Australian Life Scientist Jan/Feb 2014 by Westwick-Farrow Media

2014

Lorne

PP100009448 ISSN 1448-9791

meeting previews

Vol 11 Issue 1 • January/February 2014

G U T FE E L IN G S | PROT EO M I C S | G ROW IN G C RYS TA L S | G E N ES A N D CA N C E R

Contents FACE TO FACE

08 A neuroscientist’s view One of the founders of neuroscience in Australia, Professor Marcello Costa, reflects on a distinguished career as a research scientist, teacher, musician and philosopher.

08 LORNE CANCER

20 Op-shopping the genome yields RNA gold

LORNE PROTEOMICS

14 Vesicular ‘omics’ Using sea urchin eggs as a model system, Professor Jens Coorssen is unveiling the mechanisms that underlie vesicular release and paving the way for this pathway to be targeted in rational drug development. LORNE PROTEIN STRUCTURE AND FUNCTION

17 The magic of molybdenum Solving the structure of proteins is all in a day's work for Dr Megan Maher, who is using X-ray crystallography to further our understanding of how cells acquire and use trace metals.

REGULARS

06 08 19 31 33 34

AusBiotech Face to Face GrantWatch New products Publish or perish Events

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Research into the role of noncoding RNA molecules and gene splicing mechanisms in white blood cell development has led to the discovery of a completely unimagined mechanism of gene expression regulation in granulocyte differentiation.

24 Managing the cancer microenvironment Dr Roberta Mazzieri is passionate about understanding the complex cellular interactions that make up a tumour microenvironment and using these to combat cancer.

cancer are moving towards being trialled in animals.

28 Enzymes with alter egos Enzymes are well known for their housekeeping role in cells, but some enzymes bind RNA and might play a significant role in linking intermediary metabolism to gene expression via post-transcriptional regulation.

LORNE GENOME

26 The ‘omics’ of cancer Novel synthetic molecules that represent potential complementary therapies for late-stage basal breast

IN THE NEXT ISSUE OF ALS

• Cell biology • Regenerative medicine • Virology • Vaccines

Editorial deadline: 17/02/14 Advertising deadline: 17/02/14

Cover image:

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Neuroscience

and society

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he advances that have been made in understanding the human brain and body over the last few decades have been huge. The progress that has been made in genetic, proteomic and protein technologies has helped expand our knowledge in neuroscience, as have the numerous empirical studies that link social cognition and emotions to our physical biology or neurological system. A bridge now exists across what some have seen as an impassable abyss between the social and biological sciences. With the help of neuroimaging, using magnetic resonance imaging or functional MRI, as well as physiological measures such as heart rate or blood pressure, there is increasing consensus that biological and neurological processes influence psychological or social processes and visa versa. Neuroscience has played an important role in bridging this divide. It has brought a number of scientific disciplines together under the one umbrella - anatomy, physiology, biochemistry, molecular biology, pharmacology - and has united these with allied disciplines such as physics, computer science, philosophy, economics and political science. Only 30 or 40 years ago, disciplines such as neuroanatomy and neurophysiology were distinctly separate entities, isolated from each other as well as from other areas of biology. The interdisciplinary nature of neuroscience extends further to link the

Susan Williamson

Printed and bound by Pegasus +61 2 8822 0716 Print Post Approved PP100009448 ISSN No. 1448-9791 All material published in this magazine is published in good faith and every care is taken to accurately relay information provided to us. Readers are advised by the publishers to ensure that all necessary safety devices and precautions are installed and safe working procedures adopted before the use of any equipment found or purchased through the information we provide. Further, all performance criteria was provided by the representative company concerned and any dispute should be referred to them. Information indicating that products are made in Australia or New Zealand is supplied by the source company. Westwick-Farrow Pty Ltd does not quantify the amount of local content or the accuracy of the statement made by the source.

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physical sciences with the humanities bringing culture and law, morality and ethics, religious beliefs and politics into the mix. Rearranging scientific disciplines provides the opportunity for new interactions that potentially lead to changes in perspective and progress. Indeed, neuroscience has inspired and continues to inspire such opportunities - you will find this in action at the 2014 Australasian Neuroscience Society meeting in Adelaide at the end of January. Bridging the science and humanities divide came up when I interviewed Professor Marcello Costa for this issue (page 8), which inspired the writing of this editorial. A passionate advocate for bringing neuroscience to the public, Professor Costa is actively involved in public events that link neuroscience to the arts including theatre, painting, music and dance. We humans are basically a social species - we organise and create families, groups, cities and cultures. These social structures we create have evolved hand in hand with our neural and hormonal biology, and consequent social behaviours. Creating a dialogue between neuroscience and society is an important part of the field moving forward. Better understanding human behaviour provides the possibility of more comprehensive theories of understanding the human brain, its dysfunction and disease, not to mention deepening our basic understanding of what it means to be human.

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AUSBIOTECH | VIEWPOINT

The importance of a quality board in attracting investors Many investors will tell you that one of most important things they look at when assessing an investee company is the calibre of the management team. And increasingly, they are focusing on the membership and calibre of the board, due to the direct impact the board can have on a company’s chances of success.

eremy Curnock-Cook, managing director of BioScience Managers, said, “The board of directors is an often overlooked key asset of a biotechnology company. It needs to be well balanced, well chaired, appropriately skilled, prepared to evolve as the enterprise itself grows, develops and matures. The strength of its composition should be regarded as a critical contributor to successful outcome and impacts the quality of the investment, and the quality of that investment’s decisionmaking. Get the board right and it will often be the difference between success and failure. “Biotechnology is a complex business, requiring an unusual blend of scientific, commercial, financial, clinical and operational expertise. It is also an industry where companies often run into significant technical and commercial challenges when things don’t go as planned. But it is when things don’t go as planned that the best boards will truly prove their worth, and the weaker boards end up with a company that is out of capital and out of ideas. “Never is the competence of a board tested so much as when a technology fails to meet its nominated endpoints in a clinical trial or receives negative feedback from a regulator. We have seen a number of these cases in the Australian market over the past

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few years, and the ability of the company to recover is usually a function of the response of the board,” he said. AusBiotech has a long-held and genuine desire to support the development of boards and directors, the quality of company governance and disclosure, which in turn supports the broader industry’s development and attracts investment by increasing investor confidence. Innovative, technology-focused companies in the life science industry have different pressures, such as unique regulatory requirements and a different business cycle than many other industries. Directors of such companies therefore require additional, specialised knowledge that is not generally learned from available corporate governance materials or taught in mainstream governance courses. For the reasons outlined above and with the support of the Victorian Government, AusBiotech has recently completed the Guide for Life Science Company Directors, a resource to support and enhance the performance of boards of directors leading public and private life science companies. It is endorsed by the Australian Institute of Company Directors and its development was guided by an advisory panel of industry experts. The guide outlines, for

AU S T R A L I A N L I F E S C I E N T I S T

Dr Anna Lavelle, CEO , AusBiotec

less experienced directors or those new to life sciences, issues typical to life science companies that are generally not typical in other industry sectors. The guide makes an excellent induction resource for new directors, those new to investing in life science companies or those working in research and considering commercialisation pathways. The guide is a companion document to the Code of Best Practice for Reporting Life Sciences Companies, which was developed with the Australian Securities Exchange (ASX) to support high standards of communication and market disclosure to promote investor confidence. Originally developed in 2006, the code was revised in early 2013 and the second edition launched by Victorian Minister Gordon Rich Phillips in May. The venture capital community is looking to companies for signs that they are implementing the code and taking the support of the guide seriously. Life science company boards are urged to take advantage of these resources by recommending and distributing them to current and would-be directors and displaying the code’s ‘web button’ on company websites. The guide, code and the web button can be found at www.ausbiotech.org/ biotechboards. ALS

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FACE TO FACE | NEUROSCIENCE

A neuroscientist’s view

Susan Williamson

One of the founders of neuroscience in Australia, Professor Marcello Costa, reflects on a distinguished career as a research scientist, teacher, musician and philosopher. Australian Life Scientist: What inspired you to become a scientist? Professor Marcello Costa: I’ve really liked science since I was a boy. I migrated to Argentina with my family in 1949 after the Second World War and lived there for 11 years. I was really interested in the natural world and apart from playing soccer on the street I looked at bugs under the microscope. I even sold my bicycle to buy a microscope, which I still have. When I went back to Italy in 1960 I decided to do medicine. I was determined to become a scientist, really, and maintain my interest in human beings, so I had to go through medicine. ALS: Was it challenging moving from

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South America back to university in Europe? MC: When I returned to Italy to do medicine I was not very impressed with the slightly old-fashioned system of Italian universities, which was not surprising. In Argentina I led a life of freedom. It was hard to get used to the city of Turin, which was very civilised but very conservative. I found that pretty hard going. But I was given enough chances and I was rebellious enough to make the best of it. I did live a very interesting decade in Italy in the 1960s. I was involved in a lot of activities as a medical student - music, climbing, politics. I entered into student politics and became

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one of the leaders of a student movement in northern Italy at the time. I was already working in a lab by the time I finished medicine. I chose anatomy as a base for my studies, not because I really wanted to do anatomy but because it was the only place where they studied the nervous system. I was lucky because it was the laboratory that had generated three Nobel prize winners, although they got the Nobel prizes when they left Italy, mostly to escape the fascism in the late 1930s. In a way my studies were surrounded by the ghosts of these people who had left - perhaps this is the reason I also left. ALS: What inspired you to move to Australia? MC: In those years science in Italy was still under the heavy burden of powerful figures in the university - it wasn’t a very open system. I was in anatomy but I really wanted to do physiology. My professor was not very keen for me to work with

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NEUROSCIENCE | FACE TO FACE

people in other departments or even other universities. But I did and discovered that is possible to become multidisciplinary. Also, the main second language of Turin is French. Everybody spoke French and went to meetings of the Academy of Anatomists in France. I realised English was the universal language of science, so I considered to move, first to England and then to Australia. I met Geoff Burnstock in Venice in 1969. He was a young Professor of Zoology in Melbourne at the time and is now one of the dons of the autonomic nervous system in the world. He offered me an 18-month postdoctoral fellowship to work in his lab. I asked my girlfriend, Daniela, whether she would marry and go to Australia and we did. We came to Melbourne as migrants in 1970 and I did not expect to stay very long. But we stayed another year and then returned again in 1974. ALS: Were you involved in founding the Flinders Medical School? MC: Yes, I was a foundation lecturer and probably one of the youngest members of the school at the time. When I went back to Melbourne in 1974 I met Laurie Griffen, a dynamic young Professor of Physiology from Flinders University who was looking for young foundation lecturers. I joined Laurie’s group in Adelaide and started teaching in little apartments because the medical school at Flinders University had not been built yet. The medical school was quite modern in the sense that it abolished the distinction between the various research disciplines. Although we were in anatomy or physiology or biochemistry, the laboratories and the teaching were integrated. We started back to front in the sense that basic scientists formed the place with a strong science base and research culture - the clinicians came later. Despite the fact that it was a medical school, it was not a medical school based on the power of clinicians but on the power of young scientists, which is quite extraordinary. It was a very open and very productive place. We were free to collaborate widely and we created the conditions that remained probably the best in Australia for many years.

