Devil facial tumour disease
Devil facial tumour disease (DFTD) is an aggressive non-viral transmissible parasitic cancer—which likely originated in Schwann cells—that affects Tasmanian devils. The first "official case" was described in 1996, in Australia. In the subsequent decade the disease ravaged Tasmania's wild devils, with estimates of decline ranging from 20% to as much as a 50% of the devil population, across over 65% of the state. Affected high-density populations suffer up to 100% mortality in 12–18 months. The disease has mainly been concentrated in Tasmania's eastern half. Visible signs of DFTD begin with lesions and lumps around the mouth. These develop into cancerous tumours that may spread from the face to the entire body. Once lesions first appear devils usually die within six months owing to organ failure, secondary infection, or metabolic starvation. The tumours interfere with feeding, and the affected animal may starve to death. Devil Facial Tumour Disease affects males and females equally. At present the population has dwindled by 70% since 1996. Numbers as of 2010, 80% of population is infected. The most plausible route of transmission is through biting, particularly, when canine teeth come into direct contact with the diseased cells. Other modes of transmission include, but are not limited to, the ingesting of an infected carcass or the sharing of food, both of which involve an allogeneic transfer of cells between unrelated individuals.
Six females have been found with a partial immunity. Breeding in captivity has begun in an attempt to save the population.
Tasmanian devil cells have 14 chromosomes, while the oldest-known strain of the tumour cell contains thirteen chromosomes, nine of which are recognizable and four of which are mutated “marker” chromosomes. More recently evolved strains have an additional mutant marker chromosome, for a total of fourteen chromosomes. Researchers identified the cancer as a neuroendocrine tumour, and found identical chromosomal rearrangements in all the cancer cells. The karyotype anomalies of DFTD cells are similar to those of cancer cells from canine transmissible venereal tumour (CTVT), a cancer of dogs that is transmitted between canines by physical contact. Among the different alterations present in the tumour genome can be found several single base mutations or shorts insertions and deletions (indels) like deletions in the chromosomes 1, 2 and 3 or trisomy in 5p. Some of the mutated or deleted genes in DFTD are RET, FANCD2, MAST3 and BTNL9-like gene.
The theory that cancer cells themselves could be an infective agent (the Allograft Theory) was first supported by the researchers A-M. Pearse, K. Swift, and colleagues. In 2006, Pearse and Swift analyzed DFTD cells from several devils in different locations, and determined that all of the DFTD cells were not only genetically identical to each other, but also genetically distinct from their hosts, and from all known Tasmanian devils. Thus the cancer must have originated in a single individual and spread, rather than arising separately within each animal. In fact, it has been identified up to 21 different subclones by analyzing the genome (mitochondrial and nuclear) of 104 tumours from different Tasmanian devils. Later researchers witnessed a previously-uninfected devil develop tumours from lesions caused by an infected devil’s bites, confirming that the disease is spread by allograft, and that the normal methods of transmission include biting, scratching, and aggressive sexual activity between individuals. Since June 2005, three females have been found that are partially resistant to DFTD.
Further research from the University of Sydney has shown that the infectious facial cancer may be able to spread because of low diversity in devil immune genes (MHC class I and II). The same genes are also found in the tumours, so the devil's immune system does not recognize the tumour cells as foreign. There are at least four, and most likely more, strains of the cancer, showing that it is evolving, and may become more virulent. The strains may also complicate attempts to develop a vaccine, and the mutation of the cancer may mean that it could spread to other related species, like the quoll.
In a paper published in the January 2010 issue of Science, an international team of researchers announced that devil facial tumour disease likely originated in the Schwann cells of a single devil. Schwann cells are found in the peripheral nervous system, and produce myelin and other proteins essential for the functions of nerve cells in the peripheral nervous system. The researchers sampled 25 tumours and found that the tumours were genetically identical. Using deep sequencing technology, they then profiled the tumours' transcriptome, the set of genes that are active in tumours. The transcriptomes closely matched those of Schwann cells, revealing high activity in many of the genes coding for myelin basic protein production. Several specific markers were identified by the team, including the MBP and PRX genes, which may enable veterinarians to more easily distinguish DFTD from other types of cancer, and may eventually help identify a genetic pathway that can be targeted to treat it.
