Australian Life Scientist Jan/Feb 2013

SIR GUSTAV NOSSAL ON HIS REMARK ABLE CAREER AS RESEARCHER AND SCIENCE ADVOCATE 14

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Contents FACE TO FACE

14 The campaigner Following a 30-year stint heading up the Walter and Eliza Hall Institute, Sir Gustav Nossal is now a top level advisor helping to advance the cause of global health. Here he reflects on his long and fruitful career.

Lorne 2013 GENE REGULATION

20 Ghost in the genome It turns out genetics is not as complex as we thought. It’s even more complex. Associate Professor Kevin Morris has recently arrived from the US and is working on uncovering the startling complexity of gene regulation.

20 PHOSPHOPROTEOMICS

26 Looking at the big picture Professor David James and his team are assembling a comprehensive atlas of the phosphoproteome of insulin that is revealing new insights about diabetes.

14 CYTOKINE SIGNALLING

BREAST CANCER

30 Signal strength

38 Connecting the dots

Associate Professor Brendan Jenkins is helping to uncover the cellular signals involved in stomach cancer, which may one day be used as biomarkers or as new targets for treatment.

STEM CELLS

34 Altered states UWA’s Professor Ryan Lister’s research into the epigenome suggests the future clinical use of induced pluripotent stem cells should proceed with great caution.

Dr Stacey Edwards is uncovering the mechanism responsible for inherited disorders that occur seemingly without a clear genetic basis.

Australian Neuroscience Society 2013 COLOUR VISION

42 Vivid insight

REGULARS

IN THE NEXT ISSUE OF ALS

6 10 12 45 46

• BIO 2013 conference preview • Biotechnology State of the Nation report • Hunter Meeting • Cell biology & stem cells Editorial deadline: 19/02/13 Advertising deadline: 19/02/13

Movers and shakers GrantWatch AusBiotech Publish or perish Events

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Professor Paul Martin has his eye on uncovering the complexities of primate colour vision.

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Reaching

the herd

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eryl Dorey, head of the inaptly-named Australian Vaccination Network and recipient of the 2012 Bent Spoon Award from the Australian Skeptics and nominee for Crikey’s 2012 Arsehat of the Year - vehemently believes the risks of vaccination outweigh the benefits. And she is not shy in spreading that belief across the airwaves and the internet connections of this nation.

So vocal and so deft is Dorey with her manipulation of facts and falsehoods that her message appears to be resonating.

Vaccination rates in Australia have been dropping over the past few years, particularly in Western Australia, causing alarm amongst health workers.

This represents the first substantial reversal in vaccination rates in decades. The danger to children and the wider population of death or sickness from preventable diseases is on the rise.

The good news is, for the vast majority of the population, reason and prudence have prevailed. While a drop in vaccination rates is an alarming development, we should take solace that the drop has been relatively small. According to government figures, 92% of babies under 12 months were fully vaccinated in 2002, dropping to 91% in 2006, with the rate hovering around 91.8% today.

Western Australia trails the pack at 90.3%.

However, we should temper that news with the notion that herd immunity for many diseases kicks in at around 94-95% of the population. Anything below that and we’re asking for trouble.

The upshot is that we can’t afford to let our guard down. To those in the know, vaccination seems a no-brainer. Yet for many, particularly young parents who have never lived in a world where polio, measles and other diseases were a fact of life, and who are highly sensitive to any potential risks for their infants, vaccination may appear a risky venture.

As such, we must constantly endeavour to understand the motivations of those who express doubts about vaccines and work to reach out and engage them, reassuring them of the soundness of the science and the benefits to them and their family.

According to Sir Gustav Nossal, who has been a vocal advocate for vaccination throughout his career, and has helped bring it to millions of people in developing countries, the best approach is to use “sweet reason”. The facts, delivered clearly and simply, tell the strongest story.

You can read about Sir Gus’s thoughts on how to combat anti-vaxers, and on a great many other topics, in our interview with him on page 14. This appears in our inaugural Face to Face section, where each issue we will chat with some of the leaders and luminaries of the life sciences in Australia. I don’t doubt that if all of us were as engaging as Sir Gus, the Meryl Doreys of the world would never stand a chance.

Tim Dean

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INDEX OF ADVERTISERS ABSCIEX 48 BioStrategy 11 Cryosite 37 Eppendorf 17 GE Healthcare 19 Gene Works 23

ICP Firefly 41 Interpath Services 33, 44 Lonza 7,9 LAF Tech 47 MP Biomedicals 2 Merck 24,25

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

MilleniumScience Sartorius Sarstedt United Bio Research

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29 4 13 40

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MOVERS & SHAKERS

Genetic Technologies rattled as CEO resigns in protest Annual general meetings are often predictable affairs, but the one held in late November by Genetic Technologies proved volatile. First was the rejection of Mel Bridges and Huw Jones from the company’s board at the direction of major shareholder Mervyn Jacobson. Then, in protest to the board spill, CEO Paul MacLeman resigned, along with VP Legal and Corporate Development Dr David Sparling and fellow board member Greg Brown.

Replacing MacLeman is Alisen Mew, who was formerly the company’s COO. GTG also appointed Ben Silluzio as a Non-Executive Director.

Following the spill, Bridges said: “It is regrettable that a company with the potential of GTG was unable to reach middle ground with its major shareholder.”

Recent human evolution boosts number of disease mutations A huge genetic study led by researchers from the University of Washington in the US and published in Nature in November has shown that the massive population growth our species has enjoyed over the past five millennia has not been without cost. During that time, many mutations, including many that are associated with diseases, have entered our gene pool, and because the burgeoning population has spread across the globe, natural selection has not been able to eliminate them.

The study also found that Americans of European descent harboured more of these deleterious genetic variations than Americans of African descent, lending support to the Outof-Africa model of human history. According to this model, humans migrated out of Africa in a series of waves, subsequently fanning out through Europe and Asia to settle in all corners of the globe.

However, as non-African populations spread apart, with less interbreeding between them, there was less opportunity for negative, or purifying, selection to eliminate deleterious mutations. African populations, however, were more concentrated and the deleterious mutations were eliminated slightly faster.

As such, the deleterious variations in European Americans also emerged more recently than those in African Americans, with the average age of the former being 5200 years and the latter being 10,100 years. The study looked at over 15,000 genes in 6515 individuals, including 4298 of European and 2217of African descent, and found 1.15 million genetic variations in total.

“The recent dramatic increase in human population size, resulting in a deluge of rare functionally important variation, has important implications for understanding and predicting current and future patterns of human disease and evolution,” the authors write.

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

Graham Kelly back in charge of revamped Novogen Founder Dr Graham Kelly is back at the helm of Novogen (ASX:NRT) after a seven-year absence following the company’s acquisition of Triaxial Pharmaceuticals for $1.9 million.

Novogen is returning to focus on anticancer drugs with the acquisition, which it is billing as the culmination of a restructuring that began three years ago.

This restructuring involved the sale of multiple assets and the separation of US subsidiary MEI Pharma through a placement and share distribution. The acquisition of Triaxial gives Novogen a portfolio of technologies based on small molecule drugs. Triaxial had been engaged in applying these technologies to benzopyran compounds to create ‘super benzopyrans’ with anticancer activity.

Kelly said Novogen will be taking up the mantle from Triaxial for super benzopyran development. “[The new, Novogen’s] first drug, CS-6, has been designed specifically to cross the blood-brain barrier and to attack primary brain cancer cells,” he said.

Kelly founded Novogen in 1992 and helped bring it public on the ASX in 1994 and to the Nasdaq in 1999.

But he left the company in 2005, due largely to a difference of opinion over the strategic direction of the company.

Kelly co-founded Triaxial with two other former Novogen scientists in 2009. The three founders were largely responsible for developing Novogen’s original isoflavonoid anticancer IP. In an open letter to shareholders, Kelly expressed a “great sense of satisfaction” to again be heading up the company. He also stated that he had “unfinished business … a technology to put back on track and the faith of some long-suffering shareholders to restore”.

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MOVERS & SHAKERS

A study published in Cancer Cell in December has shed light on the epigenetic machinations of cancer, showing how tumour cells can activate genes that support their activities. The study, led by the Garvan Institute’s Professor Susan Clark and PhD student Saul Bert, used gene expression profiling and genome-wide sequencing data from prostate tumour cells to determine which parts of the genome were epigenetically activated in prostate cancer.

They found that regions that contained prostate cancer-specific genes, including PSA (prostate specific antigen) and PCA3, the most common prostate cancer markers, were epigenetically activated via DNA methylation. The study specifically found that large portions of the DNA within cancer cells - around 2% - were activated, with these regions containing key oncogenes, microRNAs and cancer biomarker genes, indicating they are crucial to the cancer’s survival and spread. These regions were activated via the hypermethylation of specific locations on the DNA, called CpG islands, which cluster near gene start sites.

“We took a whole genome approach, looking at all the gene transcription start sites that included CpG islands,” said Bert. “What we saw surprised us, because we saw gene activation at hypermethylated sites - that went against current thinking.

“We went on to show in the lab that if you methylate CpG islands that are very close to transcription start sites, but not exactly on top of them, then it’s possible to turn genes on. While the realisation that methylation can trigger gene activation represents a paradigm shift in thinking, our other finding - that the prostate cancer genome contains domains that harbour multiple gene families, tumour related genes, microRNAs and cancer biomarkers - is equally important. These domains are simultaneously switched on through significant epigenetic remodelling.”

Starpharma smarts after knockbacks Starpharma suffered a major setback in November with its VivaGel treatment for bacterial vaginosis (BV) failing © iStockphoto.com/Catalin Stefan to reach the primary end point of its phase III trial. This trial was to be instrumental in securing FDA approval for the treatment.

The results of the phase III trial showed that VivaGel demonstrated statistically significant effectiveness in days following initial treatment, but the primary end point of a clinical cure versus placebo two to three weeks later was not met. As a result, a New Drug Application will not be filed for VivaGel as a cure for BV. The company is investigating some unexpected results as a part of the trial, such as high placebo cure rates at some sites and the fact that placebo cure rates increased between time points in the trial rather than decreased as expected. However, given the trial outcomes of dramatically reducing symptoms and curing BV in some of the patients, Starpharma is continuing to pursue VivaGel as a potential prevention of BV recurrence.

“We are surprised and disappointed in not meeting the phase III FDA end point for the treatment indication in these trials, given the phase II trial results,” said CEO Dr Jackie Fairley.

On the upside, the company released results of trials showing its dendrimer platform technology boosted the effectiveness of agrochemical glyphosate as well as cancer treatment docetaxel.

Both avenues will be pursued by the company.

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© iStockphoto.com/Sergey Volko

Epigenetics shows how cancer manipulates our genes

3D printers enter the lab

A researcher in the United States has produced simple lab items using a 3D printer rather than purchasing them off the shelf, highlighting the power of the technology for producing low-cost items on demand, but also raising questions about intellectual property.

A blog post by a University of California Davis graduate student details his creation of electrophoresis combs from scratch. He starts by expressing outrage that the combs cost $51 each retail, yet were little more than a “lousy little scrap of plastic”. Yet, he noted, creating moulded “little scraps of plastic” is what 3D printers do so well.

Using an inexpensive Ultimaker 3D printer, he then designed his own electrophoresis combs in two different sizes and calculated that they cost only around 21c each to print. Not only were they cheap and producible on demand, they were tailored to his specifications and functioned even better than the off-the-shelf versions.

The production of simple items that resemble or are based on existing products raises some intellectual property implications. The copying of products that are currently protected by patents, design patents or copyright is currently illegal in many jurisdictions. So too is deriving new work from an existing product.

