Australian Life Scientist May/Jun 2013 by Westwick-Farrow Media

PP100009448 ISSN 1448-9791

Modern microscopy meets

Super-resolution optical microscopy and electron tomography are revealing some of the malaria parasite’s long-held secrets. 24

malaria Vol 10 Issue 3 • May/June 2013

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Contents FACE TO FACE

14 Timing it right Being in the right place at the right time has been an intrinsic part of Professor John Shine’s career trajectory from research scientist to head of a Californian biotech company to leader the Garvan Institute for 22 years. Here he reflects on this coordinated approach to life. PROTEOMICS

20 Big genome We talk with two prominent molecular geneticists about the claim that more than 80 per cent of the human genome is biologically active - one supports a modest and one supports a mostly functional genome.

MICROSCOPY

24 Modern microscopy addresses an age-old problem Professor Leann Tilley and colleagues are using the power and information provided by the latest techniques in super-resolution optical microscopy and electron tomography to reveal some of the malaria parasite’s longheld secrets. MICROBIOLOGY

30 Tour of microbes We preview the Australian Society for Microbiology annual meeting with some insights into the bacterial

bioremediation of contaminated sites and the concerning emergence of extensively drug resistant tuberculosis on North Queensland border. GENOMICS

35 Eliminating the genomic- discovery-to-clinical-assay bottleneck Digital gene expression technology is helping free up the translational bottleneck and decipher genomic discoveries.

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Movers and shakers GrantWatch AusBiotech Publish or perish Events

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BIOCHEMISTRY

36 Biomolecular in the bush Biomolecular chemists will head to the Blue Mountains for the annual Royal Australian Chemistry Institute Division of Biomolecular Chemistry conference in July. Here we provide a sneak preview of some of the talks on developing a new class of antibiotics and designing small molecule therapeutics for use in Parkinson’s disease.

Cover image: Immature malaria parasites (front) have a much less developed digestive system than mature parasites (back) and as a consequence are much less sensitive to artemisinin. Model generated from two electron tomograms by Dr Eric Hanssen, Advanced Microscopy Facility, Bio21 Institute, University of Melbourne.

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Big science

little science

he human genome was sequenced a decade ago now - the gene-containing part of it that is. The large-scale Human Genome Project involved collaborators from around the world in a public and privately funded effort. And despite scepticism at the time about the enormous cost of the project outweighing the potential benefits, it was successfully completed within 13 years. The announcement in April 2003 that the genome contained 20,000 genes was met with some surprise. This meant that only about 1% of our genome coded for functional proteins - most people were expecting there to be 100,000 or at least 60,000 genes. But it was not to be and the majority of our DNA has been called noncoding or â&#x20AC;&#x2DC;junk DNAâ&#x20AC;&#x2122;. Now, another big project has identified all the functional elements of the human genome sequence. This work involved annotating each nucleotide in the human genome - a colossal 3.2 billion As, Cs, Gs and Ts. The nine-year effort that went into the Encyclopedia of DNA Elements (ENCODE) project culminated in the publication of 30-odd papers towards the end of last year. The conclusion made from this work - that most (about 80%) of the human genome is biologically active - has been driving a heated debate about genes, the chunks of DNA that regulate genes and the definition of the word function. Whether this much intergenic DNA is required for the operating system of the human genome remains contentious (see page 20), but

developments in high-performance computing in genomics and proteomics will hopefully help resolve this. One good thing that comes out of these big science projects is advancements in technology, another is the production of knowledge. Our understanding of a multitude of areas from molecular medicine to forensic science to human evolution has grown immensely from these big projects, not to mention the arrival of the era of personalised medicine. But there remains a place for small-scale, investigatordriven projects. For example, a microbiology research project that involved the serendipitous discovery of a chloroform-eating bacterium which, for what appear to be good reasons, has a genome larger than others of its species (see page 30). The creativity of individual investigators is a requirement in solving scientific problems. And investigator-driven projects can benefit immensely from large-scale collaborative research. In this post-genomic era of biology, the big centres that perform high-throughput analyses, that have the infrastructure, systems and resources to take scientific knowledge and turn it into practical solutions, can support smallscale science projects in collaboration. The distance between a research discovery and its integration into a practical application, whether it be in a clinical setting or for environmental clean-up, is closer than ever and is only going to get more so.

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Superfast plant breeding

MOVERS & SHAKERS

Plant breeding projects that aim to increase food production traditionally depend on self-pollination for several generations because of the need to obtain ‘pure lines’ of plants - which can take a lot of time. But this is about to change. A team of international researchers, including researchers from the University of Western Australia

Stay away from bats ©

o elcald hu/d xc. w.s w w

Infectious diseases experts have warned people to stay away from bats after releasing details of the treatment of an 8-year-old boy in Queensland who died from infection with the Australian Bat Lyssavirus (ABLV) earlier this year. The boy was the third reported case of ABLV and the first in a child. There is no proven effective treatment for lyssavirus infection in humans. Drs Joshua Francis and Clare Nourse and colleagues, from the Mater Children’s Hospital in Brisbane, issued the warning at the Australasian Society for Infectious Diseases meeting held in March in Canberra. ABLV was first identified in Australian bats and flying foxes in 1996 and is common in these animals. Human infection is rare, but once the disease has progressed, it is almost always fatal. Two adult cases were confirmed in 1996 and 1998 - one was a woman bitten by a flying fox after trying to remove it from a child and the other was a carer who looked after these animals. ABLV is one of 12 known lyssavirus strains. Other strains circulate in bats in the USA and Europe, and multiple cases of human infection and subsequent deaths have been reported.

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(UWA), has developed a new technique that enables up to eight generations of wheat and nine generations of barley to be produced a year. Until recently, the fastest way to obtain ‘pure lines’ was to exploit differences in latitude or altitude, such as the ‘shuttle breeding’ technique developed by the ‘Father of the Green Revolution’, the late Nobel Laureate Dr Norman Borlaug. However, even this technique, which involves growing plants at different places, achieved only two or three generations a year. The team - involving researchers from CSIRO, UWA and China - has perfected a method of embryo culture. Although embryo culture has been used before, the team combined it with specially modified water, light, temperature, humidity and potting-mix management to achieve stunning results. The study was just published in the international journal Euphytica. Co-author Associate Professor Guijun Yan, from UWA’s School of Plant Biology and Institute of Agriculture, said a skilled technician in the team was able to dissect 60 plant embryos per hour from the developing grains. “By dramatically shortening times required to obtain pureline plant genotypes, our method could have wide applications in breeding and biological studies,” Associate Professor Yan said.

The 8-year-old boy was bitten during a family holiday to northern Queensland in December 2012 and did not tell his parents. Three weeks later he began to suffer convulsions, severe abdominal pain and fever, followed by progressive brain problems. Analysis of his brain and spinal fluid were normal at first, but on day 10 of his admission increased levels of lyssavirus were detected. The boy’s neurological condition deteriorated and treatment with the antiviral, amantadine, was unsuccessful. He died in February 2013. The warning applies to wherever bat or flying fox populations exist and is not just for the danger from bats themselves, but the risk, however remote, that the disease could spread between humans. “Human to human transmission of lyssaviruses has not been well documented, but it is theoretically possible,” Francis said. “Local and international guidelines recommend post-exposure prophylaxis (PEP) for anyone who has had skin or muscosal contact with saliva or neural tissue from an infected person. This involves immunoglobulin treatment and vaccination. Following the diagnosis, we identified 175 potential contacts of the boy, and of these five household members and 15 healthcare workers were offered PEP.” The lyssavirus is closely related to the rabies virus - the rabies vaccine is used to protect against ABLV infection. Only vaccinated people who have been trained in the care of bats should handle bats or flying foxes.

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MOVERS & SHAKERS

Celebrating art, science and cells

A leap ahead for frog research

Researchers from the University of Newcastle have successfully developed a cryopreservation technique to freeze frog embryonic cells. This important step will now allow for cloning and could slow the threat of extinction to hundreds of frog species. “Almost 200 frog species have been lost in the past 30 years due to disease and a further 200 species face imminent threat - this is the worst rate of extinction of any vertebrate group,” said project leader Professor Michael Mahony. The researchers have separated, isolated and frozen the embryonic cells of an Australian Ground Frog - the Striped Marsh Frog, Limnodynastes peronii. This is the first time slow-freezing techniques have been used successfully on amphibian cells. “Amphibian eggs and early embryos, unlike human eggs and embryos, are large in size and have traditionally presented a challenge to researchers attempting to cryopreserve and store frog genomes, as they would shatter during the freezing process,” said Mahony. Professor Mahony said the development would have wider implications for other species facing extinction. “Not only will it help us preserve the genetic diversity of frogs, but this discovery could also help in the conservation of other species with large embryonic cells, such as fish.” The University of Newcastle is a world leader on research into amphibian protection. This latest discovery follows on from recent work with other universities on the Lazarus project, which generated live embryos using cells from an extinct Australian frog.

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The winning images for the GE Healthcare 2012 Cell Imaging Competition were displayed in Times Square, New York, on NBC Universal’s high-definition screen over three days in April. Announced earlier this year, the three winning images included one from Australian research scientist Anushree Balachandran. Balachandran, who works at fertility clinic Genea in Sydney, won first prize in the High-Content Analysis category for her image of oligodendrocyte precursors derived from Huntington’s disease-affected human embryonic stem cells (see image below). She took the photo with a highperformance, laser-based, confocal microscope while working on differentiating precursor stem cells into adult-like cells.

Huntington’s disease is a neurodegenerative genetic disorder which affects muscle coordination and leads to cognitive decline. The two other winners were Jane Stout, from Indiana University in the United States, who won first place in the Microscopy category for her image of a metaphase epithelial cell. And Markus Posch from the University of Dundee in the UK, who was the regional winner in the microscopy category for his prometaphase human cervical carcinoma (HeLa) cell image. All three winners were also given a trip to New York to see their images displayed in Times Square in April. The competition attracted over 100 entries from researchers investigating conditions such as cancer, HIV and neurodegenerative disease at the cellular level. An expert scientific panel of five judges shortlisted the finalists for each category, which then went forward to the public vote for which over 15,000 votes were cast.

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MOVERS & SHAKERS

GrantWatch Funding boost for start-ups At least $200 million will be invested into early-stage, high-growth Australian companies by three new venture capital funds supported by the federal government’s Innovation Investment Fund (IIF). Announced by the Minister for Climate Change, Industry and Innovation, Greg Combet, at the end of March, three new funds selected under the latest tranche of the IIF will match $100 million of government funding dollar for dollar. The three private-sector investors include Carnegie Venture Capital, who will invest $40 million, GBS Venture Partners and Innovation Capital Associates, who will invest $30 million each.

The IIF is a venture capital program that supports new innovation funds and fund managers with expertise in earlystage venture capital investing. Through co-investment of government capital with private sector investment, it provides small and medium-sized enterprises access to venture capital, enabling them to grow and ultimately to commercialise research outcomes. As well as providing equity capital, the fund managers provide management expertise to a whole range of promising companies across the economy. “Venture capital helps turn ideas into successful businesses and new jobs. It is a vital part of our innovation system which is

why the government, as part of its Plan for Australian Jobs, has announced a new $350 million round of the IIF program,” said Combet. The IIF commenced in 1998. Over three rounds it has established 16 funds and has co-invested in more than 100 new companies including Seek, Bionomics, Pharmaxis and Benthic Geotech. For IIF Round 3 Tranche 4 at least $200 million of new capital will be invested in start-up businesses. More information on the IIF program is available at the AusIndustry website.