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ALS: When did discipline of neuroscience develop in Australia? MC: Neuroscience was in the air at the time I arrived in Australia. The Society for Neuroscience in the United States was founded I believe in 1969. When I came to Melbourne in 1970 I attended a meeting a few months later at Monash, along with biochemist Laurie Austin and others. We were probably the foundation group of the Australian Neuroscience Society (ANS) and we actually did use that term but it was not organised formally. It took another 10 years or so to formalise but we’d already had ongoing meetings almost every year. There was a good level of active neuroscience around Australia in the late 1970s, mostly in the fields of physiology and pharmacology. The Australian Physiological and Pharmacological Society (APPS) existed at the time and we all joined. There was eventually a split between pharmacology and physiology, and most of us migrated into the neuroscience mould intentionally, although I remain also a member of the APPS and more recently the APS. ALS: And that led to the formation of the Australian Neuroscience Society? MC: Yes, that is a piece of history. At the time the ANS was like a big collection of brothers and sisters, it was not very formal. Then in the early 1990s, I was asked to give the plenary lecture at the meeting in Melbourne. After I had given my lecture two senior colleagues on the ANS executive came to me and said ‘We think you should become president, would you like to do this?’ and I said ‘Wow, why not?!’ At the next meeting they chose me and I was very privileged to be president of the society in the big 1990s. At the time there were no elections, so it was a very closed shop in a way. It is now far more democratic and open. It has been an extraordinary group of people and it still is. I’ve been part of many societies around the world, nationally and internationally, and the ANS is distinct because we started without anyone having propriety. Neuroscience is not really a professional

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discipline and the advantage has been that there is no commitment to any profession; we are free to open our doors to anyone who wants to be part of the field. This has created a sense of camaraderie across disciplines without any other political reason to be together. It’s purely intellectual and that is marvellous. ALS: You are giving a plenary for the second time at this year’s ANS meeting. MC: Yes, the society has asked me to give a plenary lecture here in Adelaide for the second time. I don’t think it has happened before that a person has been asked twice in their lifetime. I’ve decided I will hold them to the promise to invite me again in 20 years’ time! ALS: How did the enteric nervous system become your research focus? MC: When I was a student in Italy I went to talk to the Professor of Physiology and said I’d like to study the brain. He said to go next door to the Department of Anatomy because they studied a bit of the nervous system. I went, and they were studying the development of nerves in different parts of the body and a bit of the gut. I thought I’d learn the techniques and then move into the brain. But when I started to working on the gut I realised it was a huge area of research that was totally untouched at the time. I was lucky to be given the chance to use a new histochemical technique. So, I started really with the histochemistry in the nervous system; in particular, the enteric nervous system. I developed new methods with my supervisors - we succeeded and I published, mostly in Italian and French, but Geoff Burnstock and colleagues in Australia knew about my work as a medical student. When I arrived in Melbourne I simply continued that work. I also learned pharmacology, electrophysiology and a bit of biochemistry. The Department of Zoology in Melbourne at that time was a very exciting place to be and at that time I began working with John Furness. We worked on many areas and put our eyes and hands on anything that walked, slithered, slimed or flew as well as different organs, different parts of the body. The enteric nervous system became the core of the work because it was less known and

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FACE TO FACE | NEUROSCIENCE

Left: Professor Marcello Costa and, to his left, Professor Simon Brookes with lab staff in 2003. Right: Sailing the Mediterranean.

probably more challenging. It was then I realised there was no reason to move away from that until I understood it a bit better. And I’m still doing it. When I moved to Adelaide I continued working with John, who had moved to Flinders from Melbourne University a few months before me. And we continued to work together for another 14 years. I still remember when we first got funding from the NHMRC - we were given $990. We joked at the time that we got all the money that we asked for from the NHMRC, which doesn’t happen often these days. It was only a few dollars, but we had the freedom at Flinders to start new endeavours in a small way, and with a small amount of money we could do some serious research. ALS: Are you still in the lab? MC: Yes, although there was a period from 2005 that I stopped and partially retired because I was getting a bit burnt out with running a big lab and managing too many people. About three or four years ago my younger colleagues called me back into the lab to help them a little and I realised I could still do research. I’ve returned to do experiments in the gut with a different approach and we are beginning to answer a lot of questions I had 10, 20 or 30, even 40 years ago. Simon Brookes, who was a postdoc of mine, is now my boss. I’m still collaborating with the young person I tutored and mentored, plus a number of other people at Flinders - in particular Nick Spence - but they run the labs and I am enjoying their facilities. One of my first medical students and then PhD student, David Wattchow, who is now a very senior surgeon, has kept us

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supplied with both research and precious human tissue. We have a most active and productive team at Flinders with collaborators at the CSIRO and in New Zealand, with local and national clinical colleagues extending the team beyond basic neurogastroenterology. ALS: What is the different approach you are taking in the lab? MC: We are studying in great detail the actual behaviour of the gut. We know a lot about the extensive network of neurons but we know little about what they are doing in real life. We assumed they were involved in movements, such as peristalsis, but we didn’t pay too much attention to the actual behaviour of the gut. We are developing the tools to conduct a proper physiological study of the behaviour of the intestine on the basis of our understanding of the enteric nervous system. It’s like reading the brain of the intestine when it’s actually working rather than studying its cells one by one. In the late 1990s, with my PhD student Grant Henning, I developed a way to describe the actual movement of the gut by making recordings of what are called spatio-temporal maps, which can then be analysed in a computational way. But that fell to the side when Grant left Australia and I couldn’t really maintain the level of knowledge in computer programming. With the arrival of Phil Dinning at Flinders, who is working on the human gut using bowel scopes and recording pressure using a super, novel high-definition optical pressure probe, I’ve started to study the behaviour of the gut again. We have combined the two recording methods, so now we can study gut behaviour in animals and in humans, and attribute the movements of the gut to neural or

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myogenic activity, to the pacemaker cells and to intrinsic or extrinsic neural pathways. We call our conceptual explanation of most movements the neuromechanical loop hypothesis. This means that the enteric neural activity produces a movement and the movement feeds back into the enteric neural circuits via enteric sensory neurons, which correct and adapt the changes in motor behaviour. It’s like the sensory-motor control of movements when walking on the terrain - I call it the locomotion of the intestine, whereby you have to include a loop between sensory and motor, and a bit of neural programming within the pathways, but they don't come alive until they are interacting with the intraluminal contents which are really part of the external world. We are funded by the ARC to develop our model on animals and we just received funding from the NHMRC to extend this to the human gut, which is wonderful. ALS: Has teaching been an important part of your career? MC: Teaching must always be in parallel to research in my view. I started almost single-handed a number of the neuroscience topics at the Medical School at Flinders and I’ve had the pleasure of teaching neuroscience to thousands of students over the last 20 years. I continue to teach. It gives me great pleasure because students are following a career in neuroscience and certainly some of them have become excellent neuroscientists and some may become even great ones. I still think teaching is the best way to keep my research meaningful because it compels me to read widely and keep up with the literature, which is growing all the time. Particularly being at Flinders, because

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FACE TO FACE | NEUROSCIENCE

Professor Marcello Costa and Associate Professor Nick Spence at the International Neurogastroenterology and Motility Meeting in Italy, 2012.

the teaching is across disciplines. We now get very young students because we started teaching foundation physiology from first year. I start really with the big bang - the origin of life and chemistry, physics, the biology of cells and energetics, and of course mechanisms of biological evolution. I take the view that if you know a bit about the origins of animal bodies and physiological principles then you’ll have a much better understanding of the nature of humans and the universe. That is part of my passion, linking philosophy and culture to science, and then extending that to the whole of society. ALS: Has this passion led to your extracurricular activities? MC: I am absolutely passionate about using neuroscience to overcome the traditional divide between science and humanities. It links us to everything we do, from cultural and intellectual aspects, to emotions, bodily functions and evolution. And I am passionate about teaching people about this - politicians, people in industry, school teachers and students, other academics and the public at large - there is a wealth of information and knowledge we can extract from understanding the nervous system. When I stopped doing lab research for a while I devoted my activities to learning more about the nervous system and society. I was involved in developing a community

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of neuroscientists here in South Australia and taking it nationally and abroad. Doing that I learned a lot about the social aspect of neuroscience. In the last few years I have been involved in a very exciting project called Science Outside the Square. This involves Ian Gibbins, Professor of Anatomy at Flinders - he is a fantastic keyboard improviser and I sing and play on the guitar. We combine science and music, science and art, and perform in public, taking advantage of small stints in the performing arts. Having people in pubs listening to music or seeing a famous choreographer, such as Lee Warren, dance and talk about dance enables us to take a bit of neuroscience to the public and compel them to think like we do as neuroscientists. And for us scientists to understand more how performance artists think and feel. I think that role is important. We had our peak in these activities in the last six years. I’m doing less of that now mostly because I’m back in the lab doing interesting experiments. However, there is a big carnevale of the Italo-Australian community in South Australia each year. Within the festivities a small section takes place under the auspices of the Royal Institution of Australia. Together with Susan Greenfield, a distinguished neuroscientist who was a Thinker in Residence in South Australia

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in 2004-05, I was involved in creating this institution to take science to the public. I speak during this event, which is run like a big public fair over a weekend. I have given talks on the Italian Nobel prize winner Rita Levi-Montalcini. Last year I talked about the Renaissance art and science, Galileo and science - he was also interested in the brain by the way - Leonardo da Vinci, and this year I am going to talk about Volta and Galvani, two geniuses to whom we owe respectively the discovery of the battery and the electricity in our muscle and brains. This provides me the opportunity to talk about a bit of history and the relation of science to culture, which I’m still committed to doing in any possible way. ALS: Do you think people understand the link between science and the humanities? MC: I think people still have a very stark view of science, even my colleagues in science. Many are top scientists but some are very committed to the hard sciences, and perhaps politics, and don’t always see the link between chemistry and physics and the humanities - like culture and law, morality and ethics, religious beliefs and so on. I am trying to link these areas to overcome what is, in my view, the big mistake made by our society. And that is the dual nature of us, which of course we owe to Descartes. Fundamentally, I am attempting to define under neuroscience the two worlds of matter and mind, and under the same umbrella as the humble but potential powerful neuromechanical loop hypothesis. It may resolve the apparent divide between the material world (res extensa of Descartes) and the thinking matter - the mind, the soul (res cogitans of Descartes). All that happens inside our brain is neural, that is to say electrochemical, and when it is translated into muscle movements it becomes part of the mechanics. People tend to talk about the mind and body all the time. They are really talking about the same thing and a small change in conceptual frame will put this under the same umbrella. To regard humans, as marvellous as we are with our mind, as a natural product of evolution nothing less, but nothing more. ALS