However, despite the legal uncertainties, 3D printing technology is appealing to many researchers, with several comments on the blog supporting the venture. Russell, the author of the blog post, closed his post with the following remark: “If you have a lab, and you don’t have a 3D printer, you are wasting your money. Seriously.”

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MOVERS & SHAKERS

GrantWatch NHMRC reviews Development Grants Scheme The NHMRC launched an independent report on the Development Grants Scheme in November to address the so-called funding “valley of death” that exists between publicly funded research and private investment to develop discoveries into commercial products.

The report, Evaluation of Development Grants Scheme, analyses the economic, health and industry outcomes of the grants. The scheme supports the commercial development of a product, process, procedure or service that can improve health care and/or disease prevention or provide health cost savings.

All completed and current development grants funded between 2000 to 2008 were analysed with 40 grants sampled for more in-depth assessment. Of the 40 grants, the majority had secured a commercial partner and more than half were under possible commercial development. Others had a product to market or were awaiting regulatory approval.

“The report recommended that the scheme’s current design should be retained and that NHMRC should increase consultation with investors and industry on incorporation of commercial milestones in grant approvals and reporting,” said NHMRC CEO Professor Warwick Anderson.

Funding calendar

Grant

Applications open

Applications close

NHMRC Research Fellowships

12 December 2012

13 February 2013

NHMRC Practitioner Fellowships

12 December 2012

13 February 2013

ARC Future Fellowships

19 December 2012

20 February 2013

ARC Discovery Projects 2014

16 January 2012

6 March 2013

NHMRC Career Development Fellowships

15 January 2013

6 March 2013

NHMRC Project Grants

5 December 2012

19 March 2013

ARC Discovery Early Career Researcher Award 2014

11 February 2013

27 March 2013

NHMRC Partnership Projects 2013

30 January 2013

20 April 2013

NHMRC Program Grants

6 March 2013

5 June 2013

NHMRC Development Grants

10 April 2013

10 July 2013

Victorian Government launches new voucher program The Victorian government has launched a new initiative to support innovative companies with the Technology Voucher Program. The first voucher is the Technology Development voucher, valued at up to $50,000, used to undertake technology development and/or absorption.

The second is the $250,000 Technology Implementation voucher, used to undertake substantial testing or applied development activities in order to adapt existing technology-based innovations into new practice or within new markets.

Third is the $10,000 Technology Student Accelerator, which can be used to undertake an ICT development project with a Victorian university using one or more students during the vacation period.

The TVP is primarily designed to support Victorian companies to use technologies - specifically ICT, industrial biotechnology and

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small technologies - in order to develop more competitive products, processes and services. As a secondary objective, the program will also support non-Victorian companies to access Victorian technology know-how and services where there are significant and measurable benefits likely to flow to the state.

Victorian small to medium-size companies (1-199 employees) are eligible for a Technology Development Voucher, a Technology Implementation Voucher and a Technology Student Accelerator Voucher. Large Victorian companies (200+ employees) are eligible for a Technology Development or a Technology Implementation Voucher. Interstate companies are eligible for a Technology Development or a Technology Implementation Voucher.

The vouchers are administered by the Small Technologies Cluster (STC) and the National ICT Australia Victoria Research Lab.

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

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AUSBIOTECH | VIEWPOINT

Patent review bonanza has industry under siege Dr Anna Lavelle, CEO, AusBiotech

C

onventional wisdom dictates that a strong and reliable intellectual property and patent system is a standard requirement to attract soughtafter foreign and domestic investment.

So too public policy certainty in the business environment encourages business confidence, underpinning commitments to research and development, staff expansion and the like.

The flip side is that unstable policy and an uncertainty about the patent system discourages investment, as investors and businesses hold their cash and wait for more certain times. In every corner of the business world, a lack of confidence, caused by uncertainty, is the enemy. In industries where the business model is based on large investments over long periods, like biotechnology, the impacts of uncertainty are amplified.

This instability underscores a huge problem for the biotechnology industry in Australia at present: the uncertainty that is being created by continual government consultations on the same issues, such as patents.

The passing of the ‘Raising the Bar’ Bill earlier this year was the culmination of a long and difficult process. Specifically, it addressed the concerns that patents were hampering research and enshrined in law certainty for researchers to continue their work without fear of infringing patents. It should have heralded a break in consultations, while the impacts settled and we assessed if the perceived problem was solved.

But nothing could be further from the truth. Instead, the consultations and their

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related uncertainty facing industry at present remain; mainly targeted at patents.

For example, AusBiotech has recently provided a submission to the Productivity Commission’s review into compulsory licensing in the patents system. The commission was examining whether and how to ensure access to patented technology, while maintaining the patent incentive to create and protect new technologies, and it is yet to report.

Last month, the federal government announced a review into the patenting of pharmaceuticals in Australia, when it appointed a three-person expert panel to review the “appropriateness of the extension arrangements for pharmaceutical patents”. This review is ongoing. In another recent review conducted by IP Australia, the federal government is “considering tough new standards for Innovation Patents in order to discourage large companies from abusing the IP system”.

The industry is also facing ongoing proposals to ban gene patents, despite the fact that there have already been two reviews by the Law Reform Commission, reviews by the Advisory Council on Intellectual Property and two Senate Inquiries, none of which have made a case for banning gene patents.

The cumulative effect of all these patent-related reviews is to make the business environment more uncertain, which has the potential to destabilise an industry that is returning real results to our economy and is bedding down a strong foundation for our future.

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

For example Australia’s pharmaceutical manufacturing exports are on the rise, having officially taken over as Australia’s number one export, with $4.1 billion in 2011-12. This is substantially more than the car industry at $2.8 billion and more than double the wine industry at $2 billion.

Patents on medicines only have fiveyear protection for clinical trial data submitted for regulatory approval, while most other industrialised countries offer eight to 12 years.

A new report from the UK Office of Health Economics reviewed research published over the last three decades and confirmed what the industry has know anecdotally for some time: the costs of R&D are increasing. The study shows an increase in costs from £125 million ($199 million) per new medicine in the 1970s to £1.2 billion ($1.9 billion) in the 2000s (both in 2011 prices).

The R&D cost of a new medicine identified four factors contributing to increasing R&D costs: higher company out-of-pocket costs, up nearly 600% over the period; lower success rates from clinical development as researchers tackle tougher therapeutic areas such as dementia and arthritis; increases in R&D time as science becomes more complex, from six years to 13.5 years; and increases in the cost of capital from 8 to 11%.

If Australia is serious about becoming a knowledge-based economy, the case is strong for extended and increased patent protection, and an end to the review bonanza that currently besieges us. ALS

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FACE TO FACE | SIR GUSTAV NOSSAL

The campaigner Tim Dean

Following a 30-year stint heading up the Walter and Eliza Hall Institute, Sir Gustav Nossal is now a top level advisor helping to advance the cause of global health. Here he reflects on his long and fruitful career.

Australian Life Scientist: How did you get your start in medical science?

Sir Gus Nossal: I wanted to be a doctor for as long as I can remember. Certainly at least from about the age of seven.

It probably relates to a kind of hero worship that my parents had of their own paediatrician. I was quite a sickly little boy: I had diphtheria, I had whooping cough and my parents didn’t believe in

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immunisation at that stage, this being the 1930s. It was the healing hand of the doctor to the forehead that helped me - there was not much else they could do in those days.

I think that’s where it came from.

So off I trotted to become a doctor in 1948. This was still at a time when the ex-service men and women were streaming into universities as part of their resettlement. There was no quota,

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

so anyone who matriculated and had the money for the modest fees could get into medicine. We were the first year of 600.

The universities were woefully underfunded. The staff/student ratios were execrable. I essentially wasted the first two years at this university; I didn’t learn a thing, besides from some horrible anatomy. So we started asking what were we going to do about execrable teaching?

In the third year, about a dozen or so of us so-called ‘clever kids’ decided to teach one another. We would take a subject in physiology or biology, chemistry, neuroanatomy, etc. We would study in the library, not from primary resources, but from reviews, and then give each other seminars. This worked extraordinarily

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SIR GUSTAV NOSSAL | FACE TO FACE

well. We taught each other a lot. That got me blooded in research. That thrill, particularly in biochemistry, of being there at the cutting edge.

At the end of my third year, I went to see the Dean, Sir Harold Dew - who, by the way, was a first-assistant at WEHI. I said I would like to do what was then a completely new course, only two years old: a bachelor of science medical. That meant taking a year off and becoming a research apprentice in a laboratory, and at the end of the year you were a bachelor of science medical. It was a way of getting, on the cheap, a bit of a blooding in research.

The Dean said: “No, you don’t want to do a year in biochemistry, there are

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plenty of PhDs to do that. I want you to do something of a more clinical nature. In fact, I want you to do a year of virology, with a senior lecturer with a biochemistry bent: Patrick de Burgh.”

Pat de Burgh was not a famous man, but he had very fine scientific tastes. Would you believe, Don Metcalf, Jacques Miller and I all got our start with Pat de Burgh?

So Pat took me and one other student down to Melbourne to spend a week there to see what ‘real research’ was like.

Of course I was utterly hooked. I was 21, completely naive, and the thought of being in a lab that did research at international levels, published in Nature, it was unbelievable. That, in a sense, was the sealing of my destiny.

So off I trot to Melbourne in 1957.

I walk in and Macfarlane Burnet says: “It’s great to have you; however, I have one thing to tell you: we are no longer studying viruses. We’re studying the immune system defence against viruses.”

And, of course, the bottom fell out of my world. The way we’d be taught, immunology was about the most boring subject there was. But unbeknownst to me, Burnet had identified a wave before it crested. And, of course, I became an immunologist.

ALS: In your area of immunology, a great deal has changed over the span of your career. We now have new avenues to produce vaccines, such as the DNA vaccines Ian Frazer is working on, yet a malaria vaccine remains elusive. Why is that?

GN: I’m tempted to say that the alluvial gold has gone. We’ve had a wonderful decade with the so-called conjugate vaccines for the encapsulated bacteria pneumococcus, meningococcus, even Haemophilus influenzae B - great vaccines that have virtually made meningitis a thing of the past.

In a manner of speaking, these are straightforward vaccines. What that leaves us with is the tough problems.

By these I mean disease whereby nature itself does not produce robust immunity.

Have you ever pondered the fact that a person can harbour HIV for 20 years

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

and still not become immune? Or, for that matter, tubercle bacillus, where we sometimes pacify it, but when we age or we’re under stress, out it pops.

Malaria is perhaps the doyen of these, having co-evolved with humanity for all human history. This wily foe has learned the Joseph’s Coat of Many Colours. It has learned to change itself to dodge away from immune response. Whereas people living in endemic areas develop a modest non-sterilising immunity; therefore they probably will not die of the disease, they’re never really fully immune.

What I’m saying is, in these more complex diseases, and malaria is the paradigm, we have to be smarter than nature. My old boss, Mac Burnet, used to say: “Gus, a malaria vaccine will never work because nature hasn’t meant there to be immunity.” I replied that there are lots of things like flying where we’re doing better than nature. But he was right in that we have to figure out a way to engender an immune response which the disease itself does not do.

ALS: After 30 years heading up the Walter and Eliza Hall, how did you come to make the transition from research to advocacy, working with the World Health Organization and the Bill and Melinda Gates Foundation?

GN: The decision to leave research was not an easy one. I still have a very bittersweet recurrent dream of being back in the lab doing something fiendishly difficult - I always loved technical challenges in the lab. And the dream is both happy and sad: I’m happy being in the lab, but I’m sad to know in my dreaming state that it’s not really true. I miss research frightfully. Now comes the “but” ...

When I retired as director of the Walter and Eliza Hall Institute after a 30-year period, I had a fairly fundamental decision to make. I said to myself, I can do what I’m doing on large scale, but only on a small scale. Or I could do something different.