Agricultural research receives funding

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The federal government has announced funding for 31 agricultural research projects that will focus on helping farmers adapt to a changing climate and reduce methane emissions. Announced by Federal Minister for Agriculture, Fisheries and Forestry, Joe Ludwig, 15 universities, research and development institutes and private groups will share $30 million in funding. The funding is part of the ongoing Filling the Research Gap Program to support research that helps farmers and landholders develop methods to reduce emissions. The program has initial funding of $201 million allocated over six years to 2016-17. It aims to help farmers and landholders participate in the Carbon Farming Futures Program to reduce emissions, improve their farm sustainability and diversify their farm income. The Carbon Farming initiative, in turn, sits under the government’s $1.7 billion Land Sector Package. AU S T R A L I A N L I F E S C I E N T I S T

Successful applicants in this round included the Queensland University of Technology, which will receive $1.8 million for three projects, and the University of Western Australia, which won $4.45 million to support five projects. The research will focus on reducing emissions from livestock production systems, reducing nitrous oxide emissions, increasing soil carbon, farm system design, adaptation to climate change and international collaboration. The grants build on last year’s first round, for which $47 million was awarded to 57 projects. It also progresses the work conducted under the Climate Change Research Program, which has supported the development and approval of a method relating to methane capture in piggeries. This method is now being used by piggery farmers to earn carbon credits under the Carbon Farming Initiative.

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AUSBIOTECH | VIEWPOINT

Survey prompts call for further tax reform

ppropriate support from the federal government was the top-line issue for respondents to the Biotechnology Industry Position Survey 2013, who urged the government to support access to capital via policy instruments and programs. The sentiment section of the annual Biotechnology Industry Position Survey showed that positive sentiment for the industry’s future continues, whereas the operating environment (economic and public policy) remains a concern. Consistent with AusBiotech’s previous years’ findings, the Australian operating environment (economic and public policy) remains a key concern with only 16% (24% in 2012) of respondents identifying the environment as conducive to growing a biotechnology company and 38% indicating that the operating environment works against the growth of companies. The expansionary sentiment towards employment persists, with only 7% of companies planning to decrease staff numbers during 2013, while 55% have flagged an intention to be a net hirer of staff. The R&D Tax Incentive remains a top priority for the industry, with 66% of respondents having already seen the benefits of the policy or identify that it will have a positive impact in the future. Among the most pressing public policy issues, respondents repeatedly expressed concern that the R&D Tax incentive would be reduced or withdrawn - and

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the industry urges further tax reform to provide incentives for manufacturing and to encourage long-term investment in home-grown technologies. The level of support from the federal government for the biotechnology industry, on a scale of 1 to 10 with 10 being most supportive, saw 68% of respondents indicate the level of support at 5 or lower. The competition for capital is high with 71% of companies planning to raise capital in the coming 12 months. For a significant portion of companies the requirement to raise capital in the short term is apparent with 37% of companies holding less than 12 months’ cash. With capital at a premium, it is no surprise non-cash remuneration remained a significant component of most companies’ remuneration strategy, with only 20% of companies using cashonly remuneration strategies. Of the 24 companies that raised capital in 2012, an overwhelming 87.5% did so by issuing (diluting) equity. The capital raised was for the dominant purpose of research and development, working capital or commercialisation - all critical to the survival of a biotechnology company. The industry is keen to see the government support non-diluting forms of capital, especially the in-tact preservation of the R&D Tax Incentive, and to continue beyond that with tax reform. However, biotechnology and medical technology manufacturers are not especially assisted by the R&D Tax

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Dr Anna Lavelle, CEO, AusBiotech

Incentive, as it phases out as a product or IP reaches commercialisation. This is the point at which Australian IP is most vulnerable to being sold overseas and the resulting community benefits going with it. The industry’s high-tech manufacturers are pursuing a tax incentive to support export-oriented local production. Such a program would, unlike a direct grant, require companies to actually generate economic benefits for Australia (income) before they would be eligible for the incentive. The majority of responding companies (60.7%) are manufacturing, with 37.5% manufacturing in Australia and 35.7% manufacturing overseas, with a crossover of 12.5% that manufacture both locally and overseas. Innovation, wealth creation and job creation should be bipartisan issues regardless of who wins the federal election. The R&D Tax Incentive is a great foundation for innovation to nation build for Australia, but the tax reform story is not over for innovation. Australian policymakers need to do more to make manufacturing and investing in industries of the future more competitive globally and attractive. The 2013 Biotechnology Industry Position Survey undertaken by AusBiotech and Grant Thornton Australia provides an industry snapshot, direct from the industry’s CEOs and senior managers, on three topics: sentiment, funding and public policy. ALS

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FACE TO FACE | JOHN SHINE

Timingit right John Shine reflects on his impressive career as a research scientist, including a foray or two into the world of biotech and 22 years as head of the Garvan Institute - he says it helped being in the right place at the right time. Australian Life Scientist: What drew you to science? Professor John Shine: Well, I’ve always been interested in science and the natural world. As kids growing up my brother and I had a lot of interest in birds and snakes. My brother, who is now Professor of Zoology at Sydney University, pursued the snake stuff more than I ever did. I went to veterinary science at the age of 16 at Sydney University, which was actually too young. After a couple of years I dropped out, moved to Canberra and enrolled in a science degree part-time while I worked as a technician at the National Biological Standards Laboratories.

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I ended up doing biochemistry at the Australian National University (ANU) - which was by the way the subject that I disliked most during vet science - and my lecturer was Lyn Dalgarno. He was an inspirational teacher who was very interested in this new arm of biochemistry, molecular biology, and he got me incredibly interested. That would have been the late 1960s, when molecular biology was very much in its infancy. And in 1972, when I finished the BSc, I did an honours degree and then a PhD with Lyn Dalgarno as my supervisor. It was during that period that we discovered what is now known as the

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Susan Williamson

Shine-Dalgarno sequence, which was really a big step in my career. ALS: What led to your discovery of protein termination? JS: Lyn had found that in insects the large subunit of ribosomal RNA (rRNA) had some sort of break. For this reason, when he extracted the RNA from insect cells he saw only 18s rRNA, whereas mammalian cells have a large and small subunit rRNA, which sediment at around 26s and 18s. It was one of those esoteric odd observations. So my job, as the honours student, was to investigate that break further. I showed that this was a specific break by sequencing the 3′ end of the RNA. I also found that the 3′ sequence of the smaller 18s rRNA from insects was exactly the same as the mammalian 18s rRNA, which we thought was interesting. My PhD followed on from that. Very soon it became clear that the 3′ end of the 18s rRNA from insects, worms, rabbits and

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JOHN SHINE | FACE TO FACE

humans was exactly the same sequence for about the first eight nucleotides coming back from the 3′ end. And given that the rest of the sequence then became quite different, that’s got to be functionally significant. I still remember thinking about that sequence a lot - GAUCAUUA. One day I was at home and it suddenly dawned on me that the complement of that sequence would be UAAUGA and that’s exactly two of the termination codons, UAA and UGA. UAG, the third termination codon, could also be recognised by the sequence through what’s called “wobble” - a G:U base pair, so that stretch of sequence would be the exact complement to all three termination codons. As there is no transfer RNA that recognises termination codons, we postulated that the 3′ end of the small rRNA scans the mRNA during translation and upon binding to a termination codon causes a structural change which triggers binding of protein release factors, catalysing release of the completed protein. It was one of those eureka moments, realising it was the signal that terminates protein synthesis in every organism I had looked at. So we published that in Nature and it was widely accepted. ALS: That’s a great start. What came next? JS: Yes, that was my PhD and that was a great moment. The next thing was to look at prokaryotes. So, expecting it to be the same, I sequenced Escherichia coli and found the 3′ sequence of E. coli rRNA was similar but different. The sequence started out exactly the same, UUA, then the sequence CCUCC, which was totally different, and then came UCA exactly the same as the eukaryotes. I looked at a few other bacteria and they were all pretty much the same. Up until then the rRNA had been thought of as the scaffolding that held together the proteins that form a ribosome and that’s all. But with this finding we showed that the end of the 18s rRNA scanned the mRNA looking for a termination codon - that was our model of it anyway. And it made me think well, if it’s scanning the mRNA and it’s still

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doing termination in bacteria, maybe this CCUCC is somehow recognising the initiation point of protein synthesis. The question about initiation was very topical at the time. It was known that AUG was a start codon and that in eukaryotes the ribosome recognised the first AUG and from then on off it goes. So I then looked for the complement to the CCUCC sequence. Others, especially Joan Steitz at Yale, who was a leader in this protein initiation field, had published some very nice work determining the sequences, about 30 nucleotides long, around the true AUG starting point in bacterial mRNA. When I looked at these sequences, I found the complement sequence, GGAGG, about 10 nucleotides back from the AUG. Although it wasn’t quite that simple, there were some that were just GAG or GGAG, and every other permutation. But when I looked at that it was quite amazing - it was like turning the light on. We submitted it to Proceedings of the National Academy of Sciences and Joan Steitz was a member of the academy. She apparently liked it, because it explained everything she’d been trying to do and she gave it the name the Shine-Dalgarno sequence when she made the commentary. The bottom line is the Shine-Dalgarno sequence was the signal for the initiation and termination of protein synthesis. ALS: You were one of the first to successfully clone a human gene, can you describe how your work led to this? JS: I did the usual thing and wrote off to a dozen labs overseas looking for a postdoc. I was fortunate in that I ended up with three offers that were all good - one in Boston, one in Berlin and the third in San Francisco. I chose to go to San Francisco, which I think was the best career choice I ever made. It was not because of any scientific insight that I chose San Francisco - Boston was too cold and I don’t speak German, so San Francisco seemed like a great place to go. I arrived just as the recombinant DNA stuff was starting - life’s all about timing. I went to work in a lab run by Howard Goodman, a biochemist working on RNA

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and restriction enzymes, at the University of California San Francisco. I arrived in Goodman’s lab at about the same time as two other postdocs - Axel Ullrich and Peter Seeberg, both from Germany - and Herb Heineker, a postdoc from Holland who was working in a lab headed by Herb Boyer. The four of us spent a lot of time trying to clone insulin cDNA from an RNA copy. I can still remember picking the X-ray film out of the tank. It was pretty late at night, and I held it up and I could read the sequence, and knowing the amino acid sequence in my head I could instantly recognise that it was insulin. It was the first time anyone had seen the insulin gene - we were at the forefront and that was very exciting. The end result of that postdoc was that we cloned the first insulin gene, the first human hormone gene (placental lactogen) as well as growth hormone. ALS: Was there then a natural progression into the biotech and commercialisation? JS: Up until that point I hadn’t thought about commercialisation. It just wasn’t on my radar. It was all pure, basic, curiositydriven science. And it was the same for the other postdocs in the lab. Until we cloned the insulin gene I don’t think we even knew what a patent was, it just wasn’t relevant.

A young John Shine in the lab at the Australian National University in 1980.

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FACE TO FACE | JOHN SHINE

With Postdoctoral Fellow Dr Lisa Selbie at Garvan around 1990.

