Science 2011

CONTENTS
EDITORIAL 567 Understanding the Human Brain NEWS OF THE WEEK

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Volume 334 Issue 6056
Sydney Brenner and Terrence J. Sejnowski
POLICY FORUM 603 Paying for Ecosystem Services Promise and Peril
A. P. Kinzig et al.
A roundup of the weeks top stories Panel Endorses Anthrax Vaccine Study in Children The Brains Social Network
>> Report p. 697
PERSPECTIVES
606
NEWS & ANALYSIS

577 578 579 581 582
A. D. Gitler >> Report p. 690

607
Another Reason to Exercise
Paradigm Shifts in Dye-Sensitized Solar Cells

M. D. McGehee >> Research Article p. 629
Psychologist Accused of Fraud on Astonishing Scale Large African TB Study Reveals Powerful Strategy to Curb Disease 12th International Congress of Human Genetics
Life on the Fertile Frontier X-tra Diversity for Africans Diabetes Genes Decline Out of Africa
608
M. Maroteaux and M. Mameli >> Report p. 693

610
Synaptic Switch and Social Status A Photothermoelectric Effect in Graphene Growth Signaling from Inside Ocean Effects of Blocking Running Out of Climate Space
page 584
D. Basko >> Report p. 648

611
H. Abrahamsen and H. Stenmark >> Report p. 678

612
NEWS FOCUS 584 Vigil at North Koreas Mount Doom

A Very Big Bang >> Science Podcast
T. Woollings >> Report p. 655

613
589
Sharp Insights and a Sharp Tongue
LETTERS 593 Conservation: Limits of Land Sparing

J. Fischer et al. K. Hayashi
R. Ohlemller >> Reports pp. 652 and 660
Conservation: Model Management Intensity
ESSAY 615 Eppendorf Winner: The Language of Dendrites

T. Branco
B. Phalan et al.
594 594
Response
REVIEWS
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page 600
TECHNICAL COMMENT ABSTRACTS CORRECTIONS AND CLARIFICATIONS
The Big and the Small: Challenges of Imaging the Brains Circuits
J. W. Lichtman and W. Denk V. M. Ho et al.
BOOKS ET AL. 598 The Copernican Question

600
623
The Cell Biology of Synaptic Plasticity CONTENTS continued >>
R. S. Westman, reviewed by P. Dear C. C. Mann, reviewed by J. Farmer
1493
COVER Conceptual illustration of information, in the form of electrical impulses, owing through neuronal processes within the brain. Fundamental aspects of neuroscience are based in the study of neurons and how they interact. An Editorial (p. 567) and several Reviews (pp. 618 and 623), Reports (pp. 690, 693, and 697), and Perspectives (pp. 606 and 608) highlight current and future approaches in neuroscience research.
Image: ktsimage/iStockphoto.com
DEPARTMENTS
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This Week in Science Editors Choice Science Staff New Products Science Careers
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RESEARCH ARTICLE
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Porphyrin-Sensitized Solar Cells with Cobalt (II/III)Based Redox Electrolyte Exceed 12 Percent Efciency
A. Yella et al. Simultaneous modication of the dye and redox shuttle boosts the efciency of a dye-sensitized solar cell. >> Perspective p. 607
B. Sandel et al. Regions with low glacial-interglacial climate-change velocity were essential refuges for many small-ranged species. >> Perspective p. 613; Report p. 652
664
The Inuence of Late Quaternary ClimateChange Velocity on Species Endemism
Long-Term Change in the Nitrogen Cycle of Tropical Forests
REPORTS
634
Structural Dynamics of a Catalytic Monolayer Probed by Ultrafast 2D IR Vibrational Echoes
P. Hietz et al. The ratio of stable isotopes in leaves and wood reveals an increase in nitrogen availability in Panamanian and Thai tropical forests.
666
D. E. Rosenfeld et al. A method to track fast vibrational motion in solution has been extended to catalytically important solid/liquid interfaces.
Neural Mechanisms for the Coordination of Duet Singing in Wrens

E. S. Fortune et al. The brains of duetting wrens encode the entire song and not just the contribution of the individual.
pages 610 & 648
639
A Fluoride-Derived Electrophilic Late-Stage Fluorination Reagent for PET Imaging
670
E. Lee et al. A palladium compound facilitates rapid incorporation of radioactive uoride into prospective tracers for medical imaging.
643
Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling
Ionic LiquidMediated Selective Conversion of CO2 to CO at Low Overpotentials
S. C. Shin et al. Successful development of fruit ies depends on a gut bacterium that interacts with its hosts insulin-signaling pathway.
674
B. A. Rosen et al. Carbon dioxide reduction reactions, a key step in creating fuels from this gas, can be achieved in an ionic liquid.
678
N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex
645
Wireless Solar Water Splitting Using Silicon-Based Semiconductors and Earth-Abundant Catalysts
D. C. Scott et al. Acetylation of an amino-terminal methionine is important for mediating specic protein-protein interactions.
S. Y. Reece et al. An articial water-splitting system was built using abundant materials and sunlight.
mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase
648
Hot CarrierAssisted Intrinsic Photoresponse in Graphene
CREDITS (TOP TO BOTTOM): NATHANIEL GABOR/MIT; MELISSA J. COLEMAN AND ERIC S. FORTUNE
N. M. Gabor et al. Photoexcited electrons in graphene remain thermally excited because they cannot transfer this energy to lattice vibrations. >> Perspective p. 610
R. Zoncu et al. Cellular sensing of amino acids occurs at the lysosome and is mediated by the vacuolar proton pump. >> Perspective p. 611
683
page 666
RNAP II CTD Phosphorylated on Threonine-4 Is Required for Histone mRNA 3 End Processing
652
The Pace of Shifting Climate in Marine and Terrestrial Ecosystems

M. T. Burrows et al. Ecologically relevant measures of contemporary global climate change can predict species distributions and vulnerabilities. >> Perspective p. 613; Report p. 660
686
J.-P. Hsin et al. Phosphorylation of a single conserved amino acid in higher eukaryotes plays a specic role in processing histone messenger RNA genes.
693
Bidirectional Control of Social Hierarchy by Synaptic Efcacy in Medial Prefrontal Cortex
Drosophila CENH3 Is Sufcient for Centromere Formation
655
Atmospheric Blocking and Atlantic Multidecadal Ocean Variability
M. J. Mendiburo et al. A specic histone is sufcient for the formation of a functional and heritable centromere in the fruit y.
690
F. Wang et al. Manipulation of the neural circuit determining social status allows subordinate mice to move up the hierarchy and dominant mice to go down. >> Perspective p. 608
697
S. Hkkinen et al. Changing ocean circulation patterns and sea surface temperatures affect atmospheric ow in the North Atlantic region. >> Perspective p. 612
Exercise and Genetic Rescue of SCA1 via the Transcriptional Repressor Capicua
J. D. Fryer et al. Gentle exercise can ameliorate disease severity in a mouse model of a fatal neurodegenerative disease. >> Perspective p. 606
J. Sallet et al. Executing social cognition successfully requires more brain power. >> News story p. 578; Science Podcast
Social Network Size Affects Neural Circuits in Macaques
CONTENTS continued >>
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SCIENCEONLINE
www.sciencexpress.org
SCIENCEXPRESS
SCIENCENOW
Fermi Detection of a Luminous -Ray Pulsar in a Globular Cluster
www.sciencenow.org Highlights From Our Daily News Coverage
RESEARCH ARTICLE: Lipotoxicity Causes Multisystem Organ Failure and Exacerbates Acute Pancreatitis in Obesity
The Fermi LAT Collaboration Contrary to expectations, the -rays from a distant cluster of stars are dominated by emission from a single neutron star. 10.1126/science.1207141
Happiness Associated With Longer Life
Among older people, upbeat moods could mean greater life span. http://scim.ag/_longlife
S. Navina et al. Unsaturated fatty acids cause lipotoxicity and mediate acute adverse outcomes in obese individuals with pancreatitis.
Cover Your Ears!
Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6
Musicologists learn what makes chalkboard screeches so painful. http://scim.ag/_chalkboard
RESEARCH ARTICLE: ALDH2 Activator Inhibits Increased Myocardial Infarction Injury by Nitroglycerin Tolerance
S. Klinge et al. The 3.5 angstromresolution structure provides insights into the architecture of the eukaryotic ribosome and its regulation. 10.1126/science.1211204
The Case of the Haunted Golf Club
Does the spirit of a previous owner of sports equipment carry over to new users? http://scim.ag/_golfclub
L. Sun et al. Tolerance to nitroglycerin, a drug often used to treat chest pain, increases susceptibility to injury by myocardial infarction.
SCIENCECAREERS
Entorhinal Cortex Layer III Input to the Hippocampus Is Crucial for Temporal Association Memory
SCIENCESIGNALING
J. Suh et al. A specic neural circuit integrates temporally dispersed stimuli into a coherent memory episode. 10.1126/science.1210125
www.sciencesignaling.org The Signal Transduction Knowledge Environment 1 November issue: http://scim.ag/ss110111
www.sciencecareers.org/career_magazine Free Career Resources for Scientists
Taken for Granted: Getting to Aaahhh!
RESEARCH ARTICLE: p53 and MicroRNA-34 Are Suppressors of Canonical Wnt Signaling
Inhibition of Pyruvate Kinase M2 by Reactive Oxygen Species Contributes to Cellular Antioxidant Responses
N. H. Kim et al. The tumor suppressor p53 activates miRNA-34 to inhibit Wnt signaling and colorectal cancer cell invasiveness.
B. L. Benderly A former postdoc nds a deeply satisfying career teaching high school. http://scim.ag/tfg_teaching
Conquering Information Overload
D. Anastasiou et al. The glycolytic metabolism of cancers differs from normal tissues, allowing tumor cells to survive under oxidative stress. 10.1126/science.1211485
RESEARCH ARTICLE: A CC Loop Decoy Peptide Blocks the Interaction Between Act1 and IL-17RA to Attenuate IL-17 and IL-25Induced Inammation
C. Liu et al.
S. A. Holgate How can you keep from drowning in the sea of information your work depends on? http://scim.ag/info_overload
In Person: At Summer Davos
Experimental Evolution of Reduced Sex Ratio Adjustment Under Local Mate Competition
PODCAST
E. Macke et al. Tests of Hamiltons theory on a spider mite show that populations evolve with less phenotypic plasticity in their sex ratio. 10.1126/science.1212177
X. Li and A. M. VanHook A cell-permeable peptide derived from an adaptor protein blocks cytokine receptor signaling and reduces inammation.
M. Anand At a world summit, a young scientist recognizes the importance of engaging real-world problems and the wider community. http://scim.ag/Summer_Davos
SCIENCEPODCAST
Deep Human Genealogies Reveal a Selective Advantage to Be on an Expanding Wave Front

C. Moreau et al. Women in the vanguard of range expansions, such as the European settlement of Quebec, married young and had more offspring. 10.1126/science.1212880 >> Science Podcast
RESEARCH ARTICLE: Persistent Stimulation with Interleukin-17 Desensitizes Cells Through SCF -TrCP-Mediated Degradation of Act1
P. Shi et al. Persistent inammation may be prevented by degradation of an adaptor protein required for inammatory cytokine receptor signaling.
www.sciencemag.org/multimedia/podcast Free Weekly Show On the 4 November Science Podcast: social networks and brain structure, fertility at the range front, keeping vigil at Changbai-Paektu volcano, and more.
SCIENCEINSIDER
Editorial Expression of Concern on Stapel and Lindenberg Report

B. Alberts 10.1126/science.1216027
www.sciencetranslationalmedicine.org Integrating Medicine and Science 2 November issue: http://scim.ag/stm110211
SCIENCETRANSLATIONAL MEDICINE
news.sciencemag.org/scienceinsider Science Policy News and Analysis
FOCUS: Epstein-Barr VirusAn Important Vaccine Target for Cancer Prevention
TECHNICALCOMMENTS Comment on Atmospheric Pco2 Perturbations Associated with the Central Atlantic Magmatic Province
M. R. Rampino and K. Caldeira Full text at www.sciencemag.org/cgi/content/ full/334/6056/594-b
J. I. Cohen et al. Epstein-Barr virus vaccine research should focus on the prevention of EBV-associated cancers and infectious mononucleosis.
PERSPECTIVE: Epigenetics of Nicotine Another Nail in the Coughing
SCIENCE (ISSN 0036-8075) is published weekly on Friday, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Periodicals Mail postage (publication No. 484460) paid at Washington, DC, and additional mailing ofces. Copyright 2011 by the American Association for the Advancement of Science. The title SCIENCE is a registered trademark of the AAAS. Domestic individual membership and subscription (51 issues): $149 ($74 allocated to subscription). Domestic institutional subscription (51 issues): $990; Foreign postage extra: Mexico, Caribbean (surface mail) $55; other countries (air assist delivery) $85. First class, airmail, student, and emeritus rates on request. Canadian rates with GST available upon request, GST #1254 88122. Publications Mail Agreement Number 1069624. Printed in the U.S.A. Change of address: Allow 4 weeks, giving old and new addresses and 8-digit account number. Postmaster: Send change of address to AAAS, P.O. Box 96178, Washington, DC 200906178. Single-copy sales: $10.00 current issue, $15.00 back issue prepaid includes surface postage; bulk rates on request. Authorization to photocopy material for internal or personal use under circumstances not falling within the fair use provisions of the Copyright Act is granted by AAAS to libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that $25.00 per article is paid directly to CCC, 222 Rosewood Drive, Danvers, MA 01923. The identication code for Science is 0036-8075. Science is indexed in the Readers Guide to Periodical Literature and in several specialized indexes.
N. D. Volkow Chronic nicotine exposure rewired the brains of rodents and enhanced their behavioral response to cocaine.
Response to Comment on Atmospheric Pco2 Perturbations Associated with the Central Atlantic Magmatic Province
M. F. Schaller et al. Full text at www.sciencemag.org/cgi/content/ full/334/6056/594-c
RESEARCH ARTICLE: Molecular Mechanism for a Gateway DrugEpigenetic Changes Initiated by Nicotine Prime Gene Expression by Cocaine
A. Levine et al. Nicotine changes the way brain cells respond to cocaine, possibly explaining how smoking acts as a gateway for cocaine abuse.
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Electrons Staying Hot

A silicon photovoltatic cell can produce a voltage because once electrons are photoexcited, the voltage bias created by the interface of positive and negative silicon (a p-n junction) draws these electrons in only one direction. Gabor et al. (p. 648, published online 6 October; see the Perspective by Basko) found that single- and double-layer graphene in a p-n junction generated a voltage not through the photovoltaic effect, but through a competing mechanism, whereby the excited electrons in graphene remained hotter than the rest of device, driving a photothermoelectric effect.
The Domineering Brain and Its Synapses

Social hierarchy is a fundamental organizing principle in many animal societies. The social status of an individual strongly affects its health and quality of life. Yet the underlying mechanisms determining social hierarchical status are unclear. Wang et al. (p. 693, published online 29 September; see the Perspective by Maroteaux and Mameli) determined the social hierarchy within groups of mice by using multiple behavioral tests and discovered that the social hierarchical status of an individual correlated with the synaptic strength in medial prefrontal cortical neurons. Furthermore, the hierarchical status of mice could be changed from dominant to subordinate, or vice versa, by manipulating the strength of synapses in the medial prefrontal cortex.
Echoes from the Surface

Vibrational echo spectroscopy can track rapid changes in the distribution of vibrations across a set of tumbling molecules. The technique has been used in solution-phase analysis, and Rosenfeld et al. (p. 634, published online 20 October) now extend it to the study of solidliquid interfaces, a more complex environment that requires correspondingly higher sensitivity. A layer of metal complexes tethered to a surfacea motif widely applied in catalysis was probed, which revealed distinctions in conformational dynamics depending on whether the surface was exposed or submerged in solvent.
Seeing Is Believing
Advances in neuroscience have often relied on advances in microscopy. Lichtman and Denk (p. 618) review how recent advances in microscopy have helped to elucidate the relationship between the structure of the nervous system and its function. Within the brain, synaptic plasticitythe experience-dependent change in connectivity between neuronsis thought to underlie learning and memory. Ho et al. (p. 623) review the cellular and molecular processes that are altered when a neuron responds to external stimuli, and how these alterations lead to an increase or decrease in synaptic connectivity.
CREDITS (TOP TO BOTTOM): FEI WANG, JUN ZHU, TAO ZHOU, AND HAILAN HU; ROSENFELD ET AL.
range of insights afforded by the technique, but it is a substantial synthetic challenge to append the unstable isotope to most molecular frameworks before its activity decays below the necessary threshold. Lee et al. (p. 639) devised a pair of palladium complexes that can rapidly incorporate uorine substituents into aromatic compounds. A particular advantage of the system is that it uses uoride anions to provide the unstable isotope: Fluoride anions are available in streams with more concentrated activity than reagents derived from elemental uorine.
Splitting CO2 and Water

It is possible to mimic photosynthesis in an electrochemical cell by using an external power source to induce electron transfer from water to CO2, but the instability of the initially formed CO2 anion requires a high applied driving potential. Rosen et al. (p. 643, published online 29 September) found that carrying out the CO2 reduction in an ionic liquid electrolyte substantially lowered the required potential, presumably by stabilizing the anion through complexation. Many solar cell devices can split water to generate hydrogen. However, large-scale implementation of such devices has been precluded by their need for noble metal catalysts and, in some cases, for highly alkaline or acidic electrolytes. Reece et al. (p. 645, published online 29 September) assembled thin lms of a cobalt phosphate oxygen reduction catalyst on the indium tin oxide surface of a commercially available amorphous silicon cell. A nickel-molybdenum-zinc alloy was used as a hydrogen-evolving catalyst to produce a device in which more than 60% of the output was able to drive water splitting.
Dye-namic Improvement
In a dye-sensitized solar cell, a molecular dye injects a charge into a semiconductor upon light absorption, and then a charge acceptor on the other side of the circuit shuttles it back around. The most common implementation of the cell has employed a ruthenium complex as a dye and an equilibrating system of iodide and triiodide ions as the charge shuttle. Yella et al. (p. 629; see the Perspective by McGehee) now show that the combination of a zinc porphyrin derived dye and cobalt ion shuttle substantially boosts efciency.
The Warm and the Cold of It
Rushing in Fluoride
Positron emission tomography relies on the rapid decay of radioactive isotopes (most commonly, uorine-18) embedded in tracer molecules to image biological environments. Increasing the diversity of tracer structures could expand the
In atmospheric blocking, a stationary atmospheric pressure eld impedes normal atmospheric circulation over a large region. In Europe, blocking conditions over the North Atlantic Ocean can persist for as long as 2 weeks and cause cold wintertime temperatures, as well as other weather anomalies. Hkkinen et al. (p. 655; see the Perspective by Woollings) reanalyzed 20th-century atmospheric data and found that winters with more frequent blocking in the North Atlantic region tended to persist for decades, mainly during periods in which the North Atlantic Ocean was relatively warm. These periods of warm North Atlantic surface waters were also related to wind and ocean circulation patterns and occurred in phase with the dominant mode of Atlantic multidecadal ocean variability.
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This Week in Science

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Nitrogen in Tropical Forest

Pollution from nitrogen deposition is well documented in temperate ecosystems and it has been predicted that the globalization of nitrogen deposition will eventually lead to its enrichment in tropical forests. Hietz et al. (p. 664) report a comprehensive data set showing a clear change in the nitrogen cycle in two tropical forestsone in Panama and one in Thailand. The data set provides evidence for nitrogen enrichment from stable isotope analysis of leaves collected for more than 40 years and of tree rings spanning approximately 100 years. Spatial data on nitrogen emissions and tropospheric NO2 suggest that the type of enrichment seen may be widespread in tropical forests.
Common Knowledge
Cooperative behaviors, such as dancing the tango or singing a duet, require coordination between sensory output and feedback in both participating individuals. Fortune et al. (p. 666) recorded duetting behavior in singing plain-tailed wrens and made extracellular neural recordings from the song centers of their brains. Both singers encoded the combined cooperative duet, but the timing of the singing was established by a single partner, the female. Thus, the entire duet is encoded in each individuals brain, not just by their own contribution.
Fly Guts
The microorganisms that live within animals guts are important for regulating many aspects of their hosts physiology, including immune responses, energy metabolism, development, and growth. The relatively simple gut microbiota of the fruit y Drosophila are particularly important in nutrient-poor conditions. Shin et al. (p. 670) identied a single bacterial member of the fruit ys gut community (Acetobacter pomorum) that could replace the ys vemember gut ora. The function of one bacterial gene product required for the oxidation of ethanolpyrroloquinoline quininedependent alcohol dehydrogenasewas key to the metabolic generation of acetic acid and for successful development of y larvae.
More Than a Proton Pump

When cells are running low of amino acids, they can activate autophagy to digest existing cellular components. But it has been unclear how the depletion of amino acids is sensed. The mTOR complex 1 (mTORC1) is a key regulator of this process and, when amino acids are present, becomes localized to lysosomes and inhibits autophagy. Zoncu et al. (p. 678; see the Perspective by Abrahamsen and Stenmark) used small interfering RNA techniques to search for lysosomal components necessary for mTORC1 signaling. The vacuolar H+adenosine triphosphatase, which functions to acidify lysosomes, was found also to be necessary for sensing of amino acids within the lysosome. The protein directly interacted in an amino aciddependent manner with proteins associated with the mTORC1 complex.
CREDIT (TOP AND BOTTOM): SHIN ET AL.
Exercise for Life

An unresolved issue in the eld of neurodegenerative diseases is whether exercise would have longlasting benecial effects or whether it would have deleterious long-term consequences by increasing the metabolic demands on already susceptible neuronal populations. Fryer et al. (p. 690; see the Perspective by Gitler) now show that exercise signicantly extended the life span of a mouse model for the polyglutamine neurodegenerative disorder spinocerebellar ataxia type 1 (SCA1). Exercise upregulated epidermal growth factor which caused down-regulation of Capicua (Cic), a transcriptional repressor that interacts with Ataxin-1 in vivo. In Cic mutant mice, all SCA1 phenotypes were rescued, including premature lethality, by reducing the level of Cic by 50%. www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011
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EDITORIAL
Understanding the Human Brain

Sydney Brenner is a professor at the Crick Jacobs Center for Theoretical and Computational Biology, Salk Institute for Biological Studies, La Jolla, CA.
LIKE MOST FIELDS IN BIOLOGY, NEUROSCIENCE IS SUCCUMBING TO AN EPIDOMIC OF DATA
Terrence J. Sejnowski is a Howard Hughes Medical Institute Investigator at the Salk Institute for Biological Studies, La Jolla, CA, and a professor in the Department of Biological Sciences at the University of California at San Diego, La Jolla, CA. E-mail: terry@salk.edu.
CREDITS: (LEFT TOP AND BOTTOM) THE SALK INSTITUTE; (RIGHT) ISTOCKPHOTO
collecting. There are major projects under way to completely characterize the proteomic, metabolomic, genomic, and methylomic signatures for all of the different types of neurons and glial cells in the human brain. In addition, connectomics plans to provide the complete network structure of brains, and synaptomics aims to uncover all molecules and their interactions at synapses. This is a good time to pause and ask ourselves what we expect to nd at the end of this immense omic brainbow. Linnaeuss catalog of species and the classications he imposed on them turned data into knowledge, but it did not lead to an understanding of why they were all there. That had to wait for Darwins theory of evolution and the development of genetics. All the lists that we will accumulate about the brain, although necessary, will be far from sufcient for understanding. The human brain contains an estimated 86 billion neurons and an equal number of glial cells. The complete structure of the enormously simpler 302-neuron network of the nematode worm Caenorhabditis elegans was published in 1986. But without the activities of neurons and their synapses, it was far from a complete wiring diagram. Today, with genetically encoded calcium sensors, with better knowledge of the molecules present at synapses, and by integrating the omic catalogs with developmental and dynamical data, we may nally be in sight of completing the worm wiring diagram, as required for a full understanding of this one relatively simple nervous system. The challenges to understanding the human brain are immense, and neuroscientists will require powerful technologies to meet them. Fortunately, a revolution in optical microscopy, driven by striking advances in molecular labeling and digital processing, has given us a new window into the inner lives of cells. The ability to see with unprecedented resolution the locations and trafcking of molecules and the dynamical organization of synapses has revealed extraordinarily complex control systems. The biochemical environment inside synapses is seldom in equilibrium and compartments are not well mixed, so stopped-ow biochemistry and new computational tools from physics such as Monte Carlo models will be needed. Electrical recordings from one neuron at a time deep in the brain can give us clues to what information has passed through a brain area. But neurons are not independent, interacting with each other and the world on a wide range of time scales. New techniques have been developed to simultaneously record from and manipulate many neurons in several brain areas that can give us a picture of how interacting neural populations give rise to behavior. Since the 1980s, neuroscience has received visionary nancial support from private foundations, jump-starting new elds including cognitive neuroscience and computational neuroscience based on new techniques for imaging and modeling the human brain. The global view of human brain activity thus far obtained from imaging experiments has changed the way we think about ourselves. Homo neuroeconomicus has replaced the rational-agent model of human behavior, neuroeducators want to make children better learners, and neuroethicists have been inspired by the discovery of biological links to aggression, trust, and afliation. However, individual differences often dominate. Although expensive bets are being placed on explaining the diversity of human behavior and mental disorders with genetic polymorphisms, gene mutations, and chromosomal rearrangements, the results so far have been modest. The Internet is making neuroscience more accessible to the public. The Society for Neuroscience, which convenes its annual meeting next week, will soon launch BrainFacts.org, a reliable source of information about the brain. In-depth interviews with neuroscientists can be found online at thesciencenetwork.org. And a Neuroeducation X Prize is being planned to encourage innovation in online computer games that enhance cognitive skills. Let us celebrate what our brains have discovered and what they can tell us about themselves.
10.1126/science.1215674
Sydney Brenner and Terrence J. Sejnowski
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EDITORSCHOICE
EDITED BY KRISTEN MUELLER AND JAKE YESTON
ECOLOGY
The Enemy of My Enemy Is My?

The factors permitting the coexistence of large numbers of plant species in tropical forests remain a key focus of ecologists. An established mechanism for limiting the abundance of individual tree species is density-dependent predation, whereby specialist natural enemiesespecially insect herbivorescongregate where concentrations of their preferred seeds or seedlings are high, typically close to adult reproductive trees. This process, however, which confers an advantage on rarer tree species, can be complicated by the activities of the enemies enemies. Visser et al. studied a tri-trophic interaction in a Panamanian rainforest, between a palm, its predator beetles (right, emerging from a palm seed), and the rodent predators of the beetle. As expected, infestation of palm seeds by beetle larvae increased close to adult plants, but the density-dependent effects were negated by preferential predation by squirrels on beetle-infested seeds. Such topdown control of seed predators, if repeated across other large-seeded tropical tree species, would add new layers of complexity to the conundrum of multiple species coexistence in tropical forests. AMS
Ecol. Lett. 14, 1093 (2011).
CHEMISTRY
More Mass in One Pass

Mass spectrometric imaging of complex surfaces can be achieved with ion microscopy. The analytes of different masses are usually separated by time of ight, but many detection schemes are too slow to image several masses simultaneously for the same desorption pulse from the surface. Jungmann et al. were able to achieve multiplemass imaging by intercepting the ions with a chevron array microchannel plate to generate a pixelated electron signal for the detected region. The electrons were detected with a Timepix chip derived from detectors that are used in high-energy particle detection, allowing a much greater dynamic range of signal intensity than conventional detection. The authors were able to use this instrument to measure multiple mass signals for peptides and proteins, with a range up to 78 kD. PDS
Anal. Chem. 83, 7888 (2011).
CHEMISTRY
designed with specic pore size and composition. As such, they have found use for gas adsorption, separation, and in some cases catalysis. Song et al. show that the Keggin-type POM [CuPW11O39]5 can t snugly into the pores of MOF-199, leading to a strong enhancement of
conditions but with lower turnover as the pores were blocked by sulfur accumulation. Tests with a noncopper-containing POM did not show catalytic activity, indicating that the Cu centers in the POM unit are probably the active sites. Air-based oxidations of thiols to disuldes showed excellent selectivity, although decreasing yield as the size of the thiol substituents increased, leading to steric hindrance within the MOF. MSL
J. Am. Chem. Soc. 133, 10.1021/ ja203695h (2011).
MOLECULAR BIOLOGY
CREDITS: (TOP) CHRISTIAN ZIEGLER; (RIGHT) SONG ET AL., J. AM. CHEM. SOC. 133, 10.1021/JA203695H (2011)
Unmethylating RNA
There is much excitement about the role of the Tet enzymes in the active demethylation of eukaryotic DNA, especially as DNA methyaltion plays such a central role in gene regulation and genome stability. The role of methylation in eukaryotic RNA is more enigmatic: Here, the dominant modication is the conversion of adenosine (A) to N6-methyadenosine (m6A). The fat massandobesityassociated (FTO) gene is involved in energy homeostasis, and variants in the human FTO gene are correlated with body mass. FTO has been associated with a number of enzymatic activities, including demethylation of the methylated bases m3T and m3U in single-stranded (ss) DNA and ssRNA. In vitro, Jia et al. now show that whereas FTO
Continued on page 571
A Marriage of POM and MOF

Polyoxometalates (POMs) are clusters, typically bearing a net negative charge, that assemble from multiple metal oxide units sharing oxygens and have shown useful catalytic activity for a range of organic oxidation reactions. Metal organic framework (MOF) materials are threedimensional porous structures that can be
catalytic properties for the oxidation of thiols to disuldes and the removal of hydrogen sulde. Analysis of the unit cells shows that the MOF remains intact after inclusion of the POM and associated (tetramethyl)ammonium counter-ion into adjacent large and small pores, and that 50% of the large pores remain empty, allowing for easy movement of reactants and products. Aerobic H2S oxidation was achieved from aqueous solutions at a rate of 4000 turnovers within 20 hours and was also observed under gas-phase SCIENCE VOL 334
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can indeed demethylate m3U, it is much more effective at converting m6A to A in RNA oligomers, including the sequence of a known m6A modication site. FTO had a similar effect in vivo, reducing or increasing the level of m6A in whole-cell RNA when overexpressed or knocked down, respectively. In the nucleus, FTO associated specically with speckles that contain RNA splicing factors and RNA polymerase II. Although its knockdown does not seem to affect assembly of the spliceosome, the authors suggest that FTO might yet be part of a regulatory system with parallels to DNA and histone methylation. GR
Nat. Chem. Biol. 7, 10.1038/ NCHEMBIO.687 (2011).
NEUROSCIENCE
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of humans as compared to the mouse. Furthermore, by proling 13 developing brain regions, the authors observed that many of these genes were more highly expressed in the developing neocortex, one of the most expanded regions of the human brain relative to those of other primates. On the basis of these results, the authors suggest that changes in the regulation of young genes during neural development may have been responsible for the evolutionary changes that account for our large brain size. LMZ
PLoS Biol. 9, e1001179 (2011).
CHEMISTRY
Same, But Different

Cephalopods, such as octopus and cuttlesh, are the most advanced of the invertebrates. Their nervous system is as large as that of a dog, and their behavior is complex and sophisticated. Yet the organization of their brain is much simpler than that of vertebrates. Shomrat et al. characterized and compared the memory and learning networks in the large vertical lobe of the octopus and the cuttlesh to determine whether the physiology of neuronal networks is constrained by computational considerations. Both systems consisted of a simple two-layered network with a computationally typical fan-outfan-in organization. The connectivity pattern was also the same in both animals: The rst synaptic connection was glutamatergic and the second cholinergic; however, the synaptic sites of short- and long-term plasticity and neuromodulation were different. In the octopus, the rst fan-out connection showed short- and long-term plasticity, whereas in the cuttlesh it was the second fan-in connection. These ndings may have implications for our understanding of the evolution of microcircuits in the nervous system. The same structures produce similar functions via distinct mechanisms, presumably shaped by evolutionary pressures. PRS
Curr. Biol. 21, 1 (2011).
EVOLUTION
Product Placement
Asymmetric catalysis relies on a chiral agent to bias the stereochemical outcome of a reaction. If the desired product has just one chiral center, it is usually straightforward to prepare either enantiomer by using the appropriate enantiomer of catalyst (though in cases where the catalyst is a scantly modied a natural product, it may be moderately more expensive or time-consuming to prepare its unnatural isomer). Reactions that generate two chiral centers in one product pose more of a conundrum though. There is no obvious means of modifying a catalyst to modulate the relative sense of those two centers; in some cases, it may be necessary to pursue entirely different approaches to bond construction, let alone catalyst design, in order to obtain each distinct diastereomer. Tian et al. present a rare instance in which fairly simple modications to the reaction conditions switch the diastereoselectivity of a single catalyst. Depending on the solvent and acid cocatalyst, their quinuclidinederived organocatalyst directs thiols to add in either syn or anti disposition with respect to the -alkyl substituent in ,-unsaturated ketones. Both pathways proceed with high enantioselectivity. JSY
J. Am. Chem. Soc. 133, 10.1021/ ja207847p (2011).
One more data point on why you should spend more time at membercentral.aaas.org. There you can enjoy a feast of blogs, videos, webinars, discounts, and downloads created by and for the most insatiable brains around.
Building Bigger Brains

Relative to those of other mammals, the human brain is exceptionally large, although how such a large size evolved is a mystery. Zhang et al. investigated differences in genes that are expressed in developing and adult brains in humans and in mice through gene expression proling. They found an excess of evolutionarily new genes (primate-specic), of diverse functional types, expressed in the developing brains www.sciencemag.org SCIENCE
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SENIOR EDITORIAL BOARD
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BOARD OF REVIEWING EDITORS
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Jens Rostrup-Nielsen, Haldor Topsoe Edward M. Rubin, Lawrence Berkeley National Lab Mike Ryan, Univ. of Texas, Austin Shimon Sakaguchi, Kyoto Univ. Miquel Salmeron, Lawrence Berkeley National Lab Jrgen Sandkhler, Medical Univ. of Vienna Randy Seeley, Univ. of Cincinnati Vladimir Shalaev, Purdue Univ. Joseph Silk, Univ. of Oxford Denis Simon, Univ. of Oregon Alison Smith, John Innes Centre Davor Solter, Inst. of Medical Biology, Singapore John Speakman, Univ. of Aberdeen Allan C. Spradling, Carnegie Institution of Washington Jonathan Sprent, Garvan Inst. of Medical Research Elsbeth Stern, ETH Zrich Ira Tabas, Columbia Univ. Yoshiko Takahashi, Nara Inst. of Science and Technology John Thomas, Duke Univ. Herbert Virgin, Washington Univ. Bert Vogelstein, Johns Hopkins Univ. Cynthia Volkert, Univ. of Gottingen Bruce D. Walker, Harvard Medical School Douglas Wallace, Leibniz Inst. of Marine Sciences Ian Walmsley, Univ. of Oxford David A. Wardle, Swedish Univ. of Agric Sciences David Waxman, Fudan Univ. Detlef Weigel, Max Planck Inst., Tbingen Jonathan Weissman, Univ. of California, San Francisco Sue Wessler, Univ. of California, Riverside Ian A. Wilson, The Scripps Res. Inst. Timothy D. Wilson, Univ. of Virginia Jan Zaanen, Leiden Univ. Kenneth Zaret, Univ. of Penn. School of Medicine Mayana Zatz, University of Sao Paolo Jonathan Zehr, Univ. of California, Santa Cruz Huda Zoghbi, Baylor College of Medicine Maria Zuber, MIT
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NEWS OF THE WEEK
AROUND THE WORLD

2 4 5 1 3
Ready to teach. The Okinawa Institute of Science and Techology.
Okinawa, Japan 3
Okinawa Institute Ofcially a Graduate University
Klong Luang, Pathum Thani, and Bangkok, Thailand 1
Thai Floods Hit Research Infrastructure
Thailands ooding is taking its toll on the countrys research institutes and universities. The campus of the Asian Institute of Technology in Pathum Thani province, 30 kilometers north of Bangkok, is now completely inundated under ood waters, according to the institutes Web site. A Thammasat University campus suffered similar damage while the adjacent Thailand Science Park, home to four national research institutes and 60 private labs, fared
Underwater. Flooding at the Thailand Science Park.
research and services on several [levels], says Pakit Vichyanond, deputy dean of international affairs at Mahidols Faculty of Medicine Siriraj Hospital in central Bangkok. Speculation on whether the worst is now pastas the countrys Flood Relief Operation Command told the local pressis very disputable, Vichyanond says. Milan, Italy 2
Court Endorses Vatican Rescue Of Research Center
New York, New York 4
University Teams Bid for NYC Research Campus
better. Water covers parts of the grounds but spared the buildings. Once ood waters recede, researchers should be able to quickly resume normal activities, says Noppawan Tanpipat, vice president of Thailands National Science and Technology Development Agency. Closer to Bangkok, last week Mahidol University closed its Salaya Campus, which hosts the engineering and environment departments as well as administrative ofces. Undoubtedly, this affects teaching,
http://scim.ag/SanRaffaele VOL 334
New York City received bids from at least ve university-based teams on 28 October to build a science and engineering center in the city. In July, Mayor Michael Bloomberg announced that he would put up land and $100 million in seed money for universities and corporations willing to invest in a facility aimed at turning the metropolis into a high-tech hub similar to Silicon Valley. An economic analysis conducted earlier this year suggested that such an institute could generate as many as 30,000 jobs in the city.
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The nancially troubled San Raffaele del Monte Tabor Foundation, which funds and oversees one of Italys most prestigious private biomedical research centers, may have gained a new lease on life. Last week, an Italian bankruptcy court approved an offer made by the Institute for Works of Religion, commonly known as the Vatican Bank, and Italian entrepreneur Victor Malacalza to rescue the foundation, which has reportedly accumulated close to 1.5 billion in debt due to poor nancial decisions and overly rapid expansion. Scientists at the foundations San Raffaele Institute hope the bankruptcy courts endorsement of the offer, in which the Vatican and Malacalza would take over 500 million of debt and invest 250 million more, will persuade funding bodies to resume now-frozen payments and stem a potential exodus of researchers from the institute. We have nothing to do with the money hole and San Raffaele still represents a center of excellence, says immunologist Maria Grazia Roncarolo, the institutes scientic director. The foundations creditors will next review details of the plan.
A Japanese graduate school backed by Nobel laureates Sydney Brenner, Susumu Tonegawa, Jerome Friedman, and others has cleared the last hurdle required to start teaching. On 28 October, Japans cabinet approved the law formally recognizing the Okinawa Institute of Science and Technology (OIST) Graduate University. The school will welcome its rst batch of students in September 2012. OIST supporters, including domestic politicians and scientists, want to shake up Japans universities by creating a new academic model emphasizing interdisciplinary research. OIST is also attempting to attract non-Japanese faculty members by using English for teaching and administrative affairs. The OIST Graduate University will start its academic year in September, instead of in April as is Japanese custom, to be more in sync with international norms. Jonathan Dorfan, a physicist and former director of what is now the SLAC National Accelerator Laboratory in Menlo Park, California, ofcially becomes the universitys rst president on 1 November, but has been president-elect since July 2010, overseeing the development of curriculum and the push to complete faculty recruitment.
NEWS
Eighteen partnerships representing 27 public and private groups initially expressed interest. At least ve groups have submitted a nal proposal, including three teams led by New York statebased private institutions Cornell, Columbia, and New York universities. All have partners from either academia or industry. The other two groups are led by Stanford University in Palo Alto, California, and Carnegie Mellon University in Pittsburgh, Pennsylvania, which is also partnering with New York University in its proposal. Bloomberg is expected to announce his pick by early 2012 and has said he hopes the winner will break ground on the new facility before he leaves ofce in 2013. Oak Flat, Arizona 5 Breakout Labs is the brainchild of Peter Thiel, a co-founder of PayPal and an early investor in Facebook. Thiel wants to fund revolutionary science by Thiel do-it-yourself scientists and those with start-up companies that arent far enough along to attract venture capital. Independent researchers can ll out a 10-page application at the site breakoutlabs. org. Academic researchers can apply, but must be working on the project outside the connes of a traditional university research setup, says program founder and executive director, Lindy Fishburne. The foundation hopes to make 10 to 20 awards in the rst year, ranging from around $50,000 to $350,000. The grantees must publish in open access journals. They also must agree to help support more projects, either by sharing a small portion of future
THEY SAID IT This modest-sized icy dirtballs inuence upon our planet is so incredibly minuscule that my subcompact automobile exerts a greater gravitational inuence on Earth than the comet ever would.
NASA planetary scientist Don Yeomans puzzling over why comet Elenin became the topic of doomsday prophesies last year. Elenin, which had its closest approach to Earth on 16 October, recently broke up into a stream of debris. royalties with Breakout Labs or by assigning intellectual property to the program in exchange for keeping some royalties.
Archaeology Groups Oppose Proposed Arizona Land Swap
CREDITS (TOP TO BOTTOM): YURI GRIPAS/REUTERS/NEWSCOM; NASA/BILL INGALLS
Archaeology groups are lining up against a proposal, approved 26 October by the U.S. House of Representatives, to give a copper mining company a large chunk of federal land in Arizona in exchange for private lands. The proposal would swap about 971 hectares of U.S. Forest Service land thought to sit atop a vast deposit of highquality copper for about 2144 hectares of private holdings, including 1214 hectares of ecologically important land along the lower San Pedro River. The trade would be a blatant giveaway of the nations public land to a single private stakeholder and would set a dangerous precedent because it wouldnt get normal prior review under environmental and historic preservation laws, William F. Limp, president of the Society for American Archaeology (SAA) in Washington, D.C., and colleagues argue in a 24 October letter to lawmakers signed by eight archaeology and preservation groups. That means no assurances that priceless historic and cultural resources will be protected, they say. Although the House approved the bill, the Senate has yet to take up a companion measure. http://scim.ag/AZlandswap
Earth-Observing Satellite Makes It Into Orbit

Third times the charm: NASAs National Polar-Orbiting Operational Environmental Satellite System Preparatory Project (NPP) satellite lifted off 28 October from Vandenberg Air Force Base in California. That success breaks NASAs recent trend of failed Earthobserving satellite launches, including the Orbiting Carbon Observatory in 2009 and Glory earlier this year. The $1.5 billion NPP, expected to operate for 5 years, carries scientic instruments designed to track everything from the ozone layer to ice cover, and to help researchers develop long- and short-term forecasts. It will also test-drive technologies for the National Oceanic and Atmospheric Administrations (NOAA) pending Joint Polar Satellite System (JPSS), a $10 billion, multi-spacecraft system thats been plagued with technical delays and scrutinized by budget-conscious lawmakers. The rst JPSS launch is not expected until 2016 or 2017, and NOAA Administrator Jane Lubchenco has warned that skimping on the systems funding will leave scientists with fewer tools to collect critical data.
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NEWSMAKERS
Tech Entrepreneur Offers Grants for Indie Science

Working on a radical biology project in your garage but dont have the cash for that DNA sequencer you spotted on eBay? A new program launched by a billionaire tech entrepreneur has your back.
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FINDINGS BY THE NUMBERS 90% Drop in blood lead levels worldwide due to lead removal from gasoline in 175 countries, according to the United Nations Environment Programmes Partnership for Clean Fuels and Vehicles.
Modern Humans First European Tour

Our species trekked to Englands southern coast by 41,000 to 44,000 years agoabout 6000 years earlier than expected, according to new dates for a partial jawbone and teeth described in two reports in this weeks issue of Nature. Researchers have long suspected that our ancestors migrated to Europe from Africa by about 42,000 years ago, based on Aurignacian tools thought to be crafted only by Homo sapiens. But the oldest modern human fossils in Europe, from Oase, Romania, are about 40,000 years old. Now, scientists have concluded that two molars from southern Italy dating to 43,000 to 45,000 years ago belonged to modern humans, making them the oldest known modern humans in Europe. They also now
Old bones. This jawbone belonged to a modern human who reached England at least 41,000 years ago.
Happiness Associated With Longer Life

People in better moods are 35% less likely to die in the next 5 years when taking their life situations into account, according to a study published online 31 October in the Proceedings of the National Academy of Sciences. The English Longitudinal Study of Ageing followed more than 11,000 people age 50 and older since 2002. In 2004, about 4700 subjects collected saliva samples four times in one day and, at those same times, rated how happy, excited, content, worried, anxious, and fearful they felt. The saliva samples are awaiting analysis for stress hormones, but in the new study, psychologist and epidemiologist Andrew Steptoe of University College London (UCL), and UCL colleague Jane Wardle published their results on mood and mortality. Of the 924 people who reported the least positive feelings, 7.3%, or 67, died within 5 years. For people with the most positive feelings, the rate fell in half, to 3.6%, or 50 of 1399 people. The researchers adjusted for age, sex, demographic factors, signs of depression, health (including disease diagnoses), and behaviors such as smoking and physical activity. Even with those adjustments, the risk of dying in the next 5 years was still 35% lower for the happiest people.
http://scim.ag/_happiness
Random Sample
The Fraternal Order of Fake Eye Doctors

An ancient manuscript, an indecipherable language, a secret society: These are not plot points from Dan Browns next novel, but the ndings of researchers at the University of Southern California (USC) in Los Angeles and Uppsala University in Sweden. Led by USC natural language researcher Kevin Knight, the team cracked a ciphered code to uncover the 250-year-old writings of a German secret society. Little is known about the origins of the 105page Copiale Cipher. But early this year, after giving a talk on using code-breaking techniques in language translation software, Knight received a copy of the book from a colleague. He and his team picked out patterns among 75,000 abstract symbols and fed the information into a language processing program. The coded language turned out to be German, obscured by symbols representing three-letter combinations or doubled consonants. After 4 months, a translation emerged, outlining initiation rituals for a secret society. Strangely, the text references ophthalmological equipment that would have been outdated by the 1730sapproximately when the group was active. We concluded that it was basically a group of people who got together and pretended to be eye doctors, Knight says. The code breakers presented their ndings at an Association for Computational Linguistics meeting in Portland, Oregon, in June. The text also explores the natural rights of man, but its secrecy probably owes more to secret societies being in vogue in the early 1700s than political intrigue, Knight says. If youre in a secret society, writing in code is just one of those things you do. Knights team is currently targeting other unsolved ciphers such as the Zodiac Killer letters and the 15th century Voynich manuscript.
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CREDITS (TOP TO BOTTOM): NATURAL HISTORY MUSEUM/TORQUAY MUSEUM; THE COPIALE CIPHER BY KEVIN KNIGHT, BETA MEGYESI AND CHRISTIANE SCHAEFER (2011); ISTOCKPHOTO.COM
think that transitional tools, once attributed to Neandertals, were made by modern humans instead, according to the report by Stefano Benazzi of the University of Vienna and colleagues. Using a rened carbon dating technique called ultraltration to redate animal bones associated with the jaw in England, and a separate technique to date shell beads in Italy, the Oxford Radiocarbon Accelerator Unit put modern humans in Italy and England earlier than those in Romaniaand at the same time as Neandertals. Modern humans got a lot further faster than we thought, says coauthor Chris Stringer of the Natural History Museum in London.
+/- 1 Accuracy of a navigation
methodpossibly used by Vikings based on Iceland spars, which depolarize sunlight to pinpoint a cloudhidden sun, a Proceedings of the Royal Society A study nds.
NEWS & ANALYSIS

B I OT E R R O R R E S E A R C H
NEWS & ANALYSIS

various reasons, the smallpox study never went forward. Fagbuyi finds the comparison lacking. Anthrax vaccine, he says, is totally different than Dryvax and has been tested in very large groups of people. The U.S. Centers for Disease Control and Prevention (CDC) is wrapping up a study it began 11 years ago in infant as an NFL football player, says John healthy adults, analyzing vaccine safety and Grabenstein, senior medical director for whether people can produce sufcient proadult vaccines at Merck and a member of the tective antibodies with fewer doses of the NBSB. For the hepatitis B vaccine, the dose vaccine, and by receiving it in muscle rather is lower in children; for u, it may also be than under the skin, which results in fewer lower and is sometimes given as two small side effects. This is a very safe vaccine, doses, rather than one larger one. Theres and side effects, like redness and swelling or enough idiosyncrasies that you cant brief fever, are similar to whats seen in many assume how children will react to a vac- childhood vaccines, says Nancy Messonnier, cine based on what you know in adults, says chief of CDCs bacterial vaccine branch. Like vaccines that protect against diphtheria and tetanus, for example, the anthrax vaccine is made with a protein from anthrax bacteria thats harmless on its own. None of this appeases pediatricians and bioethicists who consider an anthrax vaccine trial in children wrongheaded. These children are going to be exposed to a vacVaccine calculus. A 14-year-old gets immunized for hepatitis B, one of several cine from which they vaccines for which dosing differs between children and adults. Scientists want to have no chance of nd out whether anthrax is another. beneting. The only possible outcome is Grabenstein, who voted in favor of a clinical that nothing will happen, or theyll have trial. The only way to know is to try. a safety issue, says Paul Oft, an infectious In the bioterror world, a similar question disease specialist at the Childrens Hospital has come up at least once before. Soon after of Philadelphia in Pennsylvania, who often the 11 September attacks and the anthrax speaks out in favor of childhood vaccination. mailings, officials were concerned about Diekema agrees. Scientifically, a trial like weaponized smallpox. There wasnt enough this makes sense, because its the best way to of the existing smallpox vaccine, Dryvax, gather the necessary information. That said, to go around, so the government considered its a difcult study to justify. whether to test it in 2- to 5-year-olds to see Then theres the question of who would whether they could gain the same benet volunteer their children. Fagbuyi, who from a smaller dose, thus stretching sup- served in Iraq and received the anthrax vacplies. In some ways the proposed trial was cine himself, says that some military memmore problematic than an anthrax study, bers, rst responders, and scientists working says Douglas Diekema, a pediatrician and with anthraxmany of whom get the vaccine bioethicist at Seattle Childrens Research nowhave expressed interest in having their Institute: Smallpox vaccine is considered children vaccinated, too. They may be comriskier than anthrax vaccine, and many felt fortable enrolling them in an anthrax study, if the chance of a smallpox attack was much it gets off the ground. more remote than one using anthrax. For JENNIFER COUZIN-FRANKEL
Panel Endorses Anthrax Vaccine Study in Children

Its an ethically fraught question almost without precedent in modern medical research: Should children be enrolled in a clinical trial of the anthrax vaccine, which is almost certain not to help them and may harm them? Or should the U.S. government gamble and wait for a possible attack before exposing children to the vaccine for the very rst time? Last week, the full National Biodefense Science Board (NBSB) voted 121 in favor of a trial assuming its ethics are approved by a review board, saying that it was too uneasy to risk a mass science experiment on thousands of children after a bioterror strike, even if some consider that possibility remote. Before vaccines are given to children, they are tested in children, says Daniel Fagbuyi, the medical director of disaster preparedness and emergency management at Childrens National Medical Center in Washington, D.C. We do not want to set a new precedent. Fagbuyi chaired an NBSB working group that in September recommended a clinical trial. Last weeks vote isnt binding, and even if a study goes forward, the U.S. Department of Health and Human Services (HHS) will need to jump through many hoops to get it up and running. Although clinical trials are all about gathering information that benets others, rules for studies on children are more stringent than for those on adults. Its legally and ethically challenging to offer a child an experimental treatment thats even slightly risky if the chance of beneting is all but nonexistent, as is the case with the anthrax vaccine. The vaccine has been around for decades, and more than 2 million adults, mostly military personnel, have received it. But HHS and others who fret about a bioterror attack worry that this isnt enough. A quarter of the U.S. population is under 18, and no one has examined how children and teenagers react to the vaccinefor instance, whether they need different doses to ensure safety and effectiveness. There is considerable variation in the way vaccines are administered to people of different ages. With the tetanus vaccine, the same dose is given to a 2-month-old
CREDIT: JIM WEST/ALAMY
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Networking. Having a larger social network may increase gray matter in some parts of the brain, according to a new study.
NEUROSCIENCE
The Brains Social Network

Keeping track of an extended network of Facebook friends, Twitter followers, and LinkedIn connectionsnot to mention those old-fashioned face-to-face relationships with neighbors and ofce matescan certainly feel like a mental challenge. But does having a larger, more complex social network actually change the brain? A new study with macaques suggests it can. On page 697, a team led by neuroscientists Jrme Sallet and Matthew Rushworth of the University of Oxford in the United Kingdom reports that housing monkeys in larger groups increased the amount of gray matter in several parts of the brain involved in social cognition. Previous research has implicated these regions in a variety of tasks, from interpreting facial expressions and gestures to predicting what other individuals intend to do. The researchers also found correlations between gray matter volume and a monkeys dominance rank within its group, suggesting that beeng up neural circuitry in certain areas somehow promotes or enables social success. The work may help to resolve a quandary raised by a handful of recent studies that have correlated variations in human brain anatomy with social network size, some experts say. Those studies couldnt resolve which causes which. That is, do certain neuroanatomical features predispose people to building a large social network, or does having a large network cause certain brain regions to expand? In humans, its difcult to show anything but a correlation, says Lisa Barrett, a neuroscientist at Northeastern University in Boston. Although she has some reservations about the design of the experiment, Barrett says this clearly shows changes in gray matter in response to experience. The new monkey study and the recent human research point to many of the same regions of the temporal and frontal lobes as potential contributors to social success. As researchers continue to rene this picture of the brains social circuitry, there may be implications for hypotheses about brain evolution, such as the social brain hypothesis, which posits that the increasing complexity of social networks in primates in general and humans in particular drove the expansion of the cerebral cortex, or at least certain parts of it. In the Science study, Sallet and colleagues used magnetic resonance imaging (MRI) scans to look for variations in brain anatomy in 23 adult monkeys housed in groups of two to seven, plus one monkey housed alone. The monkeys were originally purchased in groups of different sizes and used in experiments by other researchers at Oxford, and their housing arrangements were determined by the requirements of these studies, Sallet says. Each monkey was scanned after it had been in a group of a constant size for 15 months on average.
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Before-and-after scans were not feasible due to the constraints of the various experiments, Sallet says. Another important caveat, Barrett and others say, is that the monkeys were not assigned to groups at random. To keep the peace, caretakers may have tended to put sociable animals together in larger groups and kept the mean ones alone or in pairs, says Michael Tomasello, a psychologist at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany. Preexisting characteristics of the individuals in the groups might plausibly be what is being measured, not any effects of social experience, Tomasello says. Sallet and colleagues insist that the housing assignments were made independently of any social characteristics of the animals and were as random as possible given their circumstances (although they note that one especially large group of 13 was split up to prevent ghts). Monkeys housed in larger groups had more gray matter in several regions of the temporal cortex, including the superior temporal sulcus (STS). Previous monkey studies have found that neurons in this region re in response to faces and body movements and seem to track where other individuals are directing their attention. Gray matter may increase in the STS in response to a growing need to decode the expressions, gestures, and movements of more individuals as social network size increases, the researchers suggest. Human MRI studies have also linked the STS to social network size. In a study published online 19 October in the Proceedings of the Royal Society B, Ryota Kanai and Geraint Rees of University College London and colleagues report that the right STS is one of several temporal lobe regions in which gray matter density correlates with the size of peoples Facebook networks. Sallet and colleagues also found increased gray matter in the amygdala of monkeys housed in larger groups. This structure deep in the temporal lobes is best known as a hub of emotional regulation. The recent study by Kanai and Rees implicated the amygdala as well, as did a study published online by Barrett and colleagues in December in Nature Neuroscience. They found greater amygdala volume in people with larger and more complex social networks, as measured by questionnaires. Barrett suggests that the amygdalas role in
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NEWS&ANALYSIS
social situations may be to ag individuals and events that are worth paying attention to and remembering. For example, she says, your amygdala might help you scan a crowded party for friends and foes. The Oxford researchers also found more gray matter in the rostral prefrontal cortex in monkeys housed in larger groups. In humans, regions of prefrontal cortex have been linked to theory of mind, or the recognition that other individuals have beliefs and intentions that may differ from ones own. Monkeys dont appear to possess this talent, but more gray matter in this region did seem to confer a social advantage. In a subset of 11 male monkeys, the researchers found that those with more gray matter in the rostral prefrontal cortex tended to be more dominant. The prefrontal cortex may also play a role in social success in humans. A study published in the 15 August issue of NeuroImage
SCIENTIFIC MISCONDUCT
GREG MILLER
Psychologist Accused of Fraud on Astonishing Scale

