Postdoc Methods Symposium program booklet...Methods Symposium Melbourne Program Davis Auditorium,...

1st Postdoctoral Methods Symposium Melbourne

ProgramDavis Auditorium, Walter and Eliza Hall Institute

Thursday 29 October 2015 8:30am – 5:10pm

Our research community:

A joint venture between the University of Melbourne and the Royal Melbourne Hospital

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1st Postdoctoral Methods Symposium Melbourne 1

WelcomeWe acknowledge the traditional owners and custodians of the land on which we meet today, the Wurundjeri people of the Kulin nation, and pay our respects to their elders past and present and the elders from other communities who may be here at this symposium.Welcome to our 1st Postdoctoral Methods Symposium in Melbourne! We have a high quality program that brings together postdocs to discuss the latest methods across a broad range of disciplines and institutes and universities across Melbourne. Postdocs represent an important fabric of our scientific research community. It has become increasingly important for postdocs to continuously exchange ideas, be updated with new trends in biomedical research and lead important scientific discoveries. The goal for this symposium is to strengthen the Postdoctoral research community by providing a forum for postdocs across Melbourne to meet, liaise and build collaborative links. And along the way, discover some new and interesting methods to broaden the repertoire of our scientific skills. We hope you find this symposium stimulating and we encourage you to connect with fellow postdocs and participate actively in discussions!

Nga Lam 2015 WEHI PDA president

2 1st Postdoctoral Methods Symposium Melbourne

Meeting support

Thank you…

To our sponsors for their generous support of this meeting:

To the WEHI PDA Methods Symposium Organising Committee:

Vanessa Bryant

Greg Ebert

Najoua Lalaoui

Rob Ninnis

Kim Pham

Alan Rubin


Program8.30am – 8.55am REGISTRATION

8.55am – 9.00am WELCOME

Greg Ebert & Vanessa Bryant, Methods Symposium Organising Committee

9.00am – 10.30am SESSION ONE: Animal models and Genetics

9.00am – 9.15 amClare Fedele: Using patient-derived xenografting to reveal clinically-relevant features of human melanomaPeter MacCallum Cancer Centre

9.15 am – 9.30 amNicholas Clemons: Improving the success rate of difficult to grow patient derived xenografts by intramuscular implantation Peter MacCallum Cancer Centre

9.30am – 9.45amAsolina Braun: Intralymphatic injection as a novel method for the delivery of cells and solutes in the mouse modelPeter Doherty Institute

9.45 am – 10.00 amSimona Carbone: Electrophysiological property changes of neurons after chemotherapyMonash Institute of Pharmaceutical Sciences

10.00 am – 10.15 amAndrew Kueh: The application of CRISPR/Cas9 technology in the production of gene-targeted mice Walter and Eliza Hall Institute

10.15 am – 10.30 amGenevieve Pepin: Using cGAS-STING activation to assess recombinase-mediated DNA damageHudson Institute of Medical Research

Chairs: Nikola Baschuk (LIMS), Ryan Cross (PeterMac)

10.30 am – 11.00am MORNING TEA IN TAPESTRY LOUNGE


11.00am – 1.00pm SESSION TWO: Bioinformatics, Sequencing and Flow Cytometry

11.00am – 11.15amBhupinder Pal:Microfluidic-based single-cell capture and RNAseq of mammary epithelium Walter and Eliza Hall Institute

11.15am – 11.30amDaniela Zalcenstein: High throughput single-cell RNA library preparation for Illumina-sequencing platformsWalter and Eliza Hall Institute

11.30am – 11.45amGoknur Giner: Cooking up a pathway analysis with ROAST and FryWalter and Eliza Hall Institute

11.45am – 12.00pmJarny Choi: Setting up a data analysis environment with python and its friendsWalter and Eliza Hall Institute

12.00pm – 12.15pmSaskia Freytag: brain-coX – an interactive web-tool for gene prioritisation and moreWalter and Eliza Hall Institute

12.15pm – 12.30pmTimothy Hinks: Application of topological data analysis and Bayesian belief networks to high dimensional clinical and immunological data for multidimensional phenotyping in human asthmaPeter Doherty Institute

12.30pm – 12.45pmIvan Poon: Monitoring the progression of cell death and disassembly of dying cells by flow cytometryLaTrobe University

12.45pm – 1.00pmPrajakta Gosavi: Combined application of light microscopy and flow cytometry to determine protein localization and organelle disruption in cell populationsBio21 Institute

Chairs: Christopher Flensburg (WEHI), Erika Duan (LIMS)

1.00pm – 2.00pm LUNCH IN TAPESTRY LOUNGE


2.00pm – 3.15 pm SESSION THREE: Imaging

2.00pm – 2.15pmJoachim Kloehn: Heavy water (2H2O) as a universal metabolic tracer to measure cell division, the physiological state and metabolism of cell populations in vivo – applied to characterise Leishmania parasites in infected mammalian tissuesBio21 Institute

2.15pm – 2.30pmDominic Hare: Imaging by laser ablation triple-quadruple inductively coupled plasma-mass spectrometryFlorey Institute