Due to the decreased life expectancy of the devils due to DFTD, they have begun breeding at younger ages in the wild, with reports that many only live to participate in one breeding cycle. A study has suggested that Tasmanian devils have changed their breeding habits in response to the disease. Females previously started breeding at the age of two, then annually for about three more years until dying normally. Now they commonly breed at the age of one, and die of tumours shortly thereafter. It is speculated that the disease is spread by devils biting each other during the mating season. Social interactions have been seen spreading DFTD in a local area. It is one of three known contagious cancers.
|This section requires expansion. (September 2010)|
Growth of large tumours impedes feeding, and starvation is a common cause of death in affected devils. Organ involvement and additional infections may also be a factor in death, as regional lymph node involvement and systemic metastasis is common. The cancer invades the heart.
The tumours are capable of dissolving parts of the skull.
Wild Tasmanian devil populations are being monitored to track the spread of the disease and to identify changes in disease prevalence. Field monitoring involves trapping devils within a defined area to check for the presence of the disease and determine the number of affected animals. The same area is visited repeatedly to characterise the spread of the disease over time. So far, it has been established that the short-term effects of the disease in an area can be severe. Long-term monitoring at replicated sites will be essential to assess whether these effects remain, or whether populations can recover. Field workers are also testing the effectiveness of disease suppression by trapping and removing diseased devils. It is hoped that the removal of diseased devils from wild populations should decrease disease prevalence and allow more devils to survive beyond their juvenile years and breed. A study felt that the current system of culling did not impede the disease's spread.
Two "insurance" populations of disease-free devils are being established at an urban facility in the Hobart suburb of Taroona and on Maria Island off the east coast of Tasmania. Captive breeding in mainland zoos is also a possibility. The decline in devil numbers is also seen as an ecological problem, since its presence in the Tasmanian forest ecosystem is believed to have prevented the establishment of the red fox, illegally introduced to Tasmania in 2001. It is believed that Tasmanian devil young would be vulnerable to red fox predation, as they are left alone for long periods of time.
In response to the impact of DFTD on Tasmanian devil populations, 47 devils have been shipped to mainland Australian wildlife parks to attempt to preserve the genetic diversity of the species. The largest of these efforts is the Barrington Tops "Devil Ark" in New South Wales; an initiative of the Australian Reptile Park. This project aims to create a set of one thousand genetically representative devils, and is now a major focus of the insurance policy. In addition, the Tasman peninsula is being considered as a possible "clean area" with the single narrow access point controlled by physical barriers. The Tasmanian Department of Primary Industries and Water is experimenting on culling infected animals with some signs of success.
In 2008, a devil named Cedric was thought to have a natural immunity to the disease, but in late 2008 he developed two tumours on his face. The tumours were removed and officials thought Cedric was responding well until September 2010 when it was discovered that the cancer had spread to his lungs. He was euthanized upon the discovery. A diagnostic blood test was developed in mid-2009 to screen for the disease. In early 2010, scientists found some Tasmanian devils, mostly in the north-west of Tasmania, that are genetically different enough for their bodies to recognise the cancer as foreign. They have only one Major Histocompatibility Complex, whereas the cancerous cells have both.
At present with the population reduced by 70% since 1996, if a cure is not found then scientists predict they will become extinct by 2035.
Vaccination with irradiated cancer cells has not proven successful.
Research published in the Proceedings of the National Academy of Sciences on June 27, 2011, suggests picking a genetically diverse breeding stock, defined by the genome sequence, for conservation efforts.
In 2011, Principal Investigator David Phalen, (Wildlife Health and Conservation Centre, University of Sydney) and Stephen Pyecroft (Department of Primary Industries and Water in Tasmania) along with Antony Moore and Angela Frimberger (Veterinary Oncology Consultants) were awarded a Tasmanian Government Project Management Grant for their project Investigation into Chemotherapy Agents Effective Against the Tasmanian Devil Facial Tumour.