However, once we cloned the insulin gene things changed. There was a big need for insulin and that’s why in the early days of the initial cloning experiments the Holy Grail was insulin. That was really my first inklings of the potential of commercialising work. Genentech was set up out of Herb Boyer’s lab - Herb was the cofounder of the company - and they were very interested in producing and marketing human insulin and human growth hormone. As soon as we announced we’d cloned the insulin gene and published it in Science, Genentech was very keen to work us. I consulted with Genentech a fair bit. It was very satisfying that the Shine-Dalgarno sequence and the concepts around it became so important in expressing mammalian proteins in bacteria. That got me quite interested in working with industry because it was real, and it was important for making the final protein. ALS: Were you tempted to continue working in biotech? JS: I was tempted. I came back to do research at the Research School of Biological Sciences at the ANU in 1978 and continued cloning various human genes. Then I went back in 1983. It became clear that not just the commercial side in the recombinant DNA works, but also the academic side was still mostly happening in California - especially in the burgeoning biotech activity over there. Molecular biologists were in high demand. I had maintained contact with endocrinologist John Baxter, who ran a lab at

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the University of California San Francisco. John was a typical American MD, very entrepreneurial. He started up a biotech company called California Biotechnology and convinced me to take the position as Director of Research. I was only going to go for about a year. It ended up being a bit over three years. This was a real learning experience for me in that I went from Director of Research to President of the company in the space of a couple of years. I was exposed to a lot more of the commercial side of the biotech industry - I would never have gotten that in Australia. CalBio was a science-driven, start-up biotech company but after about three years it had grown from a couple of scientists to a staff of about 300. It was really at a point that it needed a business person to run it, not a scientist. And our children were at an age where if we’d stayed in the US they would have become Americans, so we chose to come back to Australia. I came back to the Garvan as Deputy Director leading the neuroscience program. Shortly after I got back, the director, Les Lazarus, stepped aside and I was in the right place at the right time again. ALS: Did you bring that business experience into the directorship at the Garvan? JS: I think so. I hope so. Back in those early days when biotech was starting in the US, it just really wasn’t in the culture of the Australian scientific community to interact much with industry.

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Australian culture was very much the poorer the scientist, the better the science. And there’s a whole bunch of reasons for that - it’s part cultural, part the fact that we haven’t really had a research-based pharmaceutical industry in Australia in the past. We’ve got companies like CSL now, and ResMed, Cochlear, but there’s not many of them. This has now changed enormously. One of the main things that’s changed is that researchers have seen, especially with the development of biotech, that top science can be done in industry. It became clear that it wasn’t a ‘them and us’. If you look at a lot of the big biological science breakthroughs over the past couple of decades that you read in Nature or Science or Cell, these have involved researchers associated with, if not full-time employees of, biotech companies. ALS: Do you have any ideas about what’s next for the Garvan? JS: I became director in 1990 and was in that role for 22 years - I’ve enjoyed every minute of it. It’s been great to see the institute grow and it’s done incredibly well. But it was certainly time to step down, not just for me but to reinvigorate the institute. It’s a different time now and we’re fortunate to have John Mattick as the new director. I think that the big genomics, the big databases, the big computer power, having your genome totally sequenced when you’re six months old, that’s the way of the future and Garvan needs to embrace that. I see this bigger picture perspective of integrating research into the clinical

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setting as the way forward. Everyone does, translational research, it’s a bit of a buzz word. But the reality is, unlike almost any other time in the history of medical research, the distance between a research discovery and a practical clinical outcome is closer than ever and it’s only going to get more and more so. The Garvan needs to embrace this; hence, not long before I left we got the new cancer centre underway, the Kinghorn Cancer Centre. It’s really very satisfying to see that finished. ALS: And you’re back in the lab? JS: Yes, I’m back. I don’t get callouses pipetting anymore but I have a small research group here and am indulging myself as retired scientists do in doing some research. I’m really enjoying it. Over the past several years I’ve had this small research group and we’ve working with neuronal stem cells, especially olfactory stem cells. I’m looking hard at it now to see how competitive can we really

be - even though it’s going well the field is starting to grow rapidly. I’ve begun collaborating with the head of the renal unit at St Vincent’s, Dr Tim Furlong. Tim worked in my lab at the Garvan years ago and we’ve started to look at developing a genome assessment project for patients with inherited kidney disease who often end up requiring dialysis. ALS: Do you think discovery research will maintain its place? JS: A challenge is going to be to optimise the balance between ongoing basic curiositydriven research and marrying that with the enormous opportunities this genomic age has for predicting outcomes in medicine and directing treatment. There’s certainly a lot of thrust today to identify the biological markers of a particular disease. It’s all about prevention in a sense. Whether it be osteoporosis, Alzheimer’s or cardiovascular disease, if you know you’re at a risk of a certain disorder you can monitor your markers on a regular

basis and hopefully start some preventative treatment. On a broader scale, I think it’s very much up to government and funding agencies to ensure their funding schemes represent an appropriate balance of some shorter-term translational research and some longer-term, blue-sky, investigator-driven research. It’s a constant debate but it’s very much up to them to get the balance right because the funding is what determines it. However, we, as scientists, have a responsibility in that part of the debate to influence government and the funding agencies in getting that balance right. The Garvan is in a very privileged position - like any of the major institutes - in that the community provides us with a lot of support. We need to show leadership, to show that we are prepared to invest in trying to get that balance right - ensuring that we fund exciting innovative research as well as effectively translating our research into the clinic. ALS

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PROTEOMICS | GENOME FUNCTION

Graeme O’Neill

Debate continues about the claim that proteins interact with 80% of the human genome and whether this much regulation is required for a functional human being.

fter labouring nine years to catalogue every functional element in the human genome, the ENCODE (ENCyclopaedia Of DNA Elements) research consortium announced this year it has linked more than 80% of the human genome sequence to a specific biological function. The 440-odd researchers involved in the ENCODE project say they have mapped more than a million regulatory regions where proteins interact with DNA. Which begs the question: why are so many functional elements required to regulate the expression and activity of so few genes? FUNCTION OR FALLACY?

Some critics greeted the ENCODE consortium’s estimate with scepticism - four papers criticising the ENCODE consortium now exist in the scientific literature, not to mention blog postings. Molecular evolutionist Dan Graur and several colleagues from the University of Houston and Johns Hopkins University went further, veering into open derision. Their acerbic critique in Genome Biology and Evolution threw an uppercut in its title ‘On the immortality of

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television sets: “function” in the human genome according to the evolution-free gospel of ENCODE’. Graur et al wrote that the ENCODE estimate flies in the face of current estimates that under 10% of the genome is evolutionarily conserved through purifying selection. “Thus, according to the ENCODE Consortium, a biological function can be maintained indefinitely without selection, which implies that at least 80 – 10 = 70 per cent of the genome is perfectly invulnerable to deleterious mutations, either because no mutation can ever occur in these ‘functional’ regions, or because no mutation in these regions can ever be deleterious.” Graur and his colleagues maintain the rage for another 35 pages, before concluding, “… the ENCODE Consortium has, so far, failed to provide a compelling reason to abandon the prevailing understanding among evolutionary biologists according to which most of the human genome is devoid of function. The ENCODE results were predicted by one of its lead authors to necessitate the rewriting of textbooks (Pennisi 2012). We agree, many textbooks dealing with marketing, mass-media hype, and public relations may well have to be rewritten.”

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GENOME FUNCTION | PROTEOMICS

MODEST VERSUS MAJORITY FUNCTION

So, does a surprisingly small complement (~22,000) of protein-coding genes need such a huge constellation of DNA regulatory regions and RNA-encoded DNA elements for the task of assembling and operating a human being? Or is the functional genome a masterpiece whose operational complexity is conjured from a compact kernel of stringently conserved functional elements? ALS invited two leading molecular geneticists to argue for a modest versus mostly functional genome. Professor Merlin Crossley, Dean of the Faculty of Science at the University of Sydney, has specialised in the role of DNA-binding proteins - gene-transcription factors - in development and disease. He believes less than 20% of the genome is highly conserved and functional. Professor John Mattick, Director of the Garvan Medical Research Institute in Sydney, believes most of the genome is functional. Mattick has been influential in the developing field of ‘RNA-omics’, which emerged from the discovery of introns in 1979. Mattick and others have shown that the ‘junk DNA’ of introns and vast tracts of intergenic DNA harbours a host of small, functional RNA elements that effectively form a complex, multilayered operating system for the genome. JUMPING GENES HAVE NO FUNCTION

Crossley says around two thirds of the human genome is now understood to consist of dead retroviruses, in the form of transposable DNA elements - jumping genes. “I think transposable elements are essentially parasitic,” he said. “If two thirds of the genome is transposable elements, that leaves only a third that could have function. The estimates we are seeing is that only 10% is conserved.” Crossley says transposable elements will occasionally suffer replication errors and may evolve functions that will be co-opted by the host, but the vast majority won’t have any function. Alu elements are the most abundant transposable elements in primate genomes. Exclusive to primates, their numbers have expanded over the past 40-50 million years, peaking at 1.2 million in modern humans. Alu elements flank a significant number of duplicated segments in the human genome, suggesting they sometimes ‘capture’ and copy intervening DNA sequences as they replicate (see breakout box). “Gene duplication is the raw material for evolutionary change, so the fact that so a large part of our DNA consists consisting of repeated elements like Alu transposons has certainly influenced human evolution - it can affect biological function, but it doesn’t have intrinsic biological function itself. “Some of the duplicated genes end up as pseudogenes. A few may evolve separate roles, but most will remain pseudogenes with no function.” GENE SPACING

Crossley says there are many different ways that genetic accidents - point mutations, insertions, deletions and frame-shift mutations - can knock out a functioning gene, resulting in dominant negative changes in gene function. But these changes cannot be regarded as functional, only a very small number will result in new positive or dominant negative functions.

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While Crossley finds these genetic accidents fascinating, and they exhibit regulatory effects, he says their influence is incidental and they are not so common as to constitute a significant fraction of the genome. But he accepts that in some cases, the amount of spacer DNA in the genome can be critically important - changing the spacing between a remote enhancer and its target gene would probably affect the gene’s function. Does that mean the intervening DNA is functional? “I don’t know - if the gene has come to depend on it, it might,” he said. “But it would surprise me, when you look at related species with very different amounts of DNA, such as lungfish and puffer fish, or two Drosophila species. The fact that the genome sizes can be different, when the genes and the organism itself are similar, suggests the spacing between genes is not having a major effect on gene regulation.

“Lack of conservation does not mean lack of function. A functional nucleotide sequence is not like a telephone number, where every digit has to be dialled in its correct order to make the connection.” “Think of a stack of boxes - the one at the top prevents dust accumulating on the boxes beneath and the one at the bottom keeps the stack off the wet floor, but these are not ‘functions’.” Crossley believes the amount of conserved, functional DNA in the human genome is more than 10% but no higher than 20% - the rest is stitched in to the stuff that matters and has been provided with a ticket to ride through time, courtesy of the phenomenon that accounts for linkage disequilibrium. PERMISSIVE TERRITORY

John Mattick said he was surprised by the “almost vituperative” tone of the Graur commentary. He and Garvan Institute bioinformatician Marcel Dinger are seeking to publish a response in Genome Biology and Evolution. He said the Graur paper’s focus on strong conservation ignored evidence for the presence of large amounts of less stringently conserved DNA in the human genome. He focuses on “permissive territory” in the genome, where protein-coding DNA specifies active sites within the mature, folded protein. “The structure-function relation within these active sites is biochemically mediated - for example, a particular motif might form an oxygen-binding site. “Such active sites are analogue devices, and the amino acid sequences within them may be quite loose. As long as the structure of the binding site is vaguely polar, it will bind oxygen. “Within the active sites of a protein, the sequence can vary enormously across species, or even between individuals of the same species. Lack of conservation does not mean lack of function. A functional nucleotide sequence is not like a telephone number, where every digit has to be dialled in its correct order to make the connection.”