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LORNE PROTEOMICS | FUNCTIONAL PROTEOMICS

Vesicular ‘omics’ Susan Williamson

Using sea urchin eggs as a model system, Professor Jens Coorssen is unveiling the mechanisms underlying the essential cellular process of vesicular release and paving the way for this pathway to be targeted in rational drug development.

onducting fully coupled functional and molecular analyses to identify critical components involved in regulated exocytosis is, for Professor Jens Coorssen, an engaging task. Foundation chair of Molecular Physiology, and head of the Molecular Medicine Research Group at the University of Western Sydney, Coorssen has worked on secretory vesicles and the mechanism of triggered exocytosis for about 25 years and will present some of his team’s work in the vesicle symposium at this year’s Proteomics meeting. RELEASE-READY VESICLES

Coorssen’s team primarily uses releaseready cortical vesicles from unfertilised sea urchin eggs in their work on regulated exocytosis. Maturing oocytes, eggs and the early embryos of sea urchins have been used as key model systems for well over a century, providing critical cellular and molecular insights into mechanisms underlying fertilisation, development, calcium signalling, the immune response, cell cycle regulation and regulated exocytosis. “It is a highly conserved system in which the docked, release-ready and late calcium-triggered steps of exocytosis are isolated and can be quantitatively

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assessed,” said Coorssen. “We use different proteomic and lipidomic approaches to dissect what’s going on in the mechanisms of triggered release/ regulated exocytosis.” Despite decades of research, the calcium-triggered fusion mechanism involved in exocytosis remains poorly defined at the molecular level. Understanding this mechanism is a critical requirement to targeting this pathway for rational drug development. Like the majority of eukaryotic cells, sea urchin eggs contain secretory vesicles, but in this case, more importantly, stagespecific, release-ready cortical vesicles. These membrane-bound organelles fuse with the plasma membrane to release their contents into the extracellular space. This highly conserved and fundamental cellular process of regulated exocytosis forms the basis of a diversity of functions including fertilisation, intracellular trafficking, wound healing and neurotransmission. As might be expected of the close functional relationship to mammalian systems, there is also a very high degree of genetic conservation between sea urchins and humans. “The sea urchin genome has been sequenced,” added Coorssen. “There is high genetic conservation from sea urchins

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to humans, which makes it a very reliable model system in that respect. Critical and fundamental mechanisms are highly conserved or they cease to exist. Such is the nature of most critical molecular mechanisms.” A CHEMICAL PROTEOMICS APPROACH

One aim of the work in Coorssen’s team is to develop a list of key players involved in the mechanism of regulated exocytosis and assign them to direct or modulatory roles. They have been using different biophysical, (bio)chemical, proteomic and lipidomic approaches to identify the critical players and their roles. “We have a lot of tools to link the physiology and the proteomics. It’s functional proteomics, and the reason we use this model system is that it enables us to link as tightly as possible the quantitative functional and the quantitative molecular assays. That’s the only true route to dissecting mechanism.” These tools include two chemical proteomic approaches, one is based on differential protease effects. “In the past, one of the techniques we used was differential sensitivity to proteases,” recalled Coorssen. “We were able to proteolytically remove key proteins that were suggested to be essential to

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FUNCTIONAL PROTEOMICS | LORNE PROTEOMICS

the fusion mechanism and demonstrate quantitatively that we’d removed the critical cytosolic domains of those proteins. “Yet the calcium-triggered fusion mechanism still worked. As we did this quantitatively, we could confirm that these components were clearly important, but were not acting in the way they were claimed to according to the major hypothesis in the field at the time regarding the protein machine that was believed to drive fusion. They’re working upstream of triggered fusion. Thus, the docking and priming roles we postulated instead, years ago, have since been confirmed by others, in other secretory cell types.” The researchers are now integrating this approach using broad-spectrum proteases with another approach using selective inhibitors of different kinases and phosphatases to test for effects on triggered release. In this way they are targeting the identification of critical alterations to the phosphoproteome of secretory vesicle membranes, including parallel validation in synaptic preparations. “There are multiple things going on at a biological membrane and the membrane is thus chock-full of proteins that are not

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at all involved in your mechanisms of interest. One of the things the protease treatment does is remove a lot of those yet leave the triggering and release mechanisms intact, so any proteins that are removed must not be critical to mechanism. One can, to some extent, be rid of ‘background’. “The cortical vesicle model thus enables us to home in on critical proteins to better refine subsequent validation experiments in mammalian secretory cells that might otherwise take years of research funding.” The other chemical proteomic approach involves thiol-labelling. Using this technique the researchers have identified two classes of reagents that act in opposite ways - one promotes the calcium sensitivity and kinetics of vesicle release and the other inhibits it. “Using multiple fluorescent reagents we are now simultaneously modifying function while labelling the proteins involved,” explained Coorssen. “One simply can’t do such direct, coupled, quantitative analyses and identification of critical proteins with any other model system. Naturally, validation in synaptic and other clinically relevant mammalian secretory cells are also part of this long-term and well-defined research program.”

THE MEMBRANE PROTEOME

High-resolution, gel-based analyses have enabled Coorssen’s team to dissect the cortical vesicle membrane proteome. Now that they have the proteome they are studying it to understand critical underlying molecular mechanisms. “Part of the reason we do gel-based proteomics is because it directly delivers that critical quantitative aspect, as well as the necessary deeper analysis of protein species, in parallel, across several replicate samples,” said Coorssen. Using gel electrophoresis is also helping to identify critical protein interactions. Membrane protein complexes involved in vesicular docking and in triggering fusion have been resolved and, together with chemical proteomic approaches, this will enable the researchers to define the components that make up the physiological fusion machine of regulated exocytosis. This highly quantitative approach, directly coupling the functional and molecular assays, enables the researchers to analyse protein and lipid components of the system and work out which components, and which interactions, are essential to both setting up and driving the fusion of vesicles to the membrane.

Professor Jens R Coorssen (centre back) was appointed chair of Molecular Physiology in the School of Medicine at the University of Western Sydney in 2008ww and head of the new UWS Molecular Medicine Research Group in 2009. He studied biology and biophysics for his BSc (Hons) and MSc at Brock University in Canada and completed a PhD in Cellular Physiology in the Faculty of Medicine, McMaster University, Canada. He then pursued Fellowships at the Max Planck Institute for Medical Research (Heidelberg) and the National Institutes of Health (USA), as well as holding a prestigious visiting scientist position at the NIH before being recruited to the Faculty of Medicine at the University of Calgary (2000).

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LORNE PROTEOMICS | FUNCTIONAL PROTEOMICS

LIPIDOMIC ANALYSES

In parallel, Coorssen and colleagues have also been equally interested in the role of lipids in the exocytotic pathway. His team was the first to identify cholesterol as a critical component of the exocytotic mechanism. “Cholesterol is a central conserved component of exocytosis and of the final triggered fusion process, but that exact function will be difficult to fully dissect outside of the cortical vesicle, particularly with regard to membrane curvature contributions,” said Coorssen, “and that is also true of other such components. However, time-resolved electrophysiological analyses of the fusion pore are pushing the boundaries in this respect - the coupled, quantitative functionmolecular analytical approach.” In an attempt to better define the role of cholesterol, Coorssen’s team has refined automated, high-performance, thin-layer chromatographic analyses as a quantitative approach to lipidomics. Using this technique they have shown

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that cholesterol is directly involved in the triggered membrane fusion mechanism. They have also shown that reducing the cholesterol concentration in different vesicle membranes, using multiple strategies such as chemical extraction and sequestration, enzymatic reduction, and inhibition of cholesterol biosynthesis, adversely affects fusion. This approach has also identified other critical lipids, such as phosphatidylethanolamine and phosphatidylserine, and firmly established that still others (ie, polyphosphoinositides) actually have upstream modulatory roles during docking and priming. A QUANTITATIVE APPROACH

Resolving the fundamental molecular mechanisms underlying the process of vesicular release - understanding and describing the critical docking, priming, triggering, and fusion steps of this essential cellular process - will enable the rational targeting of this pathway in the treatment of a broad range of disorders. Many of these are serious and growing healthcare burdens,

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such as the neurodegenerative, diabetes and related disorders. “If you don’t do thorough quantitative work you’re never going to have more than a cartoon of your mechanisms,” said Coorssen. “And a cartoon isn’t good enough for identifying, for instance, critical targets for drug development. You need to know that the target you’re after is genuinely a target of interest.” ALS LORNE CONFERENCE LINE-UP

19th Lorne Proteomics Symposium February 6-9 www.australasianproteomics.org/ lorne-proteomics symposium-2014/ 39th Lorne Conference on Protein Structure and Function February 9-13 www.lorneproteins.org/ 26th Lorne Cancer Conference February 13-15 www.lornecancer.org/ 35th Lorne Genome Conference February 16-19 www.lornegenome.org/

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X-RAY CRYSTALLOGRAPHY | LORNE PROTEIN CONFERENCE

The magic of

molybdenum Susan Williamson

Solving the structure of proteins is all in a day’s work for Dr Megan Maher, who is using X-ray crystallography to further our understanding of how cells acquire and use trace metals.

atience, partnership and serendipity played their part for Dr Megan Maher and her team in solving the crystal structure of sulfite dehydrogenase (called SorT) and its electron acceptor. Maher heads a lab in the La Trobe Institute for Molecular Science, at La Trobe University in Victoria. Her lab’s interest is in the role transition metals play in biology, with a particular focus on the technique of X-ray crystallography. “Most people know about the requirement most organisms have for iron but fewer people are aware of the seemingly obscure elements like molybdenum and manganese, which are also required by all forms of life,” said Maher. “My lab is interested in all aspects of that requirement. Everything from how a cell or organism acquires those trace metal nutrients through to how they are used in the various cell functions.”

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The work Maher will present at the Lorne Protein meeting is part of a longterm collaboration with microbiologist/ biochemist Dr Ulrike Kappler, at the University of Queensland, that is investigating how the trace metal molybdenum is used in bacterial respiration. Kappler identifies enzymes from bacteria that use molybdenum and characterises their activities and biochemical properties, while Maher uses X-ray crystallography to determine their structures. ESSENTIAL FOR LIFE

Trace metals, such as iron, copper, magnesium and molybdenum, occur in extremely small quantities in plant and animal cells. All cells require them and they occur in soil and water in the environment. “Molybdenum is essential for all forms of life,” Maher said. “There are a few specialised bacteria that will use tungsten instead but most forms of life require molybdenum.”