2.30pm – 2.45pmPaul McMillan: Super-Resolution SolutionBio21 Institute

2.45pm – 3.00pmJennifer Walz: EEG Event Variability to Investigate Spatio-temporal Dynamics of fMRI Activations Florey Institute

3.00pm – 3.15pmAndrey Kan: Compensating acquisition effects in time lapse fluorescence microscopyWalter and Eliza Hall Institute

Chairs: Michele Veldsman (Florey), Gosavi Prajakta (Bio21)

3.15pm – 3.45 pm AFTERNOON TEA IN TAPESTRY LOUNGE


3.45pm – 5.10 pm SESSION FOUR: Proteomics

3.45pm – 4.00pmMichael Dagley: Metabolomics-based flux analysis using stable-isotope labellingBio21 Institute

4.00pm – 4.15pmLaura Edgington-Mitchell: Activity-dependent profiling of proteases in inflammatory diseasesMonash Institute of Pharmaceutical sciences

4.15 pm-4.30 pmGiuseppe Infusini: Intact protein profiling: On the hunt for wound healing factors.Walter and Eliza Hall Institute

4.30 pm-4.45 pmEmma Petrie: How to turn that gene of interest into a protein structure.Walter and Eliza Hall Institute

4.45 pm-5.00 pmJarrod Sandow: Characterising novel therapeutics using quantitative proteomicsWalter and Eliza Hall Institute

Chairs: Thomas Nebl (WEHI), Marta Encisco (Latrobe)

5.00pm- 5.10 pm Best Presentation Awards and CLOSING COMMENTS

Vanessa Bryant & Greg Ebert, Methods Symposium Organising Committee

5.30pm POSTDOC SOCIAL DRINKS AND NIBBLES at NAUGHTON’S HOTEL


SESSION ONE: ANIMAL MODELS & GENETICS

Using patient-derived xenografting to reveal clinically-relevant features of human melanomaFedele CG1,2,3 Samantha Boyle1 and Shackleton MJ1,2,3

1Peter MacCallum Cancer Centre, Melbourne Australia; 2Dept Pathology; 3Sir Peter MacCallum Dept of Oncology, University of Melbourne Australia [email protected] is the most deadly form of skin cancer. Advanced melanomas exhibit a propensity for rapid evolution, associated with widespread metastasis and acquisition of therapy‐resistance. To study fundamental features of human melanomas while conserving biological and clinical relevance we use patient derived xenografting (PDX) (Fig 1). Fresh viable human tissue from stage 1/2 primary cutaneous or stage 3/4 metastatic melanomas are obtained following surgery, and dissociated to single cell suspensions. Melanoma cells are FACS‐purified and injected into immuno‐compromised NODSCIDγIL2-/- (NSG) mice. In vivo biologies of resultant tumours, including numbers of tumorigenic cells, growth and metastatic potentials and therapy‐response can then be determined. By comparing the biologies of PDX tumours we can attempt to identify mechanisms driving specific biological traits. Further, through serial passaging of patient tumour cells in mice we can study in real time the potential of melanoma cells for spontaneous evolution, both in response to therapy and also in therapy‐naïve contexts.We applied this approach to study the significance of pigmentation in melanomas. We have found that while most early stage melanomas in patients produce the pigment, melanin, as tumours become more advanced melanin production is frequently suppressed. Significantly, nearly all pigmented patient melanomas established as PDX spontaneously suppress pigment production within 1‐3 passages in mice, associated with increased aggressiveness, mimicking clinical progression in patients (Fig 1). Using the PDX system we reveal that loss of pigment coincides with spontaneous clonal evolution. Transcription and methylation profiling of pigmented versus evolved non‐pigmented sub‐clones has uncovered potential mechanisms driving the selection of the non‐pigmented phenotype. Thus, spontaneous evolution converging on the pigment pathway is associated with disease progression in a high proportion of melanomas and may be a novel target pathway for therapeutic intervention. These studies demonstrate that in vivo PDX modelling can reveal clinically‐relevant features of human cancers above traditional high‐passage cell lines.

Fig 1. Patient-derived xenografting (PDX) reveals clinically-relevant pigment loss in human melanomas, associated with aggressive biology.


Improving success rate of difficult to grow patient derived xenografts by intramuscular implantationNicholas ClemonsCancer Biology and Surgical Oncology Laboratory, Peter MacCallum Cancer [email protected] for patients with oesophageal cancer (OC) are extremely poor with overall 5-year survival rates close to 15%, with many patients surviving less than a year after diagnosis. Standard treatment for this disease has changed little over the past few decades and there are few options for those who fail first line treatment. It is widely agreed that alternative treatment strategies are urgently needed to improve patient outcomes. However, progress is being hampered by a lack of suitable preclinical models with which to study the disease and test new therapies. Patient derived xenografts (PDX) are promising pre-clinical models for a number of cancers that are increasingly being used for drug target validation and biomarker identification and testing. However, the success rate in generating OC PDX is low, with only around 1 in 3 tumours reported to establish a PDX. We have developed a technique for improved success rate in establishing PDX from OC that involves implantation into a muscular pocked in the dorsum of mice. Using this approach we have successfully established PDX from ~80% (19/24) of tumours we have implanted, compared with 1/6 using the standard subcutaneous approach. Further, we have utilised this technique to grow other difficult tumour types including Merkel cell tumours, anal SCC, head and neck SCC and gastric adenocarcinoma. Moreover, we have demonstrated that this technique can be used to maintain non-neoplastic Barrett’s metaplasia, and is the only in vivo laboratory model of human Barrett’s metaplasia. Importantly, we have also identified that EBV-driven human B-cell lymphoma are a common occurrence following transplantation of human cancer tissues into immunocompromised mice, and we believe that this is an under-reported issue in the literature of PDX models.