That was where happenchance played a big role, because I always

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FACE TO FACE | SIR GUSTAV NOSSAL

“I was 21, completely naive, and the thought of being in a lab that did research at international levels, published in Nature, it was unbelievable.”

had a side interest in the World Health Organization. In fact, I’d been on committees since 1964, and with the march of time, the chairman of some important ones. I had been chairman for a dreadfully long period of the committee felicitously named SAGE - Strategic Advisory Group of Experts - on everything to do with vaccines and biologicals.

Someone stuck my name in front of Gates Foundation and I was made the founding chairman of the strategic advisory council of the Bill and Melinda Gates Children’s Vaccine Program back in 1997, even before the Foundation was fully established. I held that post until 2003.

So I said, gosh, why not make my night job my day job? Why not have a change of life and a complete change of scene and go into advocacy, peer group reviewing, promotion, guidance of third world health etc. And that’s what I chose to do. It was an on-balance decision and I freely admit that in my darker moments I wonder if Don Metcalf made a better choice to stay at the laboratory bench. I can’t answer that.

In any case, it has been a very great success and I have developed a second career through it.

ALS: And what are some of the wins you feel you have achieved in the advocacy role?

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GN: The biggest single success was something we called GAVI, or the Global Alliance for Vaccines and Immunisation, which is now called the GAVI Alliance.

In January 2000 we launched GAVI with a magnificent initial gift of $750 million, which has since doubled and has been buttressed by various governments around the world.

The end result is that immunisation rates in the 72 poorest countries in the world have gone up to 82%, which is still not absolutely fantastic, but is very good. Newer vaccines have been added as they’ve become available. And it’s now, I think, one of the most successful public health programs in the world. That would have to be my number one achievement that I look back on.

Number two is closer to research.

The Gates Foundation decided to launch what they call the Grand Challenges Program, which eventually morphed into Grand Challenges Explorations. This includes both very large grants and very adventurous small grants where, for the sake of a two-page application, you could get your first $100,000. That’s been a very exciting program, and at that level they can give out a lot of them. In terms of Grant Challenges, I’ve been very intimately involved from the beginning, and am still heavily involved in peer group reviewing and chairing and so forth.

As that program has matured, and is now run by a very well oiled bureaucracy, I was asked to become the chairman of a small group, the Discovery Experts Group.

Roughly, of the $3.5 billion that the Gates Foundation spends annually, only about a quarter goes to blue sky discovery-style research, a half goes to development and a quarter goes to program implementation.

For that first quarter - which, of course, has really been my life, namely discovery science - I’m guiding them on strategies and long-range perspectives. That is still

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a work in progress - I’m in my third year as chairman - but it’s extraordinarily enjoyable and very challenging.

ALS: What are some of the areas where progress hasn’t happened as fast as you would have liked?

GN: Progress for vaccines against the big three: HIV/AIDS, malaria, tuberculosis.

Not even the most optimistic person in the world could say they’re anything other than disappointed in this area. That doesn’t mean we’ve given up, but there’s obviously a long way to go before we can develop a vaccine that can face up to broad-scale use.

Second is how far we are from the Millennium Development goals as far as deaths in childbirth are concerned.

Would you believe that deaths through pregnancy or childbirth are 400 times more common in the worst country in the world versus the best. Four hundred times! The three main causes - obstructive labour, haemorrhaging and sepsis - are all eminently treatable even with fairly basic health systems. But the health systems are not advanced enough in many of these countries. That’s been very disappointing.

The third, which is gaining momentum and needs to be strongly supported, is genetically modified foods and GM staple crops to add protein, vitamin and mineral content. There’s not so much calorie malnutrition in the world, but there’s plenty of protein malnutrition, and certainly micronutrient deficiency. The big ones are vitamin A, iron, iodine and zinc. If we can get more of that into the staple foods of the developing countries, we can save a lot of lives.

ALS: You’ve long been an advocate for vaccinations. Yet it seems today that there are many societal and cultural pressures emerging that are against vaccines. What happened? And how do we turn this trend around?

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FACE TO FACE | SIR GUSTAV NOSSAL

GN: It’s a very paradoxical thing. In fact, vaccines have become the prisoners of their own success. The very fact that vaccines have been so successful means that mums and dads no longer have any first-hand evidence of what the epidemics of the past were like. They don’t see a hundred beds in an infectious disease hospital with kids with infantile paralysis in iron lungs. They don’t see the complications that arise from measles, which is a disease that everybody got when it wasn’t immunised. Until quite recently, they didn’t see whooping babies on their television sets. They certainly don’t see the devastation of meningitis today because it’s all but gone because of vaccines.

So this absence of first-hand evidence of a real rip-roaring epidemic has bred, on the one hand, complacency, and on the other, an obsession with adverse events. That hasn’t been helped by some

totally spurious claims like that the MMR vaccine causes autism.

The funny thing is, as we have one of the highest vaccination rates in the world - around 92-93% - the parents of the unimmunised kids can afford the luxury of their conscientious objection because of herd immunity.

That’s the story. The adverse events, and we never deny they do occur, are vanishingly rare. And the risk/ benefit equation is vastly in favour of having the vaccine rather than having the disease.

The only way to combat this is sweet reason. I’ve said to people: look at what’s happening in developing countries. Look at the amazing fall in mortality as immunisation takes hold.

And then look at the stats in our own country. We have very good graphs in the new Australian Academy of Science brochure on vaccines. It has the incidence

of deaths per year for something like measles or diphtheria, and a red arrow showing when the vaccine was introduced. And you see deaths falling to nothing. Fortunately, thank God, we haven’t had a measles death for quite a few years, whereas they would be numbered in hundreds in years before the vaccine. People find that persuasive.

Then you take the adverse events one by one and go through them. You show them the studies that show that the link between autism and the MMR is nonsense. If you have the time, many can be persuaded.

On the other hand, there are some people with minds made up - people who’s mind you’ll never change - and to those you give their democratic right to believe what they want believe. And if that’s only 2-3% we can probably put up with that.

But sweet reason goes a long way. ALS

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LORNE GENOME | GENE REGULATION

Ghost in the genome It turns out genetics is not as complex as we thought. It’s even more complex.

Associate Professor Kevin Morris has recently arrived from the US and is working on uncovering the startling complexity of gene regulation.

Tim Dean

W

hile the genome revolution has been underway for many years now, another one has been taking place in its shadow, one that has the potential to explode our understanding of the manifold mechanisms that manage the genes themselves. This is the study of the regulation of our gene expression, the systems that can effectively switch our genes on and off (or ramp their expression up or down), sometimes in lasting ways that can pass between generations.

Gene regulation can occur at multiple junctures, such as at the transcriptional level via DNA methylation or the post translational modification of histones. Another mechanism, discovered in the late 1990s, occurs post transcriptionally during gene expression and is mediated by a growing family of RNAs.

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It’s this latter approach that is of particular interest to Associate Professor Kevin Morris, who last year made the journey from the Scripps Institute in California to the University of New South Wales. This rising star of the RNA world was lured here by Australia’s pedigree in noncoding DNA, in the form of individuals such as the Garvan’s Professor John Mattick, Diamantina’s Dr Marcel Dinger and University of Queensland’s Dr Ryan Taft, as well as his passion for surfing.

Clearly, he’s come to the right place.

At UNSW he is embarking on a programme to understand the processes behind the RNA-directed epigenetic regulation of gene expression, with a particular eye towards some enticing therapeutic applications. He will be speaking at the Lorne Genome conference

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GENE REGULATION | LORNE GENOME

about his work and what he sees as the tremendous potential of both transcriptional gene silencing and activation.

MAKE HAY

Far from being ‘junk’, it appears the roughly 98% of our genome that is constituted by noncoding DNA is indeed treasure of sorts. It performs the essential role of regulating the transcription of our genes to produce and assemble the protein building blocks of life. While some outspoken researchers, such as John Mattick and Malcolm Simons, had been waving the flag that this noncoding DNA must serve some essential function, the general consensus over the past decade or two had been one of overwhelming scepticism.

“Even after some publications in prestigious journals stating as much, the reception from the wider genetics community was still unenthusiastic.”

Yet Morris and his team at Scripps were convinced something interesting was going on in the noncoding regions of the genome, and they showed as such in their lab. “Initially, we found that some RNAs can control transcription,” says Morris. “In subsequent follow-up work, we showed how it was working mechanistically, how the RNAs interacted with the chromatin, and that in some cases there are higher ordered RNA-RNA interaction that occur.

“We first did this using synthetic small interfering RNAs, because that’s back when siRNAs were really hot, but we didn’t know what the mechanism was. We just threw stuff at the cell and we could see the silencing effect. But we did know this form of silencing was occurring through epigenetics because we could use certain cancer drugs, which are used to turn tumour suppressor genes back on, to inhibit the process.”

It was around 2007 that Morris became sure that in human cells it wasn’t small RNAs that were responsible for the gene epigenetic-based regulation but longer forms of noncoding RNA. Yet, even after some publications in prestigious journals stating as much, the reception from the wider genetics community was still unenthusiastic.

Morris remembers discussing his situation with Professor John Mattick in a bar following a day at a conference in

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Brussels. “I was really bummed out that we’d been working our butts off, struggling to get funding, and nobody was believing us. I’d go to give talks and everybody was just sceptical. I told him how frustrating it was, and he said, ‘Kevin, listen, make hay while the sun is shining, because this is our time.’ And he was so right about that. Now I look at it and everybody’s doing it.”

The turning point in popular sentiment was the publishing of the Encyclopedia of DNA Elements, or ENCODE, which reported in September of 2012. It finally gave some concrete evidence to quash the ‘junk’ conjecture once and for all, finding that at least 80% of the genome serves some kind of biochemical function. That’s far more than the 2-3% that had been widely believed to be functional only a few years ago.

Morris felt vindicated.

LAYERS

At Scripps, Morris and his team went some way towards understanding the mechanism that underlies this process of gene regulation. “What we’ve learned is that we can make small RNAs that can turn genes off in a very stable manner that are also heritable,” he says.

“The way many RNAs are controlling transcription in human cells is epigenetically based. What that means is that it changes the chromatin code around the DNA, making it compacted or opening it up. So when we target a particular region with RNA, we can compact the DNA, and if we do that to a promoter, if we hit it for a long period of time, we can get DNA methylation. Then all these epigenetic changes in the DNA that make it resistant to being transcribed are heritable and they can be passed on to other cells.”

Effectively, Morris figured out how to target specific genes and ramp them up or suppress them in a lasting way, all without affecting the underlying genetic code. The therapeutic potential is enticing.

“So we can hit something and turn it off, and remove our signal that turned it off, and it stays off. But the even more amazing thing is this: if you target a particular gene - let’s say in a case of cystic fibrosis - there are two noncoding RNAs we’ve discovered that are regulating CFTR [cystic fibrosis transmembrane conductance regulator] gene expression, and if you knock those down or turn them off, you can increase CFTR expression four-fold.

“If you do that, you’re going to end up increasing some level of the defective CFTR receptor on the surface of the cell, which helps with the chloride ion transport even though it’s defective. If you take a cystic fibrosis patient and give them a four-fold increase in the receptor, it might be beneficial.