The worst fears came true. A Dutch committee that has spent the past 6 weeks investigating one of the Netherlands leading social psychologists has concluded in a report this week that he made up or manipulated data in dozens of papers over nearly a decade. Diederik Stapel was originally suspended from his position at Tilburg University in the Netherlands in September after three junior researchers reported that they suspected scientific misconduct in his work. After being confronted with the accusations, Stapel reportedly told university officials that some of his papers contained falsied data. The university launched an investigation, as did the University of Groningen and the University of Amsterdam, where Stapel had worked previously. The Tilburg commission released an interim report on 31 October, which includes preliminary results from all three investigations. The investigators found evidence of misconduct on an astonishing scale, the report says. Stapel made up data in several dozens of publications, the committee found, and 14 of the 21 Ph.D. theses he supervised are tainted. Stapel issued a statement the same day, saying he failed as a scientist and is ashamed of his actions. He has cooperated to an extent by giving investigators a list of papers that used fraudulent data, the report says. The ongoing investigations plan to examine more than 150 publications that Stapel has co-authored, Many of Stapels stuin cluding a paper earlier dents were simply given data this year in Science (8 April, to analyze and graduated p. 251) on the inuence of a without having ever run an messy environment on prejuexperiment, the report says. dice. (Science issued an EdiThe commission writes that torial Expression of Concern Stapel was absolute lord of about the paper this week.) the data in his collaborations. The fraud will cause huge Colleagues or students who damage, says Susan Fiske, a asked to see raw data told the social psychologist at Princcommission they were given eton University. His work is excuses or even threatened very centralor was. and insulted. Stapels studies encom- Data discredited. An investigaAt least two earlier groups passed a broad range of attention found that Diederik Stapel of whistleblowers questioned tion-catching topics, includ- forged data in dozens of studies. Stapels work, the report notes, ing how a position of power but no one followed up. On careinuences moral thinking. The committee, ful inspection, many of Stapels data sets have which interviewed dozens of Stapels for- improbable effect sizes and other statistical mer students and colleagues, concluded that irregularities, the report says. Among Stapels Stapel acted alone. The report says he would colleagues, the description of data as too good discuss experimental designs in detail with to be true was a heartfelt compliment to his collaborators and would claim to conduct skill and creativity, the report says. the surveys at high schools and universities The report recommends that the univerwith which he had special arrangements. sities look into criminal charges based on The experiments, however, never took the misuse of research funds and possible place, and Stapel gave collaborators made- harm to Stapels students resulting from the up data sets, investigators allege. In other fraud. The University of Amsterdam, where instances, the report says, he told colleagues Stapel received his Ph.D., has not yet deterthat he had an old data set lying around that mined whether his dissertation is fraudulent. he hadnt had a chance to analyze. When The committee suggests that the university Stapel did conduct actual experiments, the consider revoking Stapels degree, however, committee found evidence that he manipu- based on unbecoming conduct. lated results. GRETCHEN VOGEL
CREDIT: TILBURG UNIVERSITY
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found that people with more gray matter in the ventromedial prefrontal cortex (and a few other regions of temporal and frontal cortex) performed better on tests requiring multiple layers of mind reading, such as determining from a short story whether Sam thought Harry intentionally gave him misleading directions. That study, led by Oxford evolutionary anthropologist Robin Dunbar, also found that people with more gray matter in these regions tend to have larger social networks, which the researchers assessed by asking participants to write down the initials of everyone with whom theyd had a social interaction in the past month, using their cell phones to jog their memories. With these several studies, youre seeing a lot of the same sorts of brain areas implicated in correlations with social network size, says Robert Barton, who studies brain evolution at Durham University in the United
Kingdom. But the evolutionary implications arent yet clear, Barton says. We dont know what the relationship is between these studies that look at individual differences and the comparative studies that look at differences between species. It remains to be seen, for example, whether the same regions that are expanded in individuals with large social networks are the same ones that are expanded in large-brained social primates compared with less social species, Barton says. Dunbar sees it differently: Variations among individuals provide the platform for selection to act on. He views the recent findings as strong support for the social brain hypothesis, of which he is an ardent advocate. The ability to form cooperative networks of individuals is whats driving the evolution of big brain sizes, Dunbar says. The brain is a social tool.
NEWS&ANALYSIS
social situations may be to ag individuals and events that are worth paying attention to and remembering. For example, she says, your amygdala might help you scan a crowded party for friends and foes. The Oxford researchers also found more gray matter in the rostral prefrontal cortex in monkeys housed in larger groups. In humans, regions of prefrontal cortex have been linked to theory of mind, or the recognition that other individuals have beliefs and intentions that may differ from ones own. Monkeys dont appear to possess this talent, but more gray matter in this region did seem to confer a social advantage. In a subset of 11 male monkeys, the researchers found that those with more gray matter in the rostral prefrontal cortex tended to be more dominant. The prefrontal cortex may also play a role in social success in humans. A study published in the 15 August issue of NeuroImage
SCIENTIFIC MISCONDUCT
GREG MILLER
Psychologist Accused of Fraud on Astonishing Scale

The worst fears came true. A Dutch committee that has spent the past 6 weeks investigating one of the Netherlands leading social psychologists has concluded in a report this week that he made up or manipulated data in dozens of papers over nearly a decade. Diederik Stapel was originally suspended from his position at Tilburg University in the Netherlands in September after three junior researchers reported that they suspected scientific misconduct in his work. After being confronted with the accusations, Stapel reportedly told university officials that some of his papers contained falsied data. The university launched an investigation, as did the University of Groningen and the University of Amsterdam, where Stapel had worked previously. The Tilburg commission released an interim report on 31 October, which includes preliminary results from all three investigations. The investigators found evidence of misconduct on an astonishing scale, the report says. Stapel made up data in several dozens of publications, the committee found, and 14 of the 21 Ph.D. theses he supervised are tainted. Stapel issued a statement the same day, saying he failed as a scientist and is ashamed of his actions. He has cooperated to an extent by giving investigators a list of papers that used fraudulent data, the report says. The ongoing investigations plan to examine more than 150 publications that Stapel has co-authored, Many of Stapels stuin cluding a paper earlier dents were simply given data this year in Science (8 April, to analyze and graduated p. 251) on the inuence of a without having ever run an messy environment on prejuexperiment, the report says. dice. (Science issued an EdiThe commission writes that torial Expression of Concern Stapel was absolute lord of about the paper this week.) the data in his collaborations. The fraud will cause huge Colleagues or students who damage, says Susan Fiske, a asked to see raw data told the social psychologist at Princcommission they were given eton University. His work is excuses or even threatened very centralor was. and insulted. Stapels studies encom- Data discredited. An investigaAt least two earlier groups passed a broad range of attention found that Diederik Stapel of whistleblowers questioned tion-catching topics, includ- forged data in dozens of studies. Stapels work, the report notes, ing how a position of power but no one followed up. On careinuences moral thinking. The committee, ful inspection, many of Stapels data sets have which interviewed dozens of Stapels for- improbable effect sizes and other statistical mer students and colleagues, concluded that irregularities, the report says. Among Stapels Stapel acted alone. The report says he would colleagues, the description of data as too good discuss experimental designs in detail with to be true was a heartfelt compliment to his collaborators and would claim to conduct skill and creativity, the report says. the surveys at high schools and universities The report recommends that the univerwith which he had special arrangements. sities look into criminal charges based on The experiments, however, never took the misuse of research funds and possible place, and Stapel gave collaborators made- harm to Stapels students resulting from the up data sets, investigators allege. In other fraud. The University of Amsterdam, where instances, the report says, he told colleagues Stapel received his Ph.D., has not yet deterthat he had an old data set lying around that mined whether his dissertation is fraudulent. he hadnt had a chance to analyze. When The committee suggests that the university Stapel did conduct actual experiments, the consider revoking Stapels degree, however, committee found evidence that he manipu- based on unbecoming conduct. lated results. GRETCHEN VOGEL
CREDIT: TILBURG UNIVERSITY
www.sciencemag.org
579
Published by AAAS
found that people with more gray matter in the ventromedial prefrontal cortex (and a few other regions of temporal and frontal cortex) performed better on tests requiring multiple layers of mind reading, such as determining from a short story whether Sam thought Harry intentionally gave him misleading directions. That study, led by Oxford evolutionary anthropologist Robin Dunbar, also found that people with more gray matter in these regions tend to have larger social networks, which the researchers assessed by asking participants to write down the initials of everyone with whom theyd had a social interaction in the past month, using their cell phones to jog their memories. With these several studies, youre seeing a lot of the same sorts of brain areas implicated in correlations with social network size, says Robert Barton, who studies brain evolution at Durham University in the United
Kingdom. But the evolutionary implications arent yet clear, Barton says. We dont know what the relationship is between these studies that look at individual differences and the comparative studies that look at differences between species. It remains to be seen, for example, whether the same regions that are expanded in individuals with large social networks are the same ones that are expanded in large-brained social primates compared with less social species, Barton says. Dunbar sees it differently: Variations among individuals provide the platform for selection to act on. He views the recent findings as strong support for the social brain hypothesis, of which he is an ardent advocate. The ability to form cooperative networks of individuals is whats driving the evolution of big brain sizes, Dunbar says. The brain is a social tool.
NEWS&ANALYSIS
E P I D E M I O LO G Y
Large African TB Study Reveals Powerful Strategy to Curb Disease

Powerful combinations of drugs can cure tuberculosis but only if people know they have the disease and stick rigorously to a lengthy course of treatment. An ambitious study involving 1 million people in Zambia and South Africa explored two strategies to increase TB testing and treatment in 24 locales, asking whether either could reduce the incidence of the disease on a communitywide level. The results, reported on 30 October at a TB meeting in Lille, France, found that one approach led to a 22% drop in TB over 3 years, whereas the other had no effect on prevalence of the disease. The secret to success, which has implications for HIV/AIDS researchers who have similar communitywide studies in the works, is to target the places where most disease transmission occursin this case, the homes of people with TBrather than broadly taking campaigns to the streets. The positive result in the ZambiaSouth Africa TB and AIDS Reduction (ZAMSTAR) study is a milestone in the eld, says Peter Godfrey-Faussett, an epidemiologist at the London School of Hygiene & Tropical Medicine (LSHTM), who was one of three principal investigators. Up until now, we havent had any study that showed a community impact on TB in the era of HIV , Godfrey-Faussett says. HIV and Mycobacterium tuberculosis have an intimate relationship that has vastly complicated efforts to combat AIDS and TB. HIVs assault on the immune system transforms latent, harmless infections with M. tuberculosis into deadly, active disease and has caused huge spikes in TB cases across southern Africa. This, in turn, has led to jumps in AIDS, which is dened as HIV plus an opportunistic infection such as active TB. TB control is failing in southern Africa, Godfrey-Faussett says, noting that HIV there has led to vefold increases in TB. Gerald Friedland, a TB and HIV/AIDS researcher at Yale University, says that although the field desperately needs better diagnostics and treatments, ZAMSTAR shows that public health interventions can make dramatic headway with existing tools. This is a really great study and with very positive and important ndings, Friedland says. The $27 million ZAMSTAR study compared the impact of adding enhanced case finding and/or household interventions
with standard TB control efforts. Standard TB control programs test sputum samples with microscopy to find active cases, and they emphasize directly observed treatment, in which health care workers watch patients take their drugs for the several months required to cure the disease. Enhanced case nding used street dramas to advertise TB testing and offered sputum tests in front of health centers and at mobile collection points near peoples homes. In the household House calls. Counselors intervention, spevisited homes that had cially trained counactive TB cases. selors visited homes of people who had been diagnosed with the disease by their local TB control program. The counselors encouraged everyone living in the house to receive both TB and HIV tests, and anyone who tested positive for the AIDS virus but not tuberculosis still received TB drugs, which prevent latent infections from becoming active. The family and standard TB control programs helped people taking spread of HIV by ramping up testing and medication adhere to their treatments. then treating all infected people. There The researchers randomly assigned par- are many, many questions as to whether ticipants to receive standard care alone or test and treat will work, Godfrey-Faussett in conjunction with one or both of the other says. It should work, but the real world is strategies. To assess the impact of the inter- a messy, messy place. Chaisson, who studventions, investigators took sputum sam- ies HIV/AIDS, says the ZAMSTAR lesson ples from 5000 randomly chosen people is to target interventions. Going to settings in each community at the studys start and where youre likely to nd cases and interend. The addition of enhanced case nding vene effectively will have a higher yield than had no impact on prevalence, but study sites broadcasting to the community at large, he that used household interventions had 22% says. Putting condom baskets in grocery fewer positive sputum samples in 2009 than stores, for example, doesnt have as high a in 2006. For doing something at a popula- yield as putting them in sex clubs. tion level, thats huge, says epidemiologist Paul Nunn, who heads the Stop TB Richard Chaisson, who directs the Johns Department at the World Health OrganiHopkins Center for Tuberculosis Research zation in Geneva, Switzerland, says the in Baltimore, Maryland, and heads the Con- ZAMSTAR studys groundbreaking results sortium to Respond Effectively to the AIDS/ also present serious challenges to TB conTuberculosis Epidemic. (ZAMSTAR is one trol programs. The key issue is whether the of three trials that are part of the consortium, amount of reduction [in prevalence] is worth funded by the Bill and Melinda Gates Foun- what it would cost to implement these interdation, but Chaisson was not a study inves- ventions, Nunn says. JON COHEN
CREDIT: ZAMBART PROJECT
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tigator.) Skin tests that can detect latent TB infections also found that the incidence in children plummeted 50% during the study in communities that used household interventions; enhanced case nding again had no effect. The failure of enhanced case finding surprised the ZAMSTAR team. Weve been scratching our heads about that, says Godfrey-Faussett, who ran the project with Nulda Beyers of the Desmond Tutu TB Centre at Stellenbosch University in Tygerberg, South Africa, and Helen Ayles, also from LSHTM. The negative result offers a cautionary note to AIDS researchers, who will soon launch large studies that hope to reduce the
MEETINGBRIEFS>>
12TH INTERNATIONAL CONGRESS OF HUMAN GENETICS | 1115 OCTOBER 2011 | MONTREAL, CANADA Fruitful founders. A Quebec couple in 1876, with seven of their 14 children (below), gave rise to many descendants, as seen in a 1960 photo of one of their sons with his descendants (left).
Life on the Fertile Frontier

Ever since the rst humans migrated out of Africa almost 2 million years ago, our ancestors have crisscrossed the globe in search of food, resources, and better lives. Now a remarkable study of the genealogy of 1 million Canadians has found that the rst waves of European settlers to push into the wilderness of northeastern Quebec in the 17th and 18th centuries had more children than people who came later or stayed in central towns. The study was presented in a poster by population geneticists Claudia Moreau and Damian Labuda of the University of Montreal and Laurent Excofer of the University of Bern, and the work is published online in Science (http://scim.ag/cmoreau) this week. Some 30,000 settlers, mostly farmers, who opened frontiers starting in the 17th century passed on up to four times as many genes to living people than did immigrants who lagged behind in core towns, the researchers found. The work allows geneticists to test computational models of how populations and genes spread when humans move into new territories and multiply rapidly. It conrms what many people must have thought: that when conditions were hard, it was better to move and take their chances than to stay in place, says population geneticist Montgomery Slatkin of the University of California, Berkeley, who was not part of the study. The team used the comprehensive BALSAC population database of Quebec, which contains marriage, birth, and death records by parish for 5 million people from 1608 to 1970. The researchers were able to trace the pattern of settlement as 84 new parishes opened in the Charlevoix and SaguenayLac-Saint Jean regions of Quebec. Starting in 1608, European immigrants, mostly from France, arrived in Quebec City and began to spread northeast along the St. Lawrence River. In 1638, for example, about 15 people moved north from the town of Baie-SaintPaul, followed by other groups that created an uneven wave front rolling into the territory occupied by Algonquian tribes. By 1838, other settlers had arrived, lling in the gaps and evening the front line of settlers, whereas others remained behind in core settlements. Those families closest to the frontier had the most children. By comparing birth and marriage records, the researchers found that women on the leading edge of the wave had an average of 9.1 children, compared with 7.9 children per family at the core, a boost of about 15%. That increase was amplied in their offspring: An average of 4.9 children from front-line families married compared with 4.1 at the core, a 20% difference. Moving forward to 1960, 40% of living peoples ancestors had lived on the front wave between 1686 and 1930. Were excited that weve reconstructed what happens when you expand into a new territory, Labuda says. The researchers noticed that women on the front line got married a year earlier than those in core towns and bore children later, giving them a higher reproduction rate. So what was it about life on the frontier that boosted fertility? It could be partly due to cultural preferences, such as the benets of having a large family. Or it might be that fertility is highest at the wave front because there are
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more resources [there], explains population geneticist Henry Harpending of the University of Utah in Salt Lake City, who was not part of the study. As a place lls up, fertility declines in response to fewer resources. This paper shows that is exactly what happened.
ANN GIBBONS
X-tra Diversity For Africans

Call it the X factor. Compared with Europeans and Asians, Africans have extra diversity on their X chromosomes, according to an invited lecture at the meeting by Cornell University geneticist Andrew Clark. The lack of diversity in non-Africans may be the legacy, handed down for thousands of generations, of fewer women than men in the bands of modern humans who rst left Africa to colonize the rest of the world, Clark said. His team used new data from the 1000 Genomes Project to resolve a recent debate about diversity on the X and to begin to explain why non-Africans have so little variation on this sex chromosome compared with the other chromosomes, or autosomes. Were seeing a very consistent pattern of reduced X diversity out of Africa, Clark said. It shows a greater loss of variation than even in the autosomes out of Africa. To evolutionary geneticists, the X chromosome has always been something of a riddle. Unlike its partner the Y, the X still has its full complement of genes. But because of the way it is inheritedmen inherit only one copy from their mothers, whereas women inherit a copy from each parentthe X is
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NEWS&ANALYSIS
Diabetes Genes Decline Out of Africa

Ask a physician which ethnic group is most at risk for type 2 diabetes, and he might cite Native Americans or African-Americans. But now a thorough analysis of all known genetic variants associated with the disease reveals a surprising pattern: Its Africans themselves who face the highest known genetic risk from this epidemic, according to a new report at the meeting. In 2010, only 3% of subSaharan Africans had any kind of diabetes, compared with 12% of Americans, but type 2 diabetes has a big environmental component and is only now beginning to show up in African city dwellers who have adopted a Western diet and lifestyle. As that lifestyle spreads in the future, Africans may face a serious threat, according to a talk and poster from a Stanford University team. At the poster session, Stanford graduate student Erik Corona stood in front of a Google Earth map of the world that he nds surprising. On this map he had plotted the frequency of 12 gene variants known to be associated with type 2 diabetes in 51 populations from Australia to Zaire. It shows a clear gradient of red to green from west to east, from Africa to Asia, Corona says (see map). Something strange is going on with type 2 diabetes. Corona and Stanford bioinformaticist Rong Chen say the map shows that the genetic risk of diabetes decreased the farther modern humans had migrated out of Africa, especially into Asia although the risk rose again in Native Americans, perhaps due to adaptations to diet or the northern climate around the Bering Strait. This suggests that as modern humans spread around the world in the past 60,000 years, they lost the single-nucleotide polymorphisms (SNPs) that are known to be linked to diabetes, a pattern that has not been described before, Stanford team leader Atul Butte less diverse than autosomes in populations. So the chromosome has been a low priority for sequencers interested in disease risk, and published sequences have been of poor quality. But interest was piqued 3 years ago when teams studying frequencies of different alleles on the X got different results: One group found the X was less diverse in nonAfricans, but another group didnt. Then last year, the 1000 Genomes Project (Science, 29 October 2010, p. 574) posted its pilot data, including sequences of the complete genomes of 69 women from Africa and Europe. These data provide a much sharper view of the X, including single-nucleotide
says. Perhaps, Butte speculates, some or all of those 12 variants were once benecial, helping Africans maximize their use of sugar to create energy. Until now, few people lived long enough or ate enough sugar and fat to develop diabetes, so in prehistory the disease probably had little effect on tness. Stanford population geneticist Carlos Bustamante nds the pattern intriguing but points out that the challenge now is to nd out how much disease the associated gene variants actually cause. Diabetes is a complex disease, he says. We have to know if these variants are causative, or do they increase your risk by [a mere] 1.5%? The Stanford team argues that their genetic maps are a rst step. In his talk, Chen showed that diabetes is just one of 100 diseases for which they have plotted risk, using two new databases, Verimed and Geneworld (http://geneworld.stanford.edu/), that will eventually be available to doctors. A.G. extra effect meant that the Africans X chromosomes were twice as diverse as those of the Europeans. Because of the Xs pattern of inheritance, the reduction in diversity out of Africa is likely due to some sex-linked demographic process, Keinan says. One likely explanation is that more men than women were members of the bands who moved out of Africa, a gender imbalance also seen on the front line of some other migrations. Other researchers nd the work convincing. We are converging on an answer, says computer scientist August Woerner, who works with geneticist Michael Hammer at the University of Arizona in Tucson. Hammers lab was the one that saw more diversity on the X of non-Africans, but with whole genome sequences, they, too, have now found more diversity in Africans, resolving the debate. The simplest explanation is waves of male migrations or a few males disproportionately passing on their X chromosomes by having many children, says geneticist Joshua Akey of the University of Washington, Seattle.
CREDITS (TOP TO BOTTOM): ERIK CORONA/GOOGLE; ADAPTED FROM A. KEINAN
polymorphisms (SNPs). The 1000 Genomes data rides to the rescue, Clark quipped. Researchers have long known that Africans have more diverse genomes overall, presumably because the small bands of ancestors traveling out of Africa passed through a genetic bottleneck and lost many variants. Cornells Alon Keinan, Clark, and their colleagues devised a clever method to compare the amount of variation in SNPs on the X with that on the autosomes and applied it to 36 Yoruba women from West Africa. They found the X had about 73% of the diversity of the autosomes, as expected. But when Keinan examined the genomes of 33 European women, he found that their X chromosomes had only about 61% of the diversity of their autosomes. Because the European genomes were already less diverse overall, this
On the X trail. The X chromosome is less diverse outside Africa (faded pink), whereas the Y chromosomes diversity (blue) does not change.
X chromosome Y chromosome
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Diabetes alert. Genes associated with type 2 diabetes are common (red dots) in Africa and rarer (green dots) in Asia.
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Changbai-Paektu unleashed one of the largest eruptions in recent millennia; scientists are joining arms to discern when the volcano may roar back to life
Slumbering giant. North Korean scientists
descend a several-hundred-meter-long staircase to reach a singular research base next to Mount Paektus caldera lake.
MOUNT PAEKTU, NORTH KOREAJames Hammonds tan fedora pokes out of the tiny log cabin, followed by the rest of the 30-year-old seismologist. He crouches at the entryway and types commands into a laptop to activate a steel cylinder on a shelf inside the shelter. Give us a stomp, he asks a bemused colleague, who then gures out what he should do. Hammond frowns and ddles with the wiring. Another one! Seconds later, he grins. That was a magnitude 4.2, he says, beaming. Then the moments signicance sinks in. Weve just deployed the rst broadband seismometer in DPRK, Hammond says, referring to North Korea by its formal name, the Democratic Peoples Republic of Korea. Hammond, a researcher with Imperial College London, knew it would be a miracle if the seismometer were to record a major tremor. The instrument stayed in place on a steep slope above Paektu Bridge Volcano Research Station near DPRKs northern border for just one night in September, after which Hammond boxed it up and hauled it back to the United Kingdom. The training exercise, Hammond says, gave the North Korean researchers hands-on experience with Western instrumentation. The fleeting deployment also has a powerful sym-
bolic value: It demonstrates North Koreas sure to build, the next eruption should be openness to collaborate on a project of vital explosive, Xu says. In recent months, Chiimportance to the region. nese researchers have observed geophysical Paektu Bridge is one of several North anomalies, including elevated temperatures Korean stations monitoring Mount Paektu, a of hot springs and deation of the caldera volcano that straddles the border with China. rim. But most concur that there is no evi(Two-thirds of the mountain is in China, dence of magma rising toward the surface, where it is called Changbai.*) Historical which would signal an imminent eruption. records and ash layers indicate that ChangWhen Changbai next stirs to life, an bai explodes to life every 100 years or so, the immediate concern will be the fate of Tianlast time in 1903. One eruption chi calderas deep lake. The only in particular mesmerizes scienoutlet is a narrow valley on the tists. Around 1000 years ago, the volcanos north flank, in Chisciencemag.org volcano rained tephrapumice nese territory. A moderate-sized Slides and podcast eruption could send a lahar and ashacross 33,000 square interview with kilometers of northeast China vegetation, water, rocks, and author Richard Stone. and Korea, dumping 5 centimudhurtling down the valley, meters of ash as far away as Japan. The so- threatening 60,000 residents and expencalled millennium eruption was one of the sive infrastructure such as hydropower stalargest of the past few thousand years, rival- tions. A millennium-scale eruption, says ing the 1815 Tambora eruption in Indonesia. CEA volcanologist Wei Haiquan, would be Scientists are keeping a wary vigil. The catastrophic. Some 100,000 people would hazard is huge, says Xu Jiandong, a volca- be vulnerable to avalanches of superheated nologist at the Institute of Geology of the gas and tephra called pyroclastic ows. Ash China Earthquake Administration (CEA) would ruin crops throughout the region and in Beijing. Because Changbais silica-rich affect transpacific flights. The damage magma is viscous and gassy, allowing pres- would be unimaginable, says Kim Hang Myong, former director of the Institute of * Volcanology of the Earthquake AdministraBecause most of the volcano is in China, this article refers to it as Changbai, except when describing work in DPRK. tion of DPRK in Pyongyang.
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Vigil at North Koreas Mount Doom
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Chinese scientists have ringed their side of the mountain with broadband seismometers and other monitoring instruments. Over the next 5 years, CEA plans to spend $24 million to upgrade its facilities. But the part of the mountain in North Korea has been mostly beyond the reach of foreigners and modern equipmentuntil now. In September, Hammond, volcanologist Clive Oppenheimer of the University of Cambridge in the United Kingdom, and I were the first westerners to visit North Koreas volcano eld stations. The unprecedented encounter was facilitated by two nongovernmental organizations: Pyongyang International Information Center on New Technology and Economy, or PIINTEC, based in Pyongyang, and the Environmental Education Media Project in Beijing. The British duo came away impressed with the North Korean scientists, who have had to make do with antiquated equipment or fashion their own. And DPRKs seven Paektu observatories will pave the way for eldwork. The infrastructure is fantastic. Theyre ready for state-of-the-art equipment, Oppenheimer says. He and Hammond plan to return to Paektu next summer for research and hope to catalyze an ambitious campaign of crossborder measurements as early as 2013. In another sign of glasnost, Chinese scientists may also conduct research at Paektu next summer. Although geologists on both sides have made forays, this would mark the rst geophysics eldwork between the countries. The main aim is heightened vigilance. We hope to build up our capability to monitor the volcano and forecast eruption scenarios, says Yun Yong Kun, deputy director general of DPRKs Earthquake Administration. Toward that end, he says, we welcome scientists with open arms. Studies in North Korea could also help penetrate scientific conundrums, including the true scale of the millennium eruption. Deep scars The golden leaves of Changbai larch, a species unique to the region, glow in the pale afternoon light. A few meters ahead, one tree leans precariously over a precipice. The valley below looks as if it were coated with chocolate meringue. Clinging to the sheer walls are wispy, gravity-defying spires straight out of a Dr. Seuss story, hollow ignimbrite structures formed by escaping gas. A 120-meter-deep scar, some 30 kilometers from Changbais west ank in Chinese territory, was carved by a pyroclastic ow that barreled down the mountain, incinerating everything in its path. A deeper, longer gash runs north.
Seismic shift. Breaking new ground, James
Hammond (top left) and Clive Oppenheimer prepare to install a broadband seismometer. North Korea welcomes cooperation at Paektu, says Institute of Volcanology Director Kim Myong Song.
Volcanic eruptions are rated on a scale that depends on the volume or mass of lava and tephra they disgorge. Its comparable to the Richter scale for earthquakes. Anything above magnitude 8 rates as a supereruption. These gargantuan blasts spew more than 1000 cubic kilometers of ejecta, such as those hundreds of thousands of years ago that formed the calderas at Yellowstone in Wyoming. The most recent supereruption was New Zealands Lake Taupo volcano about 26,500 years ago. Only a few 7s have occurred during the last 11,500 years; the millennium eruption was one of them. On the North Korean side of the volcano, the wounds from that titanic blast are only slowly healing. Stop here! Oppenheimer commands. The minibus pulls over near a bone-white ridge on Paektus treeless east flank. Oppenheimer scrambles down an embankment and examines a few-meterthick layer of exposed pumice. The friable rock crumbles in his hand. This must
be from the millennium eruption, he says. Embedded in it are chunks of harder rock. These lithics were part of the vent. They were ripped out by the ascending magma, Oppenheimer says. The fragments have their own story to tell. From their size and distribution, for instance, scientists can reconstruct the plumes height; models suggest that it reached 25 kilometers. Theres a more fundamental question, Oppenheimer says. Why is Changbai here in the rst place? he asks. We dont know much about whats going on inside the Earth that leads to the kinds of lava and pumice you see there. Most active volcanoes lie at tectonic-plate boundaries, like the Ring of Fire girdling the Pacic Ocean. Subduction churns magma into their conduits, like a stoker shoveling coal in a steam locomotive. Other volcanoes are fed by mantle plumes that funnel magma to the surface; prime examples are the volcanoes that continue to shape the Hawaiian Islands. Changbai is one of a handful of big volcanoes that defy easy categorization. Although it sits about 1200 kilometers west of the Ring of Fire, it seems to be fueled by deep subduction of the West Pacific Plate, says CEA seismologist Lei Jianshe. Some experts, however, reject the notion that subduction is the driving force. Scientists have pieced together a timeline of the bafing volcanos history. Changbai began to form about 1 million years ago, after trachyte became the dominant composition of
CREDITS: R. STONE/SCIENCE

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A Very Big Bang

Changbais millennium eruption, one of the biggest in recorded history, is an enduring riddle. Piecing together what happened 1000 years ago could help scientists gauge the volcanos modern-day risk. One major uncertainty is how much material, or tephra, was ejected during the eruption. Estimates range from 30 to 172 cubic kilometers. Prevailing winds swept much of the ash eastward, blanketing the northern Korean Peninsula with the thickest millennium layers. A true picture of the eruption will come only from mapping deposits on the Korean side of the border. An intrepid German researcher, HansUlrich Schmincke, was the rst to take a stab at that. After the 1991 eruption of Mount Witness to catastrophe. Trees buried in pumice on Pinatubo in the Philippines shot ash into the Paektus east slope point to an early date of the stratosphere, causing a brief global cooling, millennium eruption. Schmincke, a volcanologist with the Leibniz Institute of Marine Science in Kiel, grew interested in probing the climate effects of past eruptions. For 2 years, he tried to get a visa to North Korea so he could study Paektu and failed. So in the summer of 1993, he and graduate student Susanne Horn set out for China for eldwork at Changbai. Back in Beijing, Schmincke dropped in on the North Korean Embassy. Im a person who never gives up, he says. He gave an impassioned spiel on Paektus threat, and they got their visas. Arriving at Paektu a few days later, Schmincke was struck by the huge layers of pumice. He persuaded their guides to allow them to sample from a wide area. We got lots of data, he says. They calculated that the tephra volume was 96 cubic kilometers and that the eruption would have triggered a marked cooling effect. North Korean volcanologists are attempting to rene estimates of tephra volume by mapping the millennium ash layer. So far, theyve drilled 170 boreholes, 127 millimeters in diameter, on the volcanos east ank and the surrounding countryside. Their drilling work is fabulous, says Clive Oppenheimer, a volcanologist at University of Cambridge in the United Kingdom. Korean researchers will begin analyzing the data over the winter. Another question is when did the millennium eruption happen? Theres no agreement on the date, says Xu Jiandong of the China Earthquake Administration (CEA). In the early 1990s, CEA volcanologist Liu Ruoxin collected chunks of carbonized wood from the volcanos north ank. Using radiocarbon analysis, he put the date at 1205 C.E. Trailblazer. Hans-Ulrich Schmincke and Horn turned back the clock. On Paektus east Schminckes eldwork ank in 1993, they sawed off wood from a larch buried in 8 meters in North Korea in 1993 of millennium ash and brought it back to Kiel for radiocarbon dat- yielded insights into ing. They pegged the eruption to the year 969 C.E. Other radiocarbon Paektus potent past. studies suggest it could have occurred a few decades earlier. Although most experts put stock in the earlier dates, Xu says the matter isnt settled. He argues that the eruption might have been two or more events spread over a few centuries. Historical documents shed little light; one Korean text from 1199 C.E. refers to white-hair rain, perhaps an oblique reference to the millennium eruption. The mute record is not surprising, says Cho Moonsup, a petrologist at Seoul National University. Long ago in Korea, he explains, scholars risked execution if they chronicled bad omens. Chinese and Japanese annals are silent. Xu hopes to have the last word on the millennium eruptions timing. A few months ago, he shipped several dozen samples from four trees buried under millennium deposits on the three Chinese anks, including the charred wood that Liu dated, to Switzerland for high-precision radiocarbon analysis using accelerated mass spectrometry. Results are due next month. R.S.
Strange signs Hoping to find clues to Changbais next move, CEA aims to build one of the worlds most ambitious volcano observatories. Plans call for installing real-time gas sensor arrays, adding to a network of 11 digital seismic stations and 16 GPS stations, and drilling a deep borehole in the volcanos ank for a suite of instruments. Our goal is to create a network thats capable of forecasting an eruption, says Xu, who leads a CEA group that monitors six of Chinas 14 known active volcanoes, dened as those that have erupted in the past 10,000 years. They have a solid foundation. Around 15 years ago, Chinese researchers established Tianchi Volcano Observatory on Changbais north ank. The station is acutely vulnerable: It sits on the Erdaobaihe Riverthe outlet from the caldera lakeand could eas-
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the magma spilling across the region. The lava grew more viscous and piled up, says geologist Song Gung Ho of DPRKs Institute of Volcanology. Then about 170,000 years ago, lava disgorged from smaller cones cooled into blood-red scoriae or cinders. An explosive new phase started with a bang 4000 years ago when a massive eruption scattered gray ash over northeastern China and Korea. Later, the millennium eruption left a similar mark: deposits of pale comenditic pumice studded with black fragments ripped from the cone. Scribes recorded minor eruptions in 1668, 1702, and 1903. The story may not end there. The most worrisome scenario is that we are in the midst of a cycle of millennium-scale eruptions, says Cho Moonsup, a petrologist at Seoul National University. Changbais history and possibly its future could be revealed in the panoply of colors and textures of pumice scattered around the caldera rim. These rocks tell so much about the volcanos past behavior, says Oppenheimer, who spends several weeks a year at Mount Erebus in Antarctica and other volcanic hot spots. By looking at deposits of different ages, you can begin to understand the cycles. For example, the amounts of CO2 and other volatiles in melt inclusions can reveal how much gas might have been expelled from the magma when the volcano erupted. Such information, Oppenheimer says, enables us to build a model of a volcanos plumbing system just before it erupts. It wont be easy. Even at volcanoes like Etna, Vesuvius, and Kilauea that have been observed for hundreds of years, Oppenheimer says, we still dont understand everything about how they work.
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thick pumice layers in North Korea attest. Chinese scientists sample deposits (right) near the caldera rim on Changbais south ank.
ily be demolished by a lahar. Several years ago, CEA built a second, safer perch, Changbaishan Volcano Observatory, 50 kilometers northwest of the caldera. But Tianchi observatory, still staffed, has a hidden asset. A short hike behind the station is a massive steel door in the side of a hill. A technician unlocks it and leads Xu and me inside. Its pitch-black; we use our cell phone display screens to nd our way. Built in the mid-1990s, the 65-meter-long chamber has a shaft at the end holding a broadband seismometer and, intriguingly, two 10-meterlong quartz tubes. Geophysicists monitor minute changes in conductivity in the crystal instruments, each a tiltmeter and tensometer, to track the mountains deformation. CEAs surveillance indicates that Changbai is biding its time. There was a urry of excitement in June 2002 when swarms of tremors racked the mountain. CEA traced the epicenter to 5 kilometers beneath the volcanos cone, where the main magma chamber is thought to lie. Over the next 3 years Changbai shuddered, sometimes experiencing more than 200 spasms in a month; the background rate is fewer than 10 per month. Activity crested on 19 March 2003, when more than 500 tremors were recorded. CEA observations showed that the mountain rose about 68 millimeters over that periodve times the rate before the shaking started. Some scientists argued that the tremors were due to gas bubbles: volcanic indigestion. Others believed magma was rising toward the cone and braced for a blast. But after a swarm in May 2005, Changbais seismicity receded to background levels. However quiet the ground may be now, there are curious changes underfoot. Over the past 2 years, the CEA team has determined that the caldera rim has slumped a few centi-
meters even as the surrounding land continues to rise. The deation has left researchers scratching their heads, as has last years 90-degree twist in the predominant land motion, from southwesterly to southeasterly. There are other anomalies. In 2008, Korean scientists discovered a new fumarole, or gas vent, near the east ank, 40 kilometers from the caldera. CEA scientists, meanwhile, observed an odd burst of sulfur dioxide last November and measured uctuations in hot springs on the north flank. Two years ago, temperatures suddenly shot up 2 to 3C on average, then went back down.
Sung-hyo of Pusan National University in South Korea agrees. We urgently need emergency response plan, he says. Others insist that fears about a looming eruption are overblown. Terra nova A detailed probe of the magma chamber might provide some answers. But no matter how many instruments the Chinese put on their side of the volcano, a denitive view of its plumbing will only come after the North Koreans wire up their side as well. There are two main methods of penetrating the interior. One is to track the speed of seismic waves from controlled explosions, revealing the composition and consistency of the rock. A low-velocity anomaly is usually interpreted as a magma chamber, for example. A second method is to survey the area with magnetotelluric (MT) sensors, which map subsurface variations in conductivity. Changbais underbelly is complex. Seismic scans have revealed globs of magma, 100 kilometers wide, lined up like a string of beads around 1000 kilometers below the surface, Wei says. As this magma ascends, he says, it mixes and changes composition. The volcanos main magma chamber appears to lie several kilometers below the surface, although as Hammond notes, its likely that magma pools at many depths in the crust. Finer imaging is needed, Lei says. The ndings so far, he says, are rough. North Korean scientists have taken a stab at mapping Paektus main magma chamber. In 2003, they built their own MT machine. Im very impressed, Hammond says. Its a complicated technique. While the approach is widely used in oil exploration and other subsurface mapping, few geophysicists are familiar with the complex devices. Asked to explain their resourcefulness, one North Korean volcanologist
FIRE IN THE HOLE

0 -5 -10 -15 -20 Depth (km) -25 -30 -35 -40 -45 -50 -55 -60 0 5 10 15 20 25 30 35 40 Distance (km) Changbai/Paektu Ohm meter (log) 3.4 3.0 2.5 2.0 1.5 1.0
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Belly of the beast. Using magnetotelluric sound-
ings, Chinese scientists have imaged what they presume is Changbais main magma chamber. North Korean MT data suggest magma is ascending.
What these phenomena augur is a matter of debate. The new evidence shows that the volcano will soon enter an active phase, argues Liu Guo Ming, deputy director of Changbaishan Volcano Observatory. Geologist Yun

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Sizing it up. The millennium eruption was one of the biggest in the past several thousand years, as
Lasting impression. Gravity-defying
spires, formed by escaping gas, cling to the walls of a pyroclastic valley gouged during the millennium eruption.
responds, simply, Juche!North Koreas The institute had been founded 3 years earcredo of self-reliance. lier. Among other urgent tasks in setting up Using their machine, the North Korean a network of observatories, Kim was searchteam has discerned what they believe is a ing for a patch of land with bedrock near the magma chamber at a depth of 6 kilome- surface, where his team could install a seisters. Their MT data indicate that since 2007, mometer. We found the perfect spot, with the depth of magma is getting shallower. little background noise, he says. He and a We believe it is ascending, says Kim Hang colleague hauled logs up the hill and built a Myong. But he acknowledges that their MT shelter for the instrument. device has wide error bars. We wonder about It also worked well for the British broadthe reliability of our data, Kim says. Hes band seismometer during its brief run right to be cautious, Oppenheimer says. The in September. As Hammond installed it, spatial resolution of MT is very coarse. Oppenheimer used duct tape to attach a white A complete picture of Changbais plumb- plastic GPS receiver to a pine tree branch ing would only come from seismic and MT to provide a time stamp for the seismic surveys traversing the whole signals, which are recorded volcano. That would require when an earthquake rattles installing on the North Korean the extremely sensitive seisside as many as eight broadmometer. Ri Gyong Song, a band seismometers to zero in seismologist at DPRKs Instion tremors, Hammond says. tute of Volcanology, who like Cross-border surveys would Director Kim studied theoretalso require explicit agreeical physics at DPRKs elite ments from both governKim Il Sung University, took ments, scientists say. China careful notes. We urgently and North Korea are allies need this kind of seismobut wary of monitoring. Last meter, he says. July, North Korea adopted a Good vibrations. Ri Gyong North Korea has deployed law requiring foreigners who Song is hoping for broadband six seismometers at Paektu, want to carry out volcano- seismometers. but only one is digital; the logical or seismic research to rest are modified Chinese get permission from the Earthquake Admin- analog seismometers. The researchers use istration, and North Korean scientists must solar panels and car batteries to power their accompany them in the field. (China has instruments and a transmitter that sends data similar regulations.) Seismic measurements to Pyongyang via a national intranet. There are sensitive for another reason. In Septem- are frequent outages in winter, when snow ber 2006, several days before its rst nuclear covers the panels, Kim Hang Myong says. test, North Korea asked China to turn off its Despite the hardships, the 200 North seismometers near the border. They repeated Korean researchers who study the volcano the request before their second test in 2009. have a jaw-dropping asset: a station operated year-round on the shore of the caldera lake. Caldera duty Its unbelievable. I cant think of anyplace In the summer of 1999, Kim Myong Song, else that has a manned station inside a crater, director of DPRKs Institute of Volcanol- Oppenheimer says. If the volcano were to ogy, conducted an arduous survey of the awaken in summertime, the sentinels at Lake steep hillside behind Paektu Bridge station. Chon Research Station could ee up a stone
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Volcanologists in arms The Chinese and Koreans devote great attention to the volcano not only because it poses a serious threat but also because it resonates deeply with their cultural identities. The Chinese consider Changbai the birthplace of the Manchu people. Koreans revere the mountain. Paektu represents our nations soul. Its our ancestral home, Kim Myong Song says. Oppenheimer and Hammond realize that getting an international research program at this iconic mountain off the ground will require more than their expertiseit will take science diplomacy, too. There are many conflicts that can arise when a foreign team of scientists comes to a volcano. They arrive, collect data, write papers, and go home, Oppenheimer says. Its important that the Koreans do this on their terms. The Koreans say they are ready. We can ensure conditions for data sharing and making joint measurements, says Pak Kwang Pam, head of foreign technical cooperation with DPRKs Seismological Bureau. All signs suggest things are off to a promising start, Oppenheimer says: I hope very much this is just the beginning.
staircase that zigzags several hundred meters to the caldera rim. By mid-October, snow and ice make the steps too treacherous to navigate. Four men overwinter in the three-room warren. If the staircase were impassable in an emergency, they would rely on their compatriots to put an Austrian-built gondola, shuttered in winter, into service. The teams main task is to track gases percolating up from the volcanos vent, which is 384 meters below Tianchis surface, on the Chinese side, and two underwater springs to the south. They head out on Tianchi by boat to take samples every 5 days until December, when the lake freezes over. From then until the thaw, they strike out on the ice on foot, sampling from all three springs, including the vent on the Chinese side. The lake is common area, a technician explains. Researchers analyze samples in a cozy room with heated linoleum oors. On the wall, a long wooden board, painted red with white Korean characters, offers encouragement: Great Leader Kim Il Sung lives together with us. With several gas sensors out of commission, technicians are only able to measure chloride and pH, uctuations of which may presage an eruption. With the volcano in a quiet phase, Chinese researchers are content to measure gases wafting from hot springs in their territory every 3 months.
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obvious to me that black people were living a very oppressed life, he says. But at age 21, studying history as an undergraduate at Harvard, Trivers suffered a mental breakdown. He would stay up all night, reading the 20th century philosopher Ludwig Wittgenstein. He was convinced that he had insights into Wittgensteins ideas that no one else had. Trivers wound up spending 3 months in a hospital. Such breakdowns would recur throughout his life and cause him immense suffering, but Trivers calls that rst one the most painful: In the beginning, I did not know who or what I was. When he got back on his feet, he applied to law school but decided against it when the schools wanted a copy of his medical records. After Trivers started to work for a company illustrating and then writing schoolbooks for fifth-graders, he discovered the beauty of evolution. Three billion years of the history of life, it is such a magnicent view, he says. So at age 24, Trivers went back to Harvard to study biology not knowing anything at all about animals. He even claims that fellow students showed him pictures of a rhinoceros and a hippopotamus and asked him which is which. I had a 50-50 chance, and I still chose wrong. Seeing life as conict Nonetheless, Trivers turned out to be an immensely original thinker in biology. His strength has been to see conflict where other people see only harmony. In the baby growing in a womb, he saw a struggle for resources between mother and child. In the romantic love between a man and a woman, he saw a pair eternally at odds because of their differential investment in their offspring. Whereas others see optimism and self-deception as a defensive strategy to stay sane and happy in a harsh world, he sees it as a psychological attack mechanism, fooling yourself to better fool others, he says. Conict has been a recurring theme not only in Triverss work but also in his life. Stories of his reckless and aggressive side abound. He loves to use the words fuck and motherfucker, calling them quite use-
PROFILE: ROBERT TRIVERS
Sharp Insights and a Sharp Tongue

Brilliant but troubled, Robert Trivers made his mark dissecting the evolution of human relationships. In a new book, he tackles deceit and self-deception
Mention the name Robert Trivers to those in the know, and the reaction you get is awe. Harvard University psychologist Steven Pinker calls him one of the great thinkers in the history of Western thought. Stuart West, a professor of evolutionary biology at the University of Oxford in the United Kingdom, recently described him as one of the most inuential evolutionary biologists since Charles Darwin. And in 1999, Time named him one of the 20th centurys 100 greatest thinkers and scientists. Yet most biology students do not even know the name. In the 1970s, Trivers penned a series of landmark papers that have been cited thousands of times and opened up the study of human relationships to biology. Looking in turn at interactions between friends, then lovers, then parent and child, he helped to lay the foundations for sociobiology, or a Darwinian social theory, as he called it. Then he disappeared from view, only to reemerge in the 1990s with a remarkable second career that has also led to his latest effort: a book called The Folly of Fools: The Logic of Deceit and Self-Deception in Human Life that might well introduce him to a larger audience. The person who has led this remarkable life is a man of 68 years, with a deep voice and an infectious laugh. He has recently had his hip replaced and does some leg exercises while being interviewed. Trivers likes to make fun of the fact that he has grown old. Did you know that the enjoyment of sex is actually correlated with sperm count in the ejaculate? he asks. So it is true that in old age you appreciate the smaller things more. There are no big things to enjoy anymore. But he also brags about his beautiful new black leather coat and has the air of a young man more interested in wine and women than lectures. Trivers followed a winding road to biology. He was interested in pure mathematics rst, having taught himself differential and integral calculus when he was 14. But by 18 he had lost interest and wanted to become a lawyer. I wanted to ght for justice, for the poor, and against racial discrimination, he says without a trace of irony. Indeed, one of his most vivid memories of childhood is his mother coming to the dinner table in tears, because a white police ofcer in Washington, D.C., had shot to death a 14-year-old black child for jaywalking. Growing up in Maryland, it was very
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ful, and he has gotten into public Trivers had gured out that spats with many people over the the difference in parental investyears. Trivers can be brutally honment is the most important difest and plain rude, as many letters ference between the sexes, one he has written to colleagues over from which all else springs. the years testify. True to form, in While the human male conhis new book, Trivers is scathtributes only a sperm that he ing of NASA and U.S. foreign can produce millions of, the policy, derides Turkeys denial female invests in a 9-month of an Armenian genocide, and pregnancy producing a 3-kiloargues that the genocide perpegram baby. Naturally, Trivers trated by Nazi Germany on Euroargued, her strategy for choospean Jews was far from unique. ing a partner had to be different The notion of the holocaust has from that of the male, leading spurred the growth of an industo a difference in psychology. try designed to extract long-ago Females are pickier and focus costs of this event, which ow not on a males genetic quality, stato the camp survivors but to their tus, and his willingness to invest distant cousins, usually nowhere Period piece. Robert Trivers (left) with Black Panther co-founder Huey in the offspring. Males compete near the camps, while serving to Newton (center) and another friend, Jay Friedheim (right), during Triverss for women and focus on physijustify Israels frequent attacks fallow years in California. cal evidence of fertility, among on its Arab neighbors, he writes. other attributes. Asked whether his discussion of Middle Harvard biomathematician Martin Nowak. For nights after this insight, Trivers East politics might not turn off some people And the implications were stunning. Recip- remembers dreaming of a long corridor whom he might otherwise convince of his rocal altruism led to cheating, that led to with two animals of each kind in it. Then ideas, he just says, Well, fuck em. defense against cheating, and that led to the in response to an unknown signal, each Ironically, Triverss rst contribution to evolution of a sense of fairness, friendship, pair separated, males heading for a door on biology was on cooperation. Evolutionary and trust, Trivers says. one side, females heading for another door biologist William Donald Hamilton had rst on the other. It felt symbolic of what I had proposed in the 1960s that helping a relative Friends, lovers, and children achieved, he says. even at a cost to oneself could be advanta- Most biologists spend their lives studying Having dissected friendship and love in geous in evolutionary terms because relatives ants, geese, or other animals and then extend quick succession, publishing key papers in share many of ones genes. Survival in the their conclusions to humans later in life. 1971 and 1972, Trivers turned in 1974 to the long term boils down to being successful at Trivers tended to start with humans. Some of relationship of parents and offspring. There passing on ones genes. Thus, in Hamiltons his creativity is to look at himself to under- was all this nonsense at the time about parview, a gene that would make an animal sac- stand. So a lot of Roberts papers say a lot ents teaching their children language and rice itself to save three of its siblings would about himself, says Harvard biologist David culture in a completely disinterested fashoutcompete other genes. Because each sib- Haig, one of Triverss closest friends. Indeed, ion and the child just being a vessel that they ling has a 50% chance of carrying this gene Haig says, Trivers was predestined to write the were lling, he scoffs. In fact, there was for family love, it would in effect be sacricing just one copy of itself to save 1.5 copies of itself, a smart choice. Similar logic I thought, Wait a minute. Kinship is obviously important, but applies to more distantly related relatives such as cousins, but more of them would friends can actually be more important. have to be savedthe number dependent on their degrees of relatednessto make the sacrice worthwhile. paper on reciprocal altruism, because it is so a battle for resources that started with the Based on his own life experiences, close to how he himself behaves, being nice if fetus growing in the mothers body. Later, Trivers realized that this nepotistic altruism someone is nice to him rst. Robert likes his the mother wants to cut down on the milk so could not be the whole story. I thought, Wait reciprocal altruism up front, he jokes. she can have her next offspring, but the child a minute. Kinship is obviously important, but Then, Trivers turned from friends to lov- wants to keep suckling, so there is weaning friends can actually be more important, he ers. Observing male pigeons hustling females conflict, he explains. Trivers termed this says. In 1971, he published a paper describ- while their own partners were caring for parent-offspring conict. ing the idea of reciprocal altruism. Helping a the eggs in the nest, but getting agitated as As with his other insights, he did not nonrelative could also be benecial, Trivers soon as another male approached its mate, elaborate on the principle. He is interested argued, if it did not cost too much and if there Trivers felt reminded of human double stan- in the big picture rather than fussing around was a likelihood that the two would meet dards in regard to sexual relationships. He with the details, says Haig, who adds that again and the other person would then recip- spent 9 months collecting 70 relevant papers, it is hard to find a similarly productive rocate. Like all great ideas in science, in which he read over the course of three intense period in evolutionary theory. Each of retrospect it seems intuitive and obvious, but days holed up in his apartment. Then I wrote those papers founded a new eld of research. at the time it was immensely original, says the paper, working 24/7 for a month, he says. It is incredible.
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Disappearing act But fame and fortune didnt follow Trivers the way they favored others. Oxfords Richard Dawkins started his road to stardom with The Selsh Gene, a book greatly inuenced by Triverss ideas. (Look up Trivers in the index, and you will nd him referenced on 30 pages, roughly a tenth of the book.) Trivers, meanwhile, felt underpaid at Harvard. The year was 1978. I was earning only $14,000 a year then, Trivers says. At the time, his wife was pregnant with twins. I was teaching about reproductive success, and the university was not paying me enough to have any of my own, he says. When Trivers applied for early tenure at Harvard, the university decided to delay the decision for 3 years. According to ish genetic elements. By then, Triverss theories on parent-offspring conict had been spectacularly conrmed by the discovery of imprinted genes, like Igf2, in which just one copy of a gene is active, not the usual two (Science, 25 September 1998, p. 1984). The Igf2 protein makes the fetus in the womb grow faster. The mother inactivates the gene in the egg to rein in growth, trying to sequester some of her resources for future pregnancies. But the fathers copy is still going strong, making the fetus grow as much as it can. The book Genes in Conflict was published in 2006 to great academic acclaim. A year later, Trivers received the Crafoord Prize. One of biologys most prestigious prizes, it was established by the Swedish rest of us, he fell down when it came to seeing through his own self-deception. Trivers has dedicated the book to Newton. A difcult mind Much of Triverss life has been overshadowed by his struggle with bipolar disorder. (It was his mentor, the famous biologist Ernst Mayr, who realized that a rst diagnosis of schizophrenia was wrong.) Ive spent almost a year of my life locked up, usually in mental institutions, sometimes in police stations, sometimes both, Trivers says. Only once, during his second breakdown, did he feel the disease actually spurred some creative thinking. That was in 1972 on a trip to East Africa. Trivers was losing sleep, thinking about parentoffspring conict, when it suddenly occurred to him that the conict extended far beyond ghting over milk. There was also a conict over how the child should behave. Because I share only half my genes with my brother, I am selected to transfer benet to him only if that benet is twice as big as the cost to me. But my mother is equally related to the two of us, so she wants to encourage me to help my brother whenever the benet is greater than the cost. So there is real psychological conict built into the parent-offspring relationship. That was a revelation to me. But that insight had its cost. After returning to the United States, Trivers was hospitalized for 10 days before he could return to work. After that, the breakdowns were uniformly bad, he says. Triverss life has not been easy, and he has sometimes made life hard for those around him. Over the years, Ive seen the periods of psychotic mania and depression. In those periods, he can become more difcult to deal with, and Ive learned to weather that, says Haig, who says Trivers is the most unusual scientist he knows. Nowak agrees: Meeting Robert is never an everyday encounter. Nor has Trivers ever been an everyday biologist. His latest theory on self-deception is sure to rufe some feathers, and University of Chicago zoologist Jerry Coyne says the book suffers from a lack of tangible zoological examples. But Triverss forte has never been to show what has happened but what could happen, he says. He calls Trivers one of those thinkers whose importance rests on inspiring a generation of researchers. There is a contradiction at the heart of his life and his work. Trivers might be a difcult character, and his life might have been rough at times, but his big ideas have always been simple and elegant.
Kai Kupferschmidt is a science writer in Berlin.
Ive spent almost a year of my life locked up, usually in mental institutions, sometimes in police stations, sometimes both.
him, it was because of his bipolar disorder. I had by then had three breakdowns, one as a faculty member, he says. Many myths revolve around why Trivers did not get early tenure. Some think he got caught up in the war over sociobiology that had erupted on the campus after E. O. Wilson published his book with that title. Harvard scientists such as Stephen Jay Gould and Richard Lewontin saw Triverss and Wilsons work trying to explain human relationships in evolutionary terms as a theory with no scientic support. Sociobiology was aimed at defending the status quo as an inevitable consequence of human nature, they wrote in a letter to The New York Review of Books. I have heard stories according to which Robert got done in by pretty much everyone here. They cannot all be true, Haig says. Well, I guess they could. In the end, Trivers decided to take an offer from the University of California, Santa Cruz. He started teaching there in 1978, befriended Huey Newton, co-founder of the Black Panther Party, wrote a textbook on social evolution that he says was ahead of its time but never sold well, and largely disappeared from view. Harvard anthropologist Irven DeVore calls it Triverss fallow period. In the 1990s, Trivers resurfaced on the East Coast. He joined the faculty of Rutgers University in New Brunswick, New Jersey, partly to be closer to his children. And he started to turn toward conict again, professionally as well as privately. Together with Austin Burt, a geneticist at Imperial College London, he began working on a book on self-
industrialist Holger Crafoord, who commercialized articial kidneys, and presented to Trivers by the queen of Sweden. At the ofcial banquet, Trivers gave a shout-out to everyone who had only one kidney for making this award possible. But the honor didnt mellow the man. Trivers is in the middle of a dispute with William Brown, now at the University of Bedfordshire in the United Kingdom, with whom he published a paper on symmetry and dance in Nature in 2005. The paper appears to show that men and women with more symmetric bodies are also better dancers, with dancing thus being a possible indicator of genetic quality. But Trivers has accused Brown, who he says was in charge of the statistics, of preselecting the dancers and changing the values on some of the dancers measures of symmetry to get that result. Trivers has even written a short book about it that he sends to whoever cites the paper. Brown will only say that Rutgers is investigating the matter, and Nature has no comment. Conicts have not slowed Trivers down, however. At Rutgers, he resumed work on a long-gestating project, the book on self-deception that he had started writing with Huey Newton in the 1980s. (Its central hypothesis is that our ability to deceive ourselves evolved in order to deceive others.) Trivers calls Newton, who was shot and killed in 1989, a master in three out of four aspects of deception and selfdeception: He was a master at propagating deception. He was a master at seeing through your deception. He was a master at beating your self-deception out of you. And like all the
KAI KUPFERSCHMIDT

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COMMENTARY
Copernican complexities Neurobiology prize essay
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edited by Jennifer Sills
Conservation: Limits of Land Sparing

ACCORDING TO B. PHALAN ET AL. (RECONCILING FOOD PRODUCTION AND BIODIVERSITY CONservation: Land sharing and land sparing compared, Reports, 2 September, p. 1289), land sparingprotecting some land and farming the rest intensivelysaves biodiversity more effectively than land sharingprotecting less land but farming the remainder with wildlifefriendly techniques. The authors qualify their conclusion, saying that it hinges on proper implementation and may not be transferable and that intensicationincreasing the harvest yield could be achieved through higher inputs of knowledge and labor rather than chemicals. These and other considerations severely limit land sparing in practice. First, many countries lack the means to effectively protect areas but do have a long record of sustainable land sharing (1, 2). Second, situations exist where both yields and biodiversity are high (3) or where biodiversity depends on agriculture (4). Third, vast regions with shallow soils or low rainfall are only suitable for non-intensive use (5). Ironically, where intensication without chemicals is possible, this comes very close to land sharingnamely, knowledgeintensive agroecological systems with multiple crops and a complex structure. The debate about land sparing versus land sharing is poorly framed as a black-and-white choice that must be made to feed the worlds people. In fact, the choice is not between one and the other (6, 7), nor does a higher quantity of food guarantee less hunger. Most famines are caused by a lack of access to food, rather than too little food (8). The simple model by Phalan et al. ignores vital social and ecological complexities, including rural livelihoods, the dependence of the worlds poor on local Land sharing. A wildlife-friendly landscape in Romania. ecosystem services, and the lack of reliable governance of many protected areas. It also ignores the fact that, in reality, use of agrochemicals is likely to be the default method to increase yields, which would have negative environmental side effects. Social and ecological complexities must not be an afterthought in analyses about food and biodiversity, because they fundamentally alter the outcome. Simple models must be balanced with holistic, eld-based approaches (9, 10). Otherwise there is a great risk that internally consistent solutions are overinterpreted as externally applicable by policy-makers, the media, and the public.
JOERN FISCHER, * PTER BATRY, KAMALJIT S. BAWA, LIJBERT BRUSSAARD, M. JAHI CHAPPELL, YANN CLOUGH,2 GRETCHEN C. DAILY,6 JOSH DORROUGH,7 TIBOR HARTEL,8 LOUISE E. JACKSON,9 ALEXANDRA M. KLEIN,1 CLAIRE KREMEN,10 TOBIAS KUEMMERLE,11 DAVID B. LINDENMAYER,12 HAROLD A. MOONEY,6 IVETTE PERFECTO,13 STACY M. PHILPOTT,14 TEJA TSCHARNTKE,2 JOHN VANDERMEER,13 THOMAS CHERICO WANGER,1,6 HENRIK VON WEHRDEN1,15
1 2 3 4 5
*To whom correspondence should be addressed. E-mail: joern.scher@uni.leuphana.de