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According to Maher, about 30% of proteins and enzymes require a transition metal component for activity. Transition metals are required for the structural integrity or stabilisation of a protein, or they are needed for an enzyme to be active. Molybdenum is incorporated into enzymes and facilitates reactivities that an enzyme would otherwise be unable to access using amino acids alone. This is related to molybdenum’s ability to redox cycle. “Molybdenum can access three oxidation states in the biological range,” explained Maher. “It can accept or donate two electrons at a time to a reaction - no other transition metal or elements can do that.” A REACTIVE SPECIES

In humans there are four enzyme systems so far characterised that rely on molybdenum, including xanthine oxidase, which is important in uric acid synthesis and sulfite

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LORNE PROTEIN CONFERENCE | X-RAY CRYSTALLOGRAPHY

Dr Megan Maher is a La Trobe Institute for Molecular Science (LIMS) Senior Research Fellow and Lab Head in the Department of Biochemistry at La Trobe University. She studied science at the University of Queensland and completed a PhD at the University of Melbourne in Inorganic Chemistry under the supervision of Professor Tony Wedd. She then pursued postdoctoral research in the Department of Biochemistry at the University of Sydney and the Division of Molecular Biosciences at Imperial College, London. Her research focuses on the structural biology of metals in biological systems, with particular emphasis on the technique of X-ray crystallography and has recently expanded to address the structures of integral membrane proteins (metal transporters in particular). Her work has led to discoveries involving the acquisition of essential iron by bacteria and the structures of components of the electron transport chain.

oxidase, which converts sulfite to sulfate. Maher’s team is working on the latter enzyme in bacteria. “The sulfite oxidase enzymes are required for sulfite detoxification in all organisms,” explained Maher. “For example, in humans, sulfite results from the breakdown of sulfur-containing amino acids in normal cellular processes of metabolism, such as cysteine and methionine. Sulfite is very reactive and needs to be quickly converted to sulfate to prevent it from damaging cells - for example, it reacts readily with protein and DNA. This is the role of sulfite oxidase and it would not be able to do it without molybdenum.

USING SULFITE FOR ENERGY PRODUCTION

Maher’s latest work has involved looking at the equivalent enzyme, SorT in the bacterium, Sinorhizobium meliloti, which is found on the root nodules of legumes. This bacterium can live on sulfur-containing compounds, using the sulfite-sulfate conversion for energy generation. “The bacterial system is simpler to work with in terms of generating the protein and purifying it to do the types of studies we want to do, rather than working on the human enzyme,” she said. Isolating the enzyme from its bacterial source does not generate very much pure protein, so Kappler cloned the enzyme into E. coli and developed a method to generate quantities of recombinant protein for Maher’s X-ray crystallography. “We need milligram quantities of pure protein to grow crystals,” Maher explained.

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THE ELECTRON TRANSFER DANCE

But it’s not just SorT Maher’s team has been trying to crystallise, they have also been looking for its partner or electron acceptor in the cycling reaction. When molybdenum accepts the two electrons during sulfite oxidation, it then interacts with another protein as an electron acceptor, enabling the enzymatic reaction to take place again. Much like the mitochondrial electron transfer chain, the fundamental process driving ATP synthesis in cells, the electrons flow from one protein to another through a chain. “Given it is such as fundamental process, we still don’t entirely understand what governs it,” said Maher. “In the electron transport chain of the mitochondria, the protein-protein complexes are located close together in the mitochondrial membrane, but in the case of SorT and its electron acceptor, they float around in the cell periplasm. “They need to find each other and interact very specifically but very transiently - the interaction cannot be so specific that they cannot separate again because electron transfer has to be very fast.” GROWING CRYSTALS

Not many electron transfer structures have been solved because of the transience of the interaction between the two molecules. But Maher’s team got lucky and successfully crystallised SorT with its electron acceptor. Now that they have solved the structure it will help us understand how this class of enzyme functions. “We were very lucky to get the structure of the complex,” Maher recalled. “We basically mixed the two proteins together,

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put them into our crystallisation experiments and crossed our fingers - and we got one crystal. “Often, because of the requirement for that fine balance between a specific but not very strong interaction, it’s very difficult to crystallise the two proteins in complex because they need to come together for some time in order to pack into a crystal.” Once they captured the transient interaction, they took their prize crystal to the Australian synchrotron where they have access to the high-intensity radiation needed for X-ray crystallography. “We wouldn’t be able to do this work without the synchrotron,” said Maher, recalling times when her research was restricted to experiments twice a year because it required visiting a synchrotron overseas. X-ray crystallography literally reveals the packing of the molecules within a crystal. A diffraction pattern is produced that can be used to generate an electron density map, which is basically a 3D contour map of where the electrons are positioned in the crystal which, in turn, is where the atoms are. “Once we generate this electron density map, it’s like a big puzzle, we then need to interpret where each atom is in this beautiful net,” said Maher. “We can actually see exactly where the atoms are and once we interpret that map we can see the structure of SorT and its electron acceptor together in the crystal.” SOLVING THE STRUCTURE

Solving the structure of the complex not only increases our understanding of the molybdenum class of enzymes, it will help the researchers better understand the electron transfer process - how two proteins can come together specifically but also transiently. This is relevant to all electron transfer processes that occur in cells because they all have the same requirement. “The more we understand about their structures and their functions, the more we understand about the important processes they carry out,” said Maher. “These results have reinforced a lot of the theory that was out there already about electron transfer complexes. It’s very nice that it made sense. A lot of theoretical work has been done and it all married up very nicely.” ALS

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MOVERS & SHAKERS

GrantWatch New medal for women in science The Australian Academy of Science has announced the new Nancy Millis Medal to recognise Australian women scientists. The medal has been struck by the academy as a tribute to the late Emeritus Professor Nancy Millis, who introduced fermentation technologies to Australia and created the first applied microbiology course taught at an Australian university. “This medal honours the contributions made to science by Professor Millis and recognises her importance as a role model for aspiring female scientists in Australia,” said Academy President Professor Suzanne Cory. A Fellow of the academy, Professor Millis completed a degree in agriculture in Melbourne in 1945, a PhD from Cambridge in 1952, became senior demonstrator the Department of Microbiology at the University of Melbourne in 1952 and lecturer in 1954. She also worked tirelessly to establish links between universities and industry. Professor Millis became the chancellor of La Trobe University in 1992, a position she also held until her retirement in 2006. The medal recognises outstanding research and exceptional leadership by early- to mid-career Australian women who have established independent research in the natural sciences. It is restricted to candidates normally a resident in Australia and for research conducted mainly in Australia. Deadline for nominations is 10 February 2014.

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Supporting Indigenous health Businessman and philanthropist Greg Poche is no stranger to gifting millions to research. The former founder and owner of Star Track Express has donated more than $105 million to causes around Australia, including $40 million to set up the Melanoma Institute of Australia in Sydney. Now Poche, and his wife Kay, have provided $10 million to The University of Western Australia (UWA) to create a WA-based centre for Indigenous health with the theme ‘Healthy Minds, Healthy Lives’. UWA’s new Poche Centre for Indigenous Health joins sister centres at Flinders University in Adelaide and the University of Sydney. The goal of Poche centres is to improve Aboriginal and Torres Strait Islander health and strengthen social, spiritual and emotional wellbeing. Although there have been recent improvements to health outcomes for indigenous Australians, the gap remains. For Indigenous people born between 2005 and 2007, life expectancy was estimated to be 67.2 years for men and 72.9 years for women, which is about 10 or 11 years less than non-Indigenous Australians. The UWA Poche Centre will work with the University’s School of Indigenous Studies and Centre for Aboriginal Medical and Dental Health and the Rural Clinical School and Combined Universities Centre for Rural Health - geographically the world’s biggest medical school. School of Indigenous Studies Dean, Winthrop Professor Jill Milroy, said closing the gap in health and life expectancy for Aboriginal people had been identified as a nationwide priority. “We are pleased that the new centre recognises the centrality of culture and wellbeing to promoting good mental health in Indigenous communities,” she said. “Aboriginal knowledge systems will be the cultural lens and framework for research, education and the delivery of programs.” The centre will tackle children’s health, disability and developmental outcomes as well as chronic disease. Professor Milroy and her team will address the overarching issue of Aboriginal culture and its relation to social and emotional wellbeing and mental health - issues that are estimated to contribute as much as 22% of the life expectancy gap for Aboriginal people.

Bowel cancer research Bowel cancer has received a generous $15 million bequest from Elwin à Beckett, a resident of Wellington, NSW. Australia has one of the highest rates of bowel cancer in the world. It is the second most common type of newly diagnosed cancer in Australia and is increasingly becoming linked to other chronic diseases. When she died in May this year, à Beckett left the bulk of her estate to the University of Sydney to advance research into bowel cancer. In honour of her much-loved brother Martin, who died from the disease in 1986, The William Arthur Martin à Beckett Cancer Research Trust has been set up to support research into better understanding bowel cancer and interrelated diseases. The university is considering using the funds to purchase essential equipment, fund postgraduate scholarships and to recruit a new Chair: the Elwin à Beckett Chair for the Prevention, Detection and Treatment of Bowel Cancer. The gift comes via the trust company, which has managed Elwin’s affairs prior to her death and is now trustee and sole executor of her estate.

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LORNE CANCER CONFERENCE | INTRON RETENTION

Op-shopping the genome yields RNA gold

Fiona Wylie

Shakespeare’s Hamlet was correct - “what a piece of work is a [hu]man! ... how infinite in faculty” - and now it seems, according to a team of researchers in Sydney, in gene expression regulation.

rofessor John Rasko established the Gene and Stem Cell Therapy Program in 1999 at the Centenary Institute in Sydney. These days the program comprises around 20 researchers whose work spans genetics, stem cells, gene transfer technologies, as well as RNA biology and cancer. The work that Rasko will discuss at the Lorne Cancer (and Genome) meetings (and published recently in Cell) started about 5 years ago and is based on over a decade of research aimed at better understanding small non-coding (snc) RNA molecules and their potential targets in white blood cells (granulocytes).

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A PUZZLE TO BE SOLVED

Until relatively recently, non-coding nucleic acid sequences all fell into the ‘junk’ category of genetic information, despite their abundance in genomes and conservation across species. But in light of the many studies revealing this assumption to be false, not least of which the microRNA story and Rasko’s own work, his team set out with a firm hypothesis about the role of non-coding RNA molecules and gene splicing mechanisms in white blood cell development (granulopoiesis). They then proceeded to establish all of the pieces needed to solve their particular puzzle.