“What gets even more fascinating about this is, in the case of cancer, you have tumour suppressor genes that control the cell cycle and cell division. What happens over time in a lot of cancers is that they get turned off, presumably by noncoding RNAs. These noncoding RNAs that are regulating them are getting out of control and turning them off, and when they’re

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Associate Professor Kevin Morris received his PhD in comparative pathology, with a particular interest in HIV, at the University of California Davis, and continued working there in the years following his PhD. In 2005 he shifted over to the Scripps Research Institute in California looking at antisense noncoding RNAs to regulate transcription of HIV-1 and genes involved in cancer. In 2012 he also took up a position at the School of Biotechnology and Biomolecular Sciences at the University of New South Wales. He has received several awards throughout his career including the 2010 Outstanding Young Investigator Award from the American Society of Gene and Cell Therapy.

off, the cell divides uncontrollably. So almost all the tumour suppressor genes have these noncoding RNAs, and if you know which ones are turned down in a particular cancer, you can target them and turn them back up.”

He has also been exploring the role of noncoding RNAs in viruses like HIV. “HIV has its own RNAs that it uses to control itself in viral latency. So if you target that noncoding RNA that regulates HIV, and you find a way to stably target it, it might be that the virus can’t go into a latent state and it’s always replicating. Then you can use drugs to inhibit it from spreading.”

“We can turn genes on and off willy nilly - pick your gene and we can do it - but the problem is getting it to the right cells that need it.”

In principle, the mechanisms for RNA-mediated gene expression that Morris is investigating could be used to hone in on problem genes and flip their expression in lasting ways. But there’s a hitch.

“The big elephant in the room is targeting,” he says. “We can turn genes on and off willy nilly - pick your gene and we can do it - but the problem is getting it to the right cells that need it.”

TARGETS AND INPUTS

Half of Morris’s lab, still based at Scripps, is working on this problem of developing targeted therapeutic approaches to delivering RNAs that can regulate gene expression, helped by a $14 million NIH grant. The other half, which he is establishing at UNSW, will focus on uncovering the basic mechanisms at work, particularly in human and primate cells, and exploring

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the complexity of RNA gene regulation, as well as its implications on evolution.

After all, if 80% of our genome appears to regulate the 2-3% of protein coding regions, and these regulatory patterns are conserved through generations, then this seems to be a ripe candidate to undergo evolution. John Mattick has already made claims that in order to understand the evolution of startlingly complex organisms like ourselves, we need to look not only at protein coding regions but the noncoding regions alike. Morris agrees.

One of the giveaways that noncoding RNAs have some link to the evolution of complexity is the fact that the number of protein coding genes has only a loose correspondence to the complexity of an organism, yet the proportion of noncoding regions in the genome increases with complexity.

As Morris points out, gene regulation gives a whole new level of plasticity and responsiveness to the environment, allowing organisms to adapt to changing conditions over generations without requiring significant changes in the protein coding regions of the genome. “So you can have different exposure to the environment causing different transcriptional paradigms, and that leads to adaptability,” he says.

The question that remains to be answered is how the noncoding regions sense changes in the environment and respond accordingly. “So now we have the connection that the RNA can control transcription, but the missing connection is how the outside stimulus is causing the activation of particular RNAs that lead to the different transcriptional changes. Input is the missing thing, but we’re working on that.”

There can be no question that it’s exciting times for genomics, but particularly for epigenomics. If Morris’s suspicions about the role and function of noncoding RNAs are correct, then they have the potential to radically shake up our understanding of our genome and could pave the way to a new era of personalised medicine. We’ve long known the genome is complex. But it’s only beginning to dawn just how complex it might truly be. ALS

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LORNE PROTEIN | PHOSPHOPROTEOMICS

© iStockphoto.com/Brian Jackson

Looking

at the big picture

Professor David James and his team are assembling a comprehensive atlas of the phosphoproteome of insulin that is revealing new insights about diabetes.

Fiona Wylie

I

nsulin is a remarkable molecule, its graceful structural symmetry hinting at the complex role it plays in maintaining metabolic equilibrium. Its function is intimately linked to the process of phosphorylation, which sparks of a cascade of activity within our cells, ultimately enabling glucose to enter the cell from the blood stream.

Understanding the complexities of this phosphorylation process has been the focus of Professor David James’s career, not only looking at how it works, but also how it can break down, causing diseases such as type 2 diabetes. The intricacies of protein phosphorylation and kinase signalling are convoluted, to say the least, and most researchers have tended to hone in on a small chunk of the process in order to bring it into focus. However, it can be difficult to get a handle on the full picture, which is something that James is hoping to correct.

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James and his team at the Garvan Institute’s Diabetes & Obesity Research Program have been labouring to develop a comprehensive atlas of the insulin-responsive phosphoproteome, which covers all the phosphate-containing proteins in adipocytes, or fat cells. It is hoped that this atlas will provide the big picture context that will help make the details of insulin’s function become clear.

The three-year effort to compile the atlas was driven largely by a PhD student in the group, Sean Humphrey, who took advantage of the ongoing advances in proteomics, mass spectrometry and bioinformatics to identify and characterise as many phosphoprotein changes as he could find in adipocyte cells following insulin treatment.

This relatively new approach of phosphoproteomics is providing novel information on the phosphorylation-based

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signalling pathways activated in a given cell under given conditions, which not only tells researchers more about the biological system being studied, but could also lead to new or better drug targets.

According to James, a major strength of their study lay in its highly quantitative nature. The impressive extent of insulinregulated changes in different phosphoproteins measured in their cells was enabled largely by the sensitivity of today’s mass spectrometers and techniques for phosphoprotein labelling.

“We seem to have a fairly complete and comprehensive dataset,” says James. “It comprises more than 37,000 phosphorylation sites, which I believe represents the largest single-cell phosphoproteome reported thus far.

“And, amazingly, in response to insulin, 15% of those phosphorylation events are changed by more than two-fold.

It is a humbling dataset that really demonstrates and confirms how insulin works. We see all the expected things plus some really exciting stuff.”

For example, the team has identified a completely novel substrate of Akt, which is an important kinase in multiple cellular processes including glucose metabolism, with topological features that suggest how Akt itself could be regulated. Indeed, James will highlight this particular finding when he speaks at Lorne in February.

“The dataset also turned up a whole lot of other events of as yet unknown significance. Finding out exactly what it all means will certainly keep us busy for a long time yet.”

TRACKING INSULIN’S TIMETABLE

The team also looked at the temporal nature of insulin action by analysing multiple time points after insulin addition, from 15 sec up to an hour, and this approach turned out to be incredibly revealing. In fact, James was quite surprised by the heterogeneous nature of the changes in phosphorylation events across the time scale.

“Some things appear almost as fast as you can kill the cell,” he says. “By 15 seconds they are on, and these tend to be the main signalling players that we already knew about, such as Akt. However, surprisingly, a lot of the other things destined to come on are definitely off in the early stages, and then boom, suddenly they turn completely on. So, it is sort of like a domino effect, and some of the time gaps from one thing turning on to the next were longer than expected in signalling terms.”

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The temporal aspect of James’s approach is important in terms of distinguishing various enzyme-mediated events such as phosphorylation (kinases) and dephosphorylation (phosphorylases). Indeed, one of the biggest issues in signal transduction has always been trying to match kinases with their substrates for a given function or endpoint, because those belonging to the same family often have overlapping consensus motifs.

“Temporal profiling therefore really adds another dimension to deciphering these insulin-related phosphorylation events and enables us to resolve some of these relationships that we couldn’t before.

“This time dimension of biological processes has become one of the stable horses of systems biology. You can’t just look at something and compare it to something else at a particular point in time or place. You have to watch things evolve over time to start to unpick the puzzle.”

James admits that this phosphoproteomic approach has certainly broadened his thinking into the realm of systems biology, a term commonly used to describe the study of whole body processes.

“This ‘big data’ is so impressive and exciting because it truly is a completely new way of doing science that requires a rearrangement in one’s way of doing and thinking about research. Even aspects like just being able to handle the damn stuff - that volume of data all at once - looking at it, analysing it and working out how to present it to everyone else in a way that makes sense.”

At the moment, James and his team are trying to take a step back from the enormity of the data and avoid getting caught up in the power of the technology. They instead want to focus on those findings that fit in best with the group’s ongoing research activities and questions, and explore them in more detail.

David James’s working life has come full circle. After launching his research career as a PhD graduate from the Garvan Institute in 1984, he now heads the Garvan’s Diabetes & Obesity Research Program. In between, James spent several successful postdoctoral years in the USA where he discovered and cloned the insulinregulated GLUT4 transporter, and several more back in Australia at the University of Queensland. His received the Glaxo Wellcome Medal in 1999 for contributions to his field, the Mary Kugel Award for services to the Juvenile Diabetes Foundation in 2000, the Kellion Award in 2006 from the Australian Diabetes Society, and admittance as a Fellow of Australia’s Academy of Sciences in 2007.

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“It is a humbling dataset that really demonstrates and confirms how insulin works. We see all the expected things plus some really exciting stuff.”

The mass spectrometrometry equipment in action.

GETTING PERSONAL

According to James, there is even plenty of data to go around, and James expects that once the work is published there will be many other researchers interested in applying some to their own activities and aims or entering into collaborations with James to work on molecules of interest to both groups.

“Actually, Sean has already set up a really nice collaboration with Jean Yang’s group at Sydney University.

She is a biostatistician with expertise in computational biology.

Together with a guy from her group, Sean worked on how to analyse and visualise the data, even things as seemingly simple as how to show the data to others.”

This is one of the important issues in systems biology, says James. “You have all this information and then have to work out how to show it to someone and make it stand up and say the things you think it says. It is not so easy.”

PhD student and cell biologist, Sean Humphrey (left) with postdoctoral computational biologist, Pengyi Yang (right).

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One of the things James is really excited about is taking these new high-tech results almost directly into the clinic.

“I believe that we are at somewhat of a turning point in the history of medical care and health delivery that will change some of the conventional procedures of symptom-based disease diagnosis. Indeed, it is already happening in some areas.

“More and more, doctors will have all of this new information at their fingertips: a patient’s genomics profile and potentially the relevant phosphorylation status, signal activation data etc. And I can see the possibility of taking data such as ours straight into the clinic to provide clinicians with a much greater capacity to make more specific and sensitive diagnoses.

“The real question all of this brings up in the clinical arena is: what actually is disease? Take diabetes, for example, which for the most part we think of as a single disease entity, when in fact it may be a thousand different diseases whose course depends on many things, such as the environment that a particular person is exposed to and so on.

“Where I see these big dataset types of experiments being so powerful when applied in the clinic is in helping us work out this personalised aspect to medicine, especially in complex diseases like diabetes.

“And genomics alone, in my opinion, will not be the one answer. You can’t just look at gene type and make predictions.

You have to look at multiple parameters that define the biology of a system. This will be the real power of true systems biology.

As a case in point, here am I, having spent the last 20 years of my research life poking around with a few molecules and then we do this relatively short project and end up with the data we have, and you just think ‘Oh my God, so that is how insulin really works!’” ALS

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LORNE INFECTION & IMMUNITY | CYTOKINE SIGNALLING

Š iStockphoto.com/Krzysztof Zmij

Signal strength Associate Professor Brendan Jenkins is helping to uncover the cellular signals involved in stomach cancer, which may one day be used as biomarkers or as new targets for treatment.

Fiona Wylie

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S

tomach cancer, after lung, breast and colorectal cancer, is the fourth most commonly diagnosed form of cancer worldwide and is one of the leading causes of cancer mortality, particularly in east Asia. Because there is no routine screen for stomach cancer, and the early symptoms are easily overlooked or mistaken for regular dyspepsia, it is often diagnosed late after it has set in and become aggressive.

Treatment often includes surgery, including a total or partial gastrectomy, including multiple cycles of chemotherapy.

But now there is new hope for better treatments for stomach cancer and other diseases emerging from cutting-edge research conducted by Associate Professor Brendan Jenkins at the Monash Institute of Medical Research (MIMR) in Melbourne that might aid in the diagnosis, monitoring and possibly also targeted treatment of these diseases.