1. L. Persha, A. Agrawal, A. Chhatre, Science 331, 1606 (2011). 2. J. Ranganathan, R. J. Ranjit Daniels, M. D. Subash Chandran, P. R. Ehrlich, G. C. Daily, Proc. Natl. Acad. Sci. U.S.A. 105, 17852 (2008). 3. Y. Clough et al., Proc. Natl. Acad. Sci. U.S.A. 108, 8311 (2011). 4. R. D. Gregory et al., Philos. Trans. R. Soc. London Ser. B 360, 269 (2005). 5. J. Dorrough, J. Moll, J. Crosthwaite, Agric. Ecosyst. Environ. 121, 222 (2007). 6. J. Fischer et al., Front. Ecol. Env. 6, 380 (2008). 7. L. Brussaard et al., Curr. Opin. Env. Sustain. 2, 34 (2010). 8. A. Sen, Poverty and Famines (Oxford Univ. Press, Oxford, 1981). 9. H. C. J. Godfray, Science 333, 1231 (2011). 10. J. Fischer et al., Trends Ecol. Evol. 24, 549 (2009).
References
CREDIT: KIMBERLIE RAWLINGS

B. PHALAN AND COLLEAGUES (RECONCILING food production and biodiversity conservation: Land sharing and land sparing compared, Reports, 2 September, p. 1289) report that land sparing would do less harm to biodiversity than land sharing and conclude that

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Institute of Ecology, Leuphana University Lueneburg, 21335 Lueneburg, Germany. 2Department of Agroecology, Georg-August-University Goettingen, 37077 Goettingen, Germany. 3Department of Biology, University of Massachusetts Boston, Boston, MA 02125, USA. 4Graduate School for Production Ecology and Resource Conservation, Wageningen University, 6700 AA Wageningen, The Netherlands. 5 School of Earth and Environmental Sciences, Washington State University Vancouver, Vancouver, WA 98686, USA. 6 Department of Biology, Stanford University, Stanford, CA 943055020, USA. 7CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia. 8Mihai Eminescu Trust, Sighisoara 545400, Romania. 9Department of Land, Air, and Water Resources, University of California, Davis, CA 95616, USA. 10 Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720 3114, USA. 11Department of Geography, HumboldtUniversitt zu Berlin, 10247 Berlin, Germany, and Potsdam Institute for Climate Impact Research, 14412 Potsdam, Germany. 12Fenner School of Environment and Society, The Australian National University, Canberra, ACT 0200, Australia. 13School of Natural Resources and Environment, University of Michigan, Ann Arbor, MI 48109, USA. 14Department of Environmental Sciences, University of Toledo, Toledo, OH 436063390, USA. 15Center for Methods, Leuphana University Lueneburg, 21335 Lueneburg, Germany.
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the best strategy for conserving biodiversity would be high-yield farming combined with natural habitat protection. However, the controlling factor in the equation is farm management intensity; changes in intensity affect both harvest yield and biodiversity density. Therefore, a model integrated with management intensity as a decision variable is necessary for reconciling food production and biodiversity conservation. Management practices, rather than land-use types, should be the driving force for the reconciliation. onciling agricultural production and conservation. Even in places where land sparing is preferable in principle, achieving its potential benets will entail addressing intricate social, political, and technical issues (1). That said, delivering the intended benets of land sharing also involves formidable challenges, despite the substantial effort that has been invested. Where land-sharing practices have been identied and incentivized, there is often inadequate monitoring of their quality and effectiveness (2). Land sharing can also have the serious unintended consequence of accelerating conversion or degradation of remaining natural habitats (35). Fischer et al. outline several situations where land sharing appears to be a better conservation strategy than land sparing. However, none of the studies they cite provides adequate data to demonstrate greater benets to biodiversity from land sharing (6). The studies rely on simplistic biodiversity metrics such as species richness from which population-level impacts cannot be assessed (710), fail to make comparisons with appropriate baseline habitats (79), do not quantify yields or other benets to people (10, 11), do not quantify outcomes from land sparing to compare with those from land sharing (811), or do not refer to agriculture at all (9). Fischer et al. correctly point out that some species now depend on agriculture. However, given that all species have thrived without it for most of their evolutionary history, whether it is essential for their persistence into the future is open to question. We agree that famine is caused largely by inequitable access to food, but our argument is independent of the need to solve world hunger. Regardless of how much food is produced, society has choices to make about how and where to grow it. Our data suggest that high-yielding, land-sparing approaches at least in southwest Ghana and northern Indiahave the greatest potential benets for biodiversity, because they give explicit attention to the importance of conserving remaining habitats. This pattern holds whether the required production of food is more or less than at present (see Fig. 2 in our Report). We believe that the best way to develop an understanding of the difcult and complex
KIYOTADA HAYASHI
National Agriculture and Food Research Organization, Kannondai 3-1-1, Tsukuba, Ibaraki 305-8666, Japan. E-mail: hayashi@affrc.go.jp
Response
FISCHER ET AL. DO NOT DISPUTE OUR FINDING that, in principle, land sparing would be more effective than land sharing for the taxa and areas we studied, but they appear to feel that land sparing is neither achievable nor desirable in practice. We agree that there are no simple, generic solutions to complex problems such as rec-
TECHNICAL COMMENT ABSTRACTS
CORRECTIONS AND CLARIFICATIONS

Books et al.: Picture perfect future past by G. Riddihough (7 October, p. 41). The rst sentence of the third paragraph should have read But, as Out of This World documents, modern science ction is more than the warmed-over imaginings of earlier generations. Reports: Three-dimensional Anderson localization of ultracold matter by S. S. Kondov et al. (7 October, p. 66). Reference 14 [F. Jendrzejewski et al., http://arxiv.org/abs/1108.0137 (2011)] should have been accompanied by a note stating that while their paper was in proof, Kondov et al. were made aware of related three-dimensional experiments that were released in this preprint. News Focus: False positive by J. Cohen and M. Enserink (23 September, p. 1694). In the last sentence in the rst column of p. 1699, the story credits the author of the blog CFS Central with a call for aggressive, ACT UPstyle protests and the quote, I believe we need to act quickly, before the FDA/NIH paper is killed. In fact, it was a patient who wrote this on the blog, not the blog author. Reports: African wild ungulates compete with or facilitate cattle depending on season by W. O. Odadi et al. (23 September, p. 1753). An incorrect e-mail address was given for the rst corresponding author. The correct address is woodadi@yahoo.com. The online HTML version has been corrected. In Table 2, the heading for the fourth column should be Dead stems (hits/100 pins). The reference callouts at the end of the last sentence beginning on page 1754, which begins We posit that..., should include reference (22), to read ...in the Serengeti ecosystem (18, 2022). The reference callout (22) in the nal paragraph on page 1755 should be (23). Reports: Activation of -catenin in dendritic cells regulates immunity versus tolerance in the intestine by S. Manicassamy et al. (13 August 2010, p. 849). Some of the arrows in Fig. 4C were incorrect, and the original images did not fully demonstrate the line in the text that referred to increases in inammatory cell inltration, edema, epithelial cell hyperplasia, and loss of goblet cells in the colon of -catDC/ mice as compared with -cat/ mice. In the revised gure shown here, the arrows have been corrected and new panels have been added. The corrected caption is: Histopathological changes in colon tissue from -cat/ or -catDC/ mice treated with or without 2% DSS treatment for 7 days. Areas of interest are inltration of inammatory cells (black arrows), edema (yellow arrows), and loss of crypts (green arrows) and goblet cells. The changes described here do not affect the Reports conclusions.
Comment on Atmospheric Pco2 Perturbations Associated with the Central Atlantic Magmatic Province
Michael R. Rampino and Ken Caldeira
Schaller et al. (Research Article, 18 March 2011, p. 1404) proposed that carbon dioxide (CO2) released by the Central Atlantic Magmatic Province eruptions over periods of about 20,000 years led to substantial increases of up to 2000 parts per million (ppm) in the concentration of atmospheric carbon dioxide (Pco2) near the Triassic-Jurassic boundary. Use of an atmosphere-ocean model coupled to a carbon-cycle model predicts Pco2 increases of less than 400 ppm from magmatic volatiles, with only a small climatic impact. Full text at www.sciencemag.org/cgi/content/full/334/6056/594-b
Morgan F. Schaller, James D. Wright, Dennis V. Kent
Rampino and Caldeira argue that the rst pulse of the Central Atlantic Magmatic Province would increase the concentration of atmospheric carbon dioxide (Pco2) by only 400 parts per million if erupted over 20,000 years, whereas we observed a doubling within this interval. In the absence of any data to the contrary, we suggest that a more rapid (1000-year) eruption is sufcient to explain this observation without relying on thermogenic degassing. Full text at www.sciencemag.org/cgi/content/full/334/6056/594-c
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LETTERS
choices involved in land-use decisions is to use theoretical models to guide collection of the critical empirical data and evaluate potential solutions based on measurable outcomes. Extending our approach to incorporate other objectives such as cultural values and ecosystem services, and to develop strategies with strong social safeguards, should be a priority. We contend that it would be premature to dismiss the potential benets of land sparing as undeliverable in practice when so little effort has been made so far to develop the right tools to implement it. Hayashi proposes extending our analytical approach to better understand how management intensity affects agricultural yields and species populations. We agree that such information could be useful for informing decisions by farmers and foresters and that it might be used to improve outcomes from land sharing, land sparing, or intermediate strategies. Our approach focused on decision-making at larger scales, for which understanding the consequences of land-use allocation is more relevant. Management intensity is shorthand for a diverse range of practices, from fertilizer use to tree husbandry to management of hunting, and it is often not clearly dened. High-yield farming need not necessarily involve intensive management in the sense of having high agrochemical inputs. Sustainable intensication using resource-efcient practices seems more likely to increase yields with minimal resource degradation and pollution (12). Research on the impacts of different management practices will produce useful insights if it can move beyond simplistic biodiversity metrics, quantify pollution and other costs, and integrate ne-scale management concerns with the need to address largescale land-use change.
1
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK. 2Royal Society for the Protection of Birds, The Lodge, Sandy SG19 2DL, UK. *To whom correspondence should be addressed. E-mail: btp22@cam.ac.uk
Letters to the Editor

Letters (~300 words) discuss material published in Science in the past 3 months or matters of general interest. Letters are not acknowledged upon receipt. Whether published in full or in part, Letters are subject to editing for clarity and space. Letters submitted, published, or posted elsewhere, in print or online, will be disqualied. To submit a Letter, go to www.submit2science.org.
1. E. H. A. Mattison, K. Norris, Trends Ecol. Evol. 20, 610 (2005). 2. D. Kleijn, M. Rundlf, J. Scheper, H. G. Smith, T. Tscharntke, Trends Ecol. Evol. 26, 474 (2011).
References

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BEN PHALAN,1* MALVIKA ONIAL,1 ANDREW BALMFORD,1 RHYS E. GREEN1,2
3. C. Tejeda-Cruz, E. Silva-Rivera, J. R. Barton, W. J. Sutherland, Ecol. Soc. 15, 13 (2010). 4. M. D. Madhusudan, Conserv. Biol. 19, 411 (2005). 5. H. R. Grau, N. I. Gasparri, T. M. Aide, Glob. Change Biol. 14, 985 (2008). 6. B. Phalan, A. Balmford, R. E. Green, J. P. W. Scharlemann, Food Policy 36, S62 (2011). 7. J. Dorrough, J. Moll, J. Crosthwaite, Agric. Ecosyst. Environ. 121, 222 (2007). 8. Y. Clough et al., Proc. Natl. Acad. Sci. U.S.A. 108, 8311 (2011). 9. L. Persha, A. Agrawal, A. Chhatre, Science 331, 1606 (2011). 10. J. Ranganathan, R. J. Ranjit Daniels, M. D. Subash Chandran, P. R. Ehrlich, G. C. Daily, Proc. Natl. Acad. Sci. U.S.A. 105, 17852 (2008). 11. R. D. Gregory et al., Philos. Trans. R. Soc. London Ser. B 360, 269 (2005). 12. Foresight, The Future of Food and Farming (Government Ofce for Science, London, UK, 2011).
TECHNICAL COMMENT Comment on Atmospheric P Co2 Perturbations Associated with the Central Atlantic Magmatic Province
Michael R. Rampino1,2* and Ken Caldeira3 Schaller et al. (Research Article, 18 March 2011, p. 1404) proposed that carbon dioxide (CO2) released by the Central Atlantic Magmatic Province eruptions over periods of about 20,000 years led to substantial increases of up to 2000 parts per million (ppm) in the concentration of atmospheric carbon dioxide (PCO2) near the Triassic-Jurassic boundary. Use of an atmosphere-ocean model coupled to a carbon-cycle model predicts PCO2 increases of less than 400 ppm from magmatic volatiles, with only a small climatic impact. challer et al. (1) proposed that large amounts of CO2 released by the Central Atlantic Magmatic Province (CAMP) (2.6 106 km3 of magma) eruptions contributed to substantial increases in atmospheric CO2 near the TriassicJurassic boundary. With an estimated volcanic efflux of 1.4 1010 kg of CO2 per km3 of basaltic magma (2), the total CO2 release for the CAMP basalts is about 3.4 1016 kg of CO2. There are three lava flow events in the Newark and Hartford Basins (1), so that each of the three lava 103 m3 s1 (or 1.1 107 kg s1), then it would be possible to produce ~106 km3 of lava in about 20 ky of semicontinuous eruption. The time over which the magma is erupted affects the amount of CO2 that stays in the atmosphere. We used an ocean-atmosphere box model coupled to a carbon-cycle model (6, 7) to simulate increases in PCO2 resulting from the CAMP eruptions for two cases: an unrealistic instantaneous release, and release over a period of 20 ky (Fig. 1). Instantaneous release of 1016 kg of CO2 results in an increase of PCO2 of about 1300 ppm over a high early Jurassic background of about 2000 ppm, similar to results of Schaller et al. (1). If the release is accomplished over a substantial time period, then one must take into account uptake of CO2 by the oceans, and if long enough, interactions with the solid earth through rock weathering. When released over 20 ky, the increase results in a peak PCO2 value of only about 400 ppm over early Jurassic background (Fig. 1). Thus, magmatic CO2 release alone, even for eruptions producing a million cubic kilometers in periods as short as 20 ky, is probably not sufficient to cause major climatic changes and mass extinction (Fig. 1). Schaller et al. (1) estimated PCO2 up to 4000 to 5000 ppm after CAMP eruptions based on pedogenic carbonates. Palaeobotanical evidence suggests that PCO2 may have increased by a factor of four across the Triassic-Jurassic boundary (8), and palynological studies have been interpreted as indicating an atmospheric PCO2 at least 10 times present levels, with temperatures rising by about 10C (9). If these estimates are correct, then an additional source of CO2 from interactions between CAMP magma and country rock (10) or release of marine hydrate deposits (11) seems to be required to explain the evidence for very high early Jurassic PCO2 at the time of the CAMP eruptions.
1. M. F. Schaller, J. D. Wright, D. V. Kent, Science 331, 1404 (2011). 2. S. Self, M. Widdowson, T. Thordarson, A. E. Jay, Earth Planet. Sci. Lett. 248, 518 (2006). 3. Intergovernmental Panel on Climate Change, Climate Change 1995, J. T. Houghton, et al., Eds. (Cambridge Univ. Press, Cambridge, 1996). 4. C. Dessert et al., Earth Planet. Sci. Lett. 188, 459 (2001). 5. V. Courtillot et al., Earth Planet. Sci. Lett. 80, 361 (1986). 6. K. Caldeira, M. R. Rampino, Geophys. Res. Lett. 17, 1299 (1990). 7. K. Caldeira, M. R. Rampino, Geophys. Res. Lett. 18, 987 (1991). 8. J. C. McElwain, D. J. Beerling, F. I. Woodward, Science 285, 1386 (1999). 9. B. van de Schootbrugge et al., Palaeogeogr. Palaeoclimatol. Palaeoecol. 244, 126 (2007). 10. H. Svensen et al., Nature 429, 542 (2004). 11. G. R. Dickens, J. R. ONeil, D. K. Rea, R. M. Owen, Paleoceanography 10, 965 (1995). 20 May 2011; accepted 5 October 2011 10.1126/science.1208653
1 Department of Biology and Environmental Studies Program, New York University, 100 Washington Square East, New York NY 10003, USA. 2NASA, Goddard Institute for Space Studies, 2880 Broadway, New York, NY 10025, USA. 3Department of Global Ecology, Carnegie Institution, 260 Panama Street, Stanford. CA 24305, USA.
*To whom correspondence should be addressed. E-mail: mrr1@nyu.edu
flows could represent an erupted volume about 0.8 106 km of magma releasing 1.1 1016 kg CO2. Schaller et al. (1) estimate that the release of this much CO2 over a period of ~20 thousand years (ky) (the resolution of orbital precession) would directly increase atmospheric partial pressure of CO2 (PCO2) by ~1400 parts per million (ppm) from a base of about 2000 ppm (using a conversion factor of 7.82 1012 kg of CO2 per ppm CO2). This is less than a doubling of PCO2 and hence a global climatic warming estimated as less than ~3C (3). An eruption rate producing 0.8 106 km3 of basaltic magma in only 20 ky would be much greater than the commonly inferred 105- to 106year duration for flood basalt volcanism (4, 5). However, if one scales up from the eruption of Laki in 1783, which produced 12 km3 of basaltic lava over about 2 months (2) at a rate of 4
References
Fig. 1. Increase in atmospheric PCO2 caused by magmatic volatiles from CAMP eruption of 1016 kg of CO2 added to the atmosphere instantaneously (blue line) and over 20 ky (red line).
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TECHNICAL COMMENT Response to Comment on Atmospheric P Co2 Perturbations Associated with the Central Atlantic Magmatic Province
Morgan F. Schaller,1* James D. Wright,1 Dennis V. Kent1,2 Rampino and Caldeira argue that the first pulse of the Central Atlantic Magmatic Province would increase the concentration of atmospheric carbon dioxide (PCO2) by only 400 parts per million if erupted over 20,000 years, whereas we observed a doubling within this interval. In the absence of any data to the contrary, we suggest that a more rapid (1000-year) eruption is sufficient to explain this observation without relying on thermogenic degassing. ur observations from the Newark Basin indicate that the first pulse of the Triassic-Jurassic Central Atlantic Magmatic Province (CAMP), represented by the Orange Mountain Basalt, was emplaced within a precession cycle and resulted in a doubling of the atmospheric partial pressure of CO2 (PCO2) above pre-eruptive background levels. A simple model with instantaneous degassing [<1 thousand years (ky), within the time scale of ocean overturning] of 2.5 1017 moles of CO2 (~1.2 1016 kg), roughly the efflux potential of the first volcanic pulse, gives a ~1400 parts per million (ppm) increase in PCO2 above the ~2000-ppm background level (1). This estimate is compatible with and (admittedly, barely) within the error of the doubling from ~2000 to 4400 T 1200 ppm observed in the Newark Basin. Rampino and Caldeira (2) present a model whereby a 20-ky release of the same magnitude produces only a ~400-ppm atmospheric PCO2 increase, which they take as an indication that an additional source of CO2 is necessary to explain the observed PCO2 increase. We do not dispute this point, but it begs qualification. The cycle stratigraphic record from the Newark Basin provides a constraint on the maximum require a similar thermogenic input if the duration of each pulse was ~20 ky, which would represent a substantial repeated flux of thermogenic CO2 to the atmosphere at discrete intervals. Therefore, we are left to speculate on the precise source of the CO2 pulse recorded in the Newark Basin, which is essentially an argument of release duration versus size. In the absence of any data to the contrary, we favor a rapid release that allows the majority of each perturbation to be volcanogenic but that does not preclude a metamorphic carbon source. The doubling of PCO2 observed after each volcanic unit in the Newark Basin is broadly consistent with other lower-resolution studies that indicate a tripling to quadrupling through the interval (1214). The continued challenge to the modeling community is to devise a scenario that conforms to these observations.
References and Notes
1 Earth and Planetary Sciences, Rutgers University, 610 Taylor Road Piscataway, NJ 08854, USA. 2Lamont-Doherty Earth Observatory of Columbia University, 61 Route 9W, Palisades, NY 10964, USA.
*To whom correspondence should be addressed. E-mail: schaller@rci.rutgers.edu
duration (<20 ky) of the first pulse of magmatism, but we are not aware of any data (e.g., weathering at the tops of individual lava flows or accumulation of sediments between flows) that preclude a much more rapid release. Therefore, these release-time constraints provide two useful end-member scenarios to explain the observed changes in PCO2: Either the CO2 release was rapid and could be almost exclusively volcanogenic, or it was more protracted, which would require nearly 10 times as much CO2 [e.g., see (3, 4)] [1017 moles atmospheric reservoir versus 1018 moles atmosphere-ocean reservoir (58)], opening the possibility that it may be thermogenic in origin. Because thermogenic evolution of CO2 from CaCO3 sediments is an unlikely source [e.g., see (9)], the next largest reactive carbon pool in Earths crust is organic, which implies that the extra CO2 needed for a protracted release would be relatively depleted in 13C. However, the organic carbon d13C measurements from the Newark Basin (1) do not indicate a substantially larger 13 C-depleted component in the overall atmospheric PCO2 increase, although there is a slight d13C decrease (~0.5 per mil) above each volcanic unit. We note that some marine sections record a potential light carbon-isotope excursion at about this time (10); however, the exact relationship of the marine d13C decrease to the CAMP eruptions remains unclear (e.g., see 11). Moreover, our observation of comparable PCO2 and d13C changes after the second and third volcanic events would
1. M. F. Schaller, J. D. Wright, D. V. Kent, Science 331, 1404 (2011). 2. M. R. Rampino, K. Caldeira, Science 334, 594 (2011); www.sciencemag.org/cgi/content/full/334/6056/ 594-b. 3. D. J. Beerling, R. A. Berner, Global Biogeochem. Cycles 16, 1036 (2002). 4. R. A. Berner, D. J. Beerling, Palaeogeogr. Palaeoclimatol. Palaeoecol. 244, 368 (2007). 5. R. A. Berner, K. Caldeira, Geology 25, 955 (1997). 6. K. Caldeira, G. H. Rau, Geophys. Res. Lett. 27, 225 (2000). 7. R. A. Berner, A. C. Lasaga, R. M. Garrels, Am. J. Sci. 283, 641 (1983). 8. K. E. Trenberth, J. Geophys. Res. 86, (C6), 5238 (1981). 9. D. M. Kerrick, J. A. D. Connolly, Nature 411, 293 (2001). 10. S. P. Hesselbo, S. A. Robinson, F. Surlyk, S. Piasecki, Geology 30, 251 (2002). 11. J. H. Whiteside, P. E. Olsen, T. Eglinton, M. E. Brookfield, R. N. Sambrotto, Proc. Natl. Acad. Sci. U.S.A. 107, 6721 (2010). 12. J. C. McElwain, D. J. Beerling, F. I. Woodward, Science 285, 1386 (1999). 13. D. Beerling, Nature 415, 386, author reply 388 (2002). 14. M. Steinthorsdottir, A. J. Jeram, J. C. McElwain, Palaeogeogr. Palaeoclimatol. Palaeoecol. 308, 418 (2011). Acknowledgments: This work was supported by National Science Foundation grant 0958867. This is Lamont-Doherty Earth Observatory Contribution 7497. 24 June 2011; accepted 6 October 2011 10.1126/science.1209422
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Copernican complexities Neurobiology prize essay
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edited by Jennifer Sills
Conservation: Limits of Land Sparing

ACCORDING TO B. PHALAN ET AL. (RECONCILING FOOD PRODUCTION AND BIODIVERSITY CONservation: Land sharing and land sparing compared, Reports, 2 September, p. 1289), land sparingprotecting some land and farming the rest intensivelysaves biodiversity more effectively than land sharingprotecting less land but farming the remainder with wildlifefriendly techniques. The authors qualify their conclusion, saying that it hinges on proper implementation and may not be transferable and that intensicationincreasing the harvest yield could be achieved through higher inputs of knowledge and labor rather than chemicals. These and other considerations severely limit land sparing in practice. First, many countries lack the means to effectively protect areas but do have a long record of sustainable land sharing (1, 2). Second, situations exist where both yields and biodiversity are high (3) or where biodiversity depends on agriculture (4). Third, vast regions with shallow soils or low rainfall are only suitable for non-intensive use (5). Ironically, where intensication without chemicals is possible, this comes very close to land sharingnamely, knowledgeintensive agroecological systems with multiple crops and a complex structure. The debate about land sparing versus land sharing is poorly framed as a black-and-white choice that must be made to feed the worlds people. In fact, the choice is not between one and the other (6, 7), nor does a higher quantity of food guarantee less hunger. Most famines are caused by a lack of access to food, rather than too little food (8). The simple model by Phalan et al. ignores vital social and ecological complexities, including rural livelihoods, the dependence of the worlds poor on local Land sharing. A wildlife-friendly landscape in Romania. ecosystem services, and the lack of reliable governance of many protected areas. It also ignores the fact that, in reality, use of agrochemicals is likely to be the default method to increase yields, which would have negative environmental side effects. Social and ecological complexities must not be an afterthought in analyses about food and biodiversity, because they fundamentally alter the outcome. Simple models must be balanced with holistic, eld-based approaches (9, 10). Otherwise there is a great risk that internally consistent solutions are overinterpreted as externally applicable by policy-makers, the media, and the public.
JOERN FISCHER, * PTER BATRY, KAMALJIT S. BAWA, LIJBERT BRUSSAARD, M. JAHI CHAPPELL, YANN CLOUGH,2 GRETCHEN C. DAILY,6 JOSH DORROUGH,7 TIBOR HARTEL,8 LOUISE E. JACKSON,9 ALEXANDRA M. KLEIN,1 CLAIRE KREMEN,10 TOBIAS KUEMMERLE,11 DAVID B. LINDENMAYER,12 HAROLD A. MOONEY,6 IVETTE PERFECTO,13 STACY M. PHILPOTT,14 TEJA TSCHARNTKE,2 JOHN VANDERMEER,13 THOMAS CHERICO WANGER,1,6 HENRIK VON WEHRDEN1,15
1 2 3 4 5
*To whom correspondence should be addressed. E-mail: joern.scher@uni.leuphana.de

1. L. Persha, A. Agrawal, A. Chhatre, Science 331, 1606 (2011). 2. J. Ranganathan, R. J. Ranjit Daniels, M. D. Subash Chandran, P. R. Ehrlich, G. C. Daily, Proc. Natl. Acad. Sci. U.S.A. 105, 17852 (2008). 3. Y. Clough et al., Proc. Natl. Acad. Sci. U.S.A. 108, 8311 (2011). 4. R. D. Gregory et al., Philos. Trans. R. Soc. London Ser. B 360, 269 (2005). 5. J. Dorrough, J. Moll, J. Crosthwaite, Agric. Ecosyst. Environ. 121, 222 (2007). 6. J. Fischer et al., Front. Ecol. Env. 6, 380 (2008). 7. L. Brussaard et al., Curr. Opin. Env. Sustain. 2, 34 (2010). 8. A. Sen, Poverty and Famines (Oxford Univ. Press, Oxford, 1981). 9. H. C. J. Godfray, Science 333, 1231 (2011). 10. J. Fischer et al., Trends Ecol. Evol. 24, 549 (2009).
References
CREDIT: KIMBERLIE RAWLINGS

B. PHALAN AND COLLEAGUES (RECONCILING food production and biodiversity conservation: Land sharing and land sparing compared, Reports, 2 September, p. 1289) report that land sparing would do less harm to biodiversity than land sharing and conclude that

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Institute of Ecology, Leuphana University Lueneburg, 21335 Lueneburg, Germany. 2Department of Agroecology, Georg-August-University Goettingen, 37077 Goettingen, Germany. 3Department of Biology, University of Massachusetts Boston, Boston, MA 02125, USA. 4Graduate School for Production Ecology and Resource Conservation, Wageningen University, 6700 AA Wageningen, The Netherlands. 5 School of Earth and Environmental Sciences, Washington State University Vancouver, Vancouver, WA 98686, USA. 6 Department of Biology, Stanford University, Stanford, CA 943055020, USA. 7CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia. 8Mihai Eminescu Trust, Sighisoara 545400, Romania. 9Department of Land, Air, and Water Resources, University of California, Davis, CA 95616, USA. 10 Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720 3114, USA. 11Department of Geography, HumboldtUniversitt zu Berlin, 10247 Berlin, Germany, and Potsdam Institute for Climate Impact Research, 14412 Potsdam, Germany. 12Fenner School of Environment and Society, The Australian National University, Canberra, ACT 0200, Australia. 13School of Natural Resources and Environment, University of Michigan, Ann Arbor, MI 48109, USA. 14Department of Environmental Sciences, University of Toledo, Toledo, OH 436063390, USA. 15Center for Methods, Leuphana University Lueneburg, 21335 Lueneburg, Germany.
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the best strategy for conserving biodiversity would be high-yield farming combined with natural habitat protection. However, the controlling factor in the equation is farm management intensity; changes in intensity affect both harvest yield and biodiversity density. Therefore, a model integrated with management intensity as a decision variable is necessary for reconciling food production and biodiversity conservation. Management practices, rather than land-use types, should be the driving force for the reconciliation. onciling agricultural production and conservation. Even in places where land sparing is preferable in principle, achieving its potential benets will entail addressing intricate social, political, and technical issues (1). That said, delivering the intended benets of land sharing also involves formidable challenges, despite the substantial effort that has been invested. Where land-sharing practices have been identied and incentivized, there is often inadequate monitoring of their quality and effectiveness (2). Land sharing can also have the serious unintended consequence of accelerating conversion or degradation of remaining natural habitats (35). Fischer et al. outline several situations where land sharing appears to be a better conservation strategy than land sparing. However, none of the studies they cite provides adequate data to demonstrate greater benets to biodiversity from land sharing (6). The studies rely on simplistic biodiversity metrics such as species richness from which population-level impacts cannot be assessed (710), fail to make comparisons with appropriate baseline habitats (79), do not quantify yields or other benets to people (10, 11), do not quantify outcomes from land sparing to compare with those from land sharing (811), or do not refer to agriculture at all (9). Fischer et al. correctly point out that some species now depend on agriculture. However, given that all species have thrived without it for most of their evolutionary history, whether it is essential for their persistence into the future is open to question. We agree that famine is caused largely by inequitable access to food, but our argument is independent of the need to solve world hunger. Regardless of how much food is produced, society has choices to make about how and where to grow it. Our data suggest that high-yielding, land-sparing approaches at least in southwest Ghana and northern Indiahave the greatest potential benets for biodiversity, because they give explicit attention to the importance of conserving remaining habitats. This pattern holds whether the required production of food is more or less than at present (see Fig. 2 in our Report). We believe that the best way to develop an understanding of the difcult and complex
KIYOTADA HAYASHI
National Agriculture and Food Research Organization, Kannondai 3-1-1, Tsukuba, Ibaraki 305-8666, Japan. E-mail: hayashi@affrc.go.jp
Response
FISCHER ET AL. DO NOT DISPUTE OUR FINDING that, in principle, land sparing would be more effective than land sharing for the taxa and areas we studied, but they appear to feel that land sparing is neither achievable nor desirable in practice. We agree that there are no simple, generic solutions to complex problems such as rec-
TECHNICAL COMMENT ABSTRACTS
CORRECTIONS AND CLARIFICATIONS

Books et al.: Picture perfect future past by G. Riddihough (7 October, p. 41). The rst sentence of the third paragraph should have read But, as Out of This World documents, modern science ction is more than the warmed-over imaginings of earlier generations. Reports: Three-dimensional Anderson localization of ultracold matter by S. S. Kondov et al. (7 October, p. 66). Reference 14 [F. Jendrzejewski et al., http://arxiv.org/abs/1108.0137 (2011)] should have been accompanied by a note stating that while their paper was in proof, Kondov et al. were made aware of related three-dimensional experiments that were released in this preprint. News Focus: False positive by J. Cohen and M. Enserink (23 September, p. 1694). In the last sentence in the rst column of p. 1699, the story credits the author of the blog CFS Central with a call for aggressive, ACT UPstyle protests and the quote, I believe we need to act quickly, before the FDA/NIH paper is killed. In fact, it was a patient who wrote this on the blog, not the blog author. Reports: African wild ungulates compete with or facilitate cattle depending on season by W. O. Odadi et al. (23 September, p. 1753). An incorrect e-mail address was given for the rst corresponding author. The correct address is woodadi@yahoo.com. The online HTML version has been corrected. In Table 2, the heading for the fourth column should be Dead stems (hits/100 pins). The reference callouts at the end of the last sentence beginning on page 1754, which begins We posit that..., should include reference (22), to read ...in the Serengeti ecosystem (18, 2022). The reference callout (22) in the nal paragraph on page 1755 should be (23). Reports: Activation of -catenin in dendritic cells regulates immunity versus tolerance in the intestine by S. Manicassamy et al. (13 August 2010, p. 849). Some of the arrows in Fig. 4C were incorrect, and the original images did not fully demonstrate the line in the text that referred to increases in inammatory cell inltration, edema, epithelial cell hyperplasia, and loss of goblet cells in the colon of -catDC/ mice as compared with -cat/ mice. In the revised gure shown here, the arrows have been corrected and new panels have been added. The corrected caption is: Histopathological changes in colon tissue from -cat/ or -catDC/ mice treated with or without 2% DSS treatment for 7 days. Areas of interest are inltration of inammatory cells (black arrows), edema (yellow arrows), and loss of crypts (green arrows) and goblet cells. The changes described here do not affect the Reports conclusions.
Comment on Atmospheric Pco2 Perturbations Associated with the Central Atlantic Magmatic Province
Michael R. Rampino and Ken Caldeira
Schaller et al. (Research Article, 18 March 2011, p. 1404) proposed that carbon dioxide (CO2) released by the Central Atlantic Magmatic Province eruptions over periods of about 20,000 years led to substantial increases of up to 2000 parts per million (ppm) in the concentration of atmospheric carbon dioxide (Pco2) near the Triassic-Jurassic boundary. Use of an atmosphere-ocean model coupled to a carbon-cycle model predicts Pco2 increases of less than 400 ppm from magmatic volatiles, with only a small climatic impact. Full text at www.sciencemag.org/cgi/content/full/334/6056/594-b
Morgan F. Schaller, James D. Wright, Dennis V. Kent
Rampino and Caldeira argue that the rst pulse of the Central Atlantic Magmatic Province would increase the concentration of atmospheric carbon dioxide (Pco2) by only 400 parts per million if erupted over 20,000 years, whereas we observed a doubling within this interval. In the absence of any data to the contrary, we suggest that a more rapid (1000-year) eruption is sufcient to explain this observation without relying on thermogenic degassing. Full text at www.sciencemag.org/cgi/content/full/334/6056/594-c
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choices involved in land-use decisions is to use theoretical models to guide collection of the critical empirical data and evaluate potential solutions based on measurable outcomes. Extending our approach to incorporate other objectives such as cultural values and ecosystem services, and to develop strategies with strong social safeguards, should be a priority. We contend that it would be premature to dismiss the potential benets of land sparing as undeliverable in practice when so little effort has been made so far to develop the right tools to implement it. Hayashi proposes extending our analytical approach to better understand how management intensity affects agricultural yields and species populations. We agree that such information could be useful for informing decisions by farmers and foresters and that it might be used to improve outcomes from land sharing, land sparing, or intermediate strategies. Our approach focused on decision-making at larger scales, for which understanding the consequences of land-use allocation is more relevant. Management intensity is shorthand for a diverse range of practices, from fertilizer use to tree husbandry to management of hunting, and it is often not clearly dened. High-yield farming need not necessarily involve intensive management in the sense of having high agrochemical inputs. Sustainable intensication using resource-efcient practices seems more likely to increase yields with minimal resource degradation and pollution (12). Research on the impacts of different management practices will produce useful insights if it can move beyond simplistic biodiversity metrics, quantify pollution and other costs, and integrate ne-scale management concerns with the need to address largescale land-use change.
1
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK. 2Royal Society for the Protection of Birds, The Lodge, Sandy SG19 2DL, UK. *To whom correspondence should be addressed. E-mail: btp22@cam.ac.uk
Letters to the Editor

Letters (~300 words) discuss material published in Science in the past 3 months or matters of general interest. Letters are not acknowledged upon receipt. Whether published in full or in part, Letters are subject to editing for clarity and space. Letters submitted, published, or posted elsewhere, in print or online, will be disqualied. To submit a Letter, go to www.submit2science.org.
1. E. H. A. Mattison, K. Norris, Trends Ecol. Evol. 20, 610 (2005). 2. D. Kleijn, M. Rundlf, J. Scheper, H. G. Smith, T. Tscharntke, Trends Ecol. Evol. 26, 474 (2011).
References

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BEN PHALAN,1* MALVIKA ONIAL,1 ANDREW BALMFORD,1 RHYS E. GREEN1,2
3. C. Tejeda-Cruz, E. Silva-Rivera, J. R. Barton, W. J. Sutherland, Ecol. Soc. 15, 13 (2010). 4. M. D. Madhusudan, Conserv. Biol. 19, 411 (2005). 5. H. R. Grau, N. I. Gasparri, T. M. Aide, Glob. Change Biol. 14, 985 (2008). 6. B. Phalan, A. Balmford, R. E. Green, J. P. W. Scharlemann, Food Policy 36, S62 (2011). 7. J. Dorrough, J. Moll, J. Crosthwaite, Agric. Ecosyst. Environ. 121, 222 (2007). 8. Y. Clough et al., Proc. Natl. Acad. Sci. U.S.A. 108, 8311 (2011). 9. L. Persha, A. Agrawal, A. Chhatre, Science 331, 1606 (2011). 10. J. Ranganathan, R. J. Ranjit Daniels, M. D. Subash Chandran, P. R. Ehrlich, G. C. Daily, Proc. Natl. Acad. Sci. U.S.A. 105, 17852 (2008). 11. R. D. Gregory et al., Philos. Trans. R. Soc. London Ser. B 360, 269 (2005). 12. Foresight, The Future of Food and Farming (Government Ofce for Science, London, UK, 2011).
BOOKS ET AL.
Greek, Arabic, and Latin, had based their work, discussed important themes concerning the order of the planets: How were the sizes of the orbs that carried them to be determined? From a central Earth, one can only measure the direction of a planet, not its disPeter Dear tance. Ptolemy had simply assumed that the homas Kuhns use of Nicolaus Coper- universe that incorporated the effects of the longer a planet appeared to take in completnicuss astronomical innovation as a heavens upon the Earth, whether in the form ing its celestial circuit again the backdrop of classic example of a revolution in of regional prognostications concerning such the stars, the further away it must be. But this science took what was already, among his- things as the weather, famines, plagues, and rule ran aground in the cases of Mercury and torians of science, the central story of the wars or predictions concernVenus, because each takes, on big Scientic Revolution in early-modern ing individual people (often the average, the same time for The Copernican Question Europe and gave it additional philosophi- called judicial astrology, Prognostication, Skepticism, its circuit: one year, the same cal signicance (1, 2). Copernicuss revo- although these categories time as that needed by the and Celestial Order lution was, on this view, all about creating and labels were endlessly Sun. Although the other planby Robert S. Westman a new understanding of the arrangement of variable). Astral effects on ets took longer, how could any University of California Press, the parts of the universe, an understanding the Earth were taken as a distinction of distance from Berkeley, 2011. 700 pp. subsequently completed by Johannes Kepler given: the great ancient the Earth be made for these $95, 65. ISBN 9780520254817. and Isaac Newton, among others. It was the authority Ptolemy had not two? Pico identied this as a thematic breadth that provided this storys only written the geocentric problem for the prognosticaappeal: Copernicus put the Earth in motion masterpiece of mathematical astronomy, the tors because knowledge of the ordering of the around the Sun instead of its being the cen- Almagest, but also the astrological Tetrabib- planets played a crucial role in determining ter and focus of the universe. Kepler then los, the latter as fundamental in its way in this the astral qualities possessed by each. As the provided previously unapproached accuracy period as the former. Copernicus, Westman Tetrabiblos showed, which bodies neighbored in his mathematical determination of plan- argues, had become immersed in the prog- which others affected the properties of each etary orbits. Galileo contributed and hence the effects of each on terrestrial the foundations of a new physaffairs. If this knowledge was compromised ics of motion; Descartes, a new by uncertainty, so would be the reliability of theory of matter and the vision the astrological forecasts made on its basis. of an indenitely large universe Westman thus makes a concern with in which the stars were other the reliability of prognostications central to suns. Finally, Newton explained Copernicuss new approach, which claimed Keplers laws using the mathas one of its great advantages the ability to ematical idea of universal gravimeasure the relative distances of the planets tation. The story left out sciences from the new center of the universe, the Sun. of life and had little to say about This could now be done by using the Earthchemistry (although a focus on Sun distance as a triangulatory baseline: Newton could bring such issues now that the Earth moved, planetary direcinto the fold), but it still possessed tions could also yield distances. Hence, one a scope that seemed appropriate of Picos serious attacks on the divinatory to something as grand as the Sci- Presenting prognostications. The duke of Ferraras astronomer- potential of the science of the stars had been entic Revolution [see, e.g., (3)]. astrologer Giovanni Bianchini offers his Tabulae Caelestium disarmed. Copernicuss close follower and Robert S. Westman has now Motuum Novae to Emperor Frederick III (1452). promoter, Georg Joachim Rheticus, provides brought us a hefty and enorWestman with good evidence for Copernimously erudite treatment of Copernican- nosticatory aspects of the science of the stars cuss concern with these questions despite ism. While nonetheless wearing its learning while a student in Bologna, and he brought the masters own silence on prognosticatory lightly, The Copernican Question presents a this understanding of the basic astronomical issues (itself convincingly explained in terms historical picture that puts Copernicus where problematic back with him to Polish Prussia. of the contemporary discourse of disciplinary he belongs: in his own time and place. CoperHe also brought back knowledge of con- subdivisions in astronomy). nicus was a 16th-century astronomer in a siderable difficulties. Giovanni Pico della But this book is about much more than European world where astronomy and astrol- Mirandolas Disputationes adversus astro- understanding how Copernicus came up ogy were not really separate in either disci- logiam divinatricem (Disputations against with his innovation. Its title, in stressing the plinary identity or respectability. The science divinatory astrology) had appeared in 1496, Copernican Question, captures the multiof the stars, as Westman compendiously dubs and the works arguments against astrologi- plicity of responses and nonresponses to this the endeavor in which Copernicus partici- cal prediction remained current for over a supposed revolution and examines them not pated, sought an understanding of the physical century. Part of Picos criticism concerned as individual interpretations but as instituthe theoretical mathematical astronomy of tionally intelligible orientations. The two cruthe planets, on which astrological forecasts cial settings for Westmans account are the The reviewer is at the Department of History, 450 McGraw depended. Ptolemys geocentric Almagest university and the aristocratic court. The rst Hall, Cornell University, Ithaca, NY 148534601, USA. E-mail: prd3@cornell.edu and Tetrabiblos, on which astronomers, was the initial institutional home for the sciHISTORY OF SCIENCE
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ence of the stars and the place where certain man discusses the case of Christopher Clavius, aspects of Copernicuss De revolutionibus a prominent Jesuit astronomer of the later 16th were adopted after its publication in 1543. A century, as an inuential teacher and writer network of Lutheran universities, especially of textbooks who restricted his approach to at Wittenberg (where Rheticus was for a time astronomy to the disciplinarily narrow tradia professor of mathematics) and Nuremberg tion of Copernicuss De revolutionibus, itself (where Copernicuss book first appeared) modeled on Prolemys Almagest. nurtured a particular reading of Copernicus The younger Galileo, as a university that stressed the practical side of astronomy: mathematician, had been on cordial terms the use of its mathematical models to gener- with Clavius. The same could not be said of ate tables of celestial motions, and ephemeri- his relations with Kepler. A substantial part des for specic locales, in turn applicable to of Westmans book is concerned with Galicalendrical and prognosticatory work. Wit- leo and Kepler, individually but also regardtenberg astronomers apart from Rheticus did ing the contrasts between their social styles not take the moving Earth seriously, largely and the way these shaped their interactions. because of its physical implausibility, but Kepler from the beginning wanted to enroll they did take De revolutionibus seriously as a Galileo in his program to promote the Coperwork of mathematical, technical astronomy; nican model of the universe, whereas Galileo they learned how to switch back and forth only wanted to engage Kepler if Galileos own between reference frames. achievements would thereby be afrmed. The second major framing of the issues Keplers physico-theological ideas for involves aristocratic patronage and courtly explaining the motions and form of the unisettings as alternatives to the disciplinarily more restrictive university. If Copernicuss work were to be more than a set of matheECOLOGICAL HISTORY matical techniques, its literal physical truth needed to be considered. Figures such as Tycho Brahe (a Danish aristocrat with strong connections to the world of the Lutheran Jared Farmer astronomers but not a part of their institutional system) found themselves able to purass extinction events get all the sue physical and astrological paths more attention, but mass redistribusuited to their higher social status. Some, tion eventsthe biotic interlike Tycho, took such aspects of Coperni- changes that occur when continents or cus seriously without always adopting them. oceans become connected or reconnected Kepler, after having been an assistant to are arguably as important in planetary hisTycho, replaced him, following his death in tory. The Trans-Beringian Interchange and 1601, as Imperial Mathematician to the Great American the Holy Roman Emperor Rudolf II. Interchange (across 1493 Galileo moved from a university to a the Panamanian IsthUncovering the New courtly setting when he became court mus) are two paleoWorld Columbus Created philosopher and mathematician to biogeographic examby Charles C. Mann the Grand Duke of Tuscany in 1610. ples of this process. In Knopf, New York, 2011. Westman argues that an intellectually the historical period, 557 pp. $30.50, C$34.50. freer social environment was a feawe have the ColumISBN 9780307265722. ture of such settings. bian Exchange. Unlike The attitude and teachings of the earlier biotic intergreat pedagogue, and Martin Luthers intel- changes, the human-facilitated Columbian lectual right-hand man, Philipp Melanchthon Exchange connected all of the continents were an important feature of the Lutheran aca- and oceans more or less simultaneously. Spedemic science of the stars in the mid-16th cen- cies, populations, and genomes escaped the tury. It was important that Melanchthon gave connes of former habitats with spectacular moderate support to astrological forecasting consequences. Sounding alarm, the Interand rejected Picos hard-line opposition. This national Union for Conservation of Nature reminds us of the religious and theological has called this ongoing process the great dimensions of the Copernican question. Of- reshuffling. Expressing wonder, Charles cial Catholic positions were generally much Mann calls it a great unicationa biotic less accommodating to astral divination than were those of non-Catholics and less open to The reviewer is at the Department of History, State UniCopernican innovations. (Galileos rst trial, versity of New York, Stony Brook, NY 11794, USA. E-mail: in 1605, was for casting horoscopes.) West- jared.farmer@stonybrook.edu verse included attempts to restructure the physical basis of astrological prognostication. But, ironically, The Copernican Question presents the decline of astrological forecast as the longdrawn-out consequence of the increasing success of Copernican astronomy in the decades following Keplers Rudolne Tables of 1627. The distinctions between astrological practice and its theoretical substructure became too problematic, too difcult for a Copernican science of the stars. Newtons work answered the Copernican question in a way that simply ignored astrology.
1. T. S. Kuhn, The Copernican Revolution: Planetary Astronomy in the Development of Western Thought (Harvard Univ. Press, Cambridge, MA, 1957). 2. T. S. Kuhn, The Structure of Scientic Revolutions (Univ. Chicago Press, Chicago, ed. 2, 1970). 3. I. B. Cohen, The Birth of a New Physics (Anchor, Garden City, NJ, 1960). 10.1126/science.1213727
References
Pangaea Penultima
recreation of Pangaea. In 1493: Uncovering the New World Columbus Created, Mann, a science journalist and popularizer of academic history, has composed a dense yet accessible overview of the rst 500 years of this unplanned ecological farrago. You might assume, based on its title, that 1493 continues the story of 1491, Manns award-winning account of indigenous America from the Pleistocene to the year before Columbus (1). Actually, Mann admits, his new book is designed as a follow-up to The Columbian Exchange and Ecological Imperialism, two seminal works (2, 3) by historian Alfred Crosby. In brief, Crosby argued that the two-way transfer of plants, animals, and pathogens between the Old World and the Americas transformed both hemispheres and aided European colonialism. In his acknowledgments, Mann recounts that when he pestered Crosby to update these classics, he got this answer: Well, if you think its such a good idea, why dont you do it? 1493 consists of four self-contained sections. The rst three complement one another nicely: Atlantic journeys narrates how tobacco commodities came to Europe via Virginia and how malaria and yellow fever came to America via Africa along with disease-resistant slaves destined for tobacco and sugar plantations. Pacic journeys chron-
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BOOKS ET AL.
ence of the stars and the place where certain man discusses the case of Christopher Clavius, aspects of Copernicuss De revolutionibus a prominent Jesuit astronomer of the later 16th were adopted after its publication in 1543. A century, as an inuential teacher and writer network of Lutheran universities, especially of textbooks who restricted his approach to at Wittenberg (where Rheticus was for a time astronomy to the disciplinarily narrow tradia professor of mathematics) and Nuremberg tion of Copernicuss De revolutionibus, itself (where Copernicuss book first appeared) modeled on Prolemys Almagest. nurtured a particular reading of Copernicus The younger Galileo, as a university that stressed the practical side of astronomy: mathematician, had been on cordial terms the use of its mathematical models to gener- with Clavius. The same could not be said of ate tables of celestial motions, and ephemeri- his relations with Kepler. A substantial part des for specic locales, in turn applicable to of Westmans book is concerned with Galicalendrical and prognosticatory work. Wit- leo and Kepler, individually but also regardtenberg astronomers apart from Rheticus did ing the contrasts between their social styles not take the moving Earth seriously, largely and the way these shaped their interactions. because of its physical implausibility, but Kepler from the beginning wanted to enroll they did take De revolutionibus seriously as a Galileo in his program to promote the Coperwork of mathematical, technical astronomy; nican model of the universe, whereas Galileo they learned how to switch back and forth only wanted to engage Kepler if Galileos own between reference frames. achievements would thereby be afrmed. The second major framing of the issues Keplers physico-theological ideas for involves aristocratic patronage and courtly explaining the motions and form of the unisettings as alternatives to the disciplinarily more restrictive university. If Copernicuss work were to be more than a set of matheECOLOGICAL HISTORY matical techniques, its literal physical truth needed to be considered. Figures such as Tycho Brahe (a Danish aristocrat with strong connections to the world of the Lutheran Jared Farmer astronomers but not a part of their institutional system) found themselves able to purass extinction events get all the sue physical and astrological paths more attention, but mass redistribusuited to their higher social status. Some, tion eventsthe biotic interlike Tycho, took such aspects of Coperni- changes that occur when continents or cus seriously without always adopting them. oceans become connected or reconnected Kepler, after having been an assistant to are arguably as important in planetary hisTycho, replaced him, following his death in tory. The Trans-Beringian Interchange and 1601, as Imperial Mathematician to the Great American the Holy Roman Emperor Rudolf II. Interchange (across 1493 Galileo moved from a university to a the Panamanian IsthUncovering the New courtly setting when he became court mus) are two paleoWorld Columbus Created philosopher and mathematician to biogeographic examby Charles C. Mann the Grand Duke of Tuscany in 1610. ples of this process. In Knopf, New York, 2011. Westman argues that an intellectually the historical period, 557 pp. $30.50, C$34.50. freer social environment was a feawe have the ColumISBN 9780307265722. ture of such settings. bian Exchange. Unlike The attitude and teachings of the earlier biotic intergreat pedagogue, and Martin Luthers intel- changes, the human-facilitated Columbian lectual right-hand man, Philipp Melanchthon Exchange connected all of the continents were an important feature of the Lutheran aca- and oceans more or less simultaneously. Spedemic science of the stars in the mid-16th cen- cies, populations, and genomes escaped the tury. It was important that Melanchthon gave connes of former habitats with spectacular moderate support to astrological forecasting consequences. Sounding alarm, the Interand rejected Picos hard-line opposition. This national Union for Conservation of Nature reminds us of the religious and theological has called this ongoing process the great dimensions of the Copernican question. Of- reshuffling. Expressing wonder, Charles cial Catholic positions were generally much Mann calls it a great unicationa biotic less accommodating to astral divination than were those of non-Catholics and less open to The reviewer is at the Department of History, State UniCopernican innovations. (Galileos rst trial, versity of New York, Stony Brook, NY 11794, USA. E-mail: in 1605, was for casting horoscopes.) West- jared.farmer@stonybrook.edu verse included attempts to restructure the physical basis of astrological prognostication. But, ironically, The Copernican Question presents the decline of astrological forecast as the longdrawn-out consequence of the increasing success of Copernican astronomy in the decades following Keplers Rudolne Tables of 1627. The distinctions between astrological practice and its theoretical substructure became too problematic, too difcult for a Copernican science of the stars. Newtons work answered the Copernican question in a way that simply ignored astrology.
1. T. S. Kuhn, The Copernican Revolution: Planetary Astronomy in the Development of Western Thought (Harvard Univ. Press, Cambridge, MA, 1957). 2. T. S. Kuhn, The Structure of Scientic Revolutions (Univ. Chicago Press, Chicago, ed. 2, 1970). 3. I. B. Cohen, The Birth of a New Physics (Anchor, Garden City, NJ, 1960). 10.1126/science.1213727
References
Pangaea Penultima
recreation of Pangaea. In 1493: Uncovering the New World Columbus Created, Mann, a science journalist and popularizer of academic history, has composed a dense yet accessible overview of the rst 500 years of this unplanned ecological farrago. You might assume, based on its title, that 1493 continues the story of 1491, Manns award-winning account of indigenous America from the Pleistocene to the year before Columbus (1). Actually, Mann admits, his new book is designed as a follow-up to The Columbian Exchange and Ecological Imperialism, two seminal works (2, 3) by historian Alfred Crosby. In brief, Crosby argued that the two-way transfer of plants, animals, and pathogens between the Old World and the Americas transformed both hemispheres and aided European colonialism. In his acknowledgments, Mann recounts that when he pestered Crosby to update these classics, he got this answer: Well, if you think its such a good idea, why dont you do it? 1493 consists of four self-contained sections. The rst three complement one another nicely: Atlantic journeys narrates how tobacco commodities came to Europe via Virginia and how malaria and yellow fever came to America via Africa along with disease-resistant slaves destined for tobacco and sugar plantations. Pacic journeys chron-
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recent past. Mann doesnt discuss, for example, the roles of airplanes, container ships, and interocean canals in species shifting. His most questionable omission concerns animals. While 1493 covers mosquito-borne malaria and guano fertilizer, it passes over domesticated and invasive faunatwo of the most important components of the Columbian Exchange. Instead, Mann devotes the books fourth and final section (Africa in the world) to the migration and genetic mixing of Homo sapiens. Here he makes some solid pointsfar into the colonial period, Left behind. Within three decades of Pizarros overthrow of Africans and Amerindians domithe Inka empire, Spanish farmers in the Canary Islands were nated the gene pool of the New exporting potatoes to France and the Netherlands. Yet most World; escaped slaves succeeded in of the hundreds of potato varieties bred by Andean natives creating large, long-lasting maroon are still seen only in South America. polities outside of colonial control; icles how imperial China grew dangerously Spanish and indigenous ruling families roudependent on Peruvian silver and New World tinely intermarried. Mann seems particularly crop plants. Europe in the world explains enamored with his notion that 17th-century how plant and animal products of South Mexico City, with its multiethnic, polyglot, American origin, namely potatoes, guano, cosmopolitan population, was the first of and rubber, undergird the industrial revo- todays modern, globalized megalopolises. lution and European imperialism despite a However, after almost 300 pages on orgafew major setbacks such as potato blight nisms and commodities, Manns detailed (the result of an invasive mold that probably treatment of intercultural relations and racial arrived on a guano ship). categories seems somewhat out of place. As Mann illustrates again and again, the Ecologists might bristle at Manns suggestion Columbian Exchange cut both ways. Silver that human creolization is equivalent to biotic from Potos stabilized the Chinese currency homogenization. system for a time but eventually weakened The book merits comparison to another the Ming Dynasty. Although sweet potatoes high-prole work: Jared Diamonds Pulitzer and maize enabled a Chinese population Prizewinning Guns, Germs, and Steel (4). boom, agricultural expansion onto marginal Compared to Diamond, Mann is a better styllands also resulted in deforestation, cata- ist and a more generous scholar. He amply strophic ooding, and social unrest. In the credits the many researchersmainly histoCaribbean, malaria and yellow fever burned rians such as J. R. McNeill, who has docuthrough indigenous populations and Euro- mented the impact of mosquito-borne disease pean settlements without reprieve. This stark (5), plus a few scientists such as paleoclimademographic reality motivated Europeans tologist William Ruddimanwhose work to import more African slaves, who toiled in he synthesizes. Diamond, by contrast, barely misery but who, in the long run, leveraged acknowledged his enormous debt to Crosby. their immunological advantage against slave- Nonetheless, whatever you may say about Diaholders in a series of anticolonial revolutions mond (historians tend to portray him as a bioin places such as Haiti. geographic determinist), he did put forward a Mann makes no attempt to be compre- provocative argument: the orientation of conhensive. He has little or nothing to say about tinents is the ultimate explanation of human the oceans, the polar realm, Australia, India, history. Guns, Germs, and Steel begged for or Africa beyond the Slave Coast. He gives debate, whereas 1493 politely invites admiramost attention to Western Europe, the Ameri- tion. Manns thesis is simply that globalizacas, and East Asia. Manns extensive use of tion has been going on a long time and that the Chinese language secondary sources sets him Columbian Exchange explains a lot. Summaapart from most environmental historians. rized this way, his book sounds less thoughtThe author makes several detours to the pres- provoking than it actually is. enthis own eld trips to China, the PhilFor fans of long-form nonction, 1493 ippines, and Brazilyet he glosses over the presents multitudinous delights in the form of absorbing stories and fascinating factoids. Mann subscribes to the chain-of-surprisingevents school of history. For example, did you know that Old World diseases caused devastating population losses in the Americas, which led to reforestation, which resulted in carbon sequestering, which probably intensied the Little Ice Age, which wreaked havoc on the Old World? Have you heard the story of British biopirate Sir Henry Alexander Wickham, who smuggled 70,000 tree seeds out of Brazil that became the basis for the rubber plantations of South Asia, which punctured the Brazilian rubber boom and later inspired Henry Ford to erect his own plantation town, Fordlndia, in the Amazon? Manns book is chock-full of such nuggets. Perhaps inevitably, the parts are greater than the whole. As a writer, Mann displays many fine qualities: evenhandedness, a sense of wonder, the gift of turning a phrase. At the same time, he sometimes resorts to clunky and clichd literary devices: Imagine you were in an airplane in 1642; what would you see from your passenger window? His rst-person travelogues seem self-indulgent and superuous. Such memoirism doesnt add anything to a book about the creation of the modern ecological world-system. Geologists may soon ofcially adopt the term Anthropocene to describe the current geologic epoch. Ecologists, who measure change on a different scale with different data, have an alternative term for our time: Homogenocene. This neologism is evocative because it suggests both anthropogenic change and biotic homogenization. Mann loves the word and adopts it as his own. As a best-selling author with a premier publisher, Mann may well turn this academic concept into general knowledge. That would be a good thing. Although 1493 is not ground-breaking, it offers a welcome corrective to media pundits and their puffery about the brave new world of globalization. In fact, as Mann skillfully details, our world has been at for over half a millennium.
1. C. C. Mann, 1491: New Revelations of the Americas Before Columbus (Knopf, New York, 2005); reviewed in (6). 2. A. W. Crosby, The Columbian Exchange: Biological and Cultural Consequences of 1492 (Greenwood, Westport, CT, 1972). 3. A. W. Crosby, Ecological Imperialism: The Biological Expansion of Europe, 9001900 (Cambridge Univ. Press, Cambridge, 1986). 4. J. M. Diamond, Guns, Germs, and Steel: The Fates of Human Societies (Norton, New York, 1987). 5. J. R. McNeill, Mosquito Empires: Ecology and War in the Greater Caribbean, 16201914 (Cambridge Univ. Press, Cambridge, 2010). 6. D. R. Snow, Science 312, 1313 (2006). 10.1126/science.1213474
References
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CREDIT: COURTESY INTERNATIONAL POTATO CENTER (PERU)
POLICYFORUM
SUSTAINABILITY
Paying for Ecosystem Services Promise and Peril