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“We amassed a vast amount of data from RNA sequencing to the whole suite of ‘omics’ - plus a full bioinformatics toolkit,” recalled Rasko. However, as so often happens in science, their initial hypothesis turned out to be completely wrong. Having invested significant time and money, they turned to a back-up plan. The result was a nifty new computer algorithm that allowed them to totally reanalyse their data and explore some fairly intractable questions about the role of introns in granulocyte differentiation. And basically, they hit gold by uncovering a completely unimagined mechanism of gene expression regulation.

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INTRON RETENTION | LORNE CANCER CONFERENCE

According to Rasko, it really was an ‘OMG!’ moment. Dr Justin Wong with his background in epigenetics and molecular biology then set about devoting the next two years to prove the idea with other members of the group.

MORE THAN ONE WAY TO SPLICE A GENE

So why look at introns? According to Rasko, mystery has always surrounded these parts of the genome. Introns are highly conserved, noncoding regions that sit among the gene exons, which are those bits of the genome that are eventually translated

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Mouse granulocytes examined by transmission electron microscopic. Magnification ×3000, scale bar 1 micron.

into proteins and, thus, long regarded the most important genetic components of cells. By looking at the non-coding regions of genomes, Rasko and his team have found that introns can and do control the expression levels of perhaps hundreds of genes that are differentially expressed during the development of white blood cells of the innate immune system. During gene transcription, the introns in most eukaryotic genes are removed from pre-messenger RNAs by the cell’s splicing machinery to produce proteintranslatable mature mRNAs. However, the introns are sometimes retained as part of the mature mRNA sequence by an alternative splicing mechanism called intron retention (IR). Most IR is thought to reflect cellular ‘mistakes’, often due to splicing

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mutations, that are dealt with by a cell’s garbage collection machinery. However, RNAs transcribed from intronic regions have been implicated in a number of processes related to the posttranscriptional control of gene expression and are tipped to regulate gene expression in viruses and some plants. How IR plays a role in normal gene expression, which tissues are affected and why it happens remains largely unknown.

THE WHITE BLOOD CELL MODEL

The final phases of granulocyte maturation in the bone marrow - from promyelocyte to myelocyte to mature granulocyte - are marked by distinct transcriptional and translational changes, accompanied by little-understood

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LORNE CANCER CONFERENCE | INTRON RETENTION

changes in the shape and size of the cell, especially the nucleus. These unique cellular stages can be easily isolated based on specific cell-surface markers and thus provide a valuable and well-studied model for cellular differentiation. Switching questions was therefore an easy decision for Rasko because it turns out that granulocytes also have quite a lot of IR going on. In fact, removal of transcribed intronic material via the cell’s nonsense-mediated decay (NMD) pathway is somehow essential for the normal development of haematopoietic stem and progenitor cells. Rasko’s group was therefore keen to examine why these cells retained so many introns and whether this phenomenon was functionally relevant to the specific and complex steps of granulocyte maturation. FINDING THE BABY IN THE BATH WATER

To approach this slightly different focus, the researchers realised that by the very nature of RNA sequencing technology and software, much of the information they might need about introns is actually thrown out. And, according to Rasko, their eventual ‘ah-ha’ moment was a direct consequence of this realisation. “When we do deep RNA sequencing, we basically ask the machine to analyse an immense catalogue of relatively short bits of sequence and match them back to unique locations on a reference genome to create the sequence map,” said Rasko. However, introns often contain degenerate or low-complexity stretches that are, statistically speaking, harder to match to a unique site in the genome. To deal with the zillion bits of data involved in sequencing a whole genome, the machine therefore has to preferentially ditch most of the sequencing data that is specific for introns. “To get around this, Dr William Ritchie developed and applied a novel and very rigorous computer algorithm, called IRFinder, to extract information solely about introns from all the data we had already collected for our different granulocyte cell populations; hopefully to pinpoint where the introns are being retained,” explained Rasko.

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Professor John Rasko AO is a clinical haematologist and director of Cell and Molecular Therapies at the Royal Prince Alfred Hospital in Sydney and heads the Gene and Stem Cell Therapy Program at the University of Sydney. He studied medicine at the University of Sydney and completed a PhD at the WEHI under Professor Don Metcalf and Dr Glenn Begley in molecular haematology. He then pursued postdoctoral research in gene therapy at the birthplace of bone marrow transplantation: the ‘Hutch’ in Seattle. His work in gene therapy, experimental haematology and cell biology has led to discoveries involving new mechanisms of leukaemia, blood hormones, stem cells RNA biology, amino acid transport and clinical trials of new therapies for cancer and bleeding disorders.

“And that is the moment when we knew we had something big, because there were all these examples, almost 100 genes even conservatively, that were retaining introns and being affected functionally and differentially by IR during normal granulopoiesis in terms of reduced protein expression. We knew we had uncovered a major mechanism of gene control that was previously ignored because people basically could not see the data for this lowcomplexity stuff.

LOTS MORE TO LEARN

Adding weight to this importance was the team’s subsequent finding that the genes affected by IR in granulopoiesis are conserved between mouse and human.

“We are obviously very excited about this work and certainly pursuing IR in this context to explore how broadly conserved it is. Just when did nature invent this mechanism of gene expression control? And what other cell and tissue types are affected by IR as a normal part of gene expression, including how early in differentiation does it appear … stem cells for instance? “Also, why some introns or genes and not others? What molecular markers are present in those genes that cause the splicing machinery to ignore them in an apparently normal physiological process? What sets it off in the first place remains an interesting and open question.” Rasko already knows from their work and that of collaborators that IR also affects lymphoid cells, and probably neuronal cell production. Moreover, it is very likely to be aberrant in disease states such as leukaemia and thus may offer new therapeutic targets - indeed, this is the subject of a new NHMRC grant for Rasko’s group. “If you had told me five years ago that we were going to discover a completely new mechanism of gene expression regulation in mammalian cells I would have laughed,” said Rasko, about the possibility of yet more to discover. “A fantastically dedicated local team along with our collaborators and funding from diverse sources have revealed that previously unimagined mechanisms are ripe for discovery … if only we keep an open mind! And we should keep looking for even more!” ALS

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“Finally, Drs Jeff Holst and Chuck Bailey in the lab showed that by perturbing IR specifically in a gene called Lmnb1, which shows a very high level of intron retention, we saw a dramatic alteration in nuclear morphology and cell numbers,” Rasko continued. “The result was unexpected and very exciting because it suggested to us that, in contrast to the prevalent view that such mechanisms are limited to mRNAs encoding aberrant proteins, IR is a critical regulatory pathway programmed into the cell to operate during normal granulocyte development. This was not some accident or some failed intron excision … these are highly specific introns in particular genes. And it is occurring when there is no disease state or mutation.”

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LORNE CANCER CONFERENCE | TUMOUR MARKERS

microenvironment M

alignant cells need to interact with and often actively recruit all other cells and structures in their surroundings to grow and prosper, and eventually to metastasise into the most deadly form of cancer. Together, these relationships make up the tumour microenvironment (TM). It is now being realised that the TM is critical for the cancer’s survival and also, in the context of therapy, a tumour’s response to treatment. Dr Roberta Mazzieri of the Diamantina Institute in Brisbane has studied TMs for the past eight years; in particular, a subpopulation of bone marrow-derived monocytes that she helped to identify as a postdoc in Italy (reported in Blood in 2007). These white blood cells are characterised by the expression of a specific cell-surface receptor, the Tie-2 angiopoiein receptor, and thus are called Tie-2-expressing monocytes or TEMs. EARLY FINDINGS REVEAL A POTENTIAL CANCER TARGET

TEM cells, which are present in the blood of mice and humans only at very low frequency, are recruited specifically to the site of a tumour. There they differentiate into macrophages, set up shop in a highly specific pattern along the walls of surrounding blood vessels and markedly upregulate their production of the Tie-2 receptor. It was these initial findings that first suggested to Mazzieri and her co-workers that TEMs, and their Tie-2activated signalling pathway, could be functionally important for maintaining the TM and thus also be potential tumour markers and targets for therapy. Mazzieri has worked mostly on breast cancer,

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but these cells have now been isolated with several tumour types.According to Mazzieri, one good thing about these TEMs is their characteristic Tie-2 expression because it makes them relatively easy to isolate and study. Gene profiling of isolated TEMs and comparison to similar cell types within the TM defined them as M2 macrophages, which are typically pro-tumourigenic cells that function in tissue remodelling. “This indicated to us that tumours might use the Tie-2 activity and remodelling abilities of TEMs to modulate their environment; for example, by promoting new blood vessel growth (angiogenesis).” Generally, tumours need to remodel the tissue in which they are growing to make space and to induce new vessel formation to supply their often-significant energy needs. Mazzieri’s next step was to ask what these cells actually do in the TM. Using an existing lentiviral expression system and a transducible suicide gene linked to the Tie-2 gene promoter, they engineered a microenvironment with or without TEMs in a mouse tumour model. “This type of specific depletion experiment showed that TEMs are absolutely required for tumour growth in vivo, because eliminating them stopped tumour growth and also strongly inhibited angiogenesis,” Mazzieri explained. “This information, together with the gene profiling, indicated that the TEMs promote tumour angiogenesis. Moreover, TEMs isolated from humans and co-injected back into a mouse could accelerate tumour growth, suggesting a potentially critical role of TEMs in human cancer progression.”