Jenkins has spent his research career trying to understand how some of the cellular signalling pathways that are activated

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CYTOKINE SIGNALLING | LORNE INFECTION & IMMUNITY

“Maybe cytokines could somehow be up-regulating the TLR system to the point that the whole inflammatory response goes into overdrive with tumour formation as the result.” by cytokines - proteins that modulate the immune system contribute to human disease. Around seven years ago, Jenkins joined MIMR in Melbourne to continue this research. Here his focus is the interleukin 6 (IL-6) family of cytokines and how their deregulation plays a role in inflammatory and malignant diseases of the lung and stomach.

“We have known for well over a decade that uncontrolled signalling by members of the IL-6 family, particularly IL-6 and IL-11, plays a part in inflammatory diseases, such as Crohn’s disease and arthritis, and in many cancers including stomach, lung, pancreatic and colon,” said Jenkins. “Indeed, about 35% of all cancers have an obvious inflammatory component, but the mechanisms by which inflammation leads to cancer are poorly understood.”

His research aims to identify genes that could be used as biomarkers for the early detection or monitoring of such diseases and as novel targets for more personalised and thus effective treatment strategies.

Jenkins’ group at MIMR uses numerous molecular biological and genetic approaches in their research, together with translational studies using clinical samples. One of their main tools of trade, and the linchpin of their recent exciting findings, is a novel knock-in mouse model of gastric cancer. These mice have a defect engineered into their IL-6/IL-11 activation pathways that leads to gastric cancer.

For the translational research, Jenkins’ group is closely aligned with clinicians at the nearby Monash Medical Centre and with clinical researchers in Singapore and Japan who have a large collection of gastric cancer biopsies. “These clinical biopsies are a very important resource for validating findings from our mouse model, such as identifying a particular mechanism that could be promoting stomach or lung cancer. It allows us to immediately realise the significance of our findings for potential clinical translation, and push through on those results.”

to pathogenic insult was demonstrated. “Having worked on gastric cancer and cytokines for about 10 years, and knowing the very strong inflammatory component to these types of cancer, I started thinking: maybe cytokines such as the IL-11 cytokine that we work with could somehow be up-regulating the TLR system to the point that the whole inflammatory response goes into overdrive with tumour formation as the result.”

With Jenkins’ hypothesis driving the work, his team started with the basics. “We took tumours from our gastric cancer model mice and another gastric cancer model called ‘Gan’ from colleagues in Japan and, using real-time PCR, we looked at the gene expression levels of all the TLRs,” says Jenkins. “And there was this one TLR gene that was always up-regulated in the mouse tumours: TLR2.”

They then used a modified mouse model, in which the stomach tumour phenotype had been rescued by lowering the level of STAT3 activation, to show that mice with no tumour formation had normal levels of TLR2 gene expression.

The next step was based on two other premises: earlier research by the group showing that STAT3 is actually a potent pro-inflammatory and oncogenic factor in its own right, albeit by an unknown mechanism; and the realisation that one of the main downstream signalling molecules from the IL-11 cytokine is STAT3. The connection was getting stronger.

The group then demonstrated very clearly that STAT3 directly up-regulates the expression of TLR2, and that this IL-11-linked event occurs only in the stomach. This single finding is easy to say, but the path to proving that TLR2 is in fact a novel STAT3 target gene involved an awful lot of hard work and PhD student angst to achieve.

However, it was a key part of the puzzle, especially given that in stomach cancer, IL-11 is often up-regulated and STAT3 is over-activated in about 50% of cases, so the clinical link between IL-11 and STAT3 in gastric malignancies was already strong.

THE TLR2 STORY

One such set of very promising results was recently published in the prestigious journal Cancer Cell and will be the subject of Jenkins’ presentation at the Lorne Infection and Immunity meeting in February. They centre on a cytokine signalling pathway activated by IL-11 and involving other key cancerrelated molecules, namely one of the toll-like cell-surface receptors, TLR2, and a downstream intracellular regulator of transcription called signal transducer activator of transcription-3, or STAT3.

Jenkins’ interest in TLRs was piqued several years ago when their critical role of driving inflammation in response

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GETTING DEFINITIVE

It was time for the definitive mouse experiments, as Jenkins explains. “We crossed our model mice that develop the tumours - the ones with high TLR2 expression - with mice genetically engineered to have no TLR2 gene expression, and the progeny mice also showed no TLR2 expression.

Strikingly, the crossed mice also showed a 50% reduction in size of their tumours and whole stomach. The beauty of this result was that it mimicked the rescue experiment mice, which had reduced STAT3 activity. “We therefore had further proof that STAT3 up-regulates TLR2 at the gene expression

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LORNE INFECTION & IMMUNITY | CYTOKINE SIGNALLING

Indeed, Opsona has recently developed a humanised version of the antibody, which could form the basis of a potential as first- or second-line adjuvant to chemotherapy for patients with stomach cancer. Because this antibody is already entering Phase I/II trials for another indication, Jenkins is hopeful that the TLR2 blocking treatment could be trialled for gastric cancer within a few years.

“The other main part of our gastric cancer work is towards early detection and screening, and that is when you really start thinking about biomarkers.

We are now in the process of trying to identify a TLR2-regulated ‘gene signature’ that could potentially translate into cancer biomarkers.

“The ideal candidates would be small secreted proteins that appear in the bloodstream in affected patients and therefore can be used in a simple blood test-based screening program.”

There is clearly a need to catch this disease in the early stages because most patients with stomach tumours do not know about it until the cancer is too well established for any treatment to be effective.

Associate Professor Brendan Jenkins’ lab is in the Centre for Innate Immunity and Infectious Diseases at MIMR. Following PhD studies at the Hanson Institute in Adelaide, Jenkins continued his research interest on the roles of cytokines in disease at the Fred Hutchinson Cancer Research Centre in Seattle, USA. In 1999 he returned to Australia to join the Ludwig Institute for Cancer Research before taking up his MIMR appointment in early 2006.

level in the mice tumours, and that it is all driven by IL-11,” says Jenkins.

Interestingly, this set of experiments also showed that the TLR2 activation only promoted tumour cell growth and not inflammation, which was present in both the presence and absence of TLR2 expression. This was an unexpected finding of the study, especially given the strong inflammatory component of gastric cancer.

Finally, they used an antibody-mediated therapeutic approach, in which the mice with the stomach tumours were treated with a TLR2-blocking antibody for two to three months.

The treated mice showed no further tumour growth compared to untreated controls, and this cemented the critical role of TLR2 in promoting the cancer’s progression.

It just remained for the team to validate the exciting mouse findings in a clinically based experimental system, and this is where the biopsy samples from their overseas colleagues came in. From the Singapore set of stomach cancer samples, around 40% had elevated levels of the TLR2 gene and, even more importantly, those patients with both high STAT3 activation and high TLR2 gene expression had a poor prognosis based on overall patient survival data (five-year rates) compared to patients with low TLR2 expression and low STAT3 activation.

“This was really nice clinical data demonstrating that our mouse disease model findings were also represented in the human condition.”

GENE SIGNATURE

From here, Jenkins and his team would like to move their results into human xenograft models of gastric cancer using a swag of cell lines already in the lab as well as lines derived from patient cells. The aim now is to establish xenografts in mice from those cells and then start testing the efficacy of this blocking TLR2 antibody, which is made by a Dublin-based pharmaceutical company, Opsona Therapeutics, with whom the group has established close ties.

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Photomicrograph from Jenkins’ lab showing a cross-section of stomach from the disease mouse model, stained to indicate increased cell proliferation in the base of the gastric epithelium (brown staining).

In this context, the TLR2 system identified by Jenkins and his team is very promising because it seems to be activated in such a large subset of gastric cancer patients. They also have a new NHMRC grant to follow up other promising STAT3regulated immune system genes that might be driving gastric tumour formation in the same way as TLR2.

With emerging evidence that such genes may be playing an important role in other inflammation-type cancers such as colon and liver, the findings from Jenkins’ lab promise hope to the many people worldwide with these lethal cancers. ALS

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LORNE GENOME | STEM CELLS

Altered

states

UWA’s Professor Ryan Lister’s research into the epigenome suggests the future clinical use of induced pluripotent stem cells should proceed with great caution.

Graeme O’Neill

W

hen a specialised skin or liver cell is coaxed into reverting to a more primitive pluripotent state, the question is not what is happening to its genome, says Professor Ryan Lister. The real question to ask is: what is happening to its epigenome?

The University of Western Australia’s new Winthrop Professor of Computational Systems Biology, an invited speaker at this year’s Lorne Genome conference, says that understanding cellular reprogramming is all about epigenetics. “The reprogramming of a somatic cell to a pluripotent state is a process of epigenetic reprogramming,” he stresses.

List returned in July last year to his alma mater from the Salk Institute for Biological Studies in San Diego, California, where he secured his first postdoctoral appointment in 2006. At the Salk, he developed new techniques for whole-genome mapping of one layer

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of the epigenome at single-base resolution throughout the Arabidopsis genome. The epigenome consists of methyl ‘tags’ placed upon cytosine bases in the genomic DNA via DNA methylation.

DNA methylation has long been known to play an essential role in transcriptional regulation, says Lister. Determining precisely where a cell places epigenetic modifications within its genome is an essential first step towards a comprehensive understanding of the epigenome’s role in regulating genes throughout the genome, in both normal and perturbed states.

“Arabidopsis is an ideal model for methylation studies in both plants and animals,” he says. “This is because it comes with excellent genetic resources and genetic control systems with which you can both develop new experimental procedures and test the biology. It’s a powerful model for exploring epigenetic processes.”

According to Lister, one key advantage with Arabidopsis is that any null mutant created by targeted gene knockout technology remains viable, including knockouts of genes for methyltransferase and demethylase enzymes. In rodent models, on the other hand, knockouts of the same enzymes are lethal during embryonic development. Plant and animal DNA methyltransferase enzymes are conserved, so the tools developed to study methylation patterns in Arabidopsis, and the biological mechanisms that they probe, were potentially applicable to mammalian cells.

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STEM CELLS | LORNE GENOME

Soon after Lister arrived at the Salk Institute, new DNA sequencing technologies became available that drove a phenomenal increase in sequencing speed and output. Sequencing costs plummeted as a result. Simultaneous advances in computing meant researchers could now compare methylation patterns not only in the Arabidopsis genome (~120 megabases), but also in the gigabase-sized genomes of mammalian cells.

EPIGENOMIC ROADMAP

The advent of the NIH Roadmap Epigenomics Project in 2008 saw the Salk Institute’s epigenetics group join a collaborative epigenome mapping centre, where Lister, working in the laboratory of Professor Joseph Ecker, begin applying the sequencing techniques for DNA methylome profiling that they had developed for Arabidopsis to the human genome. Their first aim was to compare the epigenomes of a differentiated fibroblast cell and a human embryonic stem cell, a pluripotent cell that has the capacity to differentiate into a wide variety of distinct cell types.

When it comes to human cells, a longstanding research challenge was to map DNA methylation throughout the entire genome at single-base precision. The established dogma in the field held that DNA methylation in mammalian cells occurred only in the CG context - that is, at a cytosine followed by a guanine. While there had been a handful of studies reporting DNA methylation in a non-CG context in mammalian cells, the papers were largely overlooked in the field.

The dogma was wrong, says Lister. By precisely determining which cytosines were methylated throughout the genome, Lister and colleagues found that DNA methylation also occurs in the non-CG context, but only in pluripotent embryonic stem cells. Within the stem cell genome, a substantial fraction of methylated cytosines were identified in the non-CG context, that is, a methylcytosine followed by an A, T or C. “With previous approaches to study DNA methylation, technological limitations imposed significant trade-offs. You could get single-base resolution identification of DNA methylation sites, but only in a small region of the genome, on the order of kilobases,” Lister said.