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PROTEOMICS | GENOME FUNCTION

Mattick finds linguistic analogies useful in explaining the concept of relative conservation of genome sequences. He says cognates - similar words in modern languages of common ancestry - have undergone phonetic shifts over time but retain their original function. Cognates like the German “brater” and Latin “frater” (brother), and the Latin “pater” and Sanskrit “pitar” (father) reflect common descent from proto-Indo European, the 6000-year-old mother tongue of the first farmers of Turkey’s TransCaucasus region. According to Mattick, the relative conservation of genomic DNA is evident in the ability of different peptides and proteins to bind the same receptor. The receptor is strongly conserved, maintaining the reciprocity of its core for multiple ligands, but the complementary amino acid sequences of its ligands can vary considerably without ill effect. Mattick says the Graur critique fails because it focuses on strict conservation and, as a result, makes circular assumptions and dubious comparisons. He believes Graur has made the mistake of arguing that only 10% of the genome is functional because the rest does not exhibit strong sequence conservation, when transcription across most of the genome more reliably indicates genetic function. KEEPING AN OPEN MIND

Mattick has spent much of his career identifying regulatory RNA sequences in the genome and says the sheer volume of regulatory RNA molecules challenges the traditional, protein-centric view of genetic programming. He also questions the assumption that transposable elements and the ancient, highly repeated DNA sequences shared by all mammals are neutrally evolving and thus non-functional. “Evidence is growing that this assumption is incorrect,” Mattick said. The conclusion that ancient repeats - many of them created by transposon activity - have no function ignores evidence that some protein-coding DNA sequences, and some micro-RNA sequences, have structure-function constraints. Such sequences may exhibit unrecognised patterns of mutation, different to the familiar classes of mutation that occur in cisregulatory sequences and other classes of trans-acting regulatory RNAs encoded in the genome. Professor John Mattick is Executive Director of the Garvan Institute of Medical Research; Conjoint Professor in the St Vincent’s Clinical School and Visiting Professorial Fellow, School of Biotechnology & Biomolecular Science, University of New South Wales. He obtained his PhD from Monash University and has worked at Baylor College of Medicine in Houston, the CSIRO Division of Molecular Biology in Sydney and the University of Queensland, where he was based from 1988-2011. He was Foundation Director of the Australian Genome Research Facility and the Institute for Molecular Bioscience and Professor of Molecular Biology and NHMRC Australia Fellow at the IMB, University of Queensland.

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Professor Merlin Crossley is Dean of the Faculty of Science at the University of New South Wales and an Honorary Associate at the University of Sydney. He completed his undergraduate degree at the University of Melbourne and a doctorate at Oxford University. After a postdoc in Oxford he took a research position at the Howard Hughes Medical Institute, Children’s Hospital and Dana Farber Cancer Center, Harvard Medical School. He later returned to the University of Sydney and established a laboratory investigating DNA-binding molecules and gene control.

Mattick says the major finding of the ENCODE project is that the majority of the mammalian genome is transcribed in highly cell-specific patterns. Such patterns indicate that the transcribed elements play a dynamic role in embryonal development, differentiation and disease - “Even supposed ‘gene deserts’ yield transcripts that are expressed in specific cells,” he said. Two key functions inferred for these transcripts are guiding chromatin-modifying complexes to their sites of action and supervising the precise sequence of epigenetic changes required for normal embryonal development. Mattick says it is unsurprising that science’s understanding of the processes underlying the molecular evolution of life is incomplete and predicts that developments in massive parallel computing will bring to light new and surprising mechanisms. Until then, he suggests that everyone should keep an open mind on the extent of functionality in the human genome. ALS ACCIDENTAL EVOLUTION

An example of transposons playing an accidental, yet possibly pivotal role in human evolution came to light last when Professor Evan Eichler’s team at the University of Washington, Seattle, announced in Cell it had discovered an Alu-mediated triplication of a gene called SRGAP2 that happened some 3.4 million years ago. SRGAP2 molecules pair up to form a duplex signalling protein that, in chimpanzees and other great apes, terminates the elongation of neural precursor cells and thread-like processes called filopodia that differentiate into dendrites. In humans, one of the three SRGAP2 proteins is truncated. If the truncated molecule is present in a duplex molecule, differentiation is delayed, and the neural precursors continue to elongate without differentiating. The result is a population of neurons in the embryonic brain able to make elaborate, longdistance interconnections between the brain’s functional modules. Eichler’s team believes the error may have laid the foundation for a phenomenally rapid increase in cognitive power in the hominin brain as its volume increased fourfold over the ensuing 2.5 million years. Although Alu elements caused the duplication, they were not functionally involved in the resulting gain-offunction mutation.

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MICROSCOPY AND IMAGE ANALYSIS | IMAGING LIVE CELLS

addresses an age-old problem

Fiona Wylie

In 1959, Nobel prize-winning physicist Richard Feynman wrote: “It is very easy to answer many of these fundamental biological questions; you just look at the thing!” In 2013, Melbourne cell biologist Leann Tilley is doing just that as part of the global fight against malaria … using some very 21st century ways of ‘looking’.

rofessor Leann Tilley and her colleagues, Nick Klonis and Eric Hanssen, at the Bio21 Institute in Melbourne seem to spend a lot of their time designing and playing with the latest and greatest microscopy ‘super-toys’ to produce a myriad of weird and wonderful images of very, very small living things. While obviously a lot of fun for this multidisciplinary group that includes cell biologists, mathematicians, computer dudes and even physicists, the goal of this work is deadly serious.

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For Tilley, it is part of her long-held research focus to better understand and control the ancient and ever-challenging problem of malaria, the parasitic infection that still kills up to 1 million people across the tropics each year, most of them children. And if recent results are anything to go by, the ‘fun’ is certainly time and money well spent. The power and information provided by the latest techniques in superresolution optical microscopy and electron tomography are finally revealing some of the malaria parasite’s long-held secrets.

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A FORMIDABLE ADVERSARY AND WEAKENING ARSENAL

Tilley has always been fascinated by the malaria parasite’s ability to undergo a remarkable series of morphological transformations during its life cycle through human and mosquito hosts. And à la Feynman’s famous advice, she believes that looking in great detail at these changes will help decipher the mechanisms underlying the parasite’s most intractable and problematic traits. In particular, the malaria parasite has developed resistance

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IMAGING LIVE CELLS | MICROSCOPY AND IMAGE ANALYSIS

A major stumbling block to improving the existing antimalarial drugs is that scientists ’t really know how they work in the first place. The biological mechanisms of action for all the major players on the whole remain a mystery - even for the earliest stalwarts like chloroquine, and especially for artemisinin. TARGETING PARASITIC DIGESTION

What is known is that for the malarial parasite to grow and prosper during its important proliferative stage inside the human host red blood cell (RBC), it needs a good source of amino acids … and what better source than the handiest around in the host’s own haemoglobin. It also needs to create a bit of growing room. Indeed, malaria parasites will ‘eat’ up to 75% of the host hemoglobin during an infection, using a stomach-like organelle referred to as the digestive vacuole. Work from the Tilley lab and others showed that endoperoxide antimalarials, including artemisinin, are activated by the haem released during the haemoglobin breakdown. This led to studies implicating the digestive vacuole as an important site of artemisinin activity, with the parasite’s haemoglobin digestive process the drug target. More to nearly all of the antimalarial drugs thrown at it over many years. “There are various drugs for treating malaria, but the parasite has developed resistance to most of these, so all the older ones like chloroquine are effectively useless,” said Tilley. “The most recent drug, an endoperoxide agent called artemisinin, still works although worrying evidence shows that resistance to this agent is also developing.” Basically, none of the antimalarial drugs that are currently used are 100% effective - indeed, combinations of agents are now used to optimise efficacy and slow the spread of resistance. “These combinations are now working less well,” said Tilley, “and we desperately need replacement drugs because we know that the development of resistance is inevitable and we need to save the good ones like artemisinin for as long as possible.”

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specifically, it was postulated that artemisinin is activated by interacting with the haem products, forming free radical species that react with things in the immediate vicinity of the parasite to induce cellular damage and, ultimately, cell death. To investigate this, Tilley’s team started to use whole-cell electron tomography to look at the mechanics of haemoglobin digestion. “In this work, we prepare serial sections of the malaria organisms for viewing under an electron microscope and then acquire electron tomograms for each section using a series of tilt angles. The whole thing is then put back together computationally to build up a 3D picture of the structures inside the parasite and ideally the whole parasite or cell, but at the level of resolution enabled by electron microscopy (EM).” Although incredibly labour- and time-intensive, Tilley found that this relatively new approach was fantastic for looking at the malaria parasite in human host cells … “and we could clearly see the digestive vacuole”. So, with the idea of the haemoglobin digestion and artemisinin mode of action in mind, they decided to look at it in more detail.

VIEWS FROM MELBOURNE TAKING OFF

Tilley is currently Director of the ARC Centre of Excellence for Coherent X-ray Science (CXS), having recently taken the baton from Keith Nugent, who is now DVCR at La Trobe University. The centre brings physicists, chemists and biologists together to develop fundamentally new approaches to probing biological structures and processes. Tilley was instrumental (no pun intended) in developing the CXS approach and her laboratory has worked for many years on developing and implementing a number of new imaging modalities including coherent X-ray diffraction imaging, 3D electron tomography, cryo X-ray tomography and structured illumination microscopy. Tilley currently holds an ARC Australian Professorial Fellowship. “At the Bio21 Institute, we now also have one of the first instruments in the world for live cell 3D Structure Illumination Microscopy (3D-SIM), and Trevor Smith is further developing the technology. This 3D-SIM BLAZE module from Applied Precision gives us an 8-fold increase in volume resolution compared with conventional light microscopes - that means you can see amazing details that we were not previously able to see in fluorescently labelled samples and collects the image data very rapidly to enable the 3D imaging of live cells at super resolution.”

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MICROSCOPY AND IMAGE ANALYSIS | IMAGING LIVE CELLS

THE DIVERSE APPLICATION OF EM TOMOGRAPHY

EM tomography is basically a union of transmission electron microscopy, the latest advances in tomographic imaging and some high-end algorithmic and computing power. The result is some very impressive and useful images packed with novel and previously inaccessible information. Other examples of tomographic imaging applications involving Eric Hanssen’s team at Bio21 include the structure of the surface layers of bacteria in dental health, the organisation and structure of nanoparticles and nanomaterials, the invasion of host red blood cells by malaria parasites (see image), the structure of milk component for the dairy industry, the effect of extracellular calcium phosphate crystals on inflammation, mitochondrial morphology in mitochondrial diseases (see image), the ultrastructure of collagen and muscle in muscular dystrophies and the organisation of the cell wall in plants, amongst others.