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Fiona Wylie

DELVING DEEPER INTO THE TUMOUR JUNGLE

To further investigate the Tie-2 connection, Mazzieri took advantage of an established collaboration between her group in Italy and global biopharmaceutical company AstraZeneca, who have developed an anti-angiopoietin-2 antibody as an agent to inhibit tumour angiogenesis. Angiopoietin-2 (Ang-2) is one of the main ligands for activating the Tie-2 receptor pathway and is also involved in tissue remodelling events; thus, an antibody for Ang-2 could be used to examine angiogenic pathways in the TM. “When we treated tumour-bearing mice with the antibody, we saw a really strong inhibitory effect on tumour growth, both primary and metastatic,” explained Mazzieri, “and we saw vessel regression in the treated tumours; that is, we inhibited angiogenesis.” Further experiments confirmed that the antibody therapy was indeed targeting the TEM cells. It was specifically disturbing their pro-angiogenic effects in the TM as well as their characteristic geographical association along the vessels. The researchers now knew for sure that this Ang2-Tie-2 signalling axis could be a powerful tumour target. “Interestingly, around the same time, one of our collaborators showed that Ang-2 could also modulate the immunosuppressive activity of the TEMs, which they do in addition to being pro-angiogenic,” recalled Mazzieri. Immune suppression is an important escape mechanism for tumours, helping them evade destruction by the body’s immune system and tumours do all they can to create an immunosuppressive

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Managing the cancer

TUMOUR MARKERS | LORNE CANCER CONFERENCE

Immunostaining of CD31 (endothelial cell/vessel, red), green fluorescent protein (GFP) (Tie-2-expressing monocytes (TEMs), green) and nuclei (blue) of A431 mammary tumours upon transplantation of Tie2GFP haematopoietic stem cells.

environment. And this is where the fantastic nexus between cancer and immunology going on at the Diamantina Institute (DI) comes to the fore for Mazzieri’s work. “Here at the DI, I plan to establish whether this anti-Ang-2 treatment, now under clinical testing, inhibits both these activities of TEMs in vivo. Such a finding would have important clinical implications because if we can somehow modulate or revert the immunosuppressive environment around a tumour by targeting the TEM activities, then we can think of combining this anti-angiogenic treatment with tumour immunotherapy that would not work otherwise due to the immunosuppression.” USING THEIR POWERS FOR GOOD

A cunning second half of Mazzieri’s plan is to exploit the TEMs as cellular vehicles to deliver cancer-treating drugs directly into the sites of tumours. “To do this we again took advantage of the cells’ own characteristics; that is, whatever TEMs produce in terms of an anti-tumour molecule will be at low levels systemically but high at the tumour site, and that is ideal. And because they are haematopoietic cells, you can isolate the stem cells from bone marrow, modify them ex vivo to express your molecule of choice and then transplant them back into a mouse or human where they will reconstitute the haematopoietic compartment and differentiate into mature TEMs.” Voila - tumour homing pigeons! Mazzieri has already published this gene and cell-based therapy approach as a proof of principle paper using interferon-alpha as the anti-tumourigenic drug. “By making TEMs produce interferonalpha under the control of the Tie-2

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promoter, we could inhibit the growth of primary and metastatic breast cancers in mice,” she said. However, one of the main roadblocks to immunotherapy strategies for cancer is the availability of good preclinical models that take all the different players of the typically complex, heterogeneous and evolving tumour microenvironment into account. TAKING IT INTO HUMANS

To address this, Mazzieri and her team developed a humanised delivery platform for breast cancer. Basically, they start with an immunodeficient mouse into which they transplant human haematopoietic stem cells to make a human haematopoietic compartment that produces human TEMs. The human haematopoietic stem cells can be purified and modified ex vivo for re-injection into the mice. “If we also inject human tumour cells into these mice we can test whether human TEMs are able to inhibit the growth of human tumours,” explained Mazzieri. And it works a treat, according to Mazzieri, and the paper describing this

research has just been accepted. In short, they could modify the TM by making TEMs that produce interferon-alpha, which is also a pro-inflammatory protein and can thus revert the immunosuppressive microenvironment. “Interferon-alpha activates both the innate and adaptive immune responses and we demonstrated in the mouse and in the humanised model that this seems to be enough to inhibit tumour growth,” Mazzieri said. “So, again I would like to combine this strategy with immunotherapy for future clinical use, taking advantage of the excellent tumour immunology going on here in the institute to develop a better antitumour metastasis therapy. “Overall, my work involves two different approaches. In one we are targeting the activities of TEMs and in the other we are using them as a vehicle to deliver a drug,” she continued, “and in both cases, we are modulating the immunosuppressive environment surrounding a tumour, and that is why in both cases I want to combine it with immunotherapies that are either in the clinic or under development.” DOWN THE TRACK

Mazzieri’s hopes going forward are that the recent push continues to reveal more about the complexity and heterogeneity of these TM interactions, for each tumour type and, more importantly, for each step in tumour progression. “Understanding how these interactions evolve will give us the opportunity to develop more targeted and efficient therapies, particularly in the metastatic and most lethal forms of cancer.” ALS

Dr Roberta Mazzieri obtained her PhD in Genetic Science from the University of Pavia (Italy) followed by postdoctoral positions at the New York University (USA) and the San Raffaele Scientific Institute in Milan (Italy). Since her PhD studies, she has focused on understanding the tumour microenvironment. In 2011, her work into the Tie2-expressing monocytes and their potential in tumour detection and therapy was featured as a cover article in Cancer Cell. In 2012 she was recruited by the University of Queensland to establish her own research group at the Diamantina Institute studying the predictive markers and novel metastatic therapies to successfully target cancers.

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J A N U A R Y/ F E B R U A R Y 2 014

LORNE GENOME CONFERENCE | EPIGENETIC REGULATION

Graeme O'Neill

The ‘omics’ of cancer I

t’s 42 years since President Richard Nixon declared war on cancer, hoping to trump President John F Kennedy’s successful 1962 promise to put an American on the Moon by 1970. A cure-all for cancer remains as remote a prospect as the first human colony on the Moon. Survival times for many cancers have increased because of earlier diagnosis, and the advent of life-prolonging drugs. But oncology’s greatest need - and greatest challenge - is to develop fast-acting therapies that might save the lives of patients at imminent risk of succumbing to end-stage, drugresistant cancers. Associate Professor Pilar Blancafort’s research group at the University of Western Australia has provided a glimpse of the future, in the form of two novel therapies for potential use in combination in late-stage basal breast carcinomas. These new synthetic molecules are already moving towards being trialled in animals. TRIPLE NEGATIVE CARCINOMA

Dr Blancafort, a plenary speaker at the Lorne Genome Conference, will describe her group’s progress towards developing effective treatments for late-stage basal carcinomas of the breast. Unlike most breast cancers, so-called ‘triple-negative’ carcinomas do not express estrogen, progesterone or human epidermal growth-factor 2 (Her2-neu) receptors, so they are not amenable to

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treatment with front-line endocrinereceptor antagonists like tamoxifen and herceptin. Blancafort and her colleagues hope their new molecules will provide a lifeline for women diagnosed with an uncommon and highly aggressive form of triple-negative breast carcinoma, known as late-stage inflammatory breast cancer. DIRECT PROMOTER INTERACTION AS THERAPY

Blancafort says a cell’s transformation from a normal to a cancerous state involves wholesale changes in the epigenetic deregulation of key genes that are transmitted to successive generations of daughter cells. In cancerous cells, demethylases reactivate repressed transcription factors that maintain the multipotency of progenitor cells that build specialised tissues and organs. However, in cancerous cells the direction of development is reversed: daughter cells regress to a relatively primitive, unspecialised state that, in time, becomes resistant to chemotherapy. With nothing to restrain their rapid growth and proliferation, the malignant cells spread to other organs, where they give rise to aggressive, metastatic tumours. Meanwhile, methyltransferases progressively methylate large segments of chromosomes, inactivating vital tumoursuppressor genes like P53. Without these sentries to perform quality-control checks on newly replicated DNA, daughter cells

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accumulate dangerous new mutations and malignant new methylation states that drive them towards uncontrolled cancerous growth. Modern breast cancer drugs typically target endocrine receptors that study the surface of cancerous cells. Blancafort’s group has taken a completely different tack, by designing synthetic molecules capable of being transported into the cell to interact directly with the promoters of the key genes driving cancerous growth. Their prime target in triple-negative breast cancers is the gene for the transcription factor engrailed (EN1), an early player in patterning the development of the central nervous system. SYNTHETIC INTERFERENCE PEPTIDES

In a paper published in Oncogene in 2013, Blancafort’s group reported that EN1 is selectively overexpressed in triplenegative breast carcinomas. Normally, it is expressed in neural progenitor cells. Later in life it appears to maintain the brain’s pool of long-lived dopaminergic neurons through its prosurvival, anti-apoptotic activity. Blancafort’s team showed that short interfering RNAs targeted to EN1 triggered potent, selective death of basal breast tumour cells, explaining the cancer’s propensity to develop drug resistance, and to re-emerge and metastastise, after surgery and chemotherapy. They engineered synthetic

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EPIGENETIC REGULATION | LORNE GENOME CONFERENCE

interference peptides (iPeps), containing a peptide sequence that mediates the EN1 protein’s interactions with other proteins. The iPeps also contained a cell-penetrating sequence that causes it to ‘home’ to the cell nucleus. MALDI-TOF mass spectrometry confirmed that the synthetic peptide captured proteins involved in transcriptional and post-transcriptional regulation of proteins involved in inflammatory pathways. The finding established EN1’s role as an activator of intrinsic inflammatory pathways associated with pro-survival in basal-like breast cancer. CANCER-SPECIFIC COMBINATION THERAPIES

Blancafort’s team has confirmed the potential of a similar synthetic peptide to knock down EN1, blocking its proinflammatory, pro-survival activity. “It works at micromolar concentrations, which is still a bit high. We want to chemically optimise the peptide to be effective at lower concentrations, and stabilise it to improve its half-life, and to avoid the body’s clearance systems,” Blancafort said. Blancafort’s team has also encapsulated the iPep in nanoparticles and targeted it to cancerous cells using ligands, including cell-killing drug molecules, to produce synergistic activity. “Using small amounts of the drug, you get the double benefit of knocking down the transcription factor that drives the cancer, and resensitising resistant cells to the drug’s cell-killing activity. “We’re looking at extending this approach to other transcription factors and developing cancer-specific combination therapies, so you get an interplay that modulates several signalling pathways, so you kill the cancerous cells at lower dosages." ANIMAL TRIALS

Blancafort says her team is planning animal trials to optimise the pharmacokinetics of their molecules, to ensure they combine high specificity with low toxicity. They are developing another novel therapeutic molecule

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Dr Pilar Blancafort is Associate Professor in the School of Anatomy, Physiology and Human Biology at the University of Western Australia. Born in Santa Eugenia de Berga, 100 km from Barcelona, in Spain, she studied physics and biology at the University of Barcelona, specialising in biochemistry, genetics and molecular biology. In the last year of her undergraduate studies, she was awarded an ERASMUS (European Community Action Scheme for Mobility of University Students) Fellowship to study at the Free University of Brussels where she did an Honours project with Dr Alain Ghysen, studying neural transcription factors in Drosophila - her first contact with transcription factors and development, the field she specialises in today.