A. P. Kinzig,1 C. Perrings,1 F. S. Chapin III,2 S. Polasky,3 V. K. Smith,4 D. Tilman,5 B. L. Turner II*6
Payment mechanisms designed without regard to the properties of the services they cover may be environmentally harmful.
he Millennium Ecosystem Assessment concluded that over the past 50 years, 60% of all ecosystem services (ES) had declined as a direct result of the growth of agriculture, forestry, fisheries, industries, and urban areas (1). This is not surprising: We get what we pay for. Markets exist for the products of agriculture, aquaculture, and forestry. But the benets of watershed protection (2), habitat provision (3), pest and disease regulation (4), climatic regulation (5), and hazard protection (6) are largely unpriced. Because existing markets seldom reect the full social cost of production, we have incorrect measures of the scarcity of some ES and no measures for the rest. There is growing support for using markets to induce people to take account of the environmental costs of their behavior (7). Markets that allow trading of industrial emissions or fishery harvest rights have been in use for some time (8). These have recently been supplemented by development of marketlike mechanismspayment for ES (PES) schemesthat allow governments and nongovernmental organizations to pay for environmental public goods, such as habitat provision, watershed protection, or carbon sequestration (9). Although they involve buyers and sellers in service provision contracts, PES schemes do not generally allow free exit and entry, or iteration toward a clearing price. Mechanisms of this kind promise much, but if poorly designed they can make things worse, not better. We identify both the main failings in existing environmental markets and marketlike mechanisms and the conditions that need to be satised for new mechanisms to do better.
School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA. 2Department of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, AK 99775, USA. 3 Department of Applied Economics, University of Minnesota, St. Paul, MN 55108, USA. 4W. P. Carey School of Business, Arizona State University, Tempe, AZ 85287, USA. 5Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108, USA. 6School of Geographical Sciences and Urban Planning, and School of Sustainability, Arizona State University, Tempe AZ 85287, USA.
1
Market Promise, Not Panacea
Markets, among the most durable of human institutions, tend to arise when resources are recognized to be scarce (10, 11). It is not surprising, therefore, that they initially emerged around basic ingredients for survival, nor is it surprising that they failed to emerge for resources whose impacts on well-being were simply unknown (e.g., the impact of early industrial emissions on health). What is surprising is that markets have not developed for many scarce ES long recognized to affect human well-being. The explanation lies partly in the publicgood nature of those services (e.g., climatic regulation through carbon sequestration), partly in the lack of well-dened property rights (e.g., sea areas beyond national jurisdiction), and partly in the various costs of forming markets (e.g., the cost of reversing historic pollution rights in agriculture). Many environmental markets created in recent years are intended to address these issues, but markets are not a panacea. Prices are only useful indicators of changes in resource scarcity if they capture all signicant effects of resource use (12). Mechanisms require careful design to be effective. The rst U.S. market for sulfur dioxide (SO2) emissions, for example, recently collapsed because the design addressed only one of many interacting pollutants. Court rulings acknowledging transboundary effects of these interactions led to proposals to form new market structures, but not before resulting uncertainty had driven SO2 market permit prices to zero (13).
Mechanism Design and Implementation
*Author for correspondence. E-mail: Billie.L.Turner@asu.edu
Effective mechanism design demands understanding of both the linkages among biodiversity, ecological functioning, and ES (14 16) and the incentives for private provision of these resources, created in part by their public-good nature. There are four main mechanisms for motivating people to provide scarce ESs that are public goods (17): (i) Regulation and penalty. Zoning restrictions, emission limits, or access rules are enforced through penalties for noncompliance. (ii) Cap and trade. The emerging carbon markets, for example, allow users to buy
and sell emission rights within the constraints imposed by a cap. (iii) Direct payments. Providers receive payment for supplying services. (iv) Self-regulation. Voluntary agreements and social norms encourage good behavior while penalizing noncompliance. Which mechanism is best depends on the properties of the ES in question and on prevailing socioeconomic and political conditions (see the table). Mechanisms that work for public goods that are the sum of the efforts of many countries [e.g., carbon sequestration under the Reducing Emissions from Deforestation and Forest Degradation (REDD) scheme] may not work for public goods that depend on efforts of one country (e.g., control of emergent zoonotic diseases through market interdiction). Ecosystem service markets and PES schemes, types (ii) and (iii), are emerging as the preferred mechanisms (1820). PES schemes exist for carbon sequestration in China (21) and the United Kingdom (22); watershed protection in South Africa (23) and Mexico (24); and biodiversity conservation in the United States (25), Costa Rica, and Nicaragua (26). But often these mechanisms are imposed without due regard to the properties of the services they cover (see the table). Nor are the prices they generate directly responsive to changing conditions. Some ES markets are too thin (early carbon-offset markets involved too few trades for prices to track conditions), and others suffer from design aws (the U.S. SO2 market described above). Often the science is uncertain or ignored (PES schemes for water supply through afforestation face uncertainty about the net effects of changing forest cover), or payments reect goals other than the scarcity of resources (the poverty alleviation goals in PES schemes for biodiversity protection in agricultural landscapes limit payment sensitivity to habitat condition) (27). Few schemes address multiple ES, yet interdependence between services generates unwanted feedbacks. Incentives that encourage production of one service may have adverse effects on others (28). For example, incentives for carbon sequestration under the REDD scheme may simply cause carbonemitting activities to be relocated. Incen-

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tives for biofuels production that promote conversion of tropical forests to tilled elds may reduce both carbon storage and habitat that supports biodiversity (29). Incentives for habitat protection that create corridors between protected areas may increase disease risks by increasing contact between wild and domesticated animals (30). Where ES are jointly produced, paying for only one service can be as damaging as paying for none.
Private Incentives Are Not Enough
Ecosystem service Public-good type Veriability Space Time Jurisdiction Mechanism
Providers, beneciares not colocated
Providers, beneciaries colocated
Regulation and penaltyType (i)
Depends on strongest provider
Depends on weakest provider
Benets accrue in the future
Reects the efforts of many
Reects the efforts of a few
Direct paymentsType (iii)
International (subglobal)
Payments for ES may replicate the incentive effects of markets in cases where the provision of environmental public goods depends on private activity. But there are many cases where payment systems will simply not be appropriate (e.g., where ES derive from lands or seas beyond national jurisdiction). Where it is not possible to use prices as indicators of the scarcity of ESs we need other metrics. Physical indicators of the state of ecosystems need to be integrated into national income and product accounts and made comparable to other measures of income. Progress has been made in developing satellite accounts for environmental ows through the United Nations System of Environmental and Economic Accounts (SEEA). Although separate from the national income accounts, these still allow comparison with conventional measures of economic activity and can be reproduced consistently over time (31). Proposals exist to extend the national income and product accounts to include environmental flows (32) and to develop consistent, comprehensive wealth accounts that include changes in environmental assets (33). Although the tractability of these approaches has been demonstrated, too few countries, including the United States, are doing any of this, in part because it would make implications of resource allocation transparent. Finally, for ES that may benet from payment systems, how we pay is critical. Payment schemes should capture all effects of ecosystem management (e.g., affecting multiple ES). They should consider scale (e.g., how country-to-country payments translate into within-country payments to landholders) and lead to measurable, veriable outcomes that go beyond what would have happened in the absence of the payment scheme. Most important, they should not be burdened with objectives such as income transfers that go beyond delivery of ES. This is one of the hardest lessons of decades of politically driven agricultural subsidies. One reason for the popularity of PES systems is their potential role in poverty alleviation
Characteristics of ecosystem services and payment mechanisms. The table schematizes authors impressions of the effectiveness of incentive mechanisms (column F) in providing environmental public goods. Column A classies a sample of ES as public goods (35). Column B indicates the scale(s) at which delivery of a service can be veried (20). Column C denotes the geographic location of providers relative to beneciaries (27). Column D and E indicate timing (20) and the governance level(s) needed to achieve effective outcomes (36). Darker shading in column F indicates mechanisms considered more effective for achieving the socially optimal level of provision, although effectiveness is context-dependent.
(34). Poverty reduction is a laudable goal, but it should not prevent PES schemes from signaling the scarcity of ES. Every payment system has implications for equity; although these effects may be extremely important they should be addressed separately, not through payments made under the scheme.
1. Millennium Ecosystem Assessment, Ecosystems and Human Well-Being: General Synthesis (Island Press, Washington, DC, 2005). 2. G. Heal, Ecosystems (N.Y., Print) 3, 24 (2000). 3. E. B. Barbier, J. C. Burgess, A. Grainger, Land Use Policy 27, 98 (2010). 4. R. S. Ostfeld, F. Keesing, V. T. Eviner, Infectious Disease Ecology: Effects of Ecosystems on Disease and of Disease on Ecosystems (Princeton Univ. Press, Princeton, NJ, 2008). 5. J. G. Canadell, M. R. Raupach, Science 320, 1456 (2008). 6. E. B. Barbier, Resour. Energy Econ. 30, 229 (2008). 7. G. Chichilnisky, G. Heal, Nature 391, 629 (1998). 8. R. N. Stavins, J. Econ. Perspect. 12, 69 (1998). 9. P. J. Ferraro, A. Kiss, Science 298, 1718 (2002). 10. S. Crocket et al., Econ. J. 119, 1162 (2009). 11. H. Demsetz, Am. Econ. Rev. 57, 347 (1967). 12. P. Dasgupta, Human Well-Being and the Natural Environment (Oxford Univ. Press, New York, 2001). 13. Environmental Protection Agency, Fed. Regist. 76(152), 48208 (2011). 14. S. Naeem, D. Bunker, A. Hector, M. Loreau, C. Perrings, Eds., Biodiversity, Ecosystem Functioning, and Human Wellbeing: An Ecological and Economic Perspective (Oxford Univ. Press, New York, 2009). 15. B. H. Walker, A. P. Kinzig, J. Langridge, Ecosystems (N. Y., Print) 2, 95 (1999). 16. D. Tilman, S. Polasky, C. L. Lehman, J. Environ. Econ. Manage. 49, 405 (2005). 17. R. N. Stavins, in Handbook of Environmental Economics,
18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
References
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K.-G. Mler and J. R. Vincent, Eds. (Elsevier, Amsterdam, 2003), vol. 1, pp. 355435. P. J. Ferraro, R. D. Simpson, Land Econ. 78, 339 (2002). S. Pagiola, Ecol. Econ. 65, 712 (2008). S. Wunder, S. Engel, S. Pagiola, Ecol. Econ. 65, 834 (2008). J. Liu, S. Li, Z. Ouyang, C. Tam, X. Chen, Proc. Natl. Acad. Sci. U.S.A. 105, 9477 (2008). T. L. Dobbs, J. N. Pretty, Rev. Aging Econ. 26, 220 (2004). J. K. Turpie, C. Marais, J. N. Blignaut, Ecol. Econ. 65, 788 (2008). C. Muoz-Pia, A. Guevara, J. Torres, J. Braa, Ecol. Econ. 65, 725 (2008). B. Madsen, N. Carroll, K. Moore Brands, Offset and Compensation Programs Worldwide (Ecosystem Marketplace, Washington, DC, 2010). S. Pagiola et al., Ecol. Econ. 64, 374 (2007). R. Arriagada, C. Perrings, Ambio 40, 798 (2011). J. A. Foley et al., Science 309, 570 (2005). J. Fargione, J. Hill, D. Tilman, S. Polasky, P. Hawthorne, Science 319, 1235 (2008). R. D. Horan, J. F. Shogren, B. M. Gramig, Environ. Dev. Econ. 13, 415 (2008). G.-M. Lange, Ecol. Econ. 61, 589 (2007). D. W. Jorgenson, J. S. Landefeld, in A New Architecture for the U.S. National Accounts, D. W. Jorgenson, J. S. Landefeld, W. D. Nordhaus, Eds. (Chicago Univ. Press, Chicago, 2006), pp. 13112. K. J. Arrow, P. Dasgupta, L. H. Goulder, K. J. Mumford, K. Oleson, Sustainability and the measurement of wealth (Working Paper 16599, National Bureau of Economic Research, Cambridge, MA, 2010). S. Wunder, Environ. Dev. Econ. 13, 279 (2008). T. Sandler, Global Collective Action (Cambridge Univ. Press, Cambridge, 2004). I. Kaul, I. Grunberg, M. Stern, in Global Public Goods: International Cooperation in the 21st Century, I. Kaul, I. Grunberg, M. Stern, Eds. (Oxford Univ. Press, Oxford, 1999), pp. 219. 10.1126/science.1210297
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Air-quality regulation Carbon sequestration Disease control Freshwater provision Habitat provision Marine capture sheries Storm protection Water-quality regulation
Self-regulationType (iv)
Cap and tradeType (ii)
International (global)
Benets accrue now
International
National
National
Local
Local
PERSPECTIVES
NEUROSCIENCE
Another Reason to Exercise

Aaron D. Gitler
Can exercise slow neurodegeneration?
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: gitler@mail.med.upenn.edu
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CREDIT: Y. HAMMOND/SCIENCE
odern medicine has demonstrated the virtues of healthy diet and routine exercise for cardiovascular health, preventing diabetes, and lowering cholesterol. But a role for exercise in combating neurodegenerative diseases is less well understood. On page 690 in this issue, Fryer et al. (1) present compelling evidence suggesting that exercise might have a long-lasting benecial effect on slowing neurodegenerative disease progression. Though often distinct in their clinical presentation, neurodegenerative disorders, including Alzheimers disease and Parkinsons disease, are characterized by the accumulation of insoluble protein aggregates in neurons (2). Thus, therapies that combat the deleterious effects of protein aggregation in one disease might be applicable to other neurodegenerative diseases as well. Spinocerebellar ataxia type 1 (SCA1) is one of a group of nine autosomal dominant hereditary neurodegenerative disorders caused by proteins that contain tracts of several uninterrupted glutamine residues so-called polyglutamine (polyQ) tract expansions (3). In SCA1, an unstable cytosineadenine-guanine (CAG) trinucleotide repeat (which specifies glutamine) in the coding region of the ATXN1 gene (encodes ataxin-1 protein) expands past a critical threshold, resulting in polyQ-expanded ataxin-1 (4). Like other spinocerebellar ataxias, the hallmark pathology in SCA1 is the atrophy and loss of Purkinje neurons from the cerebellar cortex. This manifests as deficits in motor coordination that affect gaze, speech, gait, and balance (3). How polyQ expansions cause disease is poorly understood (5). For example, is disease caused by loss of the normal function of ataxin-1, by a dominant-negative effect of the polyQ-expanded ataxin-1, or both? Animal models have provided key insights into SCA1 pathogenesis. Expressing the human SCA1 gene in ies results in neurodegeneration, and genetic screens for modiers of SCA1-induced neurodegeneration have illuminated potential new targets for therapeutic intervention (6). A mouse model genetically engineered to harbor an ataxin-1
Wild-type mouse
SCA1 mouse
SCA1 + exercise mouse
PolyQ expansion
EGF Cic
Ataxin 1
Transcription derepression
Ataxin-1
More derepression
Cic Transcription repression Hyperrepression

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Relief of hyperrepression Rescue from disease
Normal
Run mouse, run. Exercise [or genetically reducing the production of Capicua (Cic)] rescues disease phenotypes in the neurodegenerative disorder SCA1. SCA1 is caused by polyglutamine (polyQ) expansions in ataxin-1. Exercise increases production of epidermal growth factor (EGF), which decreases amounts of the transcriptional repressor Cic. PolyQ expansions in ataxin-1 confer decreasing Cic function at some target genes, while concomitantly enhancing its function at other loci. Reducing Cic abundance prolongs survival.
polyQ expansion recapitulates many features of the human disease (7) and has been useful for investigating disease-relevant ataxin-1 protein interactors (8, 9), the role of ataxin-1 posttranslational modifications (10), and potential therapeutic strategies (11). In addition to the benets of exercise on brain health and cognitive function (12), it may promote slowing neurodegenerative disease progression. For example, exercise slowed the decline in cognitive abilities of Alzheimers disease patients (13) and improved postural stability and balance in Parkinsons disease patients (14). However, it is unclear if exercise affects the underlying causes of the disease or offers symptomatic relief. Moreover, intense exercise might be deleterious to certain neurodegenerative diseases, perhaps by taxing already vulnerable neurons with increased activity and metabolic demands. One example is the increased risk of Italian professional football players for developing the motor neuron disease amyotrophic lateral sclerosis (15). Fryer et al. used the polyQ-expanded ataxin-1 mouse model to examine the impact
of exercise on SCA1. Remarkably, a modest exercise regimen substantially extended life span. The authors discovered increased concentrations of epidermal growth factor (EGF) in brain tissue, which persisted long after exercise. After exercise, the amount of Capicua (Cic) proteina transcription factor that interacts with ataxin-1decreased, suggesting that EGF might regulate Cic, and that Cic abundance could inuence disease progression in the SCA1 mouse model (see the gure). Reducing the abundance of Cic in the SCA1 mice by 50% improved motor impairments, as well as learning and memory decits, and markedly extended life span of the SCA1 mouse. These results were unexpected because reducing Cic abundance in y exacerbated SCA1-induced neurodegeneration (8). This raised the question of whether Cic function is increased or decreased in SCA1. Fryer et al. discovered that both scenarios are correct. Cic, a transcriptional repressor, is sometimes inhibited by polyQ-expanded ataxin-1, while at other times its function is enhanced such that Cic becomes a hyper-
PERSPECTIVES
repressor. Thus, in the SCA1 mouse model, both a Cic loss of function and gain of function appear to occur simultaneously, albeit at distinct transcriptional targets. Because the partial loss of Cic function cannot explain the disease rescue that Fryer et al. observed, they propose that polyQ-expanded ataxin-1 also confers a gain-of-function effect on Cic, and the relief of this gain of function is enough to rescue the disease phenotypes even in the face of further loss of Cic function. At the molecular level, it appears that polyQ-expanded ataxin-1 causes Cic to bind more tightly to the promoters of some genes (gain of function), while it concomitantly causes Cic to bind less well to the promoters of other genes (loss of function). How ataxin-1 converts Cic at certain loci to a hyper-repressor while inhibiting the function of Cic at other loci remains to be determined. Nonetheless, these ndings underscore the complexity of protein interactions in mediating neurodegenerative disease. The study by Fryer et al. suggests the exciting possibility that exercise, perhaps early in disease, or even before disease onset (this could be determined in SCA1 and other trinucleotide repeat diseases by genetic testing for risk alleles), could help delay the progression. And if the effects of exercise are attributed to a decrease in Cic, then lowering the amount of Cic might be an effective therapeutic approach. Given the encouraging results with exercise and SCA1, it will be of immediate interest to test the effects of exercise on other neurodegenerative diseases, for which effective treatments have been elusive and are desperately needed.
1. J. D. Fryer et al., Science 334, 690 (2011). 2. M. S. Forman, J. Q. Trojanowski, V. M. Lee, Nat. Med. 10, 1055 (2004). 3. H. T. Orr et al., Nat. Genet. 4, 221 (1993). 4. J. R. Gatchel, H. Y. Zoghbi, Nat. Rev. Genet. 6, 743 (2005). 5. H. Y. Zoghbi, H. T. Orr, J. Biol. Chem. 284, 7425 (2009). 6. P. Fernandez-Funez et al., Nature 408, 101 (2000). 7. K. Watase et al., Neuron 34, 905 (2002). 8. Y. C. Lam et al., Cell 127, 1335 (2006). 9. J. Lim et al., Nature 452, 713 (2008). 10. L. Duvick et al., Neuron 67, 929 (2010). 11. K. Watase et al., PLoS Med. 4, e182 (2007). 12. C. W. Cotman, N. C. Berchtold, L. A. Christie, Trends Neurosci. 30, 464 (2007). 13. Y. Rolland, G. Abellan van Kan, B. Vellas, J. Am. Med. Dir. Assoc. 9, 390 (2008). 14. L. E. Dibble, O. Addison, E. Papa, J. Neurol. Phys. Ther. 33, 14 (2009). 15. A. Chi, G. Benzi, M. Dossena, R. Mutani, G. Mora, Brain 128, 472 (2005). 10.1126/science.1214714
References
APPLIED PHYSICS
Paradigm Shifts in Dye-Sensitized Solar Cells

Michael D. McGehee
Efciency limitations from the iodine-based electron shuttle have been overcome by replacing it with cobalt compounds and by using dyes that block recombination.
CREDIT: P. HUEY/SCIENCE
ye-sensitized solar cells (DSCs) have captured the imagination of a wide range of people, from middle-school students who make their rst solar cell by sensitizing lms of titania nanocrystals with berry juice to scientists and engineers who are striving to solve the worlds energy problem. A schematic of a DSC is shown in the gure, panel A (1, 2). Until recently, practical DSCsones with efficiencies exceeding 11%have had to rely on dyes that are expensive in that they contain the noble metal ruthenium (Ru). Efforts to increase cell efciency by boosting the output voltage of the cell, and to decrease costs by eliminating the use of Ru, have run afoul of the enemy of all solar cellsthe recombination of charge carriers before they are delivered to the electrodes. On page 629 of this issue, Yella et al. (3) report on a specially designed redox mediator containing cobalt (Co) complexes that enables a DSC efciency of 12.3% under 1 sun of illumination. To understand how the redox mediator improves DSC efficiency, it is useful to review how these electrochemical cells work. Titania nanocrystalswhite paint pigDepartment of Materials Science, Stanford University, Stanford, CA 94305, USA. E-mail: mmcgehee@stanford.edu
ments that reect most of the incident solar energyare bonded into a lm on a transparent electrode. To absorb sunlight, titania is dyed with a light-absorbing material, the dye sensitizer (e.g., a Ru compound bearing bipyridyl ligands that help absorb light). The cell is completed with an electrolyte solution with a redox mediator, typically iodide ions, and a counterelectrode. Light absorption by
Glass Conducting oxide film Platinum I3
_
a dye molecule provides the energy to inject one of its electrons into the titania, which provides a pathway to the external circuit for energy extraction. The mediator donates an electron to the now electron-decient sensitizing dye. The now-oxidized mediator diffuses across the cell to the counterelectrode to be reunited with an electron passed through the external circuit.
TiO2 Dye Redox mediator
10-m TiO2 particle film Conducting oxide film Glass
EF
_
3I Redox electrolyte Co3 Co2

+
Dye molecule 1 nm
Light
VOC (I)
VOC (Co)
I3 / I O.35 V E0 [Co (byp)3]3 + + [Co2 (byp)3]2 O.535 V

3+ +
TiO2 particle 20 nm
Shuttle differences. (A) Schematic of a DSC solar cell. In a conventional cell, the dye is a Ru complex, and the redox shuttle is based on iodine ions. In the work of Yella et al., the dye contains an electron donor and acceptor connected by a conjugated bridging group, and the redox shuttle is based on a cobalt compound. (B) The open-circuit voltage of the cell, VOC, is the difference between the Fermi level EF of the titania and the redox potential E0 of the redox shuttle. The cobalt-based shuttle boosts VOC by 185 mV versus iodine ions. TiO2, titanium dioxide; byp, bipyridyl.

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DSCs are extremely good at separating photogenerated charge carriers and taking them to the electrodes. If an incoming photon is absorbed by the dye in a state-of-theart DSC, the probability of the charge carriers reaching the electrodes is nearly 100%. Until recently, the world record for power conversion was 11.1% (4). Although the sensitizing molecules in these cells have a relatively large energy gap, the cells do not generate as high a voltage as one could reasonably expect because the redox potential of the most commonly used redox couple, which is based on iodide ions, is too low for the best sensitizing dyes (see the gure, panel B). Energy is wasted as the redox couple reduces a chargedsensitizing dye back to its neutral state. Many researchers have been trying for more than a decade to nd a redox couple with a more ideally suited redox potential in order to increase the voltage of DSCs, but the process has been slow and frustrating. The iodide mediator is special as its oxidized form, I3, does not readily accept electrons from the titania surface, which would be an unwanted recombination process (5). The I3 ions can persist for 1 ms or more before undergoing an electron-transfer reaction in solution, which gives them sufcient time to diffuse to the counterelectrode for reduction. Several alternative metal complexes with better redox potentials have been found, but solar cells made with them typically have unacceptably high recombination rates and lower open-circuit voltages. Last year, Boschloo, Hagfeldt, and coworkers achieved a power-conversion efciency of 6.7% in DSCs with iodide-free electrolytes using Co complexes (6). The key to making good cells was adding just enough electrically inactive bulk to the periphery of the sensitizing dyes and the Co complexes to slow down recombination without blocking the necessary electrontransfer processes. The approach Yella et al. took to slowing down recombination was to use a relatively new family of dyes that connect an electron donor (D) moiety to an electron acceptor moiety (A) through a conjugated (bonded) bridge (DA) provided by a zinc porphyrin complex. These DA dyes do not contain any expensive, rare metal atoms, such as Ru, and tend to absorb light more strongly. Most important, they attached alkoxy chains to the sides of these molecules to provide a very effective barrier to recombination between electrons in the titania and holes in the Co complexes. One of the shortcomings of the Co complexes is that they diffuse through the electrolyte more slowly than the conventional iodide ions because they are larger. Yella et al. found that they could get efciency as high as 13.1% by reducing the illumination intensity by 50% because it is less important for the ions to diffuse to the electrode quickly when the carrier density is lower. This efciency might be obtained under normal solar lighting by reducing the distance the ions need to diffuse through the use of thinner lms. Complete absorption in these thinner lms could be achieved by using even better DA dyes that absorb more strongly or advanced light-trapping techniques such as plasmonics (7, 8). Long-term stability studies must be performed on Co complexes in dye-sensitized solar cells to determine if they are as stable as iodide-based electrolytes. For many years, dozens of researchers around the world tried to develop new sensitizing dyes and redox couples for DSCs but inevitably concluded that the best sensitizing dyes needed to contain Ru and the best mediators needed to contain iodide. These paradigms are now shattered. One of the next developments that needs to occur is reducing the energy gap of sensitizing dyes so that light can be harvested efficiently further into the infrared region of the spectrum. One approach would be to use sensitizing dyes to absorb infrared light and energy relay dyes that absorb visible light, and then transfer energy to the sensitizing dyes (9). As scientists around the world develop new DA dyes and redox couples and combine them with energy relay dyes and new lighttrapping techniques, we can expect to see the efciency climb toward 15%, which could make DSC technology competitive with other kinds of solar cells.
10.1126/science.1212818
NEUROSCIENCE
Synaptic Switch and Social Status

Matthieu Maroteaux and Manuel Mameli The strength of synaptic connections in the mammalian brain can inuence social status.
n 1859, Charles Darwin introduced the key concept of natural selectionthat in the struggle for survival and reproduction, individuals compete for the same resources. Hence, animals living in a social environment can establish dominance hierarchies within a short time, which remain stable during the existence of the group (1). This ranking within social communities has a fundamental advantageit eliminates conict in the group, which minimizes energy expenditure and violence, thereby allowing resource sharing ( 2). On page 693 of this issue, Wang et al. (3) demonstrate that encoding of social dominance in mice involves specic synapses in cortical regions of the brain. Wang et al. used a behavioral experimental model (4) that provides a quantitative
INSERM UMR-S 839, Institut du Fer Moulin, Universit Pierre et Marie Curie, F75005 Paris. E-mail: manuel. mameli@inserm.fr
measure of aggressiveness without allowing physical contact between competing mice, thereby preventing injuries. In each trial, one mouse forces its opponent outside of a neutral area, permitting identication of a dominant and a subordinate mouse. The authors show that in a cohort of four mice, a social hierarchy was quickly organized and once established, persisted over time. Another important aspect is the complete independence of social dominance from other factors, including sensorimotor and learning skills. This makes hierarchy formation a unique and distinct trait of animal behavior. When and where does nature decide that an individual will dominate in a community? Early in life, mice as well as humans are embedded in a community that in the simplest scenario consists of siblings. An interesting possibility is that innate traits and genetic programming that shape neuronal circuits from the postnatal period are responsible for the allocation of dominance or subor-
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1. B. ORegan, M. Gratzel, Nature 353, 737 (1991). 2. A. Hagfeldt, G. Boschloo, L. Sun, L. Kloo, H. Pettersson, Chem. Rev. 110, 6595 (2010). 3. A. Yella et al., Science 334, 629 (2011). 4. Y. Chiba et al., Jpn. J. Appl. Phys. 45, L638 (2006). 5. G. Boschloo, A. Hagfeldt, Acc. Chem. Res. 42, 1819 (2009). 6. S. M. Feldt et al., J. Am. Chem. Soc. 132, 16714 (2010). 7. H. A. Atwater, A. Polman, Nat. Mater. 9, 205 (2010). 8. I.-K. Ding et al., Adv. Energy Mater. 1, 52 (2011). 9. B. E. Hardin et al., Nat. Photonics 3, 406 (2009).
References
PERSPECTIVES
tus. Although the mPFC has an established role in social behavior, it cannot be considered the only structure where dominance is encoded. Indeed, the amygdala and lateral septum are key players in establishing animal hierarchy (9). Future studies will be necessary to determine the hierarchical organization among brain structures underlying this complex behavior. An interesting aspect arising from the study of Wang et al. concerns the mechanisms that establish glutamatergic transmission upon the rst encounter of a stranger. It is plausible that mice might be differently programmed for leadership from birth. However, it cannot be ruled out that animals may also acquire their social status based on biological factors and external cues, thereby learning to be dominant or subordinate. If that is the case, this process might share traits with experience-driven behavior, where synaptic adaptations are crucial. How does the synaptic strength adapt in conditions where dominancy is challenged? A network activity might be in part responsible for driving these processes or alternatively, synaptic scaling may take place more slowly over time (10, 11). Wang et al. demonstrate that impairing AMPA receptor trafcking to the synapse devalues the status of a dominant mouse. This raises the interesting possibility that AMPA receptors constitutively insert at synapses and maintain dominance over time. Although Wang et al. demonstrate that social rank correlates with synaptic strength, they also highlight the need to further understand the differences between individuals at the cellular level. This could depend on the number of AMPA receptors, dendritic spines, or synaptic inputsor a broader network tuning could be involved.
1. R. J. Blanchard, K. J. Flannelly, D. C. Blanchard, Physiol. Behav. 43, 1 (1988). 2. D. van Kreveld, Ned. Tijdschr. Psychol. 25, 55 (1970). 3. F. Wang et al., Science 334, 693 (2011); 10.1126/ science.1209951. 4. G. Lindzey, H. Winston, M. Manosevitz, Nature 191, 474 (1961). 5. R. M. Sapolsky, Science 308, 648 (2005). 6. M. I. Cordero, C. Sandi, Front. Neurosci. 1, 175 (2007). 7. H. W. Kessels, R. Malinow, Neuron 61, 340 (2009). 8. M. R. Bennett, J. L. Kearns, Prog. Neurobiol. 60, 545 (2000). 9. M. Timmer, M. I. Cordero, Y. Sevelinges, C. Sandi, Neuropsychopharmacology 36, 2349 (2011). 10. R. Malinow, R. C. Malenka, Annu. Rev. Neurosci. 25, 103 (2002). 11. G. Turrigiano, Annu. Rev. Neurosci. 34, 89 (2011). 12. We thank M. C. T. Brown and J.-C. Poncer for helpful comments and the Ecole de Neuroscience de Paris, INSERM, and Ville de Paris for support. 10.1126/science.1214713
AMPAR NMDAR
Spine head
Subordinate
Behavioral switch
Dominant
Synapses and rank. Excitatory synaptic drive onto cortical pyramidal neurons in the mouse brain is stronger in dominant individuals than subordinates. Modulating synaptic strength by increasing or decreasing AMPA receptormediated transmission switches the initial social ranking.
dination. But in the case of newly established cohorts, where animals meet randomly, how does social dominance adjust? In one plausible sequence of events, a dominant individual suddenly facing subordination experiences a range of emotions, including disappointment or even fear. Stress has been proposed to affect social relationships in this context (5). Indeed, by acting both on neuronal circuits implicated in cognition and emotions and on hormone levels, stress amplies memories associated with the previously established social status (6). Changes in the synaptic strength of excitatory connections in the rodent hippocampus, amygdala, and midbrain represent a cellular substrate of experience-driven behaviors (7). This suggests the engagement of similar mechanisms during the encoding of social dominance. In humans, the dorsolateral prefrontal cortex (PFC), the homolog of the rodent medial PFC (mPFC), has been implicated in hierarchy-related behaviors. Wang et al. focused on excitatory synapses onto layer V pyramidal neurons of the mPFC (the main output of this structure) and show that the strength of synaptic transmission mediated by the neurotransmitter glutamate matches social ranking in mice (see the gure). The stochastic release of individual neurotransmitter vesicles (quanta) has been used to probe the basic function of synapses in the neuromuscular junction and central nervous system (8). Wang et al. observed
that the size of quantal release onto cortical neurons was higher in dominant than subordinate adult mice (rank 1 versus rank 4 among four mice). As an outcome, higher synaptic efficiency will potentially translate into a stronger output signal from the cortex to downstream brain structures. The authors also established a causal link between strength of excitatory synapses and social dominance. Decreasing the synaptic transmission mediated by AMPA receptors (which are postsynaptically activated by the release of glutamate) switched the initial rankingthe dominant mouse became the subordinate member of the cohort (from rank 1 to rank 4). Likewise, an increase in AMPA receptormediated synaptic transmission switched the subordinate mouse to a dominant rank (rank 4 to rank 1). Thus, synaptic efcacy, specically in cortical layer V, is sufcient to tune social status. Although the amount of AMPA receptors at synapses is unlikely to be the only mechanism that discriminates a dominant versus a subordinate mouse, these ndings provide a further argument that manipulation or the synaptic strength by controlling the number or the trafcking of postsynaptic AMPA receptors directly modies specic behaviors. Wang et al. provide two conceptual advances: the idea that a neurobiological substrate for social ranking is located in the mPFC, and that synaptic efcacy represents a cellular substrate determining social sta-
CREDIT: B. STRAUCH/SCIENCE

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Pyramidal neuron
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APPLIED PHYSICS
A Photothermoelectric Effect in Graphene

Denis Basko
A curious response of graphene to light and heat may be useful for optoelectronic applications.
Universit de Grenoble 1, Centre National de la Recherche Scientique, Laboratoire de Physique et Modlisation des Milieux Condenss, UMR 5493, B.P. 166, 38042 Grenoble, France. E-mail: denis.basko@grenoble.cnrs.fr
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CREDIT: (A) ADAPTED FROM (1)
conventional thermocouple consists of two different metal wires placed in contact with each other. When the junction is heated and the free ends are kept cold, a voltage can be measured across the two free ends. If the metals are the same, then no voltage should appear. On page 648 of this issue, Gabor et al. (1) report a thermocouple effect that contradicts this conventional expectation. Upon heating a junction made of a single graphene sheet by shining laser light on it, they found that a voltage does develop across the junction. The observed photothermoelectric effect could be potentially exploited in novel optoelectronic devices. When electrons move through a metal, they carry electric charge and energy. The former is responsible for the electric current, the latter for the heat current. The electric current is determined by the total number of electrons moving from one electrode (source) to the other (drain). The heat current depends also on how the electrons are distributed among energy levels within the metals. Because both have the same physical originnamely, the motion of electrons through the metalthey are closely related. One manifestation of this is the Wiedemann-Franz law, an empirical observation that the ratio of electric and thermal conductivity is a constant. Another manifestation is the thermoelectric effect, whereby a temperature difference across a sample leads to the development of a voltage. Indeed, temperature difference produces a heat current, accompanied by electric current (carried by the same electrons), that is proportional to the voltage. The ratio between the voltage developed and the temperature difference is called the thermopower or Seebeck coefcient, which depends on the material. If a junction of two metals is heated up, a voltage is generated in each metal. The net voltage across the sample is nonzero if the two samples have different Seebeck coefcients, but is zero if they are the same. Gabor et al. used a single sheet of graphene and placed two regions of it under different conditions such that they had dif-
Laser
Measured voltage
Fermi level Source VTG
p (hole) doped Graphene Insulator Top gate Drain
n (electron) doped
Graphene in the spotlight. (A) Graphene-based device demonstrating the phototherBottom gate moelectric effect. The graphene VBG sheet is placed on an insulating substrate and electrically contacted by two electrodes (source and drain). Below the substrate, a bottom-gate electrode is placed under the whole graphene sheet. Above the right half of the sheet, separated by an insulating barrier, the top-gate electrode is placed. The electron density on the left is controlled by the bottom-gate voltage, and the density on the right by a combination of the top- and bottom-gate voltages, allowing for independent control of the densities. The junction between the two regions is illuminated by laser light, and the voltage difference between source and drain is measured. (B) A schematic of the electronic dispersion in the two regions of the graphene sheet (two Dirac cones that are vertically shifted to align the Fermi level).
ferent Seebeck coefcients. This is possible because the density of electrons in graphene can be strongly modied by placing an external gate electrode nearby and applying a constant voltage to itthe eld effect. Although there is no electrical contact between the gate and the graphene sheet, the electric eld from the gate penetrates the graphene, which then either pushes electrons away or attracts more electrons, depending on the sign of the gate voltage. In ordinary metals, this eld effect does not work because the external electric eld is screened at short distances (on the order of one atomic length), and because charge and heat transport occur in the bulk of the metal and therefore remain unaffected by the external eld. But graphene is atomically thin, permitting a giant eld effect whereby the electrical conductivity can be changed by orders of magnitude by tuning the gate voltage (2). A change in conductivity implies a change in the Seebeck coefcient (3), as was confirmed experimentally for the specific case of graphene (4). At room temperature, the observed variation of the Seebeck coefcient amounted to a few tens of microelectron volts per kelvin, which is at least an order of
magnitude higher than for a junction between two conventional metals (gold, silver, copper) and of the same order as for alloys used in thermocouples (chromel, alumel, constantan). In addition to this large value, the crucial advantage of graphene is that the electrical and thermal properties can be controlled by an external gate voltage. By using two gate electrodes with different voltages, two regions of the graphene sheet can be produced with different electron densities (see the gure). The junction of the two regions is heated up by a focused laser beam. Thus, the voltage appears upon exposure of the sample to lighta photovoltaic effect. In that case, is the device a photodiode rather than a thermocouple? In a conventional photodiode, illumination of a semiconductor p-n junction excites electrons (negative-charge carriers) and holes (positive-charge carriers), which are then pulled on the opposite sides of the junction by the electric eld present at the junction. The spatial separation of negative and positive charges results in a photovoltage. Which mechanism is responsible for the photovoltage measured by Gabor et al.? A similar question has been addressed for a junction
PERSPECTIVES
between single-layer graphene and bilayer graphene (5), where the two mechanisms would result in opposite signs of the photovoltage, and the one corresponding to the thermoelectric mechanism was observed. In the doubly gated monolayer graphene of Gabor et al., for the eld-induced carrier separation the sign of the photovoltage would be simply determined by which of the two regions has the higher electronic density. In contrast, the thermoelectric mechanism would result in a peculiar six-fold photovoltage pattern (6), as was observed by Gabor et al. The efcient design of an optoelectronic device requires an understanding of the main mechanism of the photovoltage generation in that device. Identication of the photothermoelectric effect as such a mechanism for graphene (1, 5), together with the demonstration of external control (1), thus provides an opportunity to develop graphene-based optoelectronic devices.
1. N. M. Gabor et al., Science 334, 648 (2011); 10.1126/science.1211384. 2. K. S. Novoselov et al., Science 306, 666 (2004). 3. M. Cutler, N. F. Mott, Phys. Rev. 181, 1336 (1969). 4. Y. M. Zuev, W. Chang, P. Kim, Phys. Rev. Lett. 102, 096807 (2009). 5. X. Xu, N. M. Gabor, J. S. Alden, A. M. van der Zande, P. L. McEuen, Nano Lett. 10, 562 (2010). 6. J. C. W. Song, M. S. Rudner, C. M. Marcus, L. S. Levitov, Nano Lett. 10.1021/nl202318u (2011). 10.1126/science.1214560
References
CELL BIOLOGY
Hilde Abrahamsen1,2 and Harald Stenmark1,2
CREDIT: Y. HAMMOND/SCIENCE
ll cells have the ability to sense whether nutrients are scarce or abundant so that appropriate anabolic or catabolic programs can be initiated. A key sensor of nutrient status in eukaryotes is target of rapamycin (TOR) (1), an enzyme that phosphorylates a subset of proteins that function in cell growth and metabolism (2). In mammalian cells, TOR associates with accessory proteins to form mammalian TOR complex 1 (mTORC1) (3). Amino acids are potent activators of mTORC1 (4, 5), but it has not been clear how mTORC1 senses amino acids within the cell. On page 678 of this issue, Zoncu et al. (6) describe how sensing amino acids occurs inside the lumen of lysosomes, the main degradative organelles in the cell. Lysosomes are characterized by their acidity and content of degradative enzymes (7). Earlier work indicated that physical contact between mTORC1 and its activator, Rheb (8), at the lysosome determines whether amino acids can activate mTORC1 (9). Zoncu et al. therefore reasoned that lysosomes must play an important role in the amino acid mediated activation of mTORC1. By using cultured cells from fruit ies and reducing the expression of genes with known roles in lysosomal biogenesis or functions, the authors found that vacuolar H+adenosine triphosphatase (v-ATPase), which pumps protons into lysosomes, is essential for TOR activation in response to amino acids. These observations were conrmed in cultured human cells by either chemical inhibition of the v-ATPase or reduced expression of the pump.
Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. 2Centre for Cancer Biomedicine, University of Oslo, Montebello, N-0310 Oslo, Norway. E-mail: stenmark@ulrik.uio.no
1
Earlier work revealed that amino acid mediated activation of mTORC1 depends on the correct nucleotide loading of a heterodimeric guanosine triphosphatase (GTPase) complex of the Rag family (RagA/B and RagC/D) (10, 11), which localizes to the lysosome by interaction with a Rag regulator (Ragulator) complex (9). Upon correct nucleotide loading and activation, the Rag complex can pull the cytosolic mTORC1 component Raptor from the cytosol to the lysosome membrane (see the gure). Rheb is localized exclusively to intracellular membranes and is therefore physically separated from cytosolic mTORC1 in the absence of amino acids. Zoncu et al. show that mTORC1 fails to accumulate at lysosomes after amino acid stimulation in the absence of an active v-ATPase, which suggests that the pump is part of the amino aciddriven sensory mechanism that
targets mTORC1 to the lysosome in proximity to Rheb. The v-ATPase is a multisubunit proton pump composed of one unit responsible for ATP hydrolysis (V1 sector) and one membrane domain that rotates upon ATP hydrolysis (V0) allowing protons to enter the lysosomal lumen and thereby acidify it. Semiquantitative mass spectrometry analyses and precipitation assays using recombinant proteins led to the hypothesis that Ragulator functions as a bridge linking the v-ATPase and the Rag GTPases together at the lysosome. Zoncu et al. further noted that amino acid stimulation and starvation reduces and strengthens, respectively, the interaction between Ragulator and the V1 but not the V0 sector. This suggests that amino acids control the interaction between Ragulator and the v-ATPase.
Phosphorylation
mTORC1
Inactive
mTORC1 Rag complex

V1
Structural Amino rearrangement acids
S6K1
mTORC1
Active Activate Rheb
Cell growth
Cytosol Cyt Membrane Lysosome Lysosome lumen lumen
V-ATPase
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Dissociation of V1
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High amino acid content
Amino acid sensing. (A) At low amino acid concentrations within the lysosome, Ragulator interacts with the v-ATPase and nucleotide loading of the Rag complex is incompatible with mTORC1 recruitment. (B) When amino acids are abundant, v-ATPase undergoes a structural rearrangement that alters the interaction surface between the v-ATPase and Ragulator. This changes the nucleotide loading of the Rag complex and results in recruitment of mTORC1. Rheb (in the lysosome membrane) activates mTORC1, which then phosphorylates growth-promoting targets such as S6 kinase 1.

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Growth Signaling from Inside
Sensing of amino acids inside lysosomes by a proton pump initiates a chain of events that stimulates cell growth.
PERSPECTIVES
To further tease apart the function of the v-ATPase in amino acid signaling to mTORC1, Zoncu et al. used a cell-free system to reconstruct the amino acidmediated binding of mTORC1 to Rag GTPases at lysosomes. Pure lysosomal fractions that were stimulated with amino acids could recruit purified Raptor, indicating that cytosol is dispensible for recruitment of Raptor to the lysosomal Rags. This suggests that amino acids do not act on the cytoplasmic face but function from the lysosomal lumen to alter v-ATPase structure. This reduces the pumps association with Ragulator, leading to activation of Rag GTPases and recruitment of Raptor (mTORC1). Moreover, permeabilization of the lysosomal fraction, which kept the v-ATPase interactions intact but allowed leakage of amino acids out of the organelle, abolished the amino aciddriven recruitment of Raptor. This supports the idea that lysosomes contain all the components necessary to sense amino acids and activate mTORC1. The study of Zoncu et al. suggests that amino acids signal their presence from within the lysosome and translate this presence through a domino effect that activates the Rag GTPase complex to mediate proximity between mTORC1 and Rheb at this compartment. It is still not clear how amino acids in the lysosome lumen can cause structural alterations of v-ATPase and how this translates into nucleotide loading and activation of the Rag complex. In this respect, it is noteworthy that although v-ATPase is required for mTORC1 activation, its strong interaction with Ragulator during amino acid suppression suggests an inhibitory role toward the Rag complex as well (6). One scenario is that the altered afnity between Ragulator and the v-ATPase upon amino acid sensing may alter the conformation of Rags in a way that allows nucleotide loading. Future experiments might reveal whether the v-ATPase indeed functions directly in nucleotide loading or if this requires yet unidentied factors. The central role of mTORC1 as a coordinator of nutrient responses is accompanied by a great interest in targeting mTORC1 pharmacologically. mTORC1 inhibitors are already in clinical use to treat certain cancers (12), and the fact that TOR inhibition can prevent neurodegeneration and increase the life span of model organisms has raised the possibility of using mTORC1 inhibitors as antiaging drugs in humans (1315). Advance in our understanding of how mTORC1 is activated is good news because it offers new strategies for therapeutic interventions of cancer, neurodegeneration, and aging.
1. J. Heitman, N. R. Movva, M. N. Hall, Science 253, 905 (1991). 2. S. Wullschleger, R. Loewith, M. N. Hall, Cell 124, 471 (2006). 3. R. Zoncu et al., Nat. Rev. Mol. Cell Biol. 12, 21 (2011). 4. K. Hara et al., J. Biol. Chem. 273, 14484 (1998). 5. Y. Iiboshi et al., J. Biol. Chem. 274, 1092 (1999). 6. R. Zoncu et al., Science 334, 678 (2011). 7. J. P. Luzio, P. R. Pryor, N. A. Bright, Nat. Rev. Mol. Cell Biol. 8, 622 (2007). 8. A. R. Tee, B. D. Manning, P. P. Roux, L. C. Cantley, J. Blenis, Curr. Biol. 13, 1259 (2003). 9. Y. Sancak et al., Cell 141, 290 (2010). 10. Y. Sancak et al., Science 320, 1496 (2008); 10.1126/science.1157535. 11. E. Kim, P. Goraksha-Hicks, L. Li, T. P. Neufeld, K. L. Guan, Nat. Cell Biol. 10, 935 (2008). 12. J. B. Easton, P. J. Houghton, Oncogene 25, 6436 (2006). 13. B. Ravikumar et al., Nat. Genet. 36, 585 (2004). 14. N. D. Bonawitz, M. Chatenay-Lapointe, Y. Pan, G. S. Shadel, Cell Metab. 5, 265 (2007). 15. I. Bjedov, L. Partridge, Biochem. Soc. Trans. 39, 460 (2011). 10.1126/science.1214355
References
ATMOSPHERIC SCIENCE
Ocean Effects of Blocking

Tim Woollings
Short-term weather events may drive ocean variability on time scales of several decades.
ariations in the circulations of the Atlantic Ocean and the atmosphere above it influence societies far beyond the ocean basin itself. Scientists have long tried to understand and predict the dramatic year-to-year variability of the North Atlantic Oscillation (NAO) (1), but modeling the associated ocean-atmosphere interaction remains a challenge (2). Attention has turned to longer-term warming and cooling episodes of the North Atlantic Ocean. These variationsoften referred to as Atlantic multidecadal variability (AMV)are widely assumed to arise from variability in the oceans overturning circulation (3), in which warm water ows northward near the ocean surface and returns southward at depth. In contrast, Hkkinen et al. argue on page 655 of this issue (4) that the AMV owes its existence to atmospheric events with time scales as short as a week.
Department of Meteorology, University of Reading, Earley Gate, Reading, RG6 6BB, UK. E-mail: t.j.woollings@reading. ac.uk
Central to the new work is the concept of blocking, in which the usual prevailing westerly winds (see the gure, panel A) are obstructed by a large-scale, stationary system, usually an anticyclone (panel B); rainbearing cyclones are thereby diverted from their usual path, and regional impacts in temperature and precipitation can be severe, as in the Russian heat wave of 2010 (5). Blocking is a reversal of the usual pattern of cyclonic rotation of air masses north of the Atlantic jet and anticyclonic rotation to the south. Hkkinen et al. now propose that this reversal leads to a temporary change in the wind forcing that maintains the oceans subpolar and subtropical gyre circulations. As a result of the changed wind forcing, the subpolar gyre contracts, opening up a pathway for warm saline water from the subtropics to move to higher latitudes. This has a direct effect on the temperature of the subpolar gyre and may also lead to changes in the overturning circulation. Previous studies have focused on fixed spatial patterns of atmospheric variability,
in particular, the seesaw in pressure between Iceland and the Azores known as the NAO. In contrast, Hkkinen et al. suggest that the gyre circulations can be forced by blocks occurring anywhere over the Atlantic, provided that the anticyclonic winds fall within the boundary of the subpolar gyre. Blocks over different parts of the Atlantic may then have the same effect on AMV despite having very , different relations to the atmospheric jets and patterns such as the NAO (6). By focusing on relatively fast, intraseasonal atmospheric events, this theory mirrors a recent focus on these time scales in understanding low-frequency atmospheric variability such as the NAO itself. In this view, the interannual variations of the NAO are governed by the dynamics of short-term weather events such as blocking (7), although this issue is still disputed (8). Hkkinen et al. suggest that the frequency of Atlantic blocking varies from decade to decade with the AMV. They focus on the role of blocking in driving AMV but perhaps , the biggest open questions are why blocking
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Normal conditions Blocking
H L
B
Diverting the jet stream. (A) In the normal weather pattern, the jet stream carries air from North America across the Atlantic to Europe. The wind shear on the anks of the jet acts to maintain the ocean gyre circulations. (B) During a blocking weather pattern, the strong westerly winds of the jet stream are diverted north and south around a dramatic reversal of streamlines. Hkkinen et al. propose that such short-term blocking events inuence the Atlantic oceans circulation on time scales of several decades.
itself exhibits such striking low-frequency variability and whether the AMV could in turn contribute to this. There is some modeling evidence that AMV can have a weak inuence on atmospheric circulation (9) and that blocking might be affected by sea surface temperatures (10). The simulation of blocking has long been a challenge for numerical models. Recent evidence, however, suggests that some climate models can now simulate blocking very well (11), opening up encouraging possibilities for modeling these oceanatmosphere interactions. Hkkinen et al.s results are based on the recent 20th-century reanalysis project (12), which provides gridded atmospheric circulation elds back to the late 19th century. This novel project provides not one version of recent climate variability, but 56 different versions, or ensemble members, each of which is consistent with the available observations. In regions and periods of good data coverage, the ensemble members agree remarkably well, but they diverge where data are sparse. Hkkinen et al. do not use this information on uncertainty, and it seems an essential next step to use this, and perhaps other methods, to investigate how robust the reported long-term changes in blocking are. From an ocean perspective, the theory fuels an ongoing debate over the relative importance of wind stress forcing and buoyancy changes associated with air-sea uxes for North Atlantic variability. For example, other studies (13) suggest that the delayed response to buoyancy forcing played a central role in some of the recent changes described by Hkkinen et al. The study adds further motivation to studying the dynamics, low-frequency variability, and modeling of high-impact atmospheric blocking events. There is a particular need to assess the role of diabatic processes in blocking (10), such as deep atmospheric convection over a warm ocean (14). The work by Hkkinen et al. is part of an exciting resurgence of interest in ocean-atmosphere interaction, for example, associated with decadal prediction and the importance of the intense western boundary currents (15).
New observational data sets, such as longer atmospheric reanalyses and ocean observing systems, and improved and higher-resolution climate models offer great potential for further progress in this area in the next few years.
1. M. J. Rodwell, D. P. Rowell, C. K. Folland, Nature 398, 320 (1999). 2. Y. Kushnir et al., J. Clim. 15, 2233 (2002). 3. J. R. Knight et al., Geophys. Res. Lett. 32, L20708 (2005). 4. S. Hkkinen et al., Science 334, 655 (2011). 5. M. Matsueda, Geophys. Res. Lett. 38, L06801 (2011). 6. T. Woollings et al., Q. J. R. Meteorol. Soc. 136, 856 (2010).
References
7. J. J. Benedict et al., J. Atmos. Sci. 61, 121 (2004). 8. K. J. Rennert, J. M. Wallace, J. Clim. 22, 5650 (2009). 9. G. Gastineau, C. Frankignoul, Clim. Dyn.; 10.1007/ s00382-011-1109-y (2011). 10. M. Croci-Maspoli, H. C. Davies, Mon. Weather Rev. 137, 664 (2009). 11. A. A. Scaife, T. Woollings, J. Knight, G. Martin, T. Hinton, J. Clim. 23, 6143 (2010). 12. G. P. Compo et al., Q. J. R. Meteorol. Soc. 137, 1 (2011). 13. K. Lohmann et al., Geophys. Res. Lett. 36, L15602 (2009). 14. A. Czaja, N. Blunt, Q. J. R. Meteorol. Soc. 137, 1095 (2011). 15. Y.-O. Kwon et al., J. Clim. 23, 3249 (2010). 10.1126/science.1214167
CLIMATE
Running Out of Climate Space

Ralf Ohlemller Mapping the speed of climate change helps to explain and predict biodiversity patterns on land and in the sea.
urrent biodiversity patterns across the globe are the result of the interplay between past and current climates and the degree to which species respond to these conditions (1). Climate affects both evolutionary processes, such as how fast species diversify, and ecological processes such as range shifts and species interactions. Understanding the spatial distribution of climatic conditions over time is thus crucial for understanding current and likely future species distributions and biodiversity patterns. Two reports in this issue, by Burrows et al. on page 652 (2) and Sandel et al. on page 660 (3), investigate past and future shifts in cliSchool of Biological and Biomedical Sciences, and Institute of Hazard, Risk and Resilience (IHRR), Durham University, Durham, DH1 3LE, UK. E-mail: ralf.ohlemuller@durham. ac.uk
mate space, with the aim to explain and predict biogeographical patterns. Climate space, or climate envelope, refers to the multidimensional climatic conditions of an area (see the gure). Climatic conditions change over time and these changes can be mapped in space. The extent, speed, and direction of such spatiotemporal shifts in climate space can help to explain and predict biotic responses to such changes. Burrows et al. analyzed the speed with which climate has changed locally and across geographic space over the past 50 years. The authors use the simple yet appealing measure of climate change velocity, which quanties how fast isotherms have shifted (4). In the study, high-velocity areas are those from which a large distance needs to be covered to reach areas that, by 2009, have the same temperature as the original area did in 1960.

