation, and synthetic biology number of non-canonical start codons leading to translation initiation. holds potential to revolutionize such approaches. Our group is exploring Based on these findings, we have been working towards creating an the expansion of synthetic biology into the field of tissue engineering, to orthogonal system that specifically initiates translation at UAG codons. produce novel multicellular structures, which can move and sense their An interesting feature of the standard genetic code is the dual function environment in an organism-like fashion. These multicellular structures, of AUG as both the dominant initiation codon as well as a methionine also called “pseudo-organisms”, are constructed from biological and codon internal to genes. The AUG codon duality can create issues synthetic hybrid components. We are engineering them to detoxify toxins in gene prediction and genetic design. For example, in recombinant in water by ligand-induced activation of an enzyme scavenger, and to protein design, in-frame AUG codons can behave as start codons if trigger warnings. Our approach may revolutionize aquatic detoxification upstream sequence resembles a Shine-Dalgarno site. Therefore, an systems in industry by offering a mobile scavenging system that is highly- orthogonal translation initiation system using a codon uniquely reserved specific, yet without harmful side products. Importantly, these pseudo- for translation initiation could overcome this problem as well as provide organisms cannot reproduce, representing a unique solution to ethical other engineering benefits. In the genetic code, UAG normally signals and social impact deliberations compared to use of GMOs, and creating “stop translation”. This UAG can be freed up in engineered strains where new possibilities in water management. Benefits ultimately include novel all native UAG codons are recoded to the UAA stop codon. Here, we technologies for water purification for industrial applications, such as will describe deployment of an orthogonal initiator tRNA within a UAG- reducing biochemically active pharmaceuticals in waste water and harmful less E. coli strain called C321 and effects on fitness and proteome. pesticide-run off from agricultural activities. In addition, our research is seeking to study cell-to-cell communication for signal amplification between sender and receiver cells and aims to engineer quorum sensing to initiate directional movement of multicellular structures.

SYM-12-04 SYM-13-01 BIOMACROMOLECULES WITHIN MOLLUSK USING CALCIUM PHOSPHATE LIPID-COATED SHELLS: GATEWAY TO BIOMIMETIC COMPLEX NANOPARTICLES TO DELIVER ANTISENSE SUPERSTRUCTURES OLIGONUCLEOTIDES TO MOTOR NEURONS IN MOTOR NEURONE DISEASE Agbaje O.B.A. and Jacob D.E. Vine K.L.1, 2, Watson C.1, 2, Chen L.1, 2, Saunders D.3, Morsch M.4, Department of Earth and Planetary Sciences, Macquarie University, 4 4 1, 2 NSW 2109 Australia. Chung R. , Cole N. and Yerbury J.J. 1School of Biological Sciences, Faculty Science, Medicine and Health, University of Wollongong, NSW, 2522, Australia. 2Illawarra Health and Medical Research Organisms precipitate a large variety of biominerals which are Institute, Wollongong, NSW, 2522, Australia. 3School of Medical Sciences, Faculty biogenic hierarchical nanocomposite materials consisting of inorganic- of Medicine, University of New South Wales, Sydney, 2052, Australia. 4Department organic hybrid components, each with its structural motifs, inorganic of Biomedical Sciences, Macquarie University, Sydney, NSW, 2113, Australia. crystal formation - shape, and micro- or macroscopic properties. Introduction: Abnormal accumulation of mutant superoxide dismutase I (SOD1) in Shells of mollusk, for instance, are formed with different ratios of motor neurons is a pathological hallmark of some forms of ALS. Considering that inorganic-organic materials and different structural motifs resulting in SOD1 can propagate from cell-to-cell in a prion-like fashion, potentially contributing a large variety of calcareous biocomposites with material properties to the orderly progression of the disease [1], reducing levels of SOD1 is a promising therapeutic approach. Antisense oligonucleotides (ASOs) can efficiently silence outperforming those of their synthetic counterparts. Amongst all proteins with gain-of-function mutations. However, naked ASOs have a short different shell microstructures, nacre is the most studied to date, circulation half-life and are unable to cross the blood brain barrier (BBB) warranting but comparable knowledge is lacking for non-nacre shell structures the use of a drug carrier for effective delivery. We therefore aimed to improve the such as homogeneous and crossed-lamellar structures. While the delivery of gene therapies to motor neurons in the context of ALS [2], using solid core calcium phosphate lipid-coated nanoparticles (CaP-lipid NPs), encapsulating an ASO organic matrix occluded in shells is a minor component, ca 5 wt% of directed to SOD1. Here we report the manufacture and biophysical characterization the total, significant fractions of the shell macromolecules have been of CaP-lipid NPs that encapsulate a SOD1 ASO and describe their in vitro uptake and presumed to be chitin and/or proteins. Identifying the biopolymer in vivo distribution in a zebrafish model. Methods: CaP-lipid NPs were prepared as phase is, therefore, a crucial step in improving our understanding described in our paper [3] and characterized for and size (nm), zeta potential (mV), polydispersity index (PDI), encapsulation effiencicy (%) and particle concentration. of design principles relevant to biominerals. We have used different The in vitro cellular uptake and gene silencing were assessed by confocal microscopy bioanalytical and biochemical techniques including solid state-NMR, and Western blotting, respectively. The in vivo distribution was assessed using a well- FTIR, Raman spectroscopy, scanning electron microscopy, ACQUITY established ALS zebrafish model [4]. Results: Specifically, we demonstrated that the delivery of CaP-lipid NPs is efficacious in a motor-neuron-like established cell line ultraperformance liquid chromatography and SDS-electrophoresis (NSC-34) and in primary motor neuron cultures isolated from murine spinal cords [5] to explore the composition of the organic part of mollusk shells and by confocal microscopy. Significant down-regulation of SOD1 protein expression was examine its interface with the inorganic part. The biopolymer content confirmed by immunoblotting following the delivery of SOD1 ASO-loaded CaP-lipid consists primarily of proteinaceous matter with structural motifs as NPs. We also describe for the first time nanoparticle distribution in the brain, spinal silk-like β-sheets in nacre and collagenous gel-like matrix in non-nacre cord and blood circulation of zebrafish, a powerful experimental vertebrate model for studying ALS. Conclusions: Our results suggest that CaP-lipid NPs could be an shells. Though other motifs persist in non-nacre, the composition in effective and safe system for the improved delivery of SOD1 ASOs to affected motor nacre is strikingly similar to the constituents of spider dragline silk neurons in ALS. Acknowledgements This work has been supported by the Motor but does not conform to the prevalent model that the biopolymer Neuron Disease Research Institute of Australia (2016-2017) and the US Department 1 of Defence (AL150057; 2016-2018). References [1] Grad et al. 2014 PNAS, 111 (9), component in shells consists of well-arranged polysaccharide-chitin . 1 3620-3625 [2] Foust et al. 2013 Molecular Therapy, 21(12): 2148–2159 [3] Chen et al. Y. Levi-Kalisman, G. Falini, L. Addadi, S. Weiner, J. Struct. Biol., 2001, 2017 Frontiers in Neuroscience, 11:476 [4] Morsch et al. 2017 Journal of Visualized 135, 8-17. Experiments, (120): 54983 [5] Kieran & Greensmith 2004 Neuroscience (125): 427- 439.

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SYM-13-02 SYM-13-03 A STRATEGY TO PROTECT THE HEART AGAINST LIPOSOME-BASED NANOSENSORS FOR CHEMICAL DOXORUBICIN INDUCED CARDIOTOXICITY AND BIOLOGICAL SENSING

Kok C.Y.1, Rao R.1, Ghossein G.1, Igoor S.1, Skelton R.1, Chong J.1, 2 Chandrawati R. and Kizana E.1, 2 School of Chemical Engineering and Australian Centre for 1Center for Heart Research, The Westmead Institute for Medical NanoMedicine (ACN), The University of New South Wales, Sydney, Research, The University of Sydney. 2Department of Cardiology, NSW 2052, Australia. Westmead Hospital. The detection of target chemical and biological molecules in a specific Doxorubicin is an anti-cancer drug used in treating a variety of and sensitive manner is critical for the development of disease malignancies. However, its major adverse effect is cardiotoxicity, which diagnostic devices. Membrane fusion is a key biological event that is dose dependent and can be either acute or chronic. Doxorubicin involves a highly selective recognition mechanism for molecular causes injury by DNA damage, formation of free reactive oxygen trafficking, facilitating communication between and within cells. The radicals and induction of apoptosis. Our aim is to induce expression highly evolved fusion process can occur on a sub-millisecond timescale. of the multiple drug transporter gene (MRP1) in cardiomyocytes The rapid response, specificity, and sensitivity make membrane fusion derived from human iPS cells (iPSC-CM), to determine whether this an attractive mechanism for sensing. This talk will describe our recent will allow cells to effectively remove doxorubicin. To determine the dose developments in mimicking lipid membrane fusion mechanism using of lentiviral vector required for efficient gene delivery to iPSC-CM, a liposomes for the detection of disease biomarkers. This system does dose titration of GFP vector (LV.GFP) was performed. We found that not require washing, amplification or separation steps, and present a an MOI of 20 was sufficient for transduction of >90% of cells. Having unique sensing mechanism inspired by nature. determined the optimal dose, we then generated a lentivirus vector for inducing expression of MRP1 (LV.MRP1) and validated its function in iPSC-CM by qPCR and western blot. We successfully showed increase of MRP1 mRNA and protein in transduced cells. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. In conclusion, we have optimised the conditions for gene delivery to human iPSC-CM in vitro. We have also shown that we can successfully over-express MRP1 protein in iPSC-CM, with functional transporter activity. The next step is to determine the dose of doxorubicin which induces cell toxicity, and then to assess the protective effect of MRP1 in those cells.

SYM-13-04 SYM-13-05 CELLULAR NANOVESICLES: EXOSOMES REDUCE EMERGING TECHNOLOGIES FOR POINT-OF-CARE PRIMARY TUMOR BURDEN BUT ACCELERATE DIAGNOSIS OF SOIL-TRANSMITTED HELMINTH METASTASIS INFECTIONS

Samuel M.1, Timpson P.2, Parker B.1 and Mathivanan S.1 Soni S.K.1, Ravindran V.B.1, Traub R.2 and Ball A.S.1 1La Trobe University, Melbourne. 2Garvan Institute for Medical 1School of Science, RMIT University, Melbourne. 2Faculty of Research, Sydney. Veterinary and Agricultural Sciences, Melbourne University, Melbourne. It has been proposed that exosomes from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable Parasitic worms (helminths) cause some of the most neglected in various organs, and exert phenotypic changes. Here, we orally tropical diseases. They infect ~ 1 billion people globally and contribute administered bovine milk-derived exosomes to mice and demonstrate substantially to poor physical and cognitive development in children, that milk-derived exosomes can survive the harsh degrading and poor maternal birth outcomes. No commercial vaccines are conditions of the gut and subsequently be detected in multiple organs. available and treatment relies heavily on only a small number of drugs. Interestingly, oral administration of milk-derived exosomes reduced Because drug resistance is a major threat, there is an urgent need to the primary tumor burden in various cancer models and attenuated develop new and innovative methods of diagnosis to contribute toward cancer cachexia. Intriguingly, in spite of the reduction in primary tumor achieving the Millennium Goals of the London Declaration. Currently, growth, milk-derived exosomes accelerated metastasis in breast and coproscopic diagnostic methods such as Kato Katz thick smear is pancreatic cancer mice models. Timing of exosome administration performed, however this technique is antiquated and its sensitivity was critical as oral administration after resection of the primary tumor is very poor. Although PCR-based methods are an alternative to reversed the pro-metastatic effects of milk-derived exosomes in breast coproscopy, constraints relate to the transport of samples to a central cancer. Taken together, our study provides novel context-based and laboratory, high cost and inability to estimate infection intensity. opposing role of milk-derived exosomes as metastasis inducers and as Therefore, a need for radically new and innovative tools is essential metastasis blocker. for the specific diagnosis of helminths. Here, we coupled propidium monoazide with isothermal amplification to effectively quantify the viability of Ascaris and Trichuris eggs. Also, we distinguished 3 different helminth ova based on surface acoustic wave drive microfluidics. Furthermore a highly selective and sensitive lateral flow based strip assay (Microbial Detection Assay Strips-MIDAS) also has been developed, that can detect 2 genera of STHs on 1 strip in 30 min. These studies will be validated further on a point-of-care basis to support mass treatment programs in the field.

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SYM-14-01 SYM-14-02 HOW AUTOTRANSPORTER PROTEINS MODULATE INFLAMMASOME ASSEMBLY AND ACTIVATION BACTERIA-HOST INTERACTIONS MECHANISMS Vajjhala P.R.1, Lu A.2, 3, Sagulenko V.1, Wong P.Y.1, Brown D.L.4, Fu T.M.2, 3, Paxman J.J.1, Lo A.2, Vo J.1, Martinez Ortiz C.1, Scembri M.A.2 Sester D.P.1, Stow J.L.4, Wu H.2, 3 and Stacey K.J.1 and Heras B.1 1School of Chemistry and Molecular Biosciences, University of 1Department of Biochemistry and Genetics, La Trobe Institute for Queensland, Brisbane, QLD 4072, Australia. 2Department of Biological Molecular Science, La Trobe University, Melbourne, VIC. 2Australian Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Infectious Diseases Research Centre, School of Chemistry and MA 02115, USA. 3Program in Cellular and Molecular Medicine, Boston Molecular Biosciences, The University of Queensland, Brisbane QLD Children’s Hospital, Boston, MA 02115, USA. 4Institute for Molecular 4072, Australia. Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. Pattern recognition receptors (PRR) of the innate immune system mediate Bacterial pathogens deploy an arsenal of virulence factors to establish the first line of defence against infections and cellular stress signals. The infection and cause disease. At the front line of the infection process inflammasome complex initiated by a subset of PRRs forms a platform are bacterial surface components, which are responsible for host for activation of procaspases-1 and -8 to mediate inflammation and cell colonisation and pathogen adhesion. Autotransporter (AT) proteins death responses. Inflammasomes initiated by the AIM2 PRR in response to are the largest group of surface adhesins in Gram-negative bacteria. cytosolic DNA are relevant to viral and bacterial infections and autoimmune These proteins play a central role in controlling bacterial interactions diseases caused by recognition of self DNA. Inflammasomes initiated by the with their environment; they allow bacteria to aggregate with other NLRP3 PRR, activated by many diverse stimuli, contribute to the pathology bacteria, adhere to human cells, and form biofilms­ all key facilitators of many common diseases including diabetes and atherosclerosis. of bacterial persistence and pathogenesis. We previously elucidated Inflammasome assembly is mediated by death-fold domains, which include pyrin domains (PYDs), caspase recruitment domains (CARDs) and death the mechanism by which the AT adhesin Antigen43 (Ag43) from effector domains (DEDs). Activation of the PRR induces oligomerisation uropathogenic E. coli (UPEC) promotes bacterial aggregation/biofilm and PYD clustering, which recruits the adaptor protein ASC and nucleates formation, by means of self-association between neighbouring cells. polymerization of ASC PYD into a helical filament. ASC CARDs condense We have produced the structures of a further AT proteins and we are the complex to form a speck that is synonymous with inflammasome now starting to elucidate the mechanisms of action for diverse ATs. activation and also recruit procaspase-1. We showed that ASC recruits So far, we have found that different AT adhesins promote bacterial procaspase-8 to the inflammasome to mediate apoptosis. The interaction aggregation using subtle variations in this self-association mechanism is mediated by interaction between ASC PYD and procaspase-8 DEDs compared to Ag43. We are also beginning to uncover in atomic and nucleates formation of procaspase-8 DED filaments. Studies with detail how AT adhesins like UpaB and TibA bind epithelial surfaces. reconstituted inflammasomes in HEK cells show that procaspase-8 DED TibA is a multifunctional AT from enterotoxigenic E. coli(ETEC), the filaments extend from the ASC speck while full-length procaspase-8 is leading bacterial cause of diarrhea. This surface protein was known condensed within the ASC speck suggesting inter-filament catalytic domain to be glycosylated by the cognate glycosyltransferase TibC. Our work interactions. Procaspase-1 CARD did not form extensive filaments from the is beginning to uncover how glycosylation regulates the function of ASC speck in HEK cells thus a similar mechanism cannot be presumed. multifunctional AT adhesins. This may represent a general mechanism Time course analysis of endogenous inflammasomes to characterise the for bacteria to regulate the virulence functions of the vast number of mechanism of inflammasome activation indicates rapid activation upon ATs expressed on their cell surface. Finally, we are using this new stimulation while live cell imaging indicates rapid ASC filament elongation. Our current models of inflammasome assembly and activation based on knowledge to successfully develop methods for disrupting AT function. our data and insights from structural studies of death-fold domain filaments will be presented.

SYM-14-03 SYM-14-04 IMMUNOSUPPRESSIVE ACTIVITY OF HUMAN CD52 TACKLING ANTIMICROBIAL RESISTANCE (AMR) VIA SPECIFIC SIALOFORMS USING SINGLE PARTICLE CRYOEM

Shathili A.M.1, Bandala-Sanchez E.2, Goddard-Borger E.D.2, John A.2, Belousoff M.J.1, Yonath A.3, Lupton D.2 and Lithgow T.1 Thaysen-Andersen M.1, Everest-Dass A.3, Harrison L.C.2 and Packer N.H.1, 3 1School of Microbiology, Monash University. 2School of Chemistry, 1Dept. of Molecular Sciences and ARC Centre of Excellence in Nanoscale Monash University. 3Weizmann Institute of Science, Israel. BioPhotonics, Macquarie University, Sydney, New South Wales, Australia. 2Walter & Eliza Hall institute of Medical Research and University of The World Health Organization states that by 2050, deaths from Melbourne, Parkville. Victoria, Australia. 3Institute for Glycomics, Griffith antibiotic or drug-resistant microbes could exceed all other current University, Gold Coast, Queensland, Australia. global health risks. These predictions rest on the assumption that there will be no new treatments come to the clinic. Our lab is determined to Homeostatic mechanisms are required to limit immune T-cell proliferation tackle a facet of the AMR problem by exploring an important target of and prevent autoimmune diseases. Human CD52 is a small glycoprotein antimicrobials; the bacterial ribosome. Ribosomal interfering antibiotics (12 amino acid residues), with an N-linked glycan at Asn3 and possible are used to commonly treat serious infections by methicillin resistant S. O-glycosylation. Glycosylated and soluble CD52 contributes to immune aureus (MRSA) and vancomycin resistant Enterococcus (VRE). One homeostasis by ligating the inhibitory sialic acid-binding immunoglobulin- of the more effective ribosomal interfering antibiotics is the first fully like lectin-10 (Siglec-10) receptor on the surface of activated T-cells. We synthetically produced antimicrobial; linezolid. Our work attempts to aimed to define the bioactive glycoforms of CD52. Our initial analysis of address two main questions: is it possible to understand how bacteria purified human spleen CD52 confirmed the presence of multi-antennary modify their ribosomal structure as they evolve linezolid resistance and sialylated N-glycans with abundant polyLacNAc extensions, together is it possible to use this structural information to redesign the linezolid with some O-glycan structures. To gain insight into the molecular basis chemical structure to keep it active against resistant strains of bacteria? of CD52 binding mechanism, recombinant soluble CD52-Fc expressed I will present the structure of the MRSA ribosome and the structure from HEK293 or Expi293 cells, but not the Fc alone, suppressed T-cell from a clinical isolate from the Alfred hospital which became resistant to function and was used to relate bioactivity with the CD52 glycoform linezolid during the course of treatment [1]. Our cryoEM studies clearly structure. Glycomics (porous graphitised carbon-liquid chromatography- show how the binding site of the antibiotic is drastically remodeled ESI-MS) and intact glycopeptide (high resolution C8-ESI-MS) analyses of due to a single point mutation in the resistant Staph. strain. Using the recombinant CD52 revealed that only specific type of N-glycans contributed structural information as to how MRSA evades linezolid treatment we to the bioactivity of CD52. Interestingly, the relative abundance of a specific rationally redesigned linezolid and chemically synthesised new antibiotic sialic acid linkage correlated with higher bioactivity, which was verified by derivatives which remained active against the linezolid resistant strains de- then re-sialylation experiments. Anion exchange chromatography on a of Staph. Moreover, I will present high resolution (2.8 Å resolution) MonoQ-GL column fractionated CD52-Fc into glycoforms with increased structures of these new drugs in complex with the MRSA ribosome capacity to suppress T-cell function. Fractions with suppressive activity to confirm our rational drug designs. Excitingly, we have shown that confirmed previous results. O-glycans were assigned their site localisation utilizing a pragmatic approach to antibiotic design that it is possible to in the active fractions. These findings define glycans involved in the immune make minor chemical changes to an existing antibiotic platform to keep suppressive bioactivity of CD52 and resulted in CD52 fractions with higher it active in the face of antibiotic resistance. [1] Matthew J. Belousoff, suppressive activity than the biologically isolated CD52. et al. Structural basis for linezolid binding site rearrangement in the Staphylococcus aureus ribosome. MBio, 2017, 8:e00395-17.

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SYM-14-05 SYM-15-01 HAEM AT THE INTERFACE BETWEEN PATHOGENIC CREATING OPPORTUNITIES FOR STUDENT SELF- AND COMMENSAL BACTERIAL SPECIES IN THE REFLECTION ON THE DEVELOPMENT OF GRADUATE HUMAN RESPIRATORY TRACT CAPABILITIES IN A LARGE FIRST YEAR MOLECULAR BIOLOGY SUBJECT Latham R.D.1, Del Rey M.T.2, Walshe J.2, Brianna A.1, Guss J.M.2, Mackay J.P.2, Tristram S.G.1 and Gell D.A.1 Kuit T. and Skropeta D. 1University of Tasmania, TAS, Australia. 2University of Sydney, NSW, University of Wollongong. Australia. Every university aims to develop graduates that are highly sought after Non-typeable Haemophilus influenzae (NTHi) is an important by industry. Programs of study aim for graduates to achieve a set of opportunistic pathogen of the human respiratory tract that has proved core learning outcomes that develop key skills such as communication recalcitrant to vaccine development and shows increasing prevalence and teamwork. These are often referred to as graduate capabilities. of antibiotic resistance, prompting us to search for alternative anti- These skills are then typically demonstrated to employees through microbial strategies. Haemophilus haemolyticus is closely related selection criteria. Currently, at UOW science undergraduate students to NTHi, and also colonises the respiratory tract, but is recognised have little opportunity to engage in self-reflection, which is key to as a non-pathogenic commensal species. We identified two H. effectively meeting these criteria. One way to effect this change is haemolyticus isolates secreting a 27-kDa protein that inhibited the to encourage student e-Portfolio development and maintenance. growth of NTHi in vitro, but did not inhibit a range of other respiratory E-Portfolios are a collection of digital evidence that validates learning flora that were tested, suggesting a level of species-specificity against and development through a process of self-reflection. This enhances NTHi. A gene knockout established that the gene product of interest the meaning of students’ work and encourages insight and higher was responsible for inhibitory activity. Spectroscopic and x-ray learning. Thus, a peer-assessed e-Portfolio component was designed crystallographic analysis of the recombinant protein identified a haem and successfully implemented in a molecular biology subject to a large binding site. The protein shares structural features with some non- cohort of first-year science students (700+) with diverse educational haem iron scavenging proteins, but the haem-binding site is unique. and cultural backgrounds. We successfully introduced the necessary Insights into the biological function, including NTHi inhibitory actions, skill foundations to students who inturn gained a greater understanding of this protein, based on structure, biochemistry and bioactivity assays into their own learning, particularly through the guided peer review are presented. The work is ongoing in the context that strains of H. process. In this presentation I will share the outcomes of this project haemolyticus might be developed as respiratory probiotics to combat and key lessons learnt along the way with a chance for discussion on colonisation and infection with NTHi. how others are supporting students to evidence the development of graduate capabilities.

SYM-15-02 SYM-15-03 FLIPPING THE LABORATORY IN PLANT ANATOMY, EFFECT OF CONTEXT-BASED UNDERGRADUATE BIOCHEMISTRY AND GLOBAL CHANGE BIOLOGY BIOCHEMISTRY FOR HEALTH SCIENCES (CUBHS) INSTRUCTION ON STUDENT PERCEPTION OF Loveys B.R. RELEVANCE, ACHIEVEMENT AND ATTITUDE School of Agriculture, Food and Wine, University of Adelaide, 1 2 3 4 Adelaide, South Australia. Fernandez K. , Overton T. , Thompson C. and Samarawickrema N. 1School of Chemistry/Department of Biochemistry & Molecular Biology, Monash University. 2School of Chemistry, Monash University. 3School of Practical classes are generally compulsory and all teachers hope their 4 students will arrive in the laboratory prepared for the class; the reality, Chemistry, Monash University. Department of Biochemistry & Molecular of course, is that students are often not prepared and are therefore Biology, Monash University. disengaged and confused in class. Many students do not read the There is an ongoing debate on the relevance of foundational biochemistry relevant material in their laboratory manual to ensure they understood in the health sciences. It stems from the fact that much of biochemistry of the principles they are about to learn. This makes it difficult for instruction has focused on the didactic delivery of concepts and theories students to make the link between theory and application. To address with little emphasis on clinical applications. This has resulted in a this problem interactive, online pre-class activities were developed and foundational-clinical gap and to the negative perception of biochemistry deployed in plant anatomy, biochemistry and global climate change among health science students. Notwithstanding, the inclusion of practical classes, thus Flipping the Laboratory to encourage students biochemistry in the health science curricula remains valid. As Gwee, to prepare for practical classes. Using this approach I have developed Samarasekera and Chay-Hoon (2010) assert, since clinical practice is multiple pre-practical online activities for my students on topics based on scientific knowledge, biochemistry remains indispensable. The such as plant anatomy, , photosynthetic reactions recommendation is for biochemistry to be taught in the context of clinical and carbohydrate metabolism. Central to the flipped classroom practice (1, 2). Considering this, the study aims to determine the effects learning model is that pre-class learning should be used to introduce of a researcher developed Context-based Undergraduate Biochemistry for foundational concepts and focus on lower level Blooms Taxonomy Health Sciences (CUBHS) instruction on student perception of relevance, such as remember and understand. The pre-practical activities provide achievement and attitude. It is a two-group posttest design wherein cohorts students with examples and activities to encourage them to prepare will be health science students (nursing, pharmacy, psychology and for practical sessions before class. Information is presented in a multi- medical laboratory science). The control group will be 2018 students who media format with videos of demonstrations of commonly used lab are currently studying biochemistry in its traditional didactic form while, the techniques. Check-point questions are presented by way of multiple experimental group will be 2019 students who will be studying biochemistry choice answers with unlimited attempts so student can gain confidence. in CUBHS instruction. Scores on relevance, attitude and achievement in Understanding foundational key concepts is critical for deeper learning. biochemistry will be measured through adapted tools: Message Content This presentation will show how sustained improvement in average Relevance Scale (MCRS), Attitude towards Biochemistry Inventory (ABI) and distribution of practical grades has been achieved. Failure rates measure and Biochemistry Achievement Tool (BAT). Finally, the significant have also declined despite increasing student enrolments. Students difference of scores between groups will be tested. References: (1) Gwee, indicated the pre-practical activities encouraged independent learning, M., Samarasekera, D and Chay-Hoon, T.(2010). Role of Basic Sciences in and, 70% of students enjoyed the pre-practical activities. Improved 21st Century Medical Education: An Asian Perspective. Medical Science engagement in practical classes extends beyond the laboratory. Once Educator, 20(3). (2) Bandierra, G.; Boucher, A.; Neville, A.; Kuper, A.; students are engaged in a course it is easier maintain their interest in Hodges, B. (2013). Integration and timing of basic and clinical sciences difficult and challenging content. education. Journal of Medical Teacher, 35(5).

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SYM-15-04 SYM-16-01 EMBEDDING SCIENCE COMMUNICATION SKILLS IN SHOOT BRANCHING - ROLE OF STRIGOLACTONES FIRST YEAR BIOLOGY STUDENTS USING A MULTI- AND INTERACTIONS WITH OTHER SIGNALS STAGE TEAM PROJECT Beveridge C.1, Chabikwa T.1, Kerr S.1, Han F.1, Fichtner F.2, Lunn J.2 Galea A.M., LeBard, R. and Wilson J.E. and Barbier F.1 School of Biotechnology and Biomolecular Sciences, The University 1The University of Queensland, School of Biological Sciences, of New South Wales, Sydney, NSW 2052, Australia. Brisbane, Australia. 2Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1. Communication skills, information literacy and the capacity for self- reflection are all highly desirable graduate attributes that can be Shoot branching occurs due to the regulation of the outgrowth of axillary developed and honed from the very beginning of a student’s university buds which are embryonic shoots in the axil of leaves. Long-distance program. But building the optimal learning tasks for achieving these signaling is central to this regulation and mainly involves strigolactones, goals in both general and discipline-specific contexts can be very cytokinins, auxin and sugars. The sugar role may be at least partly challenging, especially in large first year courses which are typically due to sugar signalling and to involve trehalose 6-phosphate. It also populated by students with very diverse interests and educational appears that the growth of axillary buds from a state of very slow backgrounds. Here we describe the design and implementation of a growth or dormancy, to sustained growth involves a number of stages new team-based learning activity that aims to embed a range of general during which the emerging shoots show differential sensitivity to growth and discipline-specific skills and capabilities in the students of a large stimulus and inhibition. For example, there are substantial differences first year biology course at the University of New South Wales, Sydney. in responses to different hormones at different periods after shoot tip The BABS1201 Science Communication Project is a semester-long removal. This could be due to differences in hormone signaling and group task with multiple components. Working in teams of 3 to 4 people, downstream responses as well as due to changes in the vasculature students select a modern biology topic or discovery from an extensive of the growing buds. We will present our latest unpublished findings on list of subjects that align with the core concepts and themes of the the interaction of signals during bud outgrowth. In addition to providing course. For each team, the primary objective is to design and develop a new mechanism for how plants respond to shoot tip removal, this a presentation (in any format) that will effectively communicate their work is provides a better understanding of how plants achieve diverse subject and its core biological concepts to their peers. The project is architecture in response to the environment. scaffolded by several other components, including a scientific literature essay, a project pitch with peer feedback, a project diary, a reflective essay, and peer evaluation. Creativity and innovation are encouraged, acknowledged and rewarded throughout the project. Initial outcomes and feedback from students and staff have indicated that the project effectively engages students with modern biology and permits them to develop, exercise and enhance a range of communication skills.

SYM-16-02 SYM-16-03 MIX AND MATCH: EPF/EPFL PEPTIDE SIGNALING IN IDENTIFICATION OF REGULATORY PATHWAYS PLANT DEVELOMENT CONTROLLING CELL DIFFERENTIATION DURING BARLEY GRAIN DEVELOPMENT Torii K.U.1, 2 1Investigator, Howard Hughes Medical Institute, Department of Aubert M.K.1, Shirley N.J.1, Houston K.2, Burton R.A.1 and Tucker M.R.1 Biology, University of Washington. 2Principal Investigator, Institute of 1School of Agriculture, Food and Wine, University of Adelaide, Waite Transformative Biomolecules, Nagoya University. Campus, Urrbrae, South Australia, Australia. 2Cell and Molecular Sciences, The James Hutton Institute, Dundee, UK. Plant peptide signals mediate both local cell-cell communications and long-distance root-to-shoot signaling. Secreted cysteine-rich Barley is a diploid cereal crop used in the feed and brewing industries. peptides in the EPIDERMAL PATTERNING FACTOR (EPF)- EPF The benefits of the barley grain are derived mainly from the endosperm, LIKE (EPFL) family regulate diverse aspects of plant development, which is produced after fertilisation. During early stages of seed including shoot meristem size, inflorescence architecture, vascular development, the endosperm differentiates along a radial axis to form differentiation, and stomatal development. The EPFL peptides are two prominent cell types; the peripheral aleurone and the inner starchy versatile: The same peptide could function in an autocrine or paracrine endosperm. We have been studying early grain development in barley manner. Moreover, related EPF/EPFL family members could work in with a view to understanding how aleurone differentiation is regulated. a unique, redundant, or even in an antagonistic manner, depending Microscopic assays were used to measure sub-epidermal details of on the specific developmental programs or tissue-specific contexts. grain development in a panel of ~200 barley cultivars. Association Thus far, ERECTA-family receptor kinases are the known receptors for mapping identified multiple genomic regions that contribute to variation. EPF/EPFL peptides. As such, this peptide hormone-receptor system Candidate genes underlying variation in aleurone development were poses an important question of how the same receptor could perceive identified using a combination of RNAseq profiling, laser capture multiple peptide ligands to elicit such diverse developmental outcomes. microdissection and plant transformation. The results of these assays Our recent study on the developmental functions of EPFL2 peptide will be discussed. The fundamental knowledge generated in this project revealed a feedback circuit between the peptide-receptor system and is providing insight into how different tissues and cells contribute to auxin response as a mechanism for maintaining proper auxin maxima grain development. This knowledge may be applied in future to tailor during leaf margin morphogenesis. Our study highlights the intersection specific improvements in grain composition. of localized peptide signaling and universal small chemical hormone auxin in shaping plant development and morphogenesis.

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SYM-16-04 SYM-16-05 AN APOCAROTENOID RETROGRADE SIGNAL POST- INVESTIGATING THE FUNCTIONS AND INTERACTIONS TRANSCRIPTIONALLY CONTROLS PROTEIN LEVELS, OF FLAS IN SECONDARY CELL WALLS PLASTID BIOGENESIS AND PLANT DEVELOPMENT DURING EXTENDED PERIODS OF DARKNESS Ma Y.1, 2, Zeng W.3, Bacic A.1, 2 and Johnson K.L.1, 2 1University of Melbourne. 2La Trobe University. 3Zhejiang Agriculture Cazzonelli C.I.1, Hou X.2, Alagoz Y.1, Rivers J.2, Dhami N.1, Lee J.3, and Forestry University. Shashikanth M.2 and Pogson B.2 1 Hawkesbury Institute for the Environment, University of Western Sydney, Our natural and plantation forests represents 90% of the captured Hawkesbury Campus, Bourke Street, Richmond, NSW AUSTRALIA 2753. 2Australian Research Council Centre of Excellence in Plant Energy Biology, carbon and is our most renewable bio-resource. The bulk of the biomass College of Medicine, Biology and Environment, Research School of Biology, consists of secondary cell walls, which are thick, rigid and deposited on The Australian National University, Canberra, ACT 26. 3Centre for Advanced the inner side of the primary walls in specialized tissues. Secondary Microscopy, The Australian National University, Canberra, ACT 2601, Australia. cell wall development is an area of intense interest given its importance for plant growth, water conductance and human applications such Cleavage products of cis-carotenes are reported to regulate nuclear gene as biofuels, bio-inspired materials/bio-composites, construction and expression, carotenoid homeostasis and leaf development in plants. Environmental paper. A group of plant cell wall glycoproteins, the Fasciclin-Like conditions that affect their biosynthesis, their physiological functions and regulatory Arabinogalactan proteins (FLAs) have been implicated in regulating targets remain unknown. Carotenoid (crtiso) mutants display various secondary cell wall development and influencing their biomechanical virescent phenotypes depending upon the environmental conditions. Here we properties. FLAs are a sub-class of the arabinogalactan-proteins demonstrate a physiological link between day length (extended darkness), the accumulation of a cis-carotenes and control over plastid biogenesis. Chemical (AGPs), glycoproteins implicated in cell wall sensing and signalling inhibition of carotenoid cleavage dioxygenase activity restored prolamellar body and proposed to cross-link to pectins in the wall. The fasciclin (FAS) (PLB) formation crtiso/ccr2 etioplasts during skotomorphogenesis providing domain has been shown to be involved in protein-protein interactions evidence for a novel cis-carotene derived apocarotenoid signal (ACS) that controls and development in mammals, insects and algae. Glycoproteins such plastid biogenesis A forward genetic screen identified an epistatic interaction as FLAs are therefore fascinating ‘chimeric’ molecules with a number between the ζ-carotene isomerase (ziso-155) and Arabidopsis ccr2 mutants of interesting properties; being able to potentially form protein-protein, that blocked the biosynthesis of specific cis-carotenes, restored PLB formation protein-carbohydrate and carbohydrate-carbohydrate interactions in in ccr2 etioplasts during skotomorphogenesis, and prevented leaf virescence in plants grown under shorter photoperiod. Transcriptomic analysis of ccr2 ziso- the wall. Using Arabidopsis as a model system, we have focused on a 155 mutant tissues revealed down-regulation of repressors of photomorphogenesis subset of FLAs (FLA11, FLA12 and FLA16) that are expressed in cells and up-regulation of photosynthesis associated nuclear gene (PhANG) expression undergoing secondary cell wall development. Preliminary evidence that correlated with normal plastid development. We next identified a mutation suggests these FLAs influence the amount and angle of cellulose in DEETIOLATED 1 (det1-154) that restored PLB formation in ccr2 and reduced deposition, the most abundant cell wall polymer that forms the structural specific cis-carotenes to levels below a threshold that promoted the efficient basis for all cell walls. Through mutant studies, biomechanical analysis, biogenesis of plastids in plants grown under extended periods of darkness. det1- examination of FLA location and bioinformatics we are gaining insight 154 reduced transcription of protochlorophyllide (POR) and into the function(s) of these complex glycoproteins. ACS post-transcriptionally maintained POR protein levels. We describe a model whereby a retrograde ACS acts via an alternative signaling pathway downstream of DET1 to post-transcriptionally regulate POR, ELONGATED HYPOCOTYL5 (HY5) and PHYTOCHROME-INTERACTING FACTOR3 (PIF3) thereby coordinating PhANG expression and plastid development during extended periods of darkness.

SYM-17-01 SYM-17-02 SELF-ASSEMBLING HYDROGEL SUBSTRATES FOR UNDERSTANDING FORCE SENSING MECHANISMS IN NEURAL NETWORKS MECHANOSENSITIVE PIEZO CHANNELS

Martin A.D. Cox C.D., Bavi N. and Martinac B. University of New South Wales. Victor Chang Cardiac Research Institute.

Primary neuronal cultures are a powerful tool to understand neuronal Mechanosensitive channels are essential molecular components of maturation, aging and neurodegeneration. They have been used to mechanosensory systems in all organisms. Bacterial mechanosensitive screen the effects of drugs and misfolded proteins on neural networks channels are gated directly by bilayer tension. Recent work has in vitro. However, culturing primary neurons in vitro is notoriously demonstrated that bilayer tension may also activate eukaryotic difficult, owing to their high sensitivity to their environment. Currently, mechanosensitive channels such as TREK-1/2. Using bleb-based primary neurons are cultured on glass coverslips coated with poly- electrophysiology we have shown that Piezo1 can also be gated by D-lysine (PDL). However, it is well known that significant differences membrane tension, supported by the fact that purified mouse Piezo1 exist in cell behaviour in a 2D versus 3D environment, which more can be gated in pure lipid systems. More recently we have explored accurately mimics in vivo conditions. Hydrogels have significant the influence of cytoskeletal proteins on Piezo1 and how these potential biomedical applications, including in cell culture, owing to components modulate the channels inherent tension sensitivity using their similarity to the extracellular matrix. We have previously used patch fluorometry. Moreover, we measure the mechanical properties of short peptides capped at their N-terminus with an aromatic group the cells expressing these channels concomitantly while we estimate to form biocompatible hydrogels with tuneable stiffnesses, pore the membrane tension necessary for channel activation. Changes in sizes and chemical functionalities. Here, we present a collaborative, Piezo1 sensitivity are not exclusively correlated to changes in bulk multidisciplinary effort where short peptide hydrogels which support mechanical properties of the cell. In the presence of STOML3 and the growth of primary neurons in a 2D and 3D environment have tropomyosin4.2 the membrane tension required to gate Piezo1 is ≤ been developed. Neurons cultured atop these hydrogels display initial 0.5 mN/m, which is well within a physiological range. In contrast, the development and maturation comparable to that on PDL, complete with activation threshold of Piezo1 in the presence of Filamin A increases. synapse formation and electrical activity. Neurons can also be cultured Our results shed light on the ability of different structural scaffold within the hydrogels, with these 3D neuronal cultures having potential in proteins to sensitize or desensitize Piezo1 channels to mechanical identifying neurodegenerative disease biomarkers, better screening of stimuli by modulating their tension sensitivity. This model holds drug molecules, modelling CNS damage and insights into aging. general applicability for sensitization/desensitization of all inherently mechanosensitive ion channels by cytoskeletal elements.

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SYM-17-03 SYM-17-04 SPECIALISATION OF THE GLIOMA CYTOSKELETON NEWLY IDENTIFIED MOLECULAR MECHANISM OF FOR NAVIGATING THE SOFT TISSUE ENVIRONMENT GLUCOCORTICOID ACTION IN ARTHRITIS OF THE BRAIN Achuthan A., Lupancu T., Lee M.C., Fleetwood A., Cook A. and O’Neill G.M.1, 2 Hamilton J. 1Childrens Cancer Research Unit, Kids Research Institute, The Department of Medicine, Royal Melbourne Hospital, University of Childrens Hospital at Westmead, Westmead, New South Wales, Melbourne, Australia. Australia. 2Discipline of Childhood and Adolescent Health, The University of Sydney, Sydney, New South Wales, Australia. Rheumatoid arthritis is a chronic inflammatory autoimmune disease, which leads to poor quality of life due to the debilitating effect of Glioma brain tumours are characteristically highly infiltrative into the inflammation. Clinical trials in rheumatoid arthritis targetting the surrounding healthy brain tissue, which is a mechanically soft tissue cytokine, granulocyte macrophage-colony stimulating factor (GM- environment. Thus mechanisms of cancer cell invasion that have been CSF) are showing promise although its mode of action remains largely defined using hard plastic dishes may not apply for the dissemination unknown. We have recently shown that that GM-CSF drives CCL17 of glioma brain cancer cells through brain tissue. Indeed, high grade production via a new interferon regulatory factor 4 (IRF4)-dependent gliomas characteristically lose expression of the high molecular weight pathway in human monocytes and murine macrophages, as well tropomyosins, a class of actin-associating proteins that are essential as in vivo. Importantly, in arthritis and pain models IRF4-regulated regulators of the actin stress fibres and focal adhesions that underpin CCL17 formation mediates the proinflammatory and algesic actions cell migration on hard 2-dimensional surfaces. Thus in the present of GM-CSF. Glucocorticoids (GCs) are potent anti-inflammatory and study we have investigated how loss of the high molecular weight immunosuppressive agents broadly used in anti-inflammatory therapy, tropomyosins affects glioma cell morphology using soft matrices that albeit with adverse side effects associated with long-term usage. The better recapitulate the biomechanical attributes of the brain. We show negative consequences of GC therapy provide an impetus for research that Tpm 2.1 is down-regulated in glioma cells grown on soft brain-like into gaining insights into the molecular mechanisms of GC action on environments. Next we demonstrate that Tpm 2.1 depletion by siRNA immune cells. We report here that GM-CSF-induced CCL17 expression induces cell spreading and elongation in soft 3D hydrogels, irrespective is inhibited by GCs in human monocytes and mouse macrophages. of matrix composition, and further that this mimics the effects of Rho Moreover, we provide evidence for the first time that GCs suppress kinase inhibition. Tpm 1.7, a second high molecular weight tropomyosin GM-CSF-induced IRF4 expression via regulating the expression is also down-regulated when cultured on soft brain-like surfaces and and activity of JMJD3, which demethylases trimethylated-H3K27. we show that effects of this isoform are matrix dependent, with Tpm Significantly, a recent study reported that synovial fluid from patients 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that with rheumatoid arthritis had elevated levels of CCL17 as compared the absence of Tpm 2.1 from primary patient derived high grade glioma to healthy controls. We will provide molecular evidence for the anti- cells correlates with elongated, mesenchymal invasion in 3D. We inflammatory actions of GCs in rheumatoid arthritis patient samples. propose that Tpm 2.1 down-regulation overcomes the rigidity-sensing The delineated pathway potentially provides new therapeutic options mechanism that would otherwise prevent the cells from spreading for the treatment of inflammatory diseases and their associated pain. throughout the soft brain environment. The unique organisation of the glioma actin cytoskeleton organisation that is highly suited to the soft brain like environment may provide novel therapeutic targets for arresting invasion of high grade glioma cells.

SYM-17-05 SYM-18-01 ROLE OF ARP2/3 IN BLEBBING MIGRATION OF T CRYO-EM STUDIES OF E. COLI ATP SYNTHASE LYMPHOCYTES IN VIVO Sobti M.1, Smits C.1, Wong A.S.W.2, Ishmukhametov R.3, Stock D.1, 4, Obeidy P.1, Ju L.2, Oehlers S.1, Zulkhernain N.S.1, Galeano Nino L.G.3, Sandin S.5 and Stewart A.G.1, 4 Tikoo S.1, Jackson S.P.2, Biro M.3, Roediger B.1 and Weninger W.1 1Molecular, Structural and Computational Biology Division, The Victor 1The Centenary Institute of Cancer Medicine and Cell Biology, Chang Cardiac Research Institute, Darlinghurst 2010, Australia. 2NTU Newtown, NSW 2042, Australia. 2Heart Research Institute, Charles Institute of Structural Biology, Nanyang Technological University, Perkins Centre, University of Sydney, Camperdown, NSW 2006, Singapore, Singapore. 3Department of Physics, Clarendon Laboratory, Australia. 3Cell Motility and Mechanobiology, School of Medical University of Oxford, Oxford OX1 3PU, UK. 4Faculty of Medicine, The Sciences, University of New South Wales, Sydney, 2050 NSW, University of New South Wales, Sydney 2052, Australia. 5School of Australia. Biological Sciences, Nanyang Technological University, Singapore, Singapore. Cytotoxic T lymphocytes (CTL) rely on the precise rearrangement of the actin cytoskeleton to provide immunosurveillance against Here we present our cryo-EM maps of the intact ATP synthase complex invading pathogens and malignant cells. Constant remodelling of the from Escherichia coli. This essential enzyme synthesises the bulk of cytoskeleton, par ticularly at the leading edge, is associated with ef ficient cellular ATP, the energy currency of the cell. The structures highlight T cell migration and function. The consequences of modulating Arp2/3, unique features of this ATP synthase complex, such as the bifurcation a macromolecular machine that nucleates branched actin filaments, at of the peripheral stalk homodimer and the position of the inhibitory the leading edge of migrating T cells are incompletely understood. We subunit ε. Further studies on this complex reveal a possible partially report that modulation of Arp3 (one of the main subunits of Arp2/3) active conformation, which points to the molecular events that may profoundly affects CTL actin cortex integrity, surveillance in vivo and inhibit this marvellous motor. effector function in vitro. We also demonstrate a significant reduction in the total F-actin resulting in decreased cortical tension and disruption of lamellipodia formation. As a result, Arp3 knockdown CTL switched from lamellipodia-based migration to the blebbing mode characterised by transient, membrane balloon-like protrusions at the leading edge both in vitro and in a zebrafish model. Our study established that optimal mechano-physical and biochemical properties of the actomyosin cortex, as maintained by the Arp2/3 complex, are essential for the proper functioning and effective migration of CTL. These unforeseen findings pave the way for a deeper understanding of actin nucleators in T cell cytoskeleton and are crucial for the development of improved therapies for cancer and inflammatory diseases.

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SYM-18-02 SYM-18-03 THE TUMBLEWEED: CONSTRUCTION OF A NANOSCALE DNA ORIGAMI TOOLS TO STUDY SYNTHETIC PROTEIN MOTOR MOLECULAR MACHINES

Davies R.1, Bromley E.2, Niman C.3, Blab G.4, Woolfson D.5, Wickham S. Zuckermann M.6, Forde N.6, Linke H.3 and Curmi P.1 School of Chemistry, University of Sydney, Sydney, NSW 2006, 1School of Physics, UNSW Sydney, Australia. 2Department of Physics, Australia. University of Durham, UK. 3Solid State Physics and Nanometer Structure Consortium, Lund University, Sweden. 4Molecular DNA has huge potential as a programmable building material for Biophysics, Universiteit Utrecht, The Netherlands. 5School of biocompatible nanostructures, with many applications as tools for Biochemistry, Bristol University, UK. 6Department of Physics, Simon single molecule biophysics, platforms for diagnostics and therapeutics, Fraser University, Canada. and templates for nanofabrication. DNA origami is a method for making a diverse range of shapes by folding up a long single-stranded DNA Molecular motors and machines are highly complex, multi-subunit scaffold. Custom DNA origami nanostructures can be designed as proteins that use chemical energy to perform a multitude of critical, tools for specific biophysical measurements. For example, a DNA mechanical tasks in cells. The physical mechanisms by which motor origami nanospring, to simultaneously measure force and position of proteins (such as myosin and kinesin) transduce chemical energy into the protein motor Myosin VI during stepping. DNA nanotechnology can mechanical work are still poorly understood. Traditionally, scientists also be used to build and test synthetic molecular machines, inspired by have taken a “top down” approach to addressing this question by their biological counterparts. For example a molecular motor that can determining crystal structures of motor proteins, characterizing mutants transport cargo through a maze of tracks. DNA origami structures can and making single molecule measurements of performance. The goal also serve as the substrate for future hybrid machines, which integrate of our work is to take a “bottom up” approach and design an artificial functional protein components with synthetic DNA scaffolds. motor based on non-motor protein components. In this way, we can test our understanding of motor protein operation by including components that have well characterized functional properties. Our current design, the Tumbleweed, consists of a three-legged clocked-walker protein that operates on a repetitive DNA track. Tumbleweed uses three discrete ligand-dependent DNA-binding domains (repressor proteins) to perform cyclical ligand-gated rectified diffusion along a synthetic DNA molecule. We have used a modular combination of molecular biology and synthetic biology to express and assemble the Tumbleweed motor where three different DNA-binding proteins are linked to an assembly hub via coiled-coil arms. The SpyCatcher-SpyTag system was used to create the covalently linked Tumbleweed motor. We are currently assaying Tumbleweed for motion on a DNA track.

SYM-18-04 SYM-18-05 NANOSCALE IMAGING OF PROTEIN SECRETION THE NUCLEOSOME REMODELLING AND SYSTEMS USED BY BACTERIA AND THEIR VIRUSES DEACETYLASE (NURD) COMPLEX HAS AN ASYMMETRIC, DYNAMIC AND MODULAR Hay I.D. and Lithgow T. ARCHITECTURE Monash University, Infection & Immunity Program, Biomedicine Discovery Institute & Dept of Microbiology, Clayton, VIC, Australia. Silva A .P.G.1, Low J.K.K.1, Tabar M.S.1, Torrado M.1, Webb S.R.1, Parker B.L.1, Schmidberger J.W.1, Brillault L.2, Landsberg M.J.2 and Secretins form large (~150 Å), homo-oligomeric, gated pores in the Mackay J.P.1 outer membrane (OM) of bacteria. Each secretin complex comprises 1School of Life and Environmental Sciences, The University of 12-16 copies of the secretin protein. Indicative of the quintessential Sydney, NSW, Australia. 2School of Chemistry and Molecular nature of this family of proteins, secretins are ubiquitous among all Biosciences, The University of Queensland, QLD, Australia. didermic bacterial phyla and also found in some phage genomes. They form the OM component of the Type Four Pili (T4P), the Type Two The NuRD complex is essential for normal development and regulates Secretion System (T2SS), the Type Three Secretion System (T3SS/ both gene transcription and DNA damage repair. We have used Injectisome) and are also required for the assembly and export of structural, biophysical and biochemical data to define the architecture filamentous phage (Inoviridae). The T2SS is unique in that it exports of the native mammalian complex. We showed that the complex a range of soluble folded exoproteins directly from the periplasm, displays considerable dynamics and we identified stable subcomplexes commonly hydrolytic enzymes used to degrade biopolymers (proteins, within NuRD, showing that the full complex is composed of two parts carbohydrates, lipids) for nutrient acquisition, but also far more with separable enzymatic activities. A pseudo-symmetric deacetylase nefarious virulence factors such as the Cholera toxin, ETEC Heat-labile module comprising MTA, HDAC and RBBP subunits; whereas MBD, enterotoxin, or Pseudomonas Exotoxin A. Previously referred to as the GATAD2 and CHD subunits form an asymmetric 1:1:1 arrangement terminal component of the general secretion system, it is now apparent with remodelling activity. The previously enigmatic GATAD2 both that the T2SS is a more specialised secretion machine which must controls the asymmetry in the complex and recruits the ATP-dependent selectively recruit pre-exoproteins from the densely packed periplasm. CHD remodeller. Taken together, our data define the architecture of the Here we present our recent advances in characterising bacterial T2SS intact NuRD complex, revealing its structural dynamics and functional secretins. The distribution and classification of these proteins will plasticity. be discussed and our recent structural insights into the function and assembly of these complex molecules will be provided.

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SYM-19-01 SYM-19-02 DETECTION OF FLUORESCENTLY LABELED CHLOROPLAST VOLUME IS UNDERESTIMATED FROM PROTEINS BY ELECTRON MICROSCOPY TWO-DIMENSIONAL CROSS SECTIONS

Ariotti N.1, Rae J.2, Ferguson C.2, Hall T.E.2 and Parton R.G.2 Harwood R.H. and Barbour M.B. 1Electron Microscope Unit, The University of New South University of Sydney, The Center For Carbon Water and Food, 380 Wales. 2Institute for Molecular Bioscience, The University of Werombi Rd, Brownlow Hill NSW 2570. Queensland. Cell ultrastructure is predominantly studied by transmission electron Localising the position of proteins in the cell at high-resolution is critical microscopy (TEM), providing the user detailed two-dimensional (2D) for determining cellular function. As GFP revolutionized the detection information. Recent advances in microscopy have streamlined the of proteins by light microscopy, similarly new genetic tags for electron acquisition of three-dimensional (3D) images. Scanning electron microscopy (EM) have great potential for EM visualisation of proteins in microscopy with an automated microtome (SBFSEM) produces serial cells. We have developed a modular system for enzyme-based protein micrographs that can be stacked and segmented to produce a three- tagging that facilitates the detection of fluorescently-tagged proteins in dimensional volume data set. We are using SBFSEM on leaf cells to the electron microscope by employing a modified soybean ascorbate explore organelle size, shape and position, along with cell density and peroxidase system termed APEX. This system allows for efficient packing. The 3D anatomical data produced is being used to explore analysis of subcellular protein distributions using existing GFP- and the relationship between leaf form and function. Models of key leaf mCherry-tagged protein libraries. We demonstrate we can target APEX processes, such as photosynthesis, sit at the heart of crop productivity to any GFP- or mCherry-tagged protein of interest by engineering and and climate change models but include significant assumptions genetically encoding a specific nanobody/binding peptide (BP) fused regarding the structure of leaves that ignore 3D complexity. SBFSEM directly to the APEX-tag. We show that this system is robust, rapid allows us to challenge the idea of “textbook” leaf cell. To date we and can be used in animal models. Moreover, this method compatible have focused on wheat and found that chloroplast volume estimates with correlative light and electron microscopy and can be coupled with from 2D cross sections underestimated volume by 54% in mesophyll sensitive methods for detecting protein-protein interactions through the cells and 44% in bundle sheath cells. Chloroplast in meosphyll cells use of split-GFP. The application of this method to a number of cell were 35% bigger then those in bundle sheath cells when measured biological questions will be addressed in the talk. in 3D, when estimated from 2D cross sections the difference was only 20%. Size, shape and relative distance of cells and organelles are critical in addressing leaf form and function, our research shows over simplification from 2D quantification limits our understanding of leaf function. Using volume microscopy, we can demonstrate uniform geometry assumptions can no longer be made in plant physiology.

SYM-19-03 SYM-19-04 MODULATION OF ROK MEMBRANE DISSOCIATION ADAPTIVE OPTICS AND ACTIVE PSF SHAPING RATE TRIGGERS ROK PLANAR POLARISATION ENABLE SUPER RESOLUTION FLUORESCENCE DURING MORPHOGENESIS MICROSCOPY IN TISSUES 1, 9 2, 3 4 1 1 1 2 2 Mlodzianoski M.J. , Cheng-Hathaway P.J. , Bemiller S.M. , Sidor C.M. , Stevens T.J. , Boulanger J. , Bailey M.J. , Prehoda K.E. , 4 1 1 4, 5, 6 2, 3, 4 3 1 McCray T.J. , Liu S. , Miller D.A. , Lamb B.T. , Landreth G.E. Harris T.J. and Roeper K. 1, 7, 8 1 2 and Huang F. MRC laboratory of Molecular Biology, Cambridge, UK. Department 1Weldon School of Biomedical Engineering, Purdue University, West of Chemistry and Biochemistry at the University of Oregon, Lafayette, Indiana, USA. 2Department of Anatomy and Cell Biology, Indiana USA. 3Department of Cell & Systems Biology, University of Toronto, University School of Medicine, Indianapolis, Indiana, USA. 3Department Canada. of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA. 4Stark Neurosciences Research Institute, Indiana The MyosinII activator Rok is involved in a variety of morphogenetic University School of Medicine, Indianapolis, Indiana, USA. 5Department of processes during embryonic development. We have shown previously Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA. 6Department of Neurosciences, Cleveland in the Drosophila embryonic salivary gland placode that Rok is planar 7 polarised at the tissue boundary through a negative regulation by the Clinic Lerner Research Institute, Cleveland, Ohio, USA. Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West apical polarity proteins Crumbs (Crb) and aPKC. Intriguingly, despite 8 Crb, aPKC and Rok being expressed in the whole tissue, this effect Lafayette, Indiana, USA. Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, Indiana, USA. 9current address: The Walter is specific to the boundary of the tissue. Using FRAP in embryos and Eliza Hall Institute, Melbourne, VIC, Australia. expressing endogenously tagged Rok, we find that Rok membrane dissociation rate is lower at the boundary of the tissue, where Crb and Single molecule localization requires accurate and precise localization of the aPKC membrane levels are lower. Moreover, aPKC can phosphorylate three-dimensional positions of single molecule point spread functions (PSFs) the Rok membrane association region in vitro, suggesting Rok to reconstruct the 3D volume of a structure with high fidelity. Depth and phosphorylation by aPKC might be responsible for the difference in sample induced optical aberrations make this task challenging when imaging Rok membrane dissociation rate. Finally, computer simulations show more than just a few microns beyond the coverslip surface. These aberrations that such differences in Rok membrane dissociation rate are sufficient distort the PSFs of single molecules resulting in significant worsening of the to explain Rok planar polarisation at the tissue boundary. localization precision, and therefore the resolution, while also introducing spatial localization biases. Optical aberrations can be compensated for using adaptive optics approaches, often with a deformable mirror, to restore high quality PSFs. We present here an efficient sensor-less adaptive optics approach using a deformable mirror for removal of aberrations for robust, 3D single molecule localization imaging. This method utilizes single molecule data as the base for the Nelder-Mead simplex algorithm to optimize the shape of the deformable mirror for removal of optical aberrations. We control the deformable mirror to include astigmatism for 3D localization information and adaptively control the magnitude of astigmatism to enforce a consistent, astigmatic PSF shape for a nearly uniform localization precision throughout the sample depth. We demonstrate this development by imaging through 30-μm thick brain tissue sections in order to visualize and reconstruct the 3D morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.

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SYM-19-05 SYM-20-01 MICROSTRUCTURE IMAGING FROM MACRO GROUPER IRIDOVIRUS MEDIATED INHIBITION OF RESOLUTION MRI APOPTOSIS

Bourne R.1 and Panagiotaki E.2 Banjara S.1, Mao J.1, Ryan T.M.2, Caria S.1 and Kvansakul M.1 1The University of Sydney. 2University College London. 1Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Magnetic resonance imaging is increasingly being used to non- Australia. 2SAXS/WAXS, Australian Synchrotron, 800 Blackburn invasively detect diseases that have previously been characterised Road, Clayton, VIC 3168, Australia. by biopsy and histopathology. However, the way underlying tissue microstructure affects the relatively low spatial resolution MRI signal Programmed cell death or apoptosis is a critical mechanism for the is generally poorly understood. Further, many so-called ‘advanced’ controlled removal of damaged or infected cells, and proteins of the imaging techniques are in fact extremely primitive and fail to exploit the Bcl-2 family are important arbiters of this process. Viruses have been potential of modern MRI technology. This presentation looks at two new shown to encode for functional and structural homologs of Bcl-2 to approaches that enable the estimation of specific tissue microstructure counter premature host cell apoptosis to ensure viral proliferation and/ changes from sophisticated image acquisition protocols and advanced or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to data modeling. the iridoviridae family that harbors GIV66, a putative Bcl-2 like protein. GIV66 is a mitochondrially localized inhibitor of apoptosis, however the molecular and structural basis of apoptosis inhibition is currently not understood. To gain insight into the mechanism of action we systematically evaluated the ability of GIV66 to bind peptides spanning the BH3 domain of pro-apoptotic Bcl-2 family members. Our data reveal that GIV66 harbors an unusually high level of specificity for pro- apoptotic Bcl-2, and only displays affinity for Bim. We then determined crystal structures of both GIV66 on its own as well as bound to Bim BH3. Unexpectedly, GIV66 forms dimers via an interface that results in occluded access to the canonical Bcl-2 ligand binding groove, which breaks apart upon Bim binding. These data suggest that GIV66 dimerization may impacts on the ability of GIV66 to bind host pro-death Bcl-2 protein. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single specificity pro-survival Bcl-2 protein, and identifying a pivotal role of Bim for GIV mediated inhibition of apoptosis.

SYM-20-02 SYM-20-03 ROLE OF DYNAMIC IN THE SUPER RESOLUTION: A CLOSER LOOK AT MECHANISM OF THE PLASMODIUM FALCIPARUM M17 THE PLASMODIUM FALCIPARUM VIRULENCE AMINOPEPTIDASE COMPLEX

Drinkwater N.1, Yang W.1, Riley B.T. 2, Malcolm T.R.1, Buckle A.M.2 Looker O., Blanch A., McMillan P., Liu B., Dixon M. and Tilley L. and McGowan S.1 Department of Biochemistry & Molecular Biology, The University of 1Biomedicine Discovery Institute, Department of Microbiology, Monash Melbourne, Bio21 Molecular Science and Biotechnology Institute, University, Clayton Melbourne, VIC 3800, Australia. 2Biomedicine Victoria 3010. Discovery Institute, Department of Biochemistry, Monash University, Clayton Melbourne, VIC 3800, Australia. After invading the human red blood cell (RBC), Plasmodium falciparum modifies the host cell surface by exporting proteins to the The family of hexameric M17 aminopeptidases are conserved RBC periphery. The physical properties of the RBC are altered and throughout all kingdoms of life, and are exciting drug targets for novel parasite derived structures called knobs arrive at the cell periphery antimalarial and antibacterial agents. The M17 from P. falciparum (Pf- where they are anchored to the RBC membrane skeleton. These M17) is crucial to parasite survival, and a validated antimalarial drug knobs are comprised mainly of the Knob-Associated Histidine Rich target. We were interested to probe the role the conserved hexameric Protein (KAHRP), which acts as a scaffold for the presentation of the assembly plays in the function of Pf-M17, and to investigate the inherent major virulence protein, P. falciparumErythrocyte Membrane Protein dynamics of Pf-M17 and how they contribute to function. Towards this 1 (PfEMP1), through the membrane. A method has been developed end, we undertook a comprehensive strategy composed of molecular for exposing the inner-surface of the infected RBC membrane dynamics simulations, X-ray crystallography, and mutational analyses allowing for the organisation of the RBC membrane skeleton to be to characterise the range of protein motions that Pf-M17 undergoes, visualised by scanning electron microscopy and for protein location and to probe the specific contribution of these motions to enzyme to be examined using super resolution localisation microscopy. This function. Based on these results, we propose a novel model for how has allowed us to visualise knob assembly at the RBC membrane Pf-M17 functions on an atomic level, whereby the two trimers within the skeleton. In a major advance, we have combined these two imaging hexamer operate in a mutually exclusive manner, and rely on a dynamic modalities in a CLEM (correlative light and electron microscopy) based re-arrangement of bound metal ions and flexibility of a key regulatory approach to investigate membrane remodelling and virulence complex loop. The regulatory loop possesses different characteristics in M17 assembly. The development of CLEM techniques is allowing further aminopeptidases from other organisms, suggesting that the loop has investigation into how RBC membrane skeleton remodelling facilitates undergone divergent evolution, and further, might be crucial to the knob formation and how PfEMP1 arrives at the knobs to drive parasite emergence of new functions within this large and important enzyme virulence. Through combining multiple imaging modalities with cellular family. biology and biophysical measurements we aim to understand the remodelling events that underpin parasite virulence.

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SYM-20-04 SYM-20-05 FINE MAPPING OF ADULT PLANT LEAF RUST MECHANISMS OF LIGAND SENSING IN THE RESISTANCE GENE Lr49 IN WHEAT NEWLY CHARACTERIZED, DOMINANT FAMILY OF PROKARYOTIC RECEPTORS Baranwal D., Bariana H. and Bansal U. The University of Sydney Plant Breeding Institute, School of Life Roujeinikova A. and Environmental Sciences, Faculty of Science 107 Cobbitty Road Monash University, Melbourne, VIC 3800, Australia. Cobbitty NSW 2570.*Corresponding author: deepak.baranwal@ sydney.edu.au Chemotaxis, mediated by membrane-embedded chemotaxis receptors, plays an important role in bacterial ecology and Sustainable wheat production is continuously affected by several pathogenesis. We investigate the structural basis of how the dominant biotic and abiotic stresses. Among biotic stresses, leaf rust caused family of bacterial chemoreceptors with an extracytoplasmic double by Puccinia triticina is considered as a major threat to sustain wheat Cache sensing domain (dCache SD) recognize chemical cues, production across the globe. Adult plant hypersensitive leaf rust and how they discriminate between attractants and repellents. We resistance (R) genes such as Lr12, Lr22a, Lr22b, Lr35 and Lr49 have have determined the first representative crystal structures of the been characterised by necrotic flecks or small pustules which check the characterised dCache SDs of chemoreceptors from a broad range further proliferation of causing pathogen. Later, Lr49 was characterised of bacteria of medical and biotechnological importance, including as slow rusting resistance gene and is effective against all pre- carcinogenic bacterium Helicobacter pyloriand important human and dominating pathotypes of Australia and India. SNP were identified by animal pathogen Campylobacter jejuni. Analysis of these structures, aligning the sequences of flow sorted 4B of parental lines in conjunction with mutagenesis, biophysical and molecular simulation (VL404-Lr49 and WL711-susceptible parent) and converted into KASP studies, provided an insight into diverse mechanisms of ligand markers. KASP markers sunKASP_21 and sunKASP_24, flanked recognition by this protein fold. In all previously characterised dCache Lr49 proximally and distally, respectively. Current study was planned SDs, direct sensing involved binding of the signal molecule to the to develop high-resolution map of Lr49 using 5120 F2 gametes from a membrane-distal, rather than membrane-proximal, subdomain. I will cross of VL404/Avocet S to narrow down the physical interval between present data that changes this paradigm and reveal the first example of gene of interest and flanking markers to facilitate map-based cloning. a chemoreceptor that directly recognises its ligand via the membrane- DNA was extracted from leaf tissues from 2560 F2 plants using SDS- proximal subdomain, helping H. pylori to seek out lactate. In addition, extraction method. KASP markers were tested of F2 DNA using real I will present the results of our systematic study of the attractant- time PCR and LGC genomics protocol. Seventy-two recombinants bound and repellent bound structures and discuss implications for were identified between flanking markers. These recombinants the mechanism of discrimination between atrractants and repellents. were screened against PBI leaf rust culture 539 (76-1, 3, (5), 10, 12) I will then describe examples of very specific Pseudomonas ( to confirm their disease response in F3 generation. Ref seq v 1.0 of fluorescens CtaB) versus rather promiscuous (P. fluorescens CtaA, C. Chinese Spring was used to map the flanking markers and these jejuni Tlp3) chemoreceptors with the same overall fold and discuss markers mapped in scaffold3450 representing 4Mb region. Markers the structural basis behind this phenomenon. Finally, I will present an will be developed to saturate the region. example of a dCache chemoreceptor (C. jejuni Tlp1) that recognises its signal molecule indirectly.

SYM-21-01 SYM-22-01 WRITING A TEACHING GRANT IMPROVING CRISPR-CAS9 MEDIATED GENE EDITING IN PLANTS Rowland S.1 and Kuit T.2 1The University of Queensland. 2The University of Wollongong. Naim F.1, Shand K.1, Roden S.1, Hayashi S.1, O’Brien M.2, Johnson A.2, Dugdale B.1 and Waterhouse P.1 In this interactive session Tracey Kuit and Susan Rowland will work with 1Centre for Tropical Crops and Biocommodities, Queensland you to develop ideas for a teaching grant, pointing out the tips, tricks, University of Technology, Brisbane, Australia. 2School of BioSciences, and simple fixes you can use to increase your chance of success. The The University of Melbourne, Melbourne, Australia. presenters are experienced writers and assessors of teaching grants. Bring your ideas!. CRISPR-Cas9 driven gene editing of crops is rapidly advancing agricultural biotechnology. The process relies on DNA double stranded breaks at user defined genomic loci and repair by non-homologous end joining and/or homologous recombination. However, there are limitations in achieving precise and efficient editing of genes. These limitations include optimum design rules for gRNAs and delivery of Cas9 editing cassette. There are many gRNA design rules and online algorithms available to design gRNAs targeting genes in humans. We used a number of these tools to design and test the efficiency of gRNAs in Nicotiana benthamiana, banana, rice and tomato. Our results showed that there is no consensus between predictions by these algorithms and efficiency of obtaining mutations in plants. We discovered that targeting a single gene with two gRNAs increased the frequency of gene edits. To further improve editing, we also explored delivery of Cas9 plasmid using various DNA viruses. Here we report our exploration of the techniques and progress in efficiently editing plant genes.

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SYM-22-02 SYM-22-03 REVEALING THE ROLE OF SEPALLATA-LIKE CRISPR/CAS9-MEDIATED DISRUPTION OF THE GENES IN DETERMINING CEREAL INFLORESCENCE RICE OSVIT1 AND OSVIT2 GENES TO IMPROVE ARCHITECTURE USING GENOME EDITING APPROACH GRAIN IRON DENSITY

Li G.1, Kuijer H.1, Wu D.2, Zhu W.2, Liang W.2, Burton R.1, Dreni L.2 O’Brien M.1, Kielnhofer E.O.F.1, Eftekhari F.N.2 and Johnson A.A.T.1 and Zhang D.1 1School of Biosciences, The University of Melbourne, VIC 3010, 1School of Agriculture, Food and Wine, University of Adelaide. 2School Australia. 2Centre for Tropical Crops and Biocommodities, of Life Sciences and Biotechnology, Shanghai Jiao Tong University. Queensland University of Technology, QLD 4001, Australia.

Over 50% of the global populations calories are obtained from Rice provides 3.5 billion humans with more than 20% of their daily cereal grains produced from spikelets on a flowering structure called caloric intake. The commonly consumed white rice of modern rice an inflorescence. Improvements in crop yield could be made by cultivars is inherently poor in iron (Fe). Human Fe deficiency can cause increasing spikelet number. To achieve this goal, we need to gain a mild to severe anaemia and is the most common nutritional disorder deeper understanding of the mechanisms underpinning inflorescence worldwide, estimated to af fect up to t wo billion people. It is most common architecture. The shape of an inflorescence varies between cereals, in developing countries where people depend upon micronutrient- ranging from highly branched in rice (Oryza sativa) termed a panicle, poor staple crops such as rice. This has prompted the development to a much more compact spike in barley (Hordeum vulgare) and wheat of biofortified rice varieties with enhanced grain Fe density as a low- (Triticum spp.). One SEPALLATA (SEP) MADS domain transcription cost and sustainable strategy to tackle global human Fe deficiency. factor gene OsMADS34 has recently been identified that control The rice VACUOLAR IRON TRANSPORTER 1 and 2 genes (OsVIT1/ inflorescence architecture and seed number in rice. To further OsVIT2) are ubiquitously expressed at low levels in all tissues except understand the role of SEP genes, i.e. OsMADS1, OsMADS5 and the flag leaf where they are predominantly expressed. The OsVIT1 OsMADS34 in the LOFSEP clade from rice and their counterparts in and 2 proteins are Fe transporters that are localised to the vacuole barley in regulating the inflorescence development, we are using gene- where they function to sequester Fe in the flag leaf. Disruption editing approaches in creating single mutants and high-order mutants of OsVIT1 or OsVIT2 gene through T-DNA insertion lead to an of these genes in rice and barley respectively. Our preliminary data increased in Fe concentrations in all tissues, including the grain1. In this revealed that rice LOFSEPs play collaborative role in determining rice study, we have used Agrobacterium-mediated transformation of rice panicle branching, while barley homologs of rice OsMADS1, OsMADS5 with CRISPR/Cas9 plasmids targeting the OsVIT1 and OsVIT2 genes and OsMADS34 showed conserved and divergent function in specifying to generate a collection of osvit1 and osvit2 mutants. Eleven and fifteen barley inflorescence development. independent mutant alleles were generated for OsVIT1 and OsVIT2, respectively. We present molecular and phenotypic analyses of homozygous osvit1 and osvit2 mutants, including an analysis of grain Fe concentration. 1 Zhang et al, 2012. Plant Journal 72, 400-410.

SYM-22-04 SYM-22-05 STREAMLINING CRISPR DELIVERY TO PLANTS CREATING SYNTHETIC GENE REGULATORY CIRCUITS IN PLANTS Jones B., Shueh S.Y., Soni K. and Lyu W. University of Sydney, School of Life and Environmental Sciences. Khan A., Kidd B. and Lister R. ARC Centre of Excellence in Plant Energy Biology, The University of Developments in gene editing technologies have the potential to Western Australia. transform the way we research and improve our production plant species. The recalcitrance of crop species and elite cultivars to transformation Plants have an innate ability to respond to the environment to efficiently and regeneration from explants and tissue cultured cells, however, allocate resources and regulate their development in order to survive. continues to pose a significant bottleneck in the implementation of To produce economically valuable chemicals and metabolites in plants gene editing technologies in agriculturally important species. We are and gain control of plant yield and development, new genetic tools and exploring ways of delivering CRISPR/Cas technologies to crop and regulatory switches are required that allow sophisticated and precise model plant species in order to overcome these persistent difficulties. control of plant gene expression, without impacting the endogenous genetic regulatory system and its ability to respond to stress. Existing genetic switches typically produce a control function in one direction (either on or off, but not both), and therefore there is a need for more sophisticated switches that can integrate signals from multiple inputs and enact a logic function to produce a desired outcome. Here we report our progress in constructing modular synthetic biology components for targeted control of plant gene expression with the aim of constructing orthogonal regulatory circuits that enact Boolean based logic in plants.

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SYM-23-01 SYM-23-02 A MOLECULAR DISSECTION OF NEURAL INDUCTION 14-3-3ZETA MODULATES NON CANONICAL SHH SIGNALLING TO CONTROL CORTICAL INTERNEURON Trevers K., Lu H.C., Anderson C., Strobl A.C., Palinkasova B., DEVELOPMENT Filipkova L., Perez-Campos L., Moncaut N., de Almeida I.M. and Stern C.D. Greenberg Z., Ramshaw H., Xu X. and Schwarz Q. Dept Cell & Developmental Biology, University College London, Centre for Cancer Biology, University of South Australia. United Kingdom. Dysfunction in the formation and function of GABAergic cortical Neural induction is the process, during normal development, when the interneurons has been implicated as a central pathogenic mechanism in future neural plate becomes specified and set aside from the rest of schizophrenia. 14-3-3ζ is part of a family of highly conserved intracellular the ectoderm, under the influence of signals emanating from a special proteins, that bind to the phosphoserine/theronine sites on target region of the embryo, the “organizer”. Neural induction is generally proteins and is highly expressed in the brain. Several findings in recent viewed as a switch, assuming a single event. To explore the nature of years implicate 14-3-3ζ as a candidate risk factor for schizophrenia such a developmental switch, we have undertaken a detailed spatio- including: 1) 14-3-3ζ is downregulated in post-mortem schizophrenic temporal analysis of the process at the molecular level. We establish brain samples at the mRNA level; 2) 14-3-3ζ is downregulated across that neural induction occurs over many hours and involves a hierarchical multiple neuroproteomic studies on schizophrenia patient samples; 3) cascade of more than 200 transcription factors. Their expression is linkage studies have implicated 14-3-3 family proteins in numerous regulated by cooperation between several signalling pathways acting neurodevelopmental disorders, and 4) genetic mutations in the gene both sequentially and in parallel. We also uncover the epigenetic encoding 14-3-3ζ have been found in schizophrenia patients. Previous regulation of this cascade by mapping chromatin marks and revealing studies have shown that 14-3-3ζ KO mice exhibit anatomical and many regulatory elements associated with these transcription factors. behavioural traits akin to those seen in schizophrenia. Here we identify We generate a dynamic Gene Regulatory Network, representing the a novel role for 14-3-3ζ in interneuron development. We found a specific interactions underlying the neural induction process. Therefore the reduction in parvalbumin expressing interneurons throughout the cortex switch between neural and non-neural ectoderm involves a highly of 14-3-3ζ KO mice. Through a series of molecular, biochemical and complex network of events, regulated at many levels. This provides morphological studies we further show that parvalbumin interneuron support for Waddington’s view of an “epigenetic landscape”: successive deficiency arises from defects in the specification and formation of decisions made by cells over time, which gradually commit cells to their interneurons during early brain development. Mechanistically, we found ultimate fate. that 14-3-3ζ regulates non-canonical Shh signalling via controlling the activity of Rac1. Taken together, this work provides novel insight into the role of 14-3-3ζ in controlling interneuron development and identifies a novel role of 14-3-3ζ in the pathogenesis of schizophrenia.

SYM-23-03 SYM-23-04 GETTING CONNECTED: THE ROLE OF SEZ6 FAMILY AUTISM-LIKE SOCIAL INTERACTION DEFICITS PROTEINS IN EXCITATORY SYNAPSE DEVELOPMENT CAN BE PREVENTED IN MICE HAPLOINSUFFICIENT AND MAINTENANCE FOR THE SULFATE TRANSPORTER SLC13A4 BY POSTNATAL ADMINISTRATION OF Munro K.M.1, Nash A.N.1, Teng K.S.-L.1, Carrodus N.L.1, 2, Barwood N-ACETYLCYSTEINE J.M.1, 2, Fuller S.J.1, 2, Eroglu C.3, Takeshima H.4, Power J.5 1, 2 and Gunnersen J.M. Zhang Z.1, Dawson P.2, Simmons D.1 and 1 1 Piper M. Department of Anatomy and Neuroscience, The University of 1The School of Biomedical Sciences, The University of 2 Melbourne, Parkville, 3010, Victoria, Australia. The Florey Institute Queensland. 2Mater Research Institute, The University of Queensland. of Neuroscience and Mental Health, The University of Melbourne, 3 Parkville, 3010, Victoria, Australia. Duke University Medical Mounting evidence suggests that dysregulated sulfate metabolism is Center, Durham, NC 27710, USA. 4Graduate School and Faculty of 5 associated with autism spectrum disorder (ASD). Here we reveal that Pharmaceutical Sciences, Kyoto University, Japan. Translational the sulfate transporter SLC13A4 is a critical regulator of postnatal Neuroscience Facility and Department of Physiology, School of brain development, and that haploinsufficiency of Slc13a4 leads to Medical Sciences, UNSW Australia, Sydney, 2052, NSW, Australia. deficits linked to ASD, including impaired social behaviours and altered neurogenesis. Importantly, conditional deletion of Slc13a4 demonstrates The development of efficient synaptic connections and their refinement that the absence of this transporter during a critical postnatal period, and maintenance is vital for cognition. Altered expression and/or but not afterwards, gives rise to the behavioural deficits. Excitingly, function of proteins in the Seizure-related 6 (Sez6) family is associated N-acetylcysteine, a safe, FDA-approved, amino acid derivative that can with cognitive disorders and mice lacking Sez6 throughout development be metabolized to inorganic sulfate, rescues the ASD-like behavioural exhibit fewer neocortical excitatory synapses. Whether Sez6 proteins phenotypes when administered within this defined postnatal window. are required in the adult brain to maintain excitatory synaptic function, NAC may therefore have utility for the prevention of some forms of and the extent to which the three Sez6 family members functionally autism if administered in early life. compensate for each other, is not known. These questions are highly relevant to the clinical trials of β-site amyloid precursor protein cleavage enzyme 1 (BACE1) inhibitors for Alzheimer’s disease because Sez6 proteins are major BACE substrates and, therefore, likely to contribute to mechanism-based side effects when BACE1 is chronically inhibited. The results of behavioural, electrophysiological, morphological and biochemical analyses of Sez6 family triple knockout and Sez6 conditional knockout mice reveal that Sez6 proteins are important, not only for synapse development but also for maintaining excitatory synapse structure and function in the adult brain.

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SYM-24-01 SYM-24-02 VISUALIZING FUNCTIONAL ION CHANNELS AT THE HIGH THROUGHPUT PHENOTYPING OF HERG CELL SURFACE CHANNEL MUTATIONS

Irving H.R.1, 2, Abad I.2, Nguyen D.-T.2 and Manallack D.T.2 Ng C.A.1, 2, Perry M.D.1, 2 and Vandenberg J.I.1, 2 1La Trobe Institute for Molecular Science, La Trobe University Bendigo 1Victor Chang Cardiac Research Institute, 405 Liverpool St, VIC 3550. 2Monash Institute of Pharmaceutical Sciences, Monash Darlinghurst, NSW 2010. 2St Vincent’s Clinical School, UNSW University Parkville VIC 3052. Sydney, Darlinghurst, NSW 2010.

Assembly of components of ion channels into functional heteromers The expression of hERG potassium channels at the plasma membrane at the cell surface is still mysterious. To begin to obtain clues about of cardiac myocytes is critical for the coordinated propagation of the this process we use HTR3A and HTR3C receptor subunits that have electrical signals that regulate the rhythm of the heartbeat. Reduced each been tagged with separate fluorescent proteins in the second hERG function due to mutations increases the risk of sudden cardiac intracellular loop. The subunits are transiently expressed in HEK293T arrest and death. Mutations may affect synthesis, assembly, trafficking cells and visualised by high resolution microscopy. These subunits are and/or function of hERG channels. The majority of mutants affect processed normally through endoplasmic reticulum and Golgi pathways channel trafficking and traditionally this has been assessed using to reach the plasma membrane surface. Using TIRF microscopy we Western blot assays. Manual patch clamping is the gold standard for show that the subunit heteromers are formed intracellularly. Whole cell assessing the gating phenotype of hERG mutants and can also be used patch clamp reveals that the subunits are functional at the sur face where to assess current density. However, manual patch clamp assays are far they respond to 5-HT and this response is suppressed by ondansetron. too labour intensive for them to have clinical utility in assessing large Analysis of our TIRF images has allowed us to determine the proportion numbers of mutants. In the era of precision medicine, large numbers of of different heteromers within the cell and at the surface. This finding is hERG mutations with unknown significance are going to be identified. important as it will allow a more precise analysis of how different drugs Therefore, there is a need to develop higher throughput methods that contribute to subtle changes in heteromer versus homomer function are amenable to automation to substitute for the current Western blot that in turn may be related to individual receptor components. and manual patch clamp assays. In this study, wild-type and 25 clinical hERG mutations were expressed in HEK293 cells to quantify their expression and gating phenotypes using ELISA assay and automated patch clamp assays (Syncropatch-384), respectively. The expression levels determined using the ELISA assay versus traditional Western blot analysis had a correlation coefficient of 0.86. The expression levels determined by current density measurements using the syncropatch (more than 1000 successful recordings) showed good correlation with the ELISA assays (correlation coefficient, 0.85). Our results indicate that both ELISA and the syncropatch methods can be used to assess channel expression. These methods not only have a higher throughput than traditional methods but they can also be applied to any ion channel including channels where the lack of glycosylation precludes the use of simple Western blot analysis.

SYM-24-03 SYM-24-04 CONTROL OF CELL MIGRATION AND SHAPE BY NEW INSIGHTS INTO THE POST-TRANSLATIONAL DUAL WATER AND ION CONDUCTING AQUAPORIN REGULATION OF ABC LIPID TRANSPORTERS CHANNELS Aleidi S.M.1, Yang A.1, Alrosan A.1, Sharpe L.2, Brown Yool A.J., Pei J.V. and Kourghi M. A.J.2 and Gelissen I.C.1 University of Adelaide, SA 5005. 1School of Pharmacy, Faculty of Medicine and Health, University of Sydney. 2School of Biotechnology and Biomolecular Sciences, Structure-function analyses of human aquaporin-1 (AQP1) channels University of NSW, Sydney. are defining the gating mechanisms and permeation pathways of these intriguing dual water- and ion-conducting channels, and identifying new A number of ABC transporters, including ABCA1, ABCG1 and its close pharmacological agents that differentially regulate the parallel ion and relative ABCG4, are essential regulators of cellular lipid homeostasis. water pores. The AQP1 ion conductance is necessary for rapid migration ABCA1 and ABCG1 have been studied in the context of macrophage in subt y pes of aggres sive c anc er c ells. A photosw itc hable c at i on sensing lipid homeostasis and atherosclerosis, insulin secretion in β-cells as probe developed by our team allows real-time imaging to localise well as brain lipid homeostasis, with substrates including cholesterol AQP1-mediated cation entry in migrating cells. An arylsulfonamide and phospholipids. ABCG4 on the other hand is thought to transport agent AqB011 designed by our group selectively blocks the ion channel cholesterol, oxysterols and cholesterol synthesis intermediates of AQP1 (IC50 14 μM) without altering water permeability (at doses up exclusively in the brain, and has been linked to Alzheimer’s disease to 200 μM), providing a key tool for analysing the physiological roles of due to its potential to transport amyloid-β peptides from cells. These the AQP1 cation channel conductance in the ability of cells to move and transporters are thought to be highly regulated at the post-translational maintain proper shape, in processes important for development, repair level. Our group and collaborators have previously identified two and survival in multicellular organisms. separate avenues by which these transporters can be regulated i.e. via cellular cholesterol status as well as protein ubiquitination. The rate limiting step in protein ubiquitination is carried out by E3-ubiquitin that have become of interest as potential therapeutic targets in a number of disease settings. We have identified a number of E3- ligases that are involved in the regulation of ABCA1, ABCG1 and ABCG4 (Aleidi et al 2015 and 2018). Here we describe under which circumstances these ligases regulate ABC lipid transporter protein levels and activity, and propose how these pathways may be exploited in the disease context in future.

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SYM-24-05 SYM-25-01 ENHANCING KCC2 AS A NOVEL STRATEGY FOR UNDERSTANDING THE BIOSYNTHESIS OF THE TREATING SEIZURES GLYCOPEPTIDE ANTIBIOTICS

Goulton C.S., Cheung D.L., Prikas E. and Moorhouse A.J. Cryle M.J. Department of Physiology, School of Medical Sciences, UNSW Department of Biochemistry & Molecular Biology, Monash University, Sydney, Australia. Clayton, VIC 3800.

The K+Cl--cotransporter (KCC2) plays a key role in regulating The glycopeptide antibiotics (GPAs) are a structurally complex and intracellular Cl-, influencing the efficacy of GABAa-receptor mediated medically important class of peptide natural products that include the inhibition. To investigate how elevating KCC2 expression affects clinical antibiotics vancomycin and teicoplanin. They contain a large neuronal function, we used a transgenic mouse in which forebrain- number of non-proteinogenic amino acids and are produced by a linear restricted over-expression of KCC2 in pyramidal neurons could non-ribosomal peptide synthetase (NRPS) machinery comprising be regulated by doxycycline in the diet. In acute brain slices, field seven modules. Furthermore, GPAs are extensively crosslinked late potentials were evoked from hippocampal CA1. It was found that KCC2 in their biosynthesis on the NRPS assembly line by the actions of a upregulation did not influence normal excitability, with similar input- cascade of Cytochrome P450 enzymes, a process which contributes to output relationships, paired-pulse ratios, and concentration-response the rigidity and structural complexity of these compounds. Due to the to muscimol observed. Interestingly, hyperexcitability was significantly challenge of synthesising GPAs, biosynthesis remains the only means reduced, as measured using the tetanus-induced afterdischarge and of accessing GPAs for clinical use, which makes understanding the Zero-Mg2+ seizure models. This finding was subsequently supported in biosynthesis of GPAs of key importance. Here I will detail results from vivo, where seizures induced by kainic acid (up to 50mg/kg, i.p) were our studies into the NRPS machinery, the P450-cyclisation cascade less likely to progress to status epilepticus in KCC2 upregulated mice and the interplay of these two important processes during GPA (1/5 mice vs. 15/15 mice from control cohorts). A recent study found biosynthesis. These demonstrate how selectivity is mediated through that the clinically available compound prochlorperazine (Stemetil) the careful orchestration of critical modification steps and interactions acutely increases KCC2 expression (Liabeauf et al, 2017). To explore between the peptide-producing NRPS machinery and trans-modifying the potential effects of Stemetil on hyperexcitability, we conducted enzymes. preliminary experiments using the in vitroafterdischarge seizure model. It was found that a 60 min incubation with Stemetil (10μM) reduced afterdischarge bursts (p<0.01) with no effects on basal excitability. Overall, our data support the strategy of enhancing KCC2 to reduce neuronal hyperexcitability, without negatively affecting basal synaptic transmission. This means that increasing KCC2 may enable greater Cl- homeostasis and maintain more efficacious GABAergic inhibition, hence providing a novel strategy to enhance neuronal inhibition. Further, preliminary results support continued investigation into Stemetil as a therapeutically relevant KCC2 enhancer for the treatment of seizures.

SYM-25-02 SYM-25-03 PEPTIDE-INSPIRED INHIBITORS OF DNA-BASED INHIBITORS OF THE HUMAN APOBEC3B C-MANNOSYLTRANSFERASES DNA CYTOSINE DEAMINASE 1 1 1 1, 2 4 1, 2 Barzak F. , Kvach M.V. , Harjes S. , Jameson G.B. , Aihara H. , Goddard-Borger E.D. 4, 5 1, 2 3 1, 2 1 Harris R.S. , Filichev V.V. , Harki D.A. and Harjes E. The Walter & Eliza Hall Institute of Medical Research, Parkville, 1 2 Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Victoria, Australia. Department of Medical Biology, University of Palmerston North, New Zealand. 2Maurice Wilkins Centre for Molecular Melbourne, Parkville, Victoria, Australia. Biodiscovery, The University of Auckland, Private Bag 92019, New Zealand. 3Department of Medicinal Chemistry, University of Minnesota, Tryptophan mannosylation is an unusual co-translational modification Minneapolis, MN 55455, USA. 4Department of Biochemistry, Molecular Biology, that promotes protein folding and enhances the stability of some and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. 5Howard type-I cytokine receptors. This protein modification is installed by two Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA. integral membrane enzymes that are localised to the ER and encoded by dpy19l1 and dpy19l3. Perturbing tryptophan mannosylation The APOBEC3 (A3) protein subfamily of seven proteins (A3A-H) are cytidine dramatically reduces the cell surface expression of TPOR and deaminases that attack retroviruses and other pathogens by hypermutating cytidine residues of single-stranded DNA [1] while, at the same time some APOBEC3 family IL7R: cytokine receptors whose constitutive expression drives the members are involved in carcinogenesis [2]. In particular, one member of the family, proliferation of some blood cancers (e.g. MPNs and T-ALL). We have APOBEC3B promotes mutagenesis in several cancers and contributes to tumor developed small molecule inhibitors of C-mannosyltransferases to evolution including the detrimental outcomes of metastasis and drug resistance [3- determine if pharmacological inhibition of tryptophan mannosylation 7]. Inhibition of APOBEC3B may therefore be used to augment existing anticancer might be useful for treating some blood cancers. To accomplish this, we therapies [8]. We designed and tested chemically modified APOBEC3 DNA established in vitro C-mannosyltransferase activity assays and probed substrates for their inhibitory potential and obtained the first DNA-based inhibitors the enzyme’s peptide substrate preferences in detail. SAR studies then of APOBEC3 enzymes with low micromolar inhibition constants. We found that guided us in the conversion of a tetrapeptide substrate of the enzyme neighbouring nucleotides in the DNA sequence influence the specificity of our into a non-peptide inhibitor of tryptophan mannosylation with low inhibitor which, led to the development of the first inhibitor selectively targeting micromolar activity in cells. APOBEC3B. This provides a platform for further development of APOBEC3B inhibitors with cellular activity. REFERENCES 1. Harris, R.S. and J.P. Dudley, APOBECs and virus restriction. Virology, 2015. 479: p. 131-145. 2. Swanton, C., et al., APOBEC enzymes: mutagenic fuel for cancer evolution and heterogeneity. Cancer discovery, 2015. 5(7): p. 704-712. 3. Burns, M.B., et al., APOBEC3B is an enzymatic source of mutation in breast cancer. Nature, 2013. 494(7437): p. 366-370. 4. Burns, M.B., N.A. Temiz, and R.S. Harris, Evidence for APOBEC3B mutagenesis in multiple human cancers. Nature Genetics, 2013b. 45(9): p. 977- 983. 5. Law, E.K., et al., The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer. Science advances, 2016. 2(10): p. e1601737. 6. Sieuwerts, A.M., et al., Elevated APOBEC3B correlates with poor outcomes for estrogen-receptor-positive breast cancers. Hormones and Cancer, 2014. 5(6): p. 405-413. 7. Ding, Q., et al., APOBEC3G promotes liver metastasis in an orthotopic mouse model of colorectal cancer and predicts human hepatic metastasis. The Journal of clinical investigation, 2011. 121(11). 8. Olson, M.E., R.S. Harris, and D.A. Harki, APOBEC Enzymes as Targets for Virus and Cancer Therapy. Cell chemical biology, 2017.

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SYM-25-04 SYM-25-05 GENETIC INCORPORATION OF UNNATURAL AMINO STRUCTURAL VARIANTS OF A LIVER FLUKE ACIDS INTO SINGLE CHAIN VARIABLE FRAGMENTS DERIVED GRANULIN PEPTIDE POTENTLY STIMULATE FOR GENERATION OF STABLE -IMAGING WOUND HEALING PROBE CONJUGATES FOR CANCER IMAGING Dastpeyman M.1, Bansal P.S.1, Wilson D.1, Sotillo J.1, Brindley P.J.2, Subas Satish H.P.1, 2, 3, Howard C.B.3, Huda P.3, Fletcher N.3 and Loukas A.1, Smout M.J.1 and Daly N.L.1 Thurecht K.3 1James Cook University, Cairns Australia. 2George Washington 1Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC University, Washington USA. 3052. 2University of Melbourne, Parkville, VIC 3010. 3University of Queensland, St Lucia, QLD 4072. Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver The linker chemistry utilised for the generation of antibody-imaging dye, fluke Opisthorchis viverrini, can significantly accelerate wound repair nanomaterial or drug conjugates plays a crucial role in the development in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in of stable and clinically successful antibody-targeted diagnostics or recombinant form at high yield, impeding its utility as a drug lead. A therapeutics. The incorporation of an unnatural amino acid having truncated analogue (Ov-GRN12-35_3s) promotes healing of cutaneous orthogonal chemical reactivity into single chain variable fragments wounds in mice. NMR analysis of this analogue indicates the presence (scFv) in vivo provides a chemical handle for the generation of such of multiple conformations, most likely as a result of proline cis/trans stable antibody conjugates. An unnatural amino acid having an azide isomerisation. To further investigate whether the proline residues are functional group, 4-azido phenylalanine or p-azido phenylalanine involved in adopting the multiple confirmations we have synthesised (pAzF) was incorporated into anti-EGFR scFv expressed in Escherichia analogues involving mutation of the proline residues. We have shown coli. A stable scFv-imaging dye conjugate was generated via a strong that the proline residues have a significant influence on the structure, triazole linkage formed by strain-promoted copper-free click reaction activity and folding of Ov-GRN12-35_3s. These results provide insight between the azide functional group of the pAzF anti-EGFR scFv and into improving the oxidative folding yield and bioactivity of Ov-GRN12- DBCO functionalized fluorescent dyes, cyanine 5 or cyanine 7. The 35_3s, and might facilitate the development of a novel wound healing pAzF anti-EGFR scFv showed significant binding to recombinant agent. EGFR in vitro and also, through flow cytometry, the target specificity of the pAzF anti-EGFR scFv-DBCO cy5 conjugates to native EGFR expressing MDA MB 468 breast cancer cells was validated in vitro. In addition, in vivo optical imaging studies performed with MDA MB 468 tumour xenograft models showed targeted accumulation of pAzF anti- EGFR scFv-DBCO cy7 in MDA MB 468 cancer, thereby establishing both the targeting and imaging potential of the conjugates. This study therefore demonstrates the incorporation of an unnatural amino acid with orthogonal chemical reactivity site-specificially into an scFv as well as the potential of unnatural amino acids to form stable antibody- imaging dye conjugates for use in tumour imaging.

SYM-26-01 SYM-26-02 MECHANISMS CONTROLLING METABOLISM BY USING AN OMICS APPROACH TO IDENTIFY NOVEL REGULATING PROTEIN TURNOVER REGULATORS OF HEPATIC LIPID METABOLISM

Christiano R., Farese R.J. and Walther T.C. Drew B.G.1, Parker B.L.2, Seldin M.3, Keating M.F.1, Meikle P.J.1, Harvard University/Howard Hughes Medical Institute. Tarling E.J.3, Lusis A.J.3, James D.J.2, De Aguiar Vallim T.Q.3 and Calkin A.C.1 Many metabolic reactions are tightly controlled. One mechanism for 1Baker Heart and Diabetes Institute. 2Charles Perkins Centre, such control occurs through regulating the stability of key enzymes of University of Sydney. 3University of California, Los Angeles. metabolism. Such regulation is mediated by ubiquitin ligases, sorting factors, and proteases. To elucidate regulatory pathways, we globally Background: The liver controls numerous pathways central to the investigated protein turnover a large sets of mutants in the degradative maintenance of whole body lipid metabolism. Dysregulation of these machinery in S. cerevisiae. Analysis of the resulting turnover map pathways can result in increased levels of pathological lipids that can (T-MAP) revealed targets for most ubiquitin ligases and identified the promote insulin resistance, fatty liver disease and cardiovascular primary degradation routes for most short-lived proteins. Illustrating the disease. Thus, we need a greater understanding of the pathways power of this approach, we uncovered new insights into ERAD pathways regulating hepatic lipid metabolism. Methods: We utilised a trans- omics governing sterol synthesis and numerous previously unknown nodes of approach to analyse mouse livers across 100+ strains of genetically regulation for sphingolipid synthesis. Expansion of the T-MAP strategy diverse mice, integrating genetics, phenomics, and proteomics (>8000 in yeast and mammalian cells provides a powerful tool to unravel the proteins; Orbitrap Fusion) as well as liver and plasma lipidomics contributions of protein degradation to proteostasis. (>300 lipids; API4000 Q/TRAP) to identify novel lipid signatures and pathways associated with metabolic diseases. Results: This approach has generated a powerful platform that we can interrogate to perform protein:protein correlations, which can give us insight into protein complexes and protein localisation within the cell; protein:lipid correlations, which can provide insight into novel regulators of known pathological lipids; lipid:lipid correlations, which can provide an avenue to identify plasma lipids that might predict hepatic accumulation of known pathological lipids linked to metabolic disease. Findings are validated in vitro, in preclinical models and in clinical cohorts such as the UK Biobank and the San Antonio Family Heart Study. Conclusions: We have established a high-resolution trans-omics platform for the identification of novel regulators of hepatic lipid metabolism, with implications for therapeutic intervention of metabolic diseases.

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SYM-26-03 SYM-26-04 THE ROLE OF MITOCHONDRIAL THE E3 UBIQUITIN MARCH6 IS BOOSTED BY PHOSPHATIDYLETHANOLAMINE SYNTHESIS CHOLESTEROL IN REGULATING SKELETAL MUSCLE LIPID HOMEOSTASIS Sharpe L.J., Howe V. and Brown A.J. UNSW Sydney NSW 2052. Bruce C.R. Institute for Physical Activity and Nutrition (IPAN), School of Exercise The E3 ligase membrane-associated RING finger (C3HC4) 6 and Nutrition Sciences, Deakin University, Burwood, VIC 3125, (MARCH6) helps control protein levels of the two rate-limiting enzymes Australia. in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and (SM). However, Phosphatidylethanolamine (PE) is the second most abundant little is known about how MARCH6 itself is regulated. Considering phospholipid in mammals. PE is synthesized via two pathways, cytidine MARCH6’s role in controlling cholesterol synthesis, we hypothesised diphosphate (CDP)-ethanolamine pathway located in the endoplasmic that cholesterol may regulate MARCH6. Indeed, we found that reticulum and the phosphatidylserine decarboxylase (PSD) pathway in cholesterol stabilises MARCH6 protein. Preliminary evidence suggests the mitochondria. While the CDP-ethanolamine pathway is considered that Insigs also play a role in regulating MARCH6’s stability. Our the major route for PE production in most mammalian tissues, our ongoing investigations focus on the nature of the MARCH6-Insig recent observations suggest the mitochondrial PSD pathway could be interplay,and whether other intermediary proteins are involved. This an important site of PE synthesis in muscle (Selathurai et al., 2015). work provides new insights into the complex feedback mechanisms Here, the results from studies exploring the role of the mitochondrial underlying cholesterol homeostasis. PE synthetic pathway in regulating lipid homeostasis in skeletal muscle will be discussed and will reveal unique insight into the significance of mitochondrial phospholipid synthesis in skeletal muscle. Selathurai A et al. The CDP-Ethanolamine Pathway Regulates Skeletal Muscle Diacylglycerol Content and Mitochondrial Biogenesis without Altering Insulin Sensitivity. Cell Metab 21(5):718-30, 2015.

SYM-26-05 SYM-27-01 TACKLING LIPID DIVERSITY IN MEMBRANES: THE UNDERSTANDING SPATIAL AND TEMPORAL EFFECT ON MEMBRANE AND PROTEIN FUNCTIONS CONTROL OF GPCR SIGNALLING USING HIGH RESOLUTION IMAGING Poger D., Corbett M.S.P. and Mark A.E. School of Chemistry & Molecular Biosciences, The University of Halls M.L. Queensland, Brisbane QLD 4072, Australia. Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, VIC 3052, Australia. Biological membranes regulate a myriad of cellular processes through the modulation of essential properties such as membrane fluidity and Cells endogenously express many different receptors that can activate the formation of lipid microdomains. Such differences in turn affect the the same second messenger, but with remarkably diverse physiological function of membranes and membrane proteins. The chemical and outcomes. This suggests a high degree of organisation and regulation structural diversity of lipids is only being uncovered. For example, the of intracellular signalling, which is achieved by the spatiotemporal repertoire of lipids in bacterial membranes is much broader than in compartmentalisation of signals – the restriction of second messengers eukaryotic membranes. In many if not most bacteria, membrane lipids in space and time. The development of targeted Forster Resonance include branched-chain fatty acids. Hopanoids have been identified in Energy Transfer (FRET)-based biosensors has increased the a range of bacteria. Branched-chain fatty acids have been proposed to resolution at which we can measure the spatial and temporal signalling protect membranes against hostile conditions and hopanoids have long of GPCRs. Moreover, single molecule imaging techniques such as Total been hypothesised to be surrogates of sterols, but, in fact, little is known Internal Reflection Fluorescence (TIRF) and Fluorescence Correlation about their actual effect on membranes. Using atomistic simulations, Spectroscopy (FCS) now allow us to correlate subtle changes in I showed that the different types of branching and hopanoids have receptor organisation at the plasma membrane with large changes in specific effects on membrane fluidity and structure that allow bacteria spatiotemporal signalling. The complementary use of single cell and to finely tune the sensitivity of their membranes to the environment. molecule imaging has revealed that GPCRs can activate very defined Furthermore, branched-chain lipids could affect the activity of signals in limited sub-cellular compartments by assembling focused commonly used disinfectants such as triclosan and para-chloroxylenol protein complexes. These complexes facilitate second messenger on membranes by modulating the interaction of the biocides with lipids production, the organisation and scaffolding of effectors, and co- and how deep they could insert into a membrane. The membrane ordination of regulatory elements. composition also plays a critical role in the function of proteins. In simulations of the type-I cytokine receptors for growth hormone (GHR), prolactin (PRLR) and erythropoietin (EPOR) embedded in membranes, the presence of cholesterol altered the behaviour of the transmembrane domains, suggesting a key role of cholesterol in the mechanical coupling of the receptors through the plasma membrane upon receptor activation. The lipid composition is thus critical in the function of membrane and membrane proteins.

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SYM-27-02 SYM-27-03 PROTEIN DYNAMICS AND KINETICS OF GENOME- PROBING MECHANISMS FOR FORCE-SENSING WIDE OCCUPANCY OF THE SOX18 TRANSCRIPTION IN MECHANOSENSITIVE ION CHANNELS WITH FACTOR ARTIFICIAL DROPLET BILAYER SYSTEMS

McCann A.1, Lou J.2, Blum A.3, Moustaqil M.4, Fontaine F1, Sierecki Jaggers O.1, Ridone P.2, Xiao B.3, Martinac B.2 and Baker M.A.B.1 E.4, Gambin Y.4, Meunier F.3, Liu J.Z.5, Hinde E.2 and Francois M.1 1School of Biotechnology and Biomolecular Science, UNSW. 2Victor 1Institute for Molecular Bioscience, The University of Queensland. Chang Cardiac Research Institute. 3Tsinghua University. 2Bio21 Institute, The University of Melbourne. 3Queensland Brain Institute, The University of Queensland. 4European Molecular Biology Droplet Hydrogel Bilayers enable simultaneous single channel Laboratory, University of New South Wales. 5Janelia Farm, Howard current and fluorescence measurements[1]. They have been used to Hughes Medical Institute. characterise the functionality of alpha-haemolysin for use in nucleobase recognition in DNA sequencing and they have been arranged in Cell fate determination relies on the ability of transcription factors (TFs) multiple arrays to parallelise high throughput channel measurements. to select protein partners and specific regulatory elements to instruct We recently used this platform to apply force and measure the a particular transcriptional output. Central to endothelial cell fate response of bacterial mechanosensitive ion channel MscL[2]. We are acquisition during embryonic development, the SOX18 transcription now using this platform to investigate the force sensitive ion channel factor is a key regulator of both blood vascular and lymphatic endothelial PIEZO1[3], in which single point mutations cause blood disorders such cell specification. This TF is transiently expressed in all vascular beds as xerocytosis and which is generally linked to cancer progression during embryogenesis, however, the mechanisms that drive its specific and post traumatic osteoarthritis. We observe that PIEZO1 in DHBs molecular mode of action to instruct the differentiation of distinct sub- displays the same gating activity in DHBs as in patch-liposomes, and, population of endothelial cells are currently unknown. To identify a similarly, is much more sensitive in the presence of cholesterol. We also global mechanism that could drive a differential activity of this TF in investigate the use of DNA-origami to regulate lipid-lipid interactions. discrete cell subtypes, we analysed chromatin occupancy dynamics of This serves to enable light-triggered insertion of membrane proteins SOX18 by combining Halo-tag technology with single molecule tracking into artificial bilayers and allows greater spatiotemporal control. [1.] A. in vitro. This approach identified that SOX18 binds to the chromatin via J. Heron, et al., JACS. 131, 1652-1653 (2009). [2.] K. Rosholm et al., a two-component model; which means that SOX18 binds transiently to Scientific Reports. 7, 45180 (2017). [3.] Q Zhao et al., Nature, 554, 487- several non-specific sites, before binding more stably to specific target 492 (2018). sites. Lastly, taking advantage of a non-functional SOX18 dominant- negative protein that causes the human syndrome Hypotrichosis- Lymphedema-Telangiectasia (HLT), we show that this non-functional TF interferes with the search pattern of SOX18 to identify its target genes. Our study shed light onto molecular behaviours of SOX18 TF on a genome-wide scale, and reinforces the concept that protein-protein interactions are central to govern target gene selectivity. We also uncover a global mechanism that explains at a molecular level how a dominant-negative protein disrupts TF activity and drives the aetiology a rare human vascular disease.

SYM-27-04 SYM-27-05 METAL STARVATION TRIGGERS CETZ1-DEPENDENT SCF-BTRCP MEDIATES THE DEGRADATION OF CELL SHAPE CHANGES IN HALOARCHAEA CEP68 INTERCENTROSOMAL LINKER PROTEIN TO CONTROL THE DISASSEMBLY OF THE De Silva R.T., Ithurbide S. and Duggin I.G. PERICENTRIOLAR MATERIAL IN MITOSIS University of Technology Sydney. Pagan J.1, Jones M.2 and Pagano M.3 Microbial cell shape is a significant attribute that affects survival, and 1University of Queensland, SBMS. 2Memorial Sloan Kettering Cancer many species can change morphology to adapt to environmental Center. 3New York University Medical Center. change and stress. Model haloarchaeon Haloferax volcanii cells transition from plates to rods to optimise swimming motility, which Skp1-Cul1-F-box protein (SCF) complexes are the best-characterised requires the tubulin-like cytoskeletal protein CetZ1. The signals that of the multisubunit E3 Ubiquitin ligases. Each SCF complex contains trigger cell shape change via CetZ1 are not yet clear. We analysed one of 69 exchangeable substrate-targeting subunits, called F-box cell shape changes in various nutrient-depleted media, and found proteins. Core SCF components, SKP1 and CUL1, localise to mitotic that cells developed highly irregular elongated forms in response to and interphase centrosome suggesting that SCF ligases mediate the depletion of metal nutrients. These shapes were substantially more turnover of centrosomal substrates. Indeed, several specific F-box diverse compared to the regular rod-shaped cells seen in motile cells. proteins have been recently shown to regulate the stability of key Remarkably, the addition of a solution containing 8 metals to complex centrosome proteins, including PLK4 kinase (by SCF-bTrCP), the growth medium (HvYPC) significantly improved culture growth, and the master regulator of centriole duplication. We demonstrate that SCF- cells showed a uniform plate-shaped morphology compared to non- bTrCP also mediates the degradation of Cep68 intercentrosomal supplemented HvYPC that produces mixed elongated- and plate-cell linker protein in mitosis. Cep68 degradation is initiated by PLK1 types. The formation of elongated cells during trace element limitation phosphorylation of Cep68 on Ser 332, allowing recognition by bTrCP. was dependent on CetZ1, and the cell elongation defect of an in-frame The removal of Cep68 mediated by PLK1 and bTrCP is the first in knock-out of cetZ1 was rescued by expression of CetZ1 from a plasmid. a series of relocalization and degradation mechanisms in mitosis Towards the goal of understanding how CetZ1 functions to control cell required to reset the centriole to a duplication competent configuration. shape, we have adapted a set of modern fluorescent proteins for use in H. volcanii. A CetZ1-mTurquoise2 fusion showed cell elongation capacity and revealed a highly dynamic localisation pattern associated with cell elongation during metal depletion, indicating that a dynamic remodelling of the cell envelope occurs during cell elongation. The improved tools and growth conditions can be utilized for working with H. volcanii in research and biotechnology.

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SYM-28-01 SYM-28-02 NON-CANONICAL TGF-β/SMAD SIGNALLING UNCOUPLING PKR ACTIVATION AND ENHANCES CELL-TO-CELL SPREAD DURING TRANSLATIONAL ARREST DURING NOROVIRUS VACCINIA VIRUS INFECTION INFECTION

Gowripalan A., McKenzie C.D. and Newsome T.P. Fritzlar S., Chao Y.W., Aktepe T.E. and Mackenzie J.M. School of Life and Environmental Sciences, University of Sydney, Department of Microbiology and Immunology, Peter Doherty Institute NSW, Australia, 2006. for Infection and Immunity, at the University of Melbourne.

The ability of viruses to manipulate the hostile cell microenvironment Human norovirus (HuNoV) are positive sense RNA viruses belonging to is often crucial to ensuring their prolonged survival within the host. the Caliciviridae family and are a major cause of acute gastroenteritis. The pathways they influence range from those which mediate However, the study of HuNoV is challenging due to the lack of effective apoptosis and cell proliferation, to others that control migration and tissue culture systems and small animal models. Despite its significant cytoskeletal structure. One commonly manipulated host system health burden, there are currently no effective treatments for HuNoV is the TGF-β signalling cascade. Observations in our lab suggest infections. Recently, the discovery of a closely related norovirus, Murine vaccinia virus (VACV) can exploit elements of this pathway during Norovirus (MNV) has advanced our understanding of norovirus biology infection. Specifically, we found, via luciferase assay and western and pathogenesis. Here we investigated the association between MNV blots, that VACV can potently activate the TGF-β-associated, R-Smad infection, stress granules and protein translation. We observed that transcription factors, Smad2 and Smad3, as well as the common Smad, stress granules (SG) are not induced during MNV infection. Further, Smad4. Using CRISPR-Cas9 and siRNA technologies, we have also infected cells treated with sodium arsenite, a known oxidative stressor demonstrated a role for Smad4 in a number of aspects of viral infection, to induce SG formation, were restricted in their ability to form SGs, including viral replication, cell-to-cell spread and cell migration. Global suggesting a potential viral control for delayed SG formation. We also transcriptomic analysis uncovered a number of Smad4-dependent demonstrated that during infection, there was a progressive increase in transcriptional targets which may play a role in enhancing VACV phosphorylated eukaryotic initiation factor 2 alpha (eIF2α), yet increased spread. Interestingly, it appears that activation of these Smad proteins MNV translation still occurred under these conditions implying MNV occurs entirely independently of TGF-β receptor phosphorylation in the may employ an alternative protein translation mechanism. To confirm VACV context. To our knowledge, no other microbe is able to stimulate the increasing host translation shutoff, we treated infected cells with this pathway in the same manner. VACV seems to be unique in this puromycin and showed that there is increasing stalling of translation ability, as closely related virus, Ectromelia virus is unable to activate via western blotting with anti-puromycin , which correlated this pathway. Understanding how VACV is able to induce this signalling to the increasing phosphorylation of eIF2α. Our subsequent analyses cascade independently of the TGF-β receptor, could be critical in suggested that the translational repression is mediated via Protein understanding regulation of non-canonical Smad signalling and also kinase-R (PKR), but further investigation revealed that PKR activation provide new insights into oncolytic virus research and tumour cell and translational arrest were uncoupled during infection. These results dynamics. suggest MNV may regulate the PKR-mediated response by promoting eIF2α phosphorylation but inhibits cellular protein translation by a currently unknown mechanism. These observations may provide a link between MNV infection, stress granules, PKR and translational control.

SYM-28-03 SYM-28-04 SUBCELLULAR TRAFFICKING OF HOST RNA BIOLOGICAL CONTROL OF RABBITS IN AUSTRALIA - HELICASE DDX3X MODULATES INNATE ANTIVIRAL AN ONGOING CO-EVOLUTIONARY ARMS RACE SIGNALLING AND PARAINFLUENZA VIRUS IMMUNITY Hall R.N. and Strive T. Heaton S.M., Atkinson S.A., Jans D.A., Sweeney M.N. and Borg N.A. CSIRO Health & Biosecurity, GPO Box 1700, Canberra, Australian Infection & Immunity Program, Biomedicine Discovery Institute, Capital Territory 2601, Australia. Department of Biochemistry & Molecular Biology, Monash University, Clayton, 3800, Australia. Wild European rabbits have enormous impacts on Australia’s agricultural industries and environment, currently costing over $200 DEAD-box RNA helicase 3, X-linked (DDX3X) multiply regulates the million in production losses annually, and threatening 304 native plants retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) antiviral and animals. To manage the damaging rabbit plagues of the last century, signalling cascade. Accordingly, manipulating DDX3X is crucial to the Australia introduced two biological control agents, a poxvirus, Myxoma replication strategy of a growing list of evolutionarily diverse pathogens virus, in the 1950s and a calicivirus, rabbit haemorrhagic disease including influenza A virus, HBV, HCV, dengue virus and HIV-1. To virus, in the 1990s. This has been tremendously successful, with the fulfil these host- and pathogen-directed roles, DDX3X localises to cumulative benefits of biocontrol agents to Australia’s agricultural various subcellular compartments. Interestingly, nuclear export of industries estimated at $70 billion over the last 60 years. However, DDX3X was previously attributed to an exportin-1/CRM1-dependent due to inevitable host-pathogen co-evolution, biocontrol agents must mechanism that, uniquely, required neither a nuclear export signal be used strategically to maximise and prolong their effectiveness. (NES) nor the nucleocytosolic Ran-GTP/GDP gradient. We examined Australia has adopted a pipeline approach to rabbit biocontrol, which the DDX3X-exportin-1 interaction by analytical ultracentrifugation aims to successively introduce new viruses to boost control efforts, and confocal microscopy. We probed its role in immune signalling as part of a broader integrated rabbit management program. The and human parainfluenza-3 (hPIV-3) replication in human lung introduction of biological control agents into naive populations from a A549 cells using plaque assays, luciferase gene reporter assays, single point source provides a unique opportunity to study the spread, NanoString transcriptome analysis, FACS and ELISA, then dissected establishment, and evolution of emerging pathogens. Of particular its mechanism of immune regulation by constitutively activating RLR interest is how closely related viruses interact with each other - a viral signalling proteins or treating with poly(I:C), LPS or IFN-β. Contrasting competition experiment on a continental scale. For caliciviruses in the current model, we find the region bound by exportin-1 bears no particular, our data show that recombination appears to be an important primary to any recognised protein domain in mechanism for generating genetic diversity, with multiple recombinant evolution. We show exportin-1 is the bulk nuclear export receptor for viruses emerging in the last three years. Understanding the mechanisms DDX3X, which is specifically impaired by treatment with exportin-1 behind the epidemiological fitness, or lack thereof, of different virus inhibitors, DDX3X point mutagenesis or removal of Ran-GTP. In strains enables strategic, targeted use of biocontrol agents to maximise response to viral RNA exposure during hPIV-3 infection, DDX3X the impacts of control programs. This has the potential to prolong the accumulates inside the nucleus to support IFN-β expression. However, effectiveness of existing virus strains in a manner analogous to strategic ectopic nuclear DDX3X impairs RLR signalling, deregulates dozens of use of antibiotics to delay the development of antimicrobial resistance. genes relevant to immunity and fails to suppress hPIV-3 replication. Our results redefine the molecular mechanism of DDX3X nuclear export and reveal new modes of immune regulation.

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SYM-28-05 CAPSID DEPENDENT EVASION OF INNATE IMMUNE SENSING IN MACROPHAGES DISTINGUISHES PANDEMIC HIV-1(M) AND NON-PANDEMIC HIV-1(O)

Jacques D.A.1, 2, Hilditch L.3, Rasaiyaah J.3, James L.C.2 and Towers G.J.3 1UNSW Sydney, NSW, 2052 Australia. 2MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK. 3University College London, London, WC1E 6BT, UK.

Transmission of simian immunodeficiency virus from great apes into humans has occurred on at least four separate occasions giving rise to the four distinct HlV1 groups: M, N, O, and P. The most common group, HIV1(M), was transmitted by chimpanzees and is the only group that has resulted in pandemic levels of human-to-human spread despite a similar time since introduction into human populations. The next most common group is HIV1(O), which originated from gorillas, and has resulted in significant spread (~100,000 human infections) yet has not reached the levels seen for HIV1(M) (~60,000,000 human infections) suggesting a reduced capacity to replicate in or transmit between humans. We have previously shown that the HIV1(M) capsid confers the ability to evade innate immune sensing in primary human macrophages. This evasion is dependent on the capsid’s ability to recruit host cofactors. When comparing HIV1(M) with HIV1(O), we find the HIV1(O) capsid to be less effective at evading innate immune triggering, with viral DNA activating the expression of interferon-stimulated genes. While this is analogous to HIV1(M) mutants that fail to engage host cofactors, HIV1(O) retains the ability to bind to CPSF6 and CypA demonstrating that binding per se is not responsible for the different phenotypes. Rather, the consequence of binding is altered. Phylogenetic comparison of HIV1(M) sequences and HIV1(O) CA sequences identified two key residues that split the M and O groups. Comparison of HIV1(M) and HIV1(O) CA crystal structures reveals that these two residues result in a less dynamic CA structure in HIV1(O), suggesting that it may not have the same allosteric capability as HIV1(M). This reduction in dynamics can be recapitulated in HIV1(M) through point mutation. We hypothesise that HIV1(O) capsids are less responsive to host interaction leading to premature reverse transcription, release of DNA, and innate immune triggering in macrophages. We further hypothesise that a reduction in the ability to infect these cells reduces the sexual transmissibility of HIV1(O) relative to HIV1(M), resulting in diminished pandemic potential.

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SYM-29-01 SYM-29-02 DYNAMICS OF TISSUE-SPECIFIC GENOME REGULATION SHOOT ANION LOADING VIA THE REGULATORY PROGRAMS IN THE GERMINATING ATSLAH1-ATSLAH3 COMPLEX BARLEY SEED Qiu J. and Gilliham M. Liew L.C., Chen M., Wang Y., Whelan J. and Lewsey M.G. ARC CoE PEB, University of Adelaide, South Australia, Australia. AgriBio, La Trobe University, Melbourne, VIC, Australia. AtSLAH1 and AtSLAH3, two Arabidopsis slow type anion channel- We are studying tissue-specific genome regulatory programs in associated 1 (SLAC1) homologues form a protein complex to regulate - - germinating barley seeds so as to better understand how they are root-to-shoot delivery of chloride (Cl ) and nitrate (NO3 ). Both proteins controlled by transcription factors. The roles of transcription factors are expressed in Arabidopsis root pericycle and their expression is down- in genome-wide regulation of gene expression are subject to much regulated by NaCl and ABA (Qiu et al., 2016). AtSL AH3 carries predominantly NO - when expressed by itself but has increased Cl- transport in a complex attention currently, but determing these roles is complex. Regulatory 3 events occur dynamically over short time scales (minutes to hours), with SLAH1 (Cubero-Font et al., 2016). Here, we explore the regulation of this complex using heterologous expression in Xenopus laevis oocytes and with many transcription factors interacting to regulate the output of any two-electrode voltage clamp. We observed two ABA signaling associated given gene and cascades of interacting factors operating across the protein kinases, SnRK2.2 and SnRK2.3, negatively regulate anion currents wider program. Studies have historically depended on analysis of bulk through AtSLAH1-AtSLAH3. Conversely, a positive guard cell anion channel tissue samples, due largely to the capabilites of available techniques. regulator from the same kinase subgroup, SnRK2.6 (OST1), appears not However, individual cell-types must be specified by distinct regulatory to regulate the complex. To confirm whether AtSLAH1-AtSLAH3 can be programs. To investigate cell-type specific genome regulation targeted by SnRK2s, the only SnRK2s-specific substrate motif RxxT/S during germination we have applied laser-capture microdissection in AtSLAH1 was site-mutagenized (R176K) to disrupt the recognition by RNA sequencing to three tissues of barley seeds that have distinct SnRk2s. When SLAH1R176 was co-injected with SLAH3 and SnRK2.2 the functions (plumules, radicles and scutellum). We analysed complete conductance inhibition was less pronounced compared to SLAH1-SLAH3- transcriptomes from samples of 200 cells over a 36 h time series, SnRK2.2. This result implies SnRk2.2 has an important role in mediating enabling us to observe the dynamic changes in gene expression. The root-to-shoot Cl- transport by regulating AtSLAH1-AtSLAH3 activity. data allow us to identify modules of gene expression that are unique to Previous studies have reported different protein kinases from the same and conserved between these three tissues and to identify associated subgroup might distinguish their roles with different tissue localization. As groups of potential cis-regulatory motifs. By extrapolation from the SnRK2.2 and SnRk2.3 are abundantly expressed within the plant including extensive Arabidopsis transcription factor target datasets we are also the root, while SnRK2.6 is highly expressed in the shoot but less detectable able to infer likely families of regulatory factors. The outcome is an in the root; this is consistent with why SnRk2.6 does not affect the ionic improved understanding of the direct regulation of gene expression conductance of AtSLAH1-AtSLAH3 complex. In addition, the AtSLAH1- and how this varies between tissues in a crop species with a relatively AtSLAH3-SnRK2.2 was also found to be regulated by calcium-dependent complex genome. protein kinase 21 (CPK21) by restoring the anion transport, suggesting a potential cross talk between kinases with/without Ca2+ dependency. As AtSLAH1-AtSLAH3 is involved in mediating root-to-shoot anion movement, multiple regulating mechanisms of the complex may help the plant to acclimate to stressful growth conditions by adjusted anion transfer to the shoot. Experiments to explore whether this regulation occurs in planta is underway.

SYM-29-03 SYM-29-04 A NOVEL MITOCHONDRIAL LYR PROTEIN IS DIURNAL VARIATION IN THERMAL ACCLIMATION OF REQUIRED FOR COMPLEX I ASSEMBLY IN LEAF RESPIRATION IN RICE ARABIDOPSIS Rashid F.A.A.1, Asao S.1, Taylor N.L.2, Fenske R.2 and Atkin O.K.1 Ivanova A.1, Gille-Hill M.1, Branca R.2, Kmiec B.3, Teixeira P.3 1ARC Centre of Excellence in Plant Energy Biology, Research and Murcha M.W.1 School of Biology, Building 134, The Australian National University, 1School of Chemistry and Biochemistry & The ARC Centre of Canberra, ACT 2601, Australia. 2ARC Centre of Excellence in Plant Excellence in Plant Energy Biology, The University of Western Energy Biology, School of Molecular Sciences, Bayliss Building, The Australia, 35 Stirling Highway, Crawley, Perth 6009, Australia. 2Clinical University of Western Australia, Crawley, WA 6009, Australia. Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory and Karolinska Institutet, Stockholm, Past studies using plants sampled at a single time-point during the day Sweden. 3Department of Biochemistry and Biophysics, Stockholm have shown that cold-acclimated leaves exhibit higher rates of leaf dark University, Arrhenius Laboratories for Natural Sciences, Stockholm, respiration (R) at a common temperature than their warm/hot grown Sweden. counterparts. What is unclear, however, is whether these differences are held throughout the day. To address this, we examined the effect of Mitochondrial Complex I, a proton pumping NADH;ubiquinone growth temperature on diurnal variations in leaf R at a set temperature oxidoreductase is the first and most complicated enzyme involved in (30°C) and associated metabolite pools of rice (Oryza sativa, IR64). the generation of ATP via oxidative phosphorylation. Located in the Plants were grown in three temperature controlled glasshouses (25, 30 mitochondrial inner membrane it is composed of at least 44 subunits and 35°C), with leaf R measured at 8:30 am, 1 pm, 5:30 pm, 9:30 pm and requires the co-ordination of both nuclear and mitochondrial gene and 4:30 am; metabolites were also quantified at the same time points. expression, protein import, co-factor biosynthesis and the assembly of As expected, rates of leaf R measured at 30°C were significantly higher each individual subunit into a 1000 kDa complexome. So far, only two in the 25°C grown plants than those grown at 30°C and 35°C. However, bona fide Complex I assembly factors (GLDH and INDH) have been the relative difference in leaf R between the growth temperatures identified for plants. Here we functionally characterise a novel Complex changed through the day, being greatest at the end of the day-light I assembly factor that is located in the mitochondrial matrix and contains period and least during the night hours. Underpinning this pattern were the conserved Complex I LYR Fe/S domain. Knock-out mutants lack diurnal variations in leaf R of the 25°C & 30°C grown plants (with rates the monomeric Complex I and the supercomplex I + III and exhibit the highest at 5:30 pm, and lowest at 4:30 am), whereas leaf R did not vary phenotypic characteristics typical of Complex I defective lines. BN- diurnally in the 35°C grown plants. Starch concentrations were lowest PAGE analysis shows the stalling and accumulation of the 650 kDa at the end of the night and highest at the day period – by contrast, and 850 kDa Complex I assembly intermediates, with protein-protein soluble sugar concentrations remained constant through the day-night interaction assays displaying specificity for subunits. Consequently, cycle in all three growth treatments. Thus, availability of substrate pools there is a general upregulation of mitochondrial biogenesis with to glycolysis did not account for the divergent effects of time observed regards to protein import ability and mitochondrial translation rates. Our among the growth treatments. The effect of growth temperature and data suggest that this novel LYR protein facilitates the biogenesis and time of day on glycolytic and TCA cycle metabolites will be reported. assembly of particular subunits that are essential for the formation of a Collectively, the results highlight the importance of considering time functional Complex I. of day when assessing thermal acclimation of respiratory energy metabolism in rice.

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SYM-29-05 SYM-30-1 REACTIVE OXYGEN SPECIES CONTRIBUTE TO MODELING GENE EXPRESSION VARIABILITY TO SUGAR SIGNALLING AND GROWTH IN ARABIDOPSIS UNDERSTAND STEM CELL REGULATION

Roman A.1, 2, Deng D.1, Eastmond H.2, Arshad W.2, Davey J.2, James S.2, Mar J.C.1, 2, 3 Ashton P.D. 2, Graham I.A.2 and Haydon M.J.1, 2 1Australian Institute for Bioengineering and 1School of BioSciences, The University of Melbourne, Parkville Nanotechnology. 2University of Queensland. 3Albert Einstein College VIC. 2Department of Biology, University of York, UK. of Medicine.

Plants produce sugars from photosynthesis to provide the stored energy When studying the transcriptome and its contribution to the regulation and building blocks for all living cells. Sugars also act as dynamic of stem cells, our inferences typically revolve around changes in signals to regulate growth and physiology. Thus, carbon status has average gene expression. For a wide range of stem cell populations, wide-ranging effects on plant productivity but defining specific sugar heterogeneity in gene expression is a recognized part of transcriptomic signalling pathways can be challenging in the context of photoautrophic data, and recent studies have demonstrated how modeling variability metabolism because it is difficult to separate responses to light and has revealed more information than following average trends alone. sugar. We have previously shown that sugar signals regulate circadian This talk outlines some of the approaches my group has developed rhythms in plants: exogenous sugars can initiate robust circadian to investigate how variability of gene expression contributes to our rhythms in dark-grown seedlings; and inhibition of photosynthesis in the understanding of transcriptional regulation using examples from human light can adjust phase of the circadian oscillator. To dissect contributions stem cell populations. of sugar and light signals on gene networks in Arabidopsis, we have performed an RNA-Seq time-series in dark-adapted seedlings treated with sucrose or mannitol in the dark or transferred to the light with or without an inhibitor of photosynthesis. We have used weighted gene co-expression network analysis (WCGNA) and GO-enrichment of this large dataset to identify functional classes of transcripts. As expected, inhibition of photosynthesis identified GO-enrichment for ‘circadian rhythm’ but also ‘response to absence of light’, including transcripts for major photoreceptors. The latter suggests a contribution of sugars to canonical light signalling and indicates our experimental design could deconvolute light and sugar signals in plants. The most significantly- enriched class of transcripts responding early to sugar was ‘response to oxygen-containing compound’, pointing to a potential role for reactive oxygen species (ROS) signalling. Using luciferase reporters and qPCR, we have shown that chemical inhibitors of ROS inhibit transcriptional responses to sugar. These inhibitors also inhibit promotive effects of sugars on root growth and shoot biomass, suggesting these contribute to meaningful sugar signalling pathways.

SYM-30-2 SYM-30-3 LIVER REGENERATIVE MEDICINE - TOWARDS CELL HOW: THE LONG AND SHORT OF INTESTINAL STEM AND ORGANOID THERAPIES CELLS

Yap K.1, 2 Qi J., Dominado N., Savva E., Casagranda F., Siddall N.A. and Hime G.R. 1St Vincent’s Institute, Fitzroy, Victoria. 2University of Melbourne Department of Anatomy and Neuroscience, University of Melbourne, Department of Surgery at St Vincent’s Hospital Melbourne, Fitzroy, Parkville 3010, Australia. Victoria. Intestinal stem cells play a key role in maintaining the intestinal Liver disease is on the rise, particularly fatty liver disease which is environment. Multiple signaling pathways regulate replication and commonly associated with obesity and diabetes. Chronic liver disease differentiation of intestinal stem cells (ISCs). The midgut epithelium often leads to liver failure, which is fatal without a liver transplant. of Drosophila melanogaster is comprised of similar cell types to However, liver transplantation simply cannot meet the ongoing the vertebrate intestinal epithelium and has served as a model for increase in demand. Strategies to better understand, diagnose, and identification of genes that regulate epithelial biology. We have recently treat liver disease are critically required. Our group has an interest in used Drosophila to show that an RNA-binding protein, known as HOW identifying the role and utility of cells within the liver that can contribute (Held-out wings), is also involved in regulating ISCs. HOW can be to liver regeneration, which can also be harnessed in cell and organoid found as 2 major isoforms; long and short proteins named HOW(L) therapies for liver disease. These include human liver progenitor cells and How(S) respectively. HOW(L) and HOW(S) have been shown to and liver sinusoidal endothelial cells derived from adult human liver, have opposing roles in regulation of tendon cell differentiation in the as well as induced pluripotent stem cell (iPSC) produced counterparts. wings of the fly. Flies with this mutation have their wings perpendicular Organoids generated from these cell types have been established to their body which gave rise to the name, ‘held- out wings’. The in culture and transplanted into a mouse model of liver disease, vertebrate ortholog of HOW is known as QKI (Quaking) and has been with demonstration of tissue assembly, functional maturation, and associated with regulation of colorectal cancer cell differentiation. survival in vivo. Comparison between iPSC-derived cells/organoids The QKI gene also produces multiple isoforms; 2 of which are very and human adult liver derived cells/organoids are underway, and similar to HOW(S) and HOW(L). Despite research showing that HOW recent developments include upscaling of transplantation experiments is present in intestinal stem cells, little research has been conducted from mouse to rat, representing a 10-fold increase in scale. It is hoped investigating its role and its targets. Complete loss of HOW function in insights gained from these experiments will guide the development of the midgut results in a decrease in ISC number. Ectopic expression of cell and organoid-based therapies for liver disease. HOW(L) results in an ISC increase and a corresponding decrease in the proportion of differentiated enterocytes. I aim to determine whether HOW(L) and HOW(S) have their own individual roles in intestinal stem cell maintenance or have redundant functions.

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 55 symposia TUESDAY

SYM-30-04 SYM-30-05 THE ROLE OF THE PRORENIN RECEPTOR IN MALE β-CATENIN DRIVES DISTINCT TRANSCRIPTIONAL FERTILITY NETWORKS IN REGENERATIVE AND NON- REGENERATIVE CARDIOMYOCYTES 1 2 3 4 1 Ahmady S. , Bernard P. , Merriner D.J. , Chan A. , Bagheri-Fam S. , 1, 2 1, 2 1, 2 1, 2 4 3 2 1 Quaife-Ryan G.A. , Lavers G. , Mills R.J. , Voges H.K. , Hobbs R.M. , O’Bryan M.K. , Pask A.J. and Wilhelm D. 3 1, 2 1, 4, 5 1 Ramialison M. , Hudson J.E. and Porrello E.R. Department of Anatomy & Neuroscience, University of Melbourne, 1 2 School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia. School of BioSciences, University of Melbourne, 2 3 4072, Australia. QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia. School of Biological Sciences, Monash University, 3 4 4006, Australia. Australian Regenerative Medicine Institute, Monash University, Australia. ARMI and the Department of Anatomy & Developmental Clayton, VIC, 3800, Australia. 4Murdoch Children’s Research Institute, The Royal Biology, Monash University, Australia. Children’s Hospital, Parkville, VIC, 3052, Australia. 5Department of Physiology, School of Biomedical Sciences, The University of Melbourne, Parkville, VIC, 3010, The prorenin receptor (PRR) is best known for its tole in the renin- Australia. angiotensin-system (RAS) to regulate blood pressure and salt homeostasis. However, more recently, PRR has been shown to be a The inability of the adult mammalian heart to regenerate following cardiac injury multi-functional protein, which is involved in a number of downstream represents a major limitation in the management of heart failure. In comparison, pathways including MAPK signalling, protein sorting and folding and the neonatal mouse heart regenerates following myocardial infarction (MI). We receptor-mediated endocytosis and recycling through its interaction recently compared the neonatal and adult transcriptomes of multiple cardiac cell populations and uncovered a regenerative gene network associated with Wnt/β- with the vacuolar H+-ATPase (V-ATPase), as well as canonical and catenin signalling. However, it is unclear what role Wnt/β-catenin signalling plays non-canonical WNT signalling. Given that MAPK and WNT signalling in driving the pro-regenerative network. Here, we study Wnt/β-catenin involvement is important for ovary and testis differentiation, we hypothesised that in cardiomyocyte regeneration. We found that stimulation of β-catenin signalling PRR plays a role in gonadal development and function. To test this by GSK3 inhibition (GSK3i) in immature human embryonic stem-cell-derived hypothesis, we deleted Prr specifically in gonadal somatic cells using cardiomyocytes and 3D human cardiac organoids potently induced cardiomyocyte the Nr5a1-Cre mouse. While these mice appear to develop normally proliferation. Furthermore, β-catenin inhibition abrogated GSK3i-induced and are born at the expected Mendelian ratio, both males and females cardiomyocyte proliferation in vitro. To identify direct β-catenin transcriptional are infertile. Here, we present our analysis to date of the testicular targets, we undertook RNA sequencing (RNA-seq) of GSK3i treated human phenotype of the conditional Prr-null mice. cardiomyocytes combined with chromatin-immunoprecipitation sequencing (ChIP-seq) to reveal 22 direct β-catenin/TCF7L2 target genes that were shared in common between the human and mouse regenerative networks. Consistent with these results, delivery of constitutively active β-catenin (caBCAT) in vivo stimulated neonatal cardiomyocyte proliferation. Additionally, β-catenin inhibition limited neonatal cardiomyocyte cell cycle activity in vivo and downregulated β-catenin- target genes. However, in contrast to these effects in regenerative cardiomyocytes, caBCAT delivery to the adult mouse heart following MI did not induce cardiomyocyte proliferation, although cardiac function was improved. RNA-seq of purified adult cardiomyocytes treated with caBCAT uncovered a distinct transcriptional network associated with cardioprotection and modulation of the immune response that appears to be driven by FoxO. Therefore, β-catenin drives distinct transcriptional programs in regenerative and non-regenerative cardiomyocytes. Redirection of β-catenin to its pro-proliferative target genes could be exploited for regenerative applications in the future.

SYM-31-01 SYM-31-02 INFLAMMASOMES IN SEVERE ASTHMA EPIGENETIC AND TRANSCRIPTIONAL REGULATION OF IL-4 INDUCED CCL17 PRODUCTION IN HUMAN Kim R.Y.1, Pinkerton J.W.1, Essilfie A.T.1, Robertson A.A.B.2, Baines K.J.1, MONOCYTES AND MURINE MACROPHAGES Brown A.C.1, Mayall J.R.1, Ali K.1, Starkey M.R.1, Hansbro N.G.1, Hirota J.A.3, 1 1 1 1 1 Wood L.G. , Simpson J.L. , Knight D.A. , Wark P.A. , Gibson P.G. , O’Neill 1, Hsu A.1, Lee M.1, Fleetwood A.1, Cook A.1, Hamilton J.1, 2 4 2 1 1 Lupancu T. L.A.J. , Cooper M.A. , Horvat J.C. and Hansbro P.M. and Achuthan A.1 1University of Melbourne, Department of Medicine, Royal Melbourne 1University of Newcastle, Newcastle, New South Wales, Hospital. 2Australian Institute for Musculoskeletal Science (AIMSS), Australia. 2University of Queensland, Brisbane, Queensland, Australia. The University of Melbourne and Western Health. 3University of British Columbia, Vancouver, British Columbia, Canada. 4 Trinity College Dublin, Dublin, Ireland. Interleukin- 4 (IL4) is an anti- inflammatory cytokine and alternative macrophage activator, recognized as a hallmark cytokine of Th2- Rationale: Severe, steroid-resistant (SSR) asthma is the major unmet associated diseases. On the other hand, granulocyte- macrophage need in asthma therapy. Disease heterogeneity and poor understanding of colony stimulating factor (GM - CSF) is a pro - inflammator y hematopoietic pathogenic mechanisms hampers the identification of therapeutic targets. cytokine and involved in the development of autoimmune inflammatory Excessive NLRP3 inflammasome and concomitant IL-1β responses occur diseases, such as rheumatoid arthritis. Despite the opposing in COPD, respiratory infections and neutrophilic asthma. However, the inflammatory roles of these two cytokines, GM-CSF and IL4 are used direct contributions to pathogenesis, mechanisms involved and potential for together to enhance in vitro differentiation of monocytes into dendritic therapeutic targeting are unknown in SSR asthma. Methods: We developed cells. We have previously reported that GM-CSF can induce the mouse models of Chlamydia, and Haemophilus, respiratory infection- secretion of chemokine (C-C motif) ligand 17 (CCL17) from monocytes mediated, ovalbumin-induced SSR allergic airways disease. These models in an interferon regulatory factor 4 (IRF4)-dependent manner. share the hallmark features of human disease, including elevated airway Interestingly, CCL17 is also an upregulated marker of IL4 polarised M2 neutrophils, and NLRP3 inflammasome and IL-1β responses. The roles macrophages. We investigated whether the IL4 induced CCL17 pathway and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1β shared any common elements with the established GM-CSF pathway. responses in experimental SSR asthma were examined using a highly- We report here that, like GM-CSF, IL4 induces CCL17 expression in an selective NLRP3 inhibitor, MCC950, the specific caspase-1 inhibitor, Ac- IRF4 dependent manner. IL4 also enhances the expression and activity YVAD-cho, and neutralizing anti-IL-1β antibody. Roles for IL-1β-induced of Jumanji domain-containing protein 3 (JMJD3) demethylase which neutrophilic inflammation were examined using IL-1β and anti-Ly6G. Results: epigenetically upregulates IRF4 expression. Signal transducer and Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1β activator of transcription 6 (STAT6) is activated by IL4, not GM-CSF, responses that drive steroid-resistant neutrophilic inflammation and airways and silencing the STAT6 gene led to a decrease in CCL17 formation, as hyper-responsiveness (AHR). Neutrophilic inflammation, disease severity well as that of its upstream regulators, JMJD3 and IRF4. Moreover, IL4 and steroid-resistance in human asthma correlates with NLRP3 and IL- treatment of human monocytes resulted in an increased association of 1β expression. Treatment with anti-IL-1β, Ac-YVAD-cho, and MCC950 STAT6 to the promoter regions of the CCL17, IRF4 and JMJD3 genes. suppressed IL-1β responses and the important steroid-resistant features of Thus, despite the different and almost opposing roles GM-CSF and disease in mice, whereas IL-1β administration recapitulated these features. IL4 play in inflammation, these 2 cytokines share a common signaling Neutrophil depletion suppressed IL-1β-induced steroid-resistant AHR. pathway in regulating CCL17 production in human monocytes and Conclusions: NLRP3 inflammasome responses drive experimental SSR murine macrophages. asthma and are potential therapeutic targets in this disease.

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SYM-31-03 SYM-31-04 MITOCHONDRIAL FUNCTION DURING MAMALIAN TLR4 AS AN INFLAMMATORY KEY TO SEVERE OOCYTE DEVELOPMENT: GOING BEYOND DENGUE DISEASE BIOENERGETICS Modhiran N., Vajjhala P.R., Watterson D., Young P.R. and Stacey K.J. Adhikari D.1, Liu J.1, Alzubaidi U.1, Zhang Q.H.1, Yuen W.S.1, Robker R.L.2 Australian Infectious Diseases Research Centre, School of Chemistry and Carroll J.1 and Molecular Biosciences, The University of Queensland, St Lucia, 1Department of Anatomy and Developmental Biology and Development Qld 4072. and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia. 2The Robinson Research Institute, School Toll - like rec eptor 4 ( T LR4) is t he pat ter n rec o gnit i on rec eptor resp onsible of Medicine, The University of Adelaide, Australia 5005. for mediating inflammatory effects of lipopolysaccharide (LPS), the major constituent of the Gram-negative bacterial cell wall. We have Due to limited glycolytic capabilities of mammalian oocytes, mitochondrial ATP shown that the dengue virus secreted non-structural protein NS1 also production by OXPHOS is essential for oocyte and preimplantation embryo activates TLR4, inducing the release of inflammatory cytokines from development. Mitochondrial biogenesis occurs throughout oocyte growth myeloid cells and loss of endothelial monolayer integrity. The serious during which the number of mitochondria increases from about 1000 to up complications of dengue virus infection include haemorrhage and to 500,000. Mitochondrial DNA (mtDNA) amplifies and mitochondria undergo shock, indicating a critical role for disruption of the endothelial barrier. divisions during oocyte growth. mtDNA replication is driven by upregulation We find that inhibition of TLR4 responses in dengue virus-infected mice of Transcription Factor A, Mitochondrial (TFAM) and mitochondrial fission substantially reduces vascular leak and is thus a potential therapeutic requires Dynamin-related Protein 1 (DRP1). Disruption of mitochondrial avenue. This suggests that at least in the later stages of infection, biogenesis by genetic ablation of TFAM or DRP1 during oocyte growth the host does not benefit from recognition of infection via TLR4. The does not significantly reduce the levels of ATP in the fully grown oocyte. striking similarities in cellular responses to LPS and NS1 via TLR4 Furthermore, oocyte maturation, fertilization and early embryo development suggest that NS1 is a viral counterpart of bacterial endotoxin. Similar appear normal, although embryo development does not progress beyond to the role of LPS in septic shock, NS1 may contribute to vascular leak day 15 of gestation. These findings suggest that the role of mitochondria in in dengue patients. A role for TLR4 in dengue infection opens up new oocytes extends beyond that of a simple bioenergetics hub. Mitochondria are therapeutic avenues for decreasing the severity of dengue pathology increasingly being recognized for coordinating multiple metabolic pathways using established antagonists. and regulating nuclear modifications through metabolites. However, the roles of mitochondria in oocytes beyond ATP production are poorly understood. Primary or secondary mitochondrial defects in oocytes caused by maternal environment are known to be detrimental for embryo development and offspring health. Our analyses reveal altered metabolites, nuclear epigenetic modifications and nuclear gene expression profiles in TFAM and DRP1- deleted oocytes. Thus, the function of mitochondria in oocytes can be extended to include roles in regulating the levels of key metabolites that have potential to modify essential epigenetic modifications occurring during oocyte growth. In conclusion, our results show that oocyte mitochondria have roles that extend well beyond the traditional and widely held view that mitochondria act as a simple power supply for the purposes of maintaining oocyte function.

SYM-31-05 SYM-32-01 CELL DEATH AND AUTOINFLAMMATION COMBATING ANTIBIOTIC RESISTANCE: STRUCTURAL AND BIOPHYSICAL STUDIES OF A COLISTIN Lawlor K.E.1, Feltham R.1, Yabal M.2, Jost P.J.2 and Vince J.E.1 RESISTANCE ENZYME INVOLVED IN ENDOTOXIN 1The Walter and Eliza Hall Institute of Medical Research, Parkville, MODIFICATION VIC 3052, Australia. 2III. Medical Department for Hematology and Oncology, Klinikum rechts der Isar,Technische Universität München, Vrielink A.1, Anandan A.1, Evans G.L.1, Condic-Jurkic K.2, O’Mara M.L.2 81675 Munich, Germany. and Kahler C.M.1 1University of Western Australia, 35 Stirling Highway, Crawley, WA, Cytosolic inflammasome sensor proteins activate caspase-1, resulting in 6009. 2The Australian National University, Canberra, ACT 2601. cleavage-induced maturation and secretion of the potent inflammatory cytokine Interleukin-1β (IL-1β). Consequently, activating mutations in Multiple drug resistance (MDR) in Gram-negative bacteria represents inflammasome sensor proteins result in autoinflammatory disease that one of the most intractable problems facing modern medicine. Colistin can be treated with anti-IL-1 therapeutics. Surprisingly, recent studies and polymyxin are cationic antimicrobial peptide antibiotics which have highlighted that mutations in components of the Toll-like Receptor permeabilise the bacterial outer membrane and have been used to (TLR) and TNF Receptor (TNFR) signaling machinery can also result treat infections. Resistance to these antibiotics is conferred by the in pathological IL-1β production, and autoinflammatory disease with modification of the lipid A headgroups with phosphoethanolamine symptoms reminiscent of patients with activating inflammasome (PEA) moieties resulting in a reduced negative charge of the bacterial mutations. How this aberrant TLR and TNF Receptor signaling can surface and exclusion of the drug. This modification is carried out by activate cytosolic inflammasome complexes remains unclear. In our the enzyme, lipid A PEA (EptA). Recently a mobile colistin recent studies we have explored this conundrum using a murine model resistance determinant, mcr-1, encoding an EptA homologue was of X-linked Inhibitor of Apoptosis (XIAP) deficiency, which can trigger identified in MDR Escherichia coli. The crystal structure of a full-length potentially fatal, pathogen-associated, hyperinflammation in humans. EptA from Neisseria sp. to 2.75Å resolution will be presented as well We define a novel TLR and TNF Receptor cooperative signaling as molecular dynamics and biophysical studies. These studies help mechanism that is essential for inflammasome and IL-1β activation in us to better understand the conformational flexibility of the protein and the absence of XIAP, and document how this pathway may also act to provide insights into the catalytic activity of the enzyme. generate pathological IL-1β responses in models of endotoxic shock and inflammatory arthritis.

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SYM-32-02 SYM-32-03

CIRCULAR RNAs in epithelial to mesenchymal HOMODIMERIZATION REGULATES AN transition (emt) ENDOTHELIAL SPECIFIC SIGNATURE OF THE SOX18 TRANSCRIPTION FACTOR Goodall, G.J. Moustaqil M.1, Fontaine F.2, Overman J.2, MacCann A.2, Bailey T.L.3, Rudolffi Soto P.1, Gambin Y.1, Francois M.2 and Sierecki E.1 1 ABSTRACT NOT AVAILABLE EMBL Australia Node in Single Molecule Science School of Medical Sciences, The University of New South Wales, Sydney, AT TIME OF PUBLICATION Australia. 2Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia. 3Department of Pharmacology, School of Medicine, University of Nevada, Reno, USA.

During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium. The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation. We recently found that in the SOX-F family, SOX18 alone has the ability to switch between a monomeric and a dimeric form, using in vitro binding assays and a split-GFP reporter assay in a zebrafish model system in vivo. SOX18 dimerization is driven by a newly identified motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations revealed enrichment for a SOX dimer binding motif on the chromatin, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.

SYM-32-04 SYM-32-05 ORDERED LIM DOMAINS TUNE ASSOCIATION MOLECULAR ARCHITECTURE OF AN E. FAECALIS RATES OF DISORDERED PARTNERS TO REGULATE ANTITERMINATION PROTEIN BOUND TO RNA TRANSCRIPTION FACTOR COMPLEX FORMATION Walshe J.L., Patel, K. and Ataide S.F. Robertson N.O.1, Smith N.C.1, Manakas A.1, Mahjuob M.1, McDonald G.2, University of Sydney. Kwan A.H.1 and Matthews J.M.1 1School of Life and Environmental Sciences, University of Sydney, Regulated transcription termination provides an efficient and responsive Australia. 2Centre for Translational Data Science, University of Sydney, means to control gene expression. Rho-independent termination Australia. occurs through the formation of a RNA stem-loop which disrupts the RNA polymerase elongation complex. To overcome this, bacteria may Intrinsically disordered regions (IDRs) are overrepresented among use anti-termination, whereby a mutually exclusive RNA structure is transcription factors, where they often act as protein-protein formed. In some cases this alternative structure is stabilised by ANTAR interaction motifs. Despite the importance of IDR interactions there domains of proteins, preventing termination. We have determined the is little quantitative information about the role disorder plays in the novel 3.8 Å crystal structure of the stabilising anti-terminator protein thermodynamic and kinetic parameters of binding. We have used LIM EutV, bound to RNA. Our studies highlight the key interactions between domain transcription factors to understand the role disorder can play conserved EutV residues and the RNA, as well as protein conformational in complex formation. Transcription factors containing disordered LIM changes undergone upon RNA binding. This has allowed us to propose interacting domains (LIDs) interact with the ordered tandem LIM domains a broad model for ANTAR domain anti-termination. (LIM1+2) of LIM-only (LMO) and LIM-homeodomain (LIM-HD) proteins. The resulting complexes regulate gene expression and are important mediators of cell specification, proliferation and differentiation. We have used FRET-based assays to study the binding affinities and kinetics of different LID:LIM1+2 interactions. LDB1LID association rate constants can vary by up to 3-orders of magnitude depending on the interacting LIM1+2 domains. These disparate kinetics are not accounted for by differences in electrostatic attractions; rather they reflect differences in binding mechanisms of LDB1LID. LDB1LIDcan bind single LIM domains with high association rate constants, indicating that the differences in LIM1+2 association occur after the initial encounter. We have used our equilibrium and kinetic data to model LID:LIM1+2 transcription factor complex as it occurs in motor neuron development, showing that the disparate kinetics facilitate the exchange of high affinity binding partners over time. Together, our studies suggest that ordered domains can use the inherent flexibility of IDRs generate highly differential binding kinetics, which may provide a mechanism for temporally regulating transcriptional complex formation during development.

Page 58 ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 symposia TUESDAY

SYM-33-01 SYM-33-02 REGULATION OF EXPRESSION OF VITAMIN D BONE REGULATION OF ENERGY AND GLUCOSE HYDROXYLASES METABOLISM

Morris H.A. Brennan-Speranza T.C.1, Liu X.1, Brock K.1 and Levinger I.2 School of Pharmacy and Medical Sciences, University of South 1School of Medical Sciences, University of Sydney. 2ISEAL, Victoria Australia, Adelaide, South Australia 5000. University.

Vitamin D is a secosteroid that is metabolically activated and degraded The skeleton is an endocrine organ participating in energy metabolism through the actions of three cytochrome P450 hydroxylase enzymes and glucose homeostasis via the undercarboxylated form of the bone- which are expressed in a wide range of tissues stimulating both derived protein, osteocalcin. Skeletal muscle is a major site of glucose endocrine and autocrine activities of vitamin D. Bioactivation occurs uptake and disposal in response to both insulin and exercise. It was through the sequential actions of CYP2R1 and CYP27B1 enzymes, previously shown that osteocalcin may enhance whole body insulin resulting in synthesis of the pleiotropic hormone 1,25-dihydroxyvitamin sensitivity and glucose control via its action on the pancreatic beta D (1,25D), which regulates over 100 genes whose actions include cells by increasing beta cell proliferation and insulin secretion, as well those associated with calcium homeostasis and immune responses as as affecting adipocytes by enhancing adiponectin secretion. Both the well as cellular growth, differentiation, and apoptosis. Inactivation of increase in insulin and adiponectin levels can increase skeletal muscle 1,25D occurs by C23/C24 oxidation pathways that are catalyzed by the glucose uptake. However, whether osteocalcin can enhance skeletal multifunctional CYP24A1 enzyme. Both CYP27B1 and CYP24A1 are muscle insulin sensitivity and glucose uptake at rest and following highly regulated genes whose differential expression is controlled in muscle contraction/exercise via a direct pathway is not clear. Our response to numerous cellular modulatory agents such as parathyroid recent evidence suggests that a direct pathway is plausible. In order hormone (PTH), calcitonin, interferon gamma, calcium, phosphorus, to better understand the pathway by which osteocalcin may affect and pituitary hormones as well as the secosteroid hormone 1,25D. The glucose uptake in skeletal muscle, a receptor for osteocalcin should upregulation of CYP27B1 by 1,25D has both a rapid nongenomic and be identified. The class C G protein-coupled receptor 6A (GPRC6A) a slower genomic component that are functionally linked. The rapid is the postulated receptor for osteocalcin in several tissues including response involves protein kinase C and mitogen-activated protein skeletal muscle. We now have evidence demonstrating a direct role kinase (MAPK) pathways that direct the phosphorylation of nuclear of osteocalcin in enhancing skeletal muscle insulin sensitivity and that transcription factors. The slower genomic actions are linked to the that GPRC6A may not be the only receptor for osteocalcin in this tissue. binding of 1,25D to the vitamin D receptor (VDR) and the interaction of Furthermore, recent studies in humans have reported significant the VDR-1,25D complex with its heterodimer partner retinoid-X-receptor correlations between osteoclacin levels and insulin sensitivity. and associated coactivators. The regulatory complex is assembled on vitamin D response elements in the proximal promoter of the CYP24A1 gene and functions to increase the transcription rate.

SYM-33-03 SYM-33-04 THE MEVALONATE PATHWAY IN BONE BIOLOGY EPIGENETIC REGULATION OF PROSTATE CANCER METASTASIS TO THE BONE Munoz M. and Rogers M.J. Bone Biology Division, Garvan Institute of Medical Research, Wilkinson E.J.1, 2, Malley R.1, Dickinson J.L.2 and Holloway A.F.1 Darlinghurst, NSW, Australia. 1School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia, 7000. 2Menzies Institute for The mevalonate pathway, necessary for the biosynthesis of cholesterol, Medical Research, College of Health and Medicine, University of is a conserved and ubiquitous process in eukaryotic organisms. It works Tasmania, Hobart, Tasmania, Australia, 7000. via a step-wise condensation of isoprene units to form longer-chain isoprenoid lipids. It has two branches: one leads to the synthesis of Prostate cancer is the most commonly diagnosed cancer in Australian sterols, and the other to the synthesis of isoprenoid lipids tags required males with a reported five-year survival rate of 95%. Prostate cancers, for the post-translational modification of proteins known as prenylation. however, primarily metastasise to the bone with a decreased five-year It is estimated that at least 300 proteins of the human proteome are survival rate of 3%. Current diagnostic tools are unable to distinguish prenylated, particularly signaling proteins such as Ras, Rho and indolent disease from that with a propensity to become metastatic. We Rab small GTPases. During prenylation, farnesyl or geranylgeranyl have taken complimentary single-gene and genome-wide approaches isoprenoid moieties are irreversibly added to a cysteine residue to identifying key genes that contribute to prostate cancer metastasis. at the protein’s carboxy terminus, enabling subcellular membrane Integrins, a group of adhesion receptors, are known to facilitate localisation and protein:protein interactions, and hence adequate signal metastasis to the bone, however the pathway(s) through which this transduction and regulation. The importance of the mevalonate pathway occurs are incompletely understood. Integrins, which act as receptors in bone biology emerged from the discovery that most bisphosphonate for the bone constituents laminin and collagen, are aberrantly expressed drugs, used worldwide to treat common diseases of excessive bone in prostate cancer. We have shown that methylation changes and destruction such as osteoporosis, act by blocking protein prenylation associated expression changes are observed in several key integrins, in bone-degrading osteoclasts. Bisphosphonates (BPs) are non- including ITGB4, in cell lines representative of different stages of hydrolysable analogues of naturally-occurring pyrophosphate. Having prostate tumour disease progression. Formalin-fixed paraffin tissue high avidity for calcium ions, BPs bind rapidly to bone mineral and are derived from matched localised and metastatic prostate tumours were preferentially internalised by osteoclasts. Once in the cytosol, BPs lock examined to determine whether these changes are also observed in farnesyl diphosphate synthase in an inactive state, thereby blocking the patient samples. We also performed genome-wide methylation analysis synthesis of farnesyl and geranylgeranyl isoprenoid tags and preventing using the Infinium® MethylationEPIC array in matched normal and protein prenylation. In this way, bisphosphonates interfere with cellular primary tumour samples, and matched tumour and metastasis samples processes governed by prenylated small GTPases that are essential to identify changes associated with disease progression. Preliminary for bone resorption, such as cell polarisation, cytoskeletal organisation analysis has revealed 10 significantly differentially methylated regions and vesicular trafficking. We are now using the tools developed to between matched normal and primary tumour samples, and over study the actions of bisphosphonates to provide new advances in the 2,300 significantly differentially methylated regions between matched diagnosis and pathophysiology of a human autoinflammatory disease tumour and metastasis samples. These data indicate that changes in caused by defective protein prenylation. methylation occur throughout disease progression, and suggest that wide-spread epigenetic changes accompany tumour metastasis.

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SYM-33-05 SYM-34-01 SPLICING FACTOR SRSF3 AS A NOVEL REGULATOR SYSTEMATIC FUNCTIONAL IDENTIFICATION OF OF ONCOGENIC MIRNAS CANCER DRUG RESISTANCE GENES

Ratnadiwakara M.1, Jarde T.1, 2, Engel R.1, 3, Oliva J.3, Mcmurrick P.J.3, Lau M.T. and Neely G.G. Abud H.E.1 and Anko M.L.1 The Dr. John and Anne Chong Lab for Functional Genomics, Charles 1Monash University. 2Hudson Institute of Medical Research. 3Cabrini Perkins Centre and School of Life & Environmental Sciences, The Health. University of Sydney NSW 2006, Australia.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally Cancer drug resistance is a major obstacle in cancer therapy. To by fine-tuning mRNA levels and translation. miRNA biogenesis is elucidate the genetic factors that regulate sensitivity to anti-cancer tightly regulated to maintain specific miRNA expression patterns in drugs, we performed whole genome CRISPR/Cas9 knockout screens different tissues and developmental stages, misexpression leading to for resistance to a spectrum of anti-cancer drugs of varying compositions pathological conditions. miRNAs are transcribed as longer precursors and general or targeted mechanisms of action. In addition to known that undergo multiple processing steps before the mature miRNAs targets and resistance mechanisms, this study revealed novel insights reach their target mRNAs in the cytoplasm. Although the RNA into drug mechanisms of action, including cellular transporters, drug binding proteins (RBPs) Drosha, DGCR8 and Dicer are the essential target effectors, and genes involved in target-relevant pathways. Anti- components of the miRNA processing pathway, multiple other RBPs, cancer drugs could be classified based on resistance mechanisms, and have recently been identified as critical regulators of miRNA biogenesis. we provide the first functional “phylogenecity” for these compounds. The CNNC motif bound by the SR protein splicing factor SRSF3 Importantly, we identified 49 multi-drug resistance genes, including specifies pri-miRNA hairpins and in vitro studies have demonstrated a previously uncharacterised gene named here Required for Drug- SRSF3 enhancing pri-miRNA processing in a CNNC dependant induced Death 1; RDD1greg. Loss of RDD1 resulted in resistance manner. However, the in vivo functional relevance of SRSF3 activity in to five anti-cancer drugs, primarily anti-tubulin agents, and RDD1 is miRNA processing has remained unexplored. Our analysis of SRSF3 required for an anti-tubulin drug to trigger MCL-1 degradation and cell binding sites in embryonic stem cells (ESCs) identified SRSF3 binding death. Loss of RDD1 also conferred resistance to anti-tubulin therapy at the CNNC motif particularly in miRNAs located in polycistronic in a mouse xenograft model, and clinically, low RDD1 expression was miRNA clusters. We demonstrate that SRSF3 depletion in ESCs associated with poor prognosis in multiple cancer cohorts, with the leads to reduced levels of mature miRNAs without affecting the levels strongest association for ovarian cancer patient outcome. Together, of the pri-miRNAs. Intriguingly, the processing of specific miRNAs we provide the first functional landscape of resistance mechanisms to within miRNA clusters is individually regulated, leading to differential a broad range of chemotherapeutic drugs and reveal new multi-drug expression of mature miRNAs derived from the same pri-miRNA. resistance nodes. This information can guide personalised anti-cancer Furthermore, SRSF3 is frequently overexpressed in tumour cells and therapies or instruct rational drug combinations designed to minimise misexpression of miRNAs is a characteristic of many cancers. Our acquisition of resistance. analysis of SRSF3, pri-miRNA, miRNA and miRNA-target expression in human tumour-normal pairs demonstrates that SRSF3 may confer some of its pathological properties through the control of miRNAs. Our work reveals a novel mechanism regulating the hallmark properties of cancer cells and highlights the roles of multifunctional RBPs in gene regulation in health and disease.

SYM-34-02 SYM-34-03 GENOMIC HETEROGENEITY IN COLORECTAL MIR-496 EXPRESSION IS ALTERED IN PRIMARY AND LIVER METASTASES SAMPLES OROPHARYNGEAL CANCERS BY HPV16 E6

Patch A.-M.1, Kawamata F.2, 3, Behrenbruch C.4, 5, Nones K.6, Kazakoff S.6, Mason D.1, Zang X.2, 3, Monteiro Marques T.4, Gama-Carvalho M.4 and Addala V.6, Pearson J.V.7, Hollande F.4, Waddell N.6 and Whitehall V.L.J.2 Tran N.1, 2 1Clinical Genomics, QIMR Berghofer Medical Research Institute, 1School Biomedical Engineering. Centre for health Technologies. Queensland, Australia. 2Conjoint Gastroenterology Laboratory, QIMR University of Technology Sydney, NSW, Australia. 2The Sydney Head Berghofer Medical Research Institute, Queensland, Australia. 3Hokkaido and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince University Graduate School of Medicine, Sapporo, Japan. 4Department Alfred Hospital, NSW, Australia. 3Department of Infectious Diseases of Clinical Pathology, The University of Melbourne, Victoria, and Immunology, University of Sydney, NSW, Australia. 4University Australia. 5Department of Oncology, Sir Peter MacCallum Cancer Centre, of Lisboa, Faculty of Sciences, BiolSl- Biosystems and Integrative Victoria, Australia. 6Medical Genomics, QIMR Berghofer Medical Research Sciences Institute, Campo Grande, Lisboa, Portugal. Institute, Queensland, Australia. 7Genome Informatics, QIMR Berghofer Medical Research Institute, Queensland, Australia. HPV is a major risk factor for oropharyngeal cancers(OPC), with 75% being associated with HPV16. Infection by the virus is known to regulate Colorectal cancer (CRC) is the third most common malignancy small non-coding RNAs, known as miRNAs. MiRNAs have been shown diagnosed worldwide and in Australia, with an estimated >16,500 to play a critical role in tumorigenesis. Using an LNA array we determined Australians affected in 2017. At diagnosis around 25% of CRC patients the expression levels of miRNAs in OPC positive for HPV. miR-33, miR- have metastatic (stage IV) disease and a further 25% of patients 210, miR-142-3p and miR-496 were differentially expressed between initially diagnosed at stage II/III will relapse with metastatic disease. HPV16(+) and HPV16(-) OPCs. Of these miR-496 and miR-33 were Unfortunately, 75% of patients presenting with metastatic disease will the most deregulated. Mechanistically we showed that E6, one of the have surgically unresectable disease and are incurable. The 5-year oncoproteins of HPV16 modulates the expression of miR-496 and survival of this group of patients is approximately 5%, almost ten times miR-33. We then revealed that miR-496 can regulate the transcription lower than patients with resectable disease. Treatment is palliative factor E2F2, which has been shown to be required for viral replication. A and there are essentially only three available cytotoxic agents, viral/miRNA interactome was constructed to map association between 5-fluorouracil, oxaliplatin and irinotecan. Newer targeted agents and miRNAs and E6. From this analysis, we discovered a mechanistic link immunotherapy only show benefit in a subset of patients. Different sites between miR-496, miR-33 and E2F2. We believe that E6 increases the of disease such as the primary tumour compared with the metastasis expression of SREBF2 which harbours miR-33. The increase in miR-33 will have heterogeneity in treatment response making the choice of regulates transcription factors, (BACH1, STAT5A, and GATA2) which therapy more complex. A personalised medicine strategy that enables impacts the expression of miR-496. Our study has identified a specific selection of the most effective therapy based on the tumour’s molecular regulatory pathway involving E6, miRNAs and E2F2. These events characteristics could provide more effective treatment options for may be important for viral replication and transformation of the cell. patient management especially for those with unresectable metastatic These interactions uncovered between HPV and miRNAs may provide disease. Therefore, we are investigating the heterogeneity between an insight into the cellular mechanisms in HPV positive oropharyngeal matched primary CRC and liver metastasis samples in the context of SCC, and the aetiology of the disease. targetable treatments.

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SYM-34-04 SYM-35-01 systems biology analysis of epithelial- CLINICAL BACTERIOPHAGE THERAPY IN THE 21ST mesenchymal transition in pancreatic CENTURY cancer Morales S. Nagaraj Hiriyur, S. AmpliPhi Biosciences.

It is now unanimously accepted that the world has entered a dangerous phase in the treatment of multi-resistant bacterial infection. The problem ABSTRACT NOT AVAILABLE seems to be that the speed and ability of the bacterial community to AT TIME OF PUBLICATION develop resistance far exceeds the capacity of the biomedical and scientific community to develop new antibacterials. Faced with this situation is it perhaps time to acknowledge that the current antibiotic paradigm, if not broken, is in need of review, and that new alternative treatment options are urgently needed. A new antibacterial treatment would ideally have the capability of matching, and if possible exceeding the mutation rate of the bacterial targets. In other words, a form of intelligent antibacterial that out-mutates the mutators. Bacteriophage therapy, the use of highly specific bacterial viruses to treat bacterial infections, is one alter native with the potential to achieve this. Histor ic ally, bacteriophages (phages) were discovered 100 years ago at the Pasteur Institute in Paris. The therapy was widely practised until the middle of last century, until the advent of small molecule antibiotics such as penicillin saw the practise fade into obscurity for the next 50 years, before a major renaissance in the late 1990s. This talk will concentrate on the extensive progress made since that time in understanding the biology of phages and their potential for clinical application. The results from both pre-clinical and human clinical trials will be discussed, along with the issues facing the fledgling phage therapy industry such as production, regulatory and financial constraints. Exciting new developments in the combined use of phages and antibiotics will be described which offer the tantalising possibility that phage therapy might not only be able to contain antibiotic resistant bacteria but even reverse it.

SYM-35-02 SYM-35-03 Structure, Function, and Biosynthetic Origin IN SEARCH OF NEW ANTIBIOTICS THAT EXPLOIT THE of Octapeptin Antibiotics Active against CELL WALL DEFICIENT L-FORM LIFESTYLE Extensively Drug-Resistant Gram-Negative Bacteria Max D.O., Lazenby J.J. and Whitchurch C.B. The ithree institute, University of Technology Sydney, Ultimo, NSW, Tony Velkov,1,10,11, Alejandra Gallardo-Godoy,2,10 James D. Swarbrick,3 Mark. A.T. Australia. Blaskovich,2 Alysha G. Elliott,2 Meiling Han,4 Philip E. Thompson,3 Kade D. Roberts,3 Johnny X. Huang,2 Bernd Becker,2 Mark S. Butler,2 5 2 4 Pseudomonas aeruginosa is a Gram negative bacterial pathogen that Lawrence H. Lash, Sonia Troeira Henriques, Roger L. Nation, has been identified by the WHO as in critical need for the development Sivashangarie Sivanesan,4 Marc-Antoine Sani,6 Frances Separovic,6 Haydyn Mertens,7 Dieter Bulach,8 Torsten Seemann,8 Jeremy Owen,9 Jian Li,4, of new antibiotics. P. aeruginosa has high levels of intrinsic resistance and Matthew A. Cooper2 to a broad range of antibiotics due to possession of a highly 1Department of Pharmacology & Therapeutics, School of Biomedical Sciences, impermeant outer-membrane (OM) and multiple, inducible multi-drug Faculty of Medicine, Dentistry and Health Sciences, The University of efflux pumps. We have previously shown that P. aeruginosa tolerates Melbourne, Parkville, VIC 3010, Australia.2Institute for Molecular Bioscience, β-lactam antibiotics by undergoing a rapid and reversible en masse The University of Queensland, Brisbane, QLD 4072, Australia. 3Medicinal conversion from bacilli to cell wall deficient L-forms that lack the Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, OM. We hypothesized that L-form P. aeruginosa may be susceptible 381 Royal Parade, Parkville, 3052 VIC, Australia. 4Drug Delivery, Disposition to antibacterial compounds that are normally inhibited by the Gram and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash 5 negative cell wall. Indeed we found that L-form P. aeruginosa is rapidly University, 381 Royal Parade, Parkville, 3052 VIC, Australia. Department killed by antimicrobial peptides (AMPs) that are normally ineffective of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA6 School of Chemistry, Bio21 Institute, against bacillary P. aeruginosa. These observations suggest that University of Melbourne, Parkville, VIC 3010, Australia. 7EMBL, Hamburg, drug combinations that induce L-form transitions with compounds that Germany. 8Department of Immunology and Microbiology, University of exploit L-form susceptibility may be novel therapeutic options. We have Melbourne, Parkville, VIC 3010, Australia. 9School of Biological Sciences, developed high-throughput screens that enable the identification of Victoria University, Wellington 6012, New Zealand. compounds that induce or kill L-form bacteria. We have used our L-form kill assay to screen an FDA-approved drug library and have identified Resistance to the last-resort antibiotic colistin is now widespread and new ~100 drugs with enhanced bactericidal activity against L-form P. therapeutics are urgently required. We report the first in toto chemical synthesis and aeruginosa. Interestingly, most of these drugs have not previously been pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related identified as having any antibiotic activity indicating that there are a to colistin. The octapeptin biosynthetic cluster consisted of threenon-ribosomal number of FDA-approved drugs that might be able to be repurposed peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic for use as antibiotic therapeutics that target L-form bacteria. Our antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multidrug resistant (MDR) strains in vitro, octapeptin C4 observations indicate that the ability of bacteria to transition into CWD displayed poor in vivo efficacy, most likely due to high plasma protein binding. deficient L-form lifestyle to avoid the effect of compounds that target Nuclear magnetic resonance solution structures, empirical structure-activity and the bacterial cell wall may be exploited to develop novel antibiotic structure-toxicity models were used to design synthetic octapeptins active against therapeutic approaches. MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant Pseudomonas aeruginosa clinical isolate.

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SYM-35-04 SYM-35-05 STRUCTURAL AND KINETIC CHARACTERISATION NOVEL STRUCTURAL PROPERTIES OF A HAEMOPHORE- OF CLASS III BIOTIN PROTEIN LIGASES; NOVEL ANTI LIKE PROTEIN OFFER AN AVENUE FOR TARGETED FUNGAL DRUG TARGETS ELIMINATION OF PORPHYROMONAS GINGIVALIS 1 1 2 1, 3 3 Sternicki L.M. , Nguyen S. , Pukala T.L. , Pendini N.R. , Beckham S. , 1 2 1 2 2 2 3 1 1 1 Kwan A. , Gao J. , Yammine A. , Zhou Z. , Nguyen K. , Hunter N. and Wilce M.C. , Booker G.W. , Wegener K.L. and Polyak S.W. 3 1 Gell D. School of Biological Sciences, The University of Adelaide, North 1 Terrace, Adelaide, South Australia, Australia 5005. 2School of Physical School of Life and Environmental Sciences, University of Sydney, NSW 2006, Australia. 2Institute of Dental Research, Westmead Hospital, University Sciences, The University of Adelaide, North Terrace, Adelaide, South 3 3 of Sydney, NSW 2145, Australia. School of Medicine, University of Tasmania, Australia, Australia 5005. School of Biological Sciences, Monash TAS 7005, Australia. University, Clayton, Victoria, Australia 3800. The covalent post-translational attachment of biotin is necessary for Porphyromonas gingivalis is a keystone bacterium found in chronic periodontal the activity of certain metabolic enzymes. Biotin protein ligase (BPL) is disease, which is characterised by severe inflammation and destruction of responsible for this modification and has been proposed as a novel anti- supportive tooth structures. The disease has also been linked to systemic infective target. Crystal structures of class I and II BPLs, present in archaea pathologies including cardiovascular diseases and autoimmune disorders1. P. and bacteria, have been reported and have aided the design of inhibitors gingivalis growth is dependent on the acquisition of environmental haem for against bacterial BPLs. However, the class III BPLs, found in mammals, its iron and porphyrin requirements. As such, the bacterium has an array of fungi and insects, have not been extensively characterised nor exploited for proteins for haem acquisition, including HusA (Heme uptake system protein antifungal therapeutics. These BPLs differ as they contain a large N-terminal A). In previous work2, we have shown that HusA directly binds haem and is extension that is proposed to assist selection of appropriate biotinylation essential for P. gingivalisto grow under haem-limiting conditions, such as targets. Limited structural information, including the absence of a class III those found inside plaques and in saliva. Therefore, HusA may represent BPL crystal structure, has hindered the molecular understanding of substrate a therapeutic target for treating chronic periodontitis and possibly other recognition by the N-terminal extension and the development of antifungal associated systemic diseases. To develop compounds that target HusA inhibitors. Initial kinetic characterisation of four class III BPLs, namely and P. gingivalis, we have determined the solution structure of HusA using those from the fungi Saccharomyces cerevisiae, Candida albicans, Botrytis Nuclear Magnetic Resonance (NMR) spectroscopy. Using NMR titration cinerea and Zymoseptoria tritici, revealed different KM values for the studies, mutagenesis and in silico docking, we have identified that haem binds substrates. Likewise, inhibition constants for known substrate mimics varied in a hydrophobic groove on the α-helical structure that lacks the typical iron between the enzymes. These data reveal subtle structural differences coordination seen in other haemophores. This mode of interaction allows localised around the substrate binding sites between these class III enzymes, HusA to bind a variety of abiotic and metal-free porphyrins with significantly suggesting selective inhibition may be possible. The S. cerevisiae BPL higher affinity than to haem. We have exploited the unusual porphyrin binding was more thoroughly investigated for structural insights, as crystallography activity of HusA to target a prototypical deuteroporphyrin-metronidazole of these four BPLs has so far been unsuccessful. SAXS, ion mobility MS conjugate with restricted antimicrobial specificity in a “Trojan horse” strategy and hydrogen-deuterium exchange MS are being employed to investigate that effectively kills intracellular P. gingivalis. Therefore, targeting and conformational changes and enzyme dynamics associated with ligand manipulating HusA:porphyrin interactions is a promising avenue to developing binding. Crosslinking MS is also being utilised to delineate how the N-terminal new therapeutics against P. gingivalis. 1. Seymour GJ, Ford PJ, Cullinan MP, domain facilitates interactions with substrates targeted for biotinylation. This Leishman S, Yamazaki K. (2007) Relationship between periodontal infections information will aid the refinement of homology models of S. cerevisiae BPL to and systemic disease. Clin Microbiol Infec 13:3. 2. Gao JL, Nguyen KA, Hunter provide a model of a class III BPL structure. This structural information is vital NJ. (2010) Characterization of a hemophore-like protein from Porphyromonas for the development of selective anti-fungal inhibitors that target pathogenic gingivalis. J Biol Chem 285:40028. . BPLs but not the human isoform.

SYM-36-01 SYM-36-02 SUPPORT NETWORKS FOR EMCRS HOW TO MAKE VALUABLE CONNECTIONS BETWEEN ACADEMIA AND INDUSTRY Bowden N.A.1, 2 1University of Newcastle, University Dr, Callaghan, NSW Oliver B. 2308. 2Hunter Medical Research Institute, Kookaburra Cct, New University of Technology Sydney and Woolcock Institute of Medical Lambton Heights, NSW 2305. Research, Australia.

Building a network can seem difficult and many EMCRs don’t know Industry is a broad term used for everything from spinout companies where to start. Having a support network is crucial for researchers at all with 1 employee, to large multinational companies, and how you career stages, but is particularly important when starting a career and interact with each needs to be different. Smaller companies often when transitioning into leading a team. Australia has several different paradoxically have more money to invest in partnerships and projects types of networks open to EMCRs and I will discuss how to get involved as they don’t have the capacity inhouse, but they often want their and build your own network locally, nationally and internationally. “pound of flesh”. Large companies are shrewder with their investments, but generally expect less in return (ie they are more realistic). In this short presentation, I will attempt to cover the question: So what should you do? For example, should you develop the relationship by doing Pro Bono research, quote $500,000 for 6 months work, or just not bother. Do you chase industry or do they come to you? What is that, that you have produced again - the worlds first recombinant monkey TNF? What exactly are you going to do together? I will also talk about barriers to effective interactions, e.g. oops I forgot about IP, I don’t have enough money (institutional overheads).

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SYM-36-03 SYM-36-04 SHAMELESS SELF-PROMOTION: CAN YOU FAKE IT CREATING USEFUL MENTOR RELATIONSHIPS TILL YOU MAKE IT? Georgousakis M. Bouveret R. Franklin Women, Sydney NSW 2065 University of New South Wales, Sydney, NSW 2052. Mentoring relationships are very valuable for research career Self-promotion presents real benefits but also some risks. If well progression however there is much confusion around what a mentor executed, it allows you to communicate your skills and interests and actually does and how you go about getting, and keeping, one. In this to influence how others, including employers, see you. If you can self- talk Melina will describe the different types of professional development promote and influence how others perceive and remember you, you relationships: Mentoring, Sponsorship and Coaching. She will also are more likely to get opportunities to do what you wish to do. However, share her personal experiences with each of them in her own research self-promotion is also risky. When poorly done, it is often associated career as well as lessons learnt after launching the first cross- with bragging, obnoxiousness or narcissism. In this talk, I will share organisational mentoring program in the health and medical research my own experience and views on how to build a personal brand with sector that specifically aims at support women aspiring to leadership modesty and humility. Ultimately, what matters most is not getting roles in their careers. better offers or being promoted, it is having talents and interests, and being remembered for doing well and doing good.

SYM-36-05 SYM-37-01 GOVERNANCE AND POLICY: WHY SUSTAINING HOW PLASTIC ARE THE C4 SUBTYPES? LINKS WITH GOVERNMENT IS IMPORTANT TO YOUR INVESTIGATING THE PLASTICITY OF C4 GRASSES FUTURE THROUGH EXPOSURE TO LOW LIGHT AND LOW CARBON DIOXIDE Phillips, P. Watson-Lazowski A., Sagun J., Koller F., Papanicolaou A. and Ghannoum O. Hawkesbury Institute for the Environment, Western Sydney University, ABSTRACT NOT AVAILABLE Locked Bag 1797, Penrith, NSW 2753, Australia. AT TIME OF PUBLICATION C4 photosynthesis is an essential process which accounts for ~25% of total plant productivity, generated from a relatively small subset of plant species (~3%). This increased productivity is due to the evolution of a carbon concentrating mechanism between the mesophyll and bundle sheath cells of C4 plants, which allows for an increased

concentration of carbon dioxide (CO2) around Rubisco. Three subtypes of C4 photosynthesis exist, each named after the main decarboxylase

they utilise to release CO2: NADP-ME, NAD-ME and PEPCK. A number of grass species which utilise C4 photosynthesis are essential to maintaining increasing food and energy demands, highlighting them as an important subset of species for investigation. Firstly, the genetic make-up of the C4 cycle within each subtype requires some elucidation, especially in regards to the NAD-ME subtype. Therefore, we provide an updated model for NAD-ME photosynthesis within grasses via analysis of transcript expression and gene evolution. We can then begin to understand the plasticity of each subtype, both within the C4 cycle and on a whole plant basis. Understanding this plasticity is key to identifying both beneficial traits and targets for improvement. Utilising multiple C4 grass species, spanning two independent origins

and all three subtypes, we subjected plants to ambient, low CO2 (180 ppm) and low light (200 PAR) conditions. By comparing phenotype, physiology, biochemistry and transcript expression we are able to dissect the plasticity of C4 grasses, as well as identify candidate genes which may be utilised to improve C4 crop productivity.

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SYM-37-02 SYM-37-03 PROFILING OF THYLAKOID COMPLEXES FROM THE STRATEGIES FOR IMPROVING PHOTOSYNTHETIC MESOPHYLL AND BUNDLE SHEATH CELLS OF C4, C3 ELECTRON TRANSPORT IN C4 PLANTS AND C2 PANICUM GRASSES BY BN/PAGE AND LC/MS 1 1, 2 1 2 2 Ermakova M. , Furbank R. and von Caemmerer S. Hernandez-Prieto M.A.1, Foster C. , Watson-Lazowski A. , 1 2 1 ARC Centre of Excellence for Translational Photosynthesis, Ghannoum O. and Chen M. 2 1 Australian National University, Canberra, Australia. CSIRO ARC Centre of Excellence for Translational Photosynthesis, The University Agriculture, Canberra, Australia. of Sydney, School of Life and Environmental Sciences, University of Sydney, NSW 2006. Australia. 2ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney Recent activities to improve photosynthetic performance in crop University, Locked Bag 1797, Penrith NSW 2751, Australia. plants have focused primarily on C3 photosynthesis where there are clear identified targets such as improving Rubisco kinetics, installation Photosynthetic efficiency is largely limited because of the low specificity of a CO2 concentrating mechanism and alleviating limitations in of Ribulose-1,5-bisphosphate carboxylase/ (RuBisCO). C4 chloroplast electron transport. However, C4 plants that utilise the C4 plants have higher efficiency compared to C3 plants because they use photosynthetic pathway also play a key role in world agriculture and two carboxylation cycles, physically separated into two cell types: the strategies to manipulate and enhance C4 photosynthesis thus have mesophyll (MC) and the bundle sheath cells (BSC). This permits C4 plants to potential for major agricultural impacts. The C4 photosynthetic pathway concentrate CO2 around RuBisCO, which favors the carboxylation pathway is a biochemical CO2 concentrating mechanism that requires the over oxygenation. The fact that C4 photosynthesis evolved independently coordinated functioning of mesophyll (M) and bundle sheath cells (BS) more than 60 times (Osborne and Sack, 2012) is indicative of its success. of leaves and species have evolved a complex blend of anatomy and Depending on the enzyme responsible for C4 decarboxylation, C4 plants can be divided into different biochemical subtypes: NADP-malic enzyme biochemistry to achieve this. Chloroplast electron transport in C4 plants (ME), NAD-ME, and phosphoenolpyruvate carboxykinase (PEP-CK). is shared between these two cell types and the diversity of thylakoid The plant genus Panicum, similarly to Flaveria, contains species that protein complexes of each cell type is defined by the requirements of perform typical C3 photosynthesis (P. bisulcatum), C2 intermediates (P. the metabolic sub-type of C4 photosynthesis. Our recent work with milioides), as well as representative of the main C4 biochemical subtypes: the model monocot C4 species Setaria viridis (green foxtail millet) and C4/NADP-ME (P. antidotale), C4/NAD-ME (P. miliaceum), and C4/PCK transgenic S.viridis plants with altered amount of cytochrome (Cyt) b6f (P. maximumrenamed Megathyrsus maximus). Because of the interest in complex demonstrates the link between electron transport capacity introducing some of the characteristic of C4 photosynthesis into C3 crops, of the leaves and CO2 assimilation. Overexpression of the Cyt b6f in the study of plant families representing the evolutionary and photosynthetic both M and BS allows higher rates of assimilation in transgenic plants gradient from C3 to C4 is required. An important aspect of this transition that without affecting Rubisco content. However, increasing the amount needs to be compared is the organization of the complexes involved in the of the Cyt b6f only in M, surprisingly, leads to a reduced rate of CO2 light reactions to compensate for the higher demand of ATP in C4 plants. To assimilation at low CO2. We link this observation to measurements of compare the chloroplast organization of these species, we tested different electron transport components and light harvesting capacity of BS. methods to separate BSC chloroplasts from MC chloroplasts, and optimized the thylakoid preparation to obtain comparable band patterning in Native polyacrylamide gel electrophoresis (PAGE) under non-denaturing (native) conditions. Our results, comprising the analysis of 130 bands, showed that both C4 plants of the NADP-ME subtype analyzed have a higher proportion of PSI and NDH complexes with respect to PSII in BSC than in MC. No such imbalance was observed in C4 species of the other biochemical subtypes.

SYM-37-04 SYM-37-05 UNDERSTANDING AQUAPORINS AS POTENTIAL EXPLORATION OF SEASONAL CHANGE OF THERMAL ENHANCERS OF PHOTOSYNTHESIS AND PLANT TOLERANCE AND PROTEINS IN THREE AUSTRALIAN PERFORMANCE DESERT PLANTS

Groszmann M., De Rosa A., Mani-George A., Skinner S., Milner K.V.1, Van Sluyter S.C.2, French K.3, Valenzuela S.M.1 and von Caemmerer S. and Evans J.R. Leigh A.1 1 ARC Centre of Excellence for Translational Photosynthesis. Research School of Life Sciences, University of Technology Sydney, 2 School of Biology. The Australian National University. Canberra ACT, NSW. Department of Biological Sciences, Macquarie University, 3 2601. NSW. School of Biological Sciences, University of Wollongong, NSW.

Plants are in constant exchange with the surrounding environment, Plants in Australia’s southern semi-arid zone experience a wide range absorbing and transporting nutrients and gases essential for growth. of temperatures (4.6 °C mean minimum in winter and 34.2 °C mean Aquaporins represent a major class of channel proteins that aid in maximum in summer) and show plasticity in their thermal tolerance this process by increasing the permeability of biological membranes. thresholds with the ability to shift photosynthetic thresholds upwards Different aquaporin isoforms can selectively facilitate the transmembrane 5 °C from winter to summer. An understanding of how they are able transport of not only water, but also a range of other small molecules to make these threshold adjustments is required. With a new absolute and gases necessary for optimal plant performance. Amongst these protein quantification method, we have the opportunity to identify protein changes that may explain this ability. A selection of three is carbon dioxide (CO2), a key substrate for photosynthesis. For the majority of plants, photosynthetic efficiency is limited by the amount of species from different functional groups were used to explore species differences. Acacia ligulata a widespread nitrogen-fixing shrub, CO2 diffusion from the atmosphere to the chloroplast where it is fixed into sugars. A strategy to increase photosynthetic efficiency could be Myoporum montanum a widespread shrub and Solanum oligacanthum a perennial herb or subshrub were grown in an experimental garden to use aquaporins to enhance CO2 diffusion to the chloroplasts by increasing transport across the plasma membrane and chloroplast in semi-arid South Australia. Sampling occurred in winter, spring and envelope (collectively termed mesophyll conductance). Of the few summer where the assessment of thermal tolerance included a series of temperature assays for photosynthetic thermal tolerance (T using aquaporins so far shown to enhance membrane permeability to CO2, 50 these all belong to the Plasma membrane Intrinsic Proteins (PIP) chlorophyll fluorescence) and membrane stability (Tcrit using electrical sub-family. PIP aquaporins also transport a range of other substrates conductivity). Protein identification and absolute quantitation used a necessary for optimal plant performance, making them exciting targets new extraction method and QconCAT-spiked samples coupled with MS/ for engineering crop improvements. I will present our progress towards MS and SWATH acquisition. All species were able to adjust upwardly characterising PIP aquaporin biology and their potential translational thermal tolerance thresholds from cooler to warmer months. However, use in crop species. the changes seen in proteins of interest differed depending on plant species. This early exploration of temporal protein changes provides insight into acclimatisation mechanisms Australian desert plants use to cope in a difficult climate.

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SYM-38-01 SYM-38-02 PRECISION MEDICINE IN A DISH ‘NEXTGEN’ HUMAN BRAIN ORGANOIDS USING 3D PRINTED GELATIN METHACRYLATE Palmieri M., Hirokawa Y., Leong E., Burgess A. and Sieber O. 1, 2, Zhang B.1, Bourke J.L.3, 4, Gu Q.1, Kapsa R.M.3, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Tomaskovic-Crook E. Wallace G.G.1 and Crook J.M.1, 2, 5 Parade, Parkville, VIC 3052, Australia. 1ARC Centre of Excellence for Electromaterials Science (ACES), AIIM, University of Wollongong, Innovation Campus, North Wollongong, Despite the exponential increase in our knowledge of cancer genetics, NSW. 2IHMRI, University of Wollongong, Wollongong, NSW. 3ACES, St our ability to effectively treat tumours based upon their genetics alone Vincent’s Hospital, University of Melbourne, Fitzroy, VIC. 4Dept Medicine, remains limited. While determining the sensitivity and resistance of an St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC. 5Dept organism before treatment has been the standard of care in infectious Surgery, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC. diseases for many years, therapy selection in oncology continues to be initiated largely based on tumour histology. Patient-derived tumour The generation of brain organoids derived from human pluripotent stem cells organoid (PDTO) technology holds promise for changing the current (PSCs) is a significant step towards better in vitro modelling of neurodevelopment treatment paradigm, providing a system amenable to culture and “real- and disease. Brain organoids are discerned by their cellular and structural time” drug sensitivity testing in clinical practice to inform decisions in complexity, with characteristics of developing embryonic brains. Conventional a timeframe that is useful to an individual patient. This presentation methods of organoid formation are limited to small-scale operation and require multiple handling steps following stem cell aggregation, coating with expensive will highlight the challenges and lessons learned from our efforts to TM establish high-throughput clinical and research workflows for PDTOs and undefined tumour-derived Matrigel basement membrane preparation, and arduous bioreactor based differentiation and expansion methods. We derived from human colorectal cancers. have initially demonstrated gelatin methacrylate (GelMA) to be a cell growth substrate for rapid and novel induction of brain organoids from human induced PSCs (iPSCs). GelMA is a versatile, 3D printable semisynthetic matrix that incorporates the intrinsic bioactivity of natural matrices with the fidelity of synthetic biomaterials for more defined and clinically-compliant cell support. Towards scaling up organoid production we have now 3D printed GelMA-based microwell arrays to generate large numbers of organoids for higher throughput R&D. Constructs consist of densely packed cell soma with regional divisions resembling cortical plate or rudimentary grey matter tissue with underlying white matter-like tissue, as well as hollow neural tube-like structures. With larger numbers of organoids we are progressing our understanding through immunofluorescent-based histochemistry of cortical lamination using layer- specific markers of cerebral neocortex and early progenitor regions; markers including RELN, CTIP2, TBR1, SATB2, PAX6, NES, and SOX2, as well as the forebrain specific marker, FOXG1. Moreover, we have demonstrated coordinated glutamate-responsive neural network activity of formed neurons by extracellular recordings using multi-electrode arrays (MEAs). The optimised ‘NextGen’ method provides a defined, simplified and higher throughput platform for ‘brain on a bench’ research and translation of iPSCs, neural derivatives and neural organoids, including in vitro modelling of brain development and disease, tissue engineering and regenerative medicine.

SYM-38-03 SYM-38-04 SCREENING IN HUMAN CARDIAC ORGANOIDS NEUREGULIN1 IS A KEY NICHE SIGNAL THAT IDENTIFIES A REQUIREMENT FOR THE MEVALONATE SUPPORTS INTESTINAL STEM CELL PROLIFERATION PATHWAY IN CARDIOMYOCYTE PROLIFERATION Jarde T.1, 2, 3, Rossello F.1, 2, 4, Kurian Arackal T.1, 2, Flores T.1, 2, Giraud M.1, Hudson J.E. 2, Prasko M.1, 2, Nefzger C.M.1, 2, 4, Abe S.5, Polo J.M.1, 2, 4 and Abud H.E.1, 2 QIMR Berghofer Medical Research Institute. 1Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia. 2Cancer Program, Monash Biomedicine 3 Rationale: Induction of endogenous cardiomyocyte proliferation Discovery Institute, Clayton, Australia. Centre for Cancer Research, 4 is a promising strategy for cardiac regeneration. Recent studies Hudson Institute of Medical Research, Clayton, Australia. Australian suggest that dual inhibition of glycogen synthase kinase 3 (GSK3) Regenerative Medicine Institute, Monash University, Clayton, 5 and serine/threonine protein kinase 4 (STK4 aka MST1) potently Australia. Department of Biological Sciences, Graduate School of drives cardiomyocyte proliferation but the mechanisms of action Science and Technology, Kumamoto University, Kumamoto, Japan. are not defined. Objective: Decipher the effects of GSK3 and MST1 inhibition on cardiomyocyte proliferation and identify small molecules Identifying strategies to enhance intestinal stem cell proliferation that can drive proliferation without detrimental effects on contractile may enable regeneration of the epithelium to be manipulated in force. Methods and Results: We coupled high throughput proteomics degenerative diseases and intestinal pathologies. Using intestinal and functional screening of small molecules in single miniaturised organoids grown ex vivo, we have defined Neuregulin1/ErbB signalling human cardiac organoids (hCOs) to identify key pathways driving as a strong driver of cell proliferation in the intestinal epithelium. We cardiomyocyte proliferation. We report that GSK3 inhibition activates defined the localisation of Neuregulin1 and interacting receptors in the a cell cycle network whereas MST1 inhibition drives the mevalonate small intestine using immunofluorescence and qRT-PCR. We observed pathway, with synergistic effects on proliferation. However, all GSK3 that supporting niche cells express Neuregulin1, while stem cells inhibitors tested also reduced contractile force in hCO. This was express ErbB receptors, supporting a model where Neuregulin1/ErbB overcome by screening of 105 compounds, identifying a p38 inhibitor, signalling directly regulates stem cells. The role of Neuregulin1 was also which activated a cell cycle network without reducing force. The screen investigated in vivo using both a gene knockout approach and a model also identified a TGFBR inhibitor that induced the mevalonate pathway, where activation of Neuregulin1/ErbB signalling was achieved in mice increased protein prenylation and synergised with cell cycle activators injected with 15ug Nrg1 for 5 days. Elevation of signalling increased to promote proliferation. Inhibition of the mevalonate pathway abolished cell proliferation in crypts, altered cellular differentiation and promoted the myocyte proliferative response and also reduced cell cycle activity regeneration. Loss of Neuregulin1 resulted in a significant decrease in in immature cardiomyocytes, consistent with downregulation of the cell proliferation within crypts in both stem and progenitor cells. The mevalonate pathway during cardiac maturation in vivo. Conclusions: molecular changes induced by Neuregulin1 were examined using These findings implicate the mevalonate pathway in cardiomyocyte cell RNA sequencing which defined a proliferative molecular signature in cycle control, which could have important ramifications for congenital both stem and progenitor cells. This was reinforced by examining the heart disease and the development of regenerative therapeutic ability of single intestinal stem cells to generate organoids. Neuregulin1 strategies for heart failure. significantly promotes organoid growth and the formation of colonies from single stem cells.

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SYM-38-05 SYM-39-01 NOTCH1/NRG1 CONTROL OF CARDIAC JELLY CONTROL OF RECEPTOR FUNCTION THROUGH DYNAMICS DEFINES THE BUILDING PLAN FOR MEMBRANE EMBEDDED SEQUENCES TRABECULATION 1, 2 1 2 1 3 4 Call M.J. Del Monte-Nieto G. , Ramialison M. , Adam A.A.S. , Wu B. , Aharonov A. , 1 5 5 4 3 1 Structural Biology Division, The Walter and Eliza Hall Bourke L.M. , Harten S.K. , Tzahor E. , Zhou B. and Harvey R.P. 2 1Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales Institute. Department of Medical Biology, University of Melbourne. 2010, Australia. 2Australian Regenerative Medicine Institute. Monash University, Victoria 3800, Australia. 3Albert Einstein College of Medicine To sense the external environment, cells rely on receptors that span the of Yeshiva University, Bronx, New York 10461, USA. 4Weizmann Institute membrane and transmit signals to the cell interior. Our understanding of Science, Rehovot 7610001, Israel. 5QIMR Berghofer Medical Research of how single-pass receptors transmit signals is lacking in part because Institute, Herston, Queensland 4006, Australia. we do not have full-length structures for single-pass receptors. It is becoming more apparent that single-pass receptor systems act in homo- In vertebrate hearts, ventricular trabecular myocardium develops as a sponge- and hetero-oligomeric formats and that the transmembrane domains of like network of cardiomyocytes critical for efficient cardiac contraction in the receptors play important roles in mediating signal transduction across early embryo, and for ventricular conduction, septation, and wall thickening through the process of compaction in the foetal heart. Defective trabeculation at the membrane, providing contacts around which receptor chains are early developmental stages leads to embryonic lethality whereas defects at later organised and are targets for regulation by other cellular components. stages can lead to Non-Compaction Cardiomyopathy, the third most commonly We are interrogating the function of single-pass receptors by studying diagnosed cardiomyopathy in adults. There are divergent views on when and how interactions among transmembrane domains to develop a more trabeculation is initiated in different species. In mice, the onset of trabeculation comprehensive view of receptor function and regulation. has not been formally addressed, but is proposed to begin at embryonic day (E)9.0 as cardiomyocytes form radially-oriented ribs. Endocardium-myocardium communication is essential for trabeculation, with numerous signalling pathways identified, including Neuregulin1 (Nrg1) and Notch1. Late disruption of the Notch1 pathway causes Non-Compaction Cardiomyopathy. Although mutants in cardiac jelly extracellular matrix (ECM) genes Has2 and Vcan lack trabeculae, and matrix metalloprotease ADAMTS1 activity at E14.5 promotes total cardiac jelly degradation and trabecular growth arrest, the role of ECM dynamics and its molecular regulation during early trabeculation is poorly understood. Here we present a new model of cardiac trabeculation starting as early as the heart tube forms (E8.0), integrating for the first time dynamic endocardial and myocardial cell behaviours, and ECM remodelling, and revealing new epistatic relationships for known signalling pathways. Notch1 signalling promotes ECM degradation during formation of endocardial projections critical for individualization of trabecular units, while Nrg1 promotes myocardial ECM synthesis, necessary for trabecular rearrangement and growth. These systems interconnect through Nrg1 control of Vegfa, yet act antagonistically to establish trabecular architecture. Furthermore, these insights allowed the prediction of persistent cardiac jelly and trabecular growth as a potential cause of disease in a murine Non-Compaction Cardiomyopathy model, providing new understanding of congenital heart disease pathophysiology.

SYM-39-02 SYM-39-03 TOWARDS CAPTURING AN ATOMIC VIEW OF HOW ARE SIGNALS TO DEVELOPING T CELLS THE ACTIVATION OF P-REX ONCOGENES AT THE COORDINATED AT THE MEMBRANE? MEMBRANE Russell S.1, 2, Charnley M.1, 2 and Allam A.1, 2 Lucato C.M.1, Mitchell C.A.1, Whisstock J.C.1, Halls M.L.2 and 1Peter MacCallum Cancer Centre. 2Swinburne University of Ellisdon A.M.1 Technology. 1Biomedicine Discovery Institute, Monash University. 2Monash Institute of Pharmaceutical Sciences, Monash University. T cell development in the thymus is precisely orchestrated by a interactions with thymic stroma, but the mechanisms of these P-Rex1 and 2 are guanine nucleotide exchange factors that activate interactions are not yet clear. We find that developing T cells assemble a number of Rho family GTPases that are crucial in regulating cell a signaling platform at the interface with stromal cells that is analogous growth and motility. Interestingly, P-Rex proteins regulate these cellular to the immunological synapse of mature T cells. This finding is intriguing functions downstream of a number of GPCRs and RTKs and, in doing because the developing T cells do not yet express the peptide-specific T so, function as a membrane-localised signalling node in many cancer- Cell Receptors that are essential for immunological synapse formation. associated pathways. Recently, P-Rex1 expression was shown to be Here, we describe the mechanisms by which developing T cells necessary for melanoma dissemination and has been associated with assemble an antigen-independent immunological synapse, and the increased metastatic phenotypes in both breast and prostate cancer. consequences of that synapse on subsequent T cell fate decisions. P-Rex1 is therefore a critical signal integrator in tumorigenesis and metastasis and subsequently a desirable therapeutic target, however currently there is little mechanistic understanding of its mode of activity. Crystallization and structural analysis of the catalytic DH-PH domains of P-Rex1 have allowed the elucidation of its mechanism of nucleotide exchange. This exchange activity is inhibited by interactions with P-Rex1 C-terminal domains which have been mapped with cross-linking and mass spectrometry. Interestingly, intra-domain cross-links move significantly upon effector binding and analysis of these movements has allowed for characterization of the multi-step P-Rex1 activation pathway. Further, molecular differences between autoinhibited P-Rex1 (185kDa) and effector-bound, partially activated P-Rex1 complexes (~230kDa) have been visualized with cryo-electron microscopy and resulting models have provided further molecular insight into the mode of P-Rex1 activation.

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SYM-39-04 SYM-39-05 ANNEXIN A6 DEPLETION RESCUES CHOLESTEROL RETROMER IS REQUIRED FOR THE RETROGRADE EGRESS IN NPC1 MUTANT CELLS VIA FORMATION OF SORTING OF CATION-INDEPENDENT MANNOSE ENDOSOME-ER MEMBRANE CONTACT SITES 6-PHOPHATE RECEPTOR INTO A SUBSET OF ENDOSOME TRANSPORT CARRIERS Grewal T.1, Meneses-Salas E.2, Garcia-Melero A.2, Bianco-MuñOz P.2, Egert A.1, Beevi S.S.1, Rentero C.2 and Enrich C.2 Cui Y.1, Carosi J.2, 3, Yang Z.1, Kerr M.4, Sargeant T.2, 5 and Teasdale R.1 1School of Pharmacy, University of Sydney, Sydney, NSW 2006, 1School of Biomedical Sciences, Faculty of Medicine, The University Australia. 2Departament de Biomedicina, Unitat de Biologia Cel·lular, of Queensland, Brisbane, Queensland, Australia. 2Hopwood Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, Centre for Neurobiology, Nutrition and Metabolism Theme, South 08036-Barcelona, Spain. Australian Health and Medical Research Institute, Adelaide, South Australia, Australia. 3Centre for Cancer Biology, University of South The ac c umulation of c holesterol in late endosomes (LE) / lysosomes of Niemann - Australia, Adelaide, South Australia, Australia. 4Institute for Molecular Pick type C1 (NPC1) mutant cells critically disturbs intracellular cholesterol Biosciences, The University of Queensland, Brisbane, Queensland, transport. Besides NPC1, oxysterol-binding proteins (ORP1L, ORP5), StAR Australia. 5School of Pharmacy and Medical Sciences, University of related lipid transfer domain (STARD) proteins, and several Rab proteins also South Australia, Adelaide, SA, Australia. facilitate cholesterol export from LE. Some of these proteins are engaged in the formation of membrane contact sites (MCS), defined by the apposition of two cellular membranes, providing opportunity for non-vesicular transport of lipids, Retromer is a peripheral protein complex that coordinates multiple including cholesterol. Hence, yet unidentified players or gatekeepers in LE may vesicular trafficking events within the endo-lysosomal system. Here, control activation or fine-tune alternative LE-cholesterol transport routes other we demonstrate that retromer is required for the maintenance of than NPC1. Annexin A6 (AnxA6), a member of the annexin family, has been normal lysosomal function. At the whole cell level, the knockout of implicated in endo- and exocytic pathways and cholesterol homeostasis. We retromer Vps35 subunit reduces the lysosomal proteolytic capacity, as previously demonstrated that AnxA6 is recruited to Low Density Lipoprotein a consequence of the improper processing of lysosomal , (LDL) -cholesterol enriched LE. Moreover, similar to the loss of NPC1, AnxA6 dependent on the cation-independent mannose 6-phophate receptor overexpression led to LE-cholesterol accumulation. Here, we demonstrate (CI-M6PR) trafficking. Moreover, we identify that CI-M6PR, sorted that this cellular cholesterol imbalance is due to AnxA6 recruiting TBC1D15, a via the retromer-dependent process, is incorporated into a subset Rab7-GTPase activating protein (Rab7-GAP), to cholesterol-rich LE to inhibit of endosome transport carriers (ETCs) tethered by a specific trans- Rab7 GTPase activity. In contrast, AnxA6 depletion elevated Rab7 activity, Golgi protein. Finally, we show that this retromer-dependent retrograde rescuing cholesterol export from LE in NPC1 mutant cells. This was associated cargo trafficking pathway also requires a specific retromer-associated with peripheral distribution and increased mobility of LE and enhanced lipid endosomal protein. accumulation in lipid droplets (LDs) in an acetyl-coenzyme A acetyltransferase (ACAT)-dependent manner. Moreover, StARD3 depletion compromised LE-Chol export and lipid deposition in LD in AnxA6-deficient NPC1 mutant cells. Electron microscopy revealed a significant increase of MCS between LE and the endoplasmic reticulum (ER) in NPC1 mutant cells lacking AnxA6, suggesting LE-cholesterol transfer to the ER via StARD3-dependent MCS formation. Taken together, this study identifies an annexin, AnxA6, as a novel gatekeeper that controls cellular distribution of LE-Chol via two critical biological activities: regulation of a Rab7-GAP and MCS functioning.

SYM-40-01 SYM-40-02 UNANTICIPATED PROTEOLYSIS DIVERSIFIES DISSECTING THE SUBCELLULAR SECRETORY TARGETS OF IMMUNITY GLYCOPROTEOME WITH MASS SPECTROMETRY PROTEOMICS Purcell A.W.1, 2 1Department of Biochemistry, Monash University. 2Infection and Zacchi L.F.1, Phung T.2 and Schulz B.L.1, 2 Immunity Program, Biomedicine Discovery Institute, Monash 1Centre for Biopharmaceutical Innovation, Australian Institute University. for Bioengineering and Nanotechnology, The University of Queensland. 2School of Chemistry and Molecular Biosciences, The The cellular immune response relies upon T cell recognition of peptides University of Queensland. presented on the cell surface in complex with HLA molecules. As such, it is the peptide cargo of these HLA molecules that dictates the quality of N-glycosylation is a critical post-translational modification that the immune response and ultimately the efficacy of protective immunity. influences the folding and function of 1/3rd of the cellular proteome. Relatively simplistic models have been used to explain how these The biosynthesis of N-glycoproteins begins in the endoplasmic peptide antigens are generated and selected for presentation, however, reticulum (ER), where an oligosaccharide is transferred to selected such models fail to predict and explain the diversity and complexity of asparagine residues in nascent polypeptides by the enzyme the immune response. Much of this unexplained complexity resides oligosaccharyltransferase. The presence of N-glycans at specific in degenerate and reconstructive proteolysis. We have recently sites is critical for productive protein folding in the ER, and defects in highlighted the complex role of proteolysis in the generation and this process perturb glycoprotein folding, secretion, and function at a diversification of peptide antigens displayed for T cell recognition. systems level. We have developed integrated subcellular fractionation For example, functional heterogeneity is observed during degenerate and SWATH glycoproteomic workflows to understand the causes and peptidase trimming of T cell epitopes such that peptides with ragged N- consequences of changes in the N-glycosylation biosynthetic pathway. or C-termini (nested peptides) can be presented with different immune We combined biochemical subcellular fractionation methods with outcomes. Moreover, we have generated evidence that reinforces quantitative SWATH-MS glycoproteomic and proteomic workflows to recent surprising studies that up to 30% of peptides presented by class measure the response to a range of genetic and chemical perturbations I HLA molecules are generated by post-translational proteasomal to N-glycoprotein biosynthesis. We optimized biochemical fractionation splicing (i.e. the ligation of peptide fragments within the proteasome methods in yeast to enable precise analysis of the subcellular proteome rather than peptide destruction). Using a novel bioinformatic workflow and glycoproteome. This enabled quantitative measurement of we show that these spliced peptides can be generated either from subcellular proteomes and site-specific and global profiling of glycan two regions of the same antigen (cis-splicing) or two distinct antigens occupancy and structure. We tested these methods in yeast with (trans-splicing) by retrospectively interrogating datasets for several defined defects in N-glycosylation, and then expanded our analysis common HLA allotypes. This is the first demonstration that trans- to profile the quantitative effects of combined defects in glycoprotein spliced peptides are abundantly represented in the immunopeptidome. biosynthesis and protein quality control on glycoprotein maturation. Our However, the role of spliced peptides in immune responses and the results give key insights into the effect of site-specific glycosylation on precise mechanism of their generation remains poorly understood and glycoprotein quality control processes, and our methods will be useful will be discuss. in diverse applications in industrial and medical glycobiotechnology.

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SYM-40-03 SYM-40-04 MULTI-OMIC PROFILING OF THE LIVER IN A RAT GOOD AND BAD FAT: DISCOVERING KEY MODEL OF TYPE 2 DIABETES DISTINGUISHING FEATURES WITH MULTIFACTORIAL PROTEOMICS DATA Li D.K.1, Smith L.E.1, Koay Y.C.1, 2, McEwan H.1, 3, Don A.1, 3, O’Sullivan J.1, 2, Cordwell S.J.1 and White M.Y.1 Deshpande V.1, Humphrey S.J.1, Yang P.2, Lo K.2, Healy M.E.1, Cooke K.C.1, 1University of Sydney, NSW, Australia. 2Heart Research Institute, NSW, Stoeckli J.1, Yang J.Y.H.2 and James D.E.1 Australia. 3ACRF Centenary Cancer Research Centre, NSW, Australia. 1Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia. 2School of Mathematics and Altered glucose metabolism via insulin resistance is a hallmark of type 2 Statistics, The University of Sydney, NSW 2006, Australia. diabetes (T2D), clinically observed as the inability to maintain postprandial blood glucose levels (BGL). Associated with energetic excess arising Subcutaneous (SC) and visceral (VIS) adipocytes store energy as fat from caloric overload, T2D is linked to excess non-esterified fatty acid and regulate whole body metabolism. Excessive VIS fat is associated production and rising nutrient levels, which influence metabolic processes. with insulin resistance, a precursor to Type 2 diabetes. In contrast, SC The liver plays a pivotal role in the pathogenesis of T2D, via elevated fat may be protective. Despite these important physiological functions, gluconeogenesis, whereby glycogen stores are liberated, further elevating relatively little is known about the molecular features that distinguish BGL. It is important to understand the molecular adaptations of the liver to these discrete adipose depots. We have used mass spectrometry to the metabolic flux and insulin resistance arising from T2D. To achieve this construct proteomes of mouse SC and VIS depots, which consist of we performed a multi-omic analysis including proteomics, lipidomics and >7,500 quantified proteins spanning six orders of magnitude. Our metabolomics in a rat model of T2D combining the effects of high fat diet study consists of three experimental variables: depot (SC and VIS), feeding (calorie overload) and streptozotocin (elevated BGL). To quantify diet (normal and high-fat) and sample type (adipocytes and whole alterations in protein abundance, samples were isobarically tagged prior tissue). Given this multifactorial experimental design, we devised to mass spectrometry (MS). Discovery lipidomics was achieved with a computational framework to comprehensively and systematically relative quantitation by comparison with synthetic standards. Targeted answer biological questions. This bioinformatic approach involved a metabolomics was performed using multiple reaction monitoring, in the series of ANOVA models to stratify the proteome into distinct classes presence of deuterated metabolite standards. We quantified close to 7,000 defined by the variable(s) driving the changes in protein expression. proteins, 300 lipid species and 100 metabolites in the course of this study. We verified that positive control proteins were assigned to expected Proteomics revealed increased levels of proteins regulating phospholipid classes. Our results suggest that adipocytes, rather than the local biosynthesis and fatty acid metabolism. A concurrent decrease in proteins microenvironment, drive major differences between SC and VIS depots. involved in steroid biosynthesis was observed. Lipid analysis show increased Of the total proteins, ~2% were upregulated in SC relative to VIS, and sphingomyelin levels and decreased levels of phosphatidylcholines in T2D, ~4% upregulated in VIS relative to SC. These included coenzyme Q both of which are components of cell membranes and can play a role in and lipolysis proteins in SC, and collagens and cathepsins in VIS. metabolic and apoptotic signalling. Elevated levels of branched chain amino Thus we demonstrate the utility of this proteomic resource and analytic acids as well as changes in metabolites indicative of altered energy and approach in uncovering novel insights into adipocyte biology. amino acid metabolism were detected by metabolomics. The current study has identified changes in protein, lipid composition and metabolite levels indicative of dysregulated energy utilisation and molecular adaptations that contribute to the pathogenesis of T2D.

SYM-40-05 SYM-41-01 GLYCOCIN F: A BACTERIOSTATIC, GLYCOSYLATED UNDERSTANDING EARLY TOLL-LIKE RECEPTOR BACTERIOCIN SIGNALLING THROUGH MYDDOSOME EXAMINATION 1, 2 1, 2 3 4 3, 5, 6 1, 3 2 2 2, 3 1 De Nardo D. , Balka K.R. , Cardona Gloria Y. , Rao V.R. , Latz E. Bisset S.W. , Amso Z. , Yang S.H. , Brimble M.A. , Patchett M.L. 1, 2 1, 3 and Masters S.L. and Norris G.E. 1Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, 1Institute of Fundamental Sciences, Massey University, Colombo Rd, 1G Royal Parade, Parkville, Victoria, 3052, Australia. 2Department of Medical Palmerston North 4442, New Zealand. 2School of Chemical Sciences, Biology, The University of Melbourne, Parkville 3010, Australia. 3Institute The University of Auckland, 23 Symonds St, Auckland 1142, New of Innate Immunity, University Hospital, University of Bonn, Sigmund Freud Zealand. 3Maurice Wilkins Centre for Molecular Biodiscovery, The Str. 25, 53127, Bonn, Germany. 4Inflammation and Immunology, Pfizer Inc., University of Auckland, Private Bag 92019, New Zealand. Cambridge, Massachusetts 02139, USA. 5Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 6German Center for Neurodegenerative Diseases, Bacteriocins are a class of bacterial peptides that inhibit the growth Bonn 53175, Germany. of closely-related strains or species of bacteria. Glycocin F (GccF) is a glycosylated, 43 amino acid bacteriocin (glycocin) produced Toll-like receptors (TLRs) form part of the host innate immune system, where they act as sensors of microbial and endogenous danger signals. Upon activation, the by a strain of the probiotic bacteria Lactobacillus plantarum. GccF intracellular Toll/Interleukin-1 receptor (TIR) domains of TLR dimers form a platform for contains two N-acetylglucosamine (GlcNAc) moieties required for oligomerisation of a multiprotein signalling platform comprising MyD88 and members of full anti-bacterial activity, one O-linked through serine 18, and the the IRAK family, termed the Myddosome. Formation of the Myddosome complex initiates other S-linked through the C-terminal cysteine 43 at the end of a signal transduction pathways leading to the activation of transcription factors and flexible tail. Two nested disulfide bonds are also required for activity. ultimately, the production of inflammatory cytokines. Despite the critical role Myddosome formation plays in initiating TLR-induced signalling, the molecular mechanisms Whereas most bacteriocins exhibit bactericidal effects on a narrow controlling Myddosome function remain poorly defined. Using immunoprecipitation range of species, GccF displays a potent, immediate and reversible approaches we successfully isolated Myddosome complexes from whole cell lysates bacteriostatic activity towards a range of Gram positive bacteria, and of TLR activated primary mouse bone marrow-derived macrophages (BMDMs) and at least one Gram negative strain, making it a potentially useful tool from IRAK-deficient immortalised BMDMs reconstituted with WT and mutant forms of IRAKs via retroviral transduction. Through the use of a selective IRAK4 inhibitor we were for combating antibiotic resistant bacteria. Although a GlcNAc-specific able to examine the role of IRAK4 kinase activity within the Myddosome. Immunoblot phosphoenolpyruvate:sugar phosphotransferase system (PTS) EIIC and immunofluorescence techniques were used to assess TLR signalling and NF-κB domain has been shown to be one target, there is evidence that another translocation respectively, while ELISAs were employed to examine the secretion of target may be involved. In order to understand how GccF works, we pro-inflammatory cytokines into the supernatant. Here we demonstrate the kinetics of have chemically synthesised a number of GccF analogues designed to the Myddosome upon TLR activation, revealing rapid assembly and slow disassembly. Furthermore, we show that inhibition of IRAK4 kinase activity leads to increased stability probe structure-activity relationships, have used genetic engineering of the Myddosome complex as demonstrated by greater associations between MyD88 techniques to probe potential receptor-GccF interactions, and are and IRAK4. Importantly, we found that the kinase activity of IRAK4 is dispensable for currently investigating the structure of one particularly interesting TLR-mediated NF-κB and MAPK signalling but essential for production of inflammatory analogue using NMR. A transcriptomic study of susceptible cells has cytokines. To our knowledge this is the first full examination of the kinetics of the Myddosome from macrophages. We further demonstrated that a loss of IRAK4 activity also been carried out in the absence and presence of GccF in an effort by either chemical inhibition or genetic manipulation resulted in a significantly more stable to narrow down the pathways exploited by GccF. These results will be Myddosome structure. This increase in stability is suggestive of a prominent protein presented, along with a model for the mechanism of action that best fits scaffold role of IRAK4, independent of its kinase activity, in which IRAK4 interacts with the data we have collected thus far. MyD88 and IRAK1, tethering them together into the Myddosome complex. Our findings may help explain why, to date, therapeutic targeting IRAK4 kinase activity has not been as successful as hoped, and highlights that targeting the scaffold function of IRAK4 may be an attractive alternative.

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SYM-41-02 SYM-41-03 MACROPHAGE MIGRATION INHIBITORY FACTOR IS WNT SIGNALLING AND HOST CONTROL OF REQUIRED FOR NLRP3 INFLAMMASOME ACTIVATION BACTERIAL PATHOGENS

Lang T., Lee J.P.W., Deen N.S., Morand E.F. and Harris J. Thanh-Tran T.1, Gatica Andrades M.1, Nguyen T.T.K.1, Rollo R.F.1, Centre for Inflammatory Diseases, Monash University. Zamoshnikova A.1, Taveras C.1, Wyer O.K.1, Barnett T.2, Joseph S.1 , Simpson F.1, Brown D.3, Stow J.L.3, Kling J.C.1, Begun J.4 Macrophage migration inhibitory factor (MIF) exerts multiple effects on and Blumenthal A.1 immune cells, as well as having functions outside of the immune system. 1Diamantina Institute, The University of Queensland, Brisbane, However, despite over 50 decades of research, the mechanisms by Australia. 2School of Chemistry and Molecular Biosciences, The which it does so are not well understood. MIF can promote inflammation University of Queensland, Brisbane, Australia. 3Institute for Molecular through the induction of other cytokines, including TNF, IL-6 and Bioscience, The University of Queensland, Brisbane, Australia. 4Mater IL-1 family cytokines. We have found that inhibition of MIF regulates Research Institute, The University of Queensland, Brisbane, Australia. the release of IL-1α, IL-1β and IL-18 via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and Bacterial infections remain an important clinical challenge despite the intermediate filament protein vimentin, which is critical for NLRP3 our extensive arsenal of antibiotics. This is exemplified by lengthy activation. Further, we demonstrate that MIF interacts with NLRP3, treatments of chronic infections, high mortality due to excessive indicating a role for MIF in inflammasome activation independent of inflammation, and an alarming increase in antibiotic resistance. One its role as a cytokine. These data advance our understanding of how attractive strategy for improved treatments for challenging infections MIF regulates inflammation and identify it as a factor critical for NLRP3 is to enhance the host anti-microbial defence. We and others have inflammasome activation. associated the WNT signalling pathway with bacterial infections in patients and model systems, implicating novel immune-related functions for this well-known developmental signalling pathway. However, the nature of its contribution to the host response to infection remains to be clearly defined. Focus of our work is defining infection- associated WNT responses and delineate functions of WNT signalling in tailoring host responses to acute bacterial infection.

SYM-41-04 SYM-41-05 PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE: AN INFLAMMASOME SIGNALLING IN THE HOST EMERGING CELL DEATH MEDIATOR AND IMMUNE DEFENSE AGAINST INFECTIOUS DISEASE REGULATOR Man S. Phan T.K., Lay F.T., Jarva M., Kvansakul M. and Hulett M.D. Department of Immunology and Infectious Disease, The John Curtin Department of Biochemistry and Genetics, La Trobe Institute for School of Medical Research, The Australian National University, Molecular Science, La Trobe University. Canberra, Australia.

Phosphorylated phosphatidylinositol lipids, or phosphoinositides Recognition of pathogens by the host cell is of paramount importance (PIPs), vitally regulate diverse cellular processes, including signalling for the initiation of an immune response and clearance of the pathogen. transduction, cytoskeletal reorganisation, membrane dynamics Intracellular and cytosolic bacteria must secure entry into the host and cellular trafficking. However, PIPs have been inadequately cytoplasm in order to engage activation of cytosolic immune sensors investigated in the context of cell death and inflammation, where they and the inflammasome. However, microbial ligands from extracellular are mainly regarded as secondary messengers of PI3K-Akt signalling. pathogens are also detected by cytosolic innate immune sensors. We Interestingly, recent studies implied their importance in mediating cell discuss our latest findings on the role of inflammasomes and innate death by demonstrating that PIPs, particularly phosphatidylinositol immune regulatory proteins in the recognition of bacteria. We also 4,5-bisphosphate (PIP2), are essential effectors for different forms of highlight novel anti-microbial host defense systems mediating cytosolic programmed cell death. MLKL and gasdermin D require PIP binding release of bacterial ligands for sensing by pattern-recognition receptors to execute necroptosis and pyroptosis respectively. In our studies, we and inflammasomes. reported biphasic functions of PIP2 in mediating cytokine induction and necrotic cell death in response to low and high concentration of host defense peptides, particular human β-defensins (HBDs). Using multiple biochemical and cell biological approaches, we was able to show that the interaction of HBD-3 with PIP2 is important for receptor- independent PI3K-Akt-NF-κΒ-mediated cytokine induction at its sub- cytotoxic level. Higher concentration of HBD-3 also binds to PIP2, however leading to membrane blebbing and acute cell permeabilisation in mammalian cells. Furthermore, in our most recent study, it was found that PIP2, via a different mechanism, mediates pathogenic fungal cell killing by HBD-2, a closely-related defensin, by promoting a necrotic HBD-2:PIP2 oligomeric complex. Our findings highlight the critical roles of PIPs, particularly PIP2, in (i) orchestrating various cellular processes including a novel role in immune response and (ii) mediating cell death upon in different scenarios strategically targeting both host and pathogen cells.

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SYM-42-01 SYM-42-02 MAGNIFYING THE CAREER PATH AFTER PHD NAVIGATING THE IMPACT OF CAREER DISRUPTION

Le S. Caldon E. Olympus Australia and New Zealand. Garvan Institute of Medical Research.

Upon receiving my PhD, I dived into my postdoc life. Driven by my Dr Liz Caldon is a group leader at the Garvan Institute, studying passion about microscopy and uncontrolled external factors, I joined genomic instability and drug resistance in cancer. Over the last 6 years Olympus to pursue a career outside academia. I am passionate about she has combined a 3 year career disruption with establishing her own leveraging my expertise and company resources to provide the best research group. Our current system in academia provides support for solution of advanced microscopy to support research in life science. career disruptions, but this does not always bridge important gaps A career path of a PhD graduate is always deemed narrow, which is which arise when combining career disruption with career progression. completely untrue. I would like to discuss the necessary preparation, Liz will discuss strategies to minimise the long-term disruption to career, practical steps and strategies that worked for me to transition from drawing on her personal experiences. academic role into a position in life science related industry.

SYM-42-03 SYM-42-04 SPINNING OUT: TALES FROM A UNIVERSITY START DOES AN EXPERIENCE IN THE PRIVATE SECTOR UP REALLY MAKE YOUR CAREER “NON-ACADEMIC”?

Burke C. Delerue F. School of Life Sciences, University of Technology Sydney, Broadway, Macquarie University, Sydney, NSW. NSW 2007. In 2015 in the U.S., only 42% of people awarded a PhD in Science I will talk about my experience as a founding member of UTS spin-out worked in Academia. Despite the competitiveness of the academic company Longas. From initial experiments as a post doctoral scientist world and its constant decrease in grant funding, the so-called “non- to patents and board meetings, working with a spin-out has been an traditional” career might end up being a good alternative to help exciting, stressful, illuminating but overall positive experience. I have researchers navigate their academic journey. Based on a personal learned many lessons along the way about project management, experience, this presentation details how working in a private industry strategy and the importance of maintaining a good team. can help academics achieve their goals by managing the interface between business and science.

SYM-42-05 SCIENCE TO COMMERCIALISATION — OH THE PLACES YOU’LL GO!

Chapman N. gemaker Pty Ltd, Sydney, NSW 2000.

Natalie is Co-Founder and Managing Director of gemaker, a science and technology commercialisation consultancy which provides expert advice, services and training to research organisations and innovative businesses. Natalie is also Corporate Communications Manager for ASX-listed Alkane Resources, a Director of the Commercialisation Studies Centre (CSC) and sits on the External Advisory boards for UNSW School of Chemistry and the UOW Faculty of Business. Her previous work, as General Manager of Commercialisation at the Smart Services CRC, resulted in two spin-off businesses. As Leader of Business Development and Marketing at ANSTO, she provided strategic advice and managed the commercialisation of the intellectual property portfolio, including incubating new businesses and growing technical consultancy services. In 2017, gemaker won three Asia- Pacific Stevie Awards for Innovation, and the NSW Telstra Business Award for Microbusiness and Natalie was a finalist in the Telstra Business Women’s Awards. In 2018 Natalie launched the AUSinnovates initiative to celebrate Australian technology commercialisation success and bring attention to Australian researchers and innovators who are changing our lives for the better, by creating new industries and jobs, enhancing our health and education and making the world safer.

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SYM-43-01 SYM-43-02 DEFINING INITIAL MOLECULAR MECHANISMS OF MOUSE AND HUMAN MICROGLIAL PHENOTYPES HUMAN CATARACT FORMATION USING LIGHT- IN ALZHEIMER’S DISEASE ARE CONTROLLED BY FOCUSING MICRO-LENSES PLAQUE PHAGOCYTOSIS THROUGH HIF1α

Umala Dewi C.1, Kabir M.D.H.1, Murphy P.1, Ho J.2, 3 and O’Connor M.D.1, 4 Grubman A.1,*, Choo X.Y.1,*, Chew G.2,*, Ouyang J.F.2, Sun, G.1, Croft N.P.1, 1Western Sydney University, Campbelltown, Australia. 2Victor Chang Rossello F.1, Simmons R.3, Buckberry S.3, Vargas Landin D.3, Pflueger J.3, Cardiac Research Institute, Darlinghurst, NSW, Australia. 3St. Vandekolk T.H.1, Abay Z.1, Chan J.1, Haynes J.1, Williams S.1, Chai S.1, Vincent’s Clinical School, University of New South Wales, Sydney, Wilson T.4, Lister R.3, Pouton C.W.1, Purcell A.1, Rackham O.2, Petretto E.2 NSW, Australia. 4Medical Sciences Research Group, Western Sydney and Polo J.M.1 University, Campbelltown, NSW, Australia. 1Monash University, Clayton, Australia. 2Duke NUS, Singapore. 3University of Western Australia, Perth, Australia. 4MHTP Cataract, caused by opacification of the eye’s lens, affects over 65 Medical Genomics Facility, Clayton Australia. *Equal contribution. million people worldwide. Surgery is the only effective treatment, however, there is great interest in anti-cataract drug development Microglia are brain immune cells that remove cellular and extracellular due to: i) the large number of cataract operations performed annually debris and regulate synaptic plasticity, maturation and removal. (millions); ii) the cost of these surgeries (billions of dollars); iii) the Recently altered microglial genomics, epigenomics and functions fact that, despite these surgeries, the number of people affected by emerged as key contributors to Alzheimer disease (AD). Nonetheless, cataract is increasing (50.5 million in 1990 to 65.2 million in 2015); and whether toxic microglial inflammatory cytokine secretion and aberrant iv) the relatively high incidence of vision-impairing complications that synapse overpruning outweigh the beneficial amyloid clearance arise from cataract surgery (such as posterior capsule opacification). functions of microglia in AD remains highly controversial. To address Environmental risk factors have been associated with cataract these questions, we explored whether functional differences in formation. The molecular pathologies initiated by different risk factors amyloid plaque phagocytosis in a plaque-depositing AD mouse are likely to be different, yet are poorly understood due to the inability result from or contribute to the underlying molecular and functional to access human lenses during cataract initialization. To address this, diversity of microglia in AD. Using a combination of bulk and single cell we have established methods for large-scale production of human RNA-seq, and proteomics approaches, we showed that the amyloid lens epithelial cells and light-focusing micro-lenses from pluripotent plaque phagocytic subset of microglia are molecularly distinct from cells. Extensive characterization of these human lens cells and micro- physiological microglia and from non-plaque containing microglia in AD lenses revealed significant functional, morphological and molecular brains. For instance, several later onset AD risk factors and their direct similarities to primary human lenses. An initial study demonstrated interacting partners are differentially expressed in plaque-containing the micro-lenses can be used to study clinically-relevant cataract, with microglia. We are now using stem cell derived microglia like cells to assessment of transparency and focusing ability new and quantifiable manipulate the signaling pathways involved in generating the plaque- readouts of cataract formation. Our recent studies suggest other associated microglial signature. environmental factors can be investigated using human micro-lenses. The micro-lens system also offers an opportunity to re-assess drugs that failed pre-clinical development due to an association with cataract formation in animals.

SYM-43-03 SYM-43-04 HUMAN PLURIPOTENT STEM CELL MODELS OF REGULATION OF THE SKELETAL MUSCLE STEM HEART DEVELOPMENT AND DISEASE CELL NICHE

Elliott D.A. Tajbakhsh S. Murdoch Childrens Research Institute. Developmental & Stem Cell Biology, CNRS UMR 3738, Institut Pasteur, Paris. Congenital heart disease is the most common form of birth defect, with a prevalence approaching 1 in 100 children. Although the etiologies The microenvironment is critical for the maintenance of stem cell underlying congenital heart disease and cardiovascular disease differ, populations, and it can be of cellular and non-cellular nature, including the development of new treatments for either condition will be critically secreted growth factors and extracellular matrix (ECM) as well as dependent on a detailed understanding of how the human heart is intrinsic regulators. Skeletal muscle satellite (stem) cells are quiescent formed and how it functions at the cellular and molecular level. Human during homeostasis and they are mobilised to restore tissue function pluripotent stem cell (hPSC) derived cardiomyocytes are the only after muscle injury. Although certain signalling pathways that regulate tractable platform for illuminating the fine detail of the genetic networks quiescence have been identified, the mechanisms by which niche that control human cardiomyocyte cell biology. We have developed molecules regulate stem cell properties remain largely unknown. We a cellular framework to investigate the genetic regulation of human have identified Notch signalling as a major regulator of the muscle cardiac cell lineage specification. We are now utilizing these reagents stem cell niche. Specifically, Notch/RBPJ-bound regulatory elements and technologies to study congenital heart disease using differentiating are located adjacent to specific collagen genes in adult muscle satellite hPSCs. In particular, we are examining the role of the important cardiac cells. These molecules are linked to the ECM and constitute putative transcription factor NKX2-5 to determine the molecular mechanisms niche components. Notably, satellite cell-produced collagen V (COLV) underlying congenital heart disease in individuals with NKX2- is a critical component of the quiescent niche, as conditional deletion 5 mutations. We have shown that NKX2-5 is essential for the activation of Col5a1 leads to anomalous cell cycle entry and differentiation of of the ventricular cardiomyocyte commitment marker VCAM1 and for satellite cells. Strikingly, COLV, but not collagen I and VI, specifically the coordinated contraction of hPSC derived cardiac monolayers. regulated quiescence through Calcitonin receptor mediated activity, Through RNA-seq and ChIP-seq HEY2 was identified as a downstream therefore, a Notch/COLV signalling cascade cell-autonomously mediator of NKX2-5. HEY2 was able to restore both VCAM1 expression maintains the stem cell quiescent state, and raises the possibility of a and contractile synchronicity to NKX2-5 deficient cardiomyocytes. similar reciprocal mechanism acting in diverse stem cell populations. Thus, NKX2-5 and HEY2 are components of the genetic network This novel mechanism of stem cell niche regulation implicates a controlling human ventricular cardiomyocyte differentiation. reciprocal mechanism were a mechanotransduction ECM protein acts as a signalling molecule for cell autonomous regulation of stem cell quiescence. Notch signalling also acts in an intrinsic manner to regulate a microRNA pathway to modulate cell migration and stem cell quiescence. These findings point to two distinct modulatory mechanisms for maintaining stem cell and niche stability.

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SYM-43-05 SYM-44-01 ADVANCES IN KIDNEY ORGANOID GENERATION; COMPUTATIONAL STUDIES OF IMMUNE STIMULATING INSIGHTS GAINED THROUGH NOVEL HUMAN IPS COMPLEX (ISCOM) STRUCTURE CELL REPORTER LINE APPROACHES AND GENE EXPRESSION PROFILING Chalmers D.K. Monash Institute of Pharmaceutical Sciences, Monash University. Vanslambrouck J.M.1, Howden S.E.1, 2, Wilson S.1, Tan K.S.1, Soo J.1 and Little M.H.1, 2 Immune stimulating complexes (ISCOMs) are lipid nanoparticles 1Murdoch Children’s Research Institute, Parkville, Melbourne, 3052. mixtures of cholesterol, phospholipid, protein and saponin from the bark 2Department of Pediatrics, Faculty of Medicine, Dentistry and Health of Quillaja Saponiaria (the Chilean soap bark tree). ISCOMS, and the Sciences, University of Melbourne, Parkville, 3052, Australia. simpler ISCOM matrix (IMX) which lack protein, are complex systems with intriguing physical properties and biological properties, including Development of directed differentiation techniques and stem cell- applications as vaccine adjuvants. ISCOM and IMX particles consist of derived organoid culture methods are revolutionising the fields of supramolecular structures, which appear from electron microscopy, to regenerative medicine, disease modelling and drug screening. Based be ‘holey’ spheres with a diameter of approximately 30 nm, although the on our knowledge of human kidney organogenesis, we recently precise structure of the IMX particles and the processes driving their established a robust protocol to direct the differentiation of human formation are currently not well understood. We have used large-scale pluripotent stem cells towards a kidney fate within complex, 3D kidney molecular dynamics simulations to model the formation of IMX particles organoids (Takasato et al., 2014, 2016). These organoids display and have developed models that are consistent with the experimental evidence of differentiation into many of the compartments present in information available. Our models raise the prospect for the design of early embryonic kidney, including nephrons, collecting duct, vasculature new, simpler saponin analogues for the development of biologically and interstitium. However, despite these impressive similarities, the active lipid particles. structures within kidney organoids are still developmentally immature and they lack the continuous growth pattern seen in embryonic kidneys in vivo. Whilst we know that successful differentiation of pluripotent stem cells towards kidney progenitors and subsequent nephron patterning are critically reliant on the culture media and growth conditions, progress in this area is dependent on our ability to interrogate individual cell types and modify organoid conditions accordingly. Using CRISPR/Cas9-mediated gene-editing, we have generated a range of induced pluripotent stem cell lines carrying fluorescent reporters targeted to genes that mark specific kidney cell types and nephron segments, including nephron progenitors, proximal and distal nephron and collecting duct, enabling real-time monitoring of differentiation. Through the use of these reporter lines, in combination with detailed gene expression analyses, we have now been able to examine organoid development more deeply than ever before, enabling the perturbation of culture conditions to improve organoid growth and nephron patterning.

SYM-44-02 SYM-44-03 THE EFFECT OF H3O+ ON MEMBRANE STRUCTURE UNDERSTANDING THE DYNAMIC SUBSTRATE AND HYDROGEN BONDING IN PHOSPHOLIPID BINDING MECHANISM OF HUMAN HEPARANASE BILAYERS Ahmed F.H.1, 3, Marsavelski A.1, 2, Mohamed A.E.1, Jamieson E.1, Deplazes E.1, Cranfield G.C.2, Sarami F.3, Poger D.4 and Cornell B.5, 2 Correy G.1 and Jackson C.J.1 1Curtin University, Perth, Australia. 2University of Technology 1The Australian National University, Research School of Chemistry, Sydney, Sydney, Australia. 3University of Western Australia, Perth, Sullivan’s Creek Road, Acton, ACT 2601, Australia. 2Department Australia. 4University of Queensland, Brisbane, Australia. 5SDx of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Tethered Membranes Pty. Ltd., Sydney, Australia. Croatia. 3Current address: CSIRO, Land and Water, Clunies Ross Street, Acton, ACT 2601, Australia. The cell membrane is critical for cells to adapt to changes in pH yet we know little about the molecular mechanisms of how hydronium ions Heparanase (HPSE) hydrolyzes heparan sulfate (HS) in the + extracellular matrix. It mediates the release of regulatory molecules (H3O ) affect the structure of phospholipid membranes. A recent study showed changes membrane conduction and structure as a function bound to HS, and hence is essential for processes that control cell + growth, coagulation and inflammation. Inhibition of the increased of H3O concentration. To gain molecular level insight into this effect we carried out μs-long unrestrained MD simulations of phospholipid HPSE activity observed in a variety of pathological conditions such + as tumour metastasis, angiogenesis and auto-immune diseases bilayers in the presence of 10 mM and 100 mM H3O and compared + attenuates disease progression, making HPSE a promising drug target. them to the same system in the absence of H3O . Results show + Although crystal structures of HSPE are now available, the dynamic that in the presence of H3O the membrane undergoes a significant increase in bilayer thickness accompanied by a significant decrease nature of its interaction with its flexible polysaccharide substrate HS is in the area per lipid, in agreement with experimental data. Analysis not fully understood. We have explored the ligand binding mechanism + of HPSE by analysing its conformational landscape with molecular of the density profiles shows that the H3O ions accumulate close to the hydrophobic core of the membrane where they displace water and dynamics (MD)-simulations and multi-conformer models generated by form hydrogen bonds with the carbonyl and phosphate oxygens of the crystallographic ensemble refinement. We found that HS binding is a lipids. These hydrogen bonds are, on average, shorter and longer- complex process, whereby the molecule is stabilized at the lived than hydrogen bonds with water molecules. Thus, our simulations for catalysis, but the outer regions of the enzyme:substrate complex are + dominated by ionic interactions and exhibit substantial conformational confirm the hypothesis that H3O disrupts hydrogen bonding between phospholipids and that this is likely the cause of the reduced area flexibility. This dynamic binding mechanism allows HSPE to retain high per lipid. In addition, the layer of constrained water at the water-lipid affinity while minimizing the entropic costs associated with binding interface known to influence membrane morphology and structure as highly flexible molecules. These findings have direct implications on + efforts in identifying and optimising inhibitors for this promising drug well as proton transport appears to increase in the presence H3O . target.

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SYM-44-04 SYM-44-05 DYNAMIN STUCTURE AND FUNCTION HINGING ON SUPER-RESOLUTION IMAGING OF SUBCELLULAR RYNGOS REMODELLING BY VIRAL PROTEINS

Cardoso D.1, Abdel-Hamid M.2, McCluskey A.2 and Robinson P.1 Rozario A.M.1, Zwettler F.2, Rawlinson S.3, Brice A.3, Sauer M.2, 1Cell Signalling Unit, Children’s Medical Research Institute, Whelan D.1, Moseley G.3 and Bell T.D.M.1 University of Sydney, NSW, Australia. 2School of Environmental and 1School of Chemistry, Monash University, Clayton, Victoria. Life Sciences, Faculty of Science, University of Newcastle, NSW, 2Department of Biotechnology and Biophysics, University of Australia. Wuerzburg, Bavaria, Germany. 3School of Biomedical Sciences, Monash University, Clayton, Victoria. Dynamins are GTPase enzymes responsible for performing the final scission of invaginated plasma membrane prior to completion of Viruses are microscopic infectious agents capable of evading immune endocytosis. Pharmacological targeting in relevant mouse models responses and causing fatal human disease. Investigating viral has been shown to provide therapeutic relief for ailments as diverse mechanisms and viral-host interactions using light microscopy is limited as chronic kidney disease and epilepsy. We have generated a series due to diffraction of light (~250 nm). Super-resolution fluorescence of small molecule modulators (Ryngos) which ‘lock’ dynamin into a microscopy (SRFM) can achieve resolutions as good as 20 nm, making ‘ring’ oligomer that structurally differs from the ‘helical’ state required possible observation of nanoscale changes in virally altered cell for endocytosis. Ryngos exhibit different actions on enzyme activity in structures. Lyssavirus phosphoproteins (P1-P5) interact with STAT1 vitro (Ryngo-1, mixed-mode; Ryngo-3, stimulation). Due to their to antagonize interferon-mediated antiviral responses. Previously, we chemical similarity, it can be surmised that these compounds share a observed rabies lyssavirus (RABV) P3 bind onto microtubules (MTs) common binding pocket. This study aims to establish the binding site of and induce bundling. Mutations to P3 diminish MT bundling, correlating Ryngos to allow for targeted drug design. Advanced computer modelling with improved interferon response and reduced lethality in mice models predicted lead compounds; Ryngo-1-23 and Ryngo-3-32, independently (Brice et al., Sci Rep. 2016, doi: 10.1038/srep33493). However, the localised to, and differentially interacted with Hinge 1, located between precise role of MT bundling in lyssavirus disease progression is still middle domain and bundle-signalling element of dynamin. Partial unclear. Here we report on using SRFM to analyse bundling effects overlap of residues between Ryngo-1-23 and Ryngo-3-32 suggests of P3 from other lyssaviruses, finding significant divergence in MT drug binding to different sub-regions of Hinge 1 may be capable of interactions between the related pathogens. To elucidate bundle imparting different actions (inhibition/stimulation) on dynamin activity. structure, we have developed assays for expansion microscopy (ExM), To validate this, mutagenesis of Hinge 1 residues was undertaken a method that physically enlarges samples ~4-fold for improved and mutants characterised. Functional assays largely support these imaging resolution, potentially down to <10 nm. Additionally, we have predictions (i.e. single mutations selectively lost drug action) whilst investigated pathogenic henipavirus matrix protein (HeV M) which highlighting a broader role for Hinge 1 in dynamin characteristics localises in subnucleolar puncta and binds Treacle, a protein involved (activity, oligomerisation). To account for allosteric effects of mutation, in DNA-damage response (DDR) machinery. We have imaged for the a chemically dissimilar dynamin inhibitor (Dynole-34-2) revealed loss first time using SRFM subnucleolar Treacle puncta (100-200 nm) and of Ryngo action to be specific to Hinge 1. The data supports the model effects thereon of HeV M, and show that M protein subverts Treacle of these compounds differentially interacting with a flexible hinge within and supresses rRNA synthesis to a similar extent as during a DDR dynamin, an exceptionally rare binding site in pharmacology. (Rawlinson et al., doi: https://doi.org/10.1101/219071).

SYM-45-01 SYM-45-02 THE STRUCTURAL BASIS FOR SELECTIVE METAL EXPLORING THE PHYSIOLOGICAL SUBSTRATES OF ION IMPORT THE PROTOTYPICAL PACE FAMILY EFFLUX PUMP ACEI McDevitt C.A. Research Centre for Infectious Diseases, The University of Adelaide. Hassan K.A.1, 2, 3, Naidu V.2, Liu Q.2, Edgerton J.3, Fahmy L.3, Li L.2, Mettrick K.A.1, Jackson S.M.3, Ahmad I.3, Sharples D.3, Henderson P.J.F.3 Bacterial infection involves a constant tug-of-war between host and Paulsen I.T.2 and pathogen for the essential nutrients of life. Nevertheless, how 1School of Environmental and Life Sciences, University of Newcastle, bacteria selectively acquire the essential first-row transition metal ion Callaghan, NSW, Australia. 2Department of Molecular Sciences, manganese from the host environment remains poorly understood. Macquarie University, North Ryde, NSW, Australia. 3School of BioMedical Here, we investigated the manganese importing ATP-binding cassette Sciences and Astbury Centre for Structural Molecular Biology, University (ABC) transporter PsaBCA of Streptococcus pneumoniae, the foremost of Leeds, Leeds LS2 9JT, UK. human bacterial pathogen. By combining molecular microbiological, biochemical, biophysical and structural approaches we show that Resistance to antimicrobials is one of the most pressing health issues specificity is achieved not by selective binding of manganese, but of our time. Multidrug efflux pumps have gained notoriety as a major by the inability of ions other than manganese to be released into the and highly promiscuous class of drug resistance determinants that transporter. Our data show that the metal-recruiting component of the contribute to the failure of antibiotic therapy and promote the persistence pathway, PsaA, is not restricted to binding manganese and is highly of pathogens in hospitals. Despite their widely-studied roles in drug permissive for interaction with any divalent first-row transition metal ion. resistance, for many multidrug efflux pumps drug transport is likely to However, biochemical assays and single molecule FRET studies show be a fortuitous side reaction made possible by flexible substrate binding that zinc and copper ions, which are highly abundant during infection, sites that have become beneficial to host organisms living under highly result in the formation of PsaA-metal complexes incapable of releasing drug selective conditions in hospitals. The core functions of these these non-cognate metal ions. Further, these PsaA-metal complexes pumps are likely to be linked to the physiology of the organism and the are incapable of stimulating ATP hydrolysis in proteoliposome- environments in which they evolved. This is almost certainly true for the reconstituted PsaBC. In contrast, manganese can be readily released AceI transport protein, the prototype for the novel PACE family of efflux from PsaA-metal complexes, and manganese-bound PsaA stimulates pumps. The gene encoding AceI is conserved across all Acinetobacter ATP hydrolysis in proteoliposome-reconstituted PsaBC. Collectively, baumannii strains to have had their genomes sequenced, indicating these findings provide a structural basis for how manganese ions are an ancient origin and long term pressure for gene maintenance. selectively imported by the PsaBCA importer. Paradoxically, its only characterised substrate is chlorhexidine, which, although widely used as an antiseptic today, is purely synthetic and has been produced only since last century. In this talk I will describe our progress in deciphering the core physiological functions of the AceI protein, as well as its mode of energisation.

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SYM-45-03 SYM-45-04 PROGESTERONE RECEPTOR MEMBRANE MEMBERS OF THE CHLORIDE INTRACELLULAR COMPONENT 1: A CONSERVED EUKARYOTIC PROTEIN ION CHANNEL DEMONSTRATE WITH MULTIPLE AND STRATIFIED DISEASE-RELEVANT CATALYSE PROTEIN DEGLUTATHIONYLATION FUNCTIONS IN CELL AND ORGANISMAL BIOLOGY 1 1 1 1 1, 2 1, 2 1 1 1 3 Ali H.A. , Hossain K.R. , D’Amario C. , Jiang L. and Valenzuela S.M. Thejer B.M. , Teakel S.L. , Marama M. , Fang J. , Gurusinghe S. , 1 3 3 3 1 1 School of Life Sciences, University of Technology Sydney, Sydney, Quinn J.C. , Weston P.A. , Weston L.A. , Forwood J.K. and Cahill M.A. NSW 2007, Australia,. 2Centre for Health Technologies, University of 1School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia. 2Department of Biology, College Technology Sydney, Sydney, NSW 2007Australia. of Science, University of Wasit, Wasit, Iraq. 3Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW The chloride intracellular ion channel (CLICs) proteins are atypical anion 2678, Australia. selective channel proteins, with some members also known to have enzymatic activity. Human CLIC proteins found in most tissues and cells. (1).We have recently shown that members of CLIC proteins have intrinsic PGRMC1 is the archetypical member of the Membrane Associated enzymatic activity (2, 3). Also, we have evidence that the expression of CLIC Progesterone Receptor (MAPR) family of cytochrome b5 (cytb5) domain proteins by bacterial cells could provide increased tolerance to oxidative proteins, of which animals have three gene subfamilies: pgrc, nenf (Neudesin) stress (unpublished study). Our current study demonstrates for the first time and neufc (Neuferricin). We show that all three MAPR genes were present that CLICs possesses a significant deglutathionylation activity with a model in the ancestor of Opisthokonts: the eukaryotic clade including yeasts and peptide substrate. CLIC1 dependent glutathionylation of cellular proteins animals. MAPR proteins are more closely related to archaebacterial than to can also detect in cultured cell lines. In contrast, our study found that the alpha-proteobacterial cytb5 proteins. Eukaryotic cytb5 is related to alpha- expression of CLIC1 in cultured CHO-K1 cell lines promotes the rapid proteobacterial genes. Lanosterol is the first sterol produced from squalene glutathionylation of cellular proteins. The deglutathionylation activity was cyclisation. PGRMC1-like proteins regulate CyP51A1 lanosterol 14-alpha determined via an in vitro assay using a glutathionylated model synthetic demethylase enzymes from yeast to humans. Both squalene cyclase and peptide as a substrate. Deglutathionylation of CLIC proteins was assessed CyP51A1 are of bacterial origin, whereas PGRMC1 is of archaebacterial origin. using a real-time fluorescence-based method. CLIC proteins were found Cholesterol dramatically affects the properties of mitochondrial membranes, to have a significant deglutathionylation activity. Mutating the active site and the first step of animal steroid hormone synthesis takes place there, where cysteine residue in CLICs largely eliminated their deglutathionylation activity. cholesterol is modified to the first progestogen, pregnenolone. PGRMC1 confers Furthermore, mutating one of the only two charged residues: arginine 29 progestogen-responsiveness to cells, and is also a member of the Insig/SCAP (R29A) or lysine 37 (K37A) in the putative transmembrane region of CLIC1 complex which regulates SREBP1 to induce genes associated with fatty-acid resulted in a reduction of its deglutathionylation activity. The discovery that catabolism, and SREBP2 to activate the mevalonate pathway leading to sterol CLIC proteins participate in the glutathionylation cycle and targets specific production. This suggests a model where PGRMC1 function may reflect the proteins could explain the association of CLICs with a diverse range of regulatory interplay between the proto-eukaryotic archaebacterial host cell and clinical disorders and provide a novel therapeutic target. REFERENCES 1. its proto-mitochondrial bacterial endosymbiont at the root of eukaryotic evolution. Valenzuela, S. M. etal.,(1997). Molecular cloning and expression of a chloride PGRMC1 also acquired tyrosine phosphorylation sites during animal evolution ion channel of cell nuclei.J Biol Chem 272, 12575-12582. 2. Al Khamici, H. concurrently with the appearance of striated muscle and nerve synapses, and etal., (2015). Members of the chloride intracellular ion channel protein family before bilateral body plan. PGRMC1 phosphorylation differences exist between demonstrate glutaredoxin-like enzymatic activity.PLoS One 10, e115699. cancers, and it is tyrosine phosphorylated in post-synaptic densities. Migration 3. Juan R. Hernandez-Fernaud etal, (2017).Secreted CLIC3 drives cancer of embryonic nerve cord axons requires PGRMC1 from nematodes to mammals, progression through its glutathione- dependent oxidoreductase activity. and PGRMC1 synaptic function is required for the synaptorestorative effects of TM Received 11 Aug 2016. Accepted 6 Dec 2016. Published 15 Feb 2017. http:// Elayta , a promising potential Alzheimers drug. Our data provide exciting new www.nature.com/naturecommunications. insights into PGRMC1 function in animal and disease biology.

SYM-45-05 SYM-46-01 HOW DOES P-GLYCOPROTEIN BIND SO MANY CHROMATIN INTERACTOME MAPPING IDENTIFIES DRUGS? TARGET GENES AT BREAST CANCER RISK SIGNALS

Callaghan R. Sivakumaran H., Beesley J., Marjaneh M.M., Chenevix-Trench G., Division of Biomedical Science & Biochemistry, Research School of French J.D. and Edwards S.L. Biology & Medical School, Australian National University, Canberra Cancer Division, QIMR Berghofer Medical Research Institute, 0200, ACT. Brisbane, Australia.

P-glycoprotein (P-gp) gained notoriety for its role in conferring drug Genome-wide association studies (GWAS) for breast cancer have resistance to an astonishing number of cy totoxic drugs used in oncology. identified 196 independent signals associated with increased risk. The The protein is able to confer resistance by preventing sufficient majority of risk-associated variants within these signals fall in regulatory accumulation of chemotherapy drugs within cancer cells. P-gp is one sequences, such as enhancers, that control gene expression. We of three multidrug efflux pumps in humans and their actions have been perform in situ Capture Hi-C using a high-resolution Variant Capture the focus of considerable research. Recently, an x-ray crystallography array (VCHi-C), which includes probes to cover all fine-mapped based structure has been presented for P- gp and has been touted as the candidate causal variants. We apply VCHi-C and Promoter Capture “missing piece in the puzzle” to generate a mechanistic understanding Hi-C (PCHi-C) to link risk variants to their target genes in six human of this protein. Has this been achieved yet and is there any benefit to mammary epithelial and breast cancer cell lines. We use the CHiCAGO further biochemical studies on P-gp? Our continuing research focus is pipeline to assign confidence scores, apply a strict threshold, and to provide a dynamic understanding of the drug translocation process identify between 10-27,000 interactions per cell type. Hierarchical of P-gp. In particular, we aim to locate the drug binding site(s) on the clustering of interaction scores stratifies cell lines by estrogen receptor protein and describe their communication with the energy providing status. Global analysis of promoter-interacting regions (PIRs) shows domains. This information will be used to describe the precise steps strong enrichment for cell-type specific accessible chromatin, histone involved in multidrug transport at a molecular level. marks for active enhancers and transcription factor binding, supporting the regulatory potential of many PIRs. In total, reciprocally validated CHiCAGO-identified interactions results in 647 candidate target genes. To further prioritise the CHi-C-derived chromatin interactions, we use a recently developed Bayesian framework, to fine-map the direct contacts. Importantly, the combined PCHi-C and VCHi-C contact fine- mapping enables us to prioritize 1832 out of 7375 highly-correlated risk variants and lowers the total number of target genes to 393. One example which makes evident the utility of this dual approach is the 1p22 risk region, where contact fine-mapping decreases the number of risk variants from 34 to 8, and the candidate target genes from 14 to 2. Our results demonstrate the power of combining genetics, computational genomics and molecular studies to streamline the identification of key variants and target genes at GWAS-identified risk regions.

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SYM-46-02 SYM-46-03 SUCCINATE DEHYDROGENASE AND HEREDITARY REGULATORY SMALL RNA NETWORKS AND PARAGANGLIOMA SYNDROMES: LINKING KREBS BACTERIAL PATHOGENESIS CYCLE DYSFUNCTION TO CANCER Tree J.J. Clifton-Bligh R. School of Biotechnology and Biomolecular Sciences, UNSW, Sydney. Kolling Institute University of Sydney and Department of Endocrinology, Royal North Shore Hospital. The transcriptomes of bacteria contain hundreds of short regulatory RNAs termed, small RNA (sRNA). These gene regulators control Succinate dehydrogenase (SDH) is located on the inner mitochondrial protein expression by modulating protein interactions with messanger membrane and functions in the mitochondrial respiratory chain and the RNAs (ribosomes, RNases, and termination factors), and by Krebs cycle. In the respiratory chain, SDH transports electrons to the ubiquinone pool, then to cytochrome c of complex III. In the Krebs cycle, modulating mRNA structure. Small RNAs often base-pair with SDH catalyses conversion of succinate to fumarate. Two predictable target mRNAs through regions of limited complementary making consequences therefore of SDH inactivation are succinate accumulation, computational prediction of sRNA targets difficult. Recently, we have and increased production of reactive oxygen species. Both outcomes have developed a proximity-dependant ligation technique termed RNase been suggested to contribute to cellular accumulation of hypoxia-inducible E-CLASH that allows recovery of bacterial sRNA-mRNA interactions factors (HIFs) and tumours associated with SDH deficiency display notable occurring in vivo, profiling the sRNA interactome. We have used RNase upregulation of hypoxia-responsive genes. Mitochondrial dysfunction due E-CLASH to capture the sRNA interactome of the bacterial pathogen to mutations in genes encoding the subunits of SDH (SDHA-D) leads to Enterohaemorrhagic E. coli (EHEC). We find that the major EHEC adrenal phaeochromocytomas (PCs), sympathetic and parasympathetic virulence factor, the Shiga toxin, unexpectedly encodes a regulatory paragangliomas (PGLs), renal cell carcinomas (RCCs), gastrointestinal sRNA within the toxin mRNA, that controls the general stress response stromal tumours (GISTs), and pituitary tumours. SDHB mutations in pathway. RNase-CLASH is applicable to a broad range of bacteria particular are associated with metastatic PC/PGLs. We have developed and we demonstrate that the sRNA interactome can be recovered several orthogonal models to test genotype-phenotype correlations from multi-drug resistant Staphylococcus aureus, identifying novel of SDHB variants, including immunohistochemistry and metabolomic regulators of antibiotic tolerance. assays of tumour samples, structural modeling, and in vitro localization and enzymatic assays. Loss of SDHB staining in tumours is a reliable marker for mutations in any of the SDH subunit genes. This is corroborated by measurement of succinate in tumour samples by LC/MS-MS: high succinate measurement relative to fumarate in the tumors represents a direct link to functional aspects associated with SDH-deficiency. Elevated succinate:fumarate ratios are a consistent biomolecular phenotype of SDH- deficient tumors including PC/PGLs, GISTs and RCCs. A homology model for human SDH was developed from a crystallographic structure. Structural modelling showed that many mutations within SDHB are predicted to disrupt the electron path. In vitro assessment by immunoprecipation from transfected cells demonstrated that most SDHB mutations result in impaired mitochondrial localisation and/or SDH enzymatic activity. In conclusion, studying SDH mutations represent fertile ground for understanding the association between Krebs cycle dysfunction and cancer.

SYM-46-04 SYM-46-05 TINC: A METHOD TO DISSECT TRANSCRIPTIONAL DEFINING TISSUE SPECIFIC PROTEIN-PROTEIN COMPLEXES AT SINGLE LOCUS RESOLUTION INTERACTOMES IN VIVO AND IN DISEASE Knaupp A.S.1,2,3, Larcombe M.R.1,2,3, Ford E.4,5, Nguyen T.4,5., Mohenska M.1,2,3, Williams S.M.1,2,3, Firas J.1,2,3, Chen J.1,2,3, Pflueger J.4,5, Werner H., Chojnowski A., Sobota R., Burke B. and Stewart C.L. Liu X.1,2,3, Lim S.M.1,2,3, Wong K.1,2,3, Sun Y.B.Y.1,2,3, Hodgson-Garms M.1,2,3, Institute of Medical Biology, 8A Biomedical Grove, Immunos, Holmes M.L.1,2,3, Nefzger C.M.1,2,3, Rossello F.J.1,2,3, Kleifeld O.6, Haigh J.J.3,7, Singapore 138648. Schittenhelm R.B.6, Lister R.4,5 and Polo J.M.1,2,3 1Department of Anatomy and Developmental Biology, Monash The genomic era identified and defined the composition of the genome. University, VIC, Australia. 2Development and Stem Cells Program, The post-genomic era seeks to understand how the organism’s genome Monash Biomedicine Discovery Institute, VIC, Australia. 3Australian functions in both building and maintaining the organism through the Regenerative Medicine Institute, Monash University, VIC, Australia. actions of the proteins encoded by the genome. To accomplish these 4Australian Research Council Centre of Excellence in Plant Energy ends, proteins interact extensively with each other resulting in the Biology, The University of Western Australia, WA, Australia. 5Harry establishment of complex protein-protein interaction (PPI) networks Perkins Institute of Medical Research, WA, Australia. 6 Monash or interactomes. We are using the BioID technique to describe and Biomedical Proteomics Facility, Monash University, VIC, Australia. decipher PPI to define the LaminA interactome and to understand 7Australian Centre for Blood Diseases, Monash University, VIC, how different mutations in the LMNA gene result in a range of tissue Australia. specific diseases called the laminopathies. BioID involves the fusion of a promiscuous variant of the biotinylation enzyme Bira Ligase (BirA) Being able to determine the molecular composition of protein to the protein of interest. The resulting fusion protein is expressed in complexes that assemble at specific regulatory elements to activate vivo in any cell. Proteins within the 10-20nm of the fusion protein are or repress gene transcription is essential for a better understanding potentially biotinylated, isolated by streptavidin pull down, and identified of how gene expression is controlled in normal and diseased states. by MassSpec, establishing them as potential interactors with the protein We have developed a transcription activator-like effectors (TALE) of interest. We inserted the BirA gene in frame into the N-terminus of based method termed TINC (TALE-mediated Isolation of Native the murine Lmna gene in ES cells. From these we derived a mouse Chromatin), which enables the isolation of a specific chromatin region line expressing a hybrid LaminA/C-BirA fusion protein that correctly from mammalian cells and consequent identification of associated localizes to the nuclear lamina in different tissues. Endogenous levels proteins by mass spectrometry. For proof of concept, we targeted of biotin within the various murine tissues were sufficient to result in the the Nanog proximal promoter in mouse embryonic stem cells and were biotinylation and identification of proteins that are known interactors of able to identify transcription factors known to bind to this locus and most the A-type lamins. With these mice, we are defining the tissue specific importantly novel proteins that play an essential role in pluripotency and interactome of the A-type lamins and find that the interactome varies reprogramming. As TINC does not require any genetic modification of between different tissues. We are also determining how the interactome the target sequence, a target-specific antibody nor high copy numbers is altered by lamins carrying specific mutations that result in disease. of the target sequence, we strongly believe that this method is applicable to any scientific field and has immense potential to change the concept of how we study gene regulation.

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SYM-47-01 SYM-47-02 HARNESSING THE SELF-ASSEMBLY OF PROTEINS CYBERNOSE AND CYBERTONGUE TECHNOLOGIES: FROM DIVERSE ORGANISMS TO BUILD FUNCTIONAL A MICROFLUIDIC SENSING PLATFORM MATERIALS Dacres H.1, Gel M.2, Wang J.1, Caron K.1, Anderson A.1 and Trowell S.1 Glover D. 1CSIRO Health & Biosecurity. 2CSIRO Manufacturing. UNSW Sydney, School of Biotechnology and Biomolecular Sciences. Objective quantitative measures of chemical composition are invaluable The intricate and ordered complexes that proteins adopt in nature is in many spheres of human activity, from healthcare to industrial central to many biological processes, ranging from cellular scaffolding processing. A number of very sophisticated analytical methods have provided by cytoskeletal proteins to the encapsulation of nucleic acids been developed, for separating and identifying chemicals, including in viral capsids. Exploiting this remarkable fidelity and precision in self- chromatography and mass spectroscopy. The sensitivity, resolving assembly is highly attractive for the fabrication of functional materials power and user-friendliness of these technologies are spectacular. with nanometer dimensions. This talk will highlight recent engineering However, these existing methods are, in general, expensive, slow and and standardisation of modular protein subunits for self-assembly into not very portable. They may generate large amounts of data, requiring geometrically defined templates. The central protein building block time and expertise to interpret. Therefore they have not been widely in the creation of these templates is the gamma-prefoldin (gPFD), adopted outside research or analytical laboratory environments. To a chaperone filament isolated from a hyperthermophilic archaeon. allow chemical analysis to be easily carried out in “real world” situations, Redesign of the gPFD subunit interface enabled the creation of two there is a need for faster and more portable methods that can be used and three-way connectors that can link multiple gPFD filaments into by non-specialists. Highly evolved chemical senses are critical for the macromolecular structures. These protein templates are now being survival of most modern day organisms. We have drawn inspiration applied to achieve more complex patterning, while expanding the from biological systems to develop a sensitive, fast and easy-to-use applicability of modular protein templates to diverse enzymatic systems. chemical analyser. The CYBERTONGUE/NOSE biosensing platform Fusing different enzymes to each subunit enables periodic positioning can measure a wide range of analytes, continuously and rapidly. The of multiple enzymes along the filament to catalyse sequential reactions platform’s essential features are that: (1) it uses bioluminescence and metabolic pathways. In addition, metalloproteins can be aligned resonance energy transfer (BRET) as the transduction modality; (2) it at high density along filaments to create conductive nanowires. incorporates multiple classes of biological recognition elements and, Ultimately, these strategies will enable the design of smart biomaterials within each class, many variants are possible; (3) it works in the fluid for complex applications that require multifunctionalities, such as drug phase of a multiplexed microfluidic system. I will describe the technical delivery systems, biosensors, and bioelectronic devices. basis of the technology and illustrate it with applications in food and health diagnostics.

SYM-47-03 SYM-47-04 YEAST 2.0 AND BEYOND: BUILDING THE WORLD’S WHOLE GENOME SEQUENCING IDENTIFIES FIRST SYNTHETIC EUKARYOTE CLINICALLY ACTIONABLE VARIANTS IN FAMILIES WITH CHD Paulsen I.T. and The Australian Team For Yeast 2.0. Macquarie University, Sydney Australia. Alakarage D.1, Ip E.1, Szot J.O.1, Munro J.1, Blue G.M.1, 2, 3, Pachter N.4, 6, Chapman G.1, Winlaw D.S.1, 2, 3, Giannoulatou E.1, 5 and Yeast 2.0 is an international consortium aiming to build the world’s first Dunwoodie S.L.1, 5 synthetic eukaryote by 2017. Systematic genome wide changes in the 1Victor Chang Cardiac Research Institute. 2Heart Centre for synthetic yeast include TAG/TAA stop-codon replacements, deletion 3 of subtelomeric regions, , transfer RNAs, transposons, and Children, The Children’s Hospital at Westmead. University of Sydney. 4Genetic Services of Western Australia, King Edward insertion of loxPsym recombination sites. The Australian Yeast 2.0 5 6 team is responsible for the design and synthesis of synthetic versions Memorial Hospital. University of New South Wales. University of of chromosomes 14 and 16. Construction is essentially complete at Western Australia. Macquarie, with 100% of the synthetic DNA successfully inserted. Troubleshooting and repairing errors identified through genome Congenital heart disease (CHD) affects up to 1% of live births. However, sequencing is currently ongoing. One of the most interesting features of a genetic diagnosis is not made in most cases. The purpose of this study Yeast 2.0 is the incorporation of the SCRaMbLE system for generating was to assess the outcomes of whole-genome sequencing (WGS) of combinatorial genomic diversity through rearrangements at loxPsym a heterogeneous cohort of CHD patients. 97 families, with probands recombination sites. This opens up the possibility of harnessing the born with CHD requiring surgical correction, were recruited for genome SCRaMbLE system for adaptive laboratory evolution experiments. We sequencing. At minimum, a proband-parents trio was sequenced per have developed biosensors that respond to a variety of industrially family. WGS data were analyzed via a two-tiered method: application useful metabolites, and are now seeking to use a combination of of a high-confidence gene screen (hcCHD), and comprehensive SCRaMbLE-ing and flow cytometry to identify strains that can produce analysis. Identified variants were assessed for pathogenicity using higher levels of these metabolites. the ACMG-AMP guidelines. Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families. Interrogating WGS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation will increase diagnostic yield during future reassessment of our WGS data.

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SYM-48-01 SYM-49-01 EARLY CAREER RESEARCHER — GRANT WRITING RELIABLE INTERPRETATION OF WATER-USE WORKSHOP EFFICIENCY IN CHICKPEA FROM D13C OF LEAF TISSUE Ittner, L.M. University of New South Wales Barbour M.M. and Lockhart E. The University of Sydney, 380 Werombi Road, Brownlow Hill. Grant Writing Workshop The carbon isotope composition of leaf tissue (δ13C) has been widely used for many years as a proxy for both whole plant water-use efficiency (WUE; biomass produced per unit water transpired) and leaf-intrinsic water-use efficiency (WUEi; photosynthetic rate divided by stomatal conductance), and has been successful in many crop species, notably wheat. However, there is conflicting evidence for the utility of δ13C in chickpea as a WUE proxy. Chickpea are somewhat unusual in having high levels of organic acids excreted by leaf hairs, so we tested the hypothesis that surface organic acids are responsible for the inconsistency in δ13C-WUE relationships either directly or indirectly. Across 20 chickpea genotypes, removal of surface acids altered whole leaf tissue δ13C in 3 genotypes, suggesting that acids may contribute to the lack of consistency in δ13C-WUE relationships between different genotypes. Water-soluble CHO samples were 2‰ more enriched, on average, than whole leaf tissue samples. For 2 chickpea genotypes grown in well-watered and droughted conditions, the strongest correlations were found between δ13C of CHO and WUE. Weaker correlations were found for δ13C of CHO and WUEi, and whole leaf δ13C with either WUE or WUEi. Removal of surface acids and extraction of water-soluble CHO is recommended when using leaf δ13C as a proxy for WUE and WUEi in chickpea. Previous studies describing genotypic variability and heritability of δ13C in chickpea may have identified variation in leaf acid presence, or differences in leaf chemical composition, rather than WUE.

SYM-49-02 SYM-49-03 MOLECULAR AND EVOLUTIONARY CONSERVATION NON-INVASIVE IMAGING OF HYDRAULIC FUNCTION OF ABSCISIC ACID AND BLUE LIGHT SIGNALLING IN IN LEAVES, STEMS AND ROOTS STOMATAL REGULATION Choat B.1, Peters J.M.R.1, Gauthey A.1, Carins-Murphy M.R.2, Cai S.1, 2, Chen G.2, Franks P.3 and Chen Z.H.2 Rodriguez-Dominguez C.M.2 and Brodribb T.J.2 1School of Science and Health, Western Sydney University, Locked 1Hawkesbury Institute for the Environment, Western Sydney Bag 1797, Penrith, NSW, 2751, Australia. 2College of Agriculture and University, Richmond, NSW 2753, Australia. 2School of Biological Biotechnology, Zhejiang University, Hangzhou 310058, China. 3School Sciences, University of Tasmania, Hobart, TAS, 7001, Australia. of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia. Plants have evolved a water transport system that relies on water sustaining a tensile force. Counter intuitively, this means water moves Evolutionary trajectories of land plants have led to structurally complex through the plant as a liquid under negative absolute pressures. This and functionally active stomata for terrestrial life. The unique morphology, mechanism is made possible by the intricate plumbing system that development and molecular regulation of stomata enable their rapid constitutes the xylem tissue of plants. However, water under tension environmental response. Growing evidence has shown that signals is prone to cavitation, which results in the formation of a gas bubble controlling stomatal opening and closure may have evolved in some (embolism). Embolism reduces the capacity of the xylem tissue to species of mosses. Here, comparative genomic study showed that many deliver water to the canopy, eventually causing dieback and whole plant of abscisic acid (ABA) signalling, photoreceptor, membrane transporter, mortality. Xylem embolism is exacerbated by environmental stresses and reactive oxygen species (ROS) and nitric oxide (NO) signalling, and protein is now considered one of the leading causes of plant mortality resulting kinase gene families are conserved over the evolutionary history of green from drought stress.Non-invasive imaging techniques offer the potential plants (Viridiplantae). Phylogenetic analysis indicated an evolutionarily to make direct observations on intact plants at high resolution and in conserved stomatal response to ABA. Moreover, comparative real time. In this presentation, I discuss recent exciting developments transcriptomic analysis has identified a suite of ABA responsive in the application of non-invasive imaging technologies such as X-ray differentially expressed genes encoding proteins associated with ABA Micro Computed Tomography (microCT) and optical imaging to studies biosynthesis, transport, reception, transcription, signalling, and ion and of plant vascular function. This includes visualisation of xylem networks sugar transport, which fit the general ABA signalling pathway constructed during drought stress and recovery in leaves, stems and roots. MicroCT from Arabidopsis thaliana and Hordeum vulgare. Furthermore, stomatal imaging of stems and roots indicated that significant embolism formation assays on epidermal peels showed ABA-induced stomatal closure in occurs at similar time points and levels of water stress in dehydrating two fern species (Polystichum proliferum and Nephrolepis exaltata) and plants. This result was observed in herbaceous and woody species, and is surprising given previous hydraulic measurements indicating blue light-induced stomatal opening of a wide range of fern species in that, within a plant, roots were more vulnerable to drought-induced the Orders of Polypodiales, Schizaeales, Psilotales, and Ophioglossales. embolism than stems. A newly developed optical technique indicates Stomata of fern species in the Polypodiales and Schizaeales are that leaf vasculature is also similar in vulnerability to stems and roots. more responsive to blue light, generating higher ROS production and The overlap in vulnerability suggests that induction of embolism occurs H+ pumping to the apoplast as compared to other fern species and at the same time in different organs or is propagated rapidly through the Arabidopsis. Understanding the evolution stomatal regulation will inform plant. In examining recovery from drought stress, we saw little evidence functional manipulation of water use efficiency for plant productivity of embolism refilling in the xylem of woody plants, except in cases and will benefit future efforts towards sustainable food production and where substantial root pressure is produced. These results suggest that ecological diversity. embolism refilling is less widespread than previously thought.

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SYM-49-04 SYM-49-05 PHOSPHORYLATION OF PLANT PLASMA MEMBRANE ESTIMATING STOMATAL AND BIOCHEMICAL AQUAPORINS REGULATES ION CHANNEL FUNCTION LIMITATIONS DURING PHOTOSYNTHETIC INDUCTION

McGaughey S.A. 1, Qiu J. 1, Groszmann M.2, Tyerman S.D1. and Byrt C.S1. Deans R.M., Busch F.A. and Farquhar G.D. 1ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Research School of Biology 46 Sullivans Creek Rd The Australian Food and Wine, The University of Adelaide, SA, Australia, 5006. 2ARC National University Acton ACT. Centre of Excellence for Translational Photosynthesis, Australian National Univeristy. Ross M. Deans, Florian A. Busch and Graham D. Farquhar Research School of Biology 46 Sullivans Creek Road The Australian National Phosphorylation on the C-terminal domain (CTD) of Arabidopsis University Acton ACT Understanding what limits photosynthesis under plasma membrane aquaporin AtPIP2;1 may be key to its ion channel different conditions is important for both the targeted improvement function. AtPIP2;1 has previously been identified as a Na+ permeable of photosynthesis as well as understanding ecological drivers of functional water channel. A series of phospho-mimic mutant AtPIP2;1 photosynthesis. Limitation analyses provide such a method to estimate proteins were expressed in X. laevis oocytes. Phospho-mimic photosynthetic limitations. Despite the use of limitation analyses to mutations of two CTD residues of AtPIP2;1 had significantly increased calculate biochemical and diffusional limitations at the leaf level being Na+ conductivity compared to WT AtPIP2;1. Interestingly, the apparent not new, their utility for estimating limitations during photosynthetic water permeability of the phospho-mimic AtPIP2;1 mutants were lower induction is still in its infancy. Moreover, few studies have systematically than that of WT AtPIP2;1 when PIP expressing X. laevis oocytes were assessed how well different methods estimate limitations. Timecourses transferred from isotonic to hypotonic solutions that had equivalent ion of photosynthetic induction provide a rich dataset in which to test the concentrations. These results suggest that (i) phosphorylation has an performance of limitation analyses. We compared how well two general important role in the regulation of ion channel function of ion permeable ways of estimating limitations predicted both temporal and overall plant aquaporins and (ii) that there may be an inverse relationship limitations for photosynthetic induction in response to an increase in between ion permeability and water permeability of these aquaporins. irradiance. One method sequentially removes the effect of a limitation As plant plasma membrane aquaporins are very important for water (elimination), while the other utilised a tangent plane approximation uptake and transcellular water flow in plant roots, PIPs with ion channel (differential) to estimate limitations. This talk will discuss under what functions may constitute a mechanism by which Na+ enters plant roots conditions each method works best and why. This last insight has in saline conditions. However the exact physiological role of dual water important implications for the general use of limitation analyses beyond ion plant aquaporins is yet to be elucidated. photosynthetic induction.

SYM-50-01 SYM-50-02 CILIARY SIGNALLING AND THE EXTRACELLULAR COMPRESSIVE FORCES ACTIVATE RHO/ MATRIX ROCK-MEDIATED CELLULAR PROCESSES CHARACTERISTIC OF DISEASE STATES McGlashan S.R.1, Leung S.1 and Choi Y.S.2 1University of Auckland. 2University of Western Australia. Boyle S.T.1, Kular J.1, Nobis M.2, Timpson P.2 and Samuel M.S.1 1Centre for Cancer Biology, University of South Australia and SA Nearly all cells in the body have a primary , a specialised Pathology, Adelaide, SA Australia. 2The Kinghorn Cancer Centre & compartment perfectly evolved to be a cellular probe. Primary cilia Garvan Institute of Medical Research & St. Vincent’s Clinical School, are microtubule-based organelles (1-15μm long, 200nm wide) that are Victoria St, Darlinghurst, NSW Australia. formed from the centriolar anchor known as the basal body. Primary cilia act as cellular sensors that receive diverse signals from the Mechanical forces exerted by the extracellular matrix (ECM) upon cells extracellular environment including light, growth factors and mechanical during homeostatic development are counterbalanced by intracellular stimuli in a tissue-specific manner. The cilium is also separated for the forces mediated via mechanotransduction signalling pathways that main cell body through a ciliary gate known as the transition zone. Our regulate remodelling and tension of the actomyosin cytoskeleton. This work has focussed on ECM/ cilia interactions in connective tissues phenomenon is termed mechano-reciprocity. The main regulator of such as articular cartilage and the intervertebral disc. Structurally, these cytoskeletal dynamics is myosin II, and its regulatory subunit chondrocyte cilia are structurally associated with the collagen fibres, myosin regulatory light chain-2 (MLC2) can be directly activated by the they express integrins and are mechanically deflected through ECM RHO/ROCK signalling pathway. Enhanced extracellular matrix tension interactions. We, and others, have shown that chondrocyte primary in diseased states such as cancer enhances mechanotransduction, cilia mechanosensitive, whereby cilia incidence and length are and so understanding the phenomenon of mechano-reciprocity and modulated by compressive or tensile forces, and removal of cilia results its regulation during homeostasis is key to understanding how these in in reduced mechanotransduction, and alterations in cartilage ECM processes become corrupted in disease. We have found that acute gene expression. Current research focuses on chemical, specific compressive force applied to cells and epithelial tissues is able to mechanical forces such as fluid flow, or gene cues that control cilia, activate the RHO/ROCK signalling pathway, elevating RHOA-GTP however, few studies have examined how the mechanics of the local levels and increasing regulatory myosin phosphorylation, actomyosin microenvironment influences cilia function. We have examined how cilia contractility and tension via ROCK. In consequence, cell proliferation length and incidence is influenced by different ECM environments in was increased, as was the expression of regulators of epithelial- diseases such in osteoarthritis, intervertebral disc disease and, more mesenchymal transition. Pharmacological inhibition of ROCK abrogated recently in model gel systems with controlled stiffness. Our data suggest myosin phosphorylation, whilst inhibition of either RHO or ROCK an intricate relationship between substrate stiffness, F-actin and cilia reversed the physiological effects of compression on cells. Our results length and more work is essential to understand the critical role of the strongly suggest that RHO/ROCK-mediated mechanical signalling mechanical microenvironment on ciliary signalling. during cancer, induced by compressive stress from tumour growth within a constricted space, could play a role in tumour progression.

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SYM-50-03 SYM-50-04 A NEW CLASS OF BIOACTIVE NANOPARTICLES FIBROBLAST ACTIVATION PROTEIN IN STEATOSIS FOR CAPABLE OF SPONTANEOUS, LINKER-FREE MULTIFUNCTIONALISATION Zhang H.E.1, Chowdhury S.1, Hamson E.1, Xiang M.1, Lay A.1 and Gorrell M.1, 2 1 2 Michael P.L.1, Santos M.1, 2, Hung J.1, Lam Y.T.1, 2, Bilek M.M.2 and Centenary Institute. The University of Sydney. Wise S.G.1, 2 1The Heart Research Institute, NSW 2042, Australia. 2The University Background & Aim: Better understanding of processes of glucose of Sydney, NSW 2006, Australia. intolerance, insulin resistance and steatosis in diabetes and fatty liver is needed. Fibroblast activation protein (FAP) has a unique post-proline cleaving activity. Normal FAP expression is very low, but is greatly up- The large number of proposed nanocarriers share limitations in regulated in activated liver mesenchymal cells in human liver cirrhosis. combining fine control of physical properties, low cytoxicity and Low level of FAP in human plasma strongly associates with lacking liver simple functionalization in a single platform. Multifunctional carbon- fi brosis. We aim to under st and met abolic outc omes of spec i fi c defi c iency based nanoparticles (nanoP3), manufactured in a custom-built plasma of FAP activity in diet-induced obesity (DIO). Methods: Wildtype (WT) chamber, are a new platform that achieves multiple functionalities in and FAPgki (gene-knockin; substituting the catalytic serine) mice were a single, simple step enabling delivery of multiple molecular cargos in a DIO model. Proteomics/degradomics was performed to identify into cells. Size, roughness, surface charge and chemical composition novel natural substrates of FAP. Results: FAP activity in pancreas are readily controlled, giving rise to a versatile nanoparticle platform. 3 was greater in DIO-WT than chow-fed-WT mice. Compared to DIO- Unfunctionalized nanoP penetrate a diverse range of cell types WT mice, DIO-FAPgki mice had less glucose intolerance, insulin including cancer lines, fibroblasts, vascular cells and stem cells. 3 resistance, insulin secretion, micro-vesicular steatosis, adiposity nanoP accumulate in the cytoplasm, after rapid endosomal escape with and circulating cholesterol. We previously found that FAP gene- no significant effect on viability or morphology, up to 3.1x108 particles 2 knockout mice had increased intrahepatic non-esterified free fatty per mm . The presence of long-lived radicals formed during synthesis acids, indicative of increased lipolysis and β-oxidation. Concordantly, facilitates direct covalent immobilization of biomolecules by simple lipogenic genes (Pparγ, Gck, Acc, Fasn) and hepatic triglyceride and incubation. Binding is rapid with >80% coverage within 30 seconds, fatty acid uptake genes (Cd36, Apoc3, Ldlr) were downregulated in and has been demonstrated for a range of molecules including drugs, FAPgki livers. We identified natural substrates of FAP, such as LOXL-1, imaging agents, SiRNA, enzymes and antibodies. The addition of CSF-1, CCL-2, C1qT6 and FGF21, pointing to roles of FAP in ECM- multiple functionalities is achieved by co-incubation in solution. To 3 cell interactions, metabolism and immunoregulation. The starvation exemplify one possible utility of nanoP , we delivered functional small hepatokine FGF21 was of particular interest. FGF21 was increased in interfering RNA targeted to VEGF in HUVECs, impairing the formation FAP deficient DIO compared to WT DIO mice. Thus, these metabolic of tubules in an established Matrigel assay. To our knowledge this is the changes depend upon FAP activity and FAP action on FGF-21 is likely first platform to facilitate co-delivery of multiple surface bound cargos involved. Conclusion: This is the first study showing that specific into diverse cell systems following one-step co-incubation without genetic ablation of FAP activity, which mimics a specific potent inhibitor, chemical pre-functionalization steps. This approach eliminates the is protective of DIO-driven glucose intolerance, insulin resistance and trade-off between additional functionality and complexity and could liver steatosis in mice. facilitate the upscaling of nanoparticle-based therapies into the clinic.

SYM-50-05 SYM-51-01 CLOSER TO NATURE IN VITRO: ORGAN-SPECIFIC THE BACTERIAL REPLISOME: DESIGN PRINCIPLES EXTRACELLULAR MATRIX-BASED THREE FOR A DYNAMIC MOLECULAR MACHINE DIMENSIONAL MODELS OF CANCER Dixon N.E., Spenkelink L.M., Lewis J.S., Xu X.-Q., Jergic S. and Nadort A.1, 2, Iqbal S.1, 2, Parker L.1, 2, Packer N.1, 2, Goldys E.2, 3 and van Oijen A.M. Guller A.2, 3 Molecular Horizons and School of Chemistry & Molecular Bioscience, 1Macquarie University, NSW 2109, Australia. 2ARC Centre of University of Wollongong, Wollongong, NSW 2522, Australia. Excellence for Nanoscale BioPhotonics, Australia. 3University of New South Wales, NSW, 2032, Australia. The E. coli replisome is a complex and dynamic assembly of more than 20 protein subunits that include the multi-protein Pol III chromosomal Successful clinical translation of techniques and therapies to detect replicase and the primosome (helicase/primase). The replisome works to and treat cancer needs controlled, ethical and practical lab-based ac hi eve simult ane ous c o py ing of b ot h st r ands at a rep lic at i o n fo r k at r ate s tumour models that more accurately represent the biological reality. We that approach 1000 bp/s, with near-perfect fidelity. In the textbook view, repurposed tissue engineering methodology to create a biochemically leading and lagging strand DNA replication are perfectly coordinated and structurally authentic environment for in vitro cell culturing and processes that are orchestrated to occur deterministically in discrete developed an organ-specific three-dimensional (3D) model of cancer steps in space and time. However, there is no evolutionary pressure to closely simulating real tumour tissue. We obtained acellular organ- achieve such elegance, nor do fundamental chemical principles allow it. specific tissue scaffolds with preserved extracellular matrix composition I will integrate recent structural and single-molecule biophysical studies and structure by original decellularization protocols, followed by that are being used to develop a new picture of replisomal function seeding and culturing the desired cancer cells to obtain tumour that is messier than the textbook view. In particular, coupled leading tissue engineering constructs (TECs). We focused on aggressive and and lagging strand replication assays with fully assembled replisomes problematic cancers such as high-grade brain cancer (glioblastoma containing fluorescent proteins enable protein exchange processes at mulitforme, GBM) and triple negative breast cancer (TNBC) known a replication fork to be imaged in real time at the single-molecule level. for its high rate of hepatic metastasis. Following our protocols, we We have shown, for example, that Pol III replicase complexes undergo created brain-TECs of GBM cells (human, U87 and U251) and control frequent exchange at the fork, on a time scale that depends on their undifferentiated neurons (rat, PC12), as well as liver-TECs of TNBC cells concentration in solution [Lewis et al., 2017, eLife, 6, e23932], while (human, MDA-MB-231) to mimic hepatic metastasis. We extensively single-stranded DNA-binding protein (SSB) can be retained at the fork characterized the tumour TECs and compared the hallmarks of tumour through multiple cycles of Okazaki fragment synthesis on the lagging progression, such as the growth dynamics, migration behavior, cell strand (Lisanne Spenkelink, unpublished). morphology, metastatic colonization, angiogenic potential and drug sensitivity, to 2D cultures and control TECs. Our results show a novel biologically accurate, living 3D tumour model, revealing ECM-specific cellular behavior that enables a more realistic study of tumour biology and therapeutic response. Our organ-specific tumour TECs can be used as a tool to improve the detection and treatment of cancer, and represent a sustainable approach to fill the gap between conventional 2D cell cultures, animal studies and clinical trials.

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SYM-51-02 SYM-51-03 FANCM SUPPRESSES ALT ACTIVITY BY MODULATING PROTECTING THE GENETIC CODE BLM-TOP3A-RMI COMPLEX ACTIVITY AT TELOMERES Richard D.J.1, Adams M.1, Cubeddu L.2, Gamsjaeger R.2, Bolderson E.1, Pickett H.A. Leong V.1, Burgess J.1, Pacquet N.1 and O’Byrne K.1 Children’s Medical Research Institute. 1Queensland University of Technology. 2Western Sydney University.

The collapse of stalled replication forks is one of the major drivers of Single-stranded DNA binding (SSB) proteins play a critical role in DNA genomic instability, and cells have evolved committed mechanisms to replication and repair in all three domains of life. SSB proteins all employ overcome replication stress. These pathways are particularly pertinent an oligonucleotide/oligosacharide binding (OB) fold in order to bind the at telomeres, which are long terminal repetitive DNA regions that are single-stranded DNA substrate. Human SSB1 (hSSB1, NABP2), has intrinsically prone to replication fork slowing, stalling and breakage. been demonstrated to play key roles in the DNA damage response, Cancer cells that use the Alternative Lengthening of Telomeres (ALT) especially in the process of double-stranded DNA breaks (DSBs) pathway of telomere maintenance display elevated levels of telomere- repair, at stalled DNA replication forks and in response to oxidative specific replication stress, and it is thought that ALT cells utilise these damage to DNA. In this presentation, we look at the role hSSB1 plays degenerate sites as substrates for break-induced telomere synthesis in the cellular response to oxidative DNA damage. Further, we will events. FANCM is a multifunctional protein that is central to the present data outlining the molecular mechanism through which hSSB1 Fanconi anaemia (FA) core complex, and can independently bind to recognises the genetic lesion and recruits the correct repair proteins for the BLM-TOP3A-RMI (BTR) complex. We demonstrate that FANCM the processing of the damage. Lastly, we will present data illustrating depletion provokes excessive ALT activity, evident by rapid induction the critical role hSSB1 plays in cancer cell biology and preliminary data of extrachromosomal telomeric repeat (ECTR) DNA, and increased on the first in class inhibitor of hSSB1. In summary, our presentation will firing of break-induced telomere synthesis events. This culminates in highlight the critical role hSSB1 plays in preventing the loss of genetic an ALT-specific G2/M arrest and loss of cell viability. The MM2 domain information within the human genome, a process that drives cancer of FANCM, which binds the BTR complex, suppresses this response, initiation and progression. suggesting that ALT activity is attenuated by FANCM-BTR-mediated replication fork remodelling, and that collapsed replication forks instigate ALT-mediated telomere synthesis events.

SYM-51-04 SYM-51-05 SINGLE MOLECULE SUPER-RESOLUTION MAPPING HISTONE FLIM-FRET MICROSCOPY REVEALS OF THE SPATIOTEMPORAL ORGANIZATION OF DNA SPATIOTEMPORAL REGULATION OF CHROMATIN DOUBLE STRAND BREAK REPAIR ORGANIZATION BY THE DNA DAMAGE RESPONSE

Whelan D.R.1, 2, Lee W.T.C. 2, Yin Y.2, Fenyo D.2 and Rothenberg E.2 Lou J.1, Scipioni L.2, Gaus K.3, Gratton E.2, Cesare A.4 and Hinde E.1, 3 1La Trobe Institute for Molecule Science, La Trobe University, 1Department of Biochemistry, University of Melbourne, Edwards Road, Flora Hill, Victoria, Australia, 3552. 2Department of Australia. 2Biomedical Engineering, University of California, Irvine, Biochemistry and Molecular Pharmacology, Langone Medical Center, USA. 3EMBL Australia Node in Single Molecule Science, University New York University, 550 First Ave, New York, NY, USA, 10016. of New South Wales, Sydney, Australia. 4Children’s Medical Research Institute, University of Sydney, Australia. DNA damage response (DDR) pathways are involved in both the cause and potential treatment of various cancers, auto-immune, and Here we describe a biophysical method to measure chromatin neurodegenerative diseases. Our current understanding of DDR has organisation in live cells with nucleosome level resolution. The method been elucidated over the course of several decades by combining is based on a localised phasor image correlation analysis (ICS) of biochemical and biophysical techniques, however, imaging of damage FLIM-FRET microscopy data acquired in human cells co-expressing and repair in vivo has remained challenging. This has predominantly H2B-eGFP and H2B-mCherry. This multiplexed approach produces been because of the dense and varied nature of the nuclear environment, spatiotemporal maps of nuclear wide chromatin compaction and and the diffraction limit of light. Here, we have successfully used single quantifies the stability, size and spacing between detected chromatin molecule localisation super resolution (SR) imaging to circumvent this foci. We used this method in cells where double strand breaks limit and capture spatially and temporally resolved snapshots of double (DSBs) were induced by near-infrared laser micro irradiation to strand break (DSB) repair in cells. Moreover, we specifically generated assay chromatin dynamics during the DNA damage response (DDR). individual single-ended DSBs similar to those endogenously created These experiments revealed that ATM and RNF8 directed rapid local by collapsing replication forks. The resulting repair foci could be chromatin decompaction at DSBs, coupled with formation of a stable visualized in multicolor SR by labelling nascent DNA via modified base ring of compact chromatin surrounding the repair locus. Based on these incorporation and click chemistry, DSBs via the TUNEL assay or direct data we built a longevity map of sites with high FRET indicating the time ligation, single stranded DNA via BrdU incorporation, and proteins via scale of large scale compaction events directed by ATM and RNF8. immunolabelling. The enhanced spatial and temporal resolutions and Then by use of a phasor-based ICS analysis we identified the locations the singular nature of the DSBs themselves revealed several exciting where the DDR shapes local and global chromatin dynamics and and novel insights including the dynamic interactions of proteins such demonstrate the utility of phasor ICS-FLIM analysis of histone FRET as Ku, MRE11 and RAD51 at the DSB, the redundant role of RAD52 for the study of chromatin biology. in repair, and a critical in vivo BRCA2 dependence on BRCA1. I will present these findings within the context of their importance to the genomic integrity research community, as well as the broader novelty and applicability of the SR assays we have developed.

Page 80 ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 symposia TUESDAY

SYM-52-01 SYM-52-02 USE OF VOLTA PHASE PLATE SINGLE STRUCTURAL BASIS OF TIR-DOMAIN ASSEMBLY PARTICLE CRYO-ELECTRON MICROSCOPY FOR FORMATION IN MYD88/MAL-DEPENDENT TLR4 DETERMINATION OF ACTIVE STATE G PROTEIN- SIGNALING COUPLED RECEPTOR STRUCTURE Ve T.1, 2, Vajjhala P.R.1, Hedger A.1, Croll T.3, Dimaio F.4, Horsefield S.1, 1 1 5 1 Wootten D., Liang L., Khoshouei M., Glukhova A., Draper Joyce C., Landsberg M.J. , Stacey K.J. , Egelman E.H. and Kobe B. Christopoulos A. and Sexton P.M. 1School of Chemistry and Molecular Biosciences, University of Drug Discovery Biology, Monash Institute of Pharmaceutical Queensland, Brisbane, QLD 4072, Australia. 2Institute for Glycomics, Sciences, Monash University, Parkville, Victoria. Griffith University, Southport, QLD 4222, Australia. 3Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, England. 4Department of Biochemistry, University of Cryo-electron microscopy (cryo-EM) has gained prominence as a 5 method of choice for determination of structure for difficult to crystallise Washington, Seattle, Washington, USA. Department of Biochemistry membrane proteins, including active state, transducer complexed and Molecular Genetics, University of Virginia, Charlottesville, VA G protein-coupled receptors (GPCRs). Class GPCRs bind critically 22908, USA. important physiological peptides of 30-40 amino acids, and are important targets for major diseases including diabetes, obesity and Toll-like receptor (TLR) signaling represents a key innate immunity osteoporosis. Our laboratory has recently applied Volta Phase Plate response to pathogen products. Recruitment of signaling adapters (VPP) single particle cryo-EM to determine structures of class GPCRs such as MAL/TIRAP and MyD88 to the receptors requires TIR-domain in complex with their canonical transducers, heterotrimeric Gs proteins. interactions, which remain structurally elusive. Here we show that MAL This methodology has been applied to minimally modified receptors, TIR domain spontaneously and reversibly forms filaments in vitro, including the human calcitonin receptor, the glucagon-like peptide-1 forms a co-filament with TLR4 TIR domain, and induces formation of a receptor (GLP-1R), and the calcitonin-gene-related peptide receptor MyD88 assembly. A 7 Å resolution cryo-electron microscopy structure (CGRPR), which consists of the calcitonin-related receptor and a reveals a stable MAL proto-filament consisting of two parallel strands of single pass transmembrane protein, receptor-activity modifying protein TIR-domain subunits in a BB loop-mediated head-to-tail arrangement. 1 (RAMP1). These studies reveal common macromolecular changes Residues at the interfaces important for the interaction are conserved associated with class B GPCR activation and G protein coupling, among different TIR domains. Although large filaments of TLR4, MAL or receptor specific-differences in peptide hormone binding and critical MyD88 are unlikely to form during signaling in the cell, structure-guided structural insights into GPCR modulation by RAMPs. Moreover, for the mutagenesis, combined with in vivo interaction assays, demonstrate GLP-1R we have solved structures bound to different biased peptide that the MAL interactions defined within the filament represent a agonists that reveal conformational variances within the receptor template for a conserved mode of TIR domain interaction involved in that can be linked to distinct efficacy for Gs signalling. More recently, both TLR and IL-1R signaling. we have solved the structure of the class A adenosine A1 receptor bound to its endogenous agonist adenosine and a Gi2 heterotrimeric protein, revealing novel insights regarding GPCR G protein selectivity. Collectively, this work highlights the power of VPP cryo-EM for GPCR structure determination and the similarities and diversities in modes of receptor activation between members of related subfamilies.

SYM-52-03 SYM-52-04 INVESTIGATING THE ARCHITECTURE OF A BIOPHYSICAL APPROACHES TO THE STUDY OF BACTERIOPHAGE USING CRYO-EM, SAXS, AND HETEROMERIC AMYLOID FIBRILS INVOLVED IN X-RAY CRYSTALLOGRAPHY VIRAL INHIBITION OF NECROPTOSIS

Hardy J.M.1, Dunstan R.1, Grinter R.1, Pickard D.2, Venugopal H.3, Pham C.L.L.1, Strange M.1, O’Carroll A.2, Shanmugam N.1, Sierecki E.2, Belousoff M.1, Gordon D.2, Lithgow T.1 and Coulibaly F.1 Gambin Y.2, Steain M.3 and Sunde M.1 1Biomedicine Discovery Institute, Monash University, Clayton, VIC, 1Discipline of Pharmacology, School of Medical Sciences and Sydney Australia. 2Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, Nano, University of Sydney, NSW 2006, Australia. 2EMBL Australia United Kingdom. 3Ramaciotti Centre for Cryo-Electron Microscopy, Node in Single Molecule Sciences, School of Medical Science, Monash University, Clayton, VIC, Australia. University of New South Wales, NSW 2052, Australia. 3Infectious Diseases and Immunology, Central Clinical School, Sydney Medical YSD1 is a lytic bacteriophage that infects and kills Salmonella School, University of Sydney, NSW 2006, Australia. enterica serovar Typhi, the causative agent of typhoid fever. YSD1 is a Siphoviridae homologous with the bacteriophage chi, which uses a The large DNA viruses, cytomegalovirus and herpes simplex virus, flagella-dependent infection mechanism. We have shown by electron express proteins that inhibit the host necroptosis programmed cell death microscopy (EM) that YSD1 attaches to flagella through its tail proteins. pathway. Our recent studies show that the RIP homotypic interaction Currently, there are no high-resolution structures of flagella-dependent motif (RHIM) within the viral necroptosis inhibitor proteins renders bacteriophages. To elucidate the structure of YSD1, purified virions them amyloidogenic. The viral proteins are therefore functional amyloid were isolated from cultures of Salmonella typhimurium and imaged structures, in which the biologically active form of the protein is retained by cryo-EM. A 2.8 Å x-ray crystallography structure combined with or generated in the self-assembled fibrillar form of the protein. We have the 4.7 Å EM map revealed a T=7 icosahedral capsid similar to the used a wide range of biophysical techniques, including single molecule HK-97 and T7 phages. However, in contrast to HK-97, which uses fluorescence studies, to investigate the fibrils formed by these proteins. crosslinking to reinforce the capsid, YSD1 has an additional cementing We have demonstrated that these proteins are able to form heteromeric protein stabilising the icosahedral shell formed by the major capsid amyloid fibrils through co-assembly with host proteins, RIPK1, RIPK3 protein. Helical reconstruction of the tail produced a 3.8 Å map which and ZBP1/DAI, which each contain RHIMs. Incorporation of viral revealed a C6 helical tube related to Type VI secretion systems and proteins into host amyloid fibrils alters the structure and properties of the tails of T4 and T5 phages. The YSD1 tail is composed of a central the fibrils and renders the component proteins unable to signal for cell beta-barrel domain decorated by two peripheral domains. A beta- death. sandwich domain, unique to Chi-like phages, has structural similarity with bacterial adherence factors. The C-terminal domain is flexible and not well resolved in the helical reconstruction. The Ig-like fold and organisation of this domain was determined using modelling and small- angle x-ray scattering (SAXS). The determination of the structure of YSD1 gives us an insight into the assembly and evolution of flagella- dependent bacteriophages.

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SYM-53-01 SYM-53-02 FUNCTIONAL GENOMICS OF SYMBIOTIC NITROGEN ALUMINIUM ACTIVATED MALATE TRANSPORTERS FIXATION IN LEGUMES FACILITATE GABA TRANSPORT IN PLANTS

Udvardi M., Roy S., Liu W., Nova Franco B., Espinoza M., Kang Y., Ramesh S.A., Gilliham M. and Tyerman S.D. Torres-Jerez I. and Huertas R. ARC Centre of Excellence in Plant Energy Biology, School of Noble Research Institute, Ardmore, OK, USA. Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Glen Osmond, SA 5064 Australia. Discovery of the first plant gene required for legume nodule development and symbiotic nitrogen fixation (SNF), LjNIN from Lotus Gamma-aminobutyric acid (GABA) is a neurotransmitter regulating japonicus, occured in 1999 (Schauser et al, 1999). Today, over 150 membrane potential in nerve cells. GABA rapidly accumulates in plant genes in multiple legume species have been found to be required for tissues in response to various stresses, and regulates growth. We nodule development and/or effective nitrogen fixation, via forward- identified GABA-binding sites within plant Aluminium-activated Malate genetics or by genomics-informed reverse-genetics (Roy, Liu, et al., Transporter (ALMT) proteins with homology to GABA-binding motifs unpublished). These genes have been implicated in signaling between of mammalian GABAA receptors (GABA-gated anion channels). The rhizobia and legumes, infection and accommodation of the micro- ALMTs are currently classified as anion channels but are also regulated symbiont in plant cells, nodule organogenesis, and plant metabolism by d i ve r s e s i g n a l s l e a d i n g t o a r a n g e o f p hy s i o l o g i c a l r e s p o n s e s . We h ave in support of bacterial nitrogen fixation. Although some of these genes previously demonstrated that anion flux through ALMTs is negatively appear to be indispensable for SNF in several legumes species, not regulated by GABA and its analogs with an EC50 in the low micromolar all are necessary in all species. This may reflect genetic and/or other range[1]. We also observed that activation of wheat ALMT (TaALMT1) functional redundancy within species related to either ancient genome led to a negative correlation between malate efflux and endogenous duplications or more recent tandem duplications of genes. It also GABA concentrations in root tips and heterologous expression systems. appears to reflect, to some extent, distinct co-evolution of legumes Recently we have shown that this negative correlation is a result of and specific rhizobia. Despite the large number of genes now known GABA efflux facilitated by TaALMT1[2]. Activation of TaALMT1 leads to to be required for SNF in legumes, our knowledge of the molecular GABA transport into the cells demonstrated by yeast complementation and cellular basis of nodule development and metabolism remains and 14C[GABA] uptake into Xenopus oocytes. GABA transport into fragmentary. This talk will summarize what is known about the genetics yeast was observed for all ALMTs we examined. Interestingly, mutation of the various processes that lead to and support SNF, including our of residue (TaALMT1F213C) in the GABA motif prevented GABA influx work on Medicago truncatula, and highlight some of the gaps in our and efflux in the multiple test systems we used, and abolished the knowledge in these areas. Finally, we address the question: how can negative correlation between anion efflux and GABA concentration. SNF be improved in legumes with so many genes implicated? We GABA and malate thus appear to interact with ALMTs in a complex are using natural variation in SNF in approximately 200 ecotypes manner to regulate each others transport and this is suggestive of a role of M. truncatula to identify genes that contribute to effectiveness for ALMTs in communicating metabolic status of cells. 1. Ramesh, S., et in this species, via genome-wide association studies, with a view to al., GABA signalling modulates plant growth by directly regulating the developing plant breeding strategies to improve SNF in crop legumes. activity of plant-specific anion transporters. Nature Communications, Examples of putative SNF effectiveness genes will be presented. 2015. 6: p. 7879. 2. Ramesh, S.A., et al., Aluminium-Activated Malate Transporters Can Facilitate GABA Transport. Plant Cell, 2018. 30 (5) 1147-1164; DOI: https://doi.org/10.1105/tpc.17.00864.

SYM-53-03 SYM-53-04 ORGANIC ANION EXUDATION FROM ROOTS FROM SMALL RNA TO METABOLITES - HOW INFLUENCES MINERAL NUTRITION, ROOT MICROBIOME EUCALYPTS MEET UP WITH ECTOMYCORRHIZAL FUNGI COMPOSITION AND ROOT ARCHITECTURE Wong J.W.H.1, Lutz A.2, Natera S.2, Wang M.3, Ng V.3, Grigoriev I.3, Ryan P.R. Martin F.4, Roessner U.2, Anderson I.1 and Plett J.1 CSIRO Agriculture and Food, Canberra. 1Hawkesbury Institute for the Environment, Western Sydney University, Richmond, NSW 2753, Australia. 2School of BioSciences, University 3 Plants release an estimated 10-20% of fixed carbon into the soil. of Melbourne, Parkville, Victoria, 3010, Australia. U.S. Department of Energy Joint Genome Institute, Walnut Creek, USA. 4INRA, UMR 1136, Whether released as a consequence of growth or in response to INRA-Nancy Universite, Interactions Arbres/Microorganismes, 54280 specific triggers and stresses, these exudates modify the chemistry of Champenoux, France. the rhizosphere which can benefit growth and survival. For example, the release of organic anions from roots enables some plants to cope 3+ Ectomycorrhizal (ECM) fungi are often associated with roots of forest trees, with the toxic aluminium (Al ) cations prevalent in acidic soils. Some bringing benefits to their hosts including enhanced nutrient uptake and of the genes controlling the organic anion release have been identified increased stress tolerance. How trees communicate with ECM fungi prior to in crop species including wheat, barley, rice and maize. In wheat, the physical interaction, and how this pre-symbiotic exchange of signals differs release of malate and citrate anions is controlled by an anion channel from plant-pathogen interactions, is largely unexplored. With a focus on this encoded by TaALMT1 and a co-transporter encoded by TaMATE1B, pre-symbiosis stage, we aim to decipher the communication routes between respectively. These transporters had only been characterised in the model tree Eucalyptus grandis and one of its associated ECM fungal young wheat seedlings so more recent experiments examined their species: Pisolithus microcarpus. Considering the roles of metabolites and physiology in 30 day-old plants with mature root systems. Malate RNAs in plant-microbial signalling as determined by previous studies, we release was detected from the seminal, nodal and lateral roots but examined ECM fungi-eucalypt molecular interaction with a combined use of citrate release only occurred from seminal and nodal roots. We further RNA-seq, small RNA-seq and untargeted metabolite profiling.We identified showed that citrate release affected the structure and composition of significant changes in both the metabolome and transcriptome (including the microbiome near the root apices. These genes therefore provide both small RNAs and RNAs) of eucalypt roots during this pre-symbiotic strategies for manipulating the root microbiome. More surprisingly, period with P. microcarpus. These results suggest that eucalypts are able the TaMATE1B locus was associated changes in root architecture to respond to the presence of ECM fungi prior to physical contact. We then because when wheat was grown in acid soil or hydroponic solutions further investigated whether these changes were activated by internal gene with toxic levels of aluminium, lines with the TaMATE1B locus regulation of eucalypt roots, or by the trafficking of fungal signals. With our small RNA-seq data, we observed a significant portion of small RNA reads developed 30-50% more nodal roots than near-isogenic lines without detected in eucalypt roots are originated from P. microcarpus rather than E. this locus. Finally, the constitutive release of malate anions from roots grandis. Amongst these fungal reads, we identified one species of fungal of transgenic rice lines altered the subcellular compartmentation of small RNAs that putatively targets defense-related R genes of the plant manganese in leaves. These experiments demonstrate that the release host during pre-symbiosis. Additionally, with the use of isotopic labeling in of simple organic anions such as malate and citrate from roots can conjunction to untargeted metabolite profiling, we also identified some ECM influence plant growth and physiology in multiple ways. fungi-derived metabolite signals in eucalypt roots. Therefore, by dissecting the molecular signals in eucalypt roots using a multi-omics platform, our research suggests that both small RNAs and metabolites are signals used by ECM fungi to prime their host plant during this pre-symbiotic stage of interaction.

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SYM-53-05 SYM-54-01 TISSUE-SPECIFICITY OF SODIUM TRANSPORT AND DIFFERENTIATION STATES IN THE ORIGIN EVOLUTION SEQUESTRATION REVEALS THE ROLE OF THE ROOT AND TREATMENT OF CHILDHOOD LEUKAEMIA MERISTEM AS A TENTATIVE SALT SENSOR Enver T. UCL Cancer Institute, London. Wu H.H.1, 2, Shabala L.1 and Shabala S.1 1Tasmanian Institute for Agriculture, University of Tasmania, Hobart, 2 Childhood acute lymphoblastic leukemia is thought in many cases to Tasmania 7001, Australia. Department of Botany and Plant Sciences, initiate in utero. The factors that influence the formation of the initiating University of California, Riverside, CA, U.S. 92521. lesions are not well understood although the nature of these genetic aberrations is well documented and include in approximately a quarter of The progress in plant breeding for salinity stress tolerance is children the presence of the t12;21 chromosomal translocation that fuses handicapped by the lack of understanding of the specificity of salt stress the transcription factors Tel (ETV6) and Aml1 (RUNX1). The Tel-Aml1 fusion signalling and adaptation at the cellular and tissue levels. In this study, gene produces a pre-leukemic clone but in and of itself is insufficient to we used electrophysiological, fluorescence imaging and real-time produce frank leukemic transformation. For this, additional mutations are qPCR tools to elucidate the essentiality of the cytosolic Na+ extrusion required and it remains unclear what factors influence their acquisition. in functionally different root zones (elongation; meristem; mature) in a These additional mutations tend to arise in loci that are normally involved large number of bread and durum wheat accessions. We show that in cell fate control in the B lineage. Cell fate regulators act in a context the difference in the root’s ability for vacuolar Na+ sequestration in the dependent manner and thus understanding the nature of the target cell or mature zone may explain differential salinity stress tolerance between cells in which the initiating and subsequent mutations arise is of importance. salt sensitive durum and salt tolerant bread wheat species. Bread wheat To gain insight with these issues we have been exploring the target genes genotypes also had on average 30% higher capacity for net Na+ efflux of TEL-AML1 and associated second hits as well as developing new fetal from the root elongation zone, providing the first direct evidence for the specific models in which to examine the biological impact of TEL-AML1. In essentiality of root salt exclusion trait at the cellular level. At the same this regard we have identified candidate target cells for TEL-AML1 within time, cytosolic Na+ accumulation in the root meristem was significantly the human foetal liver and modelled these using human IPS cells in vitro. higher in bread wheat, leading to suggestion that this tissue may harbor This has allowed us to understand that hematopoietic differentiation hierarchies differ in fetal vs adult life and that TEL-AML1 functions primarily a putative salt sensor. This hypothesis was then tested by investigating as a lineage deregulator. Target gene analysis further reveals how TEL- patterns of Na+ distribution and relative expression level of several AML1 alters epigenetic programs and reveals opportunities for therapeutic key genes related to Na+ transport in leaves in plants with intact roots intervention. Leukemic clones appear to evolve in a branching manner such and those in which the roots meristems were removed. We show that that at presentation the marrow is replete with multiple variegated subtypes tampering with this sensing mechanism has resulted in a salt-sensitive providing a diverse substrate for selection in response to therapy. Beyond phenotype, largely due to compromised plant’s ability to sequester Na+ genetic heterogeneity, leukemic cells exhibit epigenetic heterogeneity in in mesophyll cell vacuoles. The implications of these findings for plant respect of their immune-phenotypes and functional properties including breeding for salinity stress tolerance are discussed. cell cycle status and niche residence. To obtain a “real-time” longitudinal analysis of sub-clonal dynamics through treatment we have established an in vivo mouse model that allows a patient tumour to be independently exposed to treatment multiple times, enabling us to distinguish between deterministic and stochastic mechanisms of selection during therapy. Our results suggest that epigenetic state - including cycle and differentiation status - rather than genotype dictates resistance to chemotherapy.

SYM-54-02 SYM-54-03 ROLE OF ELK1 IN CONGENITAL AND LATE ONSET CONTROL OF DROSOPHILA MYC TRANSCRIPTION, CARDIAC DISEASE: AT THE HEART OF THE MATTER CELL GROWTH AND DEVELOPMENTAL PATTERNING BY THE SINGLE STRANDED DNA BINDING PROTEIN PSI Hallab J.C.1, Del-Monte Nieto G.2, Bouveret R.2, Varshney A.1, Huttner I.2, Santiago C.2, Miles L.3, Dworkin S.3, Kikuchi K.2, Hesselson, D.4, Fatkin, D.2, Zaytseva O.1, 2, Kim N.1, Guo L.1, 2, Mitchell N.C.1, Evers M.1, Marshall O.J.3, Harvey, R. 2 and Ramialison M.1 Hannan R.D.1, 4, Levens D.L.5 and Quinn L.M.1 1Australian Regenerative Medicine Institute, Monash University, 1The John Curtin School of Medical Research, ANU, Canberra, Melbourne. 2Victor Chang Cardiac Research Institute, Sydney, Australia. 2The University of Melbourne, Parkville, Australia. 3Menzies Australia. 3Department of Physiology, Anatomy and Microbiology, LaTrobe Institute for Medical Research, Hobart, Australia. 4Peter MacCallum University, Melbourne. 4Garvan Institute of Medical Research, Sydney, Cancer Centre, Melbourne, Australia. 5National Cancer Institute, NIH, Australia. Bethesda, Maryland, USA.

Elk1 is an ETS Class I, TCF subfamily transcription factor known as a The transcription factor MYC is upregulated in 70% of cancers and well- established downstream effector of the MAPK pathway and has been elevated MYC potently drives growth and proliferation. Thus, the implicated in the causation of a variety of cancers. Recent in vitro evidence capacity of the MYC promoter to integrate developmental signals places Elk1 in the context of the cardiogenic transcription factor network, is essential for correct growth patterning in all multicellular animals. although its in vivo role in cardiogenesis remains unexplored. We provide the Studies of the MYC promoter identified Far Upstream Binding Protein first in vivo evidence of the role of Elk1 in cardiogenesis using a zebrafish 1 (FUBP 1), a single stranded DNA binding protein, which acts to mutant with disrupted DNA binding domain (elk1-543/-543) and cardiac defects including valve displacement and elongation with hypertrophic/ activate MYC expression. Consistently with this function, FUBP1 is hyperplastic changes in the ventricular myocardium. elk1-543/-543 are upregulated in many cancers, including breast, liver, bladder, kidney predisposed to early embryonic death, with high incidence of heart looping and lung. Moreover, our recent Drosophila studies revealed that the defects and accelerated growth among survivors. RNA-sequencing (RNA- FUBP1 ortholog (Psi) interacts with the transcriptional Mediator (MED) seq) at larval stage provides insights into the basis of heart defects, indicating complex to integrate developmental signals, activate MYC and promote up-regulation of MAPK pathway genes and down-regulation of trim63a, cell and tissue growth in the wing epithelium. Paradoxically, our recent encoding a homeostatic protein involved in reducing muscle mass, dys- unpublished data demonstrate expansion of the neuroblast stem cell regulation of which is associated with Hypertrophic Cardiomyopathy (HCM) lineage in the Drosophila brain after Psi depletion. In line with tissue- in humans. MAPK pathway up-regulation is commonly associated with specific functions, FUBP1 knockout mice exhibit hypoproliferation HCM although the fundamental basis of this relationship is not completely in the embryonic blood lineage, while overgrowth occurs in the understood. We provide mechanistic insight, suggesting MAPK perturbations brain. Furthermore, in contrast to other tumour types, FUBP1 loss- could converge via the TCFs at a predicted trim63a enhancer, down- of-function ranks in the top 10% of predicted driver mutations in regulating trim63a to mediate HCM. Early developmental RNA-seq indicates oligodendroglioma, the second most common primary brain cancer in loss of Elk1 function is associated with down-regulation of tumor suppressor adults. The mechanisms behind these striking context-specific roles is genes. We hypothesize this promotes embryonic survival via non-optimal unknown; therefore our current studies in Drosophilamodels focus on pathways, and underlies observed defects. The sum of changes in elk1 -543/- identification of key Psi transcriptional targets in both wing epithelium 543 mimic a group of congenital syndromes known as “RASopathies” in humans. Our data provides important insights into the time line of molecular and neural stem cells. events underlying RASopathies/MAPK pathway defects and their relationship to molecules imperative in heart patterning and homeostasis.

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SYM-54-04 SYM-54-05 EXPLORING THE FUNCTION OF NKXUS, A NOVEL DEVELOPMENTAL DISORDERS OF HISTONE HEART-ASSOCIATED LNCRNA MODIFICATION

Altekoester A.1, 3, Schonrock N.2, Wu J.1, Kesteven S.1 and. Harvey R.P.1 Voss A. 1Victor Chang Cardiac Research Institute, Australia. 2Garvan Institute of Medical Research, Australia. 3University of Cologne, Germany.

A significant large amount of the mammalian genome previously ABSTRACT NOT AVAILABLE found to not code for proteins and considered “junk”, was found to AT TIME OF PUBLICATION actually specify a dynamic network of regulatory RNAs, termed long non-coding RNAs (lncRNAs). Here, we identified Nkx2-5UPSTREAM (NkxUS), a heart-associated lncRNA, which lies upstream of the cardiac key transcriptional regulator NKX2-5. In humans, NKX2-5 is essential for proper heart development and mutations are commonly associated with congenital heart disease. We hypothesise that NkxUS either regulates NKX2-5 itself or other processes affecting heart function and development. The aim of my study is to fully characterize NkxUS especially its function. Our studies show that NkxUS is a long, cardiac and nuclear enriched transcript. It is expressed in the heart throughout development and a similar heart specific transcript occurs in humans. We found a heart rate-associated GWAS SNP, located within human NkxUS, which lies within and disrupts a RNA structure conserved between mouse and human. When knocking down the mature transcript, genes involved in heart contraction and calcium signalling are down regulated. Mice lacking the conserved structure exhibit a higher resting heart rate with no differences in ECG parameter intervals, suggesting a possible dysfunction in the sinoatrial node (SAN), the pacemaker of the heart. Currently, we are performing calcium and voltage imaging combined with drug treatments on dissected mouse SANs to identify the basis of the phenotype. Further, we are testing the inducibility of atrial fibrillation (AF) in the same mice as the SNP was also associated with a higher risk of developing AF. Altogether, we present data on a novel cardiac enriched lncRNA and provide a detailed characterisation of this transcript to analyse how it might impact heart function and disease.

SYM-55-01 SYM-55-02 NEURAL AND DENDRITIC ACTIVITY DURING NEUROPEPTIDE F RECEPTOR ACTS IN SENSORY-BASED BEHAVIOUR THE DROSOPHILA PROTHORACIC GLAND TO REGULATE BODY SIZE AND DEVELOPMENTAL TIMING Palmer L.M. Florey Institute of Neuroscience and Mental Health, University of Kannangara J.R.1, Henstridge M.A.1, Parsons L.M.1, Kondo S.2, Mirth C.K.1 Melbourne. and Warr C.G.1 1School of Biological Sciences, Monash University, Clayton, Victoria 3800, In the living animal, sensory systems are generally not stimulated in Australia. 2Invertebrate Genetic Laboratory, National Institute of Genetics, isolation but are instead activated collectively. The task of understanding Mishima, Japan. how neurons receive and transform this sensory input is central to explaining brain function during behaviour. Pyramidal neuron dendrites Drosophila melanogaster is an excellent in which to i n t h e p r i m a r y s o m a t o s e n s o r y c o r t ex r e c e i ve b o t h f e e d f o r w a r d i n p u t f r o m study the regulation of growth, as many of the major growth factors the thalamus and feedback input from other cortical areas. Since the and signalling pathways are conserved between flies and humans. synaptic location of the different input streams are morphologically and In Drosophila, growth is primarily regulated by the steroid hormone, functionally isolated, how sensory input is integrated and computed at ecdysone, which is produced in and secreted from the prothoracic gland the level a single neuron is currently unknown. Here I will present recent in response to various environmental and genetic cues. Regulation of results investigating the activity of tuft dendrites of layer 2/3 pyramidal ecdysone biosynthesis in the gland involves a number of neuropeptides, neurons in the primary somatosensory cortex during a sensory-based for example the Drosophila insulin-like peptides, which regulate ecdysone reward association task. We find that tuft dendrites alter their activity production in response to nutrition. Here we identify the receptor for during the behavioural task when presented with multi-sensory input, Neuropeptide F (Npf), the fly homologue of mammalian NPY, as a novel increasing the occurrence of large Ca2+ events. This modulation of regulator of Drosophila growth. Knockdown of Npfrspecifically in the synaptic integration highlights the importance of feedback information prothoracic gland generates a developmental delay and increased in dendritic encoding of sensory-based behaviour. body size. Interestingly, while Npfr mutants also have a developmental delay, they instead have a smaller body size. Likewise, knocking down Npf in Npf-expressing neurons also results in a significant delay to development and smaller body size. These defects can be rescued by feeding larvae ecdysone-enriched food. Furthermore, there is reduced expression of ecdysone biosynthesis genes and reduced ecdysone levels in these animals. This suggests that Npf is involved in regulating ecdysone biosynthesis in the Drosophila prothoracic gland. NPY has been shown to interact with the insulin signalling pathway to regulate energy balance and body size in mammals, thus NPY and Npf may share conserved functions. Studies on Drosophila Npf may therefore further our understanding of human metabolic diseases.

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SYM-55-03 SYM-55-04 SITAGLIPTIN ALTERS METASTATIC PROGRESSION THE ROLE OF CDX2 AND HNF4α IN BARRETT’S AND IMMUNE RESPONSE IN AN ID8 OVARIAN METAPLASIA CANCER MOUSE MODEL Colleypriest B.J.1, 2, Burke Z.D.1, Griffiths L.P.1, 2, Slack J.M.W.1, 3 and Wilson A.L.1, 2, Wilson K.L.2, Moffitt L.R.1, 2, Bilandzic M.1, 2, Plebanski M.2, 3 Tosh D.1 and Stephens A.N.1, 2 1University of Bath, UK. 2Royal United Hospital, Bath, UK. 3Stem Cell 1Hudson Institute of Medical Research, VIC, Australia. 2Monash University, Institute, University of Minnesota, USA. VIC, Australia. 3RMIT University, VIC, Australia. Barrett’s metaplasia is the only known morphological precursor to Current methods used to treat epithelial ovarian cancer (EOC) often result oesophageal adenocarcinoma and is characterized by replacement of in relapse and acquired chemo-resistance; therefore, novel therapies for stratified squamous epithelium by columnar epithelium. The cell of origin EOC are urgently needed. Immunotherapy for EOC is gaining traction, is uncertain and the molecular mechanisms responsible for the change and recent studies have suggested that the dipeptidyl peptidase-4 (DPP4) in cellular phenotype are poorly understood. We therefore explored the inhibitor sitagliptin can activate the immune system, but this has not been role of two key developmental transcription factors, CDX2 and HNF4α demonstrated in ovarian cancer. Additionally, preclinical models that in the conversion, using primary organ cultures. Biopsy samples from accurately stage ovarian tumour growth are required for evaluating these cases of human Barrett’s metaplasia were analysed for the presence therapeutic responses in vivo, as no non-invasive techniques currently exist. of CDX2 and HNF4α. A new organ culture system for adult murine We developed a model in which ID8 mouse ovarian cancer cells stably oesophagus is described. Using this, Cdx2 and HNF4α were ectopically express a near-infrared protein (iRFP) via integration into the ROSA26 expressed by adenoviral infection. The phenotype following infection genomic region, then used this model to evaluate the anti-tumour effects was determined by a combination of PCR, immunohistochemical and of sitagliptin in vivo. C57BL/6 mice received an injection of 1x106 pROSA- morphological analyses. In the human biopsy samples we demonstrate iRFP720-ID8 EOC cells under the ovarian bursa. Mice were treated with the expression of both CDX2 and HNF4α. Our oesophageal organ sitagliptin (50mg/kg/body-weight/day) 14-days following implantation until culture system expressed markers characteristic of the normal SSQE: endpoint, and iRFP720 fluorescence was measured weekly to evaluate p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2, tumour deposition. T-cell, dendritic cell (DC), macrophage and myeloid- in these cultures induced expression of Tff3, villin, K8 and E-cadherin. derived suppressor cell (MDSC) populations in the spleen, lymph nodes So HNF4α is sufficient to induce a columnar-like phenotype in adult and blood were assessed by flow cytometry, and in tumour tissue by mouse oesophageal epithelium and is present in the human condition. immunofluorescence. We demonstrated that administration of sitagliptin These data suggest that induction of HNF4α is a key early step in the reduced tumour burden, as indicated by fluorescence and macroscopic formation of Barrett’s metaplasia and are consistent with an origin of tumour observations. Sitagliptin increased tumour-infiltrating T-effector Barrett’s metaplasia from the oesophageal epithelium. cells, decreased proliferating T-regulatory cells and decreased cytotoxic T-cell apoptosis. In addition, sitagliptin increased circulating DCs and macrophages, and decreased tumour-associated MDSCs. Taken together, these results suggest that administration of sitagliptin decreases tumour burden in an ID8 ovarian cancer model by shifting the balance toward anti- tumour immunity, and it may have therapeutic potential for epithelial ovarian cancer.

SYM-55-05 SYM-56-01 MORPHOGENESIS OF THE SEMICIRCULAR CANAL UNDERSTANDING AND ENGINEERING MICROBIAL DUCTS OF THE ZEBRAFISH INNER EAR SENSORS

Whitfield T.T. Gerth M.L. Bateson Centre and Department of Biomedical Science, University of Victoria University of Wellington, School of Biological Sciences, New Sheffield, Sheffield, S10 2TN, UK. Zealand.

The inner ear, the organ of hearing and balance, has a spectacular Bacterial chemoreceptors are remarkable examples of biological and complex epithelial morphology. The interlinked chambers and sensors: they can detect chemicals at nanomolar concentrations and ducts that make up the ear include the three orthogonally-oriented discriminate between closely related molecules. They play a central semicircular canals, which function to sense angular acceleration role in chemotaxis, allowing bacteria to detect chemical gradients and (turning movements) of the head. During embryogenesis, the otic bias their swimming behaviour in order to navigate towards favourable epithelium undergoes fusion and fission events that drive changes in environments. There are thousands of putative chemoreceptor genes tissue topology, defining the semicircular canal ducts. We are using in bacterial genomes, but for the vast majority, neither what they high-resolution confocal and light-sheet fluorescence microscopy detect nor how they detect it is understood. These chemoreceptors to image and analyse semicircular canal morphogenesis in the live represent a (largely untapped) source of modular parts for molecular zebrafish embryo. I will present a selection of our imaging studies devices. In my laboratory, we are exploring the functional and structural to illustrate the dynamic cell and tissue movements that accompany diversity of chemoreceptors. We are also engineering receptors with semicircular canal formation, both in wild-type embryos, and in mutant novel sensing capabilities. Ultimately, our goal is to incorporate these lines in which different aspects of semicircular canal development are modular domains into handle-held biosensor devices for applications disrupted. Together, these are giving insight into the molecular and in a variety of industries. cellular mechanisms underlying semicircular canal formation in the vertebrate embryo.

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SYM-56-02 SYM-56-03 STRUCTURE AND ASSEMBLY MECHANISM OF THE USING EVOLUTION TO GUIDE THE ENGINEERING TYPE III SECRETION SYSTEM NEEDLE TIP COMPLEX OF PPR PROTEINS AS CUSTOMIZABLE RNA PROCESSING TOOLS Tuckwell A.J., Xu S., Kyaw W. and Lee L.K. UNSW Sydney. Bernath-Levin K., Colas Des Francs-Small C., Gutmann B., Honkanen S., McDowell R., Melonek J., Pereira Vincis Sanglard L., Royan S., Sun Y. The type III secretion system (T3SS) is a protein superstructure, and Small I.D. consisting of hundreds of subunits, which self-assemble into a ARC Centre of Excellence in Plant Energy Biology, School of Molecular molecular syringe that injects virulence factors directly into the host cell. Sciences, University of Western Australia. The tip complex caps an extracellular needle filament and is involved in penetrating the host cell membrane. As a surface-exposed antigen, it is also an attractive target for vaccine development. High resolution Pentatricopeptide repeat (PPR) proteins are modular nucleic acid crystal structures of tip complex subunits have been determined from binding proteins that are highly prevalent in plants, with some species several species. However, their monomeric conformations appear producing thousands of different PPR proteins, each binding a different to be incompatible with EM micrographs. While these also provide target sequence. PPR proteins function almost exclusively inside information on stoichiometry, the resolution of EM micrographs are organelles, where they are involved in post-transcriptional steps at present far too low to determine the arrangement of subunits in an in gene expression, including RNA cleavage, splicing, RNA in-tact tip complex. This is in part because the tip complex can only stabilization, RNA editing and initiation of translation. Their sequence be visualised as part of the entire T3SS superstructure. Here we specificity resides in base-specific contacts made by 2-3 amino acids in combine, information from high-resolution crystal structures, solution each repeat that can be described by a simple code, making the target X-ray scattering and molecular dynamics simulations to obtain insight specificity of PPR proteins not only predictable, but programmable. into the assembly mechanism and structure of an in-tact T3SS tip Amongst the many different PPR proteins known, two groups show complex. These structural models are then used to rationally design unusual evolutionary behavior that make them particularly interesting and construct protein constructs and synthetic structural scaffolds from for protein engineering. Restorer-of-fertility factors are a small clade of DNA to artificially stabilise isolated T3SS tip complexes for structural PPR proteins showing a high degree of inter- and intra-specific variation characterisation. and strong diversifying selection on the residues that determine RNA binding specificity. We have found that this natural variability makes them ideal subjects for re-engineering and we are currently using them to make targeted knock-downs in mitochondrial gene expression. In a similar vein, but on a much larger scale, PPR RNA editing factors have undergone massive independent expansions in early-diverging land plants such as hornworts, lycophytes and ferns. Inspired by their natural functions, we are attempting to engineer these factors to control gene expression by the targeted creation and removal of start and stop codons using RNA editing.

SYM-56-04 SYM-56-05 DIHYDRODIPICOLINATE SYNTHASE IS ABSENT IN INTERCHANGEABLE REGULATORY DOMAINS: FUNGI EXPLORING MODULAR ALLOSTERY EN ROUTE TO CHORISMATE 1, John U.P.1, 2 and Perugini M.A.1 Desbois S. 1, 2, 5 2, 5 3 1, 4 1Department of Biochemistry and Genetics, La Trobe Institute Fan Y. , Cross P.J. , Jameson G.B. and Parker E.J. 1Ferrier Research Institute, Victoria University of Welington, 6140 for Molecular Science, La Trobe University, VIC 3086, 2 2 Wellington, New Zealand. Department of Chemistry, University Australia. Agriculture Victoria Research, Department of Economic 3 Development, Jobs, Transport and Resources, AgriBio, La Trobe of Canterbury, 8140 Christchurch, New Zealand. Maurice Wilkins University, VIC 3086, Australia. Centre, Institute of Fundamental Sciences, Massey University, 4442 Palmerston North, New Zealand. 4Maurice Wilkins Centre, Biomolecular Interaction Centre, University of Canterbury, 8140 Christchurch, New The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyses 5 t h e fi r s t c o m m i t te d s te p o f t h e d i a m i n o p i m e l ate ( DA P) l y s i n e b i o sy nt h e s i s Zealand. Biomolecular Interaction Centre, University of Canterbury, pathway in bacteria, archaea and plants. Despite the existence in 8140 Christchurch, New Zealand. databases of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We Engineering desired function into proteins via manipulation of the genes, report the characterisation of DHDPS candidates from fungi. Firstly, mimicking natural evolutionary processes, represents a promising the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE) was approach to synthesizing useful molecular tools. Most proteins comprise two or more domains from a limited suite of protein families. These shown to have negligible DHDPS enzyme activity. Sequence analysis domains are often rearranged in various combinations through gene of 3QFE showed that three out of the seven amino acid residues critical fusion events to evolve new protein functions, including the acquisition for DHDPS activity are absent; however, exact matches to catalytic of protein allostery through the incorporation of regulatory domains. residues from two other class I aldolases, 2-keto-3-deoxygluconate The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and were identified. The presence of both KDGA and HOGA activity in displays a diverse range of allosteric mechanisms. DAH7PSs adopt a 3QFE was confirmed in vitro using enzyme assays, the first report common architecture with a shared (β/α)8 catalytic domain which can of such dual activity. Subsequent analyses of all publically available be attached to an ACT-like or a chorismate mutase regulatory domain fungal sequences revealed that no entry contains all seven residues that operates via distinct mechanisms. These respective domains important for DHDPS function. The candidate with the highest number confer by controlling DAH7PS function in response of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi, was shown to ligand tyrosine or prephenate. Starting with contemporary DAH7PS to have trace DHDPS activity in vitro, partially restored by substitution proteins, two protein chimeras were created, with interchanged regulatory of the seventh critical residue, and to be incapable of complementing domains. Both engineered proteins were catalytically active and delivered DHDPS-deficient E. colicells. Combined with the presence of all seven new functional allostery with switched ligand specificity and allosteric sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis mechanisms delivered by their nonhomologous regulatory domains. This pathway in C. immitis and F. pedrosoi, we believe that DHDPS and interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new the DAP pathway are absent in fungi, and further, that robust informed 1 methods for annotating genes need to be implemented. bifunctional enzyme. 1. Fan, Y., Cross, P.J., Jameson, G.B. and Parker, E.J., 2018. Exploring modular allostery via interchangeable regulatory domains. Proceedings of the National Academy of Sciences, 115(12), pp.3006-3011.

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SYM-57-01 SYM-57-02 CROSSTALK BETWEEN ONCOGENIC SIGNALLING DYNAMIC ‘OMICS REDEFINES HOW INSULIN PATHWAYS REPROGRAMS LIPID METABOLISM IN SIGNALLING CHOREOGRAPHS GLUCOSE CANCER METABOLISM

Brown K.1, 2 Krycer J.1, Yugi K.2, 4, Fazakerley D.1, Humphrey S.1, Quek L.1, 1Cancer Metabolism Program and Cancer Therapeutics Hirayama A.3, Soga T.3, Kuroda S.4 and James D.1 Program, Peter MacCallum Cancer Centre, Melbourne, VIC, 1University of Sydney, Sydney, Australia. 2RIKEN IMS, Yokohama, Australia. 2Department of Biochemistry and Molecular Biology, The Japan. 3Keio University, Tsuruoka, Japan. 4University of Tokyo, University of Melbourne, Melbourne, VIC, Australia. Kashiwa, Japan.

Malignant transformation and tumour progression are dependent on Adipose (fat) tissue plays a crucial role in energy storage and release. reprogramming of cell metabolism to fulfil the unique energetic and Accordingly, adipose metabolism responds to fluctuating nutrient biosynthetic needs of cancer cells. A well-recognised, but poorly availability and hormonal cues. For instance, after a meal, insulin triggers understood, alteration in metabolism frequently observed in cancer a phosphorylation cascade to stimulate glucose uptake. This is considered cells is the activation of de novo lipogenesis. Our recent studies have insulin’s primary role, with subsequent energy storage activated by allostery focussed on the oncogenic transcriptional co-activator YAP, a master as substrates accumulate. However, our recent phosphoproteomics screen regulator of organ size and tumourigenesis. Aberrant activation in insulin-treated adipocytes identified hundreds of metabolic proteins - of YAP is widespread in human cancers. There is little knowledge could insulin signalling play a role beyond glucose uptake, coercing glucose regarding mechanisms by which YAP drives tumourigenesis, in large down specific metabolic routes? We addressed this by measuring acute, part because a limited number of YAP target genes and effectors temporal metabolomic changes upon insulin exposure, taking a dynamic approach to traditional steady-state 13C-tracer-experiments. We found have been identified. We find that YAP overexpression induces de three exciting results: (1) Insulin rapidly stimulated glucose uptake (t1/2=4 novolipogenesis in vitro and in vivo. Mechanistically, this phenomenon min), with its kinetics explaining 48% of metabolomic changes in response is dependent on the ability of YAP to induce transcriptional upregulation to insulin. Indeed, glucose was necessary for insulin-stimulated lipogenesis of serum- and glucocorticoid-regulated kinase 1 (SGK1), an effector of and suppression of fatty-acid oxidation, suggesting glucose facilitates the oncogenic phosphoinositide 3-kinase (PI3K) pathway. Downstream insulin action. (2) Despite this, flux analysis revealed glucose was primarily of YAP, SGK1 promotes mTORC1 signalling leading to activation of converted to lactate and favoured NADPH-generating pathways (e.g., the sterol regulatory element-binding proteins (SREBP1/2), master pyruvate anaplerosis, pentose phosphate pathway). This is reminiscent of regulators of lipid metabolism. Importantly, we find that inhibition of key cancer metabolism. We believe this allows glucose to facilitate anabolism enzymes in the de novo lipogenesis pathway blocks the uncontrolled beyond being a mere carbon source in (terminally-differentiated) proliferation associated with YAP-driven transformation in vitro and in adipocytes. (3) Overlaying metabolomic and phosphoproteomic data vivo. Our data reveal a mechanism of crosstalk between two important (‘transomic analysis’) revealed protein phosphorylation changed rapidly oncogenic signalling pathways and reveal a metabolic vulnerability that (<5 min), activating anabolism before substrates accumulated. Thus, can be targeted to disrupt oncogenic YAP/PI3K pathway activity. insulin creates a demand-driven system to ‘drag’ glucose down specific pathways. This complements supply-driven regulation of anabolism by substrate accumulation. Overall, this redefines how signalling coordinates metabolism, with implications for metabolic dysregulation in overactive signalling (cancer) or nutrient oversupply (diabetes).

SYM-57-03 SYM-57-04 TRANSCRIPTIONAL REPROGRAMMING OF NEUTRAL CHOLESTEROL ESTER METABOLISM BY YAP AND C-MYC IN LIVER CANCER 1 (NCEH1) REGULATES EXTRACELLULAR LDL- CHOLESTEROL METABOLISM TO INFLUENCE Cox A.G.1, 2 PROSTATE CANCER PROGRESSION 1Peter MacCallum Cancer Centre. 2University of Melbourne. Raftopulos N.L.1, Washaya T.C.1, Egert A.1,2,3, Aishah A.1, Varney B.1, Nagarajan S.R.1, Butler L.M.4,5, Grewal T.2, and Hoy A.J.1 Hepatocellular carcinoma (HCC) is the most common form of liver 1 cancer and among the most fatal cancer type, as few effective The University of Sydney, Charles Perkins Centre, Discipline of treatments are available. Although the pathophysiology of HCC has Physiology, School of Medical Sciences, Sydney, New South Wales, 2006, Australia. 2Faculty of Pharmacy, The University of Sydney, Sydney, not been fully elucidated, the process clearly arises in the context of NSW 2006, Australia. 3Universitätsklinikum Hamburg-Eppendorf, chronic liver disease brought on by environmental factors. In the lab, 20246 Hamburg, Germany. 4Adelaide Medical School and Freemasons we use an combination of transcriptomic, metabolomic and imaging Foundation Centre for Men’s Health, University of Adelaide, Adelaide, approaches in mutant and transgenic zebrafish to reveal mechanisms South Australia, 5005, Australia. 5South Australian Health and Medical by which metabolic reprogramming fuels premalignant hyperplasia Research Institute, Adelaide, South Australia, 5001, Australia. and tumourigenesis in vivo. We have recently focused our attention on the role that the oncogenic transcription factors Yap and c-Myc Prostate cancer (PCa) remains a leading cause of cancer-related death in Australian play in reprogramming metabolism. To this end, we have developed a men. Advanced PCa is initially treated by depleting androgens thereby removing a zebrafish model of Yap-driven HCC that recapitulates human HCC at critical promotor of tumour growth; however, the low androgen environment exerts the histological and genetic level. Using this model we found that Yap selective pressure on remaining PCa cells and the disease evolves into a lethal, aggressive form termed castrate resistant PCa (CRPC). Hypercholesterolemia is a remodels glucose and glutamine metabolism to enhance the anabolic major side effect of androgen deprivation and is linked to more aggressive disease biosynthesis on nucleotides required for rapid tissue growth. In parallel, and the development of CRPC. Also, high-grade PCa and metastases exhibit we have taken advantage of an inducible zebrafish model of HCC, in aberrant accumulation of esterified cholesterol. Cholesterol is stored in cytosolic w hi c h c - M yc i s ove rex p re s se d sp e c i fi c ally in hepato c y te s up o n ex p o sure lipid droplets as cholesterol esters (CE) and inhibition of CE formation impairs PCa to doxycycline. We have used these models to identify oncogenic progression. However, it is now known whether the mobilisation of cholesterol from changes in metabolism and screen potential metabolic interventions for CE influences CRPC cell proliferation. As expected, C4-2B cell (androgen receptor efficacy in suppressing Yap or c Myc driven hyperplasia and HCC. Our positive, androgen independent) grew normally in a low androgen environment long term goal is to identify oncogene-specific metabolic vulnerabilities (charcoal-stripped serum) but had lower CE levels than cells cultured in full that can be exploited therapeutically to prevent or combat liver cancer. serum. CE levels are regulated by Neutral Cholesterol Ester Hydrolase 1 (nCEH1), which hydrolyses CE to free cholesterol and a fatty acid, and inhibition of nCEH1 slowed growth in charcoal-stripped serum, suggesting that cholesterol stored in CE supports cell growth in a low androgen environment. Depletion of lipid-rich lipoproteins (LPDS) from the media reduced C4-2B cell growth and intracellular CE levels, and this was exacerbated in charcoal-stripped LPDS. Supplementation of LPDS with cholesterol-rich LDL fully restored C4-2B cell growth; however, nCEH1 inhibition ameliorated the ability of LDL supplementation to restore C4-2B cell growth in LPDS containing media. These data indicate that LDL-cholesterol promotes C4-2B PCa cell growth via an nCEH1-catalysed metabolism. Future studies aim to further characterise the mechanisms by which CE metabolism influences CRPC cell behaviour. * NLR & TCW contributed equally.

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SYM-57-05 SYM-58-01 NON-CANONICAL UBIQUITINATION SITES DIRECT EXPLOITING DIFFERENTIAL METABOLISM IN DEGRADATION OF THE SHORTEST KNOWN PARASITE LIFE CYCLES TO LIMIT THE SPREAD OF CHOLESTEROL-DEPENDENT DEGRON DRUG RESISTANCE

Chua N.K. and Brown A.J. Goodman C.D., Buchanan H.D. and McFadden G.I. School of Biotechnology and Biomolecular Sciences, UNSW Sydney, School of BioSciences, University of Melbourne. NSW 2052, Australia. Drug resistance is a major factor limiting our ability to control parasitic Squalene monooxygenase (SM) is the second rate-limiting enzyme diseases. Many medically and economically significant parasites in cholesterol synthesis. Excess cholesterol triggers SM degradation undergo immense changes in metabolic activity as they move between via the ubiquitin-proteasome system. The E3 ligase, MARCH6, is their mammalian hosts and invertebrate vectors. Consequently, drug responsible for mediating the ubiquitination of SM. Furthermore, selection for resistance during the vertebrate stage can have dire this process requires the first 100 amino acids of SM (termed SM consequences for parasite fitness during transmission. Using the N100), which represents the shortest known cholesterol-regulated malaria parasite, Plasmodium, we are investigating anti-malarial degron. Ubiquitination is one of the most common post-translational drugs whose targets are under markedly reduced selection pressure modifications and they often occur on lysine residues, but we have in the mammalian host. Resistance mutations in these targets tend shown that lysine residues are not crucial for the cholesterol-mediated to drive parasite failure during development in the mosquito vector, degradation of SM. Despite being an important rate-limiting enzyme thereby inhibiting disease transmission. This approach is particularly in cholesterol synthesis, the precise ubiquitination sites within the effective with drugs targeting the parasite organelles, due to their cholesterol-regulated degron of SM remain elusive. In this study, increased metabolic activity during the mosquito stages and the non- we mutated non-canonical ubiquitination residues to alanine. We Mendelian inheritance of their genomes. Clinically relevant mutations observed that serine residues are required for the cholesterol- conferring resistance to the widely used anti-malarial MalaroneTM arise dependent degradation of SM N100. In addition, loss of the key serine in the -encoded cytochrome b gene. These mutations residues boosted SM N100 protein levels and MARCH6 knockdown completely arrest development of infectious sporozoites in the did not further enhance protein levels. Our results reveal non-canonical mosquito stages of both P. falciparum and P. berghei, thereby blocking ubiquitination in the shortest known cholesterol-regulated degron. This transmission of these resistance genes. The macrolide antibiotic finding characterises the mechanism by which SM exerts its role as a azithromycin kills malarial parasites by inhibiting apicoplast protein rate-limiting enzyme and how cholesterol modulates the stability of SM. synthesis. We generated two azithromycin resistant lines in P. berghei (PbAZMR) harbouring mutations in the apicoplast-encoded rpl4 gene. Such mutations also confer azithromycin resistance in P.falciparum. PbAZMR parasites infect mosquitoes at a lower rate, produce fewer sporozoites, and fail to infect naive mice. Major defects in apicoplast development underly this transmission block. Our results demonstrate that resistance trapping due to differential selection between life stages is a common phenomenon. It can be exploited to develop drugs with greatly reduce levels of resistance transmission; not only in malaria but possibly in other parasites with invertebrate vectors.

SYM-58-02 SYM-58-03 IMPROVING ANTI-MALARIAL TREATMENT OF P. IMMUNE RESPONSES IN SCABIES: INSIGHTS FROM A VIVAX BY LEVERAGING SHORT AND LONG READ DNA PORCINE MODEL SEQUENCING TECHNOLOGIES Mounsey K. Charnaud S.C.1, 2, Munro J.E.1, Quah Y.W.1, Bahlo M.1, 2 and Mueller I.1, 2, 3 School of Health & Sport Sciences, University of the Sunshine Coast, 1Walter and Eliza Hall Institute, Parkville, Australia. 2University of Maroochydore QLD. Melbourne, Parkville, Australia. 3Institut Pasteur, Paris, France. Scabies is a skin disease caused by the parasitic mite Sarcoptes The Asia-Pacific region is aiming for malaria elimination by 2030, but scabiei. Critical biological questions surrounding scabies remained despite falling malaria cases Plasmodium vivax persists. The reasons unanswered due to the inability to undertake longitudinal study of for the greater persistence of P. vivax relate to its unique biology, most infestation in humans. The recent development of a porcine model importantly its ability to relapse from long-lasting, dormant liver stages has facilitated in vivo studies on scabies immunopathology. Our (hypnozoites). There are no diagnostic tests for hypnozoites and the research aims to identify factors leading to a dysregulated immune only currently available drug to clear hypnozoites is primaquine, which response in crusted scabies, a poorly understood clinical manifestation can cause severe haemolysis in people with glucose 6-phosphate characterised by an extreme proliferation of mites. We have undertaken dehydrogenase (G6PD) deficiency, and may not work in people with several experimental trials, focusing on defining cellular infiltrates and low cytochrome P450 2D6 activity. CYP2D6 is involved in some transcription of key cytokines in the skin over the course of infestation. part in the metabolism of 20-25% of all drugs in clinical use, making Recent gene expression studies show that scabies is characterised it extremely interesting to determine what drugs are likely to work or by early immune suppression, followed by dramatic upregulation of be contra-indicated in a population. The advent of novel long-read pro-inflammatory genes in later weeks of infestation, consistent with sequencing now makes it possible to sequence complex genes with the delayed appearance of clinical manifestations. Crusted scabies multiple mutations. We have developed long amplicon barcoded was associated with a higher number of down regulated genes at all sequencing protocols using PacBio for G6PD and CYP2D6. We will time points, and differential expression of Th17 associated pathways. link the genotypes with phenotypes of G6PD deficiency and CYP2D6 Transcriptional profiles cluster strongly according to clinical phenotype activity in populations in PNG and Solomon Islands. This will allow even prior to the introduction of infection, providing insights into markers rapid assessment of pharmocogenomic characteristics of malaria of individual susceptibility and resistance to scabies. Outcomes from endemic populations to tailor antihypnozoite therapy to achieve optimal this research may lead to strategies to protect vulnerable subjects safety and efficacy in each community. To determine the relatedness from contracting recurrent crusted scabies, and result in improved skin of multiple P. vivax infections over time within one person we are also health for disadvantaged communities where this parasite is endemic. developing a short amplicon based sequencing method. This will allow us to differentiate between (i) recrudescent parasites, indicating drug resistance, (ii) relapse infections from hypnozoite reactivation indicating liver stage treatment failure, and (iii) re-infections indicating high transmission. Together these sequencing methods will allow us to leverage genomics to improve current treatments and to potentially develop new treatments for P.vivax malaria.

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SYM-58-04 SYM-58-05 RATIONAL DESIGN OF A MULTI-TARGET DEFINING THE METABOLIC NETWORK OF THE ANTIMALARIAL COMPOUND WITH IN VIVO ACTIVITY MALARIA PARASITE REVEALS AN ESSENTIAL LIPID REGULATOR Drinkwater N.1, Vinh N.2, Malcolm T.1, Charman S.3, De Koning Ward T.4, Avery V. 5, Scammells P.2 and McGowan S.1 Cobbold S.A.1, Dumont L.1, Marapana D.2, Triglia T.2, Ralph S.A.1, 1Biomedicine Discovery Institute, Department of Microbiology, Monash Cowman A.F.2, Tilley L.1 and Mcconville M.J.1 University, Clayton Melbourne, VIC 3800. 2Medicinal Chemistry, 1Bio21 Institute, University of Melbourne, Melbourne, VIC, Monash Institute of Pharmaceutical Sciences, Monash University, Australia. 2Walter and Eliza Hall Institute, Melbourne, VIC, Australia. Parkville, VIC 3052. 3Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC Asexual development of the malaria parasite is associated with major 4 3052. School of Medicine, Deakin University, Geelong Waurn Ponds restructuring of both parasite and host erythrocyte metabolism. Most 5 Campus, Geelong, VIC 3216. Discovery Biology, Griffith Institute for current antimalarials target metabolic processes - so in the search Drug Discovery, Griffith University, Nathan, QLD 4111. for novel antimalarials - it is crucial to understand the parasite’s total metabolic capacity and identify which metabolic enzymes are Malaria, particularly that caused by P. falciparum and P. vivax, remains essential. To comprehensively define the metabolic changes that a global health concern. Artemisinin combination therapies, the gold occur in human erythrocytes following malaria infection we utilized an standard of treatment, have played a major role in reducing the malaria approach that approximates a global stable-isotope labelling strategy. burden. However, parasites resistant to artemisinin treatment have Using untargeted LC-MS we traced the fate of all major carbon emerged, and are spreading rapidly. We implemented an ambitious sources using 13C-labelled substrates. Differential analyses of all strategy to use rational drug discovery to develop a single compound mass/charge features between infected and uninfected cells has led capable of inhibiting two antimalarial drug targets, the M1 and M17 to a draft metabolome of the malaria parasite. The draft metabolome aminopeptidases, both key players in the blood stage of malaria infection. constitutes 43% of the expected metabolome of the parasite (via This strategy was designed to improve the efficacy of a compound genomic reconstructions), however 112 observed metabolites did by taking advantage of the synergistic effect achieved by inhibiting not match to the expected metabolome. To further explore this ‘dark’ multiple targets within the same metabolic pathway, and additionally, to metabolome and validate the accuracy of the draft metabolome we reduce the capacity of parasites to generate resistance, which occurs targeted potential enzymes with no defined function which could rapidly when parasites are treated with single-target therapeutics. We potentially participate in the dark metabolome. CRISPR/Cas9 was discovered potent dual inhibitors of M1 and M17 that show nanomolar used to generate a dual inducible knock-down system for 17 genes in vitro activity against both P. vivax and P. falciparum (including drug encoding uncharacterized metabolic enzymes, including a member resistant strains). Further, in mouse models, our most potent compound of the Haloacid Dehalogense family (HAD5). We demonstrate that is effective against P. berghei infection after oral administration (97% HAD5 is essential to normal parasite development and mediates lipid reduction in parasitemia). We have therefore developed a multi-target metabolism via dephosphorylating phosphatidate into diacylglycerol inhibitor capable of potent activity across multiple Plasmodium species, species. This lipid regulator was previously unrecognised in the malaria which represents an exciting lead for further development into a novel parasite and demonstrates how exploration of the dark metabolome of antimalarial therapeutic. the parasite could lead to novel antimalarial targets.

SYM-59-01 SYM-59-02 An integrated sensing pipeline to map UNDERSTANDING THE GENETIC BASIS OF CO2 the genetic loci associated with canopy RESPONSIVENESS UNDER CONTROLLED CONDITIONS: radiation use efficiency in sorghum DESIGN CHALLENGES IN THE GENOMICS ERA

Barbara Geoge-Jaeggli1,2, Andries Potgieter3, James Watson3, Pinkard E.A.1, Brookhouse M.2, Shimono H.3, Bush D.1 and Farquhar G.2 Emma Mace1,2, Colleen Hunt1,2, Adrian Hathorn4, Mark Eldridge1, 1CSIRO Land and Water. 2Research School of Biology, ANU. 3Faculty of Kenneth LAWS 1, Scott Chapman4,5, Andrew Borrell 1, David Jordan1 Agriculture Iwate University. and Graeme Hammer4 1 Queensland Alliance for Agriculture and Food Innovation, The The move towards large genomic experiments, and the substantial 2 University of Queensland, Warwick, Queensland, Australia. Agri- phenotyping required to support these experiments, challenges our capacity Science Queensland, Department of Agriculture & Fisheries, Warwick, to conduct reproducible experiments, and correctly identify key traits. If 3 Queensland, Australia. Queensland Alliance for Agriculture and Food not adequately addressed this can affect the interpretation of results and Innovation, The University of Queensland, Toowoomba, Queensland, potentially lead to flawed investment decisions regarding genetic selection Australia. 4Queensland Alliance for Agriculture and Food Innovation, The 5 for superior performance. In this work, we identify potential problems University of Queensland, St Lucia, Queensland, Australia. Agriculture & associated with phenotyping large G x CO2 experiments, and suggest a Food, CSIRO, St Lucia, Queensland, Australia. possible solution. We address the following matters. (1) In fast-growing, young plant material, small differences in environmental conditions or Cereal yield advances are slowing down and will have to come from initial plant size can substantially affect outcomes. (2) Though working with biomass production and resource use efficiency, such as improvements clonal material is attractive in that an identical genotypes can be placed in in photosynthetic capacity or radiation use efficiency as further different environments, propagation difficulties and plagiotropic growth can improvements in harvest index are becoming more difficult to achieve. be challenging (3) Size of controlled environment facilities rapidly becomes Sorghum, which is a C4 crop with more and more re-sequenced limiting for large plants, and controlling within-glasshouse spatial variation genomes and large phenotypic diversity, is an ideal model to study is an issue, though modern statistical analytical techniques can assist. (4) natural variation in traits related to photosynthetic capacity and biomass In genome-wide association experiments, there is often a need to include production. We have developed remote (UAV) and proximal (tractor a large number of genotypes, implying low replication within genotypes. based) sensing platforms and a data-analysis pipeline which integrates This reduces statistical power for some genetic parameter estimation and outputs from the various sensors to estimate traits related to dynamic requires correction for glasshouse spatial variation (5) The large numbers crop growth and canopy photosynthetic capacity, such as canopy of plants associated with genome-wide association experiments makes trait radiation use efficiency, for hundreds of field plots. We have used these assessment difficult to standardise in time, particularly for fast-growing tree remote-sensing platforms over the last two field seasons to screen an species. Many traits are time-consuming to measure and ongoing growth association panel largely consisting of around 700 sorghum conversion or trait changes due to plant developmental stage during the measurement lines, which are short, early-flowering lines developed from a diverse period can confound results. We discuss the application of pre-screening set of exotics through the introgression of dwarfing and photoperiod- as a tool in large genome x e[CO2] experiments, to reduce costs and insensitivity alleles. Our aim is to map the genetic loci and identify overcome issues of access to suitable CO2-enrichment facilities. In this candidate genes associated with photosynthetic capacity in sorghum approach a surrogate to e[CO2] is used to select cultivars for more detailed and potentially other related C4 cereals and use this information to analysis under e[CO2] conditions. It provides a potential mechanism to breed lines with greater resource efficiency and yield potential. screen large numbers of plants at relatively low cost, although to date there has been only limited testing.

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SYM-59-03 SYM-59-04 PHENOMICS CAPABILITY DEVELOPMENT AT RAIDERS OF THE VAVILOV ARK: SLEUTHING NEW AGRESEARCH FOR BETTER PASTURE AND FORAGE PHOTOSYNTHETIC TRAITS FROM THE PAST PRODUCTION Estavillo G.M.1, Do N.L.1, Blake C.1, Harding C.1, Zwart A.2, Voss-Fels K.P.5, Ghamkhar K. Barrero-Sanchez J.M.1, Hickey L.5, Sharwood R.3, Kramer D.M.4 and AgResearch, Grasslands Campus, Palmerston North, New Zealand. Condon A.G.1 1CSIRO Agriculture & Food, Canberra, ACT 2601, Australia. 2CSIRO New Zealand is recognised for the quality and productivity of its meat Data61, Canberra, ACT 2601, Australia. 3The Australian National University, and dairy agriculture. Due to the strategic role of forages in securing the Canberra, ACT 2601, Australia. 4Michigan State University, East Lansing, MI continuity of this high quality, accurate trait measurement is extremely 48824, USA. 5QAAFI, The University of Queensland, St Lucia, QLD 4072, i m p o r t a n t i n m o d e r n b r e e d i n g o f t h e s e f o r a g e p l a n t s . M a j o r d e v e l o p m e n t s Australia. have taken place to realise this need in recent years. Beyond automatic systems in contained environments, other promising developments Improving photosynthetic capacity is seen as an obvious target for can turn things around for high-throughput characterisation of plants increasing total crop biomass and yield. However the complexity at reasonable costs. Here, I revisit AgResearch’s new approach to the of photosynthesis and lack of high-throughput screening methods application of sensors and camera technology, describing recent use of hampers the identification of genotypes with improved carbon non-destructive techniques for accurate phenotyping of a range of traits fixation rates. We have used an affordable, hand-held chlorophyll from yield and growth measurement to weed detection and targeting. fluorescence spectrometer (PhotosynQ) to screen for genetic diversity of photosynthetic traits in a set of wheats pre-dating modern breeding (from the Vavilov collection). There was a large range in the rate of linear electron flow (LEF) among 60 selected Vavilov entries representing the whole genetic diversity of the panel. LEF varied from 21 to 80 μmol m-2 s-1 at low light, and from 72 to 167 μmol m-2 s-1 at five times higher irradiance (high light). Additionally, there was large variation in the magnitude of the change in LEF from low to high light, suggesting the presence of plasticity to light acclimation among these lines. There was also considerable variation in how light-derived energy was apportioned between photochemistry and other energy dissipation mechanisms (i.e., heat loss). Concurrent gas exchange, coupled with conventional chlorophyll fluorescence measurements and biochemical analyses were also performed on flag leaves of some selected lines. The results indicated that PhotosynQ could be deployed as a useful screening tool for photosynthetic traits and that screening novel genetic resources could enable the discovery of new traits or enhanced expression of existing traits to improve wheat photosynthesis.

SYM-59-05 SYM-60-01 TACKLING THE PHYSIOLOGICAL PHENOTYPING IMPACT OF SIGNALLING ACTIVITY ON THE BOTTLENECK WITH LOW-COST, ENHANCED- ALLOCATION OF GERM LAYER PROGENITORS: THROUGHPUT GAS EXCHANGE AND CEPTOMETRY INSIGHTS FROM THE SPATIAL TRANSCRIPTOME OF GASTRULA-STAGE EMBRYO Salter W.T.1, Gilbert M.G.2, Merchant A.1 and Buckley T.N.2 1School of Life and Environmental Sciences, Sydney Institute of Tam P.P.L .1, 2 Agriculture, The University of Sydney. 2Department of Plant Sciences, 1Embryology Unit, Children’s Medical Research Institute. 2School of University of California, Davis. Medical Sciences, Faculty of Medicine and Health, The University of Sydney. High throughput phenotyping platforms (HTPPs) are increasingly adopted in plant breeding research due to developments in sensor Gastrulation is a critical milestone of embryogenesis at which the technology, unmanned aeronautics and computing infrastructure. Most primary germ layers are formed and the multipotent embryonic cells of these platforms rely on indirect measurement techniques therefore are allocated to the progenitors of tissue lineages within the germ some physiological traits may be inaccurately estimated whilst others layers. Analysis of the spatial transcriptome of the epiblast showed that cannot be estimated at all. Unfortunately, existing methods of directly spatially delimited agonistic and antagonistic signalling activity in the measuring plant physiological traits, such as photosynthetic capacity epiblast and signalling conditions for the derivation and maintenance (Amax), have low throughput and can be prohibitively expensive, creating of the epiblast stem cells underpin the restriction of the tissue potency a bottleneck in the breeding pipeline. We have addressed this issue of epiblast cells, culminating in the generation of lineage-restricted by developing new low-cost enhanced-throughput phenotyping tools progenitors for the germ layers and the regionalization of tissue to directly measure physiological traits of wheat (Triticum aestivum). precursors of major body parts in the germ layers during gastrulation. Our eight-chamber multiplexed gas exchange system, OCTOflux, can

directly measure Amax with 5-10 times the throughput of conventional instruments, whilst our handmade ceptometers, PARbars, allow us to monitor the canopy light environment of many plots simultaneously and continuously across a diurnal cycle. By custom-building and optimizing these systems for throughput we have kept costs to a minimum, with OCTOflux costing roughly half that of commercially available single- chamber gas exchange systems and PARbars costing approximately 95% less than commercial ceptometers. We recently used these tools

to identify variation in the distribution of Amax relative to light availability in 160 diverse wheat genotypes grown in the field. In a two-week

measurement campaign we measured Amax in over 1300 leaves with OCTOflux and phenotyped the diurnal light environment of 418 plots using 68 PARbars. These tools could be readily modified for use with any plant functional type and also be useful in validating emerging HTPPs that rely on remotely sensed data to estimate photosynthetic parameters.

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SYM-60-02 SYM-60-03 EXPLOITING HEPARAN SULFATE PROTEOGLYCANS- ELUCIDATING THE IMPACT OF APP IN DS GROWTH FACTOR INTERACTIONS TO DIRECT PATHOGENESIS USING STEM CELL MODELS MESENCHYMAL STEM CELL NEUROGENESIS Griffiths R.1, Fortuna P.1, Ovchinnikov D.1, Balachandran A.1, Wells C.2, Yu C., Okolicsanyi R.K., Griffiths L.R. and Haupt L.M. Powell J.3, Mar J.1 and Wolvetang E.1 IHBI-QUT, Genomics Research Centre, School of Biomedical 11Australian Institute for Bioengineering and Nanotechnology, Sciences, 60 Musk Avenue, Kelvin Grove, Q 4059. University of Queensland, Brisbane, QLD 4072, Australia. 2Centre for Stem Cell Systems, MDHS, University of Melbourne, Melbourne, According to the World Health Organisation, neurological disorders, VIC 3010, Australia. 3Garvan Institute of Medical Research, Sydney, including trauma, affect 1 in 6 of the world population. With no current Australia. effective treatments, much current effort is focussed on the development and improvement of cellular replacement therapies. In particular, the propagation Down syndrome (DS) is due to trisomy of HSA21. Which genes on and direction of stem cells into specific neural lineages. Mesenchymal chromosome 21 are responsible for the pronounced Alzheimer’s stem cells (MSCs) are capable of neurogenic differentiation but lack high disease and neurocristopathy features of Down syndrome remains frequency differentiation efficiency, particularly following transplantation. largely unclear. Here we use CRISPR-assisted genome manipulation Heparan sulfate proteoglycans (HSPGs), including syndecans (SDCs) and to normalize the copynumber of APP or DYRK1A in DS induced glypicans (GPCs), are ubiquitous within the stem cell microenvironment and pluripotent stem cells. Comparison of cellular phenotypes and essential to numerous cellular activities, including self-renewal, proliferation transcriptomes of these isogenic euploid, DS and genome edited DS and differentiation. In this study we examine the role of HSPGs in human MSC (hMSCs) neurogenesis and as potential biomarkers for therapeutic IPSC following differentiation into cortical neurons in 2D or cerebral applications. Using two neural differentiation protocols: i.) direct terminal organoid setings or analysis neural crest cells reveals important differentiation (TD), and ii.) terminal differentiation via hMSC-induced roles for APP in amyloidigenic but not tau-pathology of Alzheimers neurosphere formation (TD via hMSC-INs), hMSC populations (n=3) were disease and the strong impact of DYRK1A dosage on neural crest cell directed towards neural lineages with growth factor supplementation for up to generation, survivial and migration. We further show that these in vitro 14 days. Brain-derived neurotrophic factor (BDNF) and platelet-derived growth models can be used to test the efficiacy of therapeutic compounds. factor (PDGF) bind to HSPGs and have been implicated in neuronal and glial lineage differentiation, respectively. HSPG and neural lineage marker gene expression profiles (focussing on the core protein SDCs and GPCs) were examined under neural specific culture conditions by Q-PCR, WB and ICC. Gene expression analysis identified BDNF-treated cultures to express higher self-renewal (NANOG, POU5F1 and SOX2 (P<0.05)), and oligodendrocyte markers (Olig1 (P<0.05) and Olig2 (P<0.05)) when compared to PDGF-treated cultures. PDGF cultures maintained the highest cell numbers and expressed mature neuronal markers (MAP2 and TUBB3). Analysis of the culture HSPG profiles identified SDCs 1-4 and GPC1, -4, and -6 are likely involved in hMSC neural differentiation. Our data suggests HSPGs, in particular members of the SDC and GPC families, may play key roles during hMSC neurogenesis and are potentially central to controlling human neurogenesis.

SYM-60-04 SYM-60-05 FROM SERVANT TO MASTER: THE RIBOSOME’S INNER EAR ORGANOIDS DERIVED FROM HUMAN INSTRUCTIVE ROLE IN HEMATOPOIETIC CELL FATE PLURIPOTENT STEM CELLS: COMPARISONS TO DETERMINATION HUMAN FOETAL INNER EAR

Chahal A.S.1, 2, Mitchell N.1, Zaytseva O.1, Geroge A.1, Hannan R.D.1 Mattei C.1, 2, Lim R.3, Drury H.R.3, Tadros M.A.3,Nasr B.1, 4, Chatterton R.1, 2, and Quinn L.M.1 Kulkarni T.1, 2, Jamshidi P.1, 2, D’Abaco G.2, Nayagam B.A.5 and Dottori M.1, 2, 6 1JCSMR, Australian National University. 2University of Melbourne. 1Centre for Neural Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia. 2Department of Biomedical Transcriptional control of gene expression is viewed as the key instructor Engineering, Melbourne School of Engineering, University of of stem cell fate. In contrast, dogma has relegated ribosome biogenesis Melbourne, Victoria, Australia. 3School of Biomedical Sciences and and mRNA translation as a necessary, but passive, contributor to cell and Pharmacy, Faculty of Health and Medicine, University of Newcastle, tissue specification during development. Intuitively, impaired ribosome NSW, Australia. 4Department of Electrical and Electronic Engineering, function should (and usually does) cause reduced tissue growth and Melbourne School of Engineering, University of Melbourne, Victoria, stunted development. Inexplicably, cancer arises more frequently in Australia. 5Departments of Audiology and Speech, Pathology & patients with ribosomal protein (RP) loss, particularly in the blood lineage. Ophthalmology, University of Melbourne, Victoria, Australia. 6Illawarra Our recently developed Drosophila hematopoietic models may provide Health and Medical Research Institute, University of Wollongong, NSW, a rationale for the prevalence of leukemia in patients with reduced RP Australia. levels. Following tissue specific depletion of the RPs (s19 and s24) most frequently mutated in the ribosomopathy Diamond Blackfan Anemia Introduction: In mammals, sensory hair cells do not regenerate. Consequently, (DBA), we observe stem cell fate defects, overproliferation and massive the derivation of inner ear tissue from human pluripotent stem cells (hPSC) overgrowth of the blood compartment. Interestingly, knockdown of RPs19 offers an opportunity to study human inner ear development and provides a and RPs24 differentially altered stem and progenitor cell differentiation, platform for drug screening and disease modelling. Methods: A dynamic three which suggests depletion of particular RPs from ribosomes might actively dimensional rotary cell culture system was used to derive inner ear organoids disrupt cell fate by modulating the classes of transcripts translated. Indeed, from human PSCs for 16 weeks in-vitro. Differentiation and mechanosensitiviy Mass Spec data from the RP deficient lymph glands revealed increased of hPSCs-derived organoids were examined using a combination of qPCR, protein abundance for several factors previously implicated in driving immunofluorescent labelling, and AM142 staining. Helium microscopy and developmental programs of growth and differentiation, including master electrophysiology compared the anatomical and physiological characteristics transcriptional regulators and chromatin remodeling machinery. As RNA- of inner ear organoids to foetal human inner ear. Results: Inner ear organoids Seq revealed mRNA transcript levels for these factors were unchanged, show temporal expression of key developmental hair cell markers including we hypothesise that their defective translation underlies the stem cell fate Pax2, Atoh1, MyosinVIIa, and CtBP2 by immunofluorescence and qPCR. defects and blood compartment overgrowth. A combination of TEM and Bi- AM143 fluorescence in organoid cells is indicative of mechanosensitivity. Cells FC analysis revealed that RP depletion did not significantly reduce mature have outward currents (350 pA to 5 nA) consistent with developing human ribosomes in the cytoplasm, suggesting that altered protein abundance is type II vestibular hair cells (12-16 weeks gestation). A subset of oganoid not due to competition for ribosomes. We are currently using mass spec cells also have sodium currents. Striking morphological similarities were to determine if RPs19 or s24 depletion alters ribosomal composition in detected between inner ear organoids and developing inner ear using helium Drosophila hematopoietic cell lines, and whether mRNA species encoding microscopy. Conclusion: We describe a novel three dimensional system for stem cell fate proteins are differentially translated using RNA-seq of modelling human inner ear development using rotary cell culture. Preliminary polysome fractions. data suggests this system is capable of generating a population of inner ear hair cells which resemble an early vestibular phenotype.

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SYM-61-01 SYM-61-02 THE COMPANION OF CELLULOSE SYNTHASE 1 DEVELOPMENT OF THREE-DIMENSIONAL CULTURE CONTROLS MICROTUBULE DYNAMICS THROUGH MODELS TO STUDY CANCER DEVELOPMENT AND A TAU-LIKE MECHANISM TO CONFER SALT METASTASIS TOLERANCE IN PLANTS Bray L.J.1, Murekatete B.1, Jaeschke A.1, Koch M.1, Tsurkan M.V.2, Kesten C.1, Wallmann A.2, Oschkinat H.2 and Persson S.1 Risbridger G.3, Werner C.2 and Hutmacher D.W.1 1School of Biosciences, University of Melbourne, Parkville 3010 VIC, 1Centre for Regenerative Medicine, Institute of Health and Melbourne, Australia. 2Leibniz-Forschungsinstitut fur Molekulare Biomedical Innovation, Queensland University of Technology, Pharmakologie (FMP), NMR-supported Structural Biology, Robert- Brisbane, Queensland, Australia. 2Leibniz Institute for Polymer Rossle-Str. 10, 13125, Berlin, Germany. Research, Max Bergmann Center of Biomaterials, Dresden, Saxony, Germany. 3Monash University, Clayton, Victoria, Australia. Microtubules are filamentous structures necessary for cell division, motility and morphology. Microtubule dynamics are critically regulated The culture of cells on rigid 2D substrates, such as tissue culture plastic, by microtubule-associated proteins (MAPs). We outline the molecular does not recreate the dynamic and highly complex tissue microenvironment, mechanism by which the MAP, COMPANION OF CELLULOSE but rather distorts cell-integrin and cell-cell interactions, affecting gene SYNTHASE1 (CC1), controls microtubule-bundling and dynamics expression, signal transduction, cell proliferation and differentiation, and in plants under salt stress conditions. CC1 contains an intrinsically thus is physiologically irrelevant. Naturally, cells are embedded in an disordered N-terminus that joins microtubules through conserved extracellular matrix (ECM) that provides not only architectural support, but hydrophobic regions at evenly distributed foci. Structural data on the also chemical and mechanical signals to cells in vivo. Hydrogels prepared microtubule-bound CC1 N-terminus and mutation studies revealed from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalised the regions, and specific amino acids, that contribute to microtubule- heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels binding. The importance of these regions and amino acids was support tri-cultures of human umbilical vein endothelial cells (HUVECs) with confirmed through in vivo live cell imaging, which also explains how mesenchymal stromal cells (MSCs) and breast or prostate cancer epithelial CC1 maintains cellulose synthesis during salt exposure. Surprisingly, cell lines. We extended this body of work to study the effects of cancer the microtubule-binding mechanism of CC1 is remarkably similar to associated fibroblasts (CAFs) on tumour angiogenesis. Also, we investigated that of the prominent neuropathology-related protein Tau. Hence, we the ability to produce a tri-culture mimicking tumour angiogenesis with outline how MAP functions have converged during evolution across epithelial cells, and tissue-specific microvascular endothelial cells and animal and plant cells. fibroblasts. Cultures were analysed via immunostaining and observed using confocal microscopy. We demonstrated the ability of starPEG- heparin hydrogels to support co- and tri-cultures of primary patient-derived prostate or breast cells, with capillary formation by the endothelial cells. Interactions were visualised between all cell types via confocal microscopy. Our results confirm the suitability of hydrogels constructed from starPEG- heparin for the co-cultivation of primary patient-derived tissue specific cells to study cell-cell and cell-matrix interactions in a 3D microenvironment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of various cancer types.

SYM-61-03 SYM-61-04 AN UNBIASED CHEMICAL SCREEN OF ACTIN ACTIN POLYMERIZATION ALTERS NUCLEAR ORGANISATION ENABLES DRUG DISCOVERY AND ARCHITECTURE IN RESPONSE TO DNA REPLICATION MECHANISTIC ANALYSES OF THE CYTOSKELETON STRESS TO MAINTAIN GENOME STABILITY

Bryce N.S., Lock J.G., Stehn J., Hardeman E.C. and Gunning P.W. Lamm-Shalem N.1, Masamsetti V.P.1, Biro M.2 and Cesare A.J.1 School of Medical Sciences, UNSW Australia, Sydney, NSW, 1Genome Integrity Unit, Children’s Medical Research Institute, Australia. University of Sydney, Westmead, NSW 2145, Australia. 2EMBL Australia Node in Single Molecule Science, School of Medical Sciences, The actin cytoskeleton is a highly organised and dynamic system that University of New South Wales, Sydney, NSW 2052, Australia. processes both external and internal signalling cues into changes in the structure of cellular architecture. Dysregulation of actin organisation Impediments that that slow the rate of D N A replic ation are c ollec tively refer red is seen in many tumour types and disease processes, however actin to as replicaiton stress. Replication stress is the main driver of genome itself is not a clinical drug target, due to cardiotoxic side-effects. Actin instability in early cancer development and is recognized as a hallmark of binding proteins are a diverse group of proteins that have distinct cancer. Actin is a cytoskeletal protein that forms filaments to provide cells functions and tissue and cellular expression profiles, and as such with mechanical support and driving force for movement. While actin is present as more attractive therapeutic targets. This project aims traditionally considered a cytoplasmic protein, recent evidence indicates to identify compounds that impact on the organisation of the actin actin polymerization can also occur inside the nucleus. However, the role cytoskeleton either through signalling pathways or direct actin binding for nuclear actin fibres, the mechanism(s) triggering their polymerization, proteins. We screened 114,400 diverse compounds in an image-based and the impact of nuclear actin on the genome remains unclear. Using live- screen using F-actin organisation as a readout, quantifying 75 features cell and super-resolution imaging, chromatin fibre analysis, biochemistry, cell and molecular biology, we discovered that actin polymerization plays a per condition and defining >2600 unknown compounds impacting actin prominent role in the replication stress response. Consistent with induced organisation. Unbiased clustering of responses delineated 27 distinct DNA replication stress, pharmacological inhibition of actin polymerization in actin phenotypes. This suggests that there are a discrete number of human cells resulted in S-phase elongation, reduced DNA replication rate, ways that polymeric actin can be reorganised in a mammalian cell. shortened distance between replication origins, and increased occurrence of We hypothesized that similar actin phenotypes may have similar drug micronuclei and anaphase abnormalities. Pharmacological replication stress targets, establishing 3 strategies to accelerate mechanism discovery: induced ATR and mTOR-dependent nuclear actin polymerization, which 1) Comparison with known actin compounds used as positive controls altered the nuclear architecture through the expansion of nuclear volume in the screen (>260 compounds identified); 2) Comparison with and directed migration of stalled replication forks along nuclear actin fibres. known genetic changes (3 talin-binding compounds defined), and; 3) Inhibiting ATR, mTOR or actin polymerization, suppressed replication stress- Retrospective integration (RetInt) of additional positive controls into the dependent nuclear alteration, prevented fork migration, and prevented the original screen data (2 ROCK inhibitors defined). The RetInt strategy restart of stalled replication forks. Preliminary data indicate co-localization may enhance accelerated drug discovery by providing near-unlimited of stalled replication forks with certain repair factors is compartmentalized capacity to extend mechanistic analyses in this and other phenotypic to the nuclear periphery, suggesting that actin polymerization facilitates screens. movement of stalled forks to the nuclear periphery for repair. Cumulatively, these data reveal a novel pathway where actin dependent forces shape the nucleus in response to replication stress to maintain genome health. These findings suggest nuclear actin polymerization may have additional nuclear roles that that impact genome function.

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SYM-61-05 SYM-62-01 ASYMMETRICAL DISASSEMBLY OF APOPTOTIC THE ARCHITECTURE OF THE MEMBRANE CELLS AND THE MECHANISMS UNDERPINNING THIS ASSOCIATED RETROMER-SORTING NEXIN COMPLEX PROCESS REVEALED BY CRYO-ELECTRON TOMOGRAPHY

Jiang L., Tixeira R., Atkin-Smith G., Caruso S., Phan K., Baxter A., Leneva N.1, Kovtun O.2, Ariotti N.3, Teasdale R.4, Owen D.1, Briggs J.2 Hulett M. and Poon I. and Collins B.4 La Trobe Institute for Molecular Science, La Trobe University. 1CIMR, Cambridge. 2MRC-LMB, Cambridge. 3UNSW, Sydney. 4UQ, Brisbane. Apoptotic bodies (ApoBD) generated from apoptotic cells is considered a major class of extracellular vesicles that could facilitate cell clearance Compartmentalisation is a defining feature of all eukaryotic cells, and and intercellular communication. Efficient disassembly and clearance we have evolved highly sophisticated protein machineries to control of dying cells is significant in avoiding accumulation of cellular debris, the flow of transmembrane molecules and membrane lipids between also the release of pro-inflammatory intracellular contents. Thus, the different organelles. The retromer protein complex is required for disassembly of apoptotic cells may also play a key role in preventing generating cargo-selective tubulovesicular carriers from endosomal autoimmune diseases such as systemic lupus erythematosus. membranes (1,2). Retromer-mediated trafficking is an essential However, the mechanisms underpinning this process is not entirely process in all eukaryotes, controlling the cellular localisation and clear yet. When observing T cells undergoing apoptosis by time-lapse homeostasis of hundreds of transmembrane proteins, and its disruption microscopy, we discovered the establishment of asymmetric form is associated with major neurodegenerative disorders (3). However, “octopus structure” during apoptosis. Moreover, by using fluorescent how retromer is assembled and how it is recruited to form membrane microscope, we discovered that cellular organelle (e.g. nucleus, tubules remains unknown. Here we describe the structure of the mitochondria, Golgi apparatus and lysosomes) and cell surface Chaetomium thermophilum trimeric retromer complex (Vps26-Vps29- markers are also distributed asymmetrically as well during apoptotic Vps35) assembled on membrane tubules with the sorting nexin protein membrane blebbing, which will then lead to asymmetrical disassembly Vps5, using X-ray crystallography, molecular modelling and cryo- and the generation of distinct apoptotic bodies with different contents electron tomography with sub-tomogram averaging at sub-nanometre and morphological characteristics. Additionally, we discovered similar resolution. The structure reveals two interwoven layers where Vps5 morphology in different cell lines during apoptosis, indicating a unified forms a membrane-associated lattice, while the outer lattice consists of cellular content sorting mechanism during apoptotic cell disassembly. arches of retromer that extend away from the membrane sur face. Vps35 Furthermore, we found that dynamic blebbing is essential for forms the legs of these arches, Vps29 sits at the apex where it is free asymmetrical distribution of nuclear content and ROCK1 inhibitor GSK to interact with regulatory factors, and the feet of the arches connect to can inhibit this process efficiently. Overall, these data uncover a novel each other through Vps26. Vps26 also forms the primary interface with characteristic of apoptotic cell disassembly and molecular regulators Vps5 via the same site previously shown to mediate Snx3 and cargo involved in this process. interactions (4), suggesting the existence of distinct retromer-sorting nexin complexes. The architecture of the tubulovesicular retromer coat revealed here provides key insights into the conserved mechanisms of retromer assembly and retromer-mediated endosomal trafficking.

SYM-62-02 SYM-62-03 STRUCTURAL INSIGHTS INTO THE ARCHITECTURE T CELL RECEPTOR CLUSTERING: A MECHANISM OF AND MEMBRANE INTERACTIONS OF THE SIGNAL TRANSDUCTION CONSERVED COMMD PROTEINS Gaus K.1, 2 Healy M.1, Hospenthal M.2, Hall R.1, Chilton M.3, Chandra M.1, Chen K.1, 1EMBL Australia Node in Single Molecule Science. 2ARC Centre of Cullen P.3, Lott S.2, Collins B.1 and Ghai R.1 Excellence in Advanced Molecular Imaging, University of New South 1Institute for Molecular Bioscience, The University of Queensland, St. Wales, Sydney, Australia. Lucia, Queensland, 4072, Australia. 2School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand. 3School Antigen recognition by the T cell receptor (TCR) is a hallmark of the of Biochemistry, Biomedical Sciences Building, University of Bristol, adaptive immune system. When the TCR engages a peptide bound Bristol BS8 1TD, UK. to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the assoc iated CD3 c omplex. H ow the ex trac ellular The COMMD proteins are a conserved family of proteins with central antigen recognition event leads to intracellular phosphorylation roles in intracellular membrane trafficking and transcription. They form remains unclear. We develop single-molecule localization microscopy oligomeric complexes with each other and act as components of a larger (SMLM) approaches and novel analysis to determine how spatial assembly called the CCC complex, which is localized to endosomal organization regulates signal initiation and propagation. For example, compartments and mediates the transport of several transmembrane we used SMLM data to map the organization of TCR-CD3 complexes cargos. How these complexes are formed however is completely into nanoscale clusters and to distinguish between triggered and non- unknown. Here, we have systematically characterised the interactions triggered receptor copies. We found that only TCR-CD3 complexes in between several human COMMD proteins, and determined structures dense clusters were phosphorylated and associated with downstream of COMMD proteins using X-ray crystallography and X-ray scattering signaling proteins, demonstrating that the molecular density within to define the core principles of their homo- and heteromeric assembly. clusters dictates signal initiation. This lead us to propose a model in All COMMD proteins possess an α-helical N-terminal domain, and a which antigen recognition is first translated into receptor clustering and highly conserved C terminal domain that forms a tightly interlocked then the density of receptor nanoclusters is translated into signaling. dimeric structure responsible for COMMD-COMMD interactions. The This model may explain how T cells can respond to both the affinity COMM domains also bind directly to components of CCC and mediate and dose of pMHC molecules with a common signal transduction non-specific membrane association. Overall these studies show that mechanism (Pageon et al. PNAS 2016). We also developed novel COMMD proteins function as obligatory dimers with conserved domain FRET sensors to monitor the rate of receptor clustering (Maet al. Nat architectures. Commun 2017) and a sensor that reports membrane charges (Ma et al. Nat Biotech 2017) to understand how biophysical properties of the plasma membrane contribute to TCR signaling.

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SYM-62-04 SYM-62-05 DEFINING ALLOSTERIC BINDING SITES AND BIASED CRYO-EM STRUCTURE OF THE HUMAN ADENOSINE AGONISM OF CLASS C G PROTEIN-COUPLED A1 RECEPTOR-GI2-PROTEIN COMPLEX BOUND TO ITS RECEPTORS ENDOGENOUS AGONIST

Gregory K.J. Draper-Joyce C.J.1, Khoshouei M.2, 3, Thal D.M.1, Liang Y.L.1, Drug Discovery Biology and Dept. of Pharmacology, Monash Institute Furness S.G.B.1, May L.T.1, Wootten D.1, Sexton P.M.1, Glukhova A.1 of Pharmaceutical Sciences, Monash University. and Christopoulos A.1 1Drug Discovery Biology and Department of Pharmacology, Monash Class C G protein-coupled receptors (GPCRs) are cell surface Institute of Pharmaceutical Sciences, Monash University, Parkville transmembrane proteins that are attractive therapeutic targets for 3052, Victoria, Australia. 2Department of Molecular Structural multiple disorders. Our work focuses on two well-characterised Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, members, metabotropic glutamate receptor 5 (mGlu5) and the Calcium- Germany. 3Norvatis Institutes for Biomedical Research, Norvatis sensing receptor (CaSR), which respond to glutamate and extracellular Pharma AG, 4002 Basel, Switzerland. Ca2+ respectively. Allosteric modulators that generally bind to sites within the 7 transmembrane-spanning domains, which are distinct from the endogenous agonists, are of significant interest due to their ability to fine- G protein-coupled (GPCRs) are responsible for the majority of cellular tune GPCR activity. Positive allosteric modulators (PAMs) enhance, whereas responses to hormones, neurotransmitters, and a variety of other small negative allosteric modulators (NAMs) inhibit the orthosteric agonist molecules. In recent years there has been an exponential growth in the response. Allosteric modulators offer greater subtype selectivity and the amount of determined inactive GPCR structures, however, there still potential to fine-tune GPCR activity. Indeed, cincalcet, a CaSR PAM, was remains a dearth of active-state, G-protein-bound, GPCRs. To date, all the first GPCR allosteric modulator to enter the clinic. Discovery programs solved active-GPCRs have been coupled to the stimulatory Gs protein. commonly rely on potency determinations when screening for allosteric The class A adenosine A1 receptor (A1R) is a GPCR that preferentially modulators, however, we have shown this approach lacks sufficient rigor couples to the inhibitory Gi/o family of heterotrimeric G-proteins, has and can result in misinterpretation of activity. We apply rigorous analytical been implicated in numerous diseases, yet remains poorly targeted. We methods and investigate multiple measures of activation to dissect the have recently solved the 3.6 Å structure of the human A1R in complex structural basis and functional consequences of class C GPCR allosteric with adenosine and heterotrimeric Gi2-protein determined by Volta modulation. We have found that Class C GPCR allosteric modulators can phase plate cryo-electron microscopy. Compared to inactive A1R, there differentially activate and/or modulate distinct signalling pathways, referred is contraction at the extracellular surface in the wide orthosteric binding to as biased agonism and biased modulation, respectively. Distinct bias site that is mediated via movement of transmembrane domains 1 and

profiles can be linked to in vivo efficacy and may be predictive of adverse 2. At the intracellular surface, the G-protein engages the A1R primarily effect liability. Moreover, previously unappreciated biased modulation has via amino acids in the C-terminus of the Gαi α5 helix, concomitant revealed that ligands originally classified as selective ligands at other class with a 10.5 Å outward movement of A R transmembrane domain 6. C GPCRs have off-target activity at mGlu5. Through structure-function 1 Comparison with the agonist-bound β2adrenergic receptor-Gs-protein analyses we are defining the key ligand-receptor interactions that govern complex reveals distinct orientations for each G-protein subtype upon these effects. Ultimately, our work will provide a better understanding of engagement with its receptor. This active A1R structure provides novel the mechanisms driving on-target therapeutic versus adverse effects and molecular insights into receptor/G-protein selectivity. provide a framework for future rational discovery campaigns for biased modulators that can fine-tune receptor activity at the pathway level.

SYM-63-01 SYM-63-02 EVALUATION OF LIVER SECRETED PROTEINS NEW PATHWAYS IN HEPATIC STEATOSIS, INSULIN IDENTIFIES NOVEL REGULATORS OF GLUCOSE RESISTANCE, AND TYPE 2 DIABETES METABOLISM Koay Y.C.1, 2, Yang P.3, Chen D.L.4, Jenkins A.B.4, Wood C.5, Li M.3, Watt M.J. and Montgomery M.K. Greenfield J.R.4, 6, 7, Samocha-Bonet D.4, 6 and O’Sullivan J.F.1, 2, 8 Department of Physiology, University of Melbourne, Melbourne, 3010. 1Sydney Medical School, The University of Sydney, Australia. 2Heart Research Institute, Sydney, Australia. 3School of Mathematics 4 Obesity is a risk factor for the development of inter-related complications and Statistics, University of Sydney, Australia. Diabetes and including dyslipidemia, non-alcoholic fatty liver disease (NALFD), Metabolism Division, Garvan Institute of Medical Research, Sydney, 5 cardiovascular disease and type 2 diabetes. An accumulation of lipid in Australia. Talented Scientists Progam, School of Biomedical Science, 6 the liver, which is clinically known as hepatic steatosis, is a pathologic The University of Sydney, Australia. Faculty of Medicine, UNSW, 7 abnormality that is common in obese and type 2 diabetes patients. Australia. Department of Endocrinology and Diabetes Centre, St 8 Hepatic steatosis occurs when fatty acid supply outweighs fatty acid Vincent’s Hospital, Sydney, Australia. Royal Prince Alfred Hospital, demand and occurs in a time-course that usually precedes the induction Department of Cardiology, Sydney, Australia. insulin resistance and type 2 diabetes. This presentation describes our laboratories use of proteomics to evaluate how ‘heptokine’ secretion is The global obesity epidemic, leading to non-alcoholic fatty liver disease altered in murine models of NAFLD and non-alcoholic steatohepatitis. (NAFLD) and Type 2 Diabetes (T2D), continues unabated despite We report on the pre-clinical validation of several liver secreted factors sustained efforts to combat it, and is our greatest modern healthcare that either cause insulin resistance and disturbances in systemic challenge. NAFLD is the commonest form of liver disease in the metabolic homeostasis or enhance glycemic control in diabetes. These Western world, affecting one in three people in the general population, studies have unravelled previously appreciated biology and pave the 90% of obese patients with type 2 diabetes (T2D), and 5.5 of 6 million way for potential therapeutic intervention for type 2 diabetes. Australians with liver disease – accounting for much of the $51 billion annual cost to our healthcare system. Although exact mechanisms remain unclear, accumulation of liver fat is a principal cause of T2D. We have recently discovered a new pathway in hepatic steatosis that independently predicts type 2 diabetes over 12 years in advance. Our subsequent work has revealed novel lipid biomarkers for NAFLD that can distinguish between liver insulin resistance and muscle insulin resistance. Three representative lipids alone can accurately classify patients according to the organ driving insulin resistance with >99% accuracy. We have also recently revealed how a common SNP (MAF.

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SYM-63-03 SYM-63-04 PRECLINICAL CHARACTERISATION OF JAK1/JAK2 THE ROLE OF TRAFFICKING REGULATOR OF GLUT4 1 INHIBITORS FOR TREATMENT OF TYPE 1 DIABETES (TRARG1) IN GLUT4 TRAFFICKING

Trivedi P.M.1, Scott N.A.1, Jenkins M.R.2, Brodnicki T.C.1, Kay T.W.1 Duan X.1, Krycer J.R.1, Cooke K.C.1, Yang G.1, James D.E.1, 2 and and Thomas H.E.1 Fazakerley D.J.1 1St Vincents Institute, Fitzroy, Victoria, Australia. 2Walter and Eliza Hall 1Charles Perkins Centre, School of Life and Environmental Sciences, Institute, Parkville, Victoria, Australia. The University of Sydney, Sydney, NSW 2006, Australia. 2Sydney Medical School, The University of Sydney, Sydney, NSW 2006, There is an opportunity to repurpose therapeutics from other diseases Australia. to type 1 diabetes, especially when there is evidence for overlapping mechanisms. We generated directed networks based on the phenotypes Insulin lowers blood glucose, in part, by enhancing glucose transport of gene knockout non-obese diabetic (NOD) mice, a mouse model into adipose and muscle tissues through the redistribution of glucose of type 1 diabetes. The JAK-STAT pathway was important in T cells transporter GLUT4 from specialised intracellular GLUT4 storage and pancreatic beta cells, the target of T cell-mediated destruction. vesicles (GSVs) to the plasma membrane. This process is impaired We tested the hypothesis that blocking the response to cytokines that in insulin resistance, which is a precursor to numerous metabolic activate the JAK-STAT pathway would prevent autoimmune diabetes disorders including Type 2 diabetes. Trafficking regulator of GLUT4 1 and tested the effects of drugs that selectively inhibit JAK1/JAK2. JAK (TRARG1) localises to GSVs and positively regulates GLUT4 trafficking inhibitors successfully blocked IFNγ-induced phosphorylation of STAT1 in response to insulin. Understanding how TRARG1 regulates GLUT4 and IFNγ-induced MHC class-I upregulation in mouse and human is of interest since lower TRARG1 expression may contribute to islets. Mouse islets were protected from CTL-mediated killing in vitro by impaired GLUT4 trafficking in insulin resistance. To begin to address JAK inhibitors. Time-lapse microscopy shows the inhibitor prevented this we have used a combination of bioinformatics prediction tools and the direct interaction between CTLs and beta cells, reducing calcium biochemical assays to define the membrane topology of the 173-amino flux in the CTL and synapse duration. JAK inhibitors blocked the effect acid mouse TRARG1. This revealed that TRARG1 contains a single of cytokines on beta cells in vivo by inhibiting MHC class I upregulation, transmembrane domain at its C-terminus with a cytosolic N-terminus and reduced infiltration of immune cells into islets. Furthermore, NOD and extracellular/luminal C-terminus. Interestingly, our studies also mice treated with JAK inhibitors were protected from autoimmune revealed a re-entrant loop that also confers TRARG1 membrane diabetes, and diabetes was reversed in newly diagnosed NOD mice. association. Based on this model of TRARG1 membrane topology, Interestingly, deficiency of receptors for all three interferons (IFNγ, our current studies are focussed on characterising TRARG1, including IFNα, IFNλ) did not prevent diabetes in NOD mice, suggesting that JAK identifying the signals that determine its localisation to GSVs, and inhibitors block cytokine signals other than interferons that are crucial determining how TRARG1 regulates GLUT4 trafficking. for diabetes development. Future work will study this. Our work provides mechanistic groundwork for re-purposing clinically approved JAK inhibitors for type 1 diabetes, and provides a platform for testing many of the >150 kinase inhibitors under development for other diseases for efficacy in autoimmune diabetes.

SYM-63-05 SYM-64-01 TREATMENT WITH NAD+ PRECURSORS IMPROVE A-TO-I RNA EDITING - NOT ALL EVENTS ARE WOUND HEALING IN DIABETIC AND OLD MICE CREATED EQUAL

Das A.1, 2, Giatsidis G.2, Garg N.2, Wu L.1, Orgill D.2 and Sinclair D.1, 2 Heraud-Farlow J.E. 1University of New South Wales, Sydney. 2Harvard Medical School, St. Vincent’s Institute of Medical Research, Fitzroy, Vic, 3065 USA. Australia.

Delayed wound healing is one of the major complications in diabetes Adenosine-to-inosine (A-to-I) editing of double-stranded RNA (dsRNA) and ageing, which may result in limb amputation. Recent reports by ADAR proteins is a highly prevalent form of RNA base modification suggest that Sirtuins, a family of NAD+-dependent protein deacylases, in higher eukaryotes. Tens of thousands of A-to-I editing events are play an important role in skin wound healing in both diabetes and defined in the mouse, yet the functional impact of most is unknown. ageing. Transgenic mice systemically overexpressing SIRT1, the Inosine is interpreted as guanosine by the ribosome and other cellular mammalian sirtuin orthologue, showed improved skin wound healing machinery and therefore editing has the ability to change the amino in old and diabetic mice. The activity of SIRT1 is dependent on the acid sequence of encoded proteins, splice sites, the binding sites of levels of NAD+ inside the cell. The NAD+levels however decrease in RNA-binding proteins as well as modify the RNA secondary structure. different tissues with ageing and diabetes, resulting in declining SIRT1 Editing causing protein recoding is the essential function of ADAR2, but activity. Boosting the levels of NAD+ using NAD+ boosters is emerging an essential role for recoding by ADAR1 has not been demonstrated. to be an effective strategy to activate and reproduce the effects of Additionally, ADAR1 has been proposed to have editing-dependent Sirtuins in ageing and diabetes. Here, we tested the efficacy of two and editing-independent functions however, the relative contribution of NAD precursors – Nicotinamide riboside (NR) and Nicotinamide these in vivo has not been clearly defined. A critical function of ADAR1 Mononucleotide (NMN) – in wound healing in old and diabetic is editing of endogenous RNA to prevent activation of innate immune mice. Topical administration of both of these compounds resulted in pathways via the dsRNA sensor MDA5 (Ifih1). Concurrent deletion of enhanced wound healing, increased wound vascularity, and significant MDA5 rescues the embryonic lethality of Adar1 editing deficient mice improvement in the ischemic neovascular response. These findings (Adar1E861A/E861A). Outside of this role however, it is uncertain how ADAR1 have implications to significantly improve quality of life of diabetic editing contributes to normal development and homeostasis. To address patients and ageing population who have severely impaired wound- this, we analysed the consequences of ADAR1 editing deficiency on healing capabilities after injuries. murine homeostasis in the absence of MDA5. Our findings indicate that outside of the editing of MDA5-targeted self dsRNA, editing by ADAR1 is dispensable for normal murine development.

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SYM-64-02 SYM-64-03 MODULATION OF EPIGENETIC PLASTICITY IN SINGLE MOLECULE INVESTIGATION OF LESION CANCER CELLS: FROM SIGNALLING PATHWAYS TO HAND-OFF DURING TRANSCRIPTION COUPLED ENGINEERED PROTEINS REPAIR

Blancafort, P. Ho H.N.1, 2, van Oijen A.M.1, 2 and Ghodke H.1, 2 1Molecular Horizons Institute and School of Chemistry, University of Wollongong, Wollongong, Australia. 2Illawarra Health and Medical Research Institute, Wollongong, Australia. ABSTRACT NOT AVAILABLE AT TIME OF PUBLICATION During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall on template DNA. Stalled transcription elongation complexes (TECs) at sites of lesions can be rescued by the transcription terminator Mfd. In this transcription-coupled repair (TCR) reaction, Mfd recognizes stalled TECs and recruits the nucleotide excision repair machinery (NER) to the site. Extensive biochemical, structural, genetic and single-molecule investigations have shed insight into various aspects of TCR. Despite these studies, the molecular mechanisms of stalled RNAP recognition by Mfd and recruitment of the NER machinery in vivo largely remain unknown. Single-molecule live- cell imaging of fluorescently labelled Mfd revealed that Mfd associates with TECs even in the absence of exogenous genotoxic stresses. This interaction requires an intact RNAP-interacting domain of Mfd, and is stabilized under conditions in which RNAP is stalled on template DNA. Binding of Mfd is highly regulated in cells, and mutations in its ATPase domain or the UvrB-homology module result in deregulated and non-specific DNA binding. Using an interval imaging approach on live-cells, we determined the lifetime of Mfd to be 18s during TCR in wild-type cells. We found that this lifetime is influenced by the presence of the NER machinery components UvrA and UvrB. Whereas absence of UvrA led to a longer lifetime of 30s, cells lacking functional UvrB exhibited Mfd foci that were arrested on DNA for 60-80s. Our findings shed insight into the recruitment and regulation of the transcription- repair coupling factor, and the execution of the TCR in cells.

SYM-64-04 SYM-64-05 MECHANISTIC DIFFERENCES IN DNA BINDING TRANSPOSON-DERIVED TRANSCRIPTION FACTOR BEHAVIOUR OF ISL1/LHX3 HOMEODOMAINS IN BINDING SITES AND PROMOTER ACTIVITIES IN TRANSCRIPTIONAL COMPLEXES BREAST CANCER

Smith N.C., Stokes P.H. and Matthews J.M. Jiang J. and Upton K.R. University of Sydney, Sydney, NSW, Australia. The University of Queensland, The School of Chemistry and Molecular Biosciences. LIM-homeodomain (LIM-HD) transcription factors act as key developmental regulators, both through their ability to bind DNA Transposons, a type of repetitive DNA, are pervasive in the human through homeodomain:DNA interactions, and through their ability genome, occupying approximately 45% of the total genomic sequence. to form higher order complexes through protein:protein interactions. Since their initial discovery by Barbara McClintock, transposons were The LIM-HD proteins Islet-1 (Isl1) and LIM homeobox protein 3 (Lhx3) predominantly viewed as junk or selfish DNA. However, recent progress have been implicated in the development of a broad range of tissues, has revealed the extensive co-option of transposons for the transcriptional most comprehensively in cell fate determination in the developing regulation of host genes. While generally inactivated in normal somatic central nervous system. These proteins, along with the protein cofactor tissues, transposons can become transcriptionally active in epithelial LIM-domain binding protein 1 (Ldb1) interact to form cell-specific cancers and may even act as promoters or enhancers that subsequently transcriptional complexes. The presence or absence of Isl1 in complex activate oncogenes and contribute to tumorigenesis. Currently, the with Lhx3 and Ldb1 is sufficient to redirect the complex to target regulatory impact of transposons in breast cancer remains largely unknown. To address this gap in knowledge, we investigated the landscape different genes. This then directs the differentiaton of neural precursors of transposon-derived oncogenic transcription factor (TF) binding in breast into motor neurons or interneurons. Recapitulating this system in cancer, predicted transposon-derived promoters, and validated these vitro, we have shown that Isl1 and Lhx3 bind differently in isolation predictions. Our results demonstrated that transposons were an abundant than they do in combination, displaying a change in specificity that is source of TF binding sites in breast cancer, where ~38% of all binding not explained by simple cooperative binding of the homeodomains. sites of MYC, E2F1 and C/EBPβ were harboured by transposons. We Through the use of comparative electrophoretic mobility shift assays identified 399 transposon subfamilies as significantly overrepresented in (EMSAs), multi angle laser light scattering (MALLS) and small angle the oncogenic TF binding sites, suggesting a widespread role of these X-ray scattering (SAXS) experiments, we we have studied the binding subfamilies in cancer transcriptional networks. TF-bound transposons behaviours of these homeodomains in different combinations with each were also associated with active histone modifications, further supporting other and with or without additional protein:protein interaction domains. the regulatory role of transposons in breast cancer. Finally, we validated Through this, we have gained new understanding of the mechanisms the promoter activity of individual transposons in triple negative breast by which these complexes can discern targets in the cell. Our results cancer cell lines. Luciferase assay results revealed that the promoter suggest that while the stoichiometry of binding is as expected, only activities of SYT1, UCA1, AK4 and PSAT1 oncogenes were significantly one homeodomain is responsible for the affinity of binding to different reduced, and in some cases, almost completely abolished following targets, while the other appears to be important in stabilising binding to transposon deletion. Transposons with strong promoter activity were also specific target sequences. Taken together, these data suggest a novel found to be epigenetically deregulated in breast cancer, characterised molecular mechanism for the combinatorial action of these transcription by hypomethylation and/or increased DNAse sensitivity. Our results factors that act to influence cell fate during development. provide an insight into the contribution of transposons to breast cancer transcriptional regulation, and will facilitate the development of novel diagnostic and prognostic biomarkers for breast cancer.

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SYM-65-01 SYM-65-02 FARMING MALLEE EUCALYPTS FOR VALUABLE PRECISION LANDSCAPE REGENERATION FOR FOOD, NATURAL PRODUCTS ECOSYSTEM AND CLIMATE SECURITY

Woodrow I.E.1, Fernando S.2, Humphries J.2 and Goodger J.Q.D.2 Borevitz J.O. 1School of Ecosystem and Forest Sciences, The University of Australian National University. Melbourne, VIC 3010, Australia. 2School of BioSciences, The University of Melbourne, VIC 3010, Australia. Global trends project a convergence of crisis in the next decades as demand for land exceeds planetary biocapacity. Agriculture depends Mallee eucalypts have several properties that are advantageous for on soil and water supplied by ecosystems which are alsol holding run producing natural products on a large scale. These include the capacity away climate change. Regenerative agro-ecosystems grow food, are to grow well on marginal land where agricultural cropping is unprofitable water resilient and build soil using perennial cropping, silvapasture and and an ability to be harvested mechanically in a short rotation coppice agroforestry approaches with biochar and enhanced rock weathering. cycle. The leaves of some mallee species contain exceptionally high When deployed at scale (1B hectares), regenerative landscapes can levels of natural products, which are stored in the extracellular lumina also stabilize the global climate by drawing down 10GtCO2e/y. I will of embedded secretory cavities, commonly known as oil glands. These outline a high tech (Genome2Phenome2Environment) experimental, glands can comprise up to 15% of total leaf volume. Two examples of modeling and social/economic research program to scale regenerative natural product production are explored in this presentation. In the first, agriculture nationally and globally by 2030. Australia’s diverse the research underpinning the development of profitable blue mallee landscape with synergistic primary industry capacity (agriculture and (Eucalyptus polybractea) plantations for pharmaceutical eucalyptus oil mafic rock mining) can contribute 10% of this solution valued at $1T production is described. Specifically, the biochemical mechanism for oil over the decade. synthesis and storage is outlined, and the development of exceptionally high yielding plantations through a clonal forestry approach is described. In the second example, the ability of mallee eucalypts to produce highly valuable flavonoids is discussed. Importantly, some of these compounds are already in the latter stages of testing for their ability to diminish the symptoms of several diseases of the central nervous system, including Alzheimer’s and Parkinson’s diseases and ischemic stroke. The potential to establish a mallee based industry for natural flavonoid production is discussed.

SYM-65-03 SYM-65-04 APPLYING GENOMICS TO CONSERVATION: RESPONSES OF ISOTOPE DISCRIMINATION AND INVESTIGATING CLIMATE ADAPTATION INTERCELLULAR RELATIVE HUMIDITY TO VAPOUR IN EUCALYPTUS MICROCARPA AND IMPLICATIONS PRESSURE DEFICIT IN WILDTYPE AND ABSCISIC FOR RESTORATION ACID INSENSITIVE POPULUS X CANESCENS

Jordan R.1, 2, Prober S.3, Dillon S.4 and Hoffmann A.1 Cernusak L.A.1, Goldsmith G.2, Arend M.3 and Siegwolf R.4 1Bio21 Institute, School of BioSciences, University of Melbourne, 30 1James Cook University, Cairns, Australia. 2Chapman University, Flemington Rd, Parkville Vic 3010, Australia. 2CSIRO Land & Water, Orange, California, USA. 3Basel University, Basel, Switzerland. 4Paul 15 College Rd, Sandy Bay Tas 7005, Australia. 3CSIRO Land & Water, Scherrer Institute, Villigen, Switzerland. 147 Underwood Ave, Floreat WA 6014, Australia. 4CSIRO Agriculture, Clunies Ross Street, Black Mountain ACT 2601, Australia. We investigated responses to vapour pressure deficit (D) in abscisic acid insensitive (abi) and wildtype (WT) Populus x canescens, in order Under continued environmental change, the simple presence of to better understand the crucial role of stomata in modulating leaf gas plant species within the landscape does not necessarily equate to exchange and stable isotope discrimination, and to test for unsaturation their long-term survival. This is especially true in highly fragmented of intercellular relative humidity (hi). Transpiration rate increased linearly regions where reductions in population size and connectivity may with D in abi plants up to D of about 2 kPa, beyond which it declined decrease the potential for future adaptation. To ensure evolutionary precipitously; this was followed by leaf death within hours of removing the resilience under changing conditions, conservation and restoration leaf from the gas exchange cuvette. On the other hand, WT leaves showed relies on capturing genetic diversity and thus adaptive potential. a steady or slightly declining transpiration rate up to D of nearly 7 kPa, Genomics offers a powerful new approach for investigating genetic and fully recovered photosynthetic function when measured the next day. Discrimination against 13CO (Δ13C) declined in response to increasing D diversity and adaptation across a species’ range. Using the test case 2 in WT plants, consistent with declining intercellular CO2 concentrations. of Eucalyptus microcarpa (Grey Box), we present an example of how 13 genomic approaches can be applied to conservation, in particular For the D range over which abi plants were measured, Δ C was higher in abi plants than in WT plants by about 6 permil on average, and did restoration of tree species under climate change. Employing DArTseq, not decrease in response to increasing D. Discrimination against C18OO a reduced-representation genomics approach, we firstly compared (Δ18O) differed markedly between WT and abi plants. In WT plants, genomic diversity between natural and revegetated sites to assess Δ18O increased with increasing D by about 2 permil kPa-1, whereas in how well current revegetation strategies capture genomic diversity and abi plants the rate of increase was about 66 permil kPa-1. This reflected thus adaptive potential. Secondly, we investigated adaptation at the progressive stomatal closure in the WT plants with increasing D. In abi genomic-level, aiming to identify genomic regions important for climate plants, in contrast, stomata remained open in response to increasing D adaptation as well as environmental factors potentially driving adaptive allowing CO2 to readily diffuse into the leaf interior, exchange oxygen diversity in this species. Through this case-study, we demonstrate atoms with the 18O enriched leaf water, and then diffuse back into the how genomics can provide deeper insight for restoration under climate air exiting the gas exchange cuvette. The coupled measurements of change; moving beyond general genetic diversity towards knowledge Δ18O and gas exchange allowed us to estimate hi. In WT leaves we saw of genomic signatures of climate adaptation and thus potential no evidence of unsaturation of hi, even at D above 6 kPa. However, in adaptive diversity. We show the power of genomics to provide in-depth abi leaves, hi decreased at a rate of about -0.25 kPa-1, thus declining to knowledge that may assist in improving seed sourcing strategies and values approaching 0.6 before the precipitous decline in transpiration rate evolutionary-resilience of future revegetation efforts. and subsequent leaf death.

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SYM-65-05 SYM-66-01 REGULATING TREE VIGOUR TO OPTIMISE THE SEPARATING THE FGF SIGNALING REQUIREMENTS LONG-TERM PRODUCTIVITY OF WALNUT (JUGLANS OF CLOCK OSCILLATION FROM WAVEFRONT REGIA L.) ORCHARDS ACTIVITY DURING SOMITOGENESIS

Simpson J.E.1, Lang M.D.2 and Lewis J.3 Lewandoski M.1, Kageyama R.2 and Anderson M.1 1NSW Department of Primary Industries, Orange Agricultural 1National Cancer Institute, NIH, USA. 2Kyoto University, Kyoto, Japan. Institute, 1447 Forest Rd, Orange, NSW. 2800. 2Webster Limited, 148 Colinroobie Road, Leeton, NSW 2725. 3NSW Department of Primary The vertebrate axial skeleton is comprised of segmented vertebrae, Industries, Yanco Agricultural Institute, Yanco, NSW, 2703. differing in size and shape, but with a pattern nearly invariant between individuals. Vertebrae form from somites, which segment from Walnut orchards in Australia are typically high-density plantings the posterior presomitic mesoderm (PSM). Somite segmentation comprising one or more cultivars of Juglans regia L. An important is controlled by two classical activities, known as the “clock” and criterion for cultivar selection is tree vigour, with greater vigour improving “wavefront”. Wavefront activity maintains the posterior PSM in an the profitability of orchards during the early years of tree production undifferentiated state; we previously showed that Fgf4 and Fgf8 which increases profitability. As trees mature unregulated growth leads each encode this activity. The clock is governed by oscillating Notch to shading of fruiting points and ultimately reduces tree productivity. This signals that segment new somites at the anterior wavefront edge. Loss study aimed to manage tree vigour with an exogenously applied plant of only Fgf4 in the PSM causes misshaped vertebrae specifically in growth regulator (PGR), and to determine the impact of reduced vigour cervical and thoracic regions. We have characterized these mutants on potential yield and nut quality in young (2-4 years) and mature (8-10 with Hairpin Chain Reaction mRNA in situ hybridization, allowing years) walnut trees. The triazole-type PGR, uniconazole-P, reduces the for direct quantification of up to four different mRNA sequences per internode length by inhibiting gibberellin biosynthesis ergo is a potential embryo. We found that wavefront gene expression is unaffected in means of managing tree vigour. Sunny PGR (active ingredient: 50 g/L Fgf4 mutants; however, clock gene oscillations are highly abnormal. uniconazole-P), was applied to the root zone of mature walnut trees at Hes7, a transcriptional repressor of Notch signaling, is reduced in Fgf4 four different rates (0, 0.75, 1.5, 3.0 L ha-1) and at two different timings mutant PSM, but only during the developmental window when future (autumn and spring) in three cultivars: Serr, Vina and Chandler. Young malformed vertebrae are generated. Due to the central role of Hes7 trees were treated in the same manner as mature trees, but in cultivar in regulating clock oscillation, we hypothesize that this reduction is Chandler only. All trials were randomised complete block designs with the cause of Fgf4 segmentation defects. We also find that defects are five-replicates, of four- and single-tree-plots in mature and young trees more extreme when one copy of Fgf8 is additionally removed and are respectively. General linear modelling will be used to describe the rescued when Fgf8 is overexpressed. We conclude that correct clock relationship between tree growth (internode length), crop yield and nut oscillations require an FGF signal that is genetically separable from the quality with PGR treatment (rate, timing) and cultivar in mature trees, FGF wavefront requirement. The FGF signal appears to be permissive and between tree growth and PGR treatment in young trees. and provides a robustness factor for somite segmentation, buffering the system to changes in Hes7 expression dynamics.

SYM-66-02 SYM-66-03 TREATMENT OF SOLID TUMOURS BY CO-TARGETING MEF2C PHOSPHORYLATION IS REQUIRED FOR BCL-2 PRO-SURVIVAL PROTEINS CHEMOTHERAPY RESISTANCE IN ACUTE MYELOID LEUKEMIA Lee E.F.1, Harris T.2, Tran S.2, Evangelista M.2, Herold M.J.3 and Fairlie W.D.2 Brown F.C.1, 2 and Kentsis A.1 1LIMS, La Trobe University. 2Olivia Newton-John Cancer Research 1Memorial Sloan Kettering Cancer Center, NY, USA. 2Monash Institute. 3Walter and Eliza Hall Institute. University, Melbourne, Australia.

Defective apoptosis signalling is a hallmark of most, if not all, cancers. In acute myeloid leukemia, chemotherapy resistance remains highly Typically this involves abnormally high expression of the pro-survival prevalent, representing the major barrier to cure in children and adults members of the Bcl-2 family of proteins. This enables damaged cell alike. We sought to investigate molecular mechanisms that may explain to survive when they should otherwise be removed. Over the last primary chemotherapy resistance in AML using targeted genomic decade, a new class of drugs (“BH3-mimetics”) has been developed sequencing and high-resolution mass spectrometry proteomics. This to target the Bcl-2 proteins and there are now molecules that enable analysis identified aberrant phosphorylation of MEF2C S222 in primary most of them to be selectively antagonised. In this presentation, I chemoresistant human AML specimens, and using an affinity-purified will discuss our recent data using a panel of these drugs on different antibody, established its prevalence and prognostic significance in a types of highly chemoresistant solid tumours. These data combined cohort of 47 patients, spanning the major biologic subtypes of human with results from experiments using protein-based ligands we have AML. MEF2C is a transcription factor required for hematopoietic cell engineered, as well as genetically engineered cell lines, have enabled fate determination. We found that Mef2cS222A/S222A knock-in mutant us to dissect the critical “survival” factors in these tumours. Critically, mice engineered to block MEF2C phosphorylation exhibited normal we show a consistent pattern of proteins that must be antagonised to hematopoiesis, but were resistant to leukemogenesis induced by provide potent and synergistic tumour cell killing Interestingly, the same MLL-AF9. MEF2C phosphorylation was required for leukemia stem pattern is also starting to emerge for other cancers types, suggesting a cell maintenance in both MLL-rearranged and non-MLL-rearranged potential standardised treatment strategy for many cancers using these mouse AML in vivo, and chemotherapy resistance and leukemogenicity drugs. of human AML cells. Transcriptome and chromatin analysis of gene expression changes and composition of MEF2C transcriptional complexes showed assembly of an active MEF2C transactivation complex specifically induced by MEF2C phosphorylation in leukemia c e l l s . M A R K 3 a c t i v a t i o n w a s s u f fi c i e n t t o c a u s e M E F 2 C p h o s p h o r y l a t ion, leading to enhanced transcription of MEF2 response elements in cells. Notably, treatment with the selective MARK inhibitor MRT199665 caused apoptosis and conferred enhanced sensitivity to cytarabine of MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.

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SYM-66-04 SYM-66-05 EXPLOITING TUMOUR ACIDITY FOR POLYMER BASED DUAL SPHINGOSINE KINASE AND BCL-2 INHIBITION DRUG DELIVERY EXHIBITS SYNERGISTIC CELL DEATH IN ACUTE MYELOID LEUKEMIA Whitty E.G.1, 2, 3, Castignolles P.2, Gaborieau M.2, 3 and Callaghan R.1 1Department of Biomedical Science and Biochemistry, Research Lewis A.C.1, Tea M.1, Wallington-Beddoe C.T.1, Anderson D.2, Creek D.2, School of Biology, The Australian National University, Canberra, Powell J.A.1 and Pitson S.M.1 ACT, 0200, Australia. 2Australian Centre for Research on Separation 1Centre for Cancer Biology, University of South Australia, CRI Building, Science (ACROSS), School of Science and Health, Western Sydney North Terrance, Adelaide, SA 5001, Australia. 2Department of Drug University, Parramatta, NSW, 2150, Australia. 3Medical Sciences Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Research Group, School of Science and Health, Western Sydney Sciences, Monash University, Parkville, Victoria, Australia. University, Parramatta, NSW, 2150, Australia. Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have An acidic micro-environment is a hallmark feature of solid tumours garnered significant interest as therapeutic targets due to their up- generated by metabolic adaptations including an increased reliance regulation in many cancers, including acute myeloid leukaemia (AML), on glycolysis and its concomitant production of lactic acid. The acidic leading to enhanced cancer cell survival. Small molecule inhibitors environment has deleterious effects on many anticancer drugs; such as the selective Bcl-2 inhibitor, Venetoclax, are very effective in however, several novel therapeutic strategies are beginning to exploit some cancers that are highly on Bcl-2, but have demonstrated poor this property. Our consortium has developed three configurations of single agent efficacy in AML due to these cells being highly dependent poly(sodium acrylate) (PNaA) as a pH-sensitive drug delivery system. on Mcl-1, which is not targeted by this agent. Sphingosine kinase 1 Initial efforts have focussed on the anticancer drug cisplatin, which (SK1) is a signalling enzyme with established roles in oncogenesis and is beset with poor stability, toxicity and pharmacokinetic properties. has recently emerged as a potential therapeutic target in leukaemia. Loading cisplatin onto a polymer may circumvent many of these issues We recently demonstrated that the selective SK1 inhibitor, MP-A08 by providing targeted delivery and increased stability. PNaA is loaded exhibits anti-leukemic activity in vitro and in vivo using patient-derived with cisplatin at pH9 and the attachment is stable, reversible and AML xenograft models. MP-A08-mediated cytotoxicity in AML cells displays a pH-dependent release process. The three configurations of correlated with a reduction in Mcl-1 levels, as well as upregulation PNaA differ in their branching structure and thereby offer distinct loading of BH3 only proteins. Here, we found that combinational therapies capacities. In the present investigation we have undertaken preliminary with MP-A08 and Venetoclax induced synergistic cell death in AML characterisation of PNaA in a monolayer cell culture system. Unloaded cell lines and patient samples. Mechanistically, MP-A08 induces polymers did not alter cell cycle progression nor where they associated transcriptional upregulation of BH3-only protein, Noxa and formation with significant levels of apoptosis. Cisplatin loaded polymers displayed of Noxa/Mcl-1 complexes. MP-A08 appears to exert its cytotoxicity in a greater potency than observed with free drug. The increase in drug AML cells through loss of Mcl-1 as a consequence of Noxa binding. potency demonstrates the viability of this polymer as a drug delivery Using patient-derived xenografts, we demonstrate that MP-A08 and system. Venetoclax treatment exhibits anti-leukemic activity in vivo providing pre-clinical evidence to target SK1 and Bcl-2 in AML.

SYM-67-01 SYM-67-02 MCL-1 INHIBITION PROVIDES A NEW WAY TO EXPLORING BACTERIAL RESISTANCE WITH SUPPRESS BREAST CANCER METASTASIS AND ANTIBIOTIC-DERIVED FLUORESCENT PROBES INCREASE SENSITIVITY TO DASATINIB Young A.I.J.1, Castillo L.1, Law A.M.K.1, Brummer T.2, 3, Lee E.F.4, 5, 6, Blaskovich M.A.T. Centre for Superbug Solutions, Institute for Molecular Bioscience, The Fairlie E.F. 4, 5, 6, Timpson P.1, 7, Gallego-Ortega D.1, 7, Ormandy C.J.1, 7 University of Queensland. and Oakes S.R.1, 7 1Cancer Research Division, Garvan Institute of Medical Research and the Kinghorn Cancer Centre, 384 Victoria Street, Darlinghurst, NSW, 2010, Bacteria are becoming resistant to every antibiotic, leading to multi- Australia. 2Centre for Biological Systems Analysis (ZBSA) and Centre for drug resistant ‘superbugs’ that will kill millions of people each year. Biological Signallling Studies, Albert-Ludwigs-University, Stefan-Meier- New antibiotics are urgently needed, but few new drugs are available Strasse 17, 79104, Freiburg, Germany. 3Spemann Graduate School for or in clinical development. It is imperative to discover and develop Biology and Medicine and Faculty of Biology, Albert-Ludwigs-University, 4 new antibiotics to fight these superbugs, but for this to occur we Stefan-Meier-Strasse 17, 79104, Freiburg, Germany. Olivia Newton-John require an improved understanding of how antibiotics work and how Cancer Research Institute, 145 Studley Rd, Heidelberg, Victoria, 3084, bacteria function and develop resistance. This requires new tools and Australia. 5School of Cancer Medicine and Department of Chemistry and Physics, La Trobe University, Melbourne, Victoria, 3086, Australia. 6The techniques to advance our knowledge of bacterial metabolism, efflux Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, pumps and other responses to antibiotics, allowing for analysis of key Parkville, Victoria, 3052, Australia. 7St. Vincent’s Clinical School, UNSW aspects of bacterial growth, division, metabolism and resistance. In Medicine, Victoria Street, Darlinghurst, NSW, 2052, Australia. order to create such tools, we have been systematically converting representatives of the major classes of antibiotics into mechanism- Metastatic disease is largely resistant to therapy and accounts for almost all cancer specific fluorescent probes that retain the biological profile of the parent deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and compound. This talk will present examples of how these probes are chemo-resistance in a wide range of malignancies, and thus its inhibition may prove able to provide information about bacterial resistance mechanisms by to be therapeutically useful. To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A assessing membrane permeability and efflux pump activity. We will expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and also show how they have helped to decipher differences in the mode MDA-MB-231 (MDA-MB-231-2A) cells. MCL-1 inhibition caused apoptosis of basal- of action of apparently similar antibiotics possessing strikingly different like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/ activity against resistant bacteria, and how they may also be useful in BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA- assessing antibiotic toxicity to human cells. MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL- 1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

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SYM-67-03 SYM-67-04 TARGETING MULTIFACTORIAL RESISTANCE DICHLOROACETATE AT CLINICALLY ACHIEVABLE MECHANISMS ASSOCIATED WITH MICROTUBULE CONCENTRATIONS CAN REDUCE pPDH AND PROTEINS IN CANCER CELLS REVERSE THE GLYCOLYTIC PHENOTYPE IN MULTIPLE MYELOMA CELLS Kavallaris M.1, 2, 3 1Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Tian D.D. and Blackburn A.C. Sydney. 2ARC Centre of Excellence in Convergent Bio-Nano ACRF Dept Cancer Biology and Therapeutics, John Curtin School Science and Technology, UNSW Sydney. 3Australian Centre for of Medical Research, Australian National University, Acton, ACT, NanoMedicine, UNSW Sydney. Australia.

Cancer is one of the major causes of morbidity and mortality in the Background: Multiple myeloma (MM) is a B-cell malignancy world. A major cause of cancer treatment failure is poor access of with a glycolytic phenotype. Dichloroacetate (DCA) is a pyruvate chemotherapeutic drugs to tumour sites and resistance to therapy. dehydrogenase (PDH) kinase (PDK) inhibitor that can reverse the Microtubules and other mitotic proteins are often deregulated in cancer glycolytic phenotype. In vitro, DCA (≥10mM) inhibited MM proliferation and are responsible for diverse effects including drug resistance, tumour and induced apoptosis, but these concentrations are above those formation and metastasis. In particular, aberrant expression of specific clinically achievable and are likely acting off-target. We examined microtubule proteins in epithelial cancers is a poor prognostic indicator whether DCA at clinically achievable levels can act on target, inhibit MM and is associated with clinical resistance. Recent data on understanding cell growth and enhance effects of other drugs. Methods: The response the mechanisms mediating these multifactorial resistance effects and of five human MM cell lines to DCA alone or combined with nutrient strategies to overcome resistance will be presented. deprivation, hypoxia, 2-deoxyglucose, dexamethasone or orlistat was measured using neutral red viability assay. pPDH/tPDH was measured by western blotting. PDK expression was measured by rt-PCR and western blotting. Cell proliferation (CFSE), cell cycle (BrdU/PI) and apoptosis (Annexin V/7AAD) were measured by FACS. Results: DCA (1-5mM) decreased cell proliferation without increasing apoptosis or inducing cell cycle arrest. This was accompanied by decreased pPDH and lactate which were dependent on the presence of glucose. DCA treatment as low as 0.1-0.3mM decreased pPDH/tPDH within 5-24hr. These concentrations are based on DCA serum peak and trough levels in MM patients in our DiCAM clinical trial (1). DCA had a greater effect on growth inhibition under hypoxia, corresponding with induction of the target PDKs. DCA combined with dexamethasone (inhibits glycolysis) or orlistat (fatty acid synthase inhibitor) had additive effects in reducing viable cells, and were accompanied by greater changes in metabolism. Conclusion: DCA can act on target in MM cells at clinically achievable levels and may be a useful therapy in combination with other low toxicity metabolism modifying drugs.

SYM-67-05 SYM-68-01 FUNCTIONAL ANALYSIS OF A CAMPYLOBACTER THE ROLES OF H3.3 AND ATRX ABNORMALITIES JEJUNI NUTRIENT TRANSPORT PROTEIN USING IN DRIVING ALTERNATIVE LENGTHENING OF PROTEOMICS AND METABOLOMICS TELOMERES AND CHROMATIN ABERRATIONS IN CANCERS Man L.1, 2, Cain J.A.1, 2, Solis N.2, Klare W.1, 2, Niewold P.1, 2, 3, Sumer-Bayraktar Z.1, 2 and Cordwell S.J.1, 2, 3 Udugama M., Voon H.P., Hii L. and Wong L.H. 1School of Life and Environmental Sciences, The University of Biomedicine Discovery Institute, Monash University. Sydney, Sydney, NSW, Australia. 2Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. 3Discipline of One striking finding in the area of cancer has been the Pathology, School of Medical Sciences, The University of Sydney, identification of mutated histone genes (oncohistones) in paediatric Sydney, NSW, Australia. glioblastomas (pGBMs). Two H3.3 mutations are found. The first mutation replaces lysine 27 with a methionine. The second one replaces Campylobacter jejuni is the leading cause of bacterial gastroenteritis glycine 34 by an arginine (G34R). H3.3G34R mutations always overlap in the developed world. Infection occurs predominantly through the with ATRX and p53 mutations, and these pGBMs are activated in the consumption of undercooked/poorly prepared commercial chicken Alternative Lengthening of Telomeres (ALT) pathway, suggesting that products. Importantly, C. jejuni exists mainly as a commensal organism H3.3G34R/ATRX/p53 mutations cooperate to drive ALT and GBM within the intestines of chickens, but is pathogenic in humans. While the development. We have created cell models carrying H3.3G34R/ATRX/ exact mechanism of this difference in pathogenicity is unknown, factors p53 mutations to recapitulate the initial driver epigenetic events that such as motility and nutrient uptake are thought to be significant. C. promote ALT. These mutants are compromised in heterochromatin jejuni is generally considered assaccharolytic and primarily utilizes formation at the telomeres, accompanied with telomere damage (and amino and organic acids as carbon sources. We conducted proteomic aberrant telomere protein binding) and formation of aberrant PML analysis of C. jejuni to identify proteins associated with growth in nuclear bodies (PML-NBs) that are irregular in size, shape and number. deoxycholate, mimicking gut bile salts encountered during human KDM4 proteins are demethylases or epigenetic erasers that remove infection. We quantified 1561 proteins, and the most significantly the methyl group from trimethylated H3K9 and H3K36. We find that the induced protein was the product of the cj0025 gene, which has been H3.3G34R inhibits KDM4 catalytic function of and drives its aberrant previously annotated as a ‘putative C4-dicarboxylate transport protein’. distribution. As a result, it induces aberrant histone methylation pattern To determine the function of Cj0025, metabolomic profiles of media and affects telomere chromatin maintenance. IDH1 (which is also inoculated with C. jejuni wild-type or a Δcj0025 deletion strain were mutated in GBM) is also known to inhibit KDM4 function. We propose compared. Our results showed that the mutant was not impaired in the KDM4 chromatin network as a major driver that promotes ALT and the uptake of amino acids or organic acids, but was for cystine, a cysteine oncogenic process in GBMs. In the H3.3G34R/ATRX mutants, we also dimer. This was reflected both in the mutant proteome, which showed a detect DNA copy loss at ATRX-bound ribosomal repeats, accompanied downregulation of sulfur-related proteins, and a growth-inhibition assay with severely reduced rRNA synthesis. ALT positive human sarcoma utilising a toxic analogue of cystine. tumour samples are substantially reduced in rDNA copy. Moreover, ALT cancer cells show increased sensitivity to RNA polymerase I transcription inhibitor, suggesting the therapeutic potential of targeting Pol I transcription in ALT cancers. Our study provides insights into chromatin defects associated with ATRX/H3.3 mutations and development of ALT cancers.

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SYM-68-02 SYM-68-03 INVESTIGATING THE ROLE OF DE NOVO DNA SINGLE-CELL EPIGENOMICS FOR ANALYSIS OF METHYLATION IN REGULATING LIVER METABOLISM HETEROGENEOUS AND RARE CELL POPULATIONS

Youngson N.A.1, Prates K.V.1, Yao S.1, McRae A.F.2 and Morris M.J.1 Lee H.J. 1School of Medical Sciences, UNSW Sydney. 2Institute for Molecular The University of Newcastle. Bioscience, The University of Queensland. Single-cell sequencing technologies are revolutionising our The de novo DNA methyltransferases are required for development as understanding of heterogeneous cell populations in development their constitutive deletion in mice results in lethality either in the late and disease. Incorporation of epigenetic information with single-cell embryonic period (Dnmt3a) or in the first weeks after birth Dnmt3b( ). transcriptomic and genomic analyses will provide valuable insights into Beyond development, post-natal DNA methylation changes have been the molecular mechanisms of gene regulation (Clark, Lee, Smallwood identified that associate with disease states or particular diets. However, et al. 2016 Genome Biol 17:72). DNA methylation occurs on cytosine despite hundreds of studies that associate de novo methylation residues of CpG dinucleotides in mammalian cells. This epigenetic changes in mammals, a functional requirement for those DNA modification is dynamically regulated during development and is methylation changes is still not proven. Uncovering the true role of globally dysregulated in many cancer types. We developed single-cell DNA methylation in disease is vital for understanding pathogenesis, bisulphite sequencing (scBS-seq) (Smallwood, Lee, et al. 2014 Nat and in directing the development of therapies. If DNA methylation Methods 11:817-202), which provides quantitative, single-nucleotide is just a consequence of, or a minor player in, disease associated information on DNA methylation for up to 50% of cytosines across transcriptional change, then future epigenetic studies in this area the genome. Extending on this work, we recently reported parallel should focus on other molecules such as histone tail modifications. To single-cell methylome and transcriptome sequencing (scM&T-seq) investigate this issue we have generated mice that are homozygous (Angermueller, Clark, Lee, Macaulay, et al. 2016 Nat Methods 13:229- mutants for both Dnmt3a and Dnmt3b in post-natal liver. These mice 32), which allows both DNA methylation and gene expression to be are viable and fertile. Here we present data on the requirement for assayed from the same single cell. To illustrate the power of integrated liver de novo methylation for systemic glucose metabolism, in control single-cell multi-omics, biological insights gained from scM&T-seq and obese mice. Our model also sheds light on how in vivo inhibition analysis of mouse embryonic stem cells will be presented. of de novo DNA methylation in liver affects other epigenetic regulatory systems, and systematic physiology and behaviour.

SYM-68-04 SYM-68-05 miRNA:miRNA INTERACTIONS IN HEAD AND NECK UNLOCKING FEMALE ES CELLS FOR RESEARCH SQUAMOUS CELL CARCINOMA Keniry A.J., Jansz N. and Blewitt M.E. Hill M.1 and Tran N.1, 2 Walter and Eliza Hall Institute. 1School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney, NSW, Australia. 2The Sydney Head Female mouse embryonic stem cells (mESCs) are significantly harder to and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince grow and expand compared to their male counterparts. This discrepancy Alfred Hospital, NSW, Australia. has resulted in the majority of mESC studies being performed in male cells, and therefore our appreciation of characteristics particular to Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth female mESCs is severely lacking. This is worrying as in addition most common form of cancer. It is commonly caused by smoking, to being harder to maintain, female mESCs have also been found to alcohol, and Human Papilloma Virus (HPV). Due to its recent increase be less karyotypically and epigenetically stable and display slower in incidence worldwide, it is important to investigate the molecular differentiation kinetics. Clearly female mESCs are very different to their mechanisms responsible for HNSCC. MicroRNAs (miRNAs) control male equivalents and demand significantly more research attention. To messenger RNA (mRNA) and have an important role in disease achieve this however, female mESCs must become more experimentally development. Normally, miRNAs perform their regulatory function by tractable through development of robust methods to both derive and binding to the 3’ untranslated region (UTR) of mRNA. However, several maintain them. It is not fully understood what makes female mESCs studies have demonstrated that miRNAs are involved in the regulation less robust in culture, however there is some evidence that the second of other miRNAs, known as a miRNA:miRNA interaction. This mode X chromosome is to blame as female XO mESCs behave similarly to of regulation is unexplored in HNSCC. Our lab has identified miR-21 male XY cells. Interestingly, female mESCs are the only female cell type and miR-499 as important regulators in HNSCC. The overexpression that has not undergone the process of X chromosome inactivation, and of miR-21 in HNSCC cells reduced the levels of mature miR-499. thus are the only cells to express from both X chromosomes, suggesting However, miR-499 did not alter miR-21 levels. To determine if miR-21 that it may be the activity of the second X, as opposed to the second overexpression has any global effect, we measured the expression chromosome per se, that makes female mESCs less amenable to culture. of 750 miRNAs. From this analysis, both miR-21 and miR-499 In order to study female mESCs we have tagged each X chromosome overexpression could regulate specific miRNAs such as miR-148, miR- with a different fluorescent reporter, such that we can monitor activity 100, and miR-31. This data was then used to create an interactome to from each X rapidly and accurately by FACs. The system allows for rapid visualise the identi fi ed miR N A :miR N A interac tions and their c onnec tions monitoring of cell fitness, karyotype and X inactivation status and has to genes. Furthermore, miR-21 overexpression dysregulated the RNA enabled us to establish robust methods for the derivation, culture and levels of the biogenesis components, Ago2, Ago3, and Dicer. This has manipulation of female mESCs. We have also experimentally proven implications on mature miRNA levels, and may affect the regulation the utility of these cells for the study of pluripotency, differentiation, of HNSCC related genes. In summary, our study provides the first induced pluripotency and X inactivation. Moreover, we have used the characterisation of miRNA:miRNA interactions in HNSCC cells, and cells to perform screens for epigenetic regulators of X inactivation. demonstrates that miR-21 can alter the levels of specific miRNAs and These screens have revealed genes from completely unexpected affect expression of the biogenesis machinery. pathways and suggest unappreciated mechanisms are required for X chromosome inactivation and indeed gene silencing more broadly. We are now moving on to validating and characterising these discoveries; first in XCI and then in other epigenetic processes.

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SYM-69-01 SYM-69-02 DEVELOPING THE NEXT-GEN THERAPEUTIC GERMINAL CENTER ANTIBODY MUTATION MONOCLONAL ANTIBODIES: NEW INSIGHTS BY TRAJECTORIES ARE DETERMINED BY RAPID SELF/ TARGETING AND MANIPULATING HUMAN FCR: FOREIGN DISCRIMINATION ANTIBODY DEPENDANT FUNCTIONS Burnett D.L., Langley D.B., Schofield P., Hermes J., Chan T.D., Hogarth P.M.1, 2, 3, Chenoweth A.M.1, Wines B.D.1, Trist H.M.1 and Jackson J., Bourne K., Brink R., Christ D. and Goodnow C. Esparon S.E.1 Garvan Institute of Medical Research. 1Immune Therapies Group, Burnet Institute, Melbourne. VIC. 2Clinical Pathology, University of Melbourne, Parkville VIC. 3Immunology and Antibodies have exquisite specificity to differentiate foreign antigens Pathology Monash University, Melbourne. that mimic “self”, but it remains unclear how such specificity is acquired. We generated B-cells displaying an antibody that cross-reacts with two The potency of many therapeutic antibodies, particularly in cancer and related protein antigens expressed on self versus foreign cells. B-cell immune agonism, requires antibody interaction with human Fc receptors. anergy was imposed by self antigen but reversed upon challenge with The FcR are specific, cell surface receptors for immunoglobulins high-density foreign antigen, leading to germinal center recruitment which provide a potent, system of activation and regulation of cell- and antibody gene hypermutation. Single-cell analysis revealed rapid based effector responses used by therapeutic antibodies. These selection for mutations that decrease self affinity and slower selection Fc-dependant reposes include antibody dependent cell mediated for epistatic mutations that specifically increase foreign affinity. cytotoxicity, phagocytosis, potent degranulation and mediator release. Crystal structures revealed the mutations exploited subtle topological In addition to therapeutic potency, the Fc:FcR interaction of antibodies differences to achieve 5,000-fold preferential binding to foreign targets or Fc fusions can also induce unwanted adverse reactions. Manipulation over self epitopes. Strikingly, resolution of antigenic mimicry drove the of the antibody Fc will provide the next generation therapeutic mabs optimal affinity maturation trajectory, highlighting the value of retaining and Fc fusions with more potent and specific actions i.e. cell or virus self-reactive clones as substrates for protective antibody responses. killing in anti-cancer mAbs or inhibitory anti-inflammatory modulation in autoimmunity and allergy or the ablation of FcR-dependant function to avoid adverse effects. Our therapeutic biologicals program uses our pioneering studies of antibody and Fc receptor structure and function of human, and non-human primates, as the basis for the engineering of next generation therapeutic antibodies for profoundly altered function and specificity. This will include our work on (i) Fc receptors forms and roles in antibody effector function. (ii)Manipulation of mAbs and Fc receptors for antibody therapy (iii)Novel methods of evaluation of antibody:FcR effector functions in immune therapy. (iv) Challenges in using non-human primates as models of human antibody function. WWW http://www.burnet.edu.au/staff_members/182_mark_hogarth.

SYM-69-03 SYM-69-04 INTERROGATING HUMAN B CELL IMMUNITY TO PATHOGEN SENSING BY THE INTRACELLULAR INFORM UNIVERSAL INFLUENZA VACCINE DESIGN ANTIBODY RECEPTOR, TRIM21, IS REGULATED BY B-BOX AUTOINHIBITION AND RING Wheatley A.K. PHOSPHORYLATION Department of Microbiology and Immunology, University of Melbourne. Dickson C.F.1, 2, Fletcher A.J.1, Vaysburd M.1, Yang J.C.1, Mallery D.L.1, Mclaughlin S.H.1, Chin J.W.1, Neuhaus D.1 and James L.C.1 Influenza inflicts significant global mortality and morbidity that can be 1MRC Laboratory of Molecular Biology, Cambridge, UK. 2SMS, School combated by effective immunisation. However, the protective efficacy of Medical Sciences, UNSW, Australia. of current vaccines is limited by the variability and rapid mutagenesis of circulating strains. There is tremendous interest in developing universal TRIM21 is a cytosolic antibody receptor that performs pathogen influenza vaccines for life-long protection. Here, we use recombinant sensing and antibody-dependant intracellular neutralisation of viruses. analogues of the viral hemagglutinin protein and flow cytometry to Both signalling and effector functions require E3 ubiquitin ligase interrogate human B cell and antibody responses to influenza. Our activity, which is provided by an N-terminal RING domain. TRIM21 approach enables the enumeration and phenotypic characterisation induces a potent antiviral immune response, however, it does not of B cell responses to seasonal influenza vaccines. In addition, constitutively signal, suggesting that the enzymatic activity of the RING we can pan the B cell repertoire for rare specificities allowing near is tightly regulated. We have used structural studies together with in universal influenza recognition and in some cases protection. Theses vitro ubiquitination assays to show that a domain of unknown function, approaches allow the identification of potentially protective epitopes the B-box domain, represses TRIM21 activity by competing with E2 and may inform the design of next-generation influenza vaccines. In ubiquitin conjugating enzymes for binding to the RING. We further addition, we can recover human monoclonal antibodies with potential demonstrated that cellular TRIM21 is phosphorylated by IKKβ and prophylactic, therapeutic, or diagnostic applications for eventual clinical TBK1 at serine 80, located at the RING-B-box interface. By introducing use. an phosphomimetic or a phosphoserine at position 80 in a recombinant RING-B-box protein we showed that phosphorylation of the interface relieves B-box mediated autoinhibition of the RING by promoting E2 binding and restoring enzymatic activity to the uninhibited state. Cells expressing TRIM21 carrying an S80E mutation constitutively activate NF-κB and produce a more robust cytokine response upon infection with DNA or RNA viruses, while an S80A mutation attenuated cytokine production. In contrast, neutralisation activity was unaffected by mutation of S80, consistent with a different threshold for these two activities. This work identifies several layers of regulation that fine tune the signalling activity of TRIM21 and highlights the importance of controlling inflammation, which unchecked has the potential to cause tissue damage and autoimmune disease.

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