that targets SOX-2, an oncogenic transcription factor overexpressed in a variety of malignancies characterised by a high recurrence rate and poor patient prognosis. Blancafort says an explosion of studies 2012 and 2013 implicated SOX-2 in the initiation, progression and recurrence of highly aggressive forms of breast and ovarian cancer, prostate cancer, glioblastomas, hepatocellular carcinoma and small cell lung cancer. In normal breast ductal cells, SOX-2 is epigenetically silenced by methylation of its promoter, but it is hypomethylated and overexpressed in nearly 50% of basal-like triple-negative breast carcinomas. Blancafort’s team identified SOX-2 as a potential key target for an experimental therapy with a synthetic zinc-finger molecule that will bind to its promoter, replicating the natural silencing effect of methylation. The suppressor molecule consists of a sequence C2H2 zinc-finger domains; each recognises and binds one unit of a repeated three base-pair DNA motif in the SOX-2 promoter region. While a three-unit ZF ‘zipper’ is probably sufficient to inactivate SOX-2, Blancafort says the six-unit molecule actually outcompetes the endogenous transcription factors and potently regulates SOX-2. With its exceptional avidity for the SOX-2 promoter, the synthetic ZF ‘off switch’ should work at safe, picomolar concentrations and provide ample time for drugs to kill the

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resensitised cells. She says the ZF ‘silencer’ system provides a model for experiments with other synthetic peptide complexes that will outcompete native ligands to occupy the promoters of other oncogenic transcription factors. A WHOLE GENOME PERSPECTIVE

Blancafort says such promoter-targeting synthetic complexes look particularly promising as prospective therapies for some of the most aggressive forms of cancer, involving cells in which wholesale disruption of normal epigenetic controls have regressed to a more primitive, stemlike state, including the capacity to grow and proliferate rapidly. Blancafort says the amplification and overexpression of oncogenes like Myc and Ras in aggressive cancers is a challenge for conventional therapies - the new synthetic silencing complexes offer the advantage that they can be designed to precisely target key oncogenes that drive these refractory cancers and displace the native factors driving their overexpression. Blancafort says cancers offer many targets for new therapeutic drugs and the future of the new synthetic molecules lies in developing combination therapies to hit multiple targets simultaneously. “We have so much ‘omics’ information now, so it’s a matter of identifying the important targets in different forms of cancers. A model system like ours helps us to understand how these molecules work, what happens over time and how they interact.” ALS

J A N U A R Y/ F E B R U A R Y 2 014

LORNE GENOME CONFERENCE | RNA-BINDING PROTEINS

Enzymes with alter egos © iStockphoto.com/vitanovski

Graeme O'Neill

nzymes are well known for their housekeeping role in cells, but some enzymes bind RNA and might play a significant role in linking intermediary metabolism to gene expression via post-transcriptional regulation. Professor Matthias Hentze, of the European Molecular Biology Laboratory (AMBL) in Heidelberg, Germany, and Professor Thomas Preiss, now Professor of RNA Biology at the Australian National University’s (ANU’s) John Curtin School of Medical Research, will be taking their longstanding research collaboration to a new level, after years of communicating via Skype. After giving his plenary lecture to the 2013 Genome meeting, Hentze, who is bringing five of his top people with him to Lorne, will head to the surf and sand of Kioloa for a working retreat with his team and members of Preiss’ team. Preiss was a postdoctoral researcher in Hentze’s laboratory from 1995 to 2002. BINDING RNA

Their main topic for discussion will be enzymes with alter egos - recent decades have seen sporadic reports that certain enzymes, as well as mediating classic metabolic reactions in cells, act as RNAbinding proteins. “We work together in two overlapping areas,” Preiss said. “One is the question of

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the regulatory connection between gene expression and cellular metabolism - we see that as one of the big challenges, because it requires us to combine the largely separate disciplines of molecular biology and biochemistry. “Over the past few decades, a number of research groups have made the curious observation that certain metabolic enzymes have an RNA-binding function, but in most cases the physiological role of this function has remained unclear.” POST-TRANSCRIPTIONAL REGULATORY NETWORKS

Preiss says that in 1987 Hentze co-discovered iron-responsive elements (IREs), the first regulatory elements found in mammalian messenger RNAs (mRNAs). They occur in the untranslated regions (UTRs) of mRNAs encoding for proteins involved in iron transport and storage and interact with iron regulatory proteins (IRPs). Remarkably, one of the IRPs, IRP1, is a bifunctional protein that in iron-replete cells ligates an iron-sulfur cluster to function as an aconitase enzyme, catalysing the stereospecific isomerisation of citrate to isocitrate. Its alter ego in iron-deficient cells adopts its RNA-binding (apo-enzyme) conformation. Preiss says that while enzymes of course have been widely studied for their regular, ‘housekeeping’ roles in cell metabolism, little attention has been paid to their RNAbinding functions.

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In an opinion article in Trends in Biochemistry in 2010, Hentze and Preiss suggested that RNA-binding enzymes might frequently be players in a class of posttranscriptional regulatory networks, linking intermediary metabolism to gene expression, through interactions between RNA, enzymes and metabolites (REMs). MRNA INTERACTOME CAPTURE

Further, they challenged the view that enzymes are mainly post-translationally regulated by direct feedback from fluctuating levels of their own metabolites, changes in nutrient availability, redox state, oxygen tension or stress. “One of the things we would like to do is to identify more broadly which enzymes in different cells can bind mRNA,” Preiss said. “Matthias came up with a clever way to identify all cellular RNA-binding proteins, that he calls ‘mRNA interactome capture’. “It uses ultraviolet light to cross-link RNA-binding proteins their RNA targets in vivo. Oligo dT beads are then used to capture mRNAs by their polyA tails, allowing the attached proteins to be recovered and identified by mass spectrometry.” Preiss says the technique, published in a recent paper in Cell, has revealed a previously unsuspected abundance of mRNA-binding proteins in eukaryotic cells, pointing to the existence of a system of RNA ‘operons’ that post-transcriptionally regulate the activity of messenger RNAs with related

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Professor Thomas Preiss (L) is Professor of RNA Biology at the Australian National University's John Curtin School of Medical Research and Professor Matthias Hentze (R) is co-director and co-founder of the Molecular Medicine Partnership Unit between the European Molecular Biology Laboratory (EMBL) and the Medical Faculty of Heidelberg University. After completing a PhD at the University of Newcastle Upon Tyne, Preiss spent seven years as a postdoctoral researcher in Professor Hentze's EMBL laboratory, before being appointed laboratory head at the Victor Chang Cardiac Research Institute at Sydney. After completing an MD at the University of Munster in North Rhine-Westphalia, Hentze undertook a postdoctoral appointment at the National Institutes of Health and then joined EMBL Heidelberg as a Group Leader in 1989. He has been a senior scientist with EMBL since 1998, specialising in metabolism, RNA biology and molecular medicine.

functions after they are released into the cytoplasm. “Conceptually, mRNA-binding protein functions are quite similar to the roles shown for microRNAs,” Preiss said. “By coordinating the functions of messenger RNAs, RNA-binding proteins endow cells with extra regulatory potential.” A NEW WINDOW ON GENETIC DISEASE

More than 1100 proteins in cultured human and other eukaryotic cells have now been identified as having RNA-binding activity - at least 350 of them were not previously known to have RNA-binding roles. Preiss says the collaboration between the ANU and EMBL teams is filling out details of the interactions within these post-translational regulatory networks by

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investigating, in specific cellular contexts, what groups of mRNAs interact with RNA-binding proteins - “RNA-binding proteins operate in parallel, and interact cooperatively”, he said. In a recent review in Trends in Genetics, Preiss and Hentze and two other colleagues, Alfredo Castello and Bernd Fischer, further described how errors in these networks may explain a range of inherited disorders. Complex systems are fraught with potential to malfunction, and the ANUEMBL team’s growing catalogue of RNAbinding proteins has opened up a new window on genetic disease. The ANU-EMBL paper notes that the mRNA interactome contains many proteins that, in mutant form, are associated with Mendelian disorders - predominantly of

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neurological and sensory systems, muscular atrophies, metabolic disorders and cancers. The specific mutations involved in such disorders are scattered through the domain architectures of the proteins. Many occur in non-classical RNA-binding domains and in unfolded epitopes. In some cases, the mutations might perturb previously unrecognised RNArelated functions of the proteins. “We also expect that mRNA interactome capture approaches will aid further exploration of RNA systems biology in varied physiological and pathophysiological settings,” the authors write. POST-TRANSCRIPTIONAL REGULATION HUBS

A wealth of literature now attests to the role of RNA-binding proteins, as well as noncoding RNAs including microRNAs, in directing and regulating the posttranscriptional fate of messenger RNAs in the nucleus and cytoplasm. RNA binding proteins variously influence splicing and the formation of the 3’ regions of mRNAs, as well as posttranscriptional editing of mRNAs, their localisation in cell compartments, translation into protein and mRNA turnover, “often in a dynamic and cell type-specific manner”. The authors say the recent discovery of widespread, regulated, alternative mRNA 3’-end formation in many cellular and disease contexts underscores the importance of 3’UTRs as hubs of post-transcriptional regulation - and, by implication, as likely foci for mutation-induced mischief. ALS

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Prof. Dr. Martin Müller Medizinische Fakultät Mannheim der Universität Heidelberg Dr. Nicola Gökbuget University Hospital Frankfurt, Frankfurt, Germany Dr. Vinay Tergaonkar Institute of Molecular and Cell Biology, Singapore National Speakers Dr Ben Kile Dr Mark Dawson Dr Kylie Mason Professor Rajiv Khanna Professor Richard D’Andrea Dr Ingrid Winkler Professor John Rasko Professor Don Metcalf Donald Metcalf Oration Professor Charles Mulligan

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PUBLISH OR PERISH

The return of our regular round-up of some of the best Australian research published each month in leading peer-reviewed journals.

Alaidarous M, Ve T, Casey LW, Valkov E, Ericsson DJ, Ullah MO, Schembri MA, Mansell A, Sweet MJ, Kobe B. Univ of Qld Mechanism of bacterial interference with TLR4 signaling by Brucella TIR-domain-containing protein TcpB.

J Biol Chem. 2013 Nov 21.

Callaghan BL, Li S, Richardson R. UNSW The elusive engram: what can infantile amnesia tell us about memory?

Trends Neurosci. 2013 Nov 25.

Christensen J, Steain M, Slobedman B, Abendroth A. Univ of Syd Varicella-zoster virus glycoprotein I is essential for spread in dorsal root ganglia and facilitates axonal localization of structural virion components in neuronal cultures.

J Virol. 2013 Dec;87(24):13719-28.

Garton FC, Seto JT, Quinlan KG, Yang N, Houweling PJ, North KN. Child’s Hosp Westmead, Sydney. α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization.

Hum Mol Genet. 2013 Nov 29.

Genovesi LA, Ng CG, Davis MJ, Remke M, Taylor MD, Adams DJ, Rust AG, Ward JM, Ban KH, Jenkins NA, Copeland NG, Wainwright BJ. IMB, Uni of Qld Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

Proc Natl Acad Sci USA. 2013 Nov 12;110(46):E4325-34.

Nature 2013 Nov 14;503(7475):198.