Intralymphatic injection as a novel method for the delivery of cells and solutes in the mouse modelAsolina Braun1, Tim Worbs2, Reinhold Förster2 1Department of Microbiology and Immunology, Peter Doherty Institute, University of Melbourne 2Institute of Immunology, Hannover Medical School, Hannover, [email protected] immune system relies on interactions between highly motile cell populations constantly patrolling the body for the presence of pathogens and mutated cells that need to be controlled. Lymphocytes constantly survey lymph nodes (LN) for the presence of foreign antigen by immigrating from the blood and exiting through efferent lymphatics. Additionally, lymphocytes and other immune cells such as dendritic cells (DC) can enter LN via the afferent lymphatics draining peripheral tissues. Immune cell entry into the LN from the circulation is well scrutinized on the molecular level. However our knowledge on how immune cells enter LN from the lymphatics is rudimentary. In order to be able to study this process, I have co-developed and utilized a novel method of the surgical application of cells via the afferent lymphatic vessel to the popliteal LN in the mouse. Briefly, the mouse is anesthetized and the skin on one of the calves is opened. Upon location and exposure of the lymphatics, a self-pulled glass capillary in conjunction with a microinjector is used to penetrate a collecting afferent lymphatic vessel and to deliver cells or solutes.Using this technique together with two-photon microscopy I could show that upon arrival via the afferent lymphatics DC and T cells use different routes to enter the LN. While DC preferentially immigrate via the interfollicular regions of the subcapsular sinus, the vast majority of naïve T cells enter the LN parenchyma from the medullary sinus.It has long been known that DC and T cells require the chemokine receptorCCR7 to enter LN from blood vessels. Having the new technique at hand, we could also show that CCR7 is equally as important for the correct positioning of DC and T cells in the paracortex when the cells arrive via the lymphatics.


Electrophysiological property changes of neurons after chemotherapy Simona E Carbone1, Kulmira Nurgali2 and Simon JH Brookes3.1Drug Discovery and Biology, Monash Institute of Pharmaceutical Sciences, Melbourne Australia; 2Enteric Neuropathy Laboratory, College of Health and Biomedicine, Victoria; University, Melbourne, Australia; 3Neurogastroenterolgy Laboratory, Discipline of Human Physiology, Flinders University, Adelaide [email protected]

Background: Our knowledge of the how the human enteric nervous system is greatly lacking. Most of our understanding on the electrophysiological properties of these neurons is inferred from studies on small laboratory animals. This is due to the limited number of electrophysiological studies of human enteric neurons (1) difficulties with dissection and poor visibility of enteric ganglia are all factors. Here, we have developed a method that can be readily used to identify recorded cells immediately after impalement and greatly facilitate recordings of human myenteric neurons in freshly dissected specimens of tissue. Methods: Specimens of human colon were obtained following colorectal resections for carcinoma. The mucosa, submucosa and circular muscles were removed to expose the myenteric plexus in wholemount preparations. Myenteric neurons were impaled with conventional microelectrodes containing 5% 5,6-carboxyfluorescein in 20mM Tris buffer and 1M KCl. Results: Carboxyfluorescein filled cells were identified as having Dogiel type I or type II morphology in situ. This labelling remained after fixation, in combination with immunohistochemical characterisation. Neurons were identified as having S or AH electrophysiological characteristics. Using this method we found the electrophysiological properties of neurons changed in specimens from patients that had received chemotherapy-treatment. Conclusions: This method allows electrophysiological characterisation with simultaneous identification of morphology. It is an invaluable technique that will aid in our understanding to the human enteric nervous system in health and disease.

1. Brookes SJ, Ewart WR, Wingate DL. Intracellular recordings from myenteric neurones in the human colon. J Physiol Lond)1987: 305-318.


The application of CRISPR/Cas9 technology in the production of gene-targeted mice Andrew J. Kueh, Lin Tai and Marco J. HeroldMolecular Genetics of Cancer Division, Walter and Eliza Hall Institute, Parkville, Victoria [email protected]

The Clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system has been demonstrated to have tremendous potential in gene-targeting applications. As part of the Melbourne Advanced Genome Editing Centre (MAGEC), based in WEHI, we have adapted the CRISPR/Cas9 system for almost all gene-targeting projects within the institute to produce gene-edited mice. Here, we present an overview of the optimization of CRISPR/Cas9 technology in our work flow, from the design of gene targeting strategies to the reliable genotyping of targeted mice on next-generation sequencing platforms.