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PERSPECTIVES
Low-velocity areas are those space; species may or may where in 2009, 1960-analogous not be able to track their suitTime period 1 Time period 2 conditions are nearby. At most able climate space, depending Climate space latitudes, climate change velocon their migration and disDisappearing Persisting ity is similar in the ocean and persal capacity. Combining Novel on land; however, in sub-Arctic climate space analyses with regions and around the equator, recent advances in commumarine velocity is two to seven nity (10) and individual and times higher than on land. Seapopulation modeling (11, 12) sonal shifts are also generally will help to address this issue, faster in the sea than on land, advancing our understanding especially in the Arctic and at of large-scale biotic responses northern mid-latitudes, where to changing environmental spring timing advanced more conditions. rapidly at sea than on land (2). Finally, under changing Temperature Temperature Under warming climate climatic conditions and over conditions, climate change Lost and new climates. Two-dimensional climate space (solid outlines) of a region time, adaptation and altered velocity varies across the globe, at two time periods. Under changing climatic conditions, species will have to adapt to interactions between species with generally low velocity in disappearing and novel climatic conditions at the edges of the regions original cli- will affect whether species mountainous regions where mate space. Two reports in this issue (2, 3) investigate the biogeography of such shifts. remain limited to the condiisotherms are tightly packed, tions in which they currently and high velocity in lowland areas (2). Loa- the fewer endemic species are found there. occur or whether they can occupy new clirie et al. have predicted very similar pat- This relationship is weakest for highly mate space (13, 14). The degree to which terns of global climate velocity for the next mobile birds and strongest for much less a taxonomic group tends to retain simi100 years (4). Thus, simple poleward shifts mobile amphibians, indicating that disper- lar ecological traits and to occupy similar of species in response to warming climate sal ability may drive this relationship. Areas niches over time (niche conservatism) is conditions are highly oversimplied, espe- of low climate velocity where analogous cli- the key process linking evolution and ecolcially in heterogeneous landscapes. It is the mates were within easy reach since the last ogy (15). Combining climate space analyexception rather than the rule that isotherms glaciation are safe havens for many species ses with phylogeographic and biodiversity show clear northward shifts. The spatial with currently small ranges (3). data can shed light on the degree, tempo, conguration and heterogeneity of shifting These results provide support for the idea and spatial patterns of niche conservatism isotherms and associated climate space are of assessing the conservation status not only versus niche evolution. crucial for understanding species responses of species but also of climates. Two studies Despite these caveats, climate space to these shifts over space and time. recently provided such an assessment for analyses such as those by Burrows et al. and When applying climate space analyses Californian climate space under future cli- Sandel et al. broaden our understanding of to ecological questions, three main things mate change (7, 9); both studies show that how environments change in space and time matter to species: the extent of areas with assessing climatic heterogeneity and the and how this in turn affects patterns of disparticular climate conditions, how far away extent of disappearing and novel climatic appearing, persisting, and novel climates, these areas are, and whether there are any conditions within and outside protected species, and ecosystems. obstacles in the direction of these areas. areas can add useful information to biodiReferences Over time, the extent of climatic niche versity conservation planning. 1. R. E. Ricklefs, D. G. Jenkins, Philos. Trans. R. Soc. Lond., space will expand or shrink; some climates Climate space analyses come with caveB 366, 2438 (2011). will disappear while other, novel combina- ats. First, they are only as good as the cli2. M. T. Burrows, Science 334, 652 (2011). 3. B. Sandel et al, Science 334, 660 (2011). tions of climatic factors will emerge (see the mate data on which they are based. Circular 4. S. R. Loarie et al., Nature 462, 1052 (2009). gure). Patterns of novel and disappearing argumentation is a real possibility, because 5. J. W. Williams, S. T. Jackson, J. E. Kutzbach, Proc. Natl. climates under future climate change have gridded climate data are often lled in using Acad. Sci. U.S.A. 104, 5738 (2007). 6. R. Ohlemller, E. S. Gritti, M. T. Sykes, C. D. Thomas, been modeled at the global (5), continental spatial interpolation, particularly in regions Glob. Ecol. Biogeogr. 15, 395 (2006). (6), and regional scale (7). The spatial extent with few weather stations. Climate is then 7. D. D. Ackerly et al., Divers. Distrib. 16, 476 (2010). of particular climatic conditions will affect a function of geographic distance (when the 8. R. Ohlemller et al., Biol. Lett. 4, 568 (2008). the geographic distribution of species occu- gridded climate data are lled in) and, in the 9. J. A. Wiens, N. E. Seavy, D. Jongsomjit, Biol. Conserv. 144, 2119 (2011). pying this niche space. For instance, there climate space analysis, geographic distance 10. K. Mokany, T. D. Harwood, J. M. Overton, G. M. Barker, is evidence that rare, small-range species is a function of climate (when velocity is calS. Ferrier, Ecol. Lett. 14, 1043 (2011). are mostly found in currently rare, spatially culated as the distance between isotherms). 11. B. J. Anderson et al., Proc. Natl. Acad. Sci. U.S.A. 276, 1415 (2009). restricted climates (8). However, many climate models do not rely 12. J. Pagel, F. M. Schurr, Glob. Ecol. Biogeogr.; Sandel et al. also report strong evidence on spatial interpolation, and increased cli10.1111/j.1466-8238.2011.00663.x (2011) that not only current but also past conditions mate observation coverage will lead to more 13. C. F. Dormann, B. Gruber, M. Winter, D. Herrmann, Biol. Lett. 6, 229 (2010). inuence biodiversity patterns of endemic ne-resolution climate data sets, reducing 14. S. A. Smith, J. M. Beaulieu, Proc. Biol. Sci. 276, 4345 species. In their study, the climate velocity the problem of circularity. (2009). of an area is inversely related to endemic Second, shifts in climate space should 15. J. J. Wiens et al., Ecol. Lett. 13, 1310 (2010). species richness: The faster isotherms have be used with caution to infer shifts in speshifted in an area since the last glaciation, cies or ecosystems currently found in this 10.1126/science.1214215
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ESSAY
EPPENDORF WINNER
The Language of Dendrites

Tiago Branco
Theoretical neurophysiology rests on certain cardinal assumptions. Their adjunctions, or synapses, are always between the axon of one neuron and the soma of another. McCulloch and Pitts, 1943
Dendrites perform spatial and temporal discrimination during input processing.
University College London, London, WC1E 6BT UK. E-mail: t.branco@ucl.ac.uk
is the conversion of input into action potentialsthe process of turning a neuron on. How is this conversion achieved? What are the rules for integrating input, and what kind of information can a single neuron interpret and process? The traditional view is that neurons sum input and, if the sum reaches a certain threshold, an action potential is triggered. The important variable is thus the amount of synaptic inputif neurons had a language and each synapse were a letter, they would only care about how long a word is. However, most neurons seem to have the ability to be more powerful than this. Contrary to the assumption of McCulloch and Pitts, synapses are not made onto the soma
Experiment
IN OUT
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Dendritic computation in pyramidal cells. (A) The original Wilfrid Rall model, where somatic potentials are larger in the IN activation sequence because of cable ltering ( , membrane time constant). (B) Somatic potentials recorded during activation of eight synapses along a dendrite (top, cortical pyramidal cell in layer 2/3 lled with a uorescent dye showing the dendrite selected for the experiment). The IN sequence is larger than the OUT, but unlike the model, differences are due to differential activation of NMDA receptors. (C) A cluster of seven synapses was selected at different regions of a dendritic branch [orange spots, distal; blue spots, proximal; bars on the right show color-code of plots in (D)]. (D) The input-output function is progressively steeper toward the distal end of the branch, and somatic potentials are less sensitive to input timing (EPSP, Excitatory postsynaptic potential). [Panels (C) and (D) originally published in (7), reprinted with permission]
Normalized EPSP
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nimal survival depends on the ability to analyze the environment and act on it: escape predators, nd food, select a mate. Understanding how the brain achieves this is one of the most fascinating and challenging problems in neuroscience. What sequence of steps converts sensory cues into behavior? In other words, how does the brain compute? In 1943, McCulloch and Pitts noted that neurons ring action potentials act like binary devices that can either be on or off. In a seminal paper (1), they showed how connected networks of neurons could represent any logical expression, and 60 years of subsequent work in theoretical neuroscience has devised models of neuronal networks that can implement computational tasks. The basic operation in these models
Eppendorf and Science are pleased to present the prize-winning essay by Tiago Branco, the 2011 winner of the Eppendorf and Science Prize for Neurobiology.
(cell body) but onto dendrites, protrusions from the cell body separating the input and the action potential initiation zone. Dendrites lter, transform, and compute thresholds of synaptic input and can, in theory, implement basic arithmetic operations by themselves (2). I rst became interested in dendrites during my Ph.D. work. Monitoring the properties of single synapses in hippocampal neurons, I found that dendrites can implement a negative feedback that regulates the amount of input each branch receives (3). Dendrites can thus independently process and regulate input information. Can these properties be used by single neurons to perform highorder computations? One of the most important features of animals and the surrounding environment is the dynamic nature of their interaction. Input to neurons arrives in temporal sequences, which depend on where the animal is and what it is doing. Can neurons distinguish between different sequences of synaptic input? Can they tell the difference between danger and garden, or are they both the same six-letter-long word? This was the rst question I asked in my postdoctoral research, together with my collaborators at University College London. We
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were not entirely nave about what to expect, as Wilfrid Rall had predicted in 1964 that dendritic ltering of synaptic potentials propagating to the soma should introduce time delays to their peak and confer some degree of sequence sensitivity (4) (gure, panel A). However, testing this experimentally required precise spatial and temporal control of synaptic activation, which only recently became technically possible with the development of two-photon laser glutamate uncaging (5). Using this technique in slices of rat brain cortex, I rst tested Ralls prediction: After selecting 8 to 10 synapses distributed over one dendritic branch, I activated them sequentially and in two opposite directions while measuring the voltage response at the soma with whole-cell patch clamp, as well as dendritic calcium levels with a second two-photon laser (6). The results showed what Rall had predictedresponses were larger in the IN than in the OUT direction (gure, panels A and B), but with an unexpected twist. If N-methyl-Daspartate (NMDA) receptors were blocked, the response was the same for both directions, suggesting that the mechanism was different from passive ltering. To understand these results, I constructed several compartmental models and found that the difference between the two sequences was due to the interaction between the impedance gradient along dendritic branches and the nonlinear voltage sensitivity of the NMDA receptors. Testing thousands of different sequences in the model led to the prediction that neurons should be able to resolve more than just the IN and OUT sequences, which I then conrmed experimentally. Further modeling and experiments extended these ndings and showed that the probability of discriminating any two random sequences is very high and that this applies to a variety of spatial input distributions. During these experiments, I became interested in a related problem. Dendrites introduce a spatial dimension to synaptic integration, with synapses activated all over the dendritic tree. Does synaptic location matter? Are the rules of integration the same for the entire dendritic tree? Using the same set of techniques, I measured how different regions of single dendritic branches integrated increasing numbers of activated synapses and how they responded to different degrees of input synchrony (7). I found that distal regions amplied synaptic input with high gain, displaying a very steep sigmoidal input-output function, and were remarkably insensitive to input timing. In contrast, moving the input toward the proximal part of the branch progressively generated more linear integration curves with small gain, which required high input synchrony for summation (gure, panels C and D). Pharmacological experiments showed that these properties rely on recruitment of voltage-gated calcium and sodium channels and crucially on NMDA receptors. Compartmental modeling showed that local differences in synaptic integration can again be explained by voltage-sensitive conductances acting on a gradient of impedance, whereby high-impedance distal regions are increasingly efcient in recruiting voltage-gated channels. Overall, the results of this research show that dendrites implement the complex computational task of discriminating temporal sequences and allow neurons to differentially process inputs depending on their location, suggesting that the same neuron can use multiple integration rules. The current challenge is to nd out how neuronal circuits exploit these properties in vivo and how we should update our set of cardinal assumptions for theoretical neurophysiology. As we begin to understand the language of dendrites, we can start eavesdropping on their conversations and learn more about how the brain accomplishes its tasks.
1. 2. 3. 4. W. McCulloch, W. Pitts, Bull. Math. Biophys. 5, 115 (1943). S. Bloomeld, Brain Res. 69, 115 (1974). T. Branco et al., Neuron 59, 475 (2008). W. Rall, in Neural Theory and Modeling, R. Reiss, Ed. (Stanford Univ. Press, Stanford, CA, 1964), pp. 7397. 5. M. Matsuzaki et al., Nat. Neurosci. 4, 1086 (2001). 6. T. Branco et al., Science 329, 1671 (2010). 7. T. Branco, M. Husser, Neuron 69, 885 (2011). 10.1126/science.1215079
2011 Grand Prize Winner

The author of the prize-winning essay, Tiago Branco, received his M.D. from Lisbon University in 2002. He then joined the Wellcome Trust FourYear Ph.D. Program in Neuroscience at University College London, where, in Yukiko Godas group, he focused on neurotransmitter release properties of individual synapses. After receiving his Ph.D., he moved to Michael Haussers laboratory, where he has been a postdoctoral research fellow since 2007. Dr. Branco has applied electrophysiological, optical, and modeling techniques to investigate how dendritic integration contributes to singleneuron computations. He plans to combine this approach with molecular methods to investigate the role of dendrites in controlling animal behavior. Aaron Gitler, for his essay A Simple Yeast Model Provides New Insight into a Complicated Human Neurodegenerative Disease. Dr. Gitler is an assistant professor in the Department of Cell and Developmental Biology at the University of Pennsylvania School of Medicine. He received his Ph.D. from the University of Pennsylvania, working with Jonathan Epstein on signaling pathways in cardiovascular development. In postdoctoral research with Susan Lindquist, at the Whitehead Institute for Biomedical Research, he performed yeast genetic screens for modiers of toxicity associated with the Parkinsons disease protein -synuclein. Dr Gitlers group at the University of Pennsylvania combines yeast and human genetics to elucidate novel pathways involved in neurodegenerative disease, focusing on the motor neuron disease amyotrophic lateral sclerosis. Roger Clem, for his essay An Uninstall Function for Fear Memory. Dr. Clem received his Ph.D. under the mentorship of Alison Barth at Carnegie Mellon University, where he investigated sensory-driven synaptic plasticity in the neocortex. During a postdoctoral fellowship with Rick Huganir at The Johns Hopkins University, Dr. Clem examined the role of glutamate receptor trafcking in emotional memory. His work explains how fear memories can be permanently weakened through behavioral training in a process akin to software uninstall routines. Dr. Clem has accepted an appointment to assistant professor of neuroscience at the Mount Sinai School of Medicine, where he will investigate synaptic mechanisms in memory formation and updating, as well as how those processes might be manipulated to treat psychiatric conditions. For the full text of nalist essays and for information about applying for next years awards, see Science Online at www.sciencemag.org/feature/data/prizes/eppendorf/.
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References

Published by AAAS
CREDITS (TOP TO BOTTOM): MARY HINKLEY/UCL; MARY LEONARD; JENNIFER BISHOP
REVIEWS The Big and the Small: Challenges of Imaging the Brains Circuits
Jeff W. Lichtman1* and Winfried Denk2* The relation between the structure of the nervous system and its function is more poorly understood than the relation between structure and function in any other organ system. We explore why bridging the structure-function divide is uniquely difficult in the brain. These difficulties also explain the thrust behind the enormous amount of innovation centered on microscopy in neuroscience. We highlight some recent progress and the challenges that remain. central theme of biology is the relation between the structure and function of things. By structure, we mean the physical form of something, a property that humans can apprehend by touch (if the object is big enough) or by sight. Right now, the leading edge of this effort is the field known by the general name structural biology but is focused narrowly on the shapes of molecules in order to provide insights into how proteins such as channels, enzymes, and transcription factors do their jobs. The x-ray crystallography approach commonly used in structural biology does not generate images per se (producing instead diffraction patterns), but with the help of computers humans can change the data into a form that is interpretable intuitively by the visual system. Indeed, all of imaging is a means of generating data about an object that depend on location and that are presented so they can be seen and hence interpreted by vision, our most powerful sense. Going back in history, of course, there was a time when structural biology dealt with larger things: Discovering the functions of whole organs was center stage (1). Imaging was a critical part of the endeavor too. With the exception of the heart, which in many animals can be functionally understood without the need for magnification, most organs require microscopical analysis of structure and functional dynamics for the cellular organization (measured in micrometers) to be visible to the eye, whose limiting resolution several orders of magnitude coarser. Spanning 350 years, the microscope-enabled exploration of the cellular structure of organs has been extremely fertile. Indeed, for all organ systems, save one notable exception, it is now pretty much settled how the structure of an organ is related to its functionthe exception being the nervous sysresearch to determine the full extent of cell-type diversity in this small part of the nervous system, because the range of cell types continues to grow as the analysis becomes more refined. Moreover, neuronal cellular architecture is so variable from one region to the next that no single area of the brain serves as a guide for anything other than itself. Few believe that learning the full extent of retinal cellular diversity will be of much use in trying to understand the diversity in the cerebral cortex. Researchers have found ways to explore this diversity by developing imaging-based modalities to categorize cells and synapses. On the basis of the molecular peculiarities of cell types, molecular biologists have engineered means of generating tissue-specific expression of fluorescent markers in a wide and ever-growing range of neuronal cell types. In some cases the rationale is clear: The enzymes responsible for the synthesis of inhibitory transmitters are expressed only in inhibitory cells (3). In other cases, the molecular markers are useful but not well understood (4). The perhaps most powerful approach to cell classification uses antibodies. The well-known field of immunohistochemistry has had an extraordinary impact on cellular and subcellular analysis. However, there are special challenges in using antibodies to identify neuronal processes. First, antibodies do not penetrate well in thick tissues. Second, even if they did, the sheer density of epitopes makes it nearly impossible to unambiguously associate the epitopes with a particular process of a particular cell. Third, the range of markers necessary to delineate the many different cell and synapse types is extraordinarily large. These problems all have the same solution. By labeling arrays of serial sections with different antibodies (Fig. 1A), the threedimensional (3D) distribution of an array of epitopes can be mapped in a volume and potentially assigned to different cells (5, 6). The z resolution is given by the section thickness of 50 to 100 nm, with the exciting possibility of a similar lateral resolution achievable by using one of the new super-resolution techniques (7) (Fig. 1, B and C). Problem No. 2: Imaging Electrical and Chemical Activity A second unique feature of the nervous system is that the structural connectivity may ultimately determine function but does not constitute a map of function. The nervous system depends on rapid reversals of membrane potential, known as action potentials, to transmit signals between one part of a neuron and another distant part and depends on smaller, slower changes in member potential at sites of synaptic contact (i.e., synaptic potentials) to mediate the exchange of information between one cell and the next. Action potentials are typically only a few milliseconds in duration, which means direct optical readout of action-potential voltage is a challenge, because, for whatever optical signal one might devise, only
1 Molecular and Cellular Biology and Center for Brain Science Harvard University, Cambridge, MA 02138, USA. 2Max-Planck Institute for Medical Research, Department of Biomedical Optics, 69120 Heidelberg, Germany.
*To whom correspondence should be addressed. E-mail: jeff@mcb.harvard.edu ( J.W.L.); Winfried.Denk@mpimfheidelberg.mpg.de (W.D.)
Problem Number 1: Immense Diversity of Cell Types One unique feature of the brain that frustrates easy understanding of the relation between its structure and function is the immense structural and functional diversity of its cells. The nervous system of Caenorhabditis elegans is miniscule, only ~300 neurons (2), and yet nearly every single cell is unique in shape and function. In the far larger vertebrate brains, there are certainly structural classes of neurons that, although not having identical morphologies, have enough similarities within a class to be easily identifiable. The retina is a good example. With its iterated tiled structure, its cell classes are easy to recognize because they are repeated at regular intervals and have particular morphologies and molecular properties. Despite this, it is an area of active VOL 334 SCIENCE
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tem, where much progress has been made at the molecular and functional level. But notwithstanding the extraordinary insights of neurobiologys foremost structural biologist, Cajal, our understanding of the relation between the structure and function of the brain remains primitive, especially when compared to other organ systems. There is no other organ system where so many common diseases and disorders have no known histological trace. There is no other organ system for which we still debate how many cell types there are. There is no other organ system for which one would seriously propose to image the entire organs volume with an electron microscope. And there is also no other organ system for which the complexity of the structure is so great that earnest arguments can be made against delving into structural analysis because such an effort might well provide an unprecedentedly gigantic, yet totally incomprehensible, mass of data. Here, we will explore why bridging the structure-function divide is uniquely difficult in the brain. These difficulties also explain the thrust behind the enormous amount of innovation centered on microscopy in neuroscience, innovation that has been motivated by the special challenges of understanding how the brains functions are related to its especially complicated structure.
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are head-fixed and virtually (13) or truly (14) freely moving. Functional imaging has received an additional boost from the development of genetically encoded calcium indicators (15), which are finally becoming competitive (16, 17) with chemically synthesized indicators in sensitivity and speed. The genetically encoded indicators have the added advantage of targetability to specific cell classes. Researchers still await equivalently powerful genetically encoded voltage sensors, because these might provide the high temporal resolution required to understand certain details of circuit function. Tantalizing new proteorhodopsin-based probes are being developed that may be useful in this regard (18). The fact that most brain structures extend over considerable volumes requires imaging into a tissue volume rather than a thin sheet, another impediment to functional imaging. Scattering of light blurs wide-field images and strongly attenuates signals based on confocal detection. The attenuation is much less for signals based on 2P excitation. Nonetheless, imaging deep into volumes remains an area requiring new innovations. Penetration toward the deeper layers of cerebral cortex has been shown to be aided by combining 2P microscopy with adaptive optics (19, 20), amplified pulses (21), and longer excitation wavelengths (22). But at best, these approaches can extend the depth to about 1 mm. The imaging of subcortical structures requires the removal of overlying brain tissue (23) or the insertion of penetrating optical elements such as gradient-index lenses (24). One recently rediscovered approach, which has some of the advantages of 2P imaging in that it excites fluorescent molecules in a restricted optical section without excitation of fluorescence above or below that region, is the so-called lightsheet microscopy, in which a plane of a volume is illuminated by a narrow sheet of light and detected via a wide-field camera (25, 26). One advantage of this approach is that it can acquire images of many cells simultaneously, giving higher throughput for functional imaging than otherwise possible (27) but with less depth penetration than 2P imaging. Activity or its suppression can be optically triggered with high time resolution in cells expressing light-activated channels or transporters with different ion selectivities (28, 29). In addition to controlling the illumination pattern, targeting to certain cell classes is possible by using the same molecular tricks used for fluorescent proteins. This allows, for example, the testing of hypotheses such as whether particular cells whose activity is correlated to a particular behavior are central or peripheral to its generation (30). Problem No. 3: Neurons Extend Over Vast Volumes Since Cajals insight that nerve cells are functionally interconnected in a directional signaling cascade, with axons playing the role of transmitter and dendrites and somata that of receivers,
Fig. 1. Fluorescence imaging modalities useful for studying circuits. (A) Array tomography immunofluorescence of mouse somatosensory cortex shows the location of several different synaptic proteins and other neural markers in one piece of brain tissue. (Left) DAPI (4,6-diamidino-2-phenylindole, gray) show nuclei and yellow fluorescent protein (YFP) (green) shows processes of a subset of pyramidal cells and presynaptic boutons labeled with synapsin (magenta). (Middle) Distribution of two glutamate (VGluT1 in red, VGluT2 in yellow) and one g-aminobutyric acid (GABA) (VGAT in cyan) transporters in presynaptic terminals. (Right) Several postsynaptic labelsGluR2 (blue), N-methyl-D-aspartate receptor 1 (NMDAR1, white), and gephyrin (orange)each next to presynaptic synapsin labeling (magenta). Scale bar indicates 10 mm. Adapted from (6). (B) Array tomography immunofluorescence imaged with STED, a super-resolution optical technique showing large and small processes in a 3D volume image of mouse cerebral cortex. Dark voids are cell somata, intimately surrounded by perinuclear baskets of non-neuritic microtubules. Also visible is a dense profusion of large and small axonal and dendritic microtubule core bundles. Volume depth = 1.3 mm. [Image provided by Forrest Collman, Emi Ling, Corrie DaCosta, and Stephen Smith, Stanford University.] (C) Another super-resolution technique, STORM (stochastic optical reconstruction microscopy), is used with array tomography to reveal a subcortical white-matter tract showing quasiparallel trajectories of numerous axons (volume depth = 2.8 mm). [Image provided by Hazen Babcock and Xiaowei Zhuang, Harvard University, and Nick Weiler, Stanford.] (D) Layer 2/3 cortical neurons labeled with brainbow transgenes (cells were transfected in utero by electroporation). [Image provided by K. Loulier and J. Livet, Institut de la Vision, Paris.]
relatively few photons would likely be associated with each impulse. Detecting action potentials from many cells in a volume simultaneously would be even more difficult. Synaptic potentials can be orders of magnitude longer in duration than action potentials but are smaller in size, which poses other technical challenges. The development of small-molecule fluorescent calcium indicators, begun in the 1980s (8), showed that the rise in intracellular calcium, a consequence of neuronal activity, could in many cases
serve as a substitute for the direct optical readout of action potential activity. Although in many cases it is only a rough approximation of the number of action potentials, functional imaging, as it is called, is a mainstay of linking neural function to individual neurons. Used often in conjunction with two-photon (2P) microscopy (9) and with its unique ability to image with high resolution and high sensitivity inside scattering tissue (10), calcium imaging is now performed routinely in vivo (11, 12), in awake animals that SCIENCE VOL 334
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it has been appreciated that neural connectivity holds the key to function. Although neuronal somata are not unusually large, a neurons synaptic connections are distributed all through their dendritic and axonal branches. These branches often extend through tissue volumes that are enormous when compared to the actual volume of the cell itself. A pyramidal neuron in the cerebral cortex can have axonal branches that cross to the other hemisphere or go down to the brain stem or even the spinal cord. The dendrites of one pyramidal cell may be circumscribed by a volume of nearly a cubic millimeter. All told, the length of all the branches of one such cell may exceed a centimeter in a mouse and more than a meter in human brain. Thus, fully describing the shape of a single cortical neuron could require sampling a substantial percentage of an entire brains volume. But if one wanted to document the sites of all its synaptic connections, the sampling would need to be done at very high resolution to identify all the fine branches containing preand postsynaptic sites. Indeed, if one wished to just image the complete cell geometry of one neuron along with the complete geometry of the set of all the neurons that are directly pre- and directly postsynaptic to it, that volume would probably require imaging neuronal processes that span nearly the entire brain volume. As already mentioned, seeing into a volume of brain, even in fixed material, is a long-standing challenge. Indeed, the confocal microscope was invented because of attempts to peer into a tissue block of Golgi-stained cerebral cortex, where out-of-focus and scattered light undermined a clear view (31). Recent progress with clearing agents may be quite useful as we try to understand ever larger volumes of brain (32, 33). But is it possible to image such a large volume at very high resolution? In general in imaging, there is a tradeoff between volume and resolution: Large-volume imaging is done with low spatial resolution techniques, and high spatial resolution is accomplished over small volumes. Magnetic resonance imaging (MRI) can provide an image of a whole brain with ~1-mm3 voxels, whereas an electron microscope can render the structure of a synapse impinging on a dendritic spine by using
Fig. 2. The brain is organized over sizes that span 6 orders of magnitude. (A) The macroscopic brain, at the cm scale, is organized into regions such as the lobes of the cortex, the cerebellum, the brainstem, and the spinal cord. (B) At the millimeter scale, it is apparent that each brain region has neurons arranged in columns, layers, or other kinds of clusters. Often the axons communicating between brain regions (e.g., in the white matter beneath the cortex, which is tinted pink in this drawing) are separated from the sites where neuronal somata and their synaptic interconnections reside (e.g., in the gray matter of the cerebral cortex that lies above the white matter). (C) At the 100-mm scale, it is apparent that within each region neurons extend dendrites locally and send axons into the same or more distant sites. The most common excitatory neuron in the cerebral cortex is the pyramidal cell (shown here), whose dendrites can span a cubic millimeter. (D) At the 10-mm scale, the structure of the individual branches of a neuron become apparent. Many dendrites are studded with small processes (spines) where the axons of other neurons establish excitatory synaptic connections. (E to G) The detailed structure of the brain visualized by using EM. Successively higher resolution images of the same region of cerebrum acquired from a 29-nm section imaged on tape with a SEM detecting secondary electrons. In (E), which spans 100 mm, many neuron cell bodies (N) and their exiting processes can be seen along with occasional blood vessels, such as the capillary in the bottom right (BV). In (F), more cellular detail is apparent. The dark rings are cross sections of myelinated axons, such as the one labeled MY. The small dark objects are mitochondria (Mi), and the large polygonal objects are dendrites in cross section (D). Occasional somata of neurons and glia are visible (S). Most of the remaining tissue falls into three categories: glial processes (one highlighted in blue), presynaptic terminals (one tinted red), and dendrite spines (green). (G) The synapse is shown in red at 10 times higher resolution. At 3 nm per pixel, the synaptic vesicles (SV) are visible, along with the synaptic cleft (SC) and a membranous spine apparatus (SA) in the postsynaptic dendritic spine. [Artwork by Julia Kuhl, and SEM images are from Richard Schalek, Ken Hayworth, and Bobby Kasthuri, Harvard University.] 4 NOVEMBER 2011 VOL 334 SCIENCE www.sciencemag.org
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voxels of less than 100 nm3, a trillion-fold smaller volume. Thus, it is an extraordinary challenge to image large brain volumes at a resolution sufficient to find all the synapses (Fig. 2). The solution requires the automation, even industrialization, of imaging. Once the imaging is accomplished by machines, without the need for direct human oversight of each step, it becomes possible to increase the throughput by running an instrument around the clock and potentially many imaging machines working in parallel. Automation also increases reliability, which is essential because loss of even a small amount of data can invalidate the entire data set. Over the past several years there have been a number of techniques developed to optimize the automation of imaging: optical ablation (34), optical (35) and electron microscopic (36, 37) block face imaging (Fig. 3), and increased automation of stage motion and imaging steps in both standard scanning light and electron microscopes. Problem No. 4: The Detailed Structure Cannot Be Resolved by Traditional Light Microscopy The critical details of neuronal connectivity occur at the level of the synapse. Synapses are densely packed, and in cerebral cortex synapses often connect axons thinner than 100 nm to dendritic spines whose necks are thinner still. The resolution of diffraction-limited light microscopy is insufficient even when used in conjunction with confocal detection, 2P excitation, orfor fast imaging in clear specimensselective-plane illumination (see above), all so-called optical sectioning techniques that eliminate signals from out-of-focus structures. Optical super-resolution techniques that truly break the diffraction barrier and rely on the strong nonlinearities of stimulated emissionbased deterministic (38) or light activationbased stochastic (39, 40) molecular switching have matured in recent years (41, 42) and are beginning to engage questions about synaptic function (43) and other neurobiological problems (7, 44). Particularly important problems that are not yet fully overcome include the ability to image multiple colors and to observe dynamic processes at subwavelength resolution, even when they are located hundreds of micrometers below the brains surface. There are also other serious limitations related to the facts that the overall signal from small structures is proportional to their size and that often labeling densities, especially in vivo, are not high enough to give a sufficiently low noise image of the smallest structures. Moreover, often it is not the physics of the imaging process per se or the label density but rather intrinsic properties of the tissue, especially refractive index inhomogeneities, that distort the wave front and degrade resolution. Adaptive optics, a trick borrowed from astronomy, in which it is used to counteract the effects of Earths atmosphere, can improve resolution, signal, and depth penetration, in particular for in vivo imaging (19, 20). Lastly all these techniques are fluorescence-based,
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Fig. 3. Volume EM data and their analysis. (A) Fly optic lobe data stack as taken with 8000 cycles of SEM and focused ion beam ablation in 7-nm increments. Image is over a 28 mm by 28 mm by 56 mm volume that starts in the medulla (bottom solid rendering) and shows the process crossings in the outer chiasm (middle) and a dozen lamina cartridges (top). [Image courtesy of Shan Xu and Harald Hess at Howard Hughes Medical Institute, Janelia Farm, and Zhiyiuan Lu and Ian Meinerzhagen at Dalhousie University.] (B) A reslice through a stack taken on a diamond knife-based serial block face electron microscope (SBEM). Tissue was from mouse retina. White circle highlights a synapse. [Image courtesy of Kevin Briggman, Max-Planck Institute for Medical Research.] (C) All retinal bipolar cells in a SBEM data set, measuring 80 mm by 120 mm by 130 mm, were reconstructed by using computeraided manual skeleton tracing. Cell bodies are in gray. [Image courtesy of Moritz Helmstaedter, MaxPlanck Institute for Medical Research.] SCIENCE VOL 334 4 NOVEMBER 2011
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which allows very selective labeling but can make us forget the structural context. Problem No. 5: Need for Dense or Saturated Reconstruction Our present notions of neural circuits are still largely informed by Cajals ideas that showed with small arrows the directional flow of information through circuits and pathways. For his insights, the intrinsic inefficiency of the Golgi stain, which labels less than 5% of the neurons, was crucial. This sparse labeling allowed him to propose correctly the law of dynamic polarization, in which axons send signals to dendrites and somata and that information is then passed along to the next neuron by a cells axon. But the sparse labeling and his inability to see the signs (inhibitory or excitatory) of connections undermined an understanding of how information was actually processed by the circuits. Such fundamental questions as the number of different inputs a cell receives and the number of target cells a neuron innervates remain unknown even a century later. A small muscle that wiggles the mouse ear is the only place in the mammalian nervous system for which, in some sense, the entire connectivity matrix has been worked out, by complete labeling of all axons using transgenic fluorescent expression. This was possible because the relatively large size and small number of axons allowed a full rendering of all the connections with use of light microscopy and computer-aided manual reconstruction (45). The randomness and sparseness of the Golgi stain and more modern approaches to label subsets of neurons mean that in very few cases both pre- and postsynaptic cells are actually labeled and, even if they were, that fact could not be discerned with certainty because of limited resolution. This problem is being solved by new transsynaptic virus techniques (46, 47) that may ensure that all labeled cells are connected to one or a small population of cells. Sparseness is one way to avoid the melding together of multiple nearby processes. Alternatives are giving different cells different colors, which allows the identity of a neurite to be determined without having to trace it (31, 43) (Fig. 1D), or increasing the resolution of the light microscope to be able to trace it even in a dense neuropil (see above). However, the highestresolution microscope is still the electron microscope (Fig. 2, E to G). In addition to its resolution, electron microscopy (EM) provides a cellular context that is difficult to obtain with fluorescence because all cell membranes and intracellular organelles can be made visible. Several ways to improve serial-section EM have (re)emerged during the last decade: improving z resolution, increasing automation, as well as reducing section distortion and loss. Because modern scanning electron microscopes yield good contrast and resolution, even at low electron energies, when imaging plastic-embedded and heavymetal stained material, imaging the block face between cuts (36, 37) or ion-beam abrasions (48) (Fig. 3) yields well-aligned volume data. Moreover, thinner cuts can be taken, crucially improving the z resolution. The serial block-face approach has been used in combination with 2P-based calcium imaging to investigate the wiring of the direction-selectivity circuit in the retina (49), showing for example a massive violation of the so-called Peters rule, which essentially says that connection probability is mainly a function of axons and dendrites passing near each other. Instead, connectivity is finely tuned to allow starburst cell dendrites to pass on their directional signals to the appropriate retinal ganglion cells (49). Imaging and analysis speeds are further serious impediments to a full brain circuit reconstruction. The first, and still only, full nervous system connectome is that of the several hundred cells in a tiny worm, and still it took a decade. At the moment a full cubic millimeter of brain volume resolved to the level of seeing every synapse would require many months or even years to image and far longer to analyze. One approach to speed the imaging step by parallelizing the imaging in a transmission electron microscope (SEM) appears promising (50). Faster scanning electron microscopy also seems to be on the horizon with better detectors and new scanning strategies. Advances in instrumentation and labeling are giving us an unprecedented flood of data, which, partly because they are not perfect, need sophisticated computer science for analysis. Computers can learn how to best analyze the data by giving them a sufficient number of examples even if we do not understand the computational function that determines whether a voxel is inside or outside a cell. In the end, artificial neural networks may help us to analyze the data with which we then understand the brain (51). As circuit analysis finally moves forward, serious questions concerning its utility will be raised. One obvious question concerns the variability in the structure of the brain at the synaptic level. During the study of the mouse ear muscle described above, it became clear that every instantiation of the wiring diagram was different from every other one (45). Some will take such variability to mean that nothing can be learned from doing this kind of tedious, data-intensive, and highly expensive work. Alternatively, it is likely that one could learn a great deal about the game of chess by watching one game, despite the fact that it is highly unlikely that any two games are identical. It is certainly possible that certain circuit motifs will be recognizable and will be interpretable in a number of contexts. The key may be to make successful use of orthogonal data, such as from functional imaging, to link structure to a cellular or organismal behavior. The links may help decipher the code by which neuronal connections underlie all that we do.
1. P. M. Motta, M. Malpighi, in VIIIth International Symposium on Morphological Sciences (Liss, Rome, Italy, 10 to 15 July 1988), vol. 295. 2. J. G. White, E. Southgate, J. N. Thomson, S. Brenner, Philos Trans. R. Soc. London Ser. B 314, 1 (1986). 3. G. Di Cristo et al., Nat. Neurosci. 7, 1184 (2004). 4. I. J. Kim, Y. Zhang, M. Yamagata, M. Meister, J. R. Sanes, Nature 452, 478 (2008). 5. K. D. Micheva, S. J. Smith, Neuron 55, 25 (2007). 6. K. D. Micheva, B. Busse, N. C. Weiler, N. ORourke, S. J. Smith, Neuron 68, 639 (2010). 7. A. Dani, B. Huang, Curr. Opin. Neurobiol. 20, 648 (2010). 8. R. Y. Tsien, Harvey Lect. 99, 77 (2003-2004). 9. W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990). 10. F. Helmchen, W. Denk, Nat. Methods 2, 932 (2005). 11. K. Svoboda, W. Denk, D. Kleinfeld, D. W. Tank, Nature 385, 161 (1997). 12. K. Ohki, S. Chung, Y. H. Chng, P. Kara, R. C. Reid, Nature 433, 597 (2005). 13. D. A. Dombeck, A. N. Khabbaz, F. Collman, T. L. Adelman, D. W. Tank, Neuron 56, 43 (2007). 14. J. Sawinski et al., Proc. Natl. Acad. Sci. U.S.A. 106, 19557 (2009). 15. A. Miyawaki et al., Nature 388, 882 (1997). 16. M. Mank et al., Nat. Methods 5, 805 (2008). 17. L. Tian et al., Nat. Methods 6, 875 (2009). 18. J. M. Kralj, D. R. Hochbaum, A. D. Douglass, A. E. Cohen, Science 333, 345 (2011). 19. M. Rueckel, J. A. Mack-Bucher, W. Denk, Proc. Natl. Acad. Sci. U.S.A. 103, 17137 (2006). 20. N. Ji, D. E. Milkie, E. Betzig, Nat. Methods 7, 141 (2010). 21. W. Mittmann et al., Nat. Neurosci. 14, 1089 (2011). 22. D. Kobat et al., Opt. Express 17, 13354 (2009). 23. D. A. Dombeck, C. D. Harvey, L. Tian, L. L. Looger, D. W. Tank, Nat. Neurosci. 13, 1433 (2010). 24. J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, M. J. Schnitzer, J. Neurophysiol. 92, 3121 (2004). 25. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E. H. K. Stelzer, Science 305, 1007 (2004). 26. H. Siedentopf, R. Zsigmondy, Ann. Phys. (Leipzig) 10, 1 (1903). 27. T. F. Holekamp, D. Turaga, T. E. Holy, Neuron 57, 661 (2008). 28. G. Nagel et al., Proc. Natl. Acad. Sci. U.S.A. 100, 13940 (2003). 29. L. Fenno, O. Yizhar, K. Deisseroth, Annu. Rev. Neurosci. 34, 389 (2011). 30. D. Lin et al., Nature 470, 221 (2011). 31. J. Livet et al., Nature 450, 56 (2007). 32. H. U. Dodt et al., Nat. Methods 4, 331 (2007). 33. H. Hama et al., Nat. Neurosci., published online 30 August 2011 (10.1038/nn.2928). 34. P. S. Tsai et al., Neuron 39, 27 (2003). 35. A. J. Ewald, H. McBride, M. Reddington, S. E. Fraser, R. Kerschmann, Dev. Dyn. 225, 369 (2002). 36. S. B. Leighton, Scan. Electron Microsc. 2 , 73 (1981). 37. W. Denk, H. Horstmann, PLoS Biol. 2, e329 (2004). 38. S. W. Hell, J. Wichmann, Opt. Lett. 19, 780 (1994). 39. E. Betzig et al., Science 313, 1642 (2006). 40. M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 3, 793 (2006). 41. S. W. Hell, Science 316, 1153 (2007). 42. B. Huang, M. Bates, X. Zhuang, Annu. Rev. Biochem. 78, 993 (2009). 43. V. Westphal et al., Science 320, 246 (2008); 10.1126/science.1154228. 44. T. A. Weissman, J. R. Sanes, J. W. Lichtman, J. Livet, Cold Spring Harbor Protocols 2011, 763 (2011). 45. J. Lu, J. C. Tapia, O. L. White, J. W. Lichtman, PLoS Biol. 7, e1000032 (2009). 46. E. M. Callaway, Curr. Opin. Neurobiol. 18, 617 (2008). 47. M. I. Ekstrand, L. W. Enquist, L. E. Pomeranz, Trends Mol. Med. 14, 134 (2008). 48. G. Knott, H. Marchman, D. Wall, B. Lich, J. Neurosci. 28, 2959 (2008). 49. K. L. Briggman, M. Helmstaedter, W. Denk, Nature 471, 183 (2011).
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50. D. D. Bock et al., Nature 471, 177 (2011). 51. V. Jain, H. S. Seung, S. C. Turaga, Curr. Opin. Neurobiol. 20, 653 (2010). Acknowledgments: We thank K. Briggman, H. Hess, J. Livet, M. Helmstaedter, and S. Smith for providing images and A. Karpova for commenting on the manuscript. Our own work was supported by the NIH, Gatsby Charitable Foundation, and a Collaborative Innovation Award (no. 43667) from Howard Hughes Medical Institute ( J.W.L.), the Deutsche Forschungsgemeinschaft (W.D.), and the Max-Planck Society (W.D.). W.D. has provided a technology license for block-face EM to Gatan, Incorporated.
10.1126/science.1209168
The Cell Biology of Synaptic Plasticity