“To look through whole genomes, you had to employ techniques such as immunoprecipitation of methylated fragments of genomic DNA and hybridisation to tiling microarrays, which required multiple arrays for the genome of interest, and limited detection of methylation to a resolution of approximately a kilobase at best.

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“The methylation of a single cytosine has been reported to modulate interaction of a protein with the genomic DNA, so to fully understand these processes it’s essential to know where the DNA methylation is at single-base resolution.”

Where methylome mapping had previously been a hard slog, the DNA sequencing revolution enabled a well-established chemical technique for identifying methylated cytosine bases, known as bisulfite conversion, to be coupled to massively parallelised shotgun sequencing.

ROUTINE EXERCISE

Now methylome sequencing became almost a routine exercise: isolate genomic DNA from a sample of interest - normal, mutant or genetically modified - then treat with sodium bisulfite under denaturing conditions. The treatment selectively converts all unmethylated cytosines to uracil. But, crucially, it does not convert methylated cytosines. When the converted DNA is subsequently sequenced, a C indicates that the base was methylated in the genome,

“When we looked closer, we found that each induced pluripotent stem cell line had aberrant methylation states in hundreds of distinct regions throughout the genome.” whereas a T sequenced at a cytosine position in the reference genome indicates that the genomic cytosine was unmethylated.

According to Lister, high-coverage sequencing with a singlebase resolution yields a strand-specific map of exactly which cytosines were methylated for over 90% of all cytosines in a genome.

This comprehensive methylome sequencing delivers reference epigenomes necessary for future comparisons with methylome patterns in distinct differentiated cell types, laboratory-induced pluripotent cells and disease states.

Researchers can now modify cell lines to determine how methylation patterns change in disease or during embryonic development, or what happens as resident pools of partially differentiated adult stem cells undergo terminal differentiation during tissue renewal and repair. Furthermore, it provides the means to explore genome-environment interactions, particularly during embryonic development.

Over the past decade, evidence has accumulated that environmental influences transduced via the placenta may modify normal methylation patterns in the embryo, giving the genome the capacity to make dynamic, and potentially adaptive, changes to the individual’s genome, which may be transmitted to the next generation.

But don’t invoke the ghost of Lamarck just yet, Lister cautions.

Research into transgenerational heritability of variable DNA methylation, or ‘epialleles’, is very much ongoing. If heritable epigenetic changes do indeed occur, they may offer a means by

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He says much of the promised land of regenerative medicine rests on the recent technological advances led by Nobel Laureate Shinya Yamanaka, who showed that the introduction of a handful of defined factors to somatic cells forces Professor Ryan Lister acquired a PhD them to revert back into a pluripotent in molecular bioscience at the University state, creating induced pluripotent stem of Western Australia in 2004. In 2006 he cells, or iPSCs. accepted a postdoctoral appointment at the Salk Institute Biological Studies in San According to Lister, the process Diego, California, where he studied the is fundamentally an epigenetic one. epigenomes of Arabidopsis and human However, before iSPCs can be used in cells. He returned to UWA to take up an a therapeutic role, researchers must appointment as Winthrop Professor of determine how closely iPSCs resemble Computational Systems Biology last July.

ES cells and whether the progeny of iPSCs that follow display the same methylation patterns when they are differentiated into the which additional information is appended to the DNA code. desired cell types. If the technology is to be viable, no rogues In 2009, Ryan was lead author on a Nature paper that presented should sprout among the cloned roses. the first genome-wide, single-base-resolution maps of methylation After identifying large-scale differences in the methylome in a mammalian genome, comparing the methylomes of human between ESCs and differentiated cells, Lister and colleagues embryonic stem cells (ESCs) and foetal fibroblasts. In addition, the began to investigate how completely the methylome is reset study compared the messenger RNA and small RNA components when a differentiated cell is reprogrammed into an iPSC. They of the transcriptomes of the two cell types and mapped several set about sequencing the methylomes of a panel of independent histone modifications genome-wide, as well as sites of DNA-protein iPSC lines. These were produced by different methodologies, interactions for several key regulatory factors. in different collaborating laboratories, from distinct and The paper identified significant, genome-wide differences differentiated parental cell types. in the composition and patterning of methylation. Nearly 25% They compared the resulting whole genome methylome of all methylated sites in the ESCs occurred in a non-CG context, maps to see how they differed from undifferentiated embryonic suggesting ESCs and differentiated cells employ different stem cells, the parental somatic cell lines and cells differentiated methylation mechanisms to affect their particular patterns of gene from the iPSCs and ESCs. regulation. Non-CG context methylation was concentrated within “In general, the methylomes of the embryonic stem cells the transcribed region of highly expressed genes, and depleted in and induced pluripotent stem cells looked very similar,” he protein-binding sites and enhancers. says. “It is quite remarkable that the reprogramming factors can “The presence of non-CG methylation in the body of highly induce such widespread changes. But when we looked closer, transcribed genes offers a tantalising model whereby the using special algorithms to search for small-scale changes at transcriptional activity of genes may be affected by non-CG high resolution, we found that each induced pluripotent stem methylation,” Lister says. In contrast, these non-CG methylation cell line had aberrant methylation states in hundreds of distinct events were completely absent in differentiated fibroblast cells. regions throughout the genome.

TANTALISING MODEL “These differentially methylated regions we identified in the He and his colleagues have identified hundreds of differentially iPS cell genomes fell into two broad categories. In the first, we methylated regions in the CG context, close to genes known to see DNA methylation of a region that differs from ES cells, but be involved in pluripotency and differentiation. Moreover, the is the same as the parental somatic cell from which the iPS cell fibroblast genomes featured widespread regions of reduced line was derived. This can be thought of as persistent epigenetic methylation associated with reduced transcriptional activity. It turns memory of the somatic cellular state. out that these partially methylated domains likely represent regions “In the second category, we see DNA methylation patterns of the genome that are physically associated with the nuclear lamina. that differ from both ES cells and the somatic progenitor cells, In their synopsis, they concluded that “these reference that we refer to as iPS-specific differential methylation. And epigenomes provide a foundation for future studies exploring this since we had sequenced several independent iPS cell lines, key epigenetic modification in human disease and development”. we were able to look at how frequently they arose in Lister was also lead author on another Nature paper in March independent reprogramming events. Most differentially 2011 that described hotspots of aberrant epigenetic reprogramming methylated regions were present in multiple independent iPS in induced pluripotent stem cells. The paper was the most highly cell lines, and a substantial fraction of them were present in all cited biology paper in the world in 2011. our iPS cell lines.

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STEM CELLS | LORNE GENOME

GENOMIC HOTSPOTS

“So it appears there are hotspots in the human genome that frequently tend to be aberrantly reprogrammed in iPS cell lines, either by the failure to reset the patterns found in the differentiated parental cells or inappropriate resetting to a state reflective of neither ES nor differentiated cells.

“About half of these aberrant DNA methylation patterns were transmitted through differentiation when the iPS cells were triggered to differentiate in vitro, indicating they may transmit an inappropriate regulatory state to somatic cells derived from the iPS cell lines.

“It appears there are hotspots in the human genome that frequently tend to be aberrantly reprogrammed in iPS cell lines.”

“So we have identified a characteristic epigenetic signature of induced pluripotent stem cells that distinguishes them from embryonic stem cells. Ideally, for use in regenerative medicine, we would like to have iPS cells identical to ES cells, unencumbered by this aberrant reprogramming and somatic epigenetic memory.

“We think we can now use these aberrant methylation patterns as markers to test the efficacy of a whole range of reprogramming conditions and develop protocols that may deliver completely reprogrammed iPS cell epigenomes.”

One potentially advantageous implication of the discovery is that any epigenetic memory that is found not to affect the function of derived somatic cell types might be used as a non-genetic molecular marker of cells that were derived from an iPS cell. These markers could be used to track the origin of cells involved in the repair process, in order to distinguish a patient’s cells from cells derived from patient-specific iPSC lines.

He expects single-base, genome-wide comparisons of DNA methylation patterns between individuals may reveal substantial, inter-individual variation which may be involved in differential activity of genomic regulatory elements.

“However, the question would be what specifies these different epigenetic patterns? Currently, we do not have a good understanding of whether they are simply derived from underlying genetic variants, as well as how specific epigenetic patterns influence gene expression.

“Very little is known about causality in the relationship between variable DNA methylation, DNA-protein interactions and changes in transcriptional activity. Examples have been described where DNA methylation state modulates protein-DNA interactions; however, the inverse has also been documented,” he says.

Recent research has identified at least two instances where DNAbinding proteins alter DNA methylation patterns when they bind to the genomic DNA. Overwhelmingly, researchers assumed that DNA methylation was causal for altering gene expression, not vice

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versa, so there is now a huge challenge to systematically understand causality with respect to these epigenetic changes and gene activity.

“If we don’t understand the basic biology in order to progress beyond the current assumptions, we’re going to head down a lot of dead ends when investigating the role of the epigenome in development and disease.

“It’s also important to remember that there are multiple flavours of epigenetic modification and complex relationships with noncoding RNAs. We need to understand how these all interact and their combined effect upon the complex and dynamic three-dimensional structure of the genome.

“We tend to envisage the genome as a set of linear structures, but some exquisite experiments recently have begun to map the higherorder structures of our genome. These experiments show that the three-dimensional structure of DNA is critical to understanding how the genome functions, and it will be important to integrate with these structures the maps of the epigenome that we can now generate.”

While iPSCs are touted to have tremendous potential in research and medicine, we clearly still have a lot to understand about the detail of their function and how the various epigenetic factors can influence their ultimate fate. ALS

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LORNE CANCER | BREAST CANCER

Connecting

the dots

Dr Stacey Edwards is uncovering the mechanism responsible for inherited disorders that occur seemingly without a clear genetic basis.

Graeme O’Neill

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BREAST CANCER | LORNE CANCER

W

hy is it that female members of some families with a history of breast cancer can test negative for mutations or deletions in BRCA1 and BRCA2, along with the other usual genetic suspects in inherited breast cancers? And why is it that extended multigeneration pedigree diagrams for such families have an excess of black circles each representing an female affected by breast cancer - which is strongly suggestive of inherited risk, but which defies explanation by Mendel’s rules of inheritance?

Breast cancer researcher Dr Stacey Edwards has been doing a spot of genomic ghost-busting in an attempt to resolve this mystery of the so-called ‘missing heritability’, where the mutations and deletions only tell a part of the story. “Inherited mutations and deletions in known breast cancer genes accounts for only 30% of familial breast cancers,” she says.

In the classic model of familial cancers, a mutation in a protein-coding region of a cancer gene disrupts its expression, leading to cancer. Yet, according to Edwards, at least some of the unexplained cases of families with an abnormally high risk of breast cancer can be put down to missing heritability in the form of DNA variants in unidentified regulatory elements or transcription factors.

She has recently been investigating this hypothesis by pouring over the data that came out of a recent study conducted by the UK-based Breast Cancer Association Consortium (BCAC), which assembled a huge data bank of genomic data from 39 breast cancer case-control sets, representing a total of 49,068 cases of breast cancer and 48,772 unaffected controls, predominantly of European ancestry.

They performed a genome-wide association study (GWAS) on the data looking for common variants associated with an increased susceptibility to breast cancer. The GWAS confirmed new breast-cancer susceptibility loci on chromosomes 9, 10 and 11. The strongest association involved a variant named rs614367, at the 11q13 locus on the long arm of chromosome 11.

The increased risk of breast cancer involving rs614367 was specific to breast cancers that expressed oestrogen receptors (ERs), and was strongest for ER-positive breast cancers that also had an excess of progesterone receptors.