In 1996, a photographer captured several images of devils with facial tumours near Mount William in Tasmania's north-east. At around the same time, farmers reported a decline in devil numbers. Menna Jones first encountered the disease in 1999 near Little Swanport, in 2001 capturing three devils with facial tumours on the Freycinet Peninsula. The devil population on the peninsula decreased dramatically. In March 2003 Nick Mooney wrote a memo to be circulated within the Parks and Wildlife Services calling for more funding for the study of the disease, but the call for funding was edited out before the memo was presented to Bryan Green, then Minister for Primary Industries, Water and Environment. In April 2003, a working group was formed by the Tasmanian Government to respond to the disease. In September 2003, Nick Mooney went to the Tasmanian daily newspaper The Mercury, informing the general public of the disease and proposing a quarantine of healthy Tasmanian devils. At the time, it was thought that a retrovirus was a possible cause. David Chadwick of the state Animal Health Laboratory said that the laboratory did not have the resources needed to research the possibility of a retrovirus. The Tasmanian Conservation Trust criticised the Tasmanian Government for not providing sufficient resources to research the disease, and suggested that the DFTD could be zoonotic, posing a threat to livestock and humans. On 14 October 2003, a workshop was held in Launceston. In 2004, Kathryn Medlock found three oddly shaped devil skulls in European museums and found a description of a devil in London Zoo dying which showed a similarity to DFTD.
A virus was initially thought to be the cause of DFTD, but no evidence of such a virus could be detected in the cancer cells. Calicivirus, 1080 poison, agricultural chemicals, and habitat fragmentation combined with a retrovirus were other proposed causes. Environmental toxins had also been suspected. In March 2006 a devil escaped from a park into an area which was infected with DFTD. She was recaptured with bite marks on her face, and returned to live with the other devils in the park. She wounded a male and by October both devils were observed to have the disease, which was subsequently transferred to two other devils. This incident helped test the viability of the allograft theory of transmission. In 2006, DFTD was classed a List B notifiable disease under the Animal Health Act 1995. Also in 2006, the strategy of developing an insurance population in captivity was developed. It was reassessed in 2008. A 2007 investigation into the immune system of the devils found that when combatting other pathogens, the response from the immune system was normal, leading to suspicion that the devils were not capable of detecting the cancerous cells as "non-self". In 2007, it was predicted that populations could become locally extinct within 10 – 15 years of DFTD occurring, and predicted that the disease would spread across the entire range of the Tasmanian devils. This study also predicted that Tasmanian devils would become extinct within 25–35 years.
In 2008, high levels of potentially carcinogenic flame retardant chemicals were found in Tasmanian devils. Preliminary results of tests ordered by the Tasmanian government on chemicals found in fat tissue from 16 devils have revealed high levels of hexabromobiphenyl (BB153) and "reasonably high" levels of decabromodiphenyl ether (BDE209).
In other animals
Transmissible cancer, caused by a clone of malignant cells rather than a virus, is extremely rare. The only other known types are canine transmissible venereal tumour (CTVT), which is passed between dogs via sexual activity and has been known to science for about 100 years, and contagious reticulum cell sarcoma of the Syrian hamster, which can be transmitted from one Syrian hamster to another by means of the bite of the mosquito Aedes aegypti. CTVT mutes the expression of the immune response, whereas the Syrian hamster disease spreads due to lack of genetic diversity.
- HeLa cells – an immortal cell line used in biomedical research
- Zimmer, Carl (December 31, 2009). "Findings on Origin of a Cancer in Tasmanian Devils". New York Times. Retrieved 2009-12-31. "The Tasmanian devil, the spaniel-size marsupial found on the Australian island of Tasmania, has been hurtling toward extinction in recent years, the victim of a bizarre and mysterious facial cancer that spreads like a plague."
- Loh, Richmond. 2003. Tasmanian Devil (Sarcophilus harrisii) facial tumour (DFT). Paper prepared for the Devil Facial Tumour Disease Workshop, Sir Raymond Ferrall Centre, University of Tasmania, Newnham, 14 October 2003, p. 2.
- "Experts tackle the devil's tumour". BBC News. 20 February 2007.
- Weiss, Rick (11 August 2006). "Study Finds That a Type of Cancer in Dogs Is Contagious". The Washington Post. Retrieved 11 August 2006.