Using cGAS-STING activation to assess recombinase-mediated DNA damageGeneviève Pépin, Jonathan Ferrand & Michael P. GantierCentre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria 3168, [email protected]

Gene-recombinase technologies such as Cre/loxP-mediated DNA recombination are important tools to study gene function, with potential side effects due to damaging activities on the DNA. Here we demonstrate that Cre-dependent DNA damage recruits the cytosolic cGAS-STING innate immune pathway. We show that measuring cGAS-STING activation is a very sensitive approach to exclude off-target phenotypes following enzymatic cleavage of nuclear DNA by Cre.


SESSION TWO: BIOINFORMATICS, SEQUENCING AND FLOW CYTOMETRY

Microfluidic-based single-cell capture and RNAseq of mammary epithelium Pal B., Chen Y., Vaillant F., Fu N.Y., Jamieson P., Liu K., Smyth G.K., Lindeman G.J. and Visvader J.E.Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Vic 3052 [email protected]

The mammary epithelium consists of functionally distinct basal and luminal cell populations including basal mammary stem cells (MaSCs) and luminal progenitors (LP) and mature cells (ML). We used microfluidic-based single-cell capture and RNA-seq library preparation method to study molecular heterogeneity and further delineate the cellular hierarchy in mouse mammary epithelium. This study identified novel cell subsets marked by unique gene signatures and cell-surface markers that were used for cell fractionation and isolation of new cell subsets for in vitro and in vivo studies.

High throughput single-cell RNA library preparation for Illumina-sequencing platformsDaniela Zalcenstein, Jaring Schreuder, Jerry Gao, Shian Su, Luyi Tian, Matt Richi, Shalin NaikMolecular Medicine Division, Walter & Eliza Hall [email protected]

High-throughput single cell RNA sequencing is not yet widely used, since commercially available kits and systems are still too expensive to allow for thousands of cells to be analyzed on a routine basis. We are setting up a single cell RNA-Seq platform, consisting of a “kit-free” sample preparation as well as an analysis pipeline to allow for high throughput at low cost processing of hundreds to thousands of single cells. The key feature of our sample preparation is early indexing and pooling of the samples, which dramatically reduces the cost of library preparation, since hundreds of cells can be simultaneously prepared in one single reaction. The pooled cells undergo linear amplification prior to library preparation to promote mRNA enrichment and ensure sufficient starting material to account for sample loss during cleanup procedures. Our protocol is based on the recently published CEL-Seq (Hashimshony et al., Cell Reports, 2012) and MARS-Seq (Jaitin et al., Science, 2014) protocols. We are developing a semi-automated approach to process FACS sorted single cells in 384 well-plates to ensure high reproducibility and fast processing times while working with sub-microliter volumes. Libraries will be optimized for sequencing on the NextSeq500 system ensuring high quality sequence data as well as fast turn-around times. In collaboration with Matt Richie’s group we are developing an analysis pipeline, which will incorporate sequencing run as well as data QC, demultiplexing of samples and transcriptome analysis. While we are currently still in the process of optimizing the procedure, we hope to share this platform with the WEHI community once it is fully operational. In addition, we are also setting-up a microfluidic based single cell RNA-Seq library preparation platform (Drop-Seq; Macosko et al., Cell, 2015) to even further increase throughput (10.000 cells per experiment) and profit from the enhanced reaction properties of microfluidic systems.


Cooking up a pathway analysis with ROAST and FryGoknur Giner1,2, Gordon K. Smyth1,3

1Walter and Eliza Hall Institute of Medical Research; 2Department of Medical Biology, University of Melbourne; 3 Department of Mathematics and Statistics, University of [email protected]

Gene set tests are often used in differential expression analyses to explore the behaviour of a group of related genes. This is useful for identifying large-scale co-regulation of genes belonging to the same biological process or molecular pathway. One of the most flexible and powerful gene set tests is the ROAST method in the limma package. ROAST uses residual space rotation as a sort of continuous version of sample permutation. Like permutation tests, it protects against false positives caused by correlations between genes in the set. Unlike permutation tests, it can be used with complex experimental design and with small numbers of replicates. It is the only gene set test method that is able to analyse complex “gene expression signatures” that incorporate information about both up and down regulated genes simultaneously.ROAST works well for individual expression signatures, but has limitations when applied to large collections of gene sets, such as the Broad Institute’s Molecular Signature Database with over 8000 gene sets. In particular, the p-value resolution is limited by the number of rotations that are done for each set. This makes it impossible to obtain very small p-values and hence to distinguish the top ranking pathways from a large collection. As with permutation tests, the p-values for each set may vary from run to run.This talk presents Fry, a very fast approximation to the complete ROAST method. Fry approximates the limiting p-value that would be obtained from performing a very large number of rotations with ROAST. Fry preserves most of the advantages of ROAST, but also provides high resolution exact p-values very quickly. In particular, it is able to distinguish the most significant sets in large collections and to yield statistically significant results after adjustment for multiple testing. This makes it an ideal tool for large-scale pathway analysis.