WATCHING AND WAITING PAYS OFF

“We started by morphologically tracking the digestive vacuole through the different stages of parasite development within the RBC, which takes about 48 hours,” explained Tilley. “Firstly, we were able to pinpoint the genesis of this organelle, which does not start until about a third of the way through the parasite RBC life cycle - so, for this time the parasite does not digest any haemoglobin.” This finding clearly had implications in terms of artemisinin action and a possible means of resistance if haem production is necessary for the drug to work. “Thus, by just looking at the different immature stages by EM, we could predict that the early stages of the parasite developing inside the RBC would be much less sensitive to artemisinin.” And when Tilley’s student, Stanley Xie, went back and did that experiment, this was indeed true. “Also, when we biochemically stopped mature parasite stages from digesting haemoglobin, they became resistant to the presence of artemisinin,” said Tilley. So if the parasite does not digest haemoglobin, artemisinin is inactive, confirming a mechanism by which the parasite could become drug-resistant. Tilley explained that this could work for the parasite because artemisinin is quite a ‘poor’ drug really, in that it only lasts in the bloodstream for a couple of hours “so all the parasite has to do is find a way to hold off maturing for that time and

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then it can go in its merry way digesting haemoglobin and growing”. Tilley then called on some mathematical colleagues, including James McCaw and Julie Simpson of the Melbourne School of Population and Global Health, to do some modelling studies of what would need to happen for these immature parasites to become resistant - that is, what level of resistance would they have to show for the treatment to fail. This very nice study confirmed that these immature parasites are the likely cause of artemisinin resistance. “We now also have data coming in from the field telling us that our modelling data indeed reflects the real situation when artemisinin fails to treat the parasite infection in patients, it fails because the immature parasites have become even more resistant than they already were. It seems that the ‘younger’ parasites can survive the presence of artemisinin for the few hours that the drug remains stable in the patient’s circulation.” STRAIGHT TO THE SOURCE

Tilley is wasting no time translating their ‘high-end’ science into the real-life situation through collaboration with field colleagues. By collecting parasites from the field and adapting them to the laboratory for examination, the researchers can put their resistance hypothesis to the test. “We want to see how these parasites behave in our lab assays and down the microscopes to see if there is any difference in their development in terms of haemoglobin digestion.”

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For these next steps, it is important to use parasites from areas where drug resistance is known to be a problem, as Tilley explained. “We get our samples from an area in Cambodia near the border with Thailand where there seems to be a kind of cradle of antimalarial drug resistance. In fact, it is the place where resistance first developed to chloroquine and other older antimalarial staples such as mefloquine, and now to artemisinin. The conditions there are obviously just right for the development of malarial resistance, although it is not entirely clear what those conditions are.” It might be the nature of the area itself, something about the people that inhabit and pass through the area, something about the endemic parasite strains or, indeed, a combination of factors. “Whatever the reason, there is something about the parasite strains circulating in that area of Cambodia that makes it easier for them to develop resistance … and of course, from there the drug resistance mechanisms spreads all around the world as happened with chloroquine.” IMMEDIATE BENEFITS FOR TREATMENT

According to Tilley, their electron tomography findings on the parasite life cycle and drug resistance could have immediate clinical applications for patients. One is in the area of drug dosing. Currently, artemisinin is administered as one dose per day for three consecutive days, a regimen largely designed on practical grounds to have the maximum effect.

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MICROSCOPY AND IMAGE ANALYSIS | IMAGING LIVE CELLS

Professor Leann Tilley completed her undergraduate studies at the University of Melbourne and her PhD at the University of Sydney, both in biochemistry. After postdoctoral fellowships at Utrecht University in the Netherlands, the College de France in Paris and at the University of Melbourne, she joined the Biochemistry Department at La Trobe University. In July 2011 she returned to the Department of Biochemistry and Molecular Biology at the University of Melbourne and is currently Director of the ARC Centre of Excellence for Coherent X-ray Science (CXS).

“However, given what we now have realised about the mechanism of drug action and the timing of the parasite metabolism, this dosing schedule may not be optimal and perhaps only one of those doses will be present at the right time to have an effect while the other two might coincide with when the parasite is at its most resistant. Indeed, people are already looking at how we might be able to get better efficacy with existing drugs by

changing the way they are administered and how such a change might be implemented in the field.” Another implication is for current drug development efforts to synthesise longerlived forms of endoperoxide antimalarials like artemisinin. “Our work would predict that such drugs will be much more effective to use against the early and resistant stages of the parasites - they can’t hold out forever and eventually will need to start digesting

haemoglobin, thus enabling the drug to become active.” For Tilley it is very exciting to see such high-end techniques like EM tomography used in a basic science context leading to a discovery that is potentially of immediate use for treating malaria in some of the world’s poorest populations. CONVERGENCE WORKING WELL

Tilley also sees the cross-disciplinary nature of her work, involving many Bio21 Institute scientists and partners, as one of the best things about it. “We work with physicists to develop the imaging methods, mathematicians to do the modelling and computer scientists to improve the image analysis. It is true ‘convergence’ - this concept of bringing the biological and physical scientists together to work on big and important questions with a cross-disciplinary approach to generate novel and useful developments. Bio21 Institute really is a great place to do this and it seems to be working very well so far.” ALS

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MICROBIOLOGY | BIOREMEDIATION AND DRUG RESISTANCE

TOUR OF

microbes

Susan Williamson

Bacterial remediation of contaminated sites and dealing with the emergence of extensively drug-resistant tuberculosis on the North Queensland border are some of the tours available at this year’s Australian Society for Microbiology meeting.

ssociate Professor Mike Manefield, in the Centre for Marine Bio-innovation and the School of Biotechnology and Biomolecular Sciences at the University of New South Wales, is tackling the legacy of unregulated industrial activity - plumes of toxic chemicals that contaminate the groundwater under our cities. Manefield and his team have been working on the bioremediation of three main organochlorines for a number of years now, and this will be the focus of his talk at the Australian Society for Microbiology conference. The first is perchloroethylene (PCE), which is used as a chemical solvent in the dry-cleaning industry. The lack of regulation before the 1970s meant that these chemicals were often disposed of by being tipped into gutters or onto the ground. The second, 1,2-dichloroethane (DCA), is a chlorinated hydrocarbon primarily used to produce vinyl chloride monomer, which is the major precursor for polyvinyl chloride

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(PVC) production - a widely used plastic. DCA is also used as a solvent in the formation of polystyrene and latex. And the third, chloroform, is a precursor to refrigerants and plastics as well as being used as an extractant. All these chemicals are toxic. They kill in acute doses (chloroform also kills at low concentrations), are known carcinogens and because of their long half-life, they are recalcitrant in the environment. But Manefield is quick to point out that despite their toxicity the organochlorides have some very useful properties. “These chemicals are soluble, volatile and stable, which are useful properties and they have utility in society,” said Manefield, “so we do not want to stop using them, but we need to handle them better and find better ways to clean them up.” The stability of these chemicals - some have an abiotic halflife of hundreds to thousands of years - means that they can be transported and stored for extensive periods, but it also means that if they are spilled it can be disastrous.

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ORGANOCHLORIDE RESPIRATION

The Botany Industrial Park is an environment where lots of improper disposal of chemicals has occurred. A major cleanup of the groundwater at the site is underway and Manefield’s research feeds into this effort. “The aquifer has been contaminated for a long time,” said Manefield. “Containment lines are in place and groundwater is pumped out of the aquifer and into a treatment plant to decontaminate the water. It is estimated this will take 200-300 years. It’s expensive and energy intensive, so we need to find a better way.” Because organochlorides are denser than water, when they are spilled into the environment they percolate into the soil, into the groundwater and form an organic phase below the water. “They are good at dissolving greases and fats, but they do not mix with water,” Manefield explained. “They just sit there in an organic phase on the bottom of the aquifer slowly dissolving into the water forming a plume downgradient they’re called DNAPLs, dense non-aqueous phase liquids.” It is this dissolved phase of the plume that the containment system prevents from entering Botany Bay. But Manefield and his team want to create a barrier of bacteria to replace the groundwater treatment plant. One problem with this is that the environment the organochlorines occupy is anaerobic, so rapid aerobic biodegradation processes aren’t useful. A second problem is that the undissolved organochlorines sitting on the bottom of the aquifer can be toxic to the bacteria. But there is a small selection of anaerobic bacteria that break down organochlorines. Manefield said it took a number of years for his team to work out how to successfully grow these bacteria, but they are now making progress. Industry has funded the development phase and continues to provide funding for this work. “The bacteria also respire the organochlorides in the gradient up the plume,” Manefield said. “The transfer of electrons to organochlorines removes the chlorine atoms, which makes these chemicals harmless. The fully dechlorinated breakdown products are abundant hydrocarbons in the environment; for example, one breakdown product is ethene, which is used to ripen fruit.” SERENDIPITY

Manefield’s team are working with the three groups of bacteria, Dehalococcoides, which breaks down PCE, Dehalobacter, which breaks down DCA and Desulfitobacterium, which degrades chloroform. “They’re known as organochlorine respiring bacteria, or ORBs,” he said. Manefield’s team takes samples from contaminated sites back to the lab to assess the activity of the microbial communities and their ability to break down pollutants. “It is a slow process to strip a culture down to the single organism you want to look at,” said Manefield.

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Dr Mike Manefield and his team in the lab assessing bacterial growth on a plate.

And they are not easy to grow. Manefield said they started working on reductive dechlorination in 2005 and it took two or three years to optimise growth conditions for these fastidious organisms. Once they got the growth conditions right, they started working on the enrichment process, which involved creating an environment as favourable as possible for the ORB and as unfavourable as possible for competing microbes, such as methanogens and homoacetogens. Chloroform is a particularly problematic organochlorine because it inhibits the ORB that can degrade other organochlorines. In another study, Dr Matthew Lee in Manefield’s team was assessing this inhibitory effect in some groundwater from the Botany site when things appeared to be going wrong. “We thought we had a leak,” said Manefield, “because the chloroform disappeared from one of our replicate cultures. But then the chloroform disappeared from the other replicates as well.” This led to the unexpected discovery of a chloroformdegrading bacterium. The researchers began subculturing samples from the Botany site to find out what was causing the chloroform to disappear. They identified a Dehalobacter species that degraded chloroform into dichloromethane and then to acetate, carbon dioxide and methane. It’s the first isolate that can completely dechlorinate chloroform. “The more we enriched the cultures the faster they consumed chloroform,” said Manefield. “And we found that this bacterium was much more tolerant to high levels of chloroform - the average bacterium can withstand 10 ppm, our culture is highly active at 200 ppm.” The team has sequenced the genome of this species and discovered that it has a larger genome than other Dehalobacter species. They also found that it had 22 reductive dehalogenase genes that code for the enzyme responsible for the breakdown of the chloroform. “This may link to its tolerance to higher concentrations of chloroform,” Manefield suggested.

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MICROBIOLOGY | BIOREMEDIATION AND DRUG RESISTANCE

Dr Mike Manefield is currently an ARC Future Fellow in the School of Biotechnology and Biomolecular Sciences at UNSW, having spent 2004 to 2010 as a Senior Research Associate in the UNSW Centre for Marine BioInnovation where he remains Deputy Director. Following a PhD on the ability of algal metabolites to inhibit bacterial quorum sensing, he spent four years in the UK developing and applying RNA stable isotope probing. He was employed from 2001 on a continuing contract as a postdoctoral scientist in the NERC Centre for Ecology and Hydrology in Oxford, UK. He returned to Australia in 2004 and was employed by UNSW to lead the bioremediation program for the Environmental Biotechnology Cooperative Research Centre.