- DPIWE. 2005. Devil Facial Tumour Disease – Update June 2005
- DPIWE. 2005. Tasmanian Devil Facial Tumour Disease, Disease Management Strategy
- DPIWE. Disease Affecting Tasmanian Devils
- Deakin, Janine E.; Belov, Katherine (2012). "A Comparative Genomics Approach to Understanding Transmissible Cancer in Tasmanian Devils". Annual Review of Genomics and Human Genetics 13: 207–222. doi:10.1146/annurev-genom-090711-163852. PMID 22657390.
- Lachish S, McCallum H, Jones M. (2009)
- Hawkins CE, McCallum H, Mooney N, Jones M, Holdsworth M. (2009). Sarcophilus harrisii. In: IUCN red list of threatened species. Version 2009.1 <www.iucnredlist.org>
- Janeway CA, Travers P, Walport M, Shlomchik M. (2001). Immunobiology. Garland Publishing, New York, NY.
- "Scientists find 'immune' Tassie Devils". ninemsn. March 10, 2010.
- "Bites spread fatal 'devil' cancer". BBC News. 2006-02-02.
- Deakin, J.E., et al. (2012). "Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of Transmissible Tumour". PLOS Genetics 8 (2): e1002483. doi:10.1371/journal.pgen.1002483.
- Pearse, A.-M., et al. (2012). "Evolution in a transmissible cancer: a study of the chromosomal changes in devil facial tumor". Cancer Genetics 205: 101–112.
- Bostanci, A. (2005). "A Devil of a Disease". Science 307 (5712): 1035. doi:10.1126/science.307.5712.1035. PMID 15718445.
- P. Murchison, Elizabeth; Schulz-Trieglaff OB, Ning Z, Alexandrov LB, Bauer MJ, Fu B, Hims M, Ding Z, Ivakhno S, Stewart C, Ng BL, Wong W, Aken B, White S, Alsop A, Becq J, Bignell GR, Cheetham RK, Cheng W, Connor TR, Cox AJ, Feng ZP, Gu Y, Grocock RJ, Harris SR, Khrebtukova I, Kingsbury Z, Kowarsky M, Kreiss A, Luo S, Marshall J, McBride DJ, Murray L, Pearse AM, Raine K, Rasolonjatovo I, Shaw R, Tedder P, Tregidgo C, Vilella AJ, Wedge DC, Woods GM, Gormley N, Humphray S, Schroth G, Smith G, Hall K, Searle SM, Carter NP, Papenfuss AT, Futreal PA, Campbell PJ, Yang F, Bentley DR, Evers DJ, Stratton MR. (17 February 2012). "Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer.". Cell 148 (4): 780–791. doi:10.1016/j.cell.2011.11.065. PMID 22341448.
- Pearse, A-M., Swift, K. (2006). "Allograft theory: Transmission of devil facial-tumour disease". Nature 439 (7076): 549. doi:10.1038/439549a. PMID 16452970.
- Tasmanian devils felled by rare cancer, New Scientist, 1 February 2006.(subscription required)
- Gramling, Carolyn: Poor Devils: Critters' fights transmit cancer, Science News, 4 February 2006.
- Obendorf, D. L. and N. D. MacGlashan (2008). "Research priorities in the Tasmanian devil facial tumor debate". European Journal of Oncology 13: 229–238.
- Dennis, Carina (2006-02-02). "Endangered species: Time to raise the devil". Nature 439 (7076): 530. doi:10.1038/439530a. PMID 16452951.
- "Tasmanian devil epidemic: cause isolated?". Cosmos Magazine. 27 June 2007.
- Leung, Chee Chee: Extinction warning for Tassie devils, The Age, 4 October 2007.
- "Scientists make progress in understanding Tasmanian devil disease". New York Times. 2007-10-09. "Australian researchers have made a breakthrough in understanding a rapidly spreading facial cancer that has killed a large percentage of the Tasmanian devil population."
- "New Tas devil tumour strands 'harder to vaccinate' – ABC News (Australian Broadcasting Corporation)". ABC.net.au. 2008-07-25. Retrieved 2010-01-03.