SPOOKY ACTION

Edwards was invited by the consortium to investigate the link between rs614367 and ER-positive cancers. Her collaborators fine-mapped the 11q13 region, again by GWAS, to determine if rs614367 was indeed the causal variant. What they found was that two quite different variants were responsible for breast cancer risk.

Edwards has proposed a model that explains the nonMendelian pattern of inheritance in breast cancers associated with variation at the 11q13 as a consequence of a long-distance interaction between two DNA elements on chromosome 11.

She has shown that the cell-cycle gene Cyclin D1 (CCND1) on chromosome 11 interacts with an enhancer that lies at a

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“The explanation for this ‘spooky action at a distance’ (to appropriate Albert Einstein) lies in the fact that chromosomal DNA is nonlinear.” substantial remove from the gene, 125 kilobases away. Four wild-type alleles of the enhancer element, featuring different SNPs, can occupy the locus.

“Genetically, any one of the four could have a causal role, but our study showed that only two of these variants alter the function of the enhancer element,” says Edwards. “When the minor alleles of these two SNPs are present at the 11q13 locus, it stops the activation of CCND1.”

The explanation for this ‘spooky action at a distance’ (to appropriate Albert Einstein) lies in the fact that chromosomal DNA is nonlinear; it is packaged as chromatin, which are long, slender threads of DNA coiled around tiny beads of histone proteins. Multiple interactions of coiling and supercoiling compact some three metres of DNA, carrying the 3.08 billion base pairs of the human genome, to fit into the tiny volume of a cell nucleus - with room to spare.

Dr Stacey Edwards completed her PhD in molecular biology and biochemistry at the University of Queensland in 2002. She received a C. J. Martin Fellowship for a postdoctoral position at the Breakthrough Breast Cancer Research Centre in London.

On returning to the School of Chemistry and Molecular Biosciences at the University of Queensland, she was awarded a prestigious National Breast Cancer Foundation (NBCF) Fellowship to identify new mutation targets in breast cancer susceptibility genes. The NBCF is a sponsor of this year’s Lorne Cancer Conference.

Using a technique called chromosome conformation capture, Edwards showed that the 3D packaging arrangement positions CCND1 close enough to its enhancer sequence to ‘talk’ across the narrow gap. In molecular genetics terms, although they lie far apart in cis (on the same DNA strand), they lie physically close enough to interact in trans (between DNA strands).

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LORNE CANCER | BREAST CANCER

“Little more than a decade ago nobody appreciated how genetically unique we are, and why one sibling - even in identical twins - develops cancer while another doesn’t.“ According to Edwards, the minor allele carrier combination is the most potent off-switch for the enhancer, and a throw of the meiotic dice determines its presence or absence in family members; only females are at risk because males do not produce enough oestrogen to fuel the growth of oestrogendependent breast cancers.

EXPLANATORY POWER

Edwards’ study of the interaction between CCND1 and its remote enhancer was one of the first of its type, and showed the value of GWAS in combination with follow-up functional studies for illuminating new mechanisms of cancer, particularly those that might account for the missing heritability.

“The chromosome conformation capture provides evidence that the CCND1 gene and its enhancer are interacting at long

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range,” she says. “I then went on to perform a series of reporter assays where I positioned the CCND1 promoter close to different alleles of the enhancer sequence.”

The technique involves adding the promoter and enhancer DNA sequences into a construct containing the luciferase gene and measuring the resulting luminescence when the nearby enhancer activates the gene. The brighter the glow, the more potent the enhancer. It turned out that some alleles acted as repressors, silencers or insulators, but the experiments confirmed for Edwards that the combined minor alleles act as a potent repressor of CCND1 expression.

She and her colleagues believe the inheritance of combinations of low-risk alleles with Cyclin D1 may explain some of the non-Mendelian, low-penetrance pattern of breast cancer in families where women are at increased risk of oestrogen-dependent breast cancers. Edwards expects the finding to be “extraordinarily valuable” for diagnostic and prognostic purposes in women whose families show a pattern of increased risk of certain oestrogen-dependent breast cancers.

She also works on the BRCA1 gene, another major player in breast cancer. “Coding mutations in BRCA1 and BRCA2 collectively account for only 5-10% of familial breast cancers,” she says. “BRCA1 mutations have only been identified in a proportion of patients with tumours suggestive of a BRCA1 defect, many of which display reduced levels of BRCA1 expression. I’m planning to investigate whether BRCA1 may have a strong enhancer that is required for normal expression.

A mutation in the enhancer could explain loss of BRCA1 expression in some cases where the gene itself is unaffected by mutation.

“Little more than a decade ago, we didn’t have a human genome sequence or SNP chips to identify individual variation.

Nobody appreciated how genetically unique we are, and why one sibling - even in identical twins - develops cancer while another doesn’t.

“We have already identified lots of genes involved in breast cancer, which is a very heterogeneous disease, and we already have some very good drugs in the clinic. The focus has been on single genes, but the future will be more about particular combinations of genes - or alleles - that confer an increased risk of breast cancer. Increasingly, the genes are going to tell us which drugs to use.”

She says the novel mechanism she has identified in breast cancer may have analogues in other diseases, such as heart disease, asthma or even Alzheimer’s disease researchers in these fields may find a fruitful line of inquiry involving genomic action at a distance. It might appear spooky at first, but once the phenomenon is explained in concrete biochemical terms, it can become a potent tool for understanding the heritability, or otherwise, of many diseases, and may even help to improve diagnostics and treatment. ALS

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ANS 2013 | COLOUR VISION

insight Vivid Tim Dean

Professor Paul Martin has his eye on uncovering the complexities of primate colour vision.

I

t seems the most effortless thing to do: to gaze upon the canvas of the world splattered with a dazzling array of colours. Yet not every creature - or every person - sees the world in the same way.

Colour is one of those things that require what philosopher Daniel Dennett calls a “strange inversion” in thinking. Colour is not ‘out there’ in the world; colour is very much a feature of what goes on ‘in here’.

And what goes on ‘in here’ depends to a great degree on the particulars of the neurobiology of vision: the ganglion cells in the retina; the neurones that pass through the thalamic relay nucleus, the lateral geniculate nucleus; the web of connections in the primary visual cortex; and the specialised processors of the higher cortex that discriminate the ‘where’ from the ‘what’.

It’s a spectacularly complex system, and while we have come a long way in unravelling the neurobiology of colour vision, there are still a few mysteries to tackle, or so says Professor Paul Martin from Sydney University and the Save Sight Institute. He will be giving this year’s Lawrie Austin Lecture at the 33rd Annual Meeting of the Australian Neuroscience Society, in Melbourne in February.

Given the complexity of the visual system, we know a surprising amount about how it functions. It starts with the photosensitive rods and cones that we all learnt about in high school. They trigger a cascade of nerve impulses in response to light, which careen through

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COLOUR VISION | ANS 2013

the lateral geniculate nucleus, or LGN, via two main pathways, the parvocelluar and the magnocellular. The parvocelluar cells respond to colour and fine details and edges, while the magnocellular cells provide the depth and motion.

These signals then travel to the primary visual cortex where the signals are synthesised to pick out edges and shapes.

The final stage of the process sees the signals sent to other regions of the cortex that hone in on the identity and position of the visible objects. And somewhere in there, a holistic picture emerges, replete

speculated that it plays a significant role in coordinating activity between cortical and subcortical brain structures.

“It is actually an old anatomical theory that we picked up on, that the koniocellular division is part of a primitive pathway that it acts like the rhythm section

higher level isn’t really higher because it feeds back to the lower level, so it influences its own input. So it’s really a two-way street, not a one-way street. It’s not just commands that are going from one place to another, but it’s more like a conversation.”

“It’s really a two-way street, not a one-way street.

It’s not just commands that are going from one place to another, but it’s more like a conversation.”

Deuteranopia

with colour, discrete objects and all in a seamless, conscious experience.

Except it’s not quite so simple, says Martin. Firstly, the parvocellular/ magnocellular pathway story is proving to be overly simplistic. It seems the pathways interact and overlap in their function with another pathway in order to produce a complete picture, as it were. “We’re gradually starting to tease out where these pathways go,” says Martin.

“We used to think the parvocellular and the magnocellular pathways did the whole job, but now we have strong evidence that the so-called primitive pathway, which also serves visual reflexes, actually has signals that seem related to conscious visual perception.”

This primitive pathway is the koniocellular pathway. Martin and his team found that this pathway exhibits ‘rhythms’ in individuals who are asleep or under anaesthesia, and they have

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in a rock band. They’re the foundation on which the rest of it works. The rhythm section may not be the biggest part of the band, but it’s a very important part.”

TWO-WAY STREET

The second spanner in the works is the idea that the visual system is a one-way street, beginning with the eye and ending in the cul-de-sac of the higher cortex.

In reality, it appears there are some fascinating feedback cycles involved throughout the process that is changing the way we think about vision.

“We used to think of it as a one-way street,” says Martin. “There’s the front end in the eye, then you go through the thalamus, then through to the cortex, and one part of the cortex feeds to the next.

Eventually you hone down to more and more complex features within the cortex.

“Yet what we’ve found is that at every level - once you get out of the eye - each

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Protanopia

Interestingly, according to Martin, this is an insight that has been known in anatomy for many decades, but it’s only recently that the more complex physiology of the process has started to be revealed in any detail.

So why the back-and-forth? Martin believes it could be something to do with directing attention. We absorb a truly vast amount of visual information by staring at a single scene, yet we’re able to direct our conscious focus around without changing our visual focus, as it were. The backward path may be the higher cortex directing attention on to particular parts of what we’re observing.

MANY RAINBOWS

Then there’s colour. How the brain teases apart the various signals flowing down the different pathways to construct a seamlessly, yet context-sensitive, polychromatic picture is another deep

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AN S 2013 | COLOUR VISION

Professor Paul Martin received his PhD in Physiology at the University of Sydney in 1986. In 1992 he returned to join faculty at the University of Sydney following postdoctoral work in Germany. In 2003 he left Sydney to take up appointment as Director of Research at the National Vision Research Institute of Australia and Professorial Research Fellow at the University of Melbourne. He returned to Sydney in 2010 to take up his current appointment as Professor of Experimental Ophthalmology.

mystery that is slowly being unravelled. One piece of the puzzle that is helping bring context to the story is looking at the evolution of trichromatic colour vision.

Martin works with marmosets, a species of monkey in which all males are red-green colour blind, while most females are trichromats like us. These monkeys may represent something of a stepping stone between the lower dichromatic primates and the upper primates with full trichromatic colour vision, and understanding how their visual system works might give us insight into the system from which ours developed.

According to Martin, one popular theory is that there was a mutation on the X chromosome that enabled females to develop trichromatic vision, and this may have given them a selective advantage when it came to navigating their environment or identifying edible fruits. The evolutionary trick was in getting that mutation over to the males, as it occurred on the X chromosome.

“The males are all colour blind because they haven’t got the two genes on their X chromosome.

Females, on the other hand, have two X chromosomes, and if they have red on one and green on other, then they can have three receptors.

The males are stuck with two, just like in many humans with colour blindness.”

But what happens when that mutation occurs, adding a third type of photoreceptor sensitive to red light? Can the brain suddenly create trichromatic vision without any further changes?

A 2009 experiment by Professors Jay and Maureen Neitz at the University of Washington suggests it can.

They injected a virus into the eyes of the monkeys that contained a gene that produced L-opsin, a protein lacked by the monkeys that is involved in detecting red and green. Startlingly, around five months after the treatment began, the monkeys appeared to be able to consistently

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discriminate red from green overnight. It was like a penny had suddenly dropped and they saw the world in an entirely different way.