LESSONS NOT LEARNED FROM HISTORY Cairns-based respiratory physician Dr Stephen Vincent is familiar with the problem of multidrug resistant tuberculosis (MDR TB), now he is preparing to deal with extensively drug resistant tuberculosis (XDR TB) as it emerges on Australia’s border. This highly lethal, mutated variant of the tuberculosis (TB) bacterium has been confirmed in Papua New Guinea (PNG), with six cases reported to date. One patient, who was brought to Cairns from PNG, recently died. “Two years ago there were no known cases of XDR TB in PNG,” said Vincent. “Now there is a reservoir of drug-resistant TB on our doorstep.” Most of the burden of resistant TB appears to fall on poor countries - XDR TB also occurs in countries across Eastern Europe and South East Asia - but there have been some outbreaks in North America as well. New York City had an epidemic of multidrug resistant (MDR) TB in the early 1990s. TOWARDS UNTREATABLE TB

TB is caused by infection with the bacterium Mycobacterium tuberculosis. It is highly contagious and primarily affects the lungs but can also affect other organs in the body, such as the central nervous system, lymphatic system and circulatory system. The disease was called ‘consumption’ in the past because of the way it consumed an infected person from within, causing a slow death. Treatments for TB became available in the 1950s. Before this people underwent surgery, sanitisation and complete isolation. Two antibiotics were developed and patients would initially respond well to these drugs but over time they would become ill again. It was then realised that combination treatment was needed to control TB. “The emergence of resistance to TB began in the 1950s and it was kept under control,” said Vincent. “But due to complacency and lack of government policy, resistance has become worse and today we are heading back to the pre-1950s when TB was untreatable.

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Poor compliance and noncompliance to medications by patients has resulted in TB developing resistance. “We now have MDR TB, XDR TB and totally drug resistant TB (TDR TB) is not too far away,” said Vincent, adding that to be properly monitored and controlled, TB needs government intervention, public awareness and clinicians with TB expertise. According to the World Health Organization, up to 4% of TB cases worldwide are resistant to more than one antituberculosis drug. XDR TB is resistant to the two most potent TB drugs, isoniazid and rifampin, as well as the broad-spectrum antibiotic fluoroquinolone and at least one of three injectable second-line antibiotics (ie, amikacin, kanamycin or capreomycin). Despite this, XDR TB can be successfully treated in about 50% of affected people. Successful outcomes depend on the extent of the drug resistance, the severity of the disease, whether the patient’s immune system is weakened and adherence to treatment. But TDR-TB is untreatable, which is a major concern. ON OUR DOORSTEP

Due to poor TB programs, PNG has a high prevalence of MDRand some cases of XDR-TB. PNG is only 3 km away from the Queensland border. Just off the tip of Cape York, two Australian islands in the Torres Strait, Saibai and Boigu Islands, separate Cape York Peninsula from PNG. And Vincent said there is a lot of movement between these islands and PNG each year. “This open border is a concern,” said Vincent. “The Australian Government doesn’t seem to realise the importance of TB. It is giving money to AusAID but PNG needs more basic infrastructure to reduce poverty and tackle the disease at this front.” The AusAID program is specifically aimed at establishing a TB program in the Western Province of PNG; however, without improving basic infrastructure such as overcrowding, sanitation and adequate food supply, communicable diseases such as TB are unlikely to ever be adequately controlled. Vincent said the people who live in the islands off West Papua, for example Daru Island, are living in poverty. “The towns of these islands are overcrowded,” he said. “They do not have adequate housing and there is no sewerage, running water or access to medical care.” This high population density and the poor sanitary conditions create a perfect environment for TB to propagate, due to the close proximity of infectious cases. A PUBLIC HEALTH DISASTER

XDR-TB is expensive to treat. And it has a low cure rate and a high death rate. “It can cost up to $1 million to treat one person over two years, and this exponentially increases as the resistance increases,” said Vincent. Treatment involves giving an infected person as many TB medications as possible, which gives them around a 50% chance of surviving. A new drug recently came onto the market for XDR

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TB, but it is not ideal because it is expensive and has a high mortality rate. “We are running out of options for treatment,” Vincent said. “And there is a big risk that XDR TB will come into Australia in 5 or 10 years. It’s best to avoid it getting in and to do this we need government support to train people in monitoring the disease.” For this to happen, a strong and visionary political commitment is needed from the federal and state governments, which Vincent said is not happening. The Queensland Government, for example, has been cost cutting, including making cuts to the public health system. This has resulted in uncertainty about the future of TB services, including monitoring TB. “We’re trying hard in North Queensland to monitor new cases in the Torres Strait, but to do this we need to maintain trained staff in the PNG/Australia border,” said Vincent, concerned the problem is being ignored. It will be a public health disaster if XDR TB spreads into the Australian population, although Vincent thinks it is inevitable that this will happen. “Dealing with this disease needs vision and future planning,” he said. “There is a lack of adequate services and we also need to maintain adequate expertise in TB around the country because this will be needed. “We need to maintain vigilance at the border and we need government commitment to do this,” he said. ALS

Daru Island, one of the Torres Strait islands off West Papua, has a high population density. This, coupled with poor living conditions, creates a perfect environment for TB to propagate. Stephen Vincent’s interest in TB began when he moved to Cairns in 1996 as one of the TB physicians involved in the Regional TB control Unit which covers the Cape and the Torres Islands. Vincent is the current director of Thoracic and Sleep Disorders Unit at Cairns Base Hospital. He maintains links with the PNG TB program via a clinical collaborative group and is also a member of the national MDR-TB working group.

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Eliminating the genomic -discovery-to-clinicalassay bottleneck

Joseph M Beechem PhD

Next generation sequencing (NGS) is generating key medical discoveries at a rate that far exceeds our ability to translate them into robust clinical assays. But now, new technology is changing this.

GS technology is (probably) the most significant and impactful ‘pure-discovery’ instrumentation-chemistryanalysis ever developed by the biotechnology industry. While microarray technology was revolutionary, it only allowed scientists to measure ‘everything-they-thought-was-there’, whereas sequencing-technology measures everything that is there - expected and unexpected. And the genomic discoveries are pouring in faster than ever before. However, the rate at which these genomic discoveries are being translated into FDA-approved diagnostics is strikingly low - roughly 1-discovery-in-10,000 currently migrate from genomic-discovery into FDA-regulated tests in the USA, but similar disparities exist worldwide. Hence, one could make a strong argument that what we need is not new genomic-discovery technologies but rather new genomicstranslational technologies (ie, solve the translational bottleneck). Digital gene-expression technology, being just ‘downstream’ (physiologically) from NGS studies, is ideally suited to utilise genomic discoveries by monitoring how genomic-DNA changes are translated by the cell (into RNA) to generate a disease phenotype. By measuring digital RNA gene signatures, better diagnostic, prognostic and therapeutics can be developed in order to improve health care

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and decrease the overall cost burden of health care - both of these items can be simultaneously accomplished. This article will focus on some of the essential features required to successfully solve the translational bottleneck, especially for the case of cancer diagnosis (and prognosis) using multiple-gene RNA signatures. To break the translational bottleneck, a technology is needed that matches (the data type) and complements the type of results output by NGS, namely one that: 1. provides digital results, 2. is multiplexed at a sufficient level to capture the dominant disease-specific features of the NGS result, and 3. is capable of generating results from historically banked (diseasespecific) tissue banks. To read the full article explaining how modern enzyme-free mid-multiplexed digital gene expression technology, exemplified by NanoString’s nCounter system, is ideally suited to translate these NGS discoveries into robust clinical assays, visit http://lifescientist. com.au/content/molecular-biology/article/eliminating-thegenomic-discovery-to-clinical-assay-bottleneck-39843673. NanoString technologies is represented in Australia and New Zealand by Bio-Strategy www.biostrategy.com ALS

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BIOCHEMISTRY | MEDICINAL CHEMISTRY RESISTANCE

Biomolecular in the bush

Susan Williamson

The RACI Division of Biomolecular Chemistry will hold its 2013 conference in the Blue Mountains in July. With themes of medicinal chemistry, chemical biology and drug discovery, here is a taste of what the meeting has in store. ÂŠ www.sxc.hu/OwnMoment

MEDICINAL CHEMISTRY | BIOCHEMISTRY

eveloping a new class of antibiotics that target bacterial virulence rather than viability is the focus of work by Professor Jennifer Martin, an ARC Australian Laureate Fellow based at the Institute for Molecular Bioscience at the University of Queensland (UQ). A structural biologist, Martin’s research focuses on understanding the interactions, function and folding of medically relevant proteins and applying this to medical problems such as antibiotic resistance. “Many antibiotics currently in use are derived from those developed in the 1950s and 1960s. Very few new classes of antibiotics have been approved in the past 20 years,” said Martin. “These current antibiotics work by killing or inhibiting the growth of bacteria, and this puts a strong selection pressure on bacteria to develop resistance. Our research takes a different approach by targeting bacterial virulence rather than viability.” ANCIENT TACTICS

Antibiotic resistance has been documented since the first antibiotics were identified and appears to have existed for millennia. “Recent work analysing 30,000-year-old bacteria in permafrost showed that ancient bacteria also had antibiotic resistance genes,” said Martin. “This is likely because many current antibiotics are derived from natural products, such as penicillin from fungus. Bacteria appear to have evolved ways of dealing with these natural products because they have been exposed to them for millennia in nature.” Antibiotic resistance is currently developing at an alarming rate and the number of new antibiotics in the pharmaceutical development pipeline is slowing to a trickle. The rapid rise in resistance to antibiotics is thought to be due to a number of factors including the use of antibiotics in agriculture, overuse and misuse in humans, and because bacteria readily share resistance genes with each other. These resistance genes may code, for example, for modified proteins that no longer bind the drugs or enzymes that degrade the drugs; thus, protecting the bacteria from the effects of the antibiotic. INHIBITING PROTEIN ASSEMBLY

Different classes of antibiotics exist based on their chemistry and mode of action. Most new antibiotics approved for use over the past 20 years are variations on these known classes, but they remain susceptible to the same resistance mechanisms. This is what makes Martin’s work so important. “Our targets are the bacterial disulfide bond (DSB)-forming proteins,” said Martin, adding that her team of collaborators is one of only a few groups working in this area. Some researchers focus on specific virulence factors produced by bacteria, such as secretion systems that are used to inject a host cell with toxins and poisonous enzymes, which helps the bacteria to enter tissues and cells and cause disease. However, Martin’s work is different. Rather than targeting the virulence factors themselves, her team targets the enzymes that produce the virulence factors.

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“This approach has merit because inhibiting the virulence factor assembly process blocks the activity of the entire arsenal of virulence factors produced by a bacteria, not just one specific virulence factor,” said Martin. Most bacterial virulence factors have disulfide bonds and the DSB-forming proteins are required for these factors to fold and function correctly. Because virulence factors exist in the harsh extracellular environment or need to withstand host defence systems, they need extra stability: the strong covalent disulfide bond adds structural bracing to the protein to help keep it folded and functional, and to protect it from breakdown by host proteases.

Professor Jenny Martin is an ARC Australian Laureate Fellow at the University of Queensland. She has held NHMRC Senior, ARC Professorial and ARC Queen Elizabeth II Fellowships. She trained as a pharmacist in Victoria, was awarded a DPhil from Oxford University and spent her postdoctoral years at Rockefeller University.