- Murchison, E. P.; Tovar, C.; Hsu, A.; Bender, H. S.; Kheradpour, P.; Rebbeck, C. A.; Obendorf, D.; Conlan, C.; Bahlo, M.; Blizzard, C. A.; Pyecroft, S.; Kreiss, A.; Kellis, M.; Stark, A.; Harkins, T. T.; Marshall Graves, J. A. M.; Woods, G. M.; Hannon, G. J.; Papenfuss, A. T. (2009). "The Tasmanian Devil Transcriptome Reveals Schwann Cell Origins of a Clonally Transmissible Cancer". Science 327 (5961): 84–87. doi:10.1126/science.1180616. PMC 2982769. PMID 20044575.
- Kinver, Mark (1 January 2010). "Tasmanian devil facial cancer origins 'identified'". BBC.
- Walsh, Bryan (1 January 2010). "Decoding the Tasmanian Devil's Deadly Cancer". Time.
- Owen and Pemberton, p. 6.
- Kinver, Mark (15 July 2008). "Tumours 'alter devils' sex lives'". BBC News.
- O'Neill, Iain D. (2010). "Tasmanian devil facial tumor disease: Insights into reduced tumor surveillance from an unusual malignancy". International Journal of Cancer 127 (7): 1637–42. doi:10.1002/ijc.25374. PMID 20473867.
- Wood, Danielle (1 September 2003). "Tassie devil under threat". The Mercury (Hobart Town, Tasmania). p. 1.
- Lachish, Shelly; McCallum, Hamish; Mann, Dydee; Pukk, Christy E.; Jones, Menna E. (2010). "Evaluation of Selective Culling of Infected Individuals to Control Tasmanian Devil Facial Tumor Disease". Conservation Biology 24 (3): 841–851. doi:10.1111/j.1523-1739.2009.01429.x. PMID 20088958.
- Zimmer, Carl (22 January 2013). "Raising Devils in Seclusion". New York Times.
- Bostanci A (2005). "A Devil of a Disease". Science 307 (5712): 1035. doi:10.1126/science.307.5712.1035. PMID 15718445.
- "DPIW – Fox Impact on Wildlife". Dpiw.tas.gov.au. 2010-05-25. Retrieved 2010-09-11.
- "The Tasmanian Devil Needs a Saviour". Devil Ark. Retrieved 2011-04-19.
- Scientific American, March 2007
- Contagious cancer: the evolution of a killer, David Quammen, Harper's Magazine, April 2008.
- BBC – Fence hope for Tasmanian Devils. Retrieved 5 January 2009.
- "Cancer-related death for Cedric the devil – ABC News (Australian Broadcasting Corporation)". ABC.net.au. 2010-09-01. Retrieved 2010-09-11.
- "A pre-tumour diagnostic test for DFTD". Tassiedevil.com.au. 2009-06-15. Retrieved 2010-09-11.
- Denholm, Matthew (2010-03-10). "Genetic study raises hopes for Tasmanian devils hit by facial tumour disease". The Australian. Retrieved 2010-09-11.
- Czarny, N.A.; Rodger, J.C. (2010). "Vitrification as a method for genome resource banking oocytes from the endangered Tasmanian devil (Sarcophilus harrisii)☆". Cryobiology 60 (3): 322–5. doi:10.1016/j.cryobiol.2010.02.007. PMID 20219455.
- "Devil cure hope in rainforest". The Mercury. 2010-06-20. Retrieved 2010-10-08.
- Miller, Webb; et al. (27 June 2011). "Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil)". Proceedings of the National Academy of Sciences 108 (30): 12348. doi:10.1073/pnas.1102838108.
- McCallum H (2008). "Tasmanian devil facial tumour disease: lessons for conservation biology". Trends Ecol Evol. 23 (11): 631–7. doi:10.1016/j.tree.2008.07.001. PMID 18715674.
- Owen and Pemberton, pp.170–171
- "Tasmanian devils threatened by facial tumour disease". Science Show (Australian Broadcasting Corporation). 2007-04-21. Retrieved 2010-10-08.
- Owen and Pemberton, p.177
- "29 October 2003, part 2". Parliamentary Debates (Hansard) (Tasmania: House of Assembly): pages 31–104. (part 2) 3:30 pm (Jeremy Rockliff, Shadow Minister for Primary Industries, Water and Environment; Nick McKim, Tasmanian Greens; Bryan Green, Minister for Primary Industries, Water and Environment).
- . Bryan Green, Minister for Primary Industries, Water and Environment. "20 August 2003, part 1". Parliamentary Debates (Hansard) (Tasmania: House of Assembly): pages 1–29. (10:58 am) (part 1)
- "Wildlife specialists concerned about Tassie Devil disease". Australian Broadcasting Corporation. 2003-10-14. Retrieved 2010-10-08.
- "Devil Disease". Stateline Tasmania. Australian Broadcasting Corporation. 2004-09-10. Retrieved 2010-10-08.
- Owen and Pemberton, p.178
- Owen and Pemberton, p.184-185
- Conservation Breeding Specialist Group (2008). "Tasmanian Devil PHVA Final Report" (PDF). Apple Valley, Minnesota: IUCN/SSC Conservation Breeding Specialist Group. Retrieved 2 October 2010.
- Woods, Gregory M.; Kreiss, Alexandre; Belov, Katherine; Siddle, Hannah V.; Obendorf, David L.; Muller, H. Konrad (2007). "The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease". EcoHealth 4 (3): 338. doi:10.1007/s10393-007-0117-1.
- McCallum, Hamish; Tompkins, Daniel M.; Jones, Menna; Lachish, Shelly; Marvanek, Steve; Lazenby, Billie; Hocking, Greg; Wiersma, Jason; Hawkins, Clare E. (2007). "Distribution and Impacts of Tasmanian Devil Facial Tumor Disease". EcoHealth 4 (3): 318. doi:10.1007/s10393-007-0118-0.
- Denholm, Matthew (2008-01-22). "Cancer agents found in Tasmanian devils". News.com.au. Retrieved 2010-03-16.
- COPPER, H.L.; MacKay, CM; Banfield, WG (1964-10-01). "CHROMOSOME STUDIES OF A CONTAGIOUS RETICULUM CELL SARCOMA OF THE SYRIAN HAMSTER". J Natl Cancer Inst 33: 691–706. PMID 14220251.
- Banfield, William G.; Woke, PA; MacKay, CM; Cooper, HL (1965-05-28). "Mosquito Transmission of a Reticulum Cell Sarcoma of Hamsters". Science 148 (3674): 1239–1240. doi:10.1126/science.148.3674.1239. PMID 14280009.
- Siddle, H. V.; Kreiss, A.; Eldridge, M. D. B.; Noonan, E.; Clarke, C. J.; Pyecroft, S.; Woods, G. M.; Belov, K. (2007). "Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial". Proceedings of the National Academy of Sciences 104 (41): 16221–6. doi:10.1073/pnas.0704580104. PMC 1999395. PMID 17911263.
- Owen, David; Pemberton, David (2005). Tasmanian Devil: A unique and threatened animal. Crows Nest, New South Wales: Allen & Unwin. ISBN 9781741143683. Retrieved 22 August 2010.
- Australian Government (February 2010) State party report on the state of conservation of the Tasmanian Wilderness World Heritage Area (Australia) pp. 29–32
- Bender, Hannah S. (2010). "Devil Facial Tumour Disease (DFTD): Using Genetics and Genomics to Investigate Infectious Disease in an Endangered Marsupial". "Marsupial Genetics and Genomics". p. 499. doi:10.1007/978-90-481-9023-2_23. ISBN 978-90-481-9022-5.
- Bode, M., C. Hawkins, T. Rout, and B. Wintle. 2009. Efficiently locating conservation boundaries: Searching for the Tasmanian devil facial tumour disease front. Biological Conservation 142:1333–1339.
- Bradshaw, C. J. A. and B. W. Brook. 2005. Disease and the devil: density-dependent epidemiological processes explain historical population fluctuations in the Tasmanian devil. Ecography 28:181–190.
- Department of primary industries, water and environment (February 2005) Tasmanian Devil Facial Tumour Disease (DFTD) Disease Management Strategy
- Jones, Menna E.; Cockburn, Andrew; Hamede, Rodrigo; Hawkins, Clare; Hesterman, Heather; Lachish, Shelly; Mann, Diana; McCallum, Hamish; Pemberton, David. (2008). "Life-history change in disease-ravaged Tasmanian devil populations". Proceedings of the National Academy of Sciences of the United States of America 105 (29): 10023–7. doi:10.1073/pnas.0711236105. PMC 2481324. PMID 18626026.