Setting up a data analysis environment with python and its friendsJarny Choi1, 2

1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. 2Department of Medical Biology, The University of Melbourne, Australia. [email protected]

There are many different ways to analyse scientific data, from the humble spreadsheet to writing advanced functions in R. In a typical biological research context, a set of computational tools for data analysis needs to:

• manage the data, including updates to the data and metadata such as sample and cell type information,

• run a suite of analysis methods, such as differential expression,• visualise some of the results using graphs and plots,• reproduce the analysis on demand, and• help to collaborate and share the data/analyses with colleagues.

In this talk, I will outline a set of tools which can be set up to address all these issues and more, providing a suitable set up for beginners and advanced users alike. While the central language of choice in this setup is python, it also uses a number of related tools such as R for full power. I will go through a list of key packages required for the setup and what they do using some examples. Topics covered include handling data matrices, plotting graphs, setting up version control and testing code.


brain-coX – an interactive web-tool for gene prioritisation and moreSaskia Freytag1, Karen Oliver2, Johann Gagnon-Bartsch3, Terence M. Speed3,4 and Melanie Bahlo1

1Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Parkville; 2Melbourne Brain Center, Austin Hospital, Heidelberg; 3Department of Statistics, University of California, Berkely; 4Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, [email protected]

Whole-exome sequencing is a valuable clinical tool for human disease. However, for many sporadic patients this approach yields hundreds of credible mutations that could be the cause of the patients’ disorder. Sifting through these mutations individually is a time-consuming and taxing process and often fails to establish a feasible list of follow-up candidates. Gene prioritisation methods promise to identify the most interesting mutations located in genes using computational methods, which are ideally applied to tissue-specific genomic data. Unfortunately, results from these methods are not usually readily available to clinicians, as the generation of such methods, and often their interpretation, requires trained bioinformaticians. We have developed an intuitive web-tool for gene prioritisation in neurological disorders: brain-coX.brain-coX incorporates and combines six large datasets on gene expression in the developing and ageing human brain. These datasets are adaptively cleaned of unwanted variation, maximising information for the disease of interest. In addition to gene prioritisation brain-coX’s functionality includes extensive network visualization options as well as interactive analysis tools to explore changes in the gene-gene network along brain development. In the future, we hope to extend brain-coX offering interactive tools that visualize differences between brain regions. Currently, our clinical collaborators and we are extensively testing brain-coX to prioritize candidate genes for patients with childhood epilepsies. We find that the use of brain-coX not only empowers clinicians and biologists with no programming or significant statistical knowledge, but also leads to better communication between collaborators. brain-coX is available via shiny.bioinf.wehi.edu.au/freytag.s


Application of topological data analysis and Bayesian belief networks to high dimensional clinical and immunological data for multidimensional phenotyping in human asthmaTimothy SC HinksPeter Doherty Institute [email protected]

Background: A major challenge in analysis of complex data sets is integrating information obtained from different platforms such as clinical data with transcriptomic data or immunological data obtained from flow cytometry studies. Furthermore in the emerging era of stratified medicine there is a need to use unbiased (unsupervised) statistical techniques to identify novel ‘endotypes’ (subgroups) from complex disease phenotype data.Methods: I performed two cross sectional studies of human asthma (n=84 and n=215) each generating over 100 different clinical, physiological, pathological and immunological parameters per subject including analysis of sputum, endobronchial biopsies and bronchoalveolar lavage using 9-colour flow cytometry, ELISA and multiplex-electrochemiluminescent assays. I applied the novel analytical approaches of topological data analysis (TDA, using Ayasdi ‘Core’ software) and Bayesian Network Analysis (BNA) (using Genie 20, Decision Systems Laboratory – freeware) to these complex datasets to identify multidimensional endotypes of asthma and to analyse complex interactions between parameters.Results: BNA and TDA identified six novel clinico-pathobiological clusters of underlying disease mechanisms, highlighting elevated mast cell mediators in severe, steroid insensitive asthma. Visually informative networks of complex interactions between parameters were generated giving novel insights into the mechanisms of this complex disease.Conclusion: I provide proof of concept for application of TDA and BNA to identification of multidimensional clinico-pathobiological endotypes, and the investigation of complex interacting networks, using software which does not require extensive biostatistical expertise and would be applicable to many clinical and immunological questions and datasets.

Topological data analysis in asthma Bayesian belief network in asthma


Monitoring the progression of cell death and disassembly of dying cells by flow cytometryLanzhou Jiang, Rochelle Tixeira, Sarah Caruso, Georgia K. Atkin-Smith, Amy A. Baxter, Stephanie Paone, Mark D. Hulett and Ivan K. H. Poon.Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. [email protected]

The use of annexin A5 and propidium iodide/7-aminoactinomycin D stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample, that is viable, apoptotic and necrotic cells. In order to study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we have established a new flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol utilises a combination of annexin A5 and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-step analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death.