SOLVING STRUCTURES

Martin’s team is targeting the structures of two DSB proteins - the soluble DsbA protein and its integral membrane protein partner DsbB. “Genetic knockouts of the DsbA and DsbB genes in pathogenic organisms has shown that without these proteins the organisms are no longer pathogenic,” said Martin. “We expect that by chemically inhibiting these proteins with drug-like molecules we will generate the same effect.” Martin’s team is collaborating with Martin Scanlon at Monash University and Professors David Fairlie, Matt Cooper and Mark Schembri at UQ in the characterisation of the DsbA and DsbB proteins with the ultimate aim of developing inhibitors of these proteins. The work has a three-pronged approach to develop inhibitors. First, they are screening libraries of drug-like fragments against DsbA proteins from human pathogens to identify potential candidates for development into drug leads. Second, they are designing peptidomimetics. This work is based on the knowledge that the two proteins, DsbA and DsbB, interact with each other. By developing small peptide-like compounds that mimic the DsbA:B interaction, the researchers hope to block DSB activity and render bacteria avirulent. And third, they are taking an in silico or virtual screening approach. This involves screening a virtual library of drug-like chemicals against the DSB protein structures using docking software

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BIOCHEMISTRY | MEDICINAL CHEMISTRY RESISTANCE to predict which chemicals will interact. This will enable potential DSB inhibitors to be identified via computer simulation. The best hits are then purchased or synthesised and assessed for activity in biochemical assays. “Our focus is on Escherichia coli, at present,” said Martin, “but we are also solving the structures of DSB proteins from a number of other human pathogens to generate a library of DSB protein structures that will be available for us and the whole community to work with to develop new drugs. “The DSB proteins appear to be excellent targets for the development of drugs that inhibit virulence rather than viability. Our goal is to generate a new chemical class of antibacterial that can be used either alone or in combination to treat infections caused by multidrug-resistant bacteria.”

The Royal Australian Chemistry Institute Division of Biomolecular Chemistry will hold its 2013 conference Biomolecular in the Bush - at the Fairmont Resort in the Blue Mountains on 14-17 July. For more informat ion, go to the conference website at www.raci-bio-conf.org.

A LIFE SENTENCE Associate Professor Matthew Piggott, from the School of Chemistry and Biochemistry at the University of Western Australia, recently received recognition for his work on the design of small molecule therapeutics to improve quality of life for sufferers of Parkinson’s disease when he received the 2012 Biota Award for Medicinal Chemistry.

Piggott first became interested in Parkinson’s disease during his honours year in 1996 when his father was diagnosed with the disorder. Since then a large part of his work in medicinal chemistry has been directed towards this debilitating disease. At age 40, Piggott’s father was a ‘young onset’ Parkinson’s disease patient - the disease usually manifests in older people, with incidence rising progressively with age. “I turn 40 next year,” said Piggott, although he clarified that his father has the idiopathic form of the disease where there is no known cause and it is rare that the disease has a simple genetic origin. A PROGRESSIVE DISEASE

Parkinson’s is a progressive neurodegenerative disease that is characterised by the death of dopaminergic neurons in the substantia nigra area of the brain. This decrease of dopamine in the brain results in people with Parkinson’s having a great deal of difficulty moving. Thus, treatment aims to increase this neurotransmitter and thereby restore normal movement. Treatment usually begins with dopamine agonists, which are initially effective at dealing with the symptoms. However, side effects have been observed in some patients. “Some patients become more compulsive or obsessive,” said Piggott. “For example, they can suddenly develop an addiction to gambling.” And, over time, patients’ dopamine agonists lose their efficacy. Most patients then go onto levodopa (L-DOPA) therapy the mainstay treatment for Parkinson’s disease. L-DOPA is the precursor to the catecholamine neurotransmitters, one of which is dopamine. Treatment with L-DOPA restores patients’ movement but over time they commonly develop side effects. The most notable of these are the involuntary movements known as L-DOPA-induced dyskinesia and a reduction in therapeutic duration or ‘on-time’. That is, a reduction in the period of time that the patient is able to move as a result of their L-DOPA treatment. When the drug runs out, the patient is ‘off’ or unable to move very well, or at all, depending on the severity of the disease. DEALING WITH SIDE EFFECTS

Associate Professor Matthew Piggott with PhD student Michael Gandy holding a model of MDMA and discussing how it might be modified. Credit: Bob Bucat.

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Piggott’s work is focused on addressing the side effects cause by L-DOPA, the main one being dyskinesia. These jerky, involuntary movements are often mistakenly thought to be a symptom of Parkinson’s disease, when in fact they are a side effect of the treatment. “This dyskinesia tends to develop faster in younger-onset patients,” added Piggott. One current treatment for dyskinesia in these patients is deep brain stimulation. This involves inserting two electrodes into the brain and electrically stimulating that part of the brain involved in creating the involuntary movements. “It has a high success rate,” said Piggott. “It can be very effective in reducing dyskinesia, but it is extremely expensive and strokes, leading to further impairment to movement, speech, or resulting in personality changes are an unlikely but real possibility.”

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BIOCHEMISTRY | MEDICINAL CHEMISTRY RESISTANCE The only other current option for these patients to get relief from these jerky, involuntary movements is by taking a drug called amantadine, which has limited efficacy, does not work for many patients and has side effects of its own. Hence, Piggott’s work on developing a pharmaceutical option to treat this problem for patients. “It has been known for some time now that the illicit drug most commonly known as ecstasy, methylenedioxymethamphetamine (MDMA), ameliorates the side effects of L-DOPA in animal models of Parkinson’s disease, and anecdotally in humans,” said Piggott. But MDMA has little therapeutic potential in this context because it makes users ‘high’, and whether it is safe to use in humans over the long term is unclear. “Although controversial, there is evidence that MDMA may be neurotoxic, or at least responsible for long-term, deleterious changes in brain chemistry.” MODIFYING MDMA

In collaboration with Dr Jonathan Brotchie, from the University Health Network in Toronto, Canada, Piggott’s team has set out to create MDMA analogues and show that it is possible to dissociate the beneficial effects of MDMA from its undesirable attributes. “The best compound, which we call UWA-101, is even more effective than MDMA at enhancing the quality of levodopa therapy. In the best animal model of Parkinson’s disease, a primate model, UWA-101 lengthened on-time by up to 30%.

“More importantly, UWA-101 increased the proportion of on-time that was of good quality (ie, without disabling dyskinesia) by 178%. If translated to a medicine, this would mean that Parkinson’s patients could take their medication less frequently and get a better quality result from it,” Piggott explained. UWA psychopharmacologist Professor Mathew MartinIverson and PhD student Zak Millar, have shown that UWA-101 is unlikely to be psychoactive, based on studies in rats. “These studies include examining how the drug affects rats’ response to startling noises and whether they can be trained to discriminate UWA-101 from MDMA,” Piggott said. In addition, UWA-101 is not toxic to a cell line used to model MDMA-induced neurotoxicity. “Longer-term studies in animals are required to confirm a lack of neurotoxicity,” Piggott admitted. An estimated 80,000 Australians live with Parkinson’s. Most people with the disease continue to live interactive and rewarding lives. Piggott attests to this with his father continuing to lead an engaging life despite his disease. But there is no doubt that Parkinson’s makes many things difficult, and some things impossible, for most sufferers. “My father turns 60 this year,” he said. “People die with this disease, not from it - that’s why it’s been described as a life sentence, not a death sentence.” ALS

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Cell microarray The DEPArray, from Silicon Biosystems, is an automated platform that is designed to isolate, characterise and collect rare cells, such as circulating tumour cells (CTCs), based on a lab-on-a-chip technology for downstream analysis. Each cell is trapped in suspension into a cage. It is numbered, assigned to a recovery group and can be individually moved around and collected through a software-calculated pathway. Images of each cell in different emission channels and bright field are acquired and fluorescent signals are measured. Relative threshold parameters can be set and associated with morphology for selection. Even as little as one single cell can be collected out of the entire suspension, thus making the technology a suitable individual cell biology tool for cancer research and information support to cancer therapy. Applications include: rare cell sorting; cell sorting of small loads; investigation of cell-cell interaction; single-cell immunophenotyping; drug delivery. Diagnostic Technology Pty Ltd Contact info and more items like this at wf.net.au/T066

Chemiluminescence Western blot scanner LI-COR Bioscience’s latest innovation is the C-DiGit Chemiluminescence Western Blot Scanner. The unit does not require film but still produces film-quality images - without the hassle and expense of film development. It has the simplicity of film exposures without the mess of the darkroom. The user’s current chemiluminescent Western blot protocol doesn’t have to change. All the same steps are performed, with the C-DiGit providing a complete digital replacement for film. Millennium Science Pty Ltd Contact info and more items like this at wf.net.au/T565

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Photometer Eppendorf has extended its range of detection instruments for life sciences applications. With the Eppendorf BioPhotometer D30, measurement data is recorded for fixed wavelengths, making the device suitable for routine applications. Data are clearly processed, making the evaluation of results fast and simple while minimising the risk of mistakes. Assistance for the user, including comparative spectra that are stored in the device, offers an additional level of security.

Spectrophotometer range Eppendorf offers a full range of spectrophotometers: from the entrylevel D30, specifically for DNA, RNA and protein quantitation, to the full-spectrum tuneable, kinetic and scanning systems - the Basic and

Furthermore, all relevant ratios are automatically determined for

Kinetic models - which read from 200-830 nm, <4 nm bandwidth, are

the corresponding applications.

fully programmable and can integrate with a PC or operate stand-alone.

The product offers the option of recording purity scans for

In addition, the Biospectrometer Fluorescence can increase the

specific applications. In a defined measuring range, additional

possibilities. The user can utilise the micro Cuvette G1 to assay as

measurement data is collected and shown extrapolated, allow-

little as 2 ÂľL of sample or make use of the companyâ&#x20AC;&#x2122;s UV-transparent

ing contamination in the sample to be visually identified quickly.

plastic UVettes. This is all from a lightweight and portable system.

Eppendorf South Pacific Pty Ltd

Point of Care Diagnostics

Contact info and more items like this at wf.net.au/T784

Contact info and more items like this at wf.net.au/T811

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Target molecule detection system According to Bio-Strategy, the NanoString Technology nCounter Analysis System is set to become a cornerstone technology of molecular biology in the same way PCR has done. It uses colour-coded molecular barcodes that hybridise to and directly detect different types of target nucleic acid target molecules (mRNA, miRNA or DNA). The novel detection system provides digital multiplex quantification of up to 800 different target molecules in a single sample. It is sensitive to the full range of biological expression, requires very little sample (as little as a single cell) and is resistant to degraded material such as formalin-fixed, paraffin embedded (FFPE) tissue. In addition to a range of application-specific kits for gene expression, copy number variation (CNV) and miRNA analysis, custom nanoString CodeSets can detect any target sequence from any organism. Compared to qPCR, results are said to be simpler to generate, more precise and more tolerant of poor sample quality. Bio-Strategy Pty Ltd Contact info and more items like this at wf.net.au/S867

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PUBLISH OR PERISH

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.