Victoria M. Ho,1 Ji-Ann Lee,2 Kelsey C. Martin2,3,4* Synaptic plasticity is the experience-dependent change in connectivity between neurons that is believed to underlie learning and memory. Here, we discuss the cellular and molecular processes that are altered when a neuron responds to external stimuli, and how these alterations lead to an increase or decrease in synaptic connectivity. Modification of synaptic components and changes in gene expression are necessary for many forms of plasticity. We focus on excitatory neurons in the mammalian hippocampus, one of the best-studied model systems of learning-related plasticity. he circuitry of the human brain is composed of a trillion (1012) neurons and a quadrillion (1015) synapses, whose connectivity underlies all human perception, emotion, thought, and behavior. Studies in a range of species have revealed that the overall structure of the nervous system is genetically hardwired but that neural circuits undergo extensive sculpting and rewiring in response to a variety of stimuli. This process of experience-dependent changes in synaptic connectivity is called synaptic plasticity. Studies of synaptic plasticity have begun to detail the molecular mechanisms that underlie these synaptic changes. This research has examined a variety of cell biological processes, including synaptic vesicle release and recycling, neurotransmitter receptor trafficking, cell adhesion, and stimulus-induced changes in gene expression within neurons. Taken together, these studies have provided an initial molecular biological understanding of how nature and nurture combine to determine our identities. As a result, research on synaptic plasticity promises to provide insight into the biological basis of many neuropsychiatric disorders in which experiencedependent brain rewiring goes awry. Here we focus on long-lasting forms of plasticity that underlie learning and memory. We consider, in turn, each component of the synapse: the presynaptic compartment, the postsynaptic compartment, and the synaptic cleft, and discuss
constraints prevent us from addressing any single mechanism in depth; instead, our aim is to provide a framework for understanding the cell biology of synaptic plasticity. Hippocampal Synaptic Plasticity The successful study of the cell biology of synaptic plasticity requires a tractable experimental model system. Ideally, such a model should consist of a defined population of identifiable neurons and be amenable to electrophysiological, genetic, and molecular cell biological manipulations. Awell-studied model system for studying plasticity in the adult vertebrate nervous system is the rodent hippocampus (Fig. 1). Critical for memory formation, the anatomy of the hippocampus renders it particularly suitable for electrophysiological investigation. It consists of three sequential synaptic pathways (perforant, mossy fiber, and Schaffer collateral pathways), each with discrete cell body layers and axonal and dendritic projections (Fig. 1). Synaptic plasticity has been studied in all three hippocampal pathways. Distinct stimuli elicit changes in synaptic
processes that undergo activity-dependent modifications to alter synaptic efficacy. Long-lasting changes in synaptic connectivity require new RNA and/or protein synthesis, and we discuss how gene expression is regulated within neurons. We concentrate on studies of learning-related plasticity at excitatory chemical synapses in the rodent hippocampus because these provide extensive evidence for the cell biological mechanisms of plasticity in the vertebrate brain. Space
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1 Interdepartmental Program in Neurosciences, University of CaliforniaLos Angeles (UCLA), BSRB 390B, 615 Charles E. Young Drive South, Los Angeles, CA 900951737, USA. 2Department of Biological Chemistry, UCLA, BSRB 310, 615 Charles E. Young Drive South, Los Angeles, CA 900951737, USA. 3Department of Psychiatry and Biobehavioral Sciences, UCLA, BSRB 390B, 615 Charles E. Young Drive South, Los Angeles, CA 900951737, USA. 4Brain Research Institute, UCLA, BSRB 390B, 615 Charles E. Young Drive South, Los Angeles, CA 900951737, USA.
Dentate gyrus
*To whom correspondence should be addressed. E-mail: kcmartin@mednet.ucla.edu
Fig. 1. Hippocampal synaptic plasticity. The rodent hippocampus can be dissected and cut into transverse slices that preserve all three synaptic pathways. In the perforant pathway (purple), axons from the entorhinal cortex project to form synapses (yellow circles) on dendrites of dentate granule cells; in the mossy fiber pathway (green), dentate granule axons synapse on CA3 pyramidal neuron dendrites; and in the Schaffer collateral pathway (brown), CA3 axons synapse on CA1 dendrites. The dentate, CA3, and CA1 cell bodies form discrete somatic layers (dark blue lines), projecting axons and dendrites into defined regions. Electrodes can be used to stimulate axonal afferents and record from postsynaptic follower cells, as illustrated for the Schaffer collateral (CA3-CA1) pathway. SCIENCE VOL 334 4 NOVEMBER 2011
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efficacy; high-frequency stimuli produce synaptic strengthening called long-term potentiation (LTP), and low-frequency stimulation produces synaptic weakening, called longterm depression (LTD). LTP and LTD can also be produced by spike timingdependent plasticity, in which the relative timing of pre- and postsynaptic spikes leads to changes in synaptic strength (1). Different patterns of stimulation elicit changes in synaptic strength that persist over various time domains, with long-lasting forms, but not short-term forms, requiring new RNA and protein synthesis (2). Hippocampal plasticity is studied in in vivo and in vitro preparations. Implanted electrodes can be used to stimulate and record from hippocampal pathways in living animals. The hippocampus can be dissected out of the brain and cut into 300- to 500mm-thick transverse slices that can be maintained and recorded from for hours (Fig. 1). Slices can also be kept as organotypic slice cultures for weeks, preserving many aspects Fig. 2. The ultrastructure of the synapse. Neurons communicate of their architecture. Finally, hippo- with one another at chemical synapses. (A) Electron micrograph campal neurons can be studied in from area CA1 in adult rat hippocampus. The CA1 dendritic shaft dissociated cultures, which are paris colorized in yellow, the spine neck and head in green, the ticularly amenable to manipulation presynaptic terminal in orange, and astroglial processes in blue. and dynamic imaging of individ- Scale bar, 0.5 mm. (B) Three-dimensional reconstruction of an ual neurons and synapses. The de- 8.5-mm-long dendrite (yellow) with the PSDs labeled in red. Note 3 velopment of genetically modified the variation in spine and PSD size and shape. Scale cube, 0.5 mm . Reproduced with permission from Elsevier (63). mice and vectors for acute manipulation of gene expression complete a rich tool-kit for studies of the cell and molecular biology of hippocampal synaptic releasable pool docked at the active zone; the recycling pool, which can be released with modplasticity. erate stimulation; and the reserve pool, which is Presynaptic Mechanisms of Plasticity only released in response to strong stimuli. A famCommunication at chemical synapses involves ily of phosphoproteins called synapsins tether the release of neurotransmitter from the presyn- synaptic vesicles to the actin cytoskeleton and aptic terminal, diffusion across the cleft, and to one another. Neuronal stimulation activates binding to postsynaptic receptors (Figs. 2 and kinases that phosphorylate synapsins to modulate 3). Chemical neurotransmission is rapid (occur- synaptic vesicle tethering and thereby alter the ring in milliseconds) and highly regulated. The number of synaptic vesicles available for release presynaptic terminal contains synaptic vesicles (3). Synapsin knockout mice have reduced refilled with neurotransmitter and a dense matrix serve pools of synaptic vesicles and demonof cytoskeleton and scaffolding proteins at the strate deficits in learning and memory as well site of release, the active zone. Varying the prob- as various forms of plasticity (4), indicating that ability of neurotransmitter release provides one activity-dependent modulation of synaptic vesmechanism for altering synaptic strength during icle mobilization is critical to neuronal and behavioral plasticity. neuronal plasticity. RIM proteins and synaptic vesicle docking Synaptic vesicle release can be subdivided into distinct steps, including vesicle mobilization, and priming. For synaptic vesicles to become docking, priming, fusion, and recycling. Although fusion competent, they must undergo docking each of these steps may be regulated by activity, and priming, in which vesicle and plasma memwe will highlight three: vesicle mobilization, brane soluble NSF-attachment protein receptor (SNARE) proteins are brought into close contact docking, and priming. Synapsins and synaptic vesicle mobilization. to allow rapid fusion following calcium influx. The population of synaptic vesicles within a pre- The Rab3-interacting molecule (RIM) family synaptic terminal exist in three states: the readily of proteins is critical for this process (5). As large, multidomain proteins, RIMs act as scaffolding proteins to cluster calcium channels in the active zone (6) and interact with Munc-13 (7), a priming factor required for efficient SNARE complex formation and membrane fusion. RIM is a substrate for phosphorylation by protein kinase A (PKA) and is required for mossy fiber LTP (8). Postsynaptic Mechanisms of Plasticity Most principle neurons in the brain are studded with membrane protuberances called dendritic spines, which are the postsynaptic compartments. Spines are heterogeneous in shape (Fig. 2), but consist of a bulbous head and a thinner neck that connects the spine to the dendritic shaft; the size of the spine head and the volume of the spine correlate with synaptic strength (9, 10), with large spine heads containing more neurotransmitter receptors, reflecting greater synaptic strength. Spines serve as compartmentalized signaling units, and the number and shape of spines change during synaptic plasticity (11). At the ultrastructural level, the postsynaptic compartment is characterized by an electron-dense postsynaptic density (PSD), which consists of neurotransmitter receptors and an extensive network of scaffolding proteins. Activation of postsynaptic kinases in the spine: CaMKII and PKMz. LTP and LTD induction are both dependent on postsynaptic increases in intracellular calcium, with LTP requiring large increases in calcium concentrations and LTD being dependent on smaller calcium increases. The increase in calcium activates multiple downstream signaling enzymes, including the kinases calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). LTP induction in the CA1 region of the hippocampus requires CaMKII activity (12, 13), and transgenic mice lacking the a isoform have defective LTP and spatial learning (14, 15). CaMKII undergoes autophosphorylation in response to increases in Ca2+-bound calmodulin, which renders the kinase autonomously active. Neuronal activity also translocates CaMKII to the PSD, where it can phosphorylate many PSD proteins, including glutamate receptors. The autophosphorylation of CaMKII is essential for LTP induction and, perhaps, its maintenance (16) [but see (17)]. The brain-restricted atypical PKC isoform, protein kinase M zeta (PKMz), is constitutively active and thus phosphorylates targets in the absence of extracellular stimulation. PKMz mRNA is targeted to dendrites where activity-dependent signaling cascades regulate its local translation during LTP and LTD (18). PKMz is sufficient and necessary for LTP maintenance and for the maintenance of long-term memories, and PKMz activation may perpetuate synaptic plasticity and memory (18, 19). Activity-dependent modulation of postsynaptic glutamate receptors. The main excitatory neurotransmitter in the brain is glutamate, which
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activates several postsynaptic receptors. Two types of ionotropic glutamate receptorsa-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) have central roles in hippocampal synaptic plasticity. Both are ligand-gated ion channels and have unique properties that subserve different phases of synaptic plasticity. NMDA-type glutamate receptors (NMDARs) are calcium permeable and, when activated, allow an influx of calcium needed for the induction of LTP. However, NMDARs do not conduct current at resting potentials because their channel pores are blocked by magnesium cations. Consequently, NMDARs have been called coincidence detectors because, to conduct current, they require both presynaptic transmitter release as well as postsynaptic depolarization to relieve the magnesium block. AMPA-type glutamate receptors (AMPARs) are important for the expression and maintenance of LTP. Unlike NMDARs, AMPARs can be activated by ligand binding at resting potentials to allow current flow. Increased conductance through AMPARs is responsible for the increase in synaptic strength during NMDAR-dependent LTP at CA1 synapses. Given the importance of AMPARs in determining synaptic strength, much effort has focused on delineating the mechanisms that regulate their function. Regulated phosphorylation can change AMPAR function by changing the open probabilities and conductances of the receptors. However, changes in channel properties are unlikely to account for the drastic changes in AMPAR function seen with LTP (20). Instead, changes in AMPAR function during synaptic plasticity are mostly due to phosphorylation-induced changes in its abundance at the synapse. AMPARs traffic constitutively to and from the plasma membrane via recycling endosomes (21) (Fig. 3). Delivery of AMPARs to synapses is believed to occur first by exocytosis at extrasynaptic sites followed by lateral diffusion within the plasma membrane to PSDs, where the mobility of the receptors is greatly reduced. During removal of synaptic AMPARs, receptors diffuse away from the PSD and then undergo clathrinmediated, dynamin-dependent endocytosis. After endocytosis, small GTP-binding proteins of the Rab family and effector proteins direct AMPARs either to early (sorting) endosomes or back to the plasma membrane (22). AMPAR trafficking occurs constitutively under basal conditions and is modulated by activity through changes in actin and myosin dynamics (23), as well as AMPAR interactions with scaffolding proteins and accessory subunits. One of these accessory subunits, Stargazin, mediates the interaction between AMPARs and the PSD protein PSD-95, and this interaction is important for synaptic localization of AMPARs (24). Activity alters the phosphorylation of Stargazin, with phosphorylated Stargazin decreasing the
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Fig. 3. Activity-dependent modulation of pre-, post-, and trans-synaptic components. Presynaptic: Neurotransmitter vesicle cycling. Neurotransmitter release starts with the filling of synaptic vesicles, which then dock and undergo priming at the active zone. Arrival of an action potential induces calcium influx through voltage-sensitive calcium channels (VSCCs), which triggers membrane fusion and exocytosis. The synaptic vesicles are then recycled via local reuse (a; kiss and stay), fast recycling (b; kiss and run), or clathrin-mediated endocytosis (c). Neurotransmitter release can be regulated during plasticity as exemplified by the regulation of synapsin phosphorylation (1) and the regulation of RIM protein phosphorylation (2). Postsynaptic: AMPA receptor trafficking. Locally and somatically synthesized AMPARs enter a pool of endosomes that undergo constitutive www.sciencemag.org SCIENCE
and regulated membrane trafficking. During potentiation, greater receptor insertion (3) increases the concentration of AMPARs at the synapse, where they are anchored by interactions at the PSD. During synaptic depression, AMPARs are endocytosed (3). The preferential location of endocytosis and exocytosis is probably extrasynaptic. Within the plasma membrane, trafficking of AMPARs between the synapse and the point of insertion or removal occurs by lateral diffusion. Extrasynaptic movement of AMPARs increases with neuronal activity (4). Receptor trafficking is modulated by phosphorylation of AMPAR subunits (5), which influences interactions with scaffolding proteins. Trans-synaptic: Synaptic cell adhesion molecules. PSA-NCAM is increased following neuronal activity (6). Lightning bolts indicate activitydependent processes. VOL 334 4 NOVEMBER 2011
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mobility of AMPARs and enhancing AMPAR function. Blocking Stargazin phosphorylation or dephosphorylation blocks LTP and LTD, respectively (25). AMPARs exist as tetramers made up of different combinations of the four subunits, GluA1 through 4. The cytoplasmic tails of each subunit contain multiple phosphorylation sites that regulate the trafficking of AMPARs. For example, PKA phosphorylation of S845 in the long cytoplasmic tail of GluA1 increases GluA1 surface expression due to both enhanced insertion and attenuated internalization (26). Conversely, LTD of dissociated cultures and brain slices results in dephosphorylation of S845 and is correlated with an increased rate of AMPAR endocytosis (27). Knock-in mice with phosphorylation-deficient mutations at both S831A and S845A display a loss of NMDA-induced AMPAR internalization, deficits in LTP and LTD, and have impaired spatial memory (28). Although studies of posttranslational modifications at individual sites have established a role for regulating GluA1 trafficking and channel properties, they do not fully account for the changes in GluA1 function observed with synaptic plasticity (29). Activity-modified residues continue to be discovered, including, for example, the highly conserved T840 phosphorylation site, the phosphorylation of which correlates remarkably well with synaptic strength (30). It is likely that complex patterns of phosphorylation and of other post-translational modifications (e.g., palmitoylation or ubiquitination) combine to regulate AMPAR localization. Trans-Synaptic Signaling; the Synaptic Cleft The synaptic cleft is a ~20-nm junction between the pre- and postsynaptic compartments, consisting of a space through which neurotransmitters diffuse to bind postsynaptic receptors, as well as a network of cell adhesion molecules (CAMs) that keeps the synapse together. These adhesive interactions are so strong that it is impossible to separate intact pre- from postsynaptic compartments biochemically. Role of CAMs in synaptic plasticity. The CAMs that localize to the synaptic cleft include members of the cadherin, integrin, and immunoglobulincontaining CAMs, as well as neurexins and neuroligins. Much research has focused on trying to understand whether and how CAMs mediate synapse specificity during neural circuit formation. Here we focus on the regulation of synaptic CAMs during experience-dependent synaptic plasticity, limiting our discussion to just two of many examples. One such example involves the addition of large sialic acid homopolymers to the neural cell adhesion molecule (NCAM) to form polysialylated NCAM (PSA-NCAM), which decreases homophilic adhesion to allow new synaptic remodeling and growth. The ratio of PSA-NCAM
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Fig. 4. Local regulation of the synaptic proteome. Synaptic plasticity modifies gene expression at many levels. Strong stimulation of synapses triggers signals that are sent to the nucleus to modify RNA synthesis. Synaptic activity also modifies protein synthesis, and has been found to act at several key steps during translation: (1) Relief of repression, e.g., RISC-mediated repression; (2) modification of translational initiation to allow 4E-4G interaction and recruitment of 40S; (3) formation of the preinitiation complex; and (4) dephosphorylation of eEF2 to allow for catalysis of ribosome translocation
during translational elongation. To counterbalance local protein synthesis, local protein degradation also occurs at synapses (5). Together, these regulated steps in protein addition and removal allow for rapid, spatially restricted control of the synaptic proteome. Lightning bolts indicate activity-dependent processes. RBP, RNA binding proteins such as exon junction complexes, RISC machinery, Staufen, CPEB, etc. (Note: Although local translation in dendrites is a well-accepted phenomenon, it has not been demonstrated to occur in spines.) SCIENCE www.sciencemag.org
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to NCAM increases following hippocampal learning tasks, and inactivation of the enzyme that adds the polysialic moieties blocks hippocampal learning and plasticity (31). The increase in PSA-NCAM is thought to promote synaptic remodeling during persistent forms of plasticity. Another family of CAMs that play a role in hippocampal plasticity includes the synaptically localized receptor tyrosine kinase ephrins and ephrin receptors (Eph receptors). Initially studied in the context of neural development, ephrins and Eph receptors have also been found to be essential for hippocampal LTP and LTD in the adult brain (32). Specific ephrins and Eph receptors regulate the localization and function of NMDA receptors, and can thereby modulate synaptic strength in response to activity. Experiments using inhibitory ephrin and Eph receptor peptides have revealed that both molecules are required, in a kinase-independent manner, for mossy fiber hippocampal LTP (33). Trans-synaptic signaling by retrograde messengers. Another means of trans-synaptic signaling involves diffusible, membrane-soluble messengers. The CB1 and CB2 cannabinoid receptors were initially identified as receptors for cannabinoid, the active ingredient of THC/marijuana. This led to the identification of endogenous CB1 and CB2 ligands, called endocannabinoids. Endocannabinoids have emerged as important modulators of plasticity, initially at inhibitory synapses, and more recently at excitatory synapses (34). Depolarization and activation of a variety of receptors have been shown to activate release of endocannabinoids from the postsynaptic compartment and binding to presynaptic CB receptors, resulting in a suppression of neurotransmitter release (and thus regulating presynaptic plasticity). This form of plasticity is called endocannabinoid-LTD, or eCB-LTD. Endocannabinoid signaling is required for extinction but not acquisition of spatial memories (35). The Tripartite Synapse: Glia and Synaptic Plasticity Once thought of as the support cells of the nervous systems, glial cells are now considered essential partners in synapse formation, synaptic transmission, and plasticity (36). Astrocytes surround the synapse (Fig. 2), forming a tripartite synapse, composed of neuronal pre- and postsynaptic compartments as well as surrounding astrocytes. Synaptically localized glia release neuroactive molecules that influence neuronal communication. For example, release of D-serine (a coactivator of the NMDA receptor) from glia is required for LTP of hippocampal Schaffer collateral synapses (37) [although see also (38)]. Ephrin and Eph receptor signaling between neurons and glia regulates the uptake of glutamate through glial glutamate transporters and thereby affects neurotransmission and synaptic plasticity (39). The release of lactate from astrocytes and uptake by neurons has also been reported to be required for long-term hippocampal memory and plasticity (40). Regulating Gene Expression Within Neurons During Plasticity Signaling from synapse to nucleus to regulate transcription. Long-lasting forms of synaptic plasticity, such as those underlying long-term memory, require new RNA synthesis (2). This indicates that synaptic signals must be relayed to the nucleus to regulate transcription. Synapse-to-nucleus signaling poses a unique set of challenges in neurons, where the distance between the synapse and nucleus can be appreciable. Neurons are specialized for rapid communication between compartments via electrochemical signaling, with depolarization at the synaptic terminal leading to depolarization at the soma in less than a millisecond. Calcium influx can occur through voltage- and ligand-gated ion channels. Cytosolic calcium can also be released from intracellular pools following activation of Gq-coupled receptors such as metabotropic GluRs (mGluRs). Each route of calcium influx induces different programs of gene induction (41). Soluble signals can also be transported from the synapse to the nucleus by slower, microtubuleand motor proteindependent pathways (42). This class of signals includes kinases and transcriptional regulators that function to alter transcription. These slower pathways of signaling to the nucleus may sustain changes in gene expression for time periods extending beyond the initial stimulus. To obtain a global view of how transcription is altered during activity-dependent plasticity, expression profiling has been used to identify changes in transcription following depolarization of cultured mouse neurons. Such studies have identified several hundred activity-regulated genes (41). Genome-wide analyses of transcription factor binding sites of the activated genes have revealed that the transcription factors CREB, MEF2, and Npas4 control the activity-dependent transcription of a large number of downstream activity-regulated genes (41). These downstream transcription factors regulate the expression of overlapping but distinct subsets of activity-regulated genes, suggesting that the precise temporal, spatial, and stimulus-specific cellular response is achieved by the combinatorial control by different transcription factors. Local protein synthesis. Despite requiring new transcription, LTP and LTD can occur in a spatially restricted manner, raising the question of how gene expression in neurons can be limited to subsets of synapses and not generalized to the entire cell. One way of locally changing the proteome in neurons is through regulated translation of localized mRNAs (Fig. 4). The existence of local translation in dendrites of mature neurons was first suggested by electron micrographic identification of polyribosomes in hippocampal dendrites (43). Studies in hippocampal slices in which dendrites had been seSCIENCE VOL 334 vered from cell bodies found that such dendrites retain the ability to express long-lasting LTP and LTD, indicating that local translation can mediate long-term modification of synaptic strength (44, 45). Studies of mRNA localization have led to the identification of cis-acting RNA elements that bind to RNA-binding proteins to undergo export from the soma into the dendrite (46). Although several dendritic localization elements have been identified, there is to date no consensus on their sequence or structure. Among the beststudied RNA binding proteins involved in dendritic mRNA localization are Staufen, Zipcode binding protein 1 (ZBP1), and hnRNPA2 (46). These proteins bind cis-acting elements and assemble transcripts into larger RNA transport granules, which travel in a kinesin-dependent manner along microtubules to their final destination. Whether localized RNAs undergo directed targeting, anchoring, or stabilization at specific sites remains an open question. In terms of translational regulation, studies have revealed activity-dependent regulation of translation initiation and elongation. A mechanism of translational regulation known to occur at synapses involves the cytoplasmic polyadenylation element binding protein (CPEB). CPEB binding to 3 untranslated regions (3UTRs) represses translation. However, CPEB undergoes phosphorylation in an activity-dependent manner to recruit other proteins that increase the polyadenylate [ poly(A)] tails of mRNAs. Subsequently, poly(A) binding protein (PABP) is recruited to the elongated poly(A) tail, which in turn recruits eukaryotic translation initiation factor 4g (eIF4G) to interact with eukaryotic translation initiation factor 4E (eIF4E) to promote translation initiation (47). CPEB localizes to synapses, where it regulates translation of dendritically localized CamKIIa mRNA (48, 49). Another activity-dependent means of regulating translation initiation involves phosphorylation of eIF4E-binding proteins (4E-BPs). Hypophosphorylated 4E-BPs bind eIF4E and prevent translation initiation; phosphorylated 4E-BP dissociates from eIF4E and relieves translational inhibition. In neurons, activity increases 4E-BP phosphorylation and stimulates translation (50). Studies in 4E-BP2 knockout mice found that E-LTP stimulation protocols could induce L-LTP in brain slices. Recently, two additional 4E-BPs have been identified in neurons: neuroguidin and the cytoplasmic FMRP interacting protein (CYFIP). Whereas 4E-BP1 and 2 are believed to affect general translation, these new 4E-BPs may preferentially affect subgroups of transcripts within dendrites (51, 52). Activity can also regulate translational elongation during synaptic plasticity. For example, the elongation factor eukaryotic translation elongation factor 2 (eEF2) undergoes activity-dependent changes in phosphorylation. Phosphorylation of eEF2 decreases the rate of translation. Whereas action potentials decrease eEF2 phosphorylation
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(thereby increasing translation), spontaneous release of neurotransmitter increases eEF2 phosphorylation and decreases translation (53). These effects occur locally at synapses, indicating that one function of spontaneous release may be to suppress local translation and thereby stabilize synapses. Translation may also be regulated through the microRNA (miRNA) pathway, where each miRNA can potentially regulate hundreds of transcripts and hence coordinate the expression of many genes. Many miRNAs are relatively more abundant in, or restricted to, the brain. While miRNAs can regulate cell-wide levels of translation, their posttranscriptional mode of action makes them especially well suited to regulating distally localized transcripts. Specific miRNAs have been found in dendrites and synapses, and components of the RNA-induced silencing complex (RISC) machinery itself have been found to be altered by activity (54). Consistent with the importance of regulating synaptic AMPAR concentrations during plasticity, the mRNAs encoding GluA1 and GluA2 have both been detected in hippocampal dendrites and found to undergo activity-dependent changes in localization and translation (55, 56). Further linking local translation with synaptic AMPAR abundance, local eEF2-dependent translation of Arc mRNA has been shown to trigger endocytosis of AMPARs during mGluR-mediated hippocampal LTD (57, 58). Local protein degradation. The local proteome is regulated not only by local translation but also by protein degradation through the ubiquitin proteasome system (Fig. 4). Both protein synthesis and degradation are required for the maintenance of late-phase LTP, suggesting that protein degradation is needed to counterbalance protein synthesis during plasticity (59). Like local translation, protein degradation can be regulated within dendrites. Ubiquitin and proteasomal subunits have been found in dendrites and at synapses, and stimulation of hippocampal neurons triggers proteasome-dependent changes in the composition of PSD proteins (60). Activitydependent degradation involves redistribution of proteasomes from dendritic shafts to spines (61). Notably, the ubiquitin proteasome pathway alters AMPAR trafficking and degradation at synapses during plasticity (62). Perspectives As the above examples illustrate, cell biological approaches have provided a detailed understanding of many aspects of activity-dependent plasticity. By focusing on molecular processes occurring within individual neurons and subcellular compartments, we now understand specific processes that are modulated by experience to change synaptic strength. These involve alterations in neurotransmitter release, trans-synaptic signaling, postsynaptic receptor dynamics, and gene expression within neurons. Distinct plasticity mechanisms are used at different types of synapses. For instance, LTP at mossy fiber synapses occurs primarily through presynaptic changes, whereas LTP at Schaffer collateral synapses occurs mostly through postsynaptic mechanisms. The end result of many of the processes we have described is to regulate the concentration of glutamate receptors, indicating that this is a major postsynaptic determinant of synaptic strength during plasticity. Together, each of these cell biological mechanisms provides potential therapeutic targets for diseases in which brain plasticity is dysfunctional. However, they fall short of elucidating how complex circuits are altered by experience to store information and alter behavior. This will require the development of tools for investigating both the dynamic nano-architecture of the synapse and the neural circuit as a whole. A particular challenge is to study plasticity in neural circuits in living animals, and to develop methods to examine, and computational frameworks to understand, how all components of a circuit are regulated to alter circuit function dynamically. The development of methodologies for superresolution time-lapse imaging of synapses, neurons, and circuits in live animals promises to move the field forward toward a more nuanced and complete understanding of the experiencedependent plastic changes in the brain that mediate learning and memory.
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RESEARCH ARTICLE Porphyrin-Sensitized Solar Cells with Cobalt (II/III)Based Redox Electrolyte Exceed 12 Percent Efficiency
Aswani Yella,1 Hsuan-Wei Lee,2 Hoi Nok Tsao,1 Chenyi Yi,1 Aravind Kumar Chandiran,1 Md.Khaja Nazeeruddin,1 Eric Wei-Guang Diau,3* Chen-Yu Yeh,2* Shaik M Zakeeruddin,1* Michael Grtzel1* The iodide/triiodide redox shuttle has limited the efficiencies accessible in dye-sensitized solar cells. Here, we report mesoscopic solar cells that incorporate a Co(II/III)tris(bipyridyl)based redox electrolyte in conjunction with a custom synthesized donor-p-bridge-acceptor zinc porphyrin dye as sensitizer (designated YD2-o-C8). The specific molecular design of YD2-o-C8 greatly retards the rate of interfacial back electron transfer from the conduction band of the nanocrystalline titanium dioxide film to the oxidized cobalt mediator, which enables attainment of strikingly high photovoltages approaching 1 volt. Because the YD2-o-C8 porphyrin harvests sunlight across the visible spectrum, large photocurrents are generated. Cosensitization of YD2-o-C8 with another organic dye further enhances the performance of the device, leading to a measured power conversion efficiency of 12.3% under simulated air mass 1.5 global sunlight. ye-sensitized solar cells (DSCs) have recently received great attention because of their ease of fabrication and cost-effectiveness compared with silicon (Si) based photovoltaic devices (1). So far, the best conversion efficiencies have been obtained with ruthenium-based dyes used together with the iodide/triiodide redox couple (210). Upon optical excitation, the dye injects an electron into the conduction band of a nanocrystalline film of a wide band gap oxide, such as titanium dioxide (TiO2), and is subsequently regenerated back to the ground state by electron donation from a redox couple present in the electrolyte. In this fashion, the DSC achieves the separation of light harvesting and charge generation from charge carrier transport, whereas all other known photovoltaic devices, including Si cells, perform both operations simultaneously. This imposes stringent demands on the puritiy of the semiconductor 99.9999% for solar grade Siresulting in high material cost and long energy payback times. The separation of the functions of light absorption and carrier transport achieved by the DSC has many advantages, key points being a large choice of light absorber and charge transport materials, which can be applied by simple screen printing methods and complexes do have this property. However, such one-electron, outer-sphere redox couples often yield shorter electron lifetimes when used in the DSC. This faster interfacial charge recombination, when compared with iodide/triiodidebased redox electrolytes (1218), lowers the photovoltage and reduces the efficiency of charge collection, decreasing the short circuit photocurrent density (Jsc) and hence the PCE (1921). Recent evidence suggests that the introduction of long-chain alkyloxy groups in the dye structure may retard the unwanted charge recombination process (22). Donor-p-bridge-acceptor (D-p-A) sensitizers, endowed with such groups, recently reached Voc values exceeding 0.8 V when used with Co(II/III)tris(bipyridyl) redox electrolytes. However, the PCEs of these devices remained in the 6.7 to 9.6% range because of their insufficient solar light harvesting, resulting in low photocurrents (2224). This rationale has inspired us to synthesize a D-p-A zinc (Zn) porphyrin dye, designated YD2-o-C8, which absorbs light over the whole visible range and is endowed with longchain alkoxy groups so as to impair interfacial back electron transfer reaction. When used in conjuntion with cobalt polypyridylbased redox electrolytes, YD2-o-C8 achieves conversion efficiencies exceeding those obtained with todays best ruthenium sensitizers. Inspired by the important role that porphyrins play in natural photosynthesis, researchers have tested numerous derivatives of this substrate class as sensitizers for DSCs, but conversion efficiencies obtained so far have largely remained below 8% (2533). An exception is the class of D-p-A porphyrin dyes, such as YD2, which has reached a PCE of 11% when used with iodide/triiodide redox electrolyte (34). A diarylamine group attached to the porphyrin ring acts as an electron donor, and an ethynylbenzoic acid moiety serves as an acceptor, anchoring the dye to the titania surface. The unique feature of these sensitizers is that the porphyrin chromophore itself constitutes the p-bridge of the D-p-A structure (3540). The judiciously tailored variant of YD2 that we report here,YD2-o-C8 (Fig. 1), incorporates two octyloxy groups in the ortho positions of each meso-phenyl ring, producing a striking
do not have to undergo costly purification or doping treatments. Although the DSC already outperforms its competitors in ambient light and indoor conditions, its validated solar-to-electric power-conversion efficiency (PCE) under standard air mass 1.5 (AM 1.5) reporting conditions (1000 W/m2 solar light intensity and 298 K) of 11.1% (2) is still a factor of 2 below that of Si solar cells. (The air mass number expresses the ratio of the path length of the solar light in the atmosphere over that corresponding to vertical position of the sun. At air mass 1.5, the sun is at an angle of 48.19 with regard to the vertical axes). Because of an excessive loss of voltage during the dye-regeneration reaction, the use of the iodide/triiodide electrolyte as a redox shuttle limits the attainable open-circuit potential, Voc, to 0.7 to 0.8 Vand is thus a drawback of current DSC embodiments. This has prevented substantial gains in PCE over the last 5 years (11). To further improve the PCE, development of redox mediators exhibiting higher reduction potentials than that of I3 is warranted; Co(II/III)tris(bipyridyl)
1 Laboratory for Photonics and Interfaces, Institute of Chemical Sciences and Engineering, cole Polytechnique Fdrale de Lausanne, Lausanne-1015, Switzerland. 2Department of Chemistry and Center of Nanoscience and Nanotechnology, National Chung Hsing University, Taichung, Taiwan 402, Peoples Republic of China. 3Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, Hsinchu, Taiwan 300, Peoples Republic of China.
*To whom correspondence should be addressed. E-mail: michael.graetzel@epfl.ch (M.G.); shaik.zakeer@epfl.ch (S.M.Z.); diau@mail.nctu.edu.tw (E.W.-G.D); cyyeh@dragon. nchu.edu.tw (C.-Y.Y.)
Fig. 1. The molecular structures of the (left) YD2 and (right) YD2-o-C8 porphyrin dyes. SCIENCE VOL 334 4 NOVEMBER 2011
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amelioration of the photo-induced charge separation in DSCs using Co(II/III)tris(bipyridyl) based redox electrolyte. High PCE values reaching 11.9% have been achieved with this molecular photovoltaic system, which produces a Voc of 965 mV, a Jsc of 17.3 mA/cm2, and a fill factor (FF) of 0.71 under standard AM 1.5 sunlight at 995 W/m2 intensity. The cosensitization of YD2-o-C8 with the previously prepared organic D-p-A dye, coded Y123, yielded an efficiency of 12.3% when used in conjunction with the Co(II/III)tris(bipyridyl)based redox electrolyte. The PCE even exceeds 13% under AM 1.5 solar light of 500 Wm2 intensity. Molecular properties and photovoltaic performance. The detailed synthetic procedure for preparing the YD2-o-C8 porphyrin is described in the supporting online material (SOM). Absorption spectra of these two porphyrin dyes measured in tetrahydrofuran (THF) solvent are shown in fig. S1, and table S1 lists the corresponding spectral properties. Both porphyrins exhibit maxima in the 400 to 500 nm and 550 to 750 nm ranges, corresponding to the Soret and Q bands, respectively, with similar molar absorption coefficients. Nevertheless, the Soret band of YD2o-C8 is slightly red-shifted, and its Q band is narrower as compared with the YD2 spectrum. This is ascribed to the electronic effect of the two electron-donating dioctyloxy substituents introduced in ortho-position of the meso-phenyl ring. The emission behavior for the two sensitizers matches their absorption spectra, the fluorescence maximum of YD2-o-C8 being blue-shifted by 13 nm relative to that of YD2. The oxidation potentials of the two dyes were determined by means of cyclic voltammetry (CV). YD2-o-C8 shows two oxidation waves at half-wave potential (E1/2) = +0.82 and +1.37 V versus normal hydrogen electrode (NHE) (fig. S2). These values are lower than the corresponding potentials of YD2 (table S1) because of the electron-donating substituents on the meso-phenyl rings. The 190-mV negative shift of the reduction potential of YD2-o-C8 corresponds to a lifting of the lowest unoccupied molecular orbital (LUMO) level and is a manifestation of the same effect. Fig. 2. (A) Comparison of the J-V curves of YD2o-C8 and YD2-sensitized solar cells under full AM 1.5 solar intensity. (B) Incident photon-toelectric current conversion efficiencies as a function of wavelength for the two dyes (YD2-o-C8, red; YD2, blue). The TiO2 film thickness is a 5-mm transparent layer and on top a 5-mm scattering layer. To gain further insight into the electron density distribution within the frontier and other closelying orbitals, we performed quantum chemical calculations on the YD2-o-C8 porphyrin using density functional theory (DFT) at the B3LYP/631G(d) level [Spartan 08 package (Wavefunction, Irvine, CA)] and compared them with the calculated electronic structure of the YD2 dye (fig. S3). In the ground state, the electron density of YD2 for the highest occupied molecular orbital (HOMO) and HOMO-1 is shared by the diphenylamine donor moiety and the p-system of the porphyrin ring. Introduction of the strongly electrondonating group (octyloxy) at the ortho-positions of phenyl rings, attached at the meso-positions of porphyrin core in YD2-o-C8, increases the electronic density on the porphyrin p-system over tert-butyl groups present at the HOMO and HOMO-1 level of the YD2 dye. Thus, for YD2o-C8 there is a considerable electronic coupling between the alkoxy groups and the porphyrin core, lifting the LUMO level higher than that of the YD2 dye. The predicted effect is an increase of the HOMO-LUMO gap of YD2-o-C8 compared with YD2 dye, which is in good agreement with the experimental observations. Photovoltaic experiments were conducted with an electrolyte by using the Co(II/III)tris(bipyridyl) tetracyanoborate complexes as redox couple. The optimized electrolyte composition, designated AY1, consists of 0.165 M [CoII(bpy)3](B(CN)4)2, 0.045 M [CoIII(bpy)3](B(CN)4)3 along with 0.8 M tert-butyl pyridine (TBP), and 0.1 M LiClO4 in acetonitrile as a solvent. Details on the optimization of the concentration for the electrolyte components are presented in fig. S4. Using the Nernst equation and standard potential (E o ) = 0.57 V versus NHE for the experimentally determined (23) standard potential of the Co(II/III)tris(bipyridyl) tetracyanoborate couple, the oxidation potential for this electrolyte is derived as 0.535 V versus NHE. Performance in a Co(II/III)tris(bipyridyl) mediated solar cell. Photocurrent density versus voltage (J-V) is shown in Fig. 2A; the curves were measured under standard photovoltaic reporting conditions (AM 1.5 global sunlight at 1000 W/m2 and a temperature of 298 K) for cells with YD2-o-C8 or YD2 sensitizers used in conjunction with the AY1 electrolyte. YD2-o-C8 gives a PCE of 11.9% compared with only 8.4% for the reference dye YD2. The key observation here is that a subtle modification of the porphyrin structure induces strikingly large Jsc and Voc improvements. This performance advantage is maintained over a large range of light intensities, ranging from 10% to full solar intensity (Table 1). The incident monochromatic photon-to electric current conversion efficiency (IPCE) as a function of wavelength and J-V curves for the same YD2 and YD2-o-C8 devices is presented in Fig. 2. YD2-o-C8 shows impressively high IPCE values over the whole visible wavelength range, exceeding 80% from 450 nm up to 680 nm, except for a narrow dip in the spectrum around 530 nm. The spectral response of the photocurrent obtained with the YD2 cell shows similar features, with peaks at around 460 and 650 nm. However, the absolute IPCE values are 20 to 30% lower for YD2 as compared with YD2-o-C8, despite its higher molar extinction coefficient, which is in keeping with the lower short-circuit photocurrent densities observed for YD2 in Fig. 2A. The Jsc values obtained from calculating the overlap integral of the IPCE spectrum with the standard AM 1.5 global spectral solar photon flux were 17.3 and 14.9 mA/cm2 for YD2o-C8 and YD2, respectively. These figures agree within 2% with the measured Jsc, showing that any spectral mismatch between the simulated and true AM 1.5 solar emission is very small with the solar simulator used in our experiments. The four factors determining the IPCE are the light-harvesting efficiency (LHE), the quantum yield of electron injection from the excited sensitizer into the TiO2 conduction band (Finj), the efficiency for dye regeneration (hreg), and the collection efficiency of the photo-generated charge carriers (hcoll): IPCE = LHE Finj hreg hcoll (1)
The charge carrier collection efficiency is determined in turn by the time constant for transport (ttrans) and recombination (trec) of the conduction
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band electrons injected into the nanocrystalline TiO2 film: hcoll = 1/(1 + ttrans/trec) (2) slightly shifted to the blue with respect to that of YD2, which matches the behavior of the absorption spectra. The LHE reaches close to 100% near the Soret and Q bands, even for the 6-mmthick transparent TiO2 film. The spectral domain where all the photons are captured by the dye-sensitized mesoscopic TiO2 film is further broadened for the 10-mm-thick double layer used in Fig. 2 because of the increase of the optical path length by light scattering from the top particle layer. This rules out that a change in the LHE causes the difference in the IPCE and Jsc values for the two porphyrins (34). Dividing the IPCE by the LHE gives the absorbed photon-toelectric current generation efficiency (APCE), presenting the true quantum yield for electric current generation from light. The APCE values in the wavelength range from 420 to 700 nm are between 80 and 100%, and 60 to 90% for YD2-o-C8 and YD2, respectively (Fig. 4). This indicates that the electron injection, dye regeneration, or charge carrier collection is less efficient for the latter than for the former sensitizer when used in conjunction with the Co(II/III)tris(bipyridyl) tetracyanoborate complexes as redox couple. We performed time-resolved luminescence experiments to unravel any differences in the dynamics of photo-induced electron injection between the two porphyrins. In fig. S5, we compare emission data in THF solution and for YD2o-C8 or YD2-covered nanocrystalline TiO2 films in contact with the same Co(II/III)tris(bipyridyl) based redox electrolyte used in the photovoltaic experiments. In solution, the fluorescence lifetimes for YD2-o-C8 and YD2 are 1.5 and 1.2 ns, respectively, whereas the emission for both sensitizers is strongly quenched in contact with TiO2. Data fitting, including reconvolution of the emission signal over the ~1 ns excitation pulse length, indicates that the lifetime of the excited singlet state of the two porphyrins in the adsorbed state is at most 100 ps. These experiments do not reveal any significant difference in the fluorescence kinetics of the two porphyrins. There is also little doubt that the very rapid quenching of luminescence leads in both cases to nearquantitative charge injection from their excited singlet state to the conduction band of the TiO2 nanocrystals, as implied by the very high APCE values observed for YD2-o-C8 in Fig. 4 and the near-unity IPCE values for YD2 obtained with iodide/triiodidebased redox electrolytes. The data obtained from intensity-modulated photo-induced absorption (PIA) measurements (41) shown in the top row of fig. S6 confirm the occurrence of oxidative quenching after light excitation of the two porphyrins adsorbed on the nanocrystalline TiO2 film. The absorption peaks at 550, 800, and 1400 nm reveal the formation of porphyrin radical cations by electron injection from the excited state in the conduction band of the titania. The two PIA spectra in the lower row show that these features disappear completely in the presence of the Co(II/III)tris(bipyridyl) electrolyte, indicating complete regeneration of the YD2 or YD2-o-C8 porphyrins through electron donation from the Co(II) form of the redox couple. Because the LHE as well as the quantum yield for electron injection and regeneration are very high for both dyes, we reasoned that the lower IPCE values for YD2 as compared with YD2-o-C8 reflect differences in the efficiency for carrier collection between the two porphyrin sensitizers. Inhibition of back electron transfer. We used transient photocurrent and photovoltage decay measurements to compare the rates of interfacial
We separately examined the four efficiency parameters of Eq. 1 in order to elucidate the reasons for the much higher IPCE values obtained with YD2-o-C8 as compared with the YD2 porphyrin. The LHE of a 6-mm-thick transparent TiO2 film loaded with a monolayer of the sensitizer is shown in Fig. 3. The LHE for the two porphyrins is very similar, the Q-band for YD2-o-C8 being
Table 1. Detailed photovoltaic parameters of the devices made with the dyes YD2 and YD2-o-C8 and cobalt-based AY1 electrolyte at different light intensities. Pin, incident intensity of AM1.5 solar light.
Dye YD2 Electrolyte AY1 Pin (mW/cm2) 9.4 51.3 99.8 9.4 51.2 99.5
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1.5 8.0 14.9 1.7 9.3 17.3
745 805 825 875 940 965
0.82 0.76 0.69 0.77 0.74 0.71
9.5 9.5 8.4 12.5 12.7 11.9
Fig. 3. LHE as a function of wavelength for the YD2 and YD2-o-C8 porphyrins adsorbed at the surface of a 6-mm-thick nanocrystalline TiO2 film.
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Fig. 4. APCE as a function of wavelength for the YD2 and YD2-o-C8 porphyrin adsorbed at the surface of a 6-mm-thick nanocrystalline TiO2 film in contact with Co(II/III)tris(bipyridyl) based electrolyte.
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Fig. 5. Electron (A) lifetime and (B) capacitance determined with photocurrent and photovoltage decay measurements of devices with YD2 and YD2-o-C8 dyes.
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Fig. 6. (A) J-V characteristics of a YD2-oC8/Y123 cosensitized DSC, measured under standard AM 1.5 global sunlight under various light intensities. The molar YD2-oC8/Y123 ratio in the dye solution was 7. (B) Spectral response of the IPCE for YD2-o-C8 (red dots), Y123 (blue triangles), and YD2-o-C8/Y123 cosensitized nanocrystalline TiO2 films (black squares)
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recombination of electrons from the TiO2 conduction band to CoIIItris(bipyridyl) (42, 43). The electron lifetime as a function of Voc for YD2 and YD2-o-C8 is plotted in Fig. 5A. The Voc was adjusted by varying the intensity of the bias light impinging on the cell. The electron lifetime is 2 to 10 times longer for YD2-o-C8 than for YD2-sensitized TiO2 films, the difference becoming larger as the Voc increases. One plausible explanation of this observation is that at open circuit, electrons in the conduction band of TiO2 (eTiO2) reacts faster with CoIIItris(bipyridyl) for films loaded with the latter than with the former dye. It appears that the specific molecular structure of YD2-o-C8in particular, the presence of the four octyloxy groupsreduces the recombination rate, most likely by inhibiting the access by CoIIItris(bipyridyl) to the TiO2 surface. The distance dependence of the back reaction is in accordance with the semiclassical electron transfer theory (44, 45). The lower rate of electron recapture by CoIIItris(bipyridyl) for YD2-o-C8sensitized nanocrystalline TiO2 films allows very high opencircuit voltages to be realized, with this sensitizer reaching a value of 965 mV in full sunlight without sacrificing short circuit photocurrent or fill factor. The voltage advantage of YD2-o-C8 over YD2 is maintained at lower light levels, down to 3 W/m2 solar intensity (fig. S7). The CoII/IIItris(bipyridyl) electrolyte yields slopes of 90 mV/decade and 80 mV/decade, corresponding to ideality factors m = 1.52 and 1.35 in the diode equation for YD2o-C8 and YD2 dyes, respectively. This suggests that surface-trapping states participate in the back-electron transfer reaction, which is similar to the findings by Hagfeldt and co-workers (22). It may be argued that the higher Voc observed with YD2-o-C8 with respect to YD2 arises from a larger dipole moment of the former as compared with the latter sensitizer in the adsorbed state, causing an upward shift of the conduction band of TiO2. This would displace the trap-state distribution function toward higher energies, rendering the density of occupied states (DOS) at a given forward bias voltage lower for YD2o-C8 than for YD2. To check this possibility, the DOS of the films loaded with sensitizer was determined from transient photocurrent decay measurements (42, 43). The chemical capacitance Cm for the YD2-o-C8 and YD2-sensitized films as a function of Voc are compared in Fig. 5B. Cm is directly proportional to the DOS: Cm = q(e) DOS, where q(e) is the charge of one electron. The Cm values are very similar for the two sensitizers, with small differences being noted only at Voc > 0.8 V. This rules out any substantial contribution of a conduction band shift to the VOL 334 SCIENCE
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observed decrease in the back-electron transfer rate. Using YD2-o-C8 as a sensitizer, we compared the photovoltaic performance of CoII/IIItris(bipyridyl) with that of a I /I3based redox electrolyte using again a (5+5) mm double-layer TiO2 film. The concentrations of I and I3 in this electrolyte, designated AY2, were identical to those of the Co2+ and Co3+ tris(bipyridyl) in AY1; the other additives, such as TBP and LiClO4, were also maintained at the same levels, as was the acetonitrile solvent. The results presented in table S2 and fig. S8 show that the photovoltaic performance of devices using the CoII/IIItris(bipyridyl) electrolyte is far superior to that of the I /I3 based devices. The Voc and Jsc values increase by 193 mV and 2.3 mA/cm2, respectively, producing a 58% gain of the PCE from 7.6 to 11.9%. To evaluate the photovoltaic performance of the newly designed YD2-o-C8 dye under conditions that are optimal for I /I3based DSCs, a thicker double-layer TiO2 film of (12+5) mm was applied along with the Z959 standard volatile electrolyte [1.0 M 1,3-dimethylimidazolium iodide (DMII), 0.03 M iodine, 0.1 M guanidinium thiocyanate, and 0.5 M tert-butylpyridine in a mixture of valeronitrile/acetonitrile (15:85 v/v)]. Under these conditions, the PCE reached 9.4% (detailed photovoltaic parameters are in table S2). These results
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Table 2. Detailed photovoltaic parameters of the devices made with AY1 electrolyte and YD2-o-C8 dye cosensitized with Y123 dye at different light intensities.
Pin (mW/cm2) 9.4 50.8 99.5 Jsc (mA/cm2) 1.83 9.72 17.66 Voc (mV) 840 910 935 FF 0.79 0.76 0.74 h (%) 13.0 13.1 12.3
B. ORegan, M. Grtzel, Nature 353, 737 (1991). Y. Chiba et al., Jpn. J. Appl. Phys. 45, L638 (2006). C.-Y. Chen et al., ACS Nano 3, 3103 (2009). W. Zeng et al., Chem. Mater. 22, 1915 (2010). Q. Yu et al., ACS Nano 4, 6032 (2010). J. M. Kroon et al., Prog. Photovolt. Res. Appl. 15, 1 (2007). 7. M. K. Nazeeruddin et al., J. Am. Chem. Soc. 127, 16835 (2005). 8. Y. Chiba, A. Islam, R. Komiya, N. Koide, L. Y. Han, Appl. Phys. Lett. 88, 223505 (2006). 9. Z. S. Wang, M. Yanagida, K. Sayama, H. Sugihara, Chem. Mater. 18, 2912 (2006). 10. M. D. Wei et al., J. Mater. Chem. 16, 1287 (2006). 11. G. Boschloo, A. Hagfeldt, Acc. Chem. Res. 42, 1819 (2009). 12. M. Wang, N. Chamberland, L. Breau, J.-E. Moser, R. H.-Baker, B. Marsan, S. M. Zakeeruddin, M. Grtzel, Nat. Chem. 2, 385 (2010). 13. D. Li et al., Adv. Funct. Mater. 20, 3358 (2010). 14. T. Daeneke et al., Nat. Chem. 3, 211 (2011). 15. Y. Bai et al., Chem. Commun. (Camb.) 47, 4376 (2011). 16. B. A. Gregg, F. Pichot, S. Ferrere, C. L. Fields, J. Phys. Chem. B 105, 1422 (2001). 17. S. Hattori, Y. Wada, S. Yanagida, S. Fukuzumi, J. Am. Chem. Soc. 127, 9648 (2005). 18. Z. Zhang, P. Chen, T. N. Murakami, S. M. Zakeeruddin, M. Grtzel, Adv. Funct. Mater. 18, 341 (2008). 19. H. Nusbaumer, S. M. Zakeeruddin, J. E. Moser, M. Grtzel, Chemistry 9, 3756 (2003). 20. S. A. Sapp, C. M. Elliott, C. Contado, S. Caramori, C. A. Bignozzi, J. Am. Chem. Soc. 124, 11215 (2002). 21. H. Nusbaumer, J. E. Moser, S. M. Zakeeruddin, M. K. Nazeeruddin, M. Gratzel, J. Phys. Chem. B 105, 10461 (2001). 22. S. M. Feldt et al., J. Am. Chem. Soc. 132, 16714 (2010). 23. H. N. Tsao et al., ChemSusChem 4, 591 (2011). 24. Z. Difei et al., Environ. Sci. 4, 2030 (2011). 25. J. N. Clifford, G. Yahioglu, L. R. Milgrom, J. R. Durrant, Chem. Commun. (Camb.) 12, 1260 (2002). 26. T. Hasobe et al., J. Am. Chem. Soc. 127, 1216 (2005). 27. T. Hasobe et al., J. Phys. Chem. B 109, 19 (2005). 28. M. Borgstrm et al., J. Phys. Chem. B 109, 22928 (2005). 29. C.-W. Lee et al., Chem. Eur. J. 15, 1403 (2009). 30. A. Huijser, T. J. Savenije, A. Kotlewski, S. J. Picken, L. D. A. Siebbeles, Adv. Mater. (Deerfield Beach Fla.) 18, 2234 (2006). 31. O. Hagemann, M. Jrgensen, F. C. Krebs, J. Org. Chem. 71, 5546 (2006). 32. G. M. Hasselman et al., J. Phys. Chem. B 110, 25430 (2006). 33. S. Eu et al., J. Phys. Chem. C 111, 3528 (2007). 34. T. Bessho, S. M. Zakeeruddin, C. Y. Yeh, E. W. G. Diau, M. Grtzel, Angew. Chem. Int. Ed. 49, 6646 (2010). 35. H.-P. Lu et al., J. Phys. Chem. C 113, 20990 (2009). 36. C.-P. Hsieh et al., J. Mater. Chem. 20, 1127 (2010). 37. S.-L. Wu et al., Environ. Sci. 3, 949 (2010). 38. J.-C. Chang, C.-J. Ma, G.-H. Lee, S.-M. Peng, C.-Y. Yeh, Dalton Trans. 8, 1504 (2005). 39. H.-P. Lu et al., Phys. Chem. Chem. Phys. 11, 10270 (2009). 40. C.-L. Mai et al., Chem. Commun. (Camb.) 46, 809 (2010). 41. H. J. Snaith et al., Nanotechnology 19, 424003 (2008). 42. B. C. ORegan, F. J. Lenzmann, J. Phys. Chem. B 108, 4342 (2004). 43. B. C. O'Regan, Bakker, J. Kroeze, H. Smit, P. Sommeling, James R. Durrant, J. Phys. Chem. B 110, 17155 (2006). 44. P. Siders, R. A. Marcus, J. Am. Chem. Soc. 103, 741 (1981). 45. J. N. Clifford, E. Martnez-Ferrero, A. Viterisi, E. Palomares, Chem. Soc. Rev. 40, 1635 (2011). 46. J. J. Nelson, T. J. Amick, C. M. Elliott, J. Phys. Chem. C 112, 18255 (2008). Acknowledgments: E.W.-G.D. and C.Y.Y. acknowledge the financial support from the National Science Council of Taiwan and Ministry of Education of Taiwan. M.G. thanks the European Research Council (ERC) for an Advanced Research Grant (ARG 247404) funded under the Mesolight project. Financial support under the European communitys 7th FWP for project 227057 (INNOVASOL) and under a grant from the Dayton U.S. Air force Research Laboratory is gratefully acknowledged. 1. 2. 3. 4. 5. 6.
confirm that for YD2-o-C8, the CoII/IIItris(bipyridyl) complex outperforms the I /I3based redox electrolyte, even under optimal conditions for the latter. Despite having excellent light-harvesting properties, the YD2-o-C8 dye lacks absorption in the 480 to 630 nm range, as evidenced by the IPCE spectra in Figs. 2B and 6B. The dip in the green spectral region reduces the JSC and hence the PCE. To avoid this loss, we used a cosensitizer coded Y123 (a D-p-A dye whose structure is shown in fig. S9) that possesses a complementary absorption spectrum to YD2-o-C8. Y123 exhibits an absorption maximum at 532 nm in THF with an extinction coefficient of 53,000 M1 cm1 (23). This coincides with the minimum in the spectral IPCE response of the YD2-o-C8 dye, the absorption maximum of which is at 644 nm (e = 31,200 M1 cm1). Enhanced performance from adding a cosensitizer. The cosensitized films achieved better photovoltaic performance than that of solar cells using a single dye. The photocurrent J-V curves measured at three different light intensities, using the AY1 redox electrolyte, are shown in Fig. 6A. The photovoltaic performance parameters are listed in Table 2. Increased shortcircuit current density and FF are observed for the cosensitized films compared with YD2-o-C8 alone. The cumulative increases of Jsc and FF give rise to an efficiency of 12.3% at AM 1.5 global full sun using the CoII/IIItris(bipyridyl)based redox electrolyte. The PCE reached an even higher value of 13.1% at 509 W/m2 solar intensity (50.9% sun). These results were independently verified by repeating the photovoltaic characterization of the cells in the Photovoltaic Laboratory at the Institute of Micro Technique (IMT), Neuchtel, Switzerland. The Wacom high-precision class AAA solar simulator system available at the IMT Photovoltaic Laboratory very closely mimics the solar spectrum in the absorption range of the cosensitized solar cells in the range of 350 to 750 nm. This avoids any substantial spectral mismatch between the simulated and true AM 1.5 solar light source. Results shown in fig. S10 and table S3 fully confirm the PCE measurements carried out in our own laboratory. The IPCE spectra of devices made with the individual and combined dyes are shown in Fig. 6B. The Jsc value obtained from integrating the product of the IPCE spectrum with the AM 1.5 global spectral solar photon flux was 18.3 mA/cm2. This value is greater than the measured Jsc value (17.6 mA/cm2) at full sunlight. The difference can be attributed to mass trans-
port limitations at full sunlight, which limit the photocurrent. This effect is corroborated by the photocurrent transient measurements by using an on/off modulation of the incident light (46). The data reported in fig. S11 show a spike in the photocurrent, which reaches a lower stationary value after a few seconds of illumination time. Our goal is to eliminate this small Jsc loss so as to attain a strictly linear response of the photocurrent up to full solar intensity. Such linear behavior already can be achieved by reducing the porphyrin content in the YD2-o-C8/Y123 containing staining solution from 7:1 to 4:1, as shown in table S4. This indicates that the mass transport limitation is also partly caused by the bulky nature of the YD2-o-C8 dye. Because the increase in Y123 content in the dye mixture also reduces the Voc value, the linear photocurrent response does not contribute to the PCE at full sunlight. The YD2-o-C8/Y123 cosensitized nanocrystalline TiO2 film exhibit an impressive panchromatic photocurrent response over the whole visible range, achieving incident photon-to-electron conversion efficiencies of >90% in a large wavelength domain below 700 nm. Although the cosensitization increases the Jsc, the Voc decreases by about 30 mV with respect to films sensitized by YD2-o-C8 alone. Transient photo-voltage decay measurements giving access to the electron lifetime were used to examine the reason for this decrease. The plots of electron lifetime versus open-circuit potential in fig. S12 show that the incorporation of Y123 reduced the lifetime of the photo-generated electrons in the TiO2 conduction band compared with YD2-o-C8 alone. Continued exposure of YD2-o-C8sensitized solar cells for 220 hours to full sunlight at 30C showed only a 10 to 15% decrease of the overall efficiency over this extended light-soaking period. This shows that the photosystem is robust and that the small decline is probably caused by losses of the volatile acetonitrile solvent. The present results provide a fertile base for further investigation, which will focus on achieving linear response of the photocurrent to full sunlight, enabling PCE values above 13% to be reached under standard reporting conditions. Further challenges to be tackled are the use replacement of the volatile electrolyte by nonvolatile ionic liquid-based systems, as well as solid-state hole conductors. Last, new anchoring groups will be introduced in the porphyrin in order to avoid any desorption on long-term heat and light exposure, along with functional groups increasing the near-infrared response of the sensitizer. SCIENCE VOL 334
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M.K.N. thanks World Class University program, Photovoltaic Materials, Department of Material Chemistry, Korea University, Chungnam 339-700, Korea, which is funded by the Ministry of Education, Science and Technology through the National Research Foundation of Korea (R31-2008-000-10035-0). We thank R. Humphry-Baker for fruitful discussions as well as optical measurements
and M. J. M. Bonnet-Eymard from the IMT Photovoltaic Laboratory for assistance with the cell photovoltaic performance measurements.
Figs. S1 to S12 Tables S1 to S4 References (47, 48)
Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6056/629/DC1 Materials and Methods 13 June 2011; accepted 23 September 2011 10.1126/science.1209688
REPORTS Structural Dynamics of a Catalytic Monolayer Probed by Ultrafast 2D IR Vibrational Echoes