Combined application of light microscopy and flow cytometry to determine protein localization and organelle disruption in cell populationsPrajakta Gosavi, Wei Hong Toh, Fiona Houghton and Paul A GleesonDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science & Biotechnology institute, University of Melbourne, 30 Flemington Road, Parkville, VIC 3010 [email protected]

Traditional approaches to determine protein localization include microscopy-based techniques such as light or electron microscopy. We have used confocal, 3D structured illumination and cryo-immuno-electron microscopy to analysis the Golgi apparatus in HeLa cells. In addition to these techniques, I will describe a novel flow cytometry based method developed by the laboratory that defines the cellular distribution of a protein as well as detects changes in organelle morphology. The technique, termed as pulse-shaped analysis (PulSA), measures pulse-width of a fluorescent signal by flow cytometry. We have utilized PulSA to monitor Golgi fragmentation arising from over expression of a trans-Golgi protein in HeLa cells. PulSA offers a rapid and quantitative high throughput method to monitor a number of cellular processes such as protein aggregation, cargo trafficking between intracellular components, effects of drug treatments on different organelles. References

1. Tracking protein aggregation and mislocalization in cells with flow cytometry. Ramdzan YM, Polling S, Chia CP, Ng IH, Ormsby AR, Croft NP, Purcell AW, Bogoyevitch MA, Ng DC, Gleeson PA, Hatters DM. Nat Methods. 2012 Mar 18;9(5):467-702. Application of flow cytometry to analyze intracellular location and trafficking of cargo in cell populations. Toh WH, Houghton FJ, Chia PZ, Ramdzan YM, Hatters DM, Gleeson PA. Methods Mol Biol. 2015;1270:227-38


SESSION THREE: IMAGING

Heavy water (2H2O) as a universal metabolic tracer to measure cell division, the physiological state and metabolism of cell populations in vivo – applied to characterise Leishmania parasites in infected mammalian tissuesJoachim Kloehn1, Berin A. Boughton2, Eleanor C. Saunders1, Sean O´Callaghan3, Malcolm J. McConville4

1Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville; 2Metabolomics Australia, School of Botany, The University of Melbourne, Parkville; 3Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville; 4Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville; Metabolomics Australia, Bio21 Institute of Molecular Science & Biotechnology, University of Melbourne, [email protected]

Stable isotope-labelled metabolites such as 13C-glucose are widely used to track the fate of metabolites and analyse changes in metabolism during disease. An alternative tracer is heavy water (2H2O). 2H2O is safe, inexpensive and easily administered in vitro or in vivo by providing 2H2O enriched water in the drinking water of laboratory animals or humans. In the presence of 2H2O, deuterium (2H) from 2H2O is incorporated into a wide range of metabolites during specific metabolic reactions/pathways, which in many cases are well defined (e.g.: deoxyribose 2H-enrichment occurs during: gluconeogenesis, the pentose phosphate pathway and the ribonucleotide reductase activity). 2H2O-labelling can hence be used to measure the turnover rate of macromolecules (DNA, RNA, protein, lipids) by determining the replacement rate of unlabelled macromolecules by newly synthesized macromolecules (composed of deuterated metabolites), as well as to measure the contribution of specific pathways such as gluconeogenesis or fatty acid synthesis. We employed this technique to measure the replication rate (DNA turnover), physiological state (RNA and protein turnover) and metabolism (fatty acid synthesis, central carbon metabolism) of the protozoan parasite Leishmania in vivo (in infected BALB/c mice). The infected animals were given 2H2O and parasites were isolated from macrophages within infected host tissues and isolated parasite macromolecules and metabolites were analysed by GC-MS. Our findings provide new insights into the biology of this medically important parasite. More recently, we combined 2H2O labelling with imaging mass spectrometry to measure the turnover of parasite specific lipids in infected tissues. The turnover of the measured surface lipids is closely linked to cell division and the analysis of 2H-enrichment in these lipids within sections of infected tissues allows for the analysis of parasite division at a spatial resolution of individual host cells, revealing the dynamics and heterogeneity of parasite populations within infected tissues. These methods are widely applicable.


[email protected]


The Super-Resolution SolutionPaul McMillan1,2, Matthew Dixon1, Oliver Looker1, Marion Hliscs1,3, Nick Katris3,4, Paul Monaghan5, Ross Waller3,4, Leann Tilley1.1Dept. Biochemistry & Molecular Biology, University of Melbourne 2Biological Optical Microscopy Platform, University of Melbourne 3School of Biosciences, University of Melbourne 4Department of Biochemistry, University of Cambridge 5Australian Animal Health Laboratory, [email protected]

Is blurry vision giving you a sore head on a Monday morning? It’s not the result of too many beers on Sunday night, but the effect of the limited resolution on your fluorescence microscope. When trying to see the sub‐cellular structures within samples, fluorescence microscopes just run out of resolution. However, help is at hand with the development of Super-Resolution Microscopy techniques that enable us to break through those old barriers. The development of these techniques resulted in three researchers being awarded Nobel Prize for Chemistry last year. We will present the application of super-resolution microscopy techniques to host-parasite/virus interactions in Malaria, Toxoplasma and Hendra infections. We will also discuss the application of this technology to other samples such as tissue sections.

The above image shows a malaria infected red blood cell imaged in a standard fluorescent microscope (left) and in the Super Res method 3D-SIM (right).