Allison CC, Ferrand J, McLeod L, Hassan M, Kaparakis-Liaskos M, Grubman A, Bhathal PS, Dev A, Sievert W, Jenkins BJ, Ferrero RL. Monash Uni Nucleotide oligomerization domain 1 enhances IFN-g signaling in gastric epithelial cells during Helicobacter pylori Infection and exacerbates disease severity.

J Immunol. 2013 Apr 1

Ashman LK. Uni of Newcastle Renal disease as a potential compounding factor in carcinogenesis experiments with Cd151-null mice.

Oncogene. 2013 Mar 11

Becker TM, Boyd SC, Mijatov B, Gowrishankar K, Snoyman S, Pupo GM, Scolyer RA, Mann GJ, Kefford RF, Zhang XD, Rizos H. Uni of Sydney at Westmead Millennium Inst, Westmead Hosp

Mutant B-RAF-Mcl-1 survival signalling depends on the STAT3 transcription factor. Oncogene. 2013 Mar 4

Denny KJ, Coulthard LG, Jeanes A, Lisgo S, Simmons DG, Callaway LK, Wlodarczyk B, Finnell RH, Woodruff TM, Taylor SM. Uni of Qld, Brisbane C5a receptor signaling prevents folate deficiencyinduced neural tube defects in mice.

J Immunol. 2013 Apr 1

Garratt M, Gaillard JM, Brooks RC, Lemaître JF. UNSW Diversification of the eutherian placenta is associated with changes in the pace of life.

Proc Natl Acad Sci USA. 2013 Apr 22

Gilmour JP, Smith LD, Heyward AJ, Baird AH, Pratchett MS. Uni of WA Oceans Inst, Perth Recovery of an isolated coral reef system following severe disturbance.

Science. 2013 Apr 5

Gonsalvez DG, Cane KN, Landman KA, Enomoto H, Young HM, Anderson CR. Uni of Melb Proliferation and cell cycle dynamics in the developing stellate ganglion.

Jegaskanda S, Weinfurter JT, Friedrich TC, Kent SJ. Uni of Melbourne Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques.

J Virol. 2013 May

Kim WS, Li H, Ruberu K, Chan S, Elliott DA, Low JK, Cheng D, Karl T, Garner B. Neuroscience Res Aust, Sydney Deletion of Abca7 increases cerebral amyloid-β accumulation in the J20 mouse model of Alzheimer’s disease.

J Neurosci. 2013 Mar 6

Koyama M, Kuns RD, Olver SD, Lineburg KE, Lor M, Teal BE, Raffelt NC, Leveque L, Chan CJ, Robb RJ, Markey KA, Alexander KA, Varelias A, Clouston AD, Smyth MJ, Macdonald KP, Hill GR. Qld Inst of Med Res, Brisbane Promoting regulation via the inhibition of DNAM-1 after transplantation.

J Neurosci. 2013 Apr 3

Blood. 2013 Apr 25

Borneman AR, Schmidt SA, Pretorius IS. Aust Wine Res Inst, Adelaide At the cutting-edge of grape and wine biotechnology.

Gratten J, Visscher PM, Mowry BJ, Wray NR. Uni of Qld, Qld Brain Inst, Brisbane

Trends Genet. 2013 Apr 29

Interpreting the role of de novo protein-coding mutations in neuropsychiatric disease. Nat Genet. 2013 Mar

Lee P, Swarbrick MM, Ho KK. Uni of Qld and Princess Alex Hosp, Brisbane; Garvan Inst of Med Res and UNSW, Sydney Brown adipose tissue in adult humans: a metabolic renaissance.

Botté CY, Yamaryo-Botté Y, Rupasinghe TW, Mullin KA, Macrae JI, Spurck TP, Kalanon M, Shears MJ, Coppel RL, Crellin PK, Maréchal E, McConville MJ, McFadden GI. Uni of Melbourne

Hayes BJ, Lewin HA, Goddard ME. Dept of Primary Ind; Dairy Futures CRC; La Trobe Uni, Vic The future of livestock breeding: genomic selection for efficiency, reduced emissions intensity, and adaptation.

Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites. Proc Natl Acad Sci USA. 2013 Apr 15 Branford S, Yeung DT, Ross DM, Prime JA, Field CR, Altamura HK, Yeoman AL, Georgievski J, Jamison BA, Phillis S, Sullivan B, Briggs NE, Hertzberg M, Seymour JF, Reynolds J, Hughes TP. SA Pathology, Adelaide Early molecular response and female sex strongly predict stable undetectable BCR-ABL1, the criteria for imatinib discontinuation in patients with CML.

Blood. 2013 Mar 20

Chan JY, Luzuriaga J, Bensellam M, Biden TJ, Laybutt DR. Garvan Inst of Med Res and St Vincent’s Hosp, Sydney Failure of the adaptive unfolded protein response in islets of obese mice is linked with abnormalities in β-cell gene expression and progression to diabetes.

Diabetes. 2013 May

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Trends Genet. 2013 Apr

Holliday EG. Uni of Newcastle Hints of unique genetic effects for type 2 diabetes in India.

Endocr Rev. 2013 Apr 2

Redfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, Foulds CE, Stuart LM, Barker A, Russell VJ, Ramsay K, Kobelke SJ, Li X, Hatchell EC, Payne C, Giles KM, Messineo A, Gatignol A, Lanz RB, O’Malley BW, Leedman PJ. WA Inst for Med Res RNA-induced silencing complex (RISC) proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

Diabetes. 2013 May

Proc Natl Acad Sci USA. 2013 Apr 16

Hor L, Dobson RC, Downton MT, Wagner J, Hutton CA, Perugini MA. La Trobe University, Vic Dimerization of bacterial diaminopimelate epimerase is essential for catalysis.

Tan JH, Ludeman JP, Wedderburn J, Canals M, Hall P, Butler SJ, Taleski D, Christopoulos A, Hickey MJ, Payne RJ, Stone MJ. Monash Uni Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1).

J Biol Chem. 2013 Mar 29

Housley GD, Morton-Jones R, Vlajkovic SM, Telang RS, Paramananthasivam V, Tadros SF, Wong AC, Froud KE, Cederholm JM, Sivakumaran Y, Snguanwongchai P, Khakh BS, Cockayne DA, Thorne PR, Ryan AF. UNSW ATP-gated ion channels mediate adaptation to elevated sound levels.

Proc Natl Acad Sci USA. 2013 Apr 16

J Biol Chem. 2013 Apr 5

van Alphen B, Yap MH, Kirszenblat L, Kottler B, van Swinderen B. Univ of Qld A dynamic deep sleep stage in Drosophila.

J Neurosci. 2013 Apr 17

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EVENTS

DATES FOR THE LIFE SCIENCES CALENDAR The coming year is packed with exciting local and international events. Here’s a taste.

ARCS Annual Scientific Congress

June 5-6, Sydney Previously the Association of Regulatory and Clinical Scientists, ARCS Australia is a not-for-profit professional development association for people working in the development of therapeutic goods. Their annual scientific congress caters to a wide audience in the therapeutic industry and will bring together clinical, regulatory, quality, medical affairs, medical information, pharmacovigilance and soft skills. Congress sessions will cover a broad range of topics from the regulation of biologicals and practical aspects of running clinical trials to medicines and devices, with something of interest for people from industry, researchers and academics.

http://arcsconferences.com

2nd Annual NHMRC Research Translation Faculty Symposium - from Bench to Bourke: improving practice, policy and commercialisation October 2-3, Sydney

www.nhmrc.gov.au/media/events/2013/2ndannual-nhmrc-symposium-researchtranslation Annual Bioprocessing Network Conference October 22-24, Gold Coast

www.bioprocessingnetwork.com.au/ Conferences.html

The Australasian Bioenergy and Bioproducts Symposium 2013 October 25, Brisbane

www.tabbs.com.au The Australasian Mycological Society Joint Conference July 10–12, Adelaide

AusMedtech 2013 May 15-16, Melbourne

www.ausbiotech.org

www.theasm.org.au

Collaborate Innovate 2013 May 15-17, Melbourne

Biomolecular in the Bush July 14-17, Leura, Blue Mountains

http://conference.crca.asn.au/

www.raci-bio-conf.org

Asian Society for Pigment Cell Research and the Australasian Society for Dermatology Research (ASPCR-ASDR) Conference 2013 May 17-19, Sydney

www.aspcr-asdr2013.org

18th NSW Stem Cell Network Workshop May 21, Sydney

www.stemcellnetwork.org.au/Workshop/ MainFrameSet.html 24th International Conference on Arabidopsis Research June 24-28, Sydney

http://www.sallyjayconferences.com.au/ icar2013/ 4th International NanoMedicine Conference July 1-3, Sydney

What’s New in Laboratory Technology? Biannual symposium July 16–17, Macquarie University, Sydney

www.raci.org.au/events

ASID Gram-Negative ‘Superbugs’ Meeting August 2-3, Gold Coast

http://www.asid.net.au/gramnegative Familial Aspects of Cancer Meeting August 25-28, Cairns

www.meeting-makers.com/fac

International Society for Gastrointestinal and Hereditary Tumours August 28-31, Cairns

www.insight-group.org

www.oznanomed.org

Tech Transfer Summit Australia 2013 September 3-4, Melbourne

Australian Society for Microbiology Annual Scientific Meeting 2013 July 7-10, Adelaide

ComBio2013 September 29-October 3, Perth

www.theasm.org.au

www.asbmb.org.au

Australian Marine Science Association Golden Jubilee conference July 7-11, Gold Coast

Australasian College for Infection Prevention and Control Conference September 30-October 2, Gold Coast

www.amsaconference.com.au/

www.ausbiotech.org

http://www.acipcconference.com.au

5th Asia-Pacific NMR Symposium and 9th Australian & New Zealand Society for Magnetic Resonance October 27-30, Brisbane

http://apnmr2013.org

15th World Conference on Lung Cancer

October 27-31, Sydney www.2013worldlungcancer.org Association of Biosafety for Australia and New Zealand 3rd Annual Conference October 29-November 1, Auckland, New Zealand

www.absanz.org.au/Conference 2013.html AusBiotech 2013 October 29-November 2, Brisbane

www.ausbiotech.org

9th World Sponge Conference November 4-8, Fremantle, Western Australia

www.aims.gov.au/web/sponge/home International Marine Biotechnology Conference November 11-15, Brisbane

www.imbc2013australia.com/

Laboratory Management & Design Conference November 18-20, Brisbane

www.labmanagers.org.au/

HPLC 2013 - 40th International Symposium on High-Performance-Liquid-Phase Separations and Related Techniques November 18-21, Hobart

www.hplc2013-hobart.org

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BioResearch

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To book a free Nucleofection™ Demonstration for your cells, please contact: Lonza Australia Pty Ltd Lonza Pharma - Bioscience Solutions Tel: +61 3 9550 0883 Local Call: 1300 657 508 Email: bioscience.australia@lonza.com

www.lonza.com/4d-nucleofector

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Send in 10 Pipettes and we’ll service 2 for FREE Now is a great time to have your Pipettes serviced and ready in time for the new financial year, with Pathtech’s EOFY Service Laboratory Offer.

Contact the Pathtech the team on 1800 069 161 and quote ‘End of Financial Year’ to receive this offer. Offer strictly available from 1st May until 30th June. Offer available on the 2 x lowest price Pipettes serviced and is applicable for labour only. Offer does not include any spare parts of equipment that may be required.

For further information, contact the Pathtech team phone 1800 069 161

pathtech.com.au 48

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