What was especially intriguing is that this change didn’t appear to require any significant rewiring of the higher cortex. Instead, once the red-green signals were sent down different pathways, the brain began discriminating between them and the distinction between the colours emerged.

“When the paper was published, Jay Neitz told me there were people queuing outside his laboratory door asking for him to give them the needle,” says Martin. Yet the gene therapy is still some way from being ready to potentially address colour blindness in humans - although, in principle, it should work.

What excites Martin is the prospect of treating a whole slew of diseases once we gain a greater understanding of our colour visual system.

“Certainly, the potential of this gene therapy is there to treat colour blindness,” he says. “But from the perspective of a vision scientist, and someone who thinks about other blinding diseases, the same method they used to get colour genes could be used to fix the cells in other ways. You could package up other restorative genes to fix up diseases such as retinal dystrophy, where the receptors degenerate, for example.”

Yet, even with the tremendous progress over the last few decades in uncovering the internal machination that produces colour vision, there are still a lot of mysteries yet to be solved. Martin and his team are currently delving deeper into the koniocellular pathway and investigating how it contributes to colour vision and brain rhythms.

They are also taking a closer look at the macula, with an eye - as it were - to determining the similarities with the monkey macula. This is particularly important if, for example, the gene therapy that worked in monkeys is to be translated into a human treatment. ALS

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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.

Del Monte G, Harvey RP. Victor Chang Cardiac Research Institute, NSW An endothelial contribution to coronary vessels.

Cell. 2012 Nov 21.

Du X, Leang L, Mustafa T, Renoir T, Pang TY, Hannan AJ. Florey Neuroscience Institutes, VIC Environmental enrichment rescues female-specific hyperactivity of the hypothalamic-pituitaryadrenal axis in a model of Huntington’s disease.

Transl Psychiatry. 2012 Jul 3.

Fiorenza S, Kenna TJ, Comerford I, McColl S, Steptoe RJ, Leggatt GR, Frazer IH. University of Queensland Diamantina Institute, QLD A Combination of Local Inflammation and Central Memory T Cells Potentiates Immunotherapy in the Skin.

J Immunol. 2012 Nov 9.

Harper CB, Popoff MR, McCluskey A, Robinson PJ, Meunier FA. University of Queensland, QLD Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors.

Trends Cell Biol. 2012 Nov 16.

Hewitt VL, Heinz E, Shingu-Vazquez M, Qu Y, Jelicic B, Lo TL, Beilharz TH, Dumsday G, Gabriel K, Traven A, Lithgow T. Monash University, VIC A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways.

Proc Natl Acad Sci U S A. 2012 Nov 14.

Hui L, Bianchi DW. University of Sydney, NSW Recent advances in the prenatal interrogation of the human fetal genome.

Trends Genet. 2012 Nov 14.

Lane JR, Sexton PM, Christopoulos A. Monash University, VIC Bridging the gap: bitopic ligands of G-proteincoupled receptors.

Trends Pharmacol Sci. 2012 Nov 20.

Lee SH, Harold D, Nyholt DR; ANZGene Consortium; ENDOGENE Consortium; the Genetic and Environmental Risk for Alzheimer’s disease (GERAD1) Consortium, Goddard ME, Zondervan KT, Williams J, Montgomery GW, Wray NR, Visscher PM. University of Queensland, QLD Estimation and partitioning of polygenic variation captured by common SNPs for Alzheimer’s disease, multiple sclerosis and endometriosis.

Hum Mol Genet. 2012 Nov 28.

Neely GG, Rao S, Costigan M, Mair N, Racz I, et al. Garvan Institute of Medical Research, NSW Construction of a Global Pain Systems Network

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Highlights Phospholipid Signaling as a Regulator of Heat Nociception.

PLoS Genet. 2012 Dec 6.

Owen DM, Williamson DJ, Magenau A, Gaus K. University of New South Wales, NSW Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution.

Nat Commun. 2012 Dec 4.

Pitcher JB, Riley AM, Doeltgen SH, Kurylowicz L, Rothwell JC, McAllister SM, Smith AE, Clow A, Kennaway DJ, Ridding MC. Robinson Institute, University of Adelaide, SA; UCL Institute of Neurology, UK; University of Westminster, UK Physiological Evidence Consistent with Reduced Neuroplasticity in Human Adolescents Born Preterm.

J Neurosci. 2012 Nov 14.

Renoir T, Pang TY, Mo C, Chan G, Chevarin C, Lanfumey L, Hannan AJ. Florey Neuroscience Institutes, VIC Differential effects of early environmental enrichment on emotionality-related behaviours in Huntington’s disease transgenic mice.

J Physiol. 2012 Nov 12.

Rossy J, Owen DM, Williamson DJ, Yang Z, Gaus K. University of New South Wales, NSW

Conformational states of the kinase Lck regulate clustering in early T cell signaling. Nat Immunol. 2012 Dec 2.

Schlipalius DI, Valmas N, Tuck AG, Jagadeesan R, Ma L, Kaur R, Goldinger A, Anderson C, Kuang J, Zuryn S, Mau YS, Cheng Q, Collins PJ, Nayak MK, Schirra HJ, Hilliard MA, Ebert PR. Agri-Science Queensland, QLD A core metabolic enzyme mediates resistance to phosphine gas.

Science. 2012 Nov 9.

Walker MJ, Beatson SA. University of Queensland, QLD Epidemiology. Outsmarting outbreaks.

Science. 2012 Nov 30.

Warner CE, Kwan WC, Bourne JA. Monash University, VIC The Early Maturation of Visual Cortical Area MT is Dependent on Input from the Retinorecipient Medial Portion of the Inferior Pulvinar.

J Neurosci. 2012 Nov 28.

Weller JL, Liew LC, Hecht VF, Rajandran V, Laurie RE, Ridge S, Wenden B, Vander

Schoor JK, Jaminon O, Blassiau C, Dalmais M, Rameau C, Bendahmane A, Macknight RC, Lejeune-Hénaut I. University of Tasmania, TAS A conserved molecular basis for photoperiod adaptation in two temperate legumes.

Proc Natl Acad Sci U S A. 2012 Dec 3.

Whitnall M, Rahmanto YS, Huang ML, Saletta F, Lok HC, Gutiérrez L, Lázaro FJ, Fleming AJ, St Pierre TG, Mikhael MR, Ponka P, Richardson DR. University of Sydney, NSW Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

Proc Natl Acad Sci U S A. 2012 Nov 20.

Xu NL, Harnett MT, Williams SR, Huber D, O’Connor DH, Svoboda K, Magee JC. Howard Hughes Medical Institute, USA; University of Queensland, QLD Nonlinear dendritic integration of sensory and motor input during an active sensing task.

Nature. 2012 Nov 11.

Biankin AV, Grimmond SM, Gibbs RA, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, Miller DK, Wilson PJ, Patch AM, Wu J, Chang DK, Cowley MJ, Gardiner BB, Song S, Harliwong I, et al. The Kinghorn Cancer Centre, NSW Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Nature. 2012 Nov 15.

Duong HT, Kamarudin ZM, Erlich RB, Li Y, Jones MW, Kavallaris M, Boyer C, Davis TP. Australian Centre for NanoMedicine, The University of New South Wales, NSW Intracellular nitric oxide delivery from stable NOpolymeric nanoparticle carriers.

Chem Commun (Camb). 2012 Nov 16.

Lin AE, Ebert G, Ow Y, Preston SP, Toe JG, Cooney JP, Scott HW, Sasaki M, Saibil SD, Dissanayake D, Kim RH, Wakeham A, You-Ten A, Shahinian A, Duncan G, Silvester J, Ohashi PS, Mak TW, Pellegrini M. University of Toronto, CAN; Walter and Eliza Hall Institute, VIC ARIH2 is essential for embryogenesis, and its hematopoietic deficiency causes lethal activation of the immune system.

Nat Immunol. 2012 Nov 25.

Purtell L, Jenkins A, Viardot A, Herzog H, Sainsbury A, Smith A, Loughnan G, Steinbeck K, Campbell LV, Sze L. Garvan Institute of Medical Research, NSW Postprandial cardiac autonomic function in prader-willi syndrome.

Clin Endocrinol (Oxf) . 2012 Oct 26.

Tell the world about your paper: email tdean@westwick-farrow.com.au

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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.

24th International Conference on Arabidopsis Research (ICAR) June 24-28, Sydney

www.sallyjayconferences.com.au/icar2013/

4th International NanoMedicine Conference July 1-3, Sydney

www.oznanomed.org

Australian Society for Microbiology ASM 2013 July 7-10, Adelaide

www.theasm.org.au

BIO 2013

April 22-25, Chicago The BIO International Convention is the largest global event for the biotechnology industry and attracts the biggest names in biotech, offers networking and partnering opportunities and provides insights on the major trends affecting the industry. Keynotes and sessions are from key policymakers, scientists, CEOs and celebrities. The convention also features the BIO Business Forum (One-on-One Partnering), hundreds of sessions covering biotech trends, policy issues and technological innovations, and the world’s largest biotechnology exhibition.

http://convention.bio.org

Biomolecular in the Bush July 14-17, Leura

www.raci-bio-conf.org

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 ANS 2013 February 3-6, Melbourne

www.ans2013.org

18th Lorne Proteomics Symposium February 7-10, Lorne

www.australasianproteomics.org

38th Lorne Conference on Protein Structure and Function February 10-14, Lorne

www.lorneproteins.org

Lorne Cancer Conference February 14-16, Lorne

www.lornecancer.org Lorne Genome 2013 February 17-19, Lorne

http://lornegenome.org Lorne Infection and Immunity Conference 2013 February 20-22, Lorne

www.lorneinfectionimmunity.org

Peter Mac Colorectal Cancer Conference 2013 March 14-15, Melbourne

http://petermaccolorectalconf.org Hunter Meeting March 19-22, Hunter Valley

http://hcbm.mtci.com.au

AusBiotech Business Development 2013 March 20, Glenelg, South Australia

Australia China Life Sciences Summit March 26-27, Melbourne

www.ausbiotech.org

Tech Transfer Summit Australia 2013 September 3-4, WEHI, Melbourne

www.ausbiotech.org

The Annual Endocrine Society of Australia Seminar 2013 April 5-7, Sunshine Coast, Queensland

ComBio2013 September 29-October 3, Perth

HGM 2013 + 21st International Congress of Genetics April 13-18, Singapore

5th Asia-Pacific NMR Symposium (APNMR5) and 9th Australian & New Zealand Society for Magnetic Resonance (ANZMAG) October 27-30, Brisbane

www.esaseminar.org.au

www.hgm2013-icg.org

Stem Cells and Cancer Symposium April 17, Parkville

www.sapphirebioscience.com/symposium BIO 2013 April 22-25, Chicago

http://convention.bio.org 12th International Symposium on Mutation in the Genome April 22-26, Lake Louise, Canada

www.mutationdetection.org AusMedtech 2013 May 15-16, Melbourne

www.ausbiotech.org ASPCR-ASDR 2013 May 17-19, Sydney

www.aspcr-asdr2013.org

www.asbmb.org.au

http://apnmr2013.org

AusBiotech 2013 October 29-November 2, Brisbane

www.ausbiotech.org

HPLC 2013 November 18-21, Hobart

www.hplc2013-hobart.org 15th International Conference on Systems Biology in Melbourne September 13-19, 2014, Melbourne

www.emblaustralia.org

ComBio2014 September 28-October 2, 2014, Canberra

www.asbmb.org.au

AusBiotech 2014 October 28-31, 2014, Gold Coast

www.ausbiotech.org

www.ausbiotech.org

Tell the world about your event: email tdean@westwick-farrow.com.au

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