EEG Event Variability to Investigate Spatio-temporal Dynamics of fMRI ActivationsJennifer M. Walz1 and Graeme D. Jackson1,2,3

1The Florey Institute of Neuroscience and Mental Health, Austin Campus, Heidelberg, Victoria, Australia. 2Florey Department of Neuroscience and Mental Health, The University of Melbourne, Austin Campus, Heidelberg, Victoria, Australia. 3Department of Medicine, The University of Melbourne, Parkville, Victoria, [email protected]

Simultaneous electro-encephalography and functional magnetic resonance imaging (EEG-fMRI) is a useful tool for pre-surgical planning in epilepsy patients. The data are normally analysed using epileptiform event timing detected by the EEG to model the simultaneously-acquired fMRI data (Fig. 1). This is a proven method to determine the brain areas that are activated (on average) during these EEG events, and thus suggest the epileptic areas of the brain to surgically resect in order to eliminate the seizures. However, the EEG events vary in amplitude due to variability in the amount of brain tissue recruited, the speed at which the activity spreads within the brain, and the particular brain areas that are activated with each event. The standard analysis method cannot identify the spatio-temporal dynamics of the events.This work incorporates the single-event variability captured by the EEG, importantly at different latencies spanning the events, and uses this information in the fMRI model design (Fig. 2). It incorporates EEG information from all electrode sites instead of only determining timing from a limited set of electrodes. A machine learning method is applied to find the most informative projection of the multi-dimensional EEG data, done so by estimating the most discriminating features of the EEG events relative to the background activity, and single event variability along that projection is utilised. This is performed independently for narrow time windows of EEG data spanning the event duration. This method has the potential to identify not only the areas involved, but the temporal order in which they are activated. This could determine the specific region where the EEG events and seizures begin, to decrease the amount of brain tissue removed during surgery. This would increase the chance of successful surgical outcome while minimising neural deficits resulting from the surgery.


Compensating acquisition effects in time lapse fluorescence microscopyLiyan Liu, Andrey Kan, John Markham, Phil Hodgkin, and Chris LeckieWalter & Eliza Hall [email protected]

Time-lapse fluorescence microscopy coupled with cell tracking has proved to be an increasingly important tool in cell biology. However, the process of image acquisition leads to distortion of the original signal. Prominent acquisition effects include autofluorescence, cross-talk, uneven illumination, and noise induced by the acquisition electronics. In order to achieve a correct interpretation of observed fluorescence levels, these effects need to be addressed. Here we focus on two of the listed challenges, namely, correcting the effects of cross-talk and uneven illumination.Various spectral unmixing methods have been adopted to solve the cross-talk problem. Evaluating the quality of unmixing usually requires reference objects: sets of pixels that are single-positive for one of the channels. However, reference objects are not always readily available, particularly in experiments that involve multi-colour fluorescence reporters. We address the challenge of missing reference objects by proposing a new approach in which the quality of unmixing is judged using properties of reconstructed single cell fluorescence time courses. We make use of the prior knowledge available for given fluorescence reporters to deduce the expected properties for the time courses, and therefore we can assess the quality of unmixing by comparing reconstructed and expected signals.Moreover, uneven illumination results in inaccurate estimates of the single-cell features such as average and total intensity. Numerous shading correction methods have been proposed, and in order to compare the performance of the methods, many quantitative measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. We address this limitation by generating synthetic, but realistic, set of images and assessing efficiency of the performance measures. We identify the most suitable measure, and propose a new algorithm that performs illumination correction by optimising for this measure.


SESSION FOUR: PROTEOMICS

Metabolomics-based flux analysis using stable-isotope labellingMichael J. Dagley and Malcolm J. McConville.Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville, Victoria, 3010, [email protected]

Metabolomics is increasingly being used to understand fundamental aspects of cell development, disease and modes of drug action in fields of research as diverse as cancer, diabetes, immunology and microbiology. While most metabolomics studies have focussed on measuring the relative or absolute changes in the metabolite levels, there is increasing interest in combining profiling methods with stable-isotope labelling approaches to delineate metabolic networks and fluxes. Stable-isotope labelling approaches can provide precise information on the fluxes through parallel and potentially redundant pathways, and greatly improve the interpretative power of static profiling data. Using this approach, differences in the rate and extent of labelling of major metabolic pathways can reveal perturbations, which would not be apparent through the measurement of metabolite abundance or other ‘-omics’ approaches. Here I will describe the use of stable-isotope labelled metabolic tracers for the analysis of polar metabolites (glycolytic intermediates, pentose phosphate pathway intermediates and amino acids) by gas chromatography-mass spectrometry to investigate parasite metabolism.

Activity-dependent profiling of proteases in inflammatory diseasesLaura Edgington-Mitchell1, Matthew Bogyo2 & Nigel W. Bunnett1

1Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia; 2Stanford University School of Medicine, California, [email protected]