Daniel E. Rosenfeld,* Zsolt Gengeliczki,* Brian J. Smith, T. D. P. Stack, M. D. Fayer Ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy has proven broadly useful for studying molecular dynamics in solutions. Here, we extend the technique to probing the interfacial dynamics and structure of a silica surface-tethered transition metal carbonyl complextricarbonyl (1,10-phenanthroline)rhenium chlorideof interest as a photoreduction catalyst. We interpret the data using a theoretical framework devised to separate the roles of structural evolution and excitation transfer in inducing spectral diffusion. The structural dynamics, as reported on by a carbonyl stretch vibration of the surface-bound complex, have a characteristic time of ~150 picoseconds in the absence of solvent, decrease in duration by a factor of three upon addition of chloroform, and decrease another order of magnitude for the bulk solution. Conversely, solvent-complex interactions increase the lifetime of the probed vibration by 160% when solvent is applied to the monolayer. ailoring surface properties by depositing molecular monolayers (1) on various solid substrates is critical to many technologies, including industrial catalysis, chemical sensors (2), fuel cells, and molecular electronics (3). The functional groups terminating the monolayer determine the hydrophobicity, chemical reactivity (4), and charge transfer properties (5) of the interface, which are strongly influenced by local structure and fast associated dynamics. Despite a long-standing need, the tools to study structural dynamics of interfacial molecules under chemically relevant conditions have been lacking (6). Commonly used microscopy and scattering techniques provide information on the size, shape, and electronic structure of particles and adsorbates, but their time resolution is generally insufficient to study molecular dynamics, and many only function under ultrahigh vacuum conditions (7). The development of ultrafast infrared (IR) spectroscopy over the past two decades has provided tools for the in-depth examination of the dynamics and structure of bulk liquids, liquids in nanoscopic environments, organic complexes, biological macromolecules, and solids (811). in time-resolved SFG renders interpretation difficult because of the included Raman process (23), which necessitates careful determination of the time correlation functions measured in the given beam geometry (25). Furthermore, SFG-based techniques are inherently insensitive because upconversion is inefficient. Techniques relying on hole-burning methods have intrinsically lower time resolution and sensitivity and produce convoluted spectra, whereas echo-based methods suffer none of these drawbacks (26). Here, we report on the application of an ultrafast 2D IR vibrational echo method to molecular monolayers that overcomes all of these challenges, because there is no intrinsic tradeoff between time and frequency resolution, and the associated heterodyne detection provides much higher sensitivity (signal-to-noise ratio) than other methods. This approach opens the way for the quantitative understanding of the effect of immobilization and solvent on the structural and vibrational dynamics of molecular monolayers on solid substrates. We applied our technique to the study of a silica-immobilized transition metal carbonyl compound of interest as a photocatalyst. Immobilized homogeneous catalysts are appealing because they maintain high molecular specificity and activity under mild conditions while precluding the need for expensive separation methods (2729). However, conditions such as the presence or absence of solvent can strongly affect catalytic activity in presently unpredictable ways (29, 30). Microscopic changes affecting molecular reactivity should manifest themselves in the dynamical characteristics of the system due to the small energy differences among states. As a first step toward resolving solvent-dependent vibrational dynamics that could ultimately assist rational catalyst optimization, we have compared spectral diffusion rates and vibrational lifetimes of bare (monolayer/air interface) versus solvated surface-bound complexes. In addition, the dynamics of the immobilized catalyst are compared to the corresponding homogeneous catalyst in bulk solution. We immobilized a compound in the LRe(CO)3X class of complexes (L, heteroaromatic bidentate ligand; X, halide/pseudohalide) that are under investigation as homogeneous photo/electro-catalysts for the reduction of CO2 to CO or formate (30). Other metal-ligandbased catalysts have been immobilized (27), and the system depicted in Fig. 1 represents a good model system for studying
Department of Chemistry, Stanford University, Stanford, CA 94305, USA. *These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: fayer@stanford.edu
IR techniques such as pump-probe absorption, transient grating, and two-dimensional (2D) IR vibrational echo spectroscopy have been used to study spectral diffusion (9), vibrational relaxation (12), chemical exchange, (8, 10), and orientational dynamics (11). The extension of these techniques to surfaces and interfaces has been a long-standing goal of the surface and ultrafast spectroscopy communities (13). Sum-frequency generation (SFG) and secondharmonic generation (SHG) form the current basis for vibrational spectroscopy of surfaces and interfaces. Frequency-domain experiments provide important information on the molecular orientation (14), vibrational coupling (15), and hydrogen-bond network at interfaces (16), whereas time-domain studies can probe reorientational (17) and translational motions (18), thermal conductance (19), vibrational relaxation (20), and spectroscopic line broadening (21). The measurement and quantitative interpretation of 2D IR spectra of molecular adsorbates has previously been limited to thick samples and attempts at 2D IR-pump SFG-probe spectroscopy (upconverted hole-burning) (22, 23). Upconverted two-pulse vibrational echoes, which measure the homogeneous component (ultrafast motionally narrowed dynamics) of the infrared absorption spectrum but cannot study spectral diffusion (time dependence of structural evolution), have been attempted as well (21, 24). The frequency upconversion VOL 334 SCIENCE
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immobilized catalysts in general. The planarity and optical transparency of the substrate facilitates optical study. We prepared the molecular monolayer by tethering fac-RePhen(CO)3Cl (Phen, phenanthroline) to an undecane monolayer through a triazole linker, created by Cu(I)-catalyzed 1,3-dipolar Huisgen cyclization between a terminal alkyne and an organoazide, popularly referred to as a click reaction (27, 31) (Fig. 1, A to C). The undecane monolayer is attached to a 100-nmthick silica layer grown on a calcium fluoride substrate [see Supporting Online Material (SOM) for details]. The fac-RePhen(CO)3Cl head group has three CO stretching modes that absorb strongly in the infrared. For the monolayer/air interface (Fig. 1D), the peaks are at 1898 cm1 (n1), 1921 cm1 (n2), and 2022 cm1 (n3). The n3 transition is a nearly symmetric stretch involving all three carbonyls. The band shifts to higher frequency and narrows when the surface is submerged in chloroform (Fig. 1E). The blue shift and peak narrowing are more pronounced in a bulk solution of the RePhen(CO)3Cl head group (Fig. 1, C and F, and Table 1). We chose to study the n3 transition because it is well resolved spectroscopically and acts as a probe of the effects of the heterogenization and solvent on the metal complex. The absorbance [optical density (OD)] for the n3 transition was ~5 104 which gave a sufficient signal-to-noise ratio in the 2D IR experiments. The monolayer was monitored for degradation by frequent Fourier transform infrared (FTIR) analysis and was found to be inert and stable over the course of all experiments. The tilt angle of the transition dipole moment for the n3 transition with respect to the surface normal was determined to be 63 T 1 at the monolayer/air interface from the polarization dependence of the FTIR spectrum when the incident angle was Brewsters angle (32, 33). When the monolayer was submerged in chloroform, the n3 transition dipole tilt shifted to 57 T 1, indicating only small structural changes upon solvation. By performing the same analysis for all three modes, the absolute orientation of the head group in the monolayer was determined. Four optically indistinguishable configurations of the ligands around the Rhenium atom were found (fig. S4). We determined the coverage of the monolayer with infrared spectroscopy using the absorbance, the molecular orientation, and the extinction coefficient. These results were confirmed with inductively coupled plasma mass spectrometry (ICPMS), which measures the total rhenium content (table S2). The surface coverage was 1.9 1014 molecules per cm2 (53 2 per molecule), which is in line with the typical surface coverage of transition metal complexes (34)
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Fig. 1. (A) The molecular structure of the heterogenized catalyst in the monolayer. (B) Schematic illustration of a monolayer of the heterogenized catalyst on silica. (C) The structure of the fac-RePhen(CO)3Cl molecule used in the bulk solution experiments. (D to F) Linear infrared absorption spectra of the three CO stretching modes of (D) the heterogenized catalyst monolayer/air interface, (E) the monolayer/chloroform (CHCl3) interface, and (F) fac-RePhen(CO)3Cl in bulk chloroform solution Table 1. Dynamic and static parameters determined from experiments. D is the full width half maximum of the n3 transition, DH is the homogeneous full width half maximum of the n3 transition, and DI is the inhomogeneous full width half maximum of the n3 transition.
Sample Monolayer, bare Monolayer, wet Bulk solution n3 (cm1) 2022 2025 2027 D (cm1) 15.2 11.9 7.9 DH (cm1) 1.8 2.4 3.5 DI (cm1) 14.3 10.6 6.5 TVR (ps) 10 T 2 16 T 2 30 T 3 TSSD (ps) 120180 4060 5T1
and indicates that we obtained a molecular submonolayer. Details of the 2D IR vibrational echo spectroscopy method have been given elsewhere (35). In the experiments, three ~100-fs IR laser pulses tuned to the frequency of the n3 mode are crossed in the sample. The three pulses generate a nonlinear polarization that emits the vibrational echo pulse (the signal) in a unique direction; this pulse is subsequently detected as a function of frequency, wm (the vertical axis in the 2D spectra) by optical heterodyning, which provides phase information and amplifies the signal. The first laser pulse starts a coherent oscillation of the vibrational oscillators, effectively labeling the initial frequency of each oscillator, wt (the horizontal axis in the 2D IR spectra). At a time t later, the second laser pulse stops this coherent oscillation and starts the population period of duration Tw (the waiting time) by driving the system into vibrational population states. The third laser pulse ends the population period and triggers the emission of the vibrational echo pulse. During the second time period of length Tw, the excited vibrations can undergo frequency evolution, vibrational relaxation, or other dynamic processes. The frequency evolution (spectral diffusion) during the Tw period can be characterized by a frequency-frequency correlation function (FFCF), which is typically modeled as a sum of exponential decays. The 2D IR spectra of the n3 mode were acquired for the monolayer (bare, Fig. 2A), the same monolayer under chloroform (wet, Fig. 2B), and the bulk chloroform solution of the RePhen(CO)3Cl head group (Fig. 2C). The large inhomogeneous components and slow spectral dynamics of the interfacial systems are reflected in the elongated shape of the absorption peaks at Tw = 0.5 ps, and their slow changes in shape with increasing Tw. The 2D IR spectra of the bulk solution show clear differences relative to the monolayer complexes: The central peak is less elongated at short time, which indicates a larger homogeneous component, and also undergoes more substantial change in shape with increasing time, demonstrating that spectral diffusion is much faster than in the monolayer samples. The rate of the spectral diffusion is quantified with the well-established center-line-slope (CLS) method (Fig. 3, A and C) (36, 37). A clear trend is observed in the CLS decay time constant, which decreased from 78 T 15 ps for the bare monolayer to 26 T 4 ps for the wet interface, both substantially slower than the 5 T 1 ps measured for the head group in bulk solution. The fast spectral diffusion time constant for the complex in bulk solution is consistent with fast structural evolution (rotation/ translation) of the solvent surrounding the complex that results in fast sampling of the heterogeneous solvation microstates. This is clearly not the case when the complex is immobilized on the surface (compare CLS decays in Fig. 3, A and C).
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The homogeneous linewidth (very fast structural fluctuations that produce motional narrowing) is associated with the difference between 1 and the value of the CLS curve at Tw = 0. Here, again, there is a clear trend. The homogeneous component of the FFCF increases from 12% for the bare monolayer to 20% at the wet interface, indicating substantial additional motional narrowing. For the bulk solution of the head group, the homogeneous component of the linewidth accounts for nearly 50% of the total absorption line broadening, indicating drastic differences in the solvation of the surface bound complex versus the bulk solution. The homogeneous and inhomogeneous linewidths can be recovered directly from the CLS method (36) and are listed in Table 1. We can conclude that the solvent has rendered some of the heterogeneous microstates of the bare monolayer spectroscopically equivalent when the monolayer is exposed to solvent. After excitation, vibrations relax to the ground vibrational state with the lifetime, TVR. Vibrational relaxation conserves energy and so requires lower frequency molecular modes, including collective modes of the surroundings (phonons), in which to deposit the energy (38). Vibrational relaxation time constants are highly sensitive to molecular structure, the local environment, and the phonon density of states. To measure the vibrational lifetime, we operated the 2D IR spectrometer in a heterodyne-detected transient grating (HDTG) configuration using a new approach, as outlined in the SOM. This method overcomes technical challenges associated with standard IR pump-probe experiments in measuring vibrational relaxation in samples with very low absorption. Figure 3, B and D, shows the HDTG decays (points) and single exponential fits (curves) for the bare and wet monolayers as well as the bulk solution. The vibrational lifetime increased steadily from the bare monolayer to the wet monolayer to the bulk solution (Table 1). The vibrational lifetime might rather be expected to decrease upon addition of solvent as the phonon density of states increases. Therefore, the observed lifetime increases are likely due to changes in the anharmonic coupling of the n3 mode to intramolecular acceptor modes. Both the frequency and the vibrational lifetime of a metal carbonyl vibration are inversely correlated with the amount of p back-bonding from the metal into p* antibonding molecular orbitals of the CO ligands (15). The n3 vibration shifts to higher frequency and relaxes more slowly when the monolayer is in contact with the chloroform liquid. The lifetime increases further and the frequency shifts further blue when the complex is fully immersed in bulk chloroform. These changes are consistent with diminished p back-bonding in the Re(CO)3 moiety on exposure to chloroform (15). The changes in p back-bonding are consistent with a CH-p interaction between the chloroform and the phenanthroline ligand, which will affect the electron density of the metal CO system (39). The electron density of the phenanthroline will be inductively drawn away from the Re, which will decrease the amount of p back-bonding. The same p back-bonding changes that affect the n3 spectra and vibrational lifetimes could influence the catalytic activity of the immobilized metal complex because increases in p back-bonding decrease the CO bond order but augment the strength of the Re-C bond (39). The 2D IR data show that the solvent significantly increases the rate of spectral diffusion and, as will be discussed below, the rate of structural evolution. However, even in the absence of solvent, there is substantial spectral diffusion. In samples with nearly full monolayer coverage, vibrational excitation transfer (Frster transfer) between molecules that are close to each other but have different frequencies can be a source of spectral diffusion (40). Therefore, both structural spectral diffusion (SSD) and excitation transferinduced spectral diffusion (ETISD) can contribute to the measured spectral diffusion. Both SSD and ETISD processes can be affected by the addition of solvent. The solvent can affect microscopic structural fluctuations that are responsible for SSD and change the homogeneous and inhomogeneous contributions to the vibrational line shape, which independently will affect ETISD. To separate the contributions of structural evolution and excitation transfer to the measured spectral diffusion, a new excitation transport theory has been developed. Vi2035 2030 2025 2020 2015 2010
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brational excitation transport depends on the spatial separation between molecules, the relative orientations of the vibrational transition dipoles, the homogeneous and inhomogeneous contributions to the vibrational line shapes, and the vibrational lifetime. We measured all of these necessary parameters. Two vibrational chromophores in spatial proximity that have significant spectral overlap of their homogeneous lines will undergo ET via the Frster mechanism at a significant rate (41). ET processes are extremely sensitive to geometry and spatial proximity due to their r6 distance dependence. That the exchange depends on the overlap of the homogeneous lines of the vibrations on two molecules is an important caveat. The overlap of the vibrational homogenous linewidths of two molecules that have different center frequencies enables conservation of energy in the excitation transfer process. Two vibrational oscillators that are close together spatially and close in frequency will undergo fast exchange, whereas two oscillators that have the same spatial separation but that are farther apart in frequency will undergo much slower exchange. Figure 4A shows the Gaussian absorption line and the underlying homogeneous Lorentzians. The slow excitation transfer for vibrations that are far apart in frequency is caused by small overlap of their homogeneous lines, which results in a small Frster radius. The inset shows calculations of the temporal decay of the probability that the initially excited molecule
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remains excited [Pin(t)] for transfer between the red spectra and between the blue spectra in the main part of the figure. In this calculation, the molecules are 8 apart. The 2D IR experiments (Figs. 2 and 3 and Table 1) provide the homogeneous linewidths, without which the ET calculations would be impossible. In addition, it is necessary to know the transition dipole (0.27 D), which was determined from the absorption spectrum, the vibrational lifetime (Fig. 3, B and D, and Table 1) to determine the quantum yield, and the direction of the transition dipole, which was measured with polarized FTIR. Finally, it is also necessary to know the surface coverage, which was determined both by FTIR absorption and ICPMS. The surface coverage sets the distance scale for the ET. The ETISD can be calculated by solving the excitation transfer problem for a given geometry and distribution of frequencies (see SOM). We examined three possible geometries conforming to the measured surface coverage: hexagonal close-packed (HCP), a loose random circle packing (LP) that has a hard disk radial distribution function, and a uniform random distribution with excluded area (RE). We are able to exclude HCP and LP based on the fact that the ETISD-only FFCF decays faster than the experimentally observed decay (see SOM). Only the RE structure is physically consistent with the experimental data. In Fig. 4B, we show the experimentally determined exponential decay of the FFCF with Tw for the bare sample (circles) and its fit (black curve), as well as two calculated curves for the RE structure. The solid curve is a complete numerical solution to the transfer problem, and the dashed curve uses the Huber cumulant expansion approximation (42), which is seen to be quite accurate. The exclusion radius in the RE model defines the excluded area that prevents the initially excited molecule from physically overlapping with a nearby acceptor molecule. Based on the size of the complexes, it is possible to bound the exclusion radius between 6.25 and 7.5 (fig. S10). In Fig. 4B, the exclusion radius is chosen to be 6.5 (a molecular radius of about 3.25 ), which is consistent with the calculated van der Waals radius of the Rhenium complex. The total spectral diffusion, which is measured experimentally, can be separated into the SSD and ETISD components using a theory that accounts for both types of spectral diffusion. In connection with Fig. 4A, we saw that the rate of excitation transfer depends on the frequency separation of the chromophores in addition to their spatial separation. Structural evolution of the system causes the frequencies of the vibrations to evolve in time, which is the source of SSD. SSD can bring two frequencies close together or can move them farther apart, resulting in a greater or lower probability of ET, respectively. Therefore, in addition to the time-independent spatial distribution of chromophores, the timedependent SSD must be included in the ET calculation. We have developed the theoretical methodology to accomplish the calculation of combined SSD and ETISD using the Huber cumulant expansion (42), which we have shown to be accurate for describing the excitation transfer in this system (see Fig. 4B and SOM). Figure 4C illustrates the theoretical methodology that we implement. Frequency trajectories from the simulated SSD stochastic process are drawn for each oscillator, and the excitation transfer problem is propagated at each time step using the instantaneous frequencies of each oscillator and the Huber cumulant approximation. Figure 4D shows the experimental FFCF for the bare monolayer (circles) and ETISD curves with the exclusion radius between 6.25 and 7.5 (shaded region). For the calculated total spectral diffusion to match the experimental data, the structural spectral diffusion time constant must fall between 120 ps and 180 ps as hard limits. The red and blue solid curves through the data correspond to these bounds, which come from the red and blue dashed curves that are the limits of the shaded region. Figure 4E shows the same information for the wet monolayer. Because the wet sample is the same monolayer/ substrate as the bare sample except that it has been immersed in the solvent, the spatial distribution is the same. However, the wet monolayer ETISD calculations include the changes in the homogeneous and inhomogeneous linewidths in going from bare to wet (Table 1). To match the total spectral diffusion with the experimental data, the SSD time constant must fall between 40 ps and 60 ps as hard limits. The structural spectral diffusion time constants, which can only be measured from 2D IR spectroscopy, quantify the structural dynamics of the interfacial layer because the structural motions of the monolayer and surrounding molecules are coupled to the vibrational frequency. One mechanism of SSD that has been demonstrated for the CO stretching mode of CO bound to Fe-heme in the protein myoglobin (43) and for the hydroxyl stretch of water (44) is the Stark effect. Motions of molecules bound to the surface can produce fluctuating electric fields that cause spectral diffusion. Therefore, the spectral diffusion of the bare sample is reporting on the time scale of motions of the surface-attached molecules. The results show that even the bare layer has relatively fast structural motions, ~150 ps. The relatively short time scale associated with the bare surface structural dynamics shows that the immobilized Re complex is far from static even in the absence of a solvent.
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Addition of the CHCl3 solvent changes the time scale of structural fluctuations from ~150 ps to ~50 ps. This time should be compared to the spectral diffusion time constant of 5 ps for the fac-RePhen(CO)3Cl head group in bulk CHCl3 solution. Therefore, the functionalized surface molecules in contact with the solvent have motions that are very different from those of a fully solvated metal complex. The increase in the rate of spectral diffusion for the wet surface sample is unlikely to be due strictly to solvent dynamics. The bulk solution sample has a very large homogeneous width (Fig. 3 CLS data and Table 1), 3.5 cm1 compared to an inhomogeneous width of 6.5 cm1. In contrast, the wet surface sample has a homogeneous width of 2.4 cm1 compared to an inhomogeneous width of 10.6 cm1. Furthermore, the wet sample vibrational lifetime is 60% shorter than the bare sample, but the bulk solution samples lifetime is 300% slower than the bare sample. The stark contrast between the solvated monolayer and the solvated bulk complex demonstrate that the solvent changes the structural dynamics of the metal complexes in the monolayer in a different manner than in the bulk. If the addition of solvent to the monolayer simply added structural fluctuations of the solvent to the SSD of the monolayer, one would expect to see a ~5-ps component, which is not present. The solvent might disentangle the carbon chains, resulting in the small observed change in head group angle and facilitating faster molecular motions. Experimental and molecular dynamics simulation studies of the dependence of the rate of spectral diffusion on the chemical nature of the solvent will help determine the role the solvent plays in the dynamics of this and other functionalized surfaces (43). Using ultrafast 2D IR vibrational echo spectroscopy, HTDG experiments, and FTIR studies, we have examined the dynamics and structure of a monolayer of molecules bound to a planar SiO2 surface. The experiments open up a longanticipated field. Our general methodology when systematically applied across solvents, monolayer coverage, and metal complexes or other vibrational dynamics labels can provide greatly increased understanding of the structural and vibrational dynamics of molecules on surfaces and at interfaces.
1. J. J. Gooding, S. Ciampi, Chem. Soc. Rev. 40, 2704 (2011). 2. J. J. Gooding, F. Mearns, W. Yang, J. Liu, Electroanalysis 15, 81 (2003). 3. J. S. Lindsey, D. F. Bocian, Acc. Chem. Res. 44, 638 (2011). 4. S. F. Bent, J. S. Kachian, J. C. F. Rodrguez-Reyes, A. V. Teplyakov, Proc. Natl. Acad. Sci. U.S.A. 108, 956 (2011). 5. D. M. Adams et al., J. Phys. Chem. B 107, 6668 (2003). 6. G. A. Somorjai, Y. Li, Proc. Natl. Acad. Sci. U.S.A. 108, 917 (2011). 7. G. A. Somorjai, J. Y. Park, Surf. Sci. 603, 1293 (2009). 8. J. R. Zheng et al., Science 309, 1338 (2005). 9. J. K. Chung, M. C. Thielges, S. E. J. Bowman, K. L. Bren, M. D. Fayer, J. Am. Chem. Soc. 133, 6681 (2011). 10. Y. S. Kim, R. M. Hochstrasser, Proc. Natl. Acad. Sci. U.S.A. 102, 11185 (2005). 11. D. E. Rosenfeld, Z. Gengeliczki, M. D. Fayer, J. Phys. Chem. B 113, 13300 (2009). 12. D. D. Dlott et al., J. Am. Chem. Soc. 118, 7853 (1996). 13. X. D. Zhu, Y. R. Shen, Appl. Phys. B 50, 535 (1990). 14. Y. Rao, M. Comstock, K. B. Eisenthal, J. Phys. Chem. B 110, 1727 (2006). 15. J. R. Hill et al., J. Phys. Chem. 100, 18023 (1996). 16. I. V. Stiopkin et al., Nature 474, 192 (2011). 17. J. A. McGuire, Y. R. Shen, Science 313, 1945 (2006). 18. E. H. G. Backus, A. Eichler, A. W. Kleyn, M. Bonn, Science 310, 1790 (2005). 19. J. A. Carter, Z. Wang, D. D. Dlott, Acc. Chem. Res. 42, 1343 (2009). 20. A. Eftekhari-Bafrooei, E. Borguet, J. Phys. Chem. Lett. 2, 1353 (2011). 21. I. M. Lane, D. A. King, H. Arnolds, J. Chem. Phys. 126, 024707 (2007). 22. W. Xiong et al., J. Am. Chem. Soc. 131, 18040 (2009). 23. J. Bredenbeck, A. Ghosh, H. K. Nienhuys, M. Bonn, Acc. Chem. Res. 42, 1332 (2009). 24. Y. J. Chabal, P. Dumas, P. Guyot-Sionnest, G. S. Higashi, Surf. Sci. 242, 524 (1991). 25. Z. Gengeliczki, D. E. Rosenfeld, M. D. Fayer, J. Chem. Phys. 132, 244703 (2010). 26. V. Cervetto, J. Helbing, J. Bredenbeck, P. Hamm, J. Chem. Phys. 121, 5935 (2004). 27. N. K. Devaraj, J. P. Collman, QSAR Comb. Sci. 26, 1253 (2007). 28. D. C. Bailey, S. H. Langer, Chem. Rev. 81, 109 (1981). 29. D. E. De Vos, M. Dams, B. F. Sels, P. A. Jacobs, Chem. Rev. 102, 3615 (2002). 30. A. J. Morris, G. J. Meyer, E. Fujita, Acc. Chem. Res. 42, 1983 (2009). 31. H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 40, 2004 (2001). 32. J. Umemura, T. Kamata, T. Kawai, T. Takenaka, J. Phys. Chem. 94, 62 (1990). 33. Y. Zhang, M. A. Firestone, T. B. Rauchfuss, P. W. Bohn, J. Phys. Chem. 100, 13804 (1996).
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Fig. 4. (A) Excitation transfer within an inhomogeneously 0.7 broadened line. Oscillators with significantly different frequencies transfer more slowly (red) than oscillators with similar frequencies (blue). (Inset) The probability of 0.6 0 1 2 3 4 5 6 7 8 9 10 the excitation remaining on the initially excited oscillator t (ps) for the red and blue cases. (B) Spectral diffusion data (circles) and fit (black curve). Calculated ETISD curves for the random distribution with excluded area effect model (solid blue line, exact numerical solution; dashed blue line, approximation). (C) Illustration of the influence of spectral diffusion on excitation transfer. Two frequency trajectories are drawn from the inhomogeneous distribution (green Gaussian), and excitation transfer can occur at each time step. The transfer is faster when the frequencies approach each other and slower when they are apart. (D) Total spectral diffusion (SSD and ETISD) for the bare monolayer (points, experimental data; black curve, fit). The shaded region is the set of possible ETISD curves. The blue and red lines show the normalized FFCF after inclusion of a 180-ps SSD decay and a 120-ps SSD, respectively, which are based on the dashed red and blue lines that bound the possible ETISD decays. (E) Total spectral diffusion (SSD and ETISD) for the wet monolayer. Blue and red lines denote 60- and 40-ps SSD decays, respectively, which are based on the dashed blue and red lines that bound the possible ETISD decays.
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34. J. P. Collman, N. K. Devaraj, T. P. A. Eberspacher, C. E. D. Chidsey, Langmuir 22, 2457 (2006). 35. S. Park, K. Kwak, M. D. Fayer, Laser Phys. Lett. 4, 704 (2007). 36. K. Kwak, S. Park, I. J. Finkelstein, M. D. Fayer, J. Chem. Phys. 127, 124503 (2007). 37. K. Kwak, D. E. Rosenfeld, M. D. Fayer, J. Chem. Phys. 128, 204505 (2008). 38. V. M. Kenkre, A. Tokmakoff, M. D. Fayer, J. Chem. Phys. 101, 10618 (1994). 39. R. K. Hocking, T. W. Hambley, Organometallics 26, 2815 (2007). 40. S. Yamamoto, A. Ghosh, H.-K. Nienhuys, M. Bonn, Phys. Chem. Chem. Phys. 12, 12909 (2010). 41. T. Frster, Radiat. Res. Suppl. 2, 326 (1960). 42. D. L. Huber, D. S. Hamilton, B. Barnett, Phys. Rev. B 16, 4642 (1977). 43. R. B. Williams, R. F. Loring, M. D. Fayer, J. Phys. Chem. B 105, 4068 (2001). 44. C. J. Fecko, J. J. Loparo, S. T. Roberts, A. Tokmakoff, J. Chem. Phys. 122, 054506 (2005). Acknowledgments: The authors thank C. Xie and B. Cui for preparation of the SiO2-coated CaF2 substrates and D. B. Wong for making the bulk solution orientational relaxation measurements. D.E.R. acknowledges the support of the Fannie and John Hertz Foundation, a National Science Foundation Graduate Research Fellowship, and a Stanford Graduate Fellowship. This material is based on work supported by the Air Force Office of Scientific Research under AFOSR grant F49620-01-1-0018 and the Department of Energy under grant DE-FG0384ER13251. In addition, B.J.S. and T.D.P.S. thank the National Institutes of Health (GM50730).

www.sciencemag.org/cgi/content/full/science.1211350/DC1 Materials and Methods SOM Text Figs. S1 to S10 Tables S1 and S2 References (4564) 18 July 2011; accepted 14 September 2011 Published online 20 October 2011; 10.1126/science.1211350
Eunsung Lee,1* Adam S. Kamlet,1* David C. Powers,1 Constanze N. Neumann,1 Gregory B. Boursalian,1 Takeru Furuya,1 Daniel C. Choi,1 Jacob M. Hooker,2,3 Tobias Ritter,1,3 The unnatural isotope fluorine-18 (18F) is used as a positron emitter in molecular imaging. Currently, many potentially useful 18F-labeled probe molecules are inaccessible for imaging because no fluorination chemistry is available to make them. The 110-minute half-life of 18F requires rapid syntheses for which [18F]fluoride is the preferred source of fluorine because of its practical access and suitable isotope enrichment. However, conventional [18F]fluoride chemistry has been limited to nucleophilic fluorination reactions. We report the development of a palladium-based electrophilic fluorination reagent derived from fluoride and its application to the synthesis of aromatic 18F-labeled molecules via late-stage fluorination. Late-stage fluorination enables the synthesis of conventionally unavailable positron emission tomography (PET) tracers for anticipated applications in pharmaceutical development as well as preclinical and clinical PET imaging. ositron emission tomography (PET) is a noninvasive imaging technology used to observe and probe biological processes in vivo (1, 2). Although several positronemitting isotopes can be used for PET imaging, fluorine-18 (18F) is the most clinically relevant radioisotope (3, 4). For example, the radiotracer [18F]fluorodeoxyglucose ([18F]FDG) has revolutionized clinical diagnosis in oncology. Despite the success of PET and decades of research, there remains a major deficiency in the ability to synthesize complex PET tracers; in fact, no general method is available to radiolabel structurally complex molecules with 18F. In organic molecules, fluorine atoms are typically attached by carbonfluorine bonds (5), yet carbon-fluorine bond formation is challenging, especially in the presence
1 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA. 3 Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Massachusetts General Hospital, Boston, MA 02114, USA.
*These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: hooker@nmr.mgh.harvard.edu ( J.M.H.); ritter@chemistry. harvard.edu (T.R.)
of the variety of functional groups commonly found in structurally complex molecules (6). For PET applications, chemical challenges are exacerbated by the short half-life of 18F (110 min), which dictates that carbon-fluorine bond formation occur at a late stage in the synthesis to avoid unproductive radioactive decay before injection in vivo. The unnatural isotope 18F is generated using a cyclotron, either as nucleophilic [18F]fluoride or as electrophilic [18F]fluorine gas ([18F]F2). [18F]Fluoride, formed from proton bombardment of oxygen-18enriched water, is easier to make and handle than [18F]F2. Moreover, [18F]F2 gas is liberated from the cyclotron with [19F]F2; 19F is the natural, PET-inactive isotope of fluorine. As a result, the 18F/19F ratio, quantified as specific activity, is substantially lower when [18F]F2 is used than when [18F]fluoride is used. High specific activity is often critical to PET imaging. If a biological target of a radiotracer is saturated with the nonpositron-emitting 19F-isotopolog of the tracer, a meaningful PET image cannot be obtained. PET tracers of low specific activity cannot be used to visualize biological targets that are of low concentration. For example, imaging neurotransmitter receptors in the brain typically necessitates tracers of high specific activity (3). SCIENCE VOL 334
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A Fluoride-Derived Electrophilic Late-Stage Fluorination Reagent for PET Imaging
Research toward PET tracer development has focused on the use of [18F]fluoride to make PET tracers with high specific activity. Incorporation of 18F still usually relies on simple nucleophilic substitution reactions, a class of reactions originally developed more than 100 years ago (7) and often not suitable to address modern challenges in imaging. Recent advances in nucleophilic fluorination (811) include a palladium-catalyzed fluorination reaction of aryl triflates with anhydrous cesium fluoride developed by the Buchwald group in which carbon-fluorine bond formation proceeds by reductive elimination from palladium(II) aryl fluoride complexes (12, 13). Challenges associated with the application of fluorination reactions to PET include the requirement of short reaction times, as well as different reaction conditions for 18 F chemistry relative to 19F chemistry. For example, extensive drying of fluoride is readily achieved for 19F chemistry but can be impractical for radiochemistry, which is typically executed on a nanomole scale. When transitioning from 19 F chemistry to 18F chemistry, the smaller ratio of fluorine to water can be problematic because hydrated fluoride has diminished nucleophilicity. As a consequence, even promising modern fluorination reactions developed for 19F chemistry are often not translated to radiochemistry. Electrophilic and nucleophilic fluorination reactions allow access to complementary sets of molecules (6), yet all electrophilic 18F-fluorination reactions developed to date use electrophilic fluorination reagents that ultimately originate from [18F]F2. In 1997, Solin developed a method to generate [18F]F2 with higher specific activity than is common for [18F]F2, by minimizing the amount of [19F]F2 used (14). By using [18F]F2 made via the Solin method, Gouverneur succeeded in synthesizing [18F]N-chloromethyl-Nfluorotriethylenediammonium bis(tetrafluoroborate) ([18F]F-TEDA), an electrophilic 18F-fluorination reagent more useful and selective than [18F]F2 (15). However, nucleophilic [18F]fluoride is currently the only practical and generally available source of fluorine to prepare PET tracers with high specific activity (3). If an electrophilic fluorination reagent were to be made from fluoride (16, 17) without the need for F2, electrophilic fluorination could become a general and widely used method to prepare PET tracers that are currently
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inaccessible via conventional nucleophilic fluorination chemistry. Previously, we reported the fluorination of palladium aryl complexes with the electrophilic fluorination reagent F-TEDA (Fig. 1) (18). F-TEDA can oxidize palladium(II) aryl complexes, which subsequently afford aryl fluorides by carbonfluorine reductive elimination from palladium(IV) aryl fluoride complexes (19, 20). Replacement of F-TEDA by a fluorination reagent that mimics the function of F-TEDA but is made from fluoride has the potential for use in the synthesis of 18 F radiotracers with high specific activity. Here, we report the design and synthesis of an organometallic complex made from fluoride (1 2, Fig. 1) that behaves as an electrophilic fluorination reagent, and the use of the reagent for the synthesis of 18F-labeled small molecules via latestage fluorination. Palladium complex 2 was envisioned to accomplish oxidative fluorine transfer by serving as an electrophile in SN2 reactions with nucleophilic attack occurring at the fluorine substituent. The complexes 1 and 2 were designed according to the following five considerations (Fig. 2): (i) The palladium center in 1 carries three formal positive charges (countercharges to two triflate anions and one negatively charged borate ligand) and should therefore capture negatively charged fluoride from solution. High fluorophilicity of the palladium complex 1 is required for radiochemistry applications because of the low effective concentration of fluoride in solution (roughly 104 M). (ii) The palladium centers in 1 and 2 are in the oxidation state +IV [Pd(IV)], a high oxidation state for palladium. Late transition metals such as palladium, when in a high oxidation state, can function as an oxidant and transfer a ligand to a nucleophile while being reduced to a lower oxidation state (21). The palladium in 2 can function as an electron acceptor, which rationalizes the reactivity of 2 as an oxidant. (iii) The supporting benzo[h]quinolyl and tetrapyrazole borate (Tp) ligands are multidentate ligands that were selected to impart stability on both 1 and 2 toward undesired reductive processes such as carbonfluorine reductive elimination. Reductive elimination from 2 would likely require dissociation of one ligand to form a pentacoordinate palladium complex (19, 20), and multidentate ligands such as Tp are less likely to dissociate and hence reduce the rate of potential reductive elimination. (iv) An octahedral Pd(IV) complex was chosen to avoid undesired nucleophilic attack at the transition metal. The palladium-fluorine bond is polarized toward fluorine, with partial negative charge on fluorine and positive charge on palladium. Solely on the basis of coulombic interactions, nucleophilic attack is expected at palladium rather than fluorine. However, the orbitals available for nucleophilic attack on octahedral (t2g)6(eg)0 Pd(IV) complexes, the dx2 y2 and dz2 orbitals, are high in energy, and nucleophilic attack on highenergy orbitals is disfavored (22). (v) The multidentate supporting ligands in 2 feature aromatic substituents to prevent nucleophilic attack on the carbon and nitrogen atoms coordinated to palladium. Complex 2 was devised to act as an electrophilic fluorination reagent through nucleophilic attack at the antibonding palladium-fluorine based orbital (s*PdF) at fluorine in an SN2 reaction. In such a putative SN2 reaction, palladium would function as a leaving group, with concomitant reduction to the oxidation state +II [Pd(II)]. Nucleophilic attack would occur at the lowest unoccupied molecular orbital (LUMO) of 2. The calculated LUMO of 2 (Fig. 2B) shows that only the lobe on fluorine points into unoccupied space; no other LUMO lobe is available for nucleophilic attack because the aromatic ligands block the trajectories. Treatment of the palladium complex 1 with nucleophilic fluoride (KF) afforded the palladium fluoride complex 2 within 5 min at 90% yield (Fig. 2A). The ability of 2 to function as an electrophilic fluorination reagent was confirmed by fluorination of Pd(II) aryl complex 3 (Fig. 2C). Fluorine transfer from 2 to 3 results in oxidation of 3 to form Pd(IV) aryl fluoride intermediate 5, assigned by 1H and 19F nuclear magnetic resonance (NMR). Carbon-fluorine reductive elimination from reactive intermediate 5 occurs at a rate comparable to oxidation of 3 with 2. Oxidation of 3 with the electrophilic fluorination reagent F-TEDA afforded 5 at a faster rate than oxidation with 2, which allowed independent synthesis and identification of 5 (fig. S2). Reduction of palladium from Pd(IV) in oxidant 2 to Pd(II) was established by isolation of Pd(II) complex 4. Oxidative fluorine transfer from 2 to 3 could proceed by SN2 reaction as designed, with nucleophilic attack of the dz2 -based orbital on palladium of 3 (fig. S17) on the s*PdF-based orbital of the LUMO of 2, or via electron transfer (23, 24) from 3 to 2 with interposed or subsequent fluorine transfer. Currently available data cannot distinguish between these mechanisms. In addition, other pathways that intercept intermediates such as 5 could potentially be used for carbon-fluorine bond formation. Palladium complex 2 is both an oxidant and a fluoride donor. The oxidation equivalents are inherent in the transition metal oxidation state, and the fluorine substituent is negatively polarized, because fluorine is the most electronegative element. But oxidation of 3 with an external oxidant followed by a reaction with an independent fluoride source could also provide 5. In such a stepwise approach, potential side reactions of putative intermediates, such as undesired reductive elimination reactions, must be prevented. Conceptually, several combinations of an oxidant and a fluoride source are conceivable for successful oxidative fluorination. The fluorination reagent 2 was subsequently evaluated for late-stage fluorination. Fluorination of the Pd(II) aryl complexes 8 to 11 with 2 afforded aryl fluorides 12 to 15 at 67 to 93% yield (Fig. 3). We propose that Pd(IV) fluoride complex 2 oxidizes the Pd(II) aryl complexes by fluorine transfer to form high-valent Pd(IV) aryl fluoride complexes, analogous to 5, from which carbon-fluorine reductive elimination can occur to form aryl fluoride products 12 to 15. The aryl fluoride molecules shown in Fig. 3 were selected on the basis of their structure and exhibition of a variety of functional groups, akin to potential smallmolecule PET tracers (25, 26). The molecules
Fig. 1. Electrophilic fluorination of palladium aryl complexes to afford aryl fluorides. Top: With the electrophilic fluorination reagent F-TEDA. Bottom: With the Pd(IV) fluoride complex 2, made from fluoride (F). Ar, aryl; Me, methyl; Tf, trifluoromethanesulfonyl. VOL 334 SCIENCE www.sciencemag.org
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are electron-rich arenes, which would be difficult to prepare by conventional fluorination reactions with [18F]fluoride. The transition from 19F chemistry to 18F chemistry is challenging because the concentration of the limiting reagent (fluoride) changes from millimolar to micromolar, and because syntheses must be performed in the presence of ionizing radiation and under a time constraint due to the 110-min half-life of 18F. Therefore, reaction chemistry applicable to 18F chemistry must be robust and as simple as possible for broad applications in medical imaging. Organometallic synthesis in a hospital setting would be impractical. However, the palladium complexes discussed here can be prepared conveniently on scale, stored, and subsequently transported to imaging sites when needed. The palladium(II) aryl complexes such as 8 to 11 can be purified by chromatography or recrystallization and can be stored and transported in air at ambient temperature. Palladium complex 1 is stable at room temperature and can be manipulated briefly in air. Palladium complex 2 is stable toward heat (no observed decomposition for 24 hours at 100C), and water (no observed decomposition in 10% aqueous acetonitrile solution after 3 hours at 23C). Thermal stability and tolerance toward water are beneficial for practical PET applications, because reaction mixtures are often heated to increase reaction rates and [18F]fluoride is made from oxygen-18enriched water. The synthesis of 18F-radiolabeled molecules using such a fluoride-derived reagent is operationally simple. Pd(IV) complex 1 reacts with conventionally prepared solutions of [18F]fluoride
Fig. 2. Reactivity and structure of 1 and 2. (A) Nucleophilic fluoride capture by Pd(IV) complex 1 to form electrophilic fluorination reagent 2. (B) X-ray structure of 2 (ORTEP drawing at 50% probability; hydrogen atoms and counteranion omitted for clarity) and calculated lowest unoccupied molecular orbital (LUMO) of 2. (C)
Fluorination of Pd(II) aryl complex 3 by reagent 2 to form Pd(IV) aryl fluoride complex 5, followed by CF reductive elimination from 5. Reductive elimination from reactive intermediate 5 to 6 proceeds at 23C; the highest yield of 6 (80%) was obtained upon heating 2 and 3 at 85C. 18-cr-6, 18-crown-6; iPr, isopropyl.
Fig. 3. Synthesis and fluorination of palladium aryl complexes. (A) Representative synthesis of a palladium aryl complex from palladium acetate complex 7. (B) Fluorination of palladium aryl complexes 8 to 11 with electrophilic fluorination reagent 2. [Pd], as shown for complex 7; Ac, acetate; Bn, benzyl; Boc, tert-butoxycarbonyl; MOM, methoxymethyl; DAM, di-(p-anisyl)methyl.
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3. S. M. Ametamey, M. Honer, P. A. Schubiger, Chem. Rev. 108, 1501 (2008). 4. P. W. Miller, N. J. Long, R. Vilar, A. D. Gee, Angew. Chem. Int. Ed. 47, 8998 (2008). 5. D. OHagan, Chem. Soc. Rev. 37, 308 (2008). 6. T. Furuya, A. S. Kamlet, T. Ritter, Nature 473, 470 (2011). 7. S. Young, J. Chem. Soc. Trans. 39, 489 (1881). 8. V. W. Pike, F. I. Aigbirhio, J. Chem. Soc. Chem. Commun. 1995, 2215 (1995). 9. H. Sun, S. G. DiMagno, Angew. Chem. Int. Ed. 45, 2720 (2006). 10. M. H. Katcher, A. G. Doyle, J. Am. Chem. Soc. 132, 17402 (2010). 11. C. Hollingworth et al., Angew. Chem. Int. Ed. 50, 2613 (2011). 12. D. A. Watson et al., Science 325, 1661 (2009); 10.1126/science.1178239. 13. T. Nol, T. J. Maimone, S. L. Buchwald, Angew. Chem. Int. Ed. 10.1002/anie.201104652 (2011). 14. J. Bergman, O. Solin, Nucl. Med. Biol. 24, 677 (1997). 15. H. Teare et al., Angew. Chem. Int. Ed. 49, 6821 (2010). 16. B. Brauner, Z. Anorg. Chem 7, 1 (1894). 17. K. O. Christe, Inorg. Chem. 25, 3721 (1986). 18. T. Furuya, H. M. Kaiser, T. Ritter, Angew. Chem. Int. Ed. 47, 5993 (2008). 19. T. Furuya, T. Ritter, J. Am. Chem. Soc. 130, 10060 (2008). 20. T. Furuya et al., J. Am. Chem. Soc. 132, 3793 (2010). 21. S. S. Stahl, J. A. Labinger, J. E. Bercaw, Angew. Chem. Int. Ed. 37, 2180 (1998). 22. F. A. Cotton, Chemical Applications of Group Theory (Wiley, New York, ed. 3, 1990), chap. 9. 23. H. Taube, H. Myers, J. Am. Chem. Soc. 76, 2103 (1954). 24. A. Haim, Prog. Inorg. Chem. 30, 273 (1983). 25. V. Pomel et al., J. Med. Chem. 49, 3857 (2006). 26. A. P. Kozikowski et al., ChemMedChem 5, 1221 (2010). 27. F. T. Chin et al., Mol. Imaging Biol. 10, 82 (2008). 28. J. Toyohara et al., Ann. Nucl. Med. 23, 301 (2009). 29. U.S. Department of Health and Human Services, Food and Drug Administration, www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/ucm078933.pdf. 30. European Medicines Agency, www.ema.europa.eu/docs/ en_GB/document_library/Scientific_guideline/2009/09/ WC500003587.pdf. 31. A. K. Buck et al., J. Nucl. Med. 51, 401 (2010). Acknowledgments: Supported by National Institute of General Medical Sciences grant GM088237, National Institute of Biomedical Imaging and Bioengineering grant EB013042, National Center for Research Resources grant 1S10RR017208-01A1, National Institute on Drug Abuse grant 5P30DA028800-02, the Richard and Susan Smith Family Foundation, the Massachusetts Life Science Center, the Harvard Catalyst, NSF Graduate Research Fellowship Program grant DGE0644491, and the Harvard Accelerator Fund. T.R. was also supported by a Sloan Research Fellowship, grants from Eli Lilly and Co. and AstraZeneca, a Camille Dreyfus Teacher-Scholar Award, and an Amgen Young Investigators Award. We thank S.-L. Zheng for x-ray crystallographic analysis. DFT computation was performed using the computer facilities at the Odyssey cluster at Harvard University. Metrical parameters for the crystal structures of compounds 1, 2, 3, 8, and S10 are available free of charge from the Cambridge Crystallographic Data Centre under reference nos. CCDC 829427, 829428, 840744, 829429, and 839058, respectively. A patent application has been filed through Harvard on methods and reagents presented in this manuscript.
Fig. 4. Late-stage fluorination to form 18F-labeled aryl fluorides. [18F]Fluoride capture by Pd(IV) complex 1 to form electrophilic fluorination reagent [ 18F]2 and subsequent fluorination of palladium aryl complexes 9 to 11. Reported radiochemical yields (RCYs) are averaged over n experiments at a 500-mCi scale. in acetone and forms the 18F reagent [18F]2 within 10 min (Fig. 4). Subsequent filtration over a polymer-supported resin and addition of the Pd(II) aryl complexes 9 to 11 yielded, upon heating for 10 min, the 18F-labeled aryl fluorides [18F]13 to [18F]15, respectively. Azeotropic drying of aqueous [18F]fluoride, the two-step reaction sequence (fluoride capture (1 [18F]2) followed by fluorine transfer (e.g., 9 [18F]13), and subsequent purification of the 18F-labeled molecules by high-performance liquid chromatography (HPLC) results in an overall synthesis time of less than 60 min. Automated syntheses with up to 1 Ci of radioactivity have been accomplished. The efficiency of radiochemical synthesis is given in radiochemical yield, which is based on decay-corrected radioactivity rather than on mass of isolated material (4). Radiochemical yields (RCYs) as high as possible are desired, but RCYs as low as 5% can provide meaningful PET imaging (27). The 18F-fluorination method described here has afforded RCYs higher than 30%. Stoichiometric amounts of precious transition metals such as palladium are typically avoided in syntheses of health care products because of toxicity and cost considerations. However, such considerations are different for the synthesis of PET tracers because of the small amount that is required (28). PET tracers with high specific activity are administered at a dose at least 2 orders of magnitude smaller than is common for pharmaceuticals because pharmacological effects are not sought (29), and purification is often straightforward because of the small amount of tracer to be purified. For example, inductively coupled plasma mass spectrometry (ICP-MS) analysis of [18F]13 (Fig. 4), purified by conventional HPLC technique (20 min), afforded material with 5 parts per billion (ppb) palladium residue, commensurate with international recommendations on the palladium impurity profile for samples injected into humans, which demand less than 1000 ppb palladium (30). Similarly, the synthesis cost for the palladium complexes is small relative to the cost of 18F-isotope infrastructure and the cost associated with clinical imaging (31). We have shown that a late-stage fluorination reaction can access 18F-labeled functionalized molecules, which would be particularly difficult to prepare with conventional fluorination reactions. The availability of a fluorination reagent with high specific activity that functions as an electrophile may find applications in 18F fluorination of pharmaceutical candidates for evaluation of their biodistribution to accelerate drug development, as well as in the development of previously unavailable 18F-PET tracers for clinical care.

www.sciencemag.org/cgi/content/full/334/6056/639/DC1 Materials and Methods Figs. S1 to S17 Tables S1 to S16 References (3261) 15 August 2011; accepted 13 September 2011 10.1126/science.1212625
1. M. E. Phelps, Proc. Natl. Acad. Sci. U.S.A. 97, 9226 (2000). 2. J. S. Fowler, A. P. Wolf, Acc. Chem. Res. 30, 181 (1997).
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Ionic LiquidMediated Selective Conversion of CO2 to CO at Low Overpotentials

Brian A. Rosen,1 Amin Salehi-Khojin,1 Michael R. Thorson,2 Wei Zhu,1 Devin T. Whipple,2 Paul J. A. Kenis,2 Richard I. Masel1* Electroreduction of carbon dioxide (CO2)a key component of artificial photosynthesishas largely been stymied by the impractically high overpotentials necessary to drive the process. We report an electrocatalytic system that reduces CO2 to carbon monoxide (CO) at overpotentials below 0.2 volt. The system relies on an ionic liquid electrolyte to lower the energy of the (CO2) intermediate, most likely by complexation, and thereby lower the initial reduction barrier. The silver cathode then catalyzes formation of the final products. Formation of gaseous CO is first observed at an applied voltage of 1.5 volts, just slightly above the minimum (i.e., equilibrium) voltage of 1.33 volts. The system continued producing CO for at least 7 hours at Faradaic efficiencies greater than 96%. n the context of artificial photosynthesis (14), considerable progress has been made toward water-splitting technology that uses solar energy or solar-derived electricity, but CO2 activation has proven to be more difficult (1, 57). Although a few homogeneous catalysts show initial activity at overpotentials of 600 mV (6, 7), most quickly lose their activity under reaction conditions. Pyridine-catalyzed conversion may be an exception (810), although performance over an extended time has not been reported. A promising catalyst for efficient CO2 conversion would need to exhibit both high energy efficiency (i.e., high Faradaic efficiency for CO production at low overpotential) as well as high current density (i.e., high rate or turnover number) (11). Twenty years ago, Bockris and co-workers proposed that high overpotentials are needed to convert CO2 (12, 13) because the first step in CO2 conversion is the formation of a CO2 intermediate. (In this context, CO2 does not necessarily denote a bare CO2 anion; instead, it is whatever species forms when an electron is added to CO2.) The equilibrium potential for (CO2) formation is very negative in water and in most common solvents (12, 13). Consequently, it is necessary to run the cathode very negative (i.e., at a high overpotential) for the reaction to occur. This is very energy-inefficient (Fig. 1). The objective of the work described here was to develop a cocatalyst that would lower the potential for formation of the CO2 intermediate, which then reacts with H+ on the silver cathode to produce CO (5). If Bockris proposal is correct, the overpotential for CO2 conversion into useful products should decrease upon lowering the free energy of formation of the CO2. For example, if a substance formed a complex with the
CO2 on the metal surface, then the reaction could follow the dashed line in Fig. 1. In that case, a complex between the solvent and the CO2, labeled EMIM-CO2 in the figure, could form quickly. Although there would still be a barrier to form the final products of the reaction, the overall barrier to reaction would be reduced (14). We chose 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIM-BF4) to test whether such a route was feasible. We first used cyclic voltammetry (CV) to characterize the reduction of CO2 in an 18 mol % EMIM-BF4 solution (see figs. S5 and S6 for CV diagrams of this process on a platinum and a silver working electrode, respectively). Barnes et al. (15) and Islam and Ohsaka (16) found that (O2) forms a complex with the cation in 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMIM-NTf2), moving the potential for (O2) formation in the positive direction by 0.65 V. CO2 is also known to form weak complexes with BF4 anions (1721). We reasoned that if CO2 and (O2) form complexes with EMIM-BF4 and BMIM-BF4, then (CO2) could do so as well, thereby shifting CO2 conversion to less negative potentials, as suggested
by Fig. 1. Also, the binding of CO2 in EMIMBF4 is weaker than in many other ionic liquids. Ideally, the CO2 complex should be bound strongly enough to facilitate CO2 reduction, but not so strongly that the CO2 is unreactive (14). We tested whether the overpotential for CO2 would be reduced as predicted. The experiments used a flow cell reported previously (22, 23) (fig. S1). The cell was constructed from a platinum anode and a silver cathode, with liquid in between. In such a setup, CO2 flows into the cell, and the products present in the gaseous stream flowing out are analyzed by gas chromatography (GC). When we originally ran the experiment, we found that the platinum anode was quickly poisoned by CO created on the cathode, so we placed a Nafion 117 membrane between the anode and the cathode to isolate the anode from the ionic liquid (23). The anode compartment contained 100 mM aqueous sulfuric acid flowing at 0.5 ml/min. The cathode compartment contained 18 mol % EMIM-BF4 in water at the same flow rate. Measurements indicated that the platinum anode had an electrochemical surface area of 500 cm2 and the silver cathode had an electrochemical surface area of 6 cm2. The procedures for the surface area measurements of both the anode (CO stripping) and cathode (underpotential deposition of lead) can be found in the supporting online material. During the experiments, we held the voltage on the cell constant, and measured the products of the reaction by GC. We observed only three products: hydrogen and CO on the cathode and oxygen on the anode. Other products may have been present at concentrations below 3 parts per million, the GC detection limit. Figure 2 shows the how the CO peak in the GC trace varies with the applied voltage, in experiments in which we held the voltage constant and waited until we found steady performance. We began to see CO at an applied potential of 1.5 V. By comparison, when we ran the cell under identical conditions but in the absence of
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1 Dioxide Materials, 60 Hazelwood Drive, Champaign, IL 61820, USA. 2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
*To whom correspondence should be addressed. E-mail: rich.masel@dioxidematerials.com
Fig. 1. A schematic of how the free energy of the system changes during the reaction CO2 + 2H+ + 2e CO + H2O in water or acetonitrile (solid line) or EMIM-BF4 (dashed line). SCIENCE VOL 334
Fig. 2. The CO peak observed by gas chromatography as a function of the total potential applied to the cell (TCD, thermal conductivity detector; O.C., open cell). Gas-phase CO production is observed at an applied potential of 1.5 V (slightly above the equilibrium potential for the reaction, 1.33 V).
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Fig. 3. A plot of the Faradaic efficiency of the process to form the desired CO and the undesired hydrogen, and the turnover rate as a function of applied cell potential.
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1. A. T. Bell, U.S. Department of Energy report PNNL17214, www.anl.gov/catalysisscience/publications/CAT_rpt.pdf (2008). 2. N. S. Lewis, D. G. Nocera, Proc. Natl. Acad. Sci. U.S.A. 103, 15729 (2006). 3. J. Tollefson, Nature 466, 541 (2010). 4. L. Hammarstrm, S. Hammes-Schiffer, Acc. Chem. Res. 42, 1859 (2009). 5. Y. Hori, in Modern Aspects of Electrochemistry, Vol. 42 (Springer, New York, 2008), pp. 89189. 6. M. Rakowski Dubois, D. L. Dubois, Acc. Chem. Res. 42, 1974 (2009). 7. D. L. DuBois, in Encyclopedia of Electrochemistry (Wiley-VCH, Weinheim, Germany, 2006), vol. 7a, pp. 202225. 8. A. J. Morris, R. T. McGibbon, A. B. Bocarsly, ChemSusChem 4, 191 (2011). 9. E. Barton Cole et al., J. Am. Chem. Soc. 132, 11539 (2010). 10. G. Seshadri, C. Lin, A. B. Bocarsly, J. Electroanal. Chem. 372, 145 (1994). 11. D. T. Whipple, P. J. A. Kenis, J. Phys. Chem. Lett. 1, 3451 (2010). 12. J. O. M. Bockris, J. C. Wass, J. Electrochem. Soc. 136, 2521 (1989). 13. K. Chandrasekaran, J. O. M. Bockris, Surf. Sci. 185, 495 (1987). 14. R. I. Masel, Chemical Kinetics and Catalysis (Wiley, New York, 2001). 15. A. S. Barnes et al., J. Phys. Chem. C 112, 13709 (2008). 16. M. M. Islam, T. Ohsaka, J. Phys. Chem. C 112, 1269 (2008). 17. B.-H. Lim et al., Korean J. Chem. Eng. 26, 1130 (2009). 18. A. Maiti, ChemSusChem 2, 628 (2009). 19. M. Shokouhi, M. Adibi, A. H. Jalili, M. Hosseini-Jenab, A. Mehdizadeh, J. Chem. Eng. Data 55, 1663 (2010). 20. A. N. Soriano, B. T. Doma Jr., M.-H. Li, J. Chem. Eng. Data 53, 2550 (2008). 21. J. Tang, Y. Shen, M. Radosz, W. Sun, Ind. Eng. Chem. Res. 48, 9113 (2009). 22. D. T. Whipple, E. C. Finke, P. J. A. Kenis, Electrochem. Solid State Lett. 13, B109 (2010). 23. A. S. Hollinger et al., J. Power Sources 195, 3523 (2010). Acknowledgments: Supported in part by the U.S. Department of Energy (DOE) under grant DE-SC0004453 and by Dioxide Materials. B.A.R. was supported in part by an award from the DOE Office of Science Graduate Fellowship Program (DOE SCGF). The DOE SCGF Program was made possible in part by the American Recovery and Reinvestment Act of 2009 and is administered by the Oak Ridge Institute for Science and Education (ORISE) for DOE. ORISE is managed by Oak Ridge Associated Universities (ORAU) under DOE contract DE-AC05-06OR23100. The following patent applications are related to the work here: 12/830338, Novel Catalyst Mixtures (R.I.M.); 13/174365, Novel Catalyst Mixtures (R.I.M.); PCT/US11/42809, Novel Catalyst Mixtures (R.I.M.); PCT/US/11/30098, Novel Catalyst Mixtures (R.I.M. and B.A.R.); and 61/499225, Inexpensive Carbon Dioxide Sensor (R.I.M. and B.A.R.). All opinions expressed in this paper are those of the authors and do not necessarily reflect the policies and views of DOE, ORAU, or ORISE. Author contributions: R.I.M. conceived the project and led the work; B.A.R., R.I.M., A.S.-K., P.J.A.K., and M.R.T. planned the experiments; D.T.W., P.J.A.K., and R.I.M. designed the cell; B.A.R. took all of the data that appear in the paper; all of the measurements in this paper were done at Dioxide Materials; A.S.-K., W.Z., and M.R.T. took confirming data; and R.I.M. and P.J.A.K. supervised the work. The paper was drafted by R.I.M., A.S.-K., and P.J.A.K. with other authors offering critical comments. We thank G. Kaul for assistance in the electrochemical work.
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Faradaic efficiency was always greater than 96%. We did observe a little hydrogen formation from electrolysis of water, but the hydrogen formation was always less than 3% of the Faradaic efficiency. Figure 3 also shows the energy efficiency of the process, calculated from the equation Faradaic efficiency1:33 V applied voltage 2 The energy efficiency is 87% at low voltage (1.5 V) and drops as voltage increases because of energy loss due to resistive losses in the membrane and solutions. To ascertain the catalytic rate and robustness, we ran the cell for 7 hours and obtained about 26,000 turnovers in which the turnovers were calculated according to the electrochemical surface area of our cathode catalysts. The plot of the data in Fig. 4 exhibits some curvature because the membrane resistance is increasing over time, but clearly the setup is capable of many turnovers. The one weakness of the system at present is that our observed rates are lower than what is needed for a commercial process. Typically, commercial electrochemical processes run at a turnover rate of about 1 to 10 per second, in contrast with the rate of 1 per second or less that we observe here. Further development of the reactor configuration and exact operating conditions for example, to overcome some mass transport issuesis expected to increase the turnover number. Indeed, we observed a rate of 60 turnovers per second with a rotating-disk electrode at a cathode potential equivalent to that observed when the cell potential is about 2 V (fig. S7). Also, scale-up needs to be done. At present, our cathode has an electrochemical surface area of only 6 cm2, whereas commercial electrochemical cells for the chlor-alkali process have electrochemical surface areas on the order of 109 cm2. At 2 V, our cell produces CO at a rate of only ~1 mol/min, whereas commercial processes require thousands of moles per minute per cell. energy efficiency VOL 334 SCIENCE
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Fig. 4. The total number of turnovers accumulated on the catalyst as a function of time at an applied potential of 2.5 V. The curvature in the plot is due to a gradual increase in membrane resistance. the ionic liquid, CO was not detected until a cell potential of 2.1 V was applied. This control experiment was carried out using 500 mM KCl electrolyte. The CO peak grew slightly as we increased the potential, but the increases were small because the membrane resistance to mass transfer was large, hence the current was mass transfer limited. The data in Fig. 2 show that most of the increased potential went toward polarizing the membrane or anode, rather than polarizing the cathode. To put the data in perspective, the equilibrium potential for the reaction CO2 CO 1 =2 O2 1
is 1.33 V, so the fact that we could observe gasphase CO formation at an applied potential of 1.5 V implies that we could form CO with a cell overpotential of only 0.17 V. Normally, an anode overpotential would also have to be accounted for, but in our cell the electrochemical surface area of the anode is about 80 times that of the cathode. We also used GC to measure the Faradaic efficiency of CO formation (i.e., the fraction of the electrons going to the CO product, as opposed to the hydrogen by-product); the results are shown in Fig. 3. We found that the

www.sciencemag.org/cgi/content/full/science.1209786/DC1 Materials and Methods Figs. S1 to S7 References (2427) 14 June 2011; accepted 1 September 2011 Published online 29 September 2011; 10.1126/science.1209786
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Wireless Solar Water Splitting Using Silicon-Based Semiconductors and Earth-Abundant Catalysts
Steven Y. Reece,1* Jonathan A. Hamel,1 Kimberly Sung,1 Thomas D. Jarvi,1* Arthur J. Esswein,1 Joep J. H. Pijpers,2,3 Daniel G. Nocera2* We describe the development of solar water-splitting cells comprising earth-abundant elements that operate in near-neutral pH conditions, both with and without connecting wires. The cells consist of a triple junction, amorphous silicon photovoltaic interfaced to hydrogen- and oxygen-evolving catalysts made from an alloy of earth-abundant metals and a cobalt|borate catalyst, respectively. The devices described here carry out the solar-driven water-splitting reaction at efficiencies of 4.7% for a wired configuration and 2.5% for a wireless configuration when illuminated with 1 sun (100 milliwatts per square centimeter) of air mass 1.5 simulated sunlight. Fuel-forming catalysts interfaced with light-harvesting semiconductors afford a pathway to direct solar-to-fuels conversion that captures many of the basic functional elements of a leaf. lthough solar photovoltaic (PV) cells normally generate electricity, they can be used to generate fuels such as hydrogen from water, thus providing a storage mechanism for sunlight (1). Such schemes mimic the photosynthetic process within a leaf that converts the energy of sunlight into chemical energy by splitting water to produce O2 and hydrogen equivalents (2). The primary steps of natural photosynthesis involve the absorption of sunlight and its conversion into spatially separated electronhole pairs. The holes of this wireless current are captured by the oxygen-evolving complex (OEC) of Photosystem II (PSII) to split H2O to O2. The electrons and protons produced as the by-products of the OEC reaction are transferred to Photosystem I (PSI) to produce a reduced form of hydrogen in the form of NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate) (2). The separation of light collection/conversion from catalysis is compulsory to the photosynthetic function because electron/hole pairs are generated one at a time and the water splitting reaction is a four electron-hole process (3). The multielectron catalysts of PSII and PSI are therefore needed to bridge the light-driven one electron-hole wireless current of the light collection and conversion apparatus of the leaf to the four electron-hole chemistry of water splitting. A general approach for mimicking photosynthesis is to generate O2 and H2 with inorganic materials using fuel-forming catalysts interfaced with light-harvesting semiconductors (46). Sunlight is absorbed by the semiconductor and generates spatially separated electron-hole pairs. The electron-hole pairs of
Sun Catalytix, Cambridge, MA 02139, USA. 2Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 021394307, USA. 3Foundation for Fundamental Research on Matter, Institute for Atomic and Molecular Physics, Science Park 104, 1098 XG, Amsterdam, Netherlands. *To whom correspondence should be addressed. E-mail: nocera@mit.edu (D.G.N.); streece@suncatalytix.com (S.Y.R.); tjarvi@suncatalytix.com (T.D.J.)
this wireless current are captured with two catalysts that drive the water-splitting reaction under near-neutral pH conditions. A system of this type must generate electrons and holes with enough energy to overcome both the energetic barrier of water oxidation (1.23 V at standard conditions) and any overpotentials needed to drive catalysis. Wireless photochemical cells (7, 8) and wired photoelectrochemical cells (PECs) (914) for solar-powered water splitting have been realized, but practical problems remain. Schemes for solar photochemical production of H2 and O2 from water at reasonable efficiency have relied on the use of prohibitively expensive light-absorbing materials [e.g., (Al)GaAs and GaInP], and/or fuel-forming catalysts (e.g., Pt, RuO2, IrO2), and strongly acidic or basic reaction media, which are corrosive and expensive to manage over the large areas required for light harvesting. A focus of current research has been to mimic photosynthesis with materials composed of earth-abundant elements in electrolytes near neutral pH conditions (15). The success of this approach will enable novel PEC and other light-harvesting (e.g., wireless) architectures to be engineered to produce solar fuel at more practical cost targets. Silicon is an attractive materials choice for constructing an artificial leaf because of its earthabundance and prevalence in the electronics and PV industries. The realization of a direct solar-tofuel device based on silicon, however, must overcome the inherent corrosion of this semiconductor in nonacid electrolytes (16). Previous stand-alone, water-splitting PEC configurations have physically shielded the silicon from the electrolyte and used wires (7, 11) or a conductive oxide (12, 13) to connect the semiconductor to the hydrogenand/or oxygen-generating electrodes. Tunneling oxide layers have also been explored recently to stabilize wired silicon photoanodes (17). These approaches limit the application of Si in photochemical water splitting to a traditional, wired PEC SCIENCE VOL 334
panel geometry, which has traditionally proved too costly for commercialization. We show that water-splitting catalysts comprising earth-abundant materials can be integrated with amorphous silicon with minimal engineering to enable direct solar-to-fuels conversion based on water splitting. For the O2evolving catalyst, we use a cobalt catalyst (18), Co-OEC, that self-assembles upon oxidation of Co2+ (19), self-heals (20), and can operate in buffered electrolyte with pure or natural water at room temperature (21, 22). These attributes are similar to those of the OEC found in photosynthetic organisms. Moreover, x-ray absorption spectroscopy (23, 24) has established that the Co-OEC is a structural relative of Mn3CaO4Mn cubane (2527) of the OEC of PSII, where Co replaces Mn and the cubane is extended in a corner-sharing head-to-tail dimer (28). It has been established that the Co-OEC, when interfaced to semiconductors, enhances the efficiency of solar-assisted water splitting (2933). The H2evolving catalyst is a ternary alloy, NiMoZn. These catalysts have been interfaced directly with a commercial triple-junction amorphous silicon (3jn-a-Si) solar cell (Xunlight Corp.) in wired and wireless configurations. For either, the cell uses stacked amorphous silicon and amorphous silicon-germanium alloy junctions deposited on a stainless steel substrate and coated with a 70-nm layer of indium tin oxide (ITO) (34). Although the abundance of Ge may be a source of debate (35), the use of a silicon-based light absorber represents a major step toward a device composed of all earth-abundant materials for solar water splitting. Co-OEC is deposited directly onto the ITO layer (the illuminated side of the cell). The NiMoZn alloy H2 catalyst was used in two configurations: (i) deposited on a Ni mesh substrate that is wired to the 3jn-a-Si solar cell and (ii) deposited directly on the opposing stainless steel surface of the 3jn-a-Si solar cell as a wireless device. The devices, which have not been optimized for performance, may operate out of an open container of water containing borate electrolyte and with overall direct solar-to-fuels efficiencies of 2.5% (wireless) and 4.7% (wired) when driven by a solar cell of 6.2% and 7.7% light-toelectricity efficiency, respectively. The overall conversion efficiency of the wired cell indicates that a majority of the power from the solar cell can be converted directly to solar fuels and that a simply engineered, functional artificial leaf comprising earth-abundant materials may be realized. The PEC properties of the unmodified solar cell were characterized by operation of the cell as a photoanode in a three-electrode voltammetry configuration (3jn-a-Si working electrode, Pt counter electrode, and Ag/AgCl reference electrode). Figure 1A plots the current densities obtained from the photoanode as a function of the applied potential, illumination, and electrolyte conditions. In the absence of light (Fig. 1A, gray trace), a low anodic current density ( j < 0.05 mA /cm2) was observed upon sweeping the 3jn-
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a-Si anode from negative to positive potentials for E > 0.02 V versus the reversible hydrogen electrode (RHE) (36). The onset potential (Eon) of this sweep may be defined as the potential at which the current changes from positive (cathodic) to negative (anodic) values. Upon illumination of the cell with 1 sun (100 mW/cm2 ) of air mass (AM) 1.5 simulated sunlight ( Fig. 1A, black trace) in 1 M potassium borate electrolyte ( pH 9.2), Eon shifted to more negative values and the magnitude of the anodic current ( jan) increased slightly (Eon = 0.14 V versus RHE, and jmax,an = 0.39 mA /cm2 at 0.55 V versus RHE, the most positive potential of the sweep). The photoanode current of the ITO-coated solar cell was limited under illumination in this configuration because water splitting does not occur appreciably in the absence of catalysts. Upon addition of 0.25 mM Co2+(aq) to the borate electrolyte, Eon shifted to a more negative value (0.37 V versus RHE) and the anodic current density increased dramatically ( jan = 4.17 mA/cm2 at 0.55 V versus RHE) (Fig. 1A, red trace). Illumination (AM 1.5) of the cell under these conditions at fixed potential (E = 0.26 V versus RHE) caused a thin film to form on the electrode, and the current-time trace observed in Fig. 1B was obtained. The photocurrent rose and reached a plateau at a value of 1.5 mA/cm2 during the 12-min course of the experiment, concomitant with the evolution of bubbles at both the photoanode surface and the Pt wire counter electrode. Gas chromatography experiments identified the evolved gases to be H2 and O2. Upon completion of the photoelectrolysis experiment, the surface coloration of the cell changed from purple to light blue, which we ascribed to the formation of a thin film of the Co-OEC on the surface of the cell (see below). We attribute the rise in current in Fig. 1B to the deposition of the Co-OEC catalyst and to water splitting. The activity of the photoanode increased with Co-OEC loading, but a tradeoff existed as the catalyst layer grew thicker and blocked more of the incident radiation. Thus, short deposition times (~5 min) yielded photoanodes with optimum performance; the presence of borate maintains the optimal film thickness and preserves the selfhealing properties of the catalyst. The Co-OEC | 3jn-a-Si photoelectrodes were characterized by scanning electron microscopy (SEM) and energy-dispersive x-ray (EDX) analysis. We coated solar cells with both thin (5-min deposition time) and thick (1-hour deposition time) Co-OEC film layers. SEM analysis of cross sections (Fig. 2) allowed for determination of the thickness of the dried catalyst layer (thin film average, 85 nm; thick film average, 200 nm) and the thickness of the 3jn-a-Si layer (~1 mm); the average film thicknesses were provided from several measurements. Low-energy EDX analysis was performed to estimate the elemental composition of the substrate surface, as compared to the pristine 3jn-a-Si cell (fig. S1). Cobalt was observed only for solar cells coated with Co-OEC films, and the signal for Co was more intense for the sample with thicker Co-OEC films. We characterized the performance of the photoanode with a Co-OEC film in the absence of Co2+ in the electrolyte. The performance of the 3jn-a-Si coated with the 85-nm-thick films of Co-OEC was assessed in solutions containing 1 M potassium borate electrolyte. The Co-OEC | 3jn-a-Si cell exhibits a negative shift in the onset potential (Eon = 0.40 V versus RHE) and enhanced anodic photocurrents ( jan = 4.4 mA/cm2), relative to an uncatalyzed 3jn-a-Si cell (Eon = 0.14 V versus RHE; jan = 0.4 mA /cm2 ). The degree to which the Co-OEC film blocked incoming light and inhibited the PV performance of the cell was assessed by measuring the currentvoltage curve in air under AM 1.5 illumination (fig. S2). Under these conditions, the 3jn-a-Si cell functioned as a pure PV cell and not a PEC. Due to deposition of the Co-OEC film, the shortcircuit current ( jSC = current at zero applied bias) decreased from 6.5 to 5.9 mA/cm2, and the fill factor decreased from 0.57 to 0.50. Thus, the catalyst film of 85 nm decreased the PV performance by ~9% (8.0% to 7.3% for light-toelectricity conversion efficiency). The precision of our measurements for a given experiment is high (<1% error); the data presented are for the highest performing cells. Stand-alone operation of the cell with no external applied potential from an electrical power source (i.e., unassisted) was performed by using the Co-OEC | 3jn-a-Si photoanode in conjunction with the NiMoZn cathode for H2 production. This earth-abundant H2 evolution catalyst was electrodeposited as described in the supporting online material (SOM). Figure S3 compares the activity of the ternary alloy, as deposited (37), and bare Ni metal in 1 M potassium borate (pH 9.2). Over the potential range of the experiment, the alloy generated 50 times as much current as smooth Ni metal for the same geometric surface area. When used in conjunction with the Co-OEC | 3jn-a-Si photoanode, the NiMoZn was deposited on a Ni wire mesh substrate that was wired to the steel substrate of the 3jn-a-Si cell and placed between the light source and the photoanode. The cell, which is schematically depicted in Fig. 3A, was illuminated with AM 1.5 solarsimulated light, and the solar-to-fuels efficiency
Fig. 1. (A) Current-voltage plot of the 3jn-a-Si photoanode: in the dark (gray trace); under AM 1.5 illumination (1 sun) (black trace); in the presence of 0.25 mM Co2+ in the dark (pink trace) and (red trace) under 1 sun; and coated with a Co-OEC film under 1 sun (blue trace). The 3jn-a-Si cell was the working electrode of a three-electrode configuration (Pt counter electrode, Ag/AgCl reference electrode, 1 M potassium borate electrolyte, pH 9.2). Potentials were scanned from negative to positive to negative values. (B) Bulk photoelectrolysis plot (current density versus time) during photodeposition of the Co-OEC film from 0.25 mM Co2+ and 1 M potassium borate (pH 9.2) under 1 sun illumination. The 3jn-a-Si photoanode was held at 0.26 V versus RHE.
A
Co-OEC a-Si
L = 82 nm
B
Co-OEC a-Si
L = 225 nm L = 1.0 m
L = 945 nm
Steel
2 m
Steel
2 m
Fig. 2. SEM of cross sections of the Co-OEC | 3jn-a-Si cell after (A) 5 min and (B) 1 hour deposition of the Co-OEC film. VOL 334 SCIENCE www.sciencemag.org
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(SFE) for conversion of light and water into H2 and O2 was calculated using Eq. 1 (15), SFE(%) = j DE / S 100% = J (mA/cm2) 1.23 V / 100 (mW/cm2) 100% (1) not been corrected for light blocking by the mesh). Electrolysis efficiencies improved slightly upon operation of the cell in 0.1 M KOH electrolyte because of an increase in catalyst activity (Fig. 3A); however, the use of this electrolyte resulted in a rapid and catastrophic decline in activity after 1 hour of photolysis (Fig. 3A, red trace), concomitant with visible dissolution of the 3jn-a-Si layer. This phenomenon had been previously observed and attributed to pitting corrosion of the ITO coating by the KOH electrolyte (12). Conversely, the Co-OEC | 3jn-a-Si | NiMoZn cell exhibited significantly enhanced stability in borate electrolyte (Figure 3A, black trace). O2 yields were measured with a phosphorescence-based O2 sensor (fig. S4) and showed that virtually all of the electron-holes created during photoelectrolysis were used to convert water into O2 at the anode (within the 5% error of the measurement). The SFE may also be expressed as a direct product of the efficiency of the solar conversion of the PV cell, (PV), and fuel generation efficiency of water-splitting electrolysis, (WS), which includes losses that arise from catalyst overpotentials and Ohmic resistances, SFE (%) = (PV) (WS) (2)
Fig. 3. (A) Plot of the efficiency versus time for Co-OEC | 3jn-a-Si | NiMoZn PEC cell (left) in 1 M potassium borate (pH 9.2, black trace) and in 0.1 M KOH (pH 13, red trace) under AM 1.5 illumination. The traces are for solar cells of 7.7% PV efficiency. The cells were operated in a two-electrode cell configuration. (B) MS signal and SFE values for a wireless Co-OEC | 3jn-a-Si | NiMoZn cell under AM 1.5 illumination in 1 M KBi (red trace) and in 0.5 M KBi and 1.5 M KNO3 (blue trace). The cell was illuminated over the 2 hours of the experiment; MS signal corresponds to the concentration of O2 in the carrier gas of the cell. The spikes in the data originate from sudden release of gas bubbles that were adhered to the cells, resulting in a temporary increase of the O2 concentration in the headspace. SFE values were calculated as described in the SOM. www.sciencemag.org SCIENCE VOL 334
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where j is the current density at the photoelectrode, DE is the stored energy of the watersplitting reaction, and S is the total incident solar irradiance, which is provided by the AM 1.5 light source at 100 mW/cm2. Figure 3A plots the efficiency and stability of Co-OEC | 3jn-a-Si | NiMoZn PEC cells operated in 1 M KBi (black trace) and 0.1 M KOH (red trace) electrolyte. The overall performance of the water-splitting cells was directly correlated to their intrinsic performance of the specific underlying PV sample. In Fig. 3A, the 3jn-a-Si PV solar cell was 7.7% efficient and yielded an overall PEC cell efficiency of 4.7% (Fig. 3A, black trace). We note that light passed through the mesh, which has a transmittance of ~ 85% (the efficiency reported here has
Thus, (WS) is calculated to be ~ 60%. This value compares well with cell efficiencies based on 3jn-a-Si PVs in which the a-Si is isolated from the electrolyte [SFE = 6% for (PV) = 10%] (7, 11, 12) and for higher-efficiency systems using expensive PV materials [SFE = 18% for (PV) = 28%] (79). We note that, based on (WS), higher overall cell efficiencies (>10%) may be readily achieved through the use of more efficient PVs (38). A wireless cell was constructed by deposition of the H2-evolving catalyst, NiMoZn, onto the steel-backing substrate of the 3jn-a-Si cell. The overall device architecture is illustrated in Fig. 3B. Movie S1 shows the operation of a 1 2 cm2 wireless Co-OEC | 3jn-a-Si | NiMoZn wafer that was immersed in an open container of electrolyte (1 M potassium borate, pH 9.2) and illuminated with 1 sun, AM 1.5 simulated sunlight. The cell architecture dictated that O2 bubbles evolved from the illuminated anode at the front face (5 to 47 s of movie S1) and bubbles of H2 evolved from the cathode at the back of the wireless cell (47 to 102 s of movie S1). Oxygen yields (Fig. 3B) were determined through operation in an electrochemical cell in a closed configuration, in which the produced gases were analyzed using a mass spectrometer (MS). For this experiment, an Ar carrier gas was flowed over the headspace of the cell at a constant flow rate. The MS signal corresponds to the concentration of O2 in the carrier gas, which was used to determine the SFE for the wireless cell (see SOM for experimental details); a SFE = 1.75% was measured for a 3jn-a-Si solar cell with (PV) = 6.2%. Based on the PEC cell of (PV) = 7.7%, we expect that minimal efficiencies of 4.7% may be obtained from a properly engineered wireless cell. For instance, in the present wireless cell configuration, protons generated at the front face of the anode must move around to the back side of the cell, where they are reduced at the cathode to H2. These relatively long distances for ion transport impose substantial Ohmic losses in the cell, resulting in lower (WS). These losses may be mitigated by increasing the conductance of the solution. For instance, substituting the 1 M KBi electrolyte (specific conductivity = 26 mS/cm) with a mixture of 0.5 M KBi and 1.5 M KNO3 (specific conductivity = 126 mS/cm) resulted in an increase in the SFE from 1.75 to 2.5% (Fig. 3B). In addition, future designs (e.g., flow cell or perforated Si cell) could increase the SFE further by decreasing the anode-cathode ion transport distance and bring the wireless cell performance closer to that of the wired PEC cell, which has a 1-mm gap between cathode and anode (Fig. 3A). The stability of the wireless cells was assessed by monitoring the O2 MS signal of a wireless
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cell operating in 1 M KBi (fig. S5A). The cell was stable for 10 hours, after which its performance gradually declined to ~80% of its initial value over 24 hours. We have found that the stability of the cell is directly related to the nature of and preparative method for the transparent conductive oxide barrier layer. For example, fluorine-doped tin oxide (FTO), when prepared and annealed on crystalline Si at high temperatures (see SOM for experimental details), results in a PEC cell with stable performance over 30 hours of testing (fig. S5B), suggesting that cells using crystalline Si with the catalysts described here have great practical value. Similar strategies may be applied for protection of the 3jn-a-Si cell; however, we note that they must be compatible with the low-temperature manufacturing conditions of 3jn-a-Si. The integration of earth-abundant watersplitting catalysts with photovoltaic silicon cells captures the functional elements of energy capture and storage by a leaf. The ability to drive water splitting directly without the use of wires under a simply engineered configuration opens new avenues of exploration. For instance, the design described here could be adapted from a panel geometry to one based on freestanding (nano)particles in solution. Moreover, owing to the low solubility of O2 and H2 in water, the solar-to-fuels conversion process may be driven in the absence of a membrane. The H2 produced by photochemical water splitting may be collected directly and used or combined with carbon dioxide in a synthetic liquid fuels process external to the cell. By constructing a simple, stand-alone device composed of silicon-based light absorbers and earth-abundant catalysts, the results described here provide a first step down a path aligned with the low-cost systems engineering and manufacturing (39) that is required for inexpensive direct solar-to-fuels systems.
1. T. R. Cook et al., Chem. Rev. 110, 6474 (2010). 2. J. Barber, Phil. Trans. Roy. Soc. A 365, 1007 (2007). 3. T. A. Betley, Q. Wu, T. Van Voorhis, D. G. Nocera, Inorg. Chem. 47, 1849 (2008). 4. N. S. Lewis, D. G. Nocera, Proc. Natl. Acad. Sci. U.S.A. 103, 15729 (2006). 5. D. G. Nocera, ChemSusChem 2, 387 (2009). 6. D. Abbott, Proc. IEEE 98, 42 (2010). 7. O. Khaselev, A. Bansal, J. A. Turner, Int. J. Hydrogen Energy 26, 127 (2001). 8. G. H. Lin, M. Kapur, R. C. Kainthla, J. O. M. Bockris, Appl. Phys. Lett. 55, 386 (1989). 9. O. Khaselev, J. A. Turner, Science 280, 425 (1998). 10. S. Licht et al., J. Phys. Chem. B 104, 8920 (2000). 11. R. E. Rocheleau, E. L. Miller, A. Misra, Energy Fuels 12, 3 (1998). 12. N. A. Kelly, T. L. Gibson, Int. J. Hydrogen Energy 31, 1658 (2006). 13. T. L. Gibson, N. A. Kelly, U.S. Patent 7,052,587 (2003). 14. A. E. Delahoy, S. C. Gau, O. J. Murphy, M. Kapur, J. O. M. Bockris, Int. J. Hydrogen Energy 10, 113 (1985). 15. M. G. Walter et al., Chem. Rev. 110, 6446 (2010). 16. X. G. Zhang, Silicon and Its Oxide (Kluwer Academic, New York, 2001). 17. Y. W. Chen et al., Nat. Mater. 10, 539 (2011). 18. M. W. Kanan, D. G. Nocera, Science 321, 1072 (2008). 19. Y. Surendranath, M. W. Kanan, D. G. Nocera, J. Am. Chem. Soc. 132, 16501 (2010). 20. D. A. Lutterman, Y. Surendranath, D. G. Nocera, J. Am. Chem. Soc. 131, 3838 (2009). 21. Y. Surendranath, M. Dinc, D. G. Nocera, J. Am. Chem. Soc. 131, 2615 (2009). 22. A. J. Esswein, Y. Surendranath, S. Y. Reece, D. G. Nocera, Energy Environ. Sci. 4, 499 (2011). 23. M. Risch et al., J. Am. Chem. Soc. 131, 6936 (2009). 24. M. W. Kanan et al., J. Am. Chem. Soc. 132, 13692 (2010). 25. J. Barber, Inorg. Chem. 47, 1700 (2008). 26. E. M. Sproviero, J. A. Gascon, J. P. McEvoy, G. W. Brudvig, V. S. Batista, J. Am. Chem. Soc. 130, 6728 (2008). 27. Y. Umena, K. Kawakami, J.-R. Shen, N. Kamiya, Nature 473, 55 (2011). 28. M. D. Symes, Y. Surendranath, D. A. Lutterman, D. G. Nocera, J. Am. Chem. Soc. 133, 5174 (2011). 29. J. A. Seabold, K. S. Choi, Chem. Mater. 23, 1105 (2011). 30. E. M. P. Steinmiller, K. S. Choi, Proc. Natl. Acad. Sci. U.S.A. 106, 20633 (2009). 31. D. K. Zhong, J. Sun, H. Inumaru, D. R. Gamelin, J. Am. Chem. Soc. 131, 6086 (2009). 32. D. K. Zhong, D. R. Gamelin, J. Am. Chem. Soc. 132, 4202 (2010).

www.sciencemag.org/cgi/content/full/science.1209816/DC1 Materials and Methods Figs. S1 to S5 Movie S1 15 June 2011; accepted 15 September 2011 Published online 29 September 2011; 10.1126/science.1209816
Hot CarrierAssisted Intrinsic Photoresponse in Graphene

Nathaniel M. Gabor,1 Justin C. W. Song,1,2 Qiong Ma,1 Nityan L. Nair,1 Thiti Taychatanapat,1,3 Kenji Watanabe,4 Takashi Taniguchi,4 Leonid S. Levitov,1 Pablo Jarillo-Herrero1* We report on the intrinsic optoelectronic response of high-quality dual-gated monolayer and bilayer graphene p-n junction devices. Local laser excitation (of wavelength 850 nanometers) at the p-n interface leads to striking six-fold photovoltage patterns as a function of bottom- and top-gate voltages. These patterns, together with the measured spatial and density dependence of the photoresponse, provide strong evidence that nonlocal hot carrier transport, rather than the photovoltaic effect, dominates the intrinsic photoresponse in graphene. This regime, which features a long-lived and spatially distributed hot carrier population, may offer a path to hot carrierassisted thermoelectric technologies for efficient solar energy harvesting. he photoresponse of semiconductors, which determines the performance of optoelectronic devices, is governed by energy relaxation pathways of photoexcited electronhole pairs: Energy transferred to the lattice is lost
eration. Novel relaxation pathways that result from electron confinement have been demonstrated in nanocrystal quantum dots and carbon nanotubes (2, 3). In graphene, energy relaxation pathways are strongly altered by the vanishing electronic density of states (46). After initial relaxation of photoexcited carriers (as a result of electron-electron scattering and optical phonon emission), electron-lattice energy relaxation can be quenched (4), which creates a bottleneck that limits further energy redistribution into the lattice. With electron-to-lattice energy relaxation quenched, a novel transport regime is reached in which thermal energy is redistributed solely
1 Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 2Harvard School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA. 3Department of Physics, Harvard University, Cambridge, MA 02138, USA. 4National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan.
as heat, while energy transported through charge carriers may be used to drive an optoelectronic circuit (1). Nanoscale systems can offer various ways to control energy relaxation pathways, potentially resulting in more efficient device opVOL 334 SCIENCE
*To whom correspondence should be addressed. E-mail: pjarillo@mit.edu
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33. D. K. Zhong, M. Cornuz, K. Sivula, M. Grtzel, D. R. Gamelin, Energy Environ. Sci. 4, 1759 (2011). 34. X. Deng, E. A. Schiff, in Handbook of Photovoltaic Science and Engineering, A. Luque, S. Hegedus, Eds. (Wiley, Chichester, England, 2003), pp. 505565. 35. Germanium commodity summary, U.S. Geological Survey; available online at http://minerals.usgs.gov/minerals/ pubs/commodity/germanium/mcs-2011-germa.pdf. 36. The RHE potential is that of the standard hydrogen electrode (SHE) adjusted for the pH of the solution. Potentials (versus RHE) were calculated from the measured values (versus Ag/AgCl) according to the equation E(RHE) = E(Ag/AgCl) + 0.197V + (0.059V pH). 37. Mo leaches from alloys to furnish high-surface-area materials; thus, the activity of the alloy increases with leaching time [see (40, 41)]. After 9 days, leaching ceases and a very active, high-surface-area material is obtained. 38. J. J. H. Pijpers, M. T. Winkler, Y. Surendranath, T. Buonassisi, D. G. Nocera, Proc. Natl. Acad. Sci. U.S.A. 108, 10056 (2011). 39. B. D. James, G. N. Baum, J. Perez, K. N. Baum, U.S. Department of Energy, Dec. 2009. Available online at: http://www1.eere.energy.gov/hydrogenandfuelcells/ pdfs/pec_technoeconomic_analysis.pdf. 40. B. E. Conway, L. Bai, Int. J. Hydrogen Energy 11, 533 (1986). 41. J. Z. O. Stachurski, D. Pouli, J. A. Ripa, G. F. Pokrzyk, U.S. Patent 4,354,915 (1982). Acknowledgments: Sun Catalytix acknowledges Xunlight Corporation for providing the 3jn-a-Si cells and ARPA-E (DE-AR0000036) for funding. D.G.N. acknowledges support with grants from the National Science Foundation (CHE-0533150), AFOSR FA9550-09-1-0689, and the Chesonis Family Foundation. J.J.H.P. performed work as part of the Fellowships for Young Energy Scientists program of the Foundation for Fundamental Research on Matter (FOM), which is financially supported by the Netherlands Organization for Scientific Research (NWO). NWO is also gratefully acknowledged for supplying a Rubicon grant to J.J.H.P. J. Turner and E. Miller are acknowledged for helpful discussions.
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among electronic charge carriers. The photogenerated carrier population remains hot while the lattice stays cool. In graphene, hot carriers should play a key role in optoelectronic response (6), yet measurements have not clearly determined the photocurrent generation mechanism. Numerous initial studies suggested that photocurrent generated at graphene-metal contacts (711) results from the photovoltaic (PV) effect, in which a built-in electric field accelerates photogenerated charge carriers to the electronic contacts. More recent studies have looked at monolayer-bilayer interfaces (12) and highly controlled chemical potential gradients at gate voltagecontrolled p-n interfaces (13), and their results indicate that photothermoelectric (PTE) effects may play an important role. The PTE effect arises from a light-induced temperature difference resulting in a thermoelectric voltage. Although PTE physics has been invoked to explain photocurrent measured in graphene (12, 13), an experimental proof of its role in determining the intrinsic photoresponse has been lacking. We report optoelectronic measurements of dual gate voltagecontrolled graphene p-n junction devices in the presence of local laser excitation. Dual gate control allows us to explore the rich structure of photoresponse as a function of carrier polarity, varied independently in two adjacent regions. Our measurements indicate that hot electronic carriers dominate graphenes intrinsic optoelectronic response at temperatures ranging from room temperature down to 10 K and in the linear optical power regime. The hot carrier regime manifests as a strong PTE effect that results in a striking six-fold photovoltage pattern as a function of gate voltages. Additionally, the spatial and charge density dependence of the photoresponse establishes a strong connection between thermal energy transport and electronic charge carriers. We studied p-n junctions in both monolayer and bilayer graphene devices integrated with global bottom gates and local top gates, as shown in Fig. 1A and described in (14). Tuning the bottomand top-gate voltages, VBG and VTG respectively, allows independent control of carrier density of electrons (n-type carriers) and holes (p-type carriers) in each region (1517). By applying voltages of opposite polarity on VBG and VTG, we formed a p-n junction at the interface of p- and n-type regions in a single graphene sample (Fig. 1C, bottom). The gate configurations in our devices are similar to those in (1517), with p-n junctions estimated to be narrower than ~80 nm. We first characterized our devices via electronic transport measurements of resistance R as a function of VBG and VTG. Figure 1B shows R versus VBG and VTG for a monolayer graphene (MLG) p-n junction. The resistance exhibited four characteristic regions, a distinctive behavior that reflects gate voltagetunable charge density and a sharp density gradient at the p-n interface (1517). When the charge density nc was tuned near the neutrality point in each gated region, R increased because of the low density of conduction carriers. When two gates were present, this resulted in two intersecting lines of relatively high resistance, and a maximum resistance Rmax at their intersection, the global charge neutrality point (CNP). The two lines divided the resistance map into four regions: p-n, n-n, n-p, and p-p, labeled according to the carrier doping induced in the bottom-gated and dual-gated regions (regions 1 and 2, respectively). As the carrier density increased away from the CNP, the resistance decreased monotonically from Rmax. Devices were placed in a low-temperature optical cryostat that combined electronic transport with mirror-controlled scanning laser excitation (Fig. 1C and fig. S1). Figure 1D shows a photocurrent image obtained by scanning a focused laser spot 1 mm in diameter (l = 850 nm) over the device at VSD = 0 V while measuring the current I. At the sharply defined junction, we observed a strong photocurrent that was an order of magnitude greater than the photocurrent at the contacts and increased linearly with optical power. The maximum photocurrent responsivity reached 5 mA/Wat low temperatures, which is greater than previously reported values of 1 mA /W at room temperature (10, 13). As a function of distance, the photocurrent exhibited a triangular shape centered at the junction and extending several micrometers to the contacts (solid line in Fig. 1E). A triangular profile indicates that the charge carriers remained hot throughout the device and contributed to photocurrent (see below). Unlike the resistance measurements, in which we observed four gate voltage regions, optoelectronic measurements with the laser fixed at the junction exhibited a striking photovoltage pattern, with six regions of alternating photovoltage sign as a function of VBG and VTG (Fig. 2). Because the photoconductance in graphene is negligible (9), the photovoltage established across the device can be determined from the measured IPH according to the relation VPH = IPHR. If the photocurrent originates from the PV effect, in which the photovoltage is proportional to the electric field induced at the junction, then we would
Fig. 1. Device geometry, band structure, and optoelectronic characteristics of the graphene p-n junction. (A) Optical microscope image of the dual-gated device incorporating boron nitride top-gate dielectric. The current I is measured at fixed bias voltage VSD. Dashed white lines mark the boundaries of graphene. MLG, monolayer graphene; VTG, top-gate voltage. (B) Resistance (in kilohms) versus VBG and VTG at VSD = 1.4 mV and T = 175 K. Four regions are labeled according to carrier doping, p-type or n-type, in the bottom- and top-gated regions, respectively. CNP, charge neutrality point. (C) Experimental schematic (top) and schematic of monolayer graphenes band structure in the p-n junction (bottom), showing electron bands (blue) and hole bands (red). The dashed line represents the electron chemical potential (or Fermi energy) EF. (D) Spatially resolved photocurrent map at T = 40 K with laser wavelength l = www.sciencemag.org SCIENCE
850 nm and optical power = 50 mW (VBG = 5 V, VTG = 2 V, VSD = 0 V). White lines mark location of gold contact and gate electrodes; white triangle is measurement point for Fig. 2A. (E) Photocurrent line trace taken at dashed line in (D). Laser position = 0 corresponds to the edge of the top-gate electrode; the dashed line is a fit to the data (see text). VOL 334 4 NOVEMBER 2011
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Fig. 2. Photovoltage and thermoelectric response of the graphene p-n junction. (A) Photovoltage (VPH) versus VBG and VTG at T = 40 K, measured at the location marked by the triangle in Fig. 1D. Gray dashed lines are lines of high resistance from transport characteristics. The white dashed line is described in the text. Photovoltage line traces were taken along the vertical (left) and diagonal (bottom) gray dashed lines. (B) Absolute value of photovoltage near the CNP. (C) Resistance and thermovoltage versus VBG at VSD = 0 V, VTG = 2.0 V, and T = 40 K (optical power = 1 mW, laser at triangle in Fig. 1D). (D) Schematic of the Seebeck coefficient as a function of Fermi energy for typical resistance characteristics of graphene (inset).
expect only a single photovoltage sign change and a monotonic dependence on increasing charge density. However, VPH as a function of VBG and VTG (Fig. 2A) exhibited multiple sign changes (6). Moreover, the photovoltage line traces extracted along the high-resistance ridges (gray lines), along which charge density increases away from the CNP, exhibited strong nonmonotonic gate voltage dependence (Fig. 2A, left and bottom). The shape of the photovoltage node lines (i.e., lines at VPH = 0) indicates that whereas R decreased monotonically away from Rmax at the CNP, the photovoltage showed nonmonotonic dependence on gate voltages. In the PV effect, changing the direction of the electric field as the relative carrier densities are changed in regions 1 and 2 would change the sign of the photovoltage across a single nodal line. This line, along which the carrier density in the two regions is equal (white dashed line in Fig. 2A), divides the p-p and n-n regions (6). Figure 2B shows the photovoltage nodes determined by plotting the absolute value of photovoltage |VPH| versus VBG and VTG. We observed three photovoltage nodes that deviated substantially from lines of high resistance. Near the CNP, where R decreased monotonically away from Rmax, the photovoltage displayed a vertically oriented Z-shaped nodal line and two points toward which the three nodal lines converged (arrows in Fig. 2B). Such nonmonotonic behavior of transport properties is reminiscent of the sign change and nonmonotonic charge density dependence of thermoelectric voltage in graphene (1821). Thermoelectric transport measurements on the same sample showed that the thermovoltage VT measured as a function of VBG exhibited features similar to the photovoltage nodes along the highresistance ridges. By laser-heating the gold contact far from the graphene-metal junction (triangle in Fig. 1D),we introduced a fixed uncalibrated temperature gradient across the device and measured the resulting thermovoltage (fig. S2). Figure
Fig. 3. Fourier analysis of the photovoltage response. (A) Fourier transform |F{VPH}| of photovoltage versus VBG and VTG from Fig. 2A. NB and NT label two components of the Fourier transform masked to extract the bottom- and top-gated photovoltage components. Upper inset: Photovoltage map from Fig. 2A. Lower inset: Sum of the individual Fourier components of the photovoltage. (B) Bottom- and top-gated photovoltage components VPH[NT] and VPH[NB] as a function of VBG and VTG calculated by inverse Fourier transforming along the masked directions NT and NB. Lower panel: Photovoltage line traces from VPH[NB] and VPH[NT]. The VBG axis has been shifted by the CNP voltage for VPH[NB]. (C) Ratio of the photovoltage VPH[NB] to the thermovoltage VT, and resistance as a function of VBG measured along the white dashed line in Fig. 2A. 2C shows VT and R as a function of VBG. Whereas R decreased monotonically away from the CNP, VT exhibited a nonmonotonic gate voltage dependence with peaks that corresponded closely to the gate voltages at which the three nodal lines of the photovoltage converged near the CNP (arrows in Fig. 2B). VOL 334 SCIENCE We attribute the intricate photovoltage pattern in graphene to the PTE effect. After optical excitation, electron-hole pairs decay rapidly (~10 to 150 fs) into a thermal distribution of electronic carriers (22, 23) with a local effective temperature Tel that remains out of equilibrium with the lattice. The difference in temperature with its
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Fig. 4. Photoresponse in the bilayer graphene p-n-p photodetector. (A) Optical microscope image of the BLG device. The current I is measured at fixed bias voltage VSD. White dashed line marks the boundary of BLG. (B) Photocurrent map at T = 40 K, l = 850 nm, and optical power = 200 mW (VBG = 15 V, VTG = 10 V, VSD = 0 V). White lines indicate location of gold contact electrodes. (C) Photovoltage at the n-p junction [diamond in (B)] versus VBG and VTG at T = 40 K. Gray dashed lines are lines of high resistance taken from transport characteristics. White dashed line is as in Fig. 2A. (fig. S3). A strong photoresponse was observed in the p-n-p configuration, with photocurrent that peaked at the p-n junctions formed near the top-gate edge (Fig. 4B). The six-fold pattern in photovoltage versus VBG and VTG indicates the presence of a strong PTE effect (Fig. 4C). The hot carrierassisted photoresponse in graphene is characterized by a long-lived and spatially distributed hot electronic population resulting from local excitation. The PTE photoresponse (Eq. 1) depends directly on the electronic temperature, which can be strongly enhanced by long cooling times t. Because the cooling length x t (24), long cooling times manifest as long cooling lengths. Using solutions to the heat equation (14), we fitted the photocurrent profile (dashed line in Fig. 1E) and obtained excellent fits for x > 2 mm (fig. S4). From the cooling length x > 2 mm, we estimated a lower bound of the hot carrier cooling time t Cel x2/k 100 ps, where Cel is the electronic heat capacity and k is the thermal conductivity of graphene (14). This cooling time is consistent with very slow cooling dynamics recently observed in graphene (23). The dimensions of the device allow us to establish only a lower bound for the cooling length. In contrast, other scattering mechanisms, such as optical phonon scattering, occur on far shorter time scales of ~1 ps (22, 23) and are ineffective at cooling low-energy carriers. Graphenes intrinsic photoresponse also exhibits charge density dependence that confirms hot carrierassisted transport of thermal energy. For excitation fixed at the p-n interface, the steady state value of DT (14) at the laser spot is determined (in the limit of long cooling length) by DT dQ=dt K1 K2 3
surroundings results in a thermal current that is accompanied by a charge current, the magnitude and sign of which is sensitive to carrier type and density (i.e., a p-type sample exhibits charge current with an opposite sign to that of an n-type sample). In unbiased homogeneous graphene, an isotropic thermal current flowing away from the excitation spot is not accompanied by a similar isotropic charge current. However, in a p-n junction this symmetry is broken, and photoexcitation can result in nonzero net PTE current. The sign and magnitude of PTE voltage resulting from this current depends on the Seebeck coefficient (12, 24) in each region and can be written as V PTE S 2 S 1 DT 1
where S is thermoelectric power (Seebeck coefficient) in the bottom- and dual-gated regions, and DT taken at the junction is proportional to the electron temperature difference between the area of optical excitation and its surroundings (6). From the Mott formula (24), we can write S as S p2 k 2 T 1 dR dV G B 3e R dV G dE E EF
where T is the sample temperature, kB is the Boltzmann constant, and EF is the Fermi energy (12, 1821). Whereas R decreased monotonically with VG away from the CNP, S changed nonmonotonically (Fig. 2D). The nonmonotonic dependence of S1 and S2 resulted in multiple sign reversals for the quantity (S1 S2), which occurred along three nodal lines and gave rise to the six-fold pattern (Fig. 2A) (6), characteristic of a PTE effect in graphene.
We obtained more direct insight into the origin of the six-fold photovoltage patterns by extracting the two components of the PTE voltage individually. A Fourier analysis technique (14) can extract S1 and S2 because they depend on different combinations of VBG and VTG. The Fourier transform of the data (Fig. 3A), exhibited a pair of streaks. The orientations of these streaks, horizontal (NB) and diagonal (NT), are described by the capacitance matrix of regions 1 and 2 [see (25)]. The relations between these streaks and the individual properties of the two regions are clarified by masking one of the streaks and performing an inverse Fourier transform of the other streak. This procedure revealed two contributions, each behaving as a function of density in the corresponding regions, VPH[NB] and VPH[NT] (Fig. 3B, top). Furthermore, the nonmonotonic density dependence of each contribution was similar to the behavior of the Seebeck coefficient given by the Mott formula (Fig. 3B, bottom). The sum VPH[NB] + VPH[NT] reproduces the photovoltage pattern (Fig. 3A, lower inset). The nonmonotonic behavior of each term produced polarity reversal on three nodal lines and gave rise to a six-fold pattern. Six-fold patterns were also observed in the bilayer graphene (BLG) photoresponse. Composed of two Bernal-stacked sheets of monolayer graphene, BLG has hyperbolic bands that touch at zero energy, resulting in the absence of a band gap (2628). Figure 4 shows the photoresponse of a BLG p-n junction photodetector. Using a top-gated configuration, two back-to-back junctions could be tuned by gate voltages. The resistance behaves similarly to the MLG junction, showing four gate voltage regions separated by lines of high resistance (gray dashed lines in Fig. 4C) SCIENCE VOL 334
where dQ/dt is the rate of heat entering the system, and K1 and K2 are respectively the electronic contributions to the thermal conductance of regions 1 and 2. Because hot carriers proliferate throughout the system, the Wiedemann-Franz re2 lation, K = p2kBT/3e2 (1/R), holds. As a consequence, DT 1/K R (24). To isolate the density dependence of DT, we take the ratio of the photovoltage VPH to the thermovoltage VT obtained from a fixed temperature difference (14). This ratio, VPH[NB]/VT DT, is shown in Fig. 3C and clearly exhibits a density dependence that mimics the device resistance. The strong density dependence peaking at the CNP clearly demonstrates the electronic origin of DT. Transport of hot electronic carriers results in a novel nonlocal transport regime that may enable increased power conversion efficiency in energy-harvesting devices. At present, graphene is considered to be an excellent candidate for energy-harvesting optoelectronics, in part because of the presence of high-responsivity photodetection with high internal quantum efficiency (6, 9). Here, we have shown that hot carriers dominate the intrinsic photoresponse and have
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demonstrated devices with Seebeck coefficients as high as 12 mV/K (at T = 40 K) and high electronic temperature DT ~ 4.5 K (14). Hot carrier assisted photoresponse is predicted to improve the power conversion efficiency of energy-harvesting devices beyond standard limits (29). Our findings, together with new design principles for next-generation solar thermoelectric devices (30), make graphene-based systems viable for energyharvesting applications.
1. S. M. Sze, Physics of Semiconductor Devices (Wiley, London, ed. 2, 1981). 2. V. Sukhovatkin, S. Hinds, L. Brzozowski, E. H. Sargent, Science 324, 1542 (2009). 3. N. M. Gabor, Z. Zhong, K. Bosnick, J. Park, P. L. McEuen, Science 325, 1367 (2009). 4. R. Bistritzer, A. H. MacDonald, Phys. Rev. Lett. 102, 206410 (2009). 5. T. Winzer, A. Knorr, E. Malic, Nano Lett. 10, 4839 (2010). 6. J. C. Song, M. S. Rudner, C. M. Marcus, L. S. Levitov, Nano Lett. 10.1021/nl202318u (2011). 7. E. J. Lee, K. Balasubramanian, R. T. Weitz, M. Burghard, K. Kern, Nat. Nanotechnol. 3, 486 (2008). 8. J. Park, Y. H. Ahn, C. Ruiz-Vargas, Nano Lett. 9, 1742 (2009). 9. F. Xia, T. Mueller, Y. M. Lin, A. Valdes-Garcia, P. Avouris, Nat. Nanotechnol. 4, 839 (2009). 10. T. Mueller, F. Xia, P. Avouris, Nat. Photonics 4, 297 (2010). 11. G. Nazin, Y. Zhang, L. Zhang, E. Sutter, P. Sutter, Nat. Phys. 6, 870 (2010). 12. X. Xu, N. M. Gabor, J. S. Alden, A. M. van der Zande, P. L. McEuen, Nano Lett. 10, 562 (2010). 13. M. C. Lemme et al., Nano Lett. 10.1021/nl2019068 (2011). 14. See supporting material on Science Online. 15. B. Huard et al., Phys. Rev. Lett. 98, 236803 (2007). 16. J. R. Williams, L. Dicarlo, C. M. Marcus, Science 317, 638 (2007). 17. B. zyilmaz et al., Phys. Rev. Lett. 99, 166804 (2007). 18. E. H. Hwang, E. Rossi, S. Das Sarma, Phys. Rev. B 80, 235415 (2009). 19. P. Wei, W. Bao, Y. Pu, C. N. Lau, J. Shi, Phys. Rev. Lett. 102, 166808 (2009). 20. Y. M. Zuev, W. Chang, P. Kim, Phys. Rev. Lett. 102, 096807 (2009). 21. J. Checkelsky, N. P. Ong. Phys. Rev. B 80, 081412(R) (2009). 22. D. Sun et al., Phys. Rev. Lett. 101, 157402 (2008). 23. J. H. Strait et al., Nano Lett. 10.1021/nl202800h (2011). 24. N. W. Ashcroft, N. D. Mermin, Solid State Physics (Thomson Learning Inc., London, 1976). 25. In the top-gated region, both VBG and VTG contribute to nc, resulting in the slope of the diagonal line m = DVTG/DVBG = CBG/CTG 0.05, where CBG and CTG are respectively the bottom- and top-gate capacitances to graphene. NB and NT in the Fourier transform are perpendicular to the minimum resistance lines (gray dashed lines) in Fig. 2A. 26. E. McCann, Phys. Rev. B 74, 161403 (2006). 27. T. Ohta, A. Bostwick, T. Seyller, K. Horn, E. Rotenberg, Science 313, 951 (2006). 28. E. V. Castro et al., Phys. Rev. Lett. 99, 216802 (2007). 29. R. T. Ross, A. J. Nozik, J. Appl. Phys. 53, 3813 (1982). 30. D. Kraemer et al., Nat. Mater. 10, 532 (2011). Acknowledgments: We thank M. Baldo, P. McEuen, P. Kim, and A. Yacoby for valuable discussions. Supported by the Air Force Office of Scientific Research, a NSF Early Career Award (P.J.H.), and the Packard Foundation. Sample fabrication was performed at the NSF-funded MIT Center for Materials Science and Engineering and Harvard Center for Nanoscale Science.
19 July 2011; accepted 26 September 2011 Published online 6 October 2011; 10.1126/science.1211384
The Pace of Shifting Climate in Marine and Terrestrial Ecosystems

Michael T. Burrows, * David S. Schoeman, Lauren B. Buckley, Pippa Moore, Elvira S. Poloczanska,7 Keith M. Brander,8 Chris Brown,7,9 John F. Bruno,4 Carlos M. Duarte,10,11 Benjamin S. Halpern,12 Johnna Holding,11 Carrie V. Kappel,12 Wolfgang Kiessling,13 Mary I. OConnor,14 John M. Pandolfi,15 Camille Parmesan,16 Franklin B. Schwing,17 William J. Sydeman,18 Anthony J. Richardson7,9,19 Climate change challenges organisms to adapt or move to track changes in environments in space and time. We used two measures of thermal shifts from analyses of global temperatures over the past 50 years to describe the pace of climate change that species should track: the velocity of climate change (geographic shifts of isotherms over time) and the shift in seasonal timing of temperatures. Both measures are higher in the ocean than on land at some latitudes, despite slower ocean warming. These indices give a complex mosaic of predicted range shifts and phenology changes that deviate from simple poleward migration and earlier springs or later falls. They also emphasize potential conservation concerns, because areas of high marine biodiversity often have greater velocities of climate change and seasonal shifts. limate warming is a global threat to biodiversity (1). Key mechanisms allowing species to cope with warming include shifting biogeographic ranges and altering phenology (the synchronous timing of ecological events) (2, 3) to accommodate spatial and seasonal changes in ambient temperature. Considerable variation in species responses exists: Average range shifts have been reported as 6.1 km/decade for terrestrial communities (2), from 1.4 to 28 km/decade for marine communities (4), and 16.1 km/decade for a combination of both (5), whereas spring phenology has been reported as advancing on average by 2.3 to 2.8 days/decade on land (2, 6) and by 4.3 days/decade at sea (3, 7). One reason for variability in estimates of responses could be that patterns of climate change are dynamic and highly heterogeneous across Earth. Different
1 2,3 4 5,6
on a 1-by-1 grid (7). The velocity of climate change (10) (in km/year) was calculated as the ratio of the long-term temperature trend (in C/year)
1 Department of Ecology, Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll, PA37 1QA, Scotland, UK. 2Environmental Sciences Research Institute, School of Environmental Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland. 3Department of Zoology, Post Office Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth, 6031, South Africa. 4Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 5Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DA, UK. 6 Centre for Marine Ecosystems Research, Edith Cowan University, Perth, 6027, Australia. 7Climate Adaptation Flagship, Commonwealth Scientific and Industrial Research Organisation Marine and Atmospheric Research, Ecosciences Precinct, General Post Office Box 2583, Brisbane, Queensland 4001, Australia. 8National Institute of Aquatic Resources, Technical University of Denmark, Charlottenlund Slot, Jgersborg All1, Charlottenlund, Denmark. 9School of Biological Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. 10 The University of Western Australia Oceans Institute, University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia. 11Department of Global Change Research, Instituto Mediterrneo de Estudios Avanzados (Consejo Superior de Investigaciones Cientficas, University of the Balearic Islands), Esporles, 07190, Spain. 12National Center for Ecological Analysis and Synthesis, University of California Santa Barbara, 735 State Street, Suite 300, Santa Barbara, CA 93101, USA. 13Museum fr Naturkunde at Humboldt University, Invalidenstrasse 43, 10115 Berlin, Germany. 14Department of Zoology and Biodiversity Research Centre, University of British Columbia, Vancouver, Canada V6T 1Z4. 15School of Biological Sciences, Australian Research Council Centre of Excellence for Coral Reef Studies, University of Queensland, Brisbane, Queensland 4072, Australia. 16Section of Integrative Biology, 1 University Station C0930, University of Texas, Austin, TX 78712, USA. 17Southwest Fisheries Science Center, National Oceanic and Atmospheric Administration Fisheries Service, 1352 Lighthouse Avenue, Pacific Grove, CA 93950, USA. 18Farallon Institute for Advanced Ecosystem Research, Post Office Box 750756, Petaluma, CA 94975, USA. 19Centre for Applications in Natural Resource Mathematics, School of Mathematics and Physics, University of Queensland, St Lucia, Queensland 4072, Australia.
regions are warming or even cooling at different rates, and air temperatures are rising faster than those of upper ocean waters (8), so uniform responses across the globe to climate change should not be anticipated. Instead, organism responses are expected to track the rate of isotherm migration over space and seasons to maintain their thermal niches (9, 10). Although organisms may respond to aspects of climate change other than temperature, our aim was to generate predictions for shifts in distribution and phenology from physical descriptions of the global thermal environment, and to compare predictions among regions across the land and ocean. We used global surface temperatures (11, 12) over 50 years (19602009) to calculate the distribution of the velocity and seasonal shifts of isotherm migration over land and ocean VOL 334 SCIENCE
*To whom correspondence should be addressed. E-mail: michael.burrows@sams.ac.uk
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to the two-dimensional spatial gradient in temperature (in C/km, calculated over a 3-by-3 grid), oriented along the spatial gradient. We introduced the seasonal climate shift (in days/decade) as the ratio of the long-term temperature trend (C/year) to the seasonal rate of change in temperature (C/day). We present seasonal shifts for spring and fall globally using April and October temperatures. The median rate of warming since 1960 has been more than three times faster on land (0.24C/decade) than at sea (0.07C/decade, Fig. 1A and table S1). At the scale of our analysis, median spatial gradients in temperature on land (0.0082C/km, Fig. 1B and table S1) are greater than those at sea (0.0030C/km) because of the greater latitudinal and topographical temperature differences on land, whereas large-scale currents tend to reduce small-scale variability in ocean surface temperatures. When spatial gradients are combined with rates of long-term temperature change, the resulting median velocity of isotherms across the ocean (21.7 km/decade) is 79% of that on land (27.3 km/decade), but when comparing only those latitudes where both land and ocean are present (50S to 80N), velocities in the ocean (27.5 km/decade) are similar to those on land (27.4 km/decade). The frequency distribution of velocities in the ocean is bimodal (Fig. 2A), with a broader spread of positive values in the ocean than on land and many negative values in cooling areas, including the Southern Ocean and Eastern Boundary Current regions with increased upwelling (Fig. 1, A and C, and fig. S1D). The relative proportions of warming and cooling areas influence the land/ocean comparison (table S1): With less cooling, median velocity in the Northern Hemisphere ocean is 37.3 km/decade but only 30.3 km/decade on land, whereas in the Southern Hemisphere median velocities are 17.6 and 14.6 km/decade for land and ocean, respectively. The velocity of climate change is two to seven times faster in the ocean than on land in the sub-Arctic and within 15 of the equator (Fig. 1C), but ocean and land velocities are similar at most other latitudes (20 to 50S and 15 to 45N). At the scales studied, the velocity of climate change is very patchy on land, whereas the ocean
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areas, and generally faster in areas of shallow spatial gradients. (D) Seasonal shift (days/decade) is the change in timing of monthly temperatures, shown for April, representing Northern Hemisphere spring and Southern Hemisphere fall: positive where timing advances, negative where timing is delayed. Cross-hatching shows areas with small seasonal temperature change (<0.2C/month), where seasonal shifts may be large. See fig. S3 for October seasonal shifts.
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surface has homogenous surface temperatures with shallow spatial gradients separated by relatively sharp thermal boundaries (Fig. 1, B and C). Areas of high velocity therefore extend across broader regions in the ocean than on land, and this may help explain the 1.5 to 5 times faster average range shifts reported in the ocean (4, 13) than on land (2, 4), albeit from few taxa in mainly temperate regions (24, 6) in areas where the velocities of climate change are similar across land and ocean. Geographical variation in the velocity of climate change may explain much of the variation among range shifts of individual species (5), and shifts relative to our predictions may indicate sensitivity to temperature. This may explain some of the differences in reported averages from marine and terrestrial meta-analyses. Despite faster warming on land, change in seasonal timing is generally greater in the ocean because of smaller seasonal thermal variation (fig. S2, A to C). Shifts in the timing of spring temperatures (19602009) were 30 to 40% faster in the ocean than on land (table S1). Spring ocean temperatures arrived earlier by 2.08 days/decade in the Northern Hemisphere and 2.52 days/decade in the Southern Hemisphere (median values excluding equatorial regions), but by 1.46 and 2.15 days/decade, respectively, on land. Equatorial regions were excluded from these estimates because small seasonal temperature change gave high values for shifts (fig. S2, B and C) and because the lack of strong annual variation (fig. S2A) suggests that temperature is not a driver of tropical phenology. A greater advance in spring timing in the ocean than on land was evident in the Arctic (>66N, Fig. 1D) and in mid-latitudes (40 to 60N). Considerable variability exists in the Southern Ocean, with delayed springs in cooling areas and advanced springs in warming areas (fig. S3). Delays in the arrival of colder fall temperatures were also greater in the ocean than on land in both hemispheres (medians for the northern ocean were 1.73 days/decade and for land 1.20 days/decade; for the southern ocean, 2.28 days/decade and for land 1.21 days/decade, table S1). The land/ocean difference was consistent across most latitudes in the Southern Hemisphere (Fig. 1D) but was only greater in the ocean in the Northern Hemisphere beyond 45N (fig. S3). Large areas of land have negligible seasonal change, particularly in central Asia. Later springs in central North America are associated with a spring cooling trend. Estimates of spring seasonal shift are close to reported global average values for earlier timing of boreal spring events [2.3 days/decade (2), 5.1 days/decade (14), and 2.8 days/decade (6)]. Rates of spring advance exceed this in the North Atlantic and North Pacific, the Arctic Ocean, and much of Europe, North America, and eastern Asia (Fig. 1D). Our indices of change relate to almost all terrestrial species but only to those marine species that live at or near the surface. Many marine species, however, depend on surface conditions, using light for energy or plants for food or having surface-living planktonic stages. For terrestrial species, the option exists to move to greater altitude to track thermal conditions, but depth changes have been reported for only a few marine organisms, such as fish (1517) and hydroids (18). For species that cannot adjust their depth, range shifts may be limited by the availability of suitable habitat. Where such habitat is not aligned with the velocity of climate change, as happens on east-west coastlines (19), the velocity along the axis of the habitat could be much faster (7). Considerable geographical heterogeneity exists in rates of velocity of climate change and magnitude of seasonal shift, with consequences for life on land (10) and in the ocean that depend on biophysical attributes of regions (Fig. 3, fig. S4, and table S2). Land/ocean boundaries constrain movement, as in the Mediterranean, where species may be forced toward the European continent, away from potential escape routes (Fig. 3C and table S2). Adjacent areas of rapid poleward and equatorward velocities exist, as seen near the Antarctic Peninsula (Fig. 3E), and spring conditions can be both advanced and delayed within small regions (Fig. 3, B and D). Thus, subregional and habitat-specific patterns in temperature may run counter to the simple expectation of poleward range shift and shifts toward earlier spring and later fall, and biological responses will vary accordingly (20). Accumulated negative effects of high velocities (21) and rapid seasonal shifts on many species may threaten biodiversity, especially where such conditions coincide with species-rich equatorial and coastal regions (22, 23), as in the Coral Triangle (Fig. 3A and table S2). For example, in tropical areas where spatial gradients are shallow, the rapid velocity of climate change may be of special concern: No communities of organisms from even warmer regions exist to replace those moving out (24). Likewise, as polar areas warm, resident organisms might lose their current thermal niches altogether. In contrast, areas of low
Fig. 3. Within-region patterns of seasonal shifts and velocity of climate change in the ocean. Arrows illustrate the magnitude and direction of velocity, perpendicular to isotherms (white lines); circles indicate velocities <1 km/decade. (A) Rapid divergent velocities in the Coral Triangle. (B) Spring temperatures arrived 5 to 10 days/decade earlier in the North Sea but by less in the Mediterranean and were delayed in the Black Sea. (C) Poleward velocities in the Mediterranean are constrained by the European landmass. (D) Spring temperatures arrived 10 to 20 days/decade earlier to the east of the Antarctic Peninsula but were delayed by a similar amount in adjacent areas of cooling. (E) Poleward velocities exist alongside equatorward velocities in the same region, suggesting location-specific responses of species ranges. VOL 334 SCIENCE www.sciencemag.org
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velocity or slow seasonal shifts, where residence times of climate would be longest (10), may be important repositories for biodiversity. The global distribution of the velocity and seasonal shift of climate change over the past 50 years can be used to generate predictions for comparison with observed biological changes. Despite slower ocean warming, the velocity of climate change and seasonal shift in the ocean are as high as on land and often deviate from simple expectations of poleward migration and earlier springs/later falls. Direct effects of climate warming are therefore likely to be as great in the oceans as on land at comparable latitudes and greater around the equator. Maps of the velocity of climate change and seasonal shift show the areas where the threat to biodiversity from organisms need to rapidly track thermal conditions by shifting distributions and retiming seasonal thermal events may be greatest; these areas may coincide with high biodiversity, especially in the oceans.
1. O. E. Sala et al., Science 287, 1770 (2000). 2. C. Parmesan, G. Yohe, Nature 421, 37 (2003). 3. S. J. Thackeray et al., Glob. Change Biol. 16, 3304 (2010). 4. W. W. L. Cheung et al., Fish Fish. 10, 235 (2009). 5. I.-C. Chen, J. K. Hill, R. Ohlemller, D. B. Roy, C. D. Thomas, Science 333, 1024 (2011). 6. C. Parmesan, Glob. Change Biol. 13, 1860 (2007). 7. Full methods are presented in the supporting material on Science Online. 8. K. E. Trenberth et al., in Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, S. Solomon et al., Eds. (Cambridge Univ. Press, Cambridge, 2007), pp. 235336. 9. D. D. Ackerly et al., Divers. Distrib. 16, 476 (2010). 10. S. R. Loarie et al., Nature 462, 1052 (2009). 11. UK Meteorological Office Hadley Centre, Natural Environment Research Councils National Centre for Atmospheric Science (NCAS) (British Atmospheric Data Centre, 2010); http://badc.nerc.ac.uk/view/badc. nerc.ac.uk__ATOM__dataent_hadisst. 12. University of East Anglia Climate Research Unit, NCAS (British Atmospheric Data Centre, 2010); http://badc.nerc.ac.uk/view/badc.nerc.ac. uk__ATOM__dataent_1256223773328276. 13. C. J. B. Sorte, S. L. Williams, J. T. Carlton, Glob. Ecol. Biogeogr. 19, 303 (2010). 14. T. L. Root et al., Nature 421, 57 (2003). 15. A. L. Perry, P. J. Low, J. R. Ellis, J. D. Reynolds, Science 308, 1912 (2005). 16. N. K. Dulvy et al., J. Appl. Ecol. 45, 1029 (2008). 17. J. A. Nye, J. S. Link, J. A. Hare, W. J. Overholtz, Mar. Ecol. Prog. Ser. 393, 111 (2009). 18. S. Puce, G. Bavestrello, C. G. Di Camillo, F. Boero, Mar. Ecol. (Berlin) 30, 313 (2009). 19. N. Mieszkowska, S. J. Hawkins, M. T. Burrows, M. A. Kendall, J. Mar. Biol. Assoc. U.K. 87, 537 (2007). 20. B. Helmuth et al., Ecol. Monogr. 76, 461 (2006). 21. H. M. Pereira et al., Science 330, 1496 (2010). 22. P. H. Williams, K. J. Gaston, C. J. Humphries, Proc. R. Soc. London Ser. B 264, 141 (1997). 23. D. P. Tittensor et al., Nature 466, 1098 (2010). 24. R. K. Colwell, G. Brehm, C. L. Cardels, A. C. Gilman, J. T. Longino, Science 322, 258 (2008). Acknowledgments: This work was conducted as a part of the Towards Understanding Marine Biological Impacts of Climate Change Working Group supported by the National Center for Ecological Analysis and Synthesis, a Center funded by NSF (grant no. EF-0553768); the University of California, Santa Barbara; and the State of California. M.T.B. thanks the UK Natural Environment Research Council Oceans 2025 program, J.P. thanks the Australian Research Council Centre of Excellence for Coral Reef Studies for additional support, and A.J.R. was supported by the Australian Research Council Discovery Grant DP0879365 and Future Fellowship Grant FT0991722. Data used in analyses are available from the University of East Anglia Climate Research Unit and the UK Meteorological Office Hadley Centre, with online access at the British Atmospheric Data Centre.

www.sciencemag.org/cgi/content/full/334/6056/652/DC1 Materials and Methods SOM Text Figs. S1 to S5 Tables S1 and S2 References (2545) 24 June 2011; accepted 20 September 2011 10.1126/science.1210288
Atmospheric Blocking and Atlantic Multidecadal Ocean Variability

Sirpa Hkkinen,1* Peter B. Rhines,2 Denise L. Worthen3 Atmospheric blocking over the northern North Atlantic, which involves isolation of large regions of air from the westerly circulation for 5 days or more, influences fundamentally the ocean circulation and upper ocean properties by affecting wind patterns. Winters with clusters of more frequent blocking between Greenland and western Europe correspond to a warmer, more saline subpolar ocean. The correspondence between blocked westerly winds and warm ocean holds in recent decadal episodes (especially 1996 to 2010). It also describes much longer time scale Atlantic multidecadal ocean variability (AMV), including the extreme pregreenhouse-gas northern warming of the 1930s to 1960s. The space-time structure of the wind forcing associated with a blocked regime leads to weaker ocean gyres and weaker heat exchange, both of which contribute to the warm phase of AMV. he climate of the global oceans undergoes natural variability with many time scales, as well as greenhouse-induced change. We discuss the relation between periods of warm subpolar ocean and patterns of blocking in the atmospheric circulation above in the North Atlantic sector. Blocking occurs when the high-latitude jet stream develops large, nearly stationary mean-
1 National Aeronautics and Space Administration (NASA) Goddard Space Flight Center, Code 615, Greenbelt, MD 20771, USA. 2University of Washington, Box 357940, Seattle, WA 98195, USA. 3Wyle Information Systems/NASA Goddard Space Flight Center, Code 615, Greenbelt, MD 20771, USA.
*To whom correspondence should be addressed. E-mail: sirpa.hakkinen@nasa.gov
ders, essentially breaking Rossby waves with precursors upwind, over North America. These trap an air mass equatorward of an anticyclonic pressure ridge. We show here that clusters of frequent blocking over the subpolar North Atlantic coincide with warm and saline ocean in the 1960s, late 1970s, and early 2000s (1). Further back in time, a new reconstruction of surface atmospheric pressure field for the entire 20th century (2, 3) encompasses the most dramatic climate event of the century: the pregreenhouse-gas northern warming that began in the 1920s and lasted until the 1960s. Sea surface temperatures (SST) of the North Atlantic also show this widespread warming, whereas the South Atlantic Ocean is cooler SCIENCE VOL 334
than normal. This longer time scale variability is known as Atlantic Multidecadal Variability (AMV, or Atlantic Meridional Oscillation, AMO) (4, 5). AMV has been linked to many multidecadal climate processes: Atlantic hurricanes, northeast Brazil and Sahel rainfall, North American and European summer climate (57). We show that warm ocean and frequent atmospheric blocking coincide over multidecadal AMV time scales. More than a local response to the atmosphere, invasions of warm ocean water northward from the subtropics, extending well below the surface, are involved (1, 8). Atmospheric blocking involves shifts in Atlantic storm tracks lasting several days. Blocking over Greenland is associated with the negative phase of the North Atlantic Oscillation (NAO) and a southward shift of the storm track (9) (fig. S1). The blocking also occurs further east over western Europe with an anticyclone over the northern subpolar gyre (fig. S2). Such blocking is accompanied by cold winter temperatures in western Europe, for example, winters in 2005/2006 (10), 1963, and 2009/2010 (11). The climatological maximum in winter blocking days is located over western Europe, with a secondary maximum over Greenland connected by a ridge of increased blocking (fig. S3), if blocking is defined by absolute geopotential height index [(12), which is an extension of (13, 14)]. Thus, the east-west location of Atlantic blocking anticyclones varies, but in 61 of 110 cases investigated in (9), European blocks immediately precede Greenland blocks.
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Changes in the storm track affect the ocean circulation through surface wind stress and windstress curl. Wind-stress curl causes downward (in the subtropics) or upward (subpolar) pumping of surface waters, driving the circulation gyres. The particularly strong episode of subpolