Steven M. Reppert

Steven M. Reppert (born September 4, 1946) is an American neuroscientist known for his contributions to the fields of chronobiology and neuroethology. His research has focused primarily on the physiological, cellular, and molecular basis of circadian rhythms in mammals and more recently on the navigational mechanisms of migratory monarch butterflies. He is the Higgins Family Professor of Neuroscience at the University of Massachusetts Medical School, and from 2001 to 2013 was the founding chair of the Department of Neurobiology. Reppert stepped down as chair in 2014 to focus exclusively on his research activities. He now holds the additional title of Distinguished Professor of Neurobiology.

Steven M. Reppert
Born (1946-09-04) September 4, 1946
Sioux City, IA
Residence Auburndale, MA
Citizenship United States
Fields
Institutions
Alma mater
Known for
  • Fetal Circadian Clock
  • Melatonin Receptors
  • Circadian Clock Mechanism in Mammals
  • Monarch Butterfly Sun Compass

Biography

Early life

Steven Reppert grew up in the village of Pender, Nebraska, and graduated from Pender Public High School in 1964. His interest in science began in childhood with the cecropia moth—an insect made famous by Harvard biologist Carroll M. Williams, who used the moth in his pioneering work on the role of juvenile hormone in molting and metamorphosis.[1] Reppert continues to rear cecropia from egg to adult each summer, and over his career has published two papers on the circadian system of the cecropia moth.[2][3]

Education and career

Reppert received his BS and MD in 1973 (with distinction) from the University of Nebraska College of Medicine and was elected as a medical student to the Alpha Omega Alpha Honor Medical Society. From 1973 to 1976 he did an internship and residency in pediatrics at the Massachusetts General Hospital. From 1976 to 1979 Reppert was a posdoctoral fellow in neuroendocrinology at the National Institute of Child Health and Human Development in Bethesda, Maryland, in David C. Klein's laboratory, which focuses on the pineal gland and circadian biology.[4] Reppert was on the faculty at the Massachusetts General Hospital and Harvard Medical School beginning in 1979 and was promoted to professor in 1993; he directed the Laboratory of Developmental Chronobiology at the Massachusetts General Hospital from 1983 to 2001, when he moved to the University of Massachusetts Medical School.[5]

Awards and honors

Reppert has been a recipient of a Charles King Trust Research Fellowship, a Basil O’Connor Early Scholar Award from the March of Dimes Foundation, and a five-year Established Investigatorship of the American Heart Association. From 2002 to 2004, he served as president of the Society for Research on Biological Rhythms.[6] Other research honors include the E. Mead Johnson Award for Outstanding Research;[7] the NIH-NICHD MERIT Award; the Gregor J. Mendel Honorary Medal for Merit in the Biological Sciences from the Academy of Sciences of the Czech Republic;[8] and an honorary doctorate from the University of South Bohemia.[9] He is also fellow of the American Association for the Advancement of Science.[10]

Research

Reppert has published more than 190 papers with over 21,000 citations and an h-index of 77.[11] He is the principal inventor on seven patents derived from his research.[12]

Fetal circadian clocks

Rodent studies have shown that the master brain clock in the suprachiasmatic nucleus (SCN) is functional in the fetus before the fetal brain is capable of registering the presence of light. Reppert and colleagues reported that the fetal SCN is entrained to the light-dark cycle before the retinohypothalamic pathway innervates the SCN from the eye.[13] This finding indicates that the mother, and her entrainment to ambient light-dark cycles, provides the necessary information to the fetus for synchronization. As Reppert states, “Mom is functioning as the transducer for the fetal circadian system. She takes in light information to her circadian system, and then that is communicated to the fetal circadian system.”[14] This fetal entrainment persists into the postnatal period and ensures that neonatal behavioral patterns are properly tuned with the environment. Dopamine and melatonin can both act as perinatal maternal entraining signals.[15]

Mammalian circadian clocks

Steven Reppert and colleagues have made seminal contributions that provide insight into the mammalian circadian clock mechanism.

Cell autonomy in the SCN

Reppert and colleagues discovered that the SCN contains a large population of autonomous, single-cell circadian oscillators.[16] They cultured cells from neonatal rat SCN on fixed microelectrode array that allowed them to monitor individual SCN neuron activity in culture. Circadian rhythms expressed by neurons in the same culture were not synchronized, indicating that they functioned independently of one another.

Functions of mouse clock genes: PERIOD2 and PERIOD3

Reppert and coworkers also discovered the mouse clock genes mPer2 and mPer3 and defined their functions. They found that the mPER2 and mPER3 proteins, as well as the previously discovered mPER1, share several regions of homology with one another and with Drosophila PER.[17][18] Reppert and coworkers found different light responses among the three Per genes.[18] Unlike mPer1 and mPer2 mRNA levels, mPer3 mRNA levels are not acutely altered by light exposure during the subjective night. They also found that mPer1–3 are widely expressed in tissues outside the brain, including the liver, skeletal muscles, and testis. To determine the function of mPER1–3, Reppert and colleagues disrupted the three genes encoding them.[19] Using double-mutant mice, they showed that mPER3 functions outside the core circadian clockwork, whereas both mPER1 and mPER2 are necessary for rhythmicity.

Negative transcriptional feedback loop

Reppert and colleagues discovered that the two mouse cryptochromes, mCRY1 and mCRY2, function as the primary transcriptional repressors of clock gene expression, and the mPER proteins are necessary for CRY nuclear translocation.[20] This work provided the first portrayal of a negative transcriptional feedback loop as the major gear driving the mouse molecular clock.[21]

Interlocking transcriptional feedback loops

Reppert and colleagues found that the core mechanisms for the SCN in mammals consist of interacting positive and negative transcriptional feedback loops.[22] The first loop is an autoregulatory negative transcriptional feedback loop in which the mCRY proteins negatively regulate mCry and mPer gene transcription. The second interlocking feedback loop involves the rhythmic regulation of Bmal1. Rhythmicity of Bmal1 is not necessary for clockwork function, but it helps modulate the robustness of rhythmicity.

CLOCK and NPAS2

Reppert and colleagues discovered that the transcription factors CLOCK and NPAS2 have overlapping roles in the SCN, revealing a new and unexpected role for NPAS2.[23] His lab observed that CLOCK-deficient mice continue to have behavioral and molecular rhythms, which showed that CLOCK is not essential for circadian rhythm in locomotor activity in mice. They then determined, by investigating CLOCK-deficient mice, that NPAS2 is a paralog of CLOCK and can functionally substitute CLOCK by dimerizing with BMAL1. Finally, they found—by investigating CLOCK-deficient, NPAS2-deficient, and double-mutant mice—that circadian rhythms in peripheral oscillators require CLOCK.[23] Thus, there is a fundamental difference between CLOCK and NPAS2 that is tissue dependent.

Mammalian melatonin receptors

In 1994, Reppert cloned human and sheep Mel1a melatonin receptor, the first in a family of GPCRs that bind the pineal hormone melatonin, and localized its expression in the mammalian brain to the SCN and the hypophyseal pars tuberalis.[24] Mel1a is believed to be responsible for the circadian effects of melatonin and the reproductive actions in seasonal breeding mammals.[24]

In 1995, Reppert cloned and characterized the Mel1b melatonin receptor. He and colleagues found that the receptor was predominantly expressed in the retina, where it is believed to modify light-dependent retinal functions.[25] They identified outbred populations of Siberian hamsters that lacked functional Mel1b but maintained circadian and reproductive responses to melatonin;[26] these data indicate that Mel1b is not necessary for the circadian and reproductive actions of melatonin, which instead depend on Mel1a.

Elucidation of the molecular nature of the melatonin receptors has facilitated definition of their ligand-binding characteristics and aided the development of melatonin analogs that are now used to treat sleep disorders and depression.[24]

Insect cryptochromes

In 2003, Reppert began investigating the functional and evolutionary properties of the CRY protein in the monarch butterfly. He identified two Cry genes in the Monarch, Cry1 and Cry2.[27] His work demonstrated that the monarch CRY1 protein is functionally analogous to Drosophila CRY, the blue-light photoreceptor necessary for photoentrainment in the fly. He also demonstrated that monarch CRY2 is functionally analogous to vertebrate CRYs and that monarch CRY2 acts as a potent transcriptional repressor in the circadian clock transcriptional translation feedback loop of the butterfly, as his group previously showed for the two mouse CRYs.[20] These data propose the existence of a novel circadian clock unique to some non-drosophilid insects that possesses mechanisms characteristic of both the Drosophila and the mammalian clocks.[28] Other insects, such as bees and ants, possess only a vertebrate-like CRY, and their circadian clocks are even more vertebrate like.[29] Drosophila is the only known insect that does not possess a vertebrate-like CRY.

In 2008, Reppert discovered the necessity of Cry for light-dependent magnetoreception responses in Drosophila. He also showed that magnetoreception requires UVA/blue light, the spectrum corresponding with the action spectrum of Drosophila CRY.[30] These data were the first to genetically implicate CRY as a component of the input pathway or the chemical-based pathway of magnetoreception. Applying these findings to his work with the monarch, Reppert has shown that both monarch CRY1 and CRY2 proteins, when transgenically expressed in CRY-deficient flies, successfully restore magnetoreception function. These results propose the presence of a CRY-mediated magnetosensitivity system in monarchs that may act in concordance with the sun compass to aid navigation. In 2011, Reppert also discovered that human CRY2 can substitute as a functional magnetoreceptor in CRY-deficient flies, a discovery that warrants additional research into magnetosensitivity in humans.[31][32]

Monarch butterfly migration

Since 2002, Reppert and coworkers have pioneered the study of the biological basis of monarch butterfly migration.[33][34] Each fall, millions of monarchs from the eastern United States and southeastern Canada migrate as much as 4,000 km to overwinter in roosts in Central Mexico.[34] Monarch migration is not a learned activity, given that migrants flying south are at least two generations removed from the previous year's migrants.[35] Thus, migrating monarchs must have some genetically based navigational mechanism.

Reppert and colleagues have focused on a novel circadian clock mechanism and its role in time-compensated sun compass orientation, a major navigational strategy that butterflies use during their fall migration.[34] Using clock-shift experiments, they showed that the circadian clock must interact with the sun compass to enable migrants to maintain a southerly flight direction as the sun moves daily across the sky.[36]

Clockwork mechanism

The monarch clockwork model, which has both Drosophila-like and mammal-like aspects, is unique because it employs two distinct CRY proteins. As presented in a 2010 review paper,[34] the clock mechanism, on a gene/protein level, operates as follows:

Antennal clocks

Reppert’s lab expanded upon Fred Urquhart's postulation that antennae play a role in monarch migration. In 2009 Reppert’s lab reported that, despite previous assumptions that the time-compensation clocks are located exclusively in the brain, there are also clocks located in the antennae, which "are necessary for proper time-compensated sun compass orientation in migratory monarch butterflies.”[37] They concluded this by comparing the sun compass orientation of monarch migrants with intact antennae and those whose antennae had been removed.[37] Reppert's lab also studied antennae in vitro and found that antennal clocks can be directly entrained by light and can function independently from the brain.[37] Further research is needed, however, on the interaction between the circadian clocks in monarch butterfly's antennae and the sun compass in the brain.

In 2012, Reppert and colleagues determined that only a single antenna is sufficient for sun compass orientation. They did so by painting one antenna black to cause discordant light exposure between the two antennae; the single not-painted antenna was sufficient for orientation. All four clock genes (per, tim, cry1, and cry2) were expressed in the various studied areas of the antenna, suggesting that “light entrained circadian clocks are distributed throughout the length of the monarch butterfly antenna.” [38]

In 2013, Reppert and colleagues showed that spring remigrants also use an antenna-dependent time-compensated sun compass to direct their northward flight from Mexico to the southern United States.[39]

Sun compass

Using anatomical and electrophysiological studies of the monarch butterfly brain, Reppert and colleagues have indicated that the central complex, a midline structure in the central brain, is likely the site of the sun compass.[40]

Temperature

Reppert and coworkers showed that fall migrants prematurely exposed to overwintering-like coldness reverse their flight orientation to the north. The temperature microenvironment at the overwintering site is essential for successful completion of the migration cycle: without cold exposure, aged migrants continue to orient to the south. The discovery that coldness triggers the northward flight direction in spring remigrants underscores how vulnerable the migration may be to climate change.[41][42]

Monarch butterfly genome

In 2011, Reppert and colleagues presented the draft sequence of the monarch butterfly genome and a set 16,866 protein-coding genes. This is the first characterized genome of a butterfly and of a long-distance migratory species.[43][44][45]

In 2012, Reppert and colleagues established MonarchBase, an integrated database for the genome of Danaus plexippus. The goal of the project was to make genomic and proteomic information about monarch butterflies accessible to biological and lepidopteran communities.[46]

In 2013, Reppert and coworkers developed a novel gene-targeting approach in monarchs that uses a zinc finger nuclease strategy to define the essential nature of CRY2 for clockwork function in lepidopterans.[47] Targeted mutagenesis of Cry2 indeed resulted in the in vivo disruption of circadian behavior and the molecular clock mechanism. The ZFN strategy is a powerful tool for targeting additional clock genes in monarchs and other members of Lepidoptera.

References

  1. ↑ Pappenheimer, A.M. Jr (1995). "Carroll Milton Williams: December 2, 1916–October 11, 1991". NAS Biographical Memoirs 68: 413–434.
  2. ↑ Reppert, Steven M.; Tsai, Tony; Roca, Alfred L.; Sauman, Ivo (1994). "Cloning of a structural and functional homolog of the circadian clock gene period from the giant silkmoth antheraea pernyi". Neuron 13 (5): 1167–76. doi:10.1016/0896-6273(94)90054-X. PMID 7946353.
  3. ↑ Sauman, Ivo; Reppert, Steven M (1998). "Brain Control of Embryonic Circadian Rhythms in the Silkmoth Antheraea pernyi". Neuron 20 (4): 741–8. doi:10.1016/S0896-6273(00)81012-0. PMID 9581765.
  4. ↑ "Neuroscience@NIH". NIH. Retrieved April 24, 2013.
  5. ↑ http://www.umassmed.edu/neuroscience/faculty/reppert.cfm[]
  6. ↑ http://www.srbr.org/Pages/past_meetings.aspx[]
  7. ↑ http://www.aps-spr.org/spr/Awards/EMJ.htm
  8. ↑ Czech News Agency (ČTK). "Academy of Science awards American researcher Reppert". Prague Daily Monitor.
  9. ↑ "Neurobiolog Steven M. Reppert převzal čestný doktorát Jihočeské univerzity". University of South Bohemia.
  10. ↑ http://www.aaas.org/aboutaaas/fellows/[]
  11. ↑ "Thomson Reuters Web of Science: Citation Report, Reppert SM". Thomson Reuters. Retrieved May 24, 2013.
  12. ↑ http://www.patentbuddy.com/Inventor/Reppert-Steven-M/1437735[]
  13. ↑ Reppert, Steven M. (1985). "Maternal Entrainment of the Developing Orcadian Systema". Annals of the New York Academy of Sciences 453: 162–9. doi:10.1111/j.1749-6632.1985.tb11808.x. PMID 3865580.
  14. ↑ Klinkenborg, Verlyn. "Awakening to Sleep". New York Times.
  15. ↑ Davis, Fred C.; Reppert, Steven M. (2001). "Development of Mammalian Circadian Rhythms". Handbook of Behavioral Neurobiology. Handbook of Behavioral Neurobiology 12. pp. 247–90. doi:10.1007/978-1-4615-1201-1_10. ISBN 978-0-306-46504-8.
  16. ↑ Welsh, David K; Logothetis, Diomedes E; Meister, Markus; Reppert, Steven M (1995). "Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms". Neuron 14 (4): 697–706. doi:10.1016/0896-6273(95)90214-7. PMID 7718233.
  17. ↑ Shearman, Lauren P.; Zylka, Mark J.; Weaver, David R.; Kolakowski Jr, Lee F.; Reppert, Steven M. (1997). "Two period Homologs: Circadian Expression and Photic Regulation in the Suprachiasmatic Nuclei". Neuron 19 (6): 1261–9. doi:10.1016/S0896-6273(00)80417-1. PMID 9427249.
  18. 1 2 Zylka, Mark J; Shearman, Lauren P; Weaver, David R; Reppert, Steven M (1998). "Three period Homologs in Mammals: Differential Light Responses in the Suprachiasmatic Circadian Clock and Oscillating Transcripts Outside of Brain". Neuron 20 (6): 1103–10. doi:10.1016/S0896-6273(00)80492-4. PMID 9655499.
  19. ↑ Bae, Kiho; Jin, Xiaowei; Maywood, Elizabeth S.; Hastings, Michael H.; Reppert, Steven M.; Weaver, David R. (2001). "Differential Functions of mPer1, mPer2, and mPer3 in the SCN Circadian Clock". Neuron 30 (2): 525–36. doi:10.1016/S0896-6273(01)00302-6. PMID 11395012.
  20. 1 2 Kume, Kazuhiko; Zylka, Mark J; Sriram, Sathyanarayanan; Shearman, Lauren P; Weaver, David R; Jin, Xiaowei; Maywood, Elizabeth S; Hastings, Michael H; Reppert, Steven M (1999). "MCRY1 and mCRY2 Are Essential Components of the Negative Limb of the Circadian Clock Feedback Loop". Cell 98 (2): 193–205. doi:10.1016/S0092-8674(00)81014-4. PMID 10428031.
  21. ↑ Kreeger, Karen Young. "Collecting Clues to the Mammalian Clock". The Scientist.
  22. ↑ Shearman, L. P.; Sriram, S; Weaver, DR; Maywood, ES; Chaves, I; Zheng, B; Kume, K; Lee, CC; Van Der Horst, GT; Hastings, MH; Reppert, SM (2000). "Interacting Molecular Loops in the Mammalian Circadian Clock". Science 288 (5468): 1013–9. doi:10.1126/science.288.5468.1013. PMID 10807566.
  23. 1 2 Debruyne, Jason P; Weaver, David R; Reppert, Steven M (2007). "CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock". Nature Neuroscience 10 (5): 543–5. doi:10.1038/nn1884. PMC 2782643. PMID 17417633.
  24. 1 2 3 Reppert, Steven M.; Weaver, David R.; Ebisawa, Takashi (1994). "Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses". Neuron 13 (5): 1177–85. doi:10.1016/0896-6273(94)90055-8. PMID 7946354.
  25. ↑ Reppert, S. M.; Godson, C.; Mahle, C. D.; Weaver, D. R.; Slaugenhaupt, S. A.; Gusella, J. F. (1995). "Molecular characterization of a second melatonin receptor expressed in human retina and brain: The Mel1b melatonin receptor". Proceedings of the National Academy of Sciences 92 (19): 8734–8. doi:10.1073/pnas.92.19.8734. PMC 41041. PMID 7568007.
  26. ↑ Weaver, DR; Liu, C; Reppert, SM (1996). "Nature's knockout: The Mel1b receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamsters". Molecular Endocrinology 10 (11): 1478–87. doi:10.1210/me.10.11.1478. PMID 8923472.
  27. ↑ Zhu, Haisun; Yuan, Quan; Briscoe, Oren; Froy, Amy; Casselman, Steven M.; Reppert, SM (2005). "The two CRYs of the butterfly". Current Biology 15 (23): R953–4. doi:10.1016/j.cub.2005.11.030. PMID 16332522.
  28. ↑ Zhu, Haisun; Sauman, Ivo; Yuan, Quan; Casselman, Amy; Emery-Le, Myai; Emery, Patrick; Reppert, Steven M. (2008). "Cryptochromes Define a Novel Circadian Clock Mechanism in Monarch Butterflies That May Underlie Sun Compass Navigation". PLoS Biology 6: e4. doi:10.1371/journal.pbio.0060004. PMC 2174970. PMID 18184036.
  29. ↑ Yuan, Q.; Metterville, D.; Briscoe, A. D.; Reppert, S. M. (2007). "Insect Cryptochromes: Gene Duplication and Loss Define Diverse Ways to Construct Insect Circadian Clocks". Molecular Biology and Evolution 24 (4): 948–55. doi:10.1093/molbev/msm011. PMID 17244599.
  30. ↑ Gegear, Robert J.; Casselman, Amy; Waddell, Scott; Reppert, Steven M. (2008). "Cryptochrome mediates light-dependent magnetosensitivity in Drosophila". Nature 454 (7207): 1014–8. doi:10.1038/nature07183. PMC 2559964. PMID 18641630.
  31. ↑ Foley, Lauren E.; Gegear, Robert J.; Reppert, Steven M. (2011). "Human cryptochrome exhibits light-dependent magnetosensitivity". Nature Communications 2 (6): 356–. doi:10.1038/ncomms1364. PMC 3128388. PMID 21694704.
  32. ↑ Matt Ridley (February 1, 2013). "Insects that put Google Maps to shame". Wall Street Journal. Retrieved September 28, 2013.
  33. ↑ Kyriacou, Charalambos P (2009). "Clocks, cryptochromes and Monarch migrations". Journal of Biology 8 (6): 55. doi:10.1186/jbiol153. PMC 2737371. PMID 19591650.
  34. 1 2 3 4 Reppert, Steven M.; Gegear, Robert J.; Merlin, Christine (2010). "Navigational mechanisms of migrating monarch butterflies". Trends in Neurosciences 33 (9): 399–406. doi:10.1016/j.tins.2010.04.004. PMC 2929297. PMID 20627420.
  35. ↑ Brower, L (1996). "Monarch butterfly orientation: Missing pieces of a magnificent puzzle". The Journal of Experimental Biology 199 (1): 93–103. PMID 9317405.
  36. ↑ Froy, O.; Gotter, AL; Casselman, AL; Reppert, SM (2003). "Illuminating the Circadian Clock in Monarch Butterfly Migration". Science 300 (5623): 1303–5. doi:10.1126/science.1084874. PMID 12764200.
  37. 1 2 3 Merlin, C.; Gegear, R. J.; Reppert, S. M. (2009). "Antennal Circadian Clocks Coordinate Sun Compass Orientation in Migratory Monarch Butterflies". Science 325 (5948): 1700–4. doi:10.1126/science.1176221. PMC 2754321. PMID 19779201.
  38. ↑ Guerra, Patrick A.; Merlin, Christine; Gegear, Robert J.; Reppert, Steven M. (2012). "Discordant timing between antennae disrupts sun compass orientation in migratory monarch butterflies". Nature Communications 3 (7): 958. doi:10.1038/ncomms1965. PMID 22805565.
  39. ↑ Guerra, Patrick A.; Reppert, Steven M. (2013). "Coldness Triggers Northward Flight in Remigrant Monarch Butterflies". Current Biology 23 (5): 419–23. doi:10.1016/j.cub.2013.01.052. PMID 23434279.
  40. ↑ Heinze, Stanley; Florman, Jeremy; Asokaraj, Surainder; El Jundi, Basil; Reppert, Steven M. (2013). "Anatomical basis of sun compass navigation II: The neuronal composition of the central complex of the monarch butterfly". Journal of Comparative Neurology 521 (2): 267–98. doi:10.1002/cne.23214. PMID 22886450.
  41. ↑ Nayantara Narayanan; ClimateWire (February 22, 2013). "Climate change may disrupt monarch butterfly migration". Scientific American. Retrieved September 28, 2013.
  42. ↑ Meghan Rosen (March 23, 2013). "Chill turns monarchs north". ScienceNews. Retrieved September 28, 2013.
  43. ↑ Zhan, Shuai; Merlin, Christine; Boore, Jeffrey L.; Reppert, Steven M. (2011). "The Monarch Butterfly Genome Yields Insights into Long-Distance Migration". Cell 147 (5): 1171–85. doi:10.1016/j.cell.2011.09.052. PMC 3225893. PMID 22118469.
  44. ↑ Stensmyr, Marcus C.; Hansson, Bill S. (2011). "A Genome Befitting a Monarch". Cell 147 (5): 970–2. doi:10.1016/j.cell.2011.11.009. PMID 22118454.
  45. ↑ Johnson, Carolyn Y. (23 November 2011). "Monarch butterfly genome sequenced". The Boston Globe (Boston, MA). Retrieved 9 January 2012.
  46. ↑ Zhan, S.; Reppert, S. M. (2012). "MonarchBase: The monarch butterfly genome database". Nucleic Acids Research 41 (Database issue): D758–63. doi:10.1093/nar/gks1057. PMC 3531138. PMID 23143105.
  47. ↑ Merlin, C.; Beaver, L. E.; Taylor, O. R.; Wolfe, S. A.; Reppert, S. M. (2012). "Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases". Genome Research 23 (1): 159–68. doi:10.1101/gr.145599.112. PMC 3530676. PMID 23009861.

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