Lim SC, Friemel M, Marum JE, Tucker EJ, Bruno DL, Riley LG, Christodoulou J, Kirk EP, Boneh A, DeGennaro CM, Springer M, Mootha VK, Rouault TA, Leimkühler S, Thorburn DR, Compton AG.Murdoch Child Res Inst, Royal Child's Hosp, Vic Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes.

Hum Mol Genet. 2013 Nov 15;22(22):4460-73.

Mahar JE, Donker NC, Bok K, Talbo GH, Green KY, Kirkwood CD.Royal Child’s Hosp, Vic Identification and characterization of antibodybinding epitopes on the norovirus GII.3 capsid.

J Virol. 2013 Nov 27.

McConville MJ, Ralph SA. Univ of Melb Chronic arsenic exposure and microbial drug resistance.

Proc Natl Acad Sci USA 2013 Dec 3;110(49):1966-7. Park HY, Light A, Lahoud MH, Caminschi I, Tarlinton DM, Shortman K. WEHI, Vic Evolution of B cell responses to Clec9A-targeted antigen.

J Immunol. 2013 Nov 15;191(10):4919-25.

Pera MF.Univ of Melb, WEHI, and Florey Neurosci and Mental Hlth Inst, Vic Epigenetics, vitamin supplements and cellular reprogramming.

Nat Genet. 2013 Nov 25;45(12):1412-3.

Groszmann M, Greaves IK, Fujimoto R, James Peacock W, Dennis ES. CSIRO, Plant Ind, ACT The role of epigenetics in hybrid vigour.

Trends Genet. 2013 Dec;29(12):684-90.

Jefferson OA, Köllhofer D, Ehrich TH, Jefferson RA.Cambia, ACT and QUT, Brisbane Transparency tools in gene patenting for informing policy and practice.

Nat Biotechnol. 2013 Dec 6;31(12):1086-93.

Job ER, Bottazzi B, Short KR, Deng YM, Mantovani A, Brooks AG, Reading PC. Univ of Melb A single amino acid substitution in the hemagglutinin of H3N2 subtype influenza A viruses is associated with resistance to the long pentraxin PTX3 and enhanced virulence in mice.

J Immunol. 2013 Dec 4.

Kartopawiro J, Bower NI, Karnezis T, Kazenwadel J, Betterman KL, Lesieur E, Koltowska K, Astin J, Crosier P, Vermeren S, Achen MG, Stacker SA, Smith KA, Harvey NL, François M, Hogan BM. IMB, Univ of Qld Arap3 is dysregulated in a mouse model of hypotrichosis-lymphedema-telangiectasia and regulates lymphatic vascular development.

Hum Mol Genet. 2013 Nov 11.

Licinio J, Wong ML.SAMRI and Flinders Univ, SA. Research infrastructure: US shutdown should spur other nations.

Perry CJ, Barron AB. Macquarie Univ, Sydney Honey bees selectively avoid difficult choices.

Proc Natl Acad Sci USA 2013 Nov 19;110(47):19155-9.

Pouliopoulos J, Chik WW, Kanthan A, Sivagangabalan G, Barry MA, Fahmy PN, Midekin C, Lu J, Kizana E, Thomas SP, Thiagalingam A, Kovoor P. Westmead Hosp, Univ of Sydney, and Westmead Mill Inst, Sydney Intramyocardial adiposity after myocardial infarction: new implications of a substrate for ventricular tachycardia.

Circulation 2013 Nov 19;128(21):2296-308.

Puccini J, Shalini S, Voss AK, Gatei M, Wilson CH, Hiwase DK, Lavin MF, Dorstyn L, Kumar S. SA Pathology Loss of caspase-2 augments lymphomagenesis and enhances genomic instability in Atm-deficient mice.

Proc Natl Acad Sci USA 2013 Dec 3;110(49):19920-5. Puri R, Nissen SE, Libby P, Shao M, Ballantyne CM, Barter PJ, Chapman MJ, Erbel R, Raichlen JS, Uno K, Kataoka Y, Nicholls SJ. UNSW, SAHMRI and collaborators C-reactive protein, but not low-density lipoprotein cholesterol levels, associate with coronary

atheroma regression and cardiovascular events after maximally intensive statin therapy.

Circulation 2013 Nov 26;128(22):2395-403.

Reilly MT, Faulkner GJ, Dubnau J, Ponomarev I, Gage FH. Mater Med Res Inst, Qld The role of transposable elements in health and diseases of the central nervous system.

J Neurosci. 2013 Nov 6;33(45):17577-86.

Secco D, Jabnoune M, Walker H, Shou H, Wu P, Poirier Y, Whelan J. Univ of WA Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery.

Plant Cell. 2013 Nov 18.

Shan L, Saxena A, McMahon R, Newcomb A. St Vincent’s Hosp, Vic and SE Syd and Illawarra Hlth Ntwk, NSW Coronary artery bypass graft surgery in the elderly: a review of postoperative quality of life.

Circulation 2013 Nov 19;128(21):2333-43.

Sprent J. Garvan, Sydney The power of dilution: using adoptive transfer to study TCR transgenic T cells.

J Immunol. 2013 Dec 1;191(11):5325-6.

Trejo CL, Green S, Marsh V, Collisson EA, Iezza G, Phillips WA, McMahon M. Peter Mac, Univ of Melb, Vic Mutationally activated PIK3CAH1047R cooperates with BRAFV600E to promote lung cancer progression.

Cancer Res. 2013 Nov 1;73(21):6448-61.

Vinkhuyzen AA, Wray NR, Yang J, Goddard ME, Visscher PM. Univ of Qld, Qld Brain Inst Estimation and partition of heritability in human populations using whole-genome analysis methods.

Annu Rev Genet. 2013 Nov 23;47:75-95.

White CW, Choong YT, Short JL, Exintaris B, Malone DT, Allen AM, Evans RJ, Ventura S. Monash Univ, Vic and collaborators Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice.

Proc Natl Acad Sci USA 2013 Dec 2.

Yip YY, Yeap YY, Bogoyevitch MA, Ng DC. Univ of Melb cAMP-dependent protein kinase and c-Jun N-terminal kinase mediate stathmin phosphorylation for the maintenance of interphase microtubules during osmotic stress.

J Biol Chem. 2013 Dec 3.

Zheng H, Zhang W, Zhang L, Zhang Z, Li J, Lu G, Zhu Y, Wang Y, Huang Y, Liu J, Kang H, Chen J, Wang L, Chen A, Yu S, Gao Z, Jin L, Gu W, Wang Z, Zhao L, Shi B, Wen H, Lin R, Jones MK, Brejova B, Vinar T, Zhao G, McManus DP, Chen Z, Zhou Y, Wang S. QIMR Berghofer and Chinese collaborators The genome of the hydatid tapeworm Echinococcus granulosus.

Nat Genet. 2013 Oct;45(10):1168-75.

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EVENTS

The coming year is packed with exciting local and international events. Here’s a taste.

The World Congress of Cardiology 2014 (WCC 2014) May 4-7, Melbourne

www.worldcardiocongress.org/

ACMM23 and ICONN 2014

February 2-6, Adelaide, South Australia The 23rd Australian Conference on Microscopy and Microanalysis and the 2014 International Conference on Nanoscience and Nanotechnology will be jointly held in Adelaide at the beginning of February. ICONN 2014 will bring together researchers, industry, students and early-career scientists in the burgeoning area of nanotechnology. The meeting will include presentations on the latest discoveries in nanomaterials, nanophotonics, nanobiotechnology and nanoelectronics, as well as investigations into nanoethics, nanosafety and industry applications. The Australian Conference for Microscopy and Microanalysis is the largest meeting of its type in the Southern Hemisphere and a key meeting for researchers, practitioners and industrial scientists involved in microscopy and microanalysis. The meeting will showcase the latest developments in the instrumentation, specimen preparation and integration of microscopy and microanalysis of different areas of physical and biological sciences. www.aomevents.com/ACMMICONN

The Fifth International Conference on the Development of Biomedical Engineering June 16-18, Ho Chi Minh City, Vietnam

http://csc.hcmiu.edu.vn/BME2014

5th Congress of the International Society for Applied Phycology 2014 June 22-27, Sydney

www.isap2014.com

International Union for the Study of Social Insects international congress July 13-18, Cairns

http://www.iussi2014.com/

AIDS 2014 - 20th International AIDS Conference July 20-25, Melbourne

www.aids2014.org/ 34th Annual Meeting of the Australasian Neuroscience Society January 28-31, Adelaide

Pathology update 2014 February 21-23, Melbourne

19th Proteomics Symposium 2014 February 6-9, Lorne, Vic

The Future of Experimental Medicine Conference - Inflammation in disease and ageing March 16-19, Manly Beach, Sydney

www.aomevents.com/ANS2014

www.australasianproteomics.org/

www.rcpa.edu.au/Continuing/ PathologyUpdate/PathologyUpdate2014.htm

39th Lorne Conference on Protein Structure and Function February 9-13, Lorne, Vic

http://femc.mtci.com.au

25th Lorne Cancer Conference 2014 February 13-15, Lorne, Vic

www.worldbrainmapping.org/11th-annualcongress/welcome-message

35th Lorne Genome Conference 2014 February 16-18, Lorne, Vic

5th New Directions in Leukaemia Research (NDLR) meeting March 30-April 2, Noosa, Queensland

www.lorneproteins.org

www.lornecancer.org

www.lornegenome.org

Australia China Life Science Summit 2014 February 18-19, Sydney

www.auschinasummit.com.au/ Lorne Infection and Immunity Conference 2014 February 19-21, Lorne, Vic

www.lorneinfectionimmunity.org

11th Annual Conference of the Society for Brain Mapping and Therapeutics March 17-19, Sydney

http://sapmea.asn.au/conventions/ndlr2014 AusMedtech 2014 April 1-2, Melbourne

www.ausmedtech.com.au Practice-Based Education Summit 2014, The promises of university education: Blending, including and integrating for future practice April 9-10, Sydney

http://csusap.csu.edu.au/~areport/pbe_ summit_2014.htm

2014 International Biophysics Congress August 3-7, Brisbane

www.iupab2014.org

10th Australasian Mutation Detection Meeting September 1-4, Daydream Island, Whitsundays

http://wired.ivvy.com/event/MD2014/ Joint International Symposium on the Nutrition of Herbivores/International Symposium on Ruminant Physiology International Conference September 8-12, Canberra

http://www.herbivores2014.com/

15th International Conference on Systems Biology September 13-19, Melbourne

www.emblaustralia.org

ComBio2014 September 28-October 2, Canberra

www.asbmb.org.au

AusBiotech 2014 October 28-31, Gold Coast

www.ausbiotech.org

Australian Health and Medical Research Congress November 16-19, Melbourne

www.ahmrcongress.org.au/

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