Proteases are enzymes whose primary function is to cleave peptide bonds of protein substrates. In addition to controlling protein homeostasis, proteases also contribute to numerous physiological processes, including hormone regulation, antigen presentation, matrix degradation, etc., and dysregulated protease activity is a hallmark of many diseases. Proteases are synthesized as inactive zymogens, and once active, they are also subject to regulation by endogenous inhibitors. In order to study the function of proteases, tools that can directly assess their activity levels are necessary. Traditional tools to study proteins, such as antibodies for western blotting or immunohistochemistry, survey total protein levels and are unable to report on activity. To overcome this caveat, we have developed a series of fluorescently quenched activity-based probes (ABPs) that bind to proteases in an activity-dependent manner. ABPs are small molecules containing a reactive warhead that binds covalently and irreversibly to the nucleophilic active site of active proteases. Upon binding, a quenching group is released, allowing for detection of light emitted from a fluorophore. Probe fluorescence can then be detected by whole animal imaging, ex vivo imaging of tissues, fluorescence microscopy, and flow cytometry. Since probe binding is covalent, the identity of the proteases that produce the fluorescence can be subsequently verified by fluorescent SDS-PAGE or mass spectrometry analyses. This is a unique feature of our probes that is not possible with commercially available fluorogenic protease probes.Our current efforts are focused on applying quenched activity-based probes to study the activity of cysteine and serine proteases in mouse models of inflammation, including ulcerative colitis, Crohn’s Disease, and pancreatitis. We have identified several proteases whose activity is increased in macrophages of inflamed tissues. In combination with specific protease inhibitors, we are using ABPs to dissect the contribution of these proteases to inflammation, to assess their potential as therapeutic targets, and to corroborate these findings in human tissues.


Intact protein profiling: On the hunt for wound healing factors.Guiseppe InfusiniWalter and Eliza Hall [email protected]

Proteoform profiling, in which intact proteins are characterized and quantified, is the only technique that can overcome the inherent limitations of bottom-up proteomics. The ultimate goal in proteomics is to map all of the proteins expressed in their native state, providing quantitative information on gene-level variability, polymorphism, splicing variation, and post-translational modification. This will undoubtedly be a powerful tool to unveil new mechanisms in biology and diseases. In this work we set out to understand how the endometrium can undergo rapid, scar-free healing repair each month after menstruation. The factors that mediate such repair are currently unknown and their identification would pave the way for development of novel wound healing treatments for other tissues such as skin. A great challenge that the analysis of intact proteoform presents is the wide range of chemico-phisical properties that affect both chromatographic separation and ionization. Also the conflicting acquisition requirements for accurate MS1 detection and comprehensive MS2 fragmentation require acquisition and data analysis strategies that are quite different from the ordinary bottom-up approaches. In order to overcome this challenges and to characterize the highest number of proteoforms possible we decided against a shot-gun approach, similar to bottom-up proteomics, but in favour of separating the MS1 quantitative profiling step from the MS2 fragmentation and protein identification. We are also in the process of developing a new feature detection analysis that will make use of existing imaging software and techniques applied to mass spectrometry data. The approach described allowed us to observe over a 1000 proteoforms in the mass range between 5 and 50KDa. The differential expression analysis has highlighted those proteoforms that are significantly overexpressed in the menstrual fluid and a few were confirmed to be involved in tissue repair.

How to turn that gene of interest into a protein structure.Emma J Petrie1,2, James M Murphy1,2

1The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia, 2Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, [email protected]

Keep seeing the same protein upregulated in your proteomics screen? CRISPR screen found the gene of dreams? Pull down something unexpected? Want to design a drug to block your pathway of interest? Sounds like you need a protein structure and function study!With the advances of technologies such as CRISPR, proteomics and bioinformatics potential protein interactions are being identified daily. The challenge then remains to validate these potential interactions and/or to characterise the structure and function of key proteins. The expression and purification of recombinant protein can open the door to many studies from the generation of antibodies and pull down assays to more involved programs characterising protein interactions using biophysical techniques to measure binding affinities, or even the determination of a protein structure. While the first steps of expressing and purifying a recombinant protein sounds simple enough, this initial stage can make or break a project. This talk will discuss the initial considerations around construct design, choice of expression system and purification strategy within the context of structural characterisation using X-ray crystallography, Small Angle X-ray Scattering and Nuclear Magnetic Resonance.The outcomes of structure/function studies can facilitate insights into the molecular mechanisms of activation, identify interaction sites and facilitate drug design.


Characterising novel therapeutics using quantitative proteomicsJarrod J Sandow, Giuseppe Infusini, Che A Stafford, Ueli Nachbur, Andrew I WebbWalter and Eliza Hall [email protected]

Small molecule therapeutics are being developed to modify the activity of a diverse range of target proteins. Although most therapeutics are designed to target a specific protein there are inevitably additional off-target effects. Characterising the specificity of a novel compound can be time consuming, expensive and often incomplete. Here we describe a number of quantitative proteomic techniques to define the protein binding partners of a novel RIPK2 inhibitor, WEHI-345. Using immobilised WEHI-345 we were able to purify RIPK2 and additional off-target proteins from our cells of interest, demonstrating a high specificity for RIPK2. We then extended our analysis using both intact and peptide mass spectrometry to identify a large number of concurrent phosphorylation sites on RIPK2 and defined which of these phosphorylated RIPK2 species preferentially bound to WEHI-345. In addition to small molecule enrichment analysis we have also characterised the global proteome changes that occur when disease cells are treated with therapeutics. This has identified a number of signalling pathways that are activated in response to drug treatment and highlighted the events that precede apoptosis in our target cells. This comprehensive array of proteomic techniques has allowed us to rapidly characterise small molecule therapeutics from both a molecular and a global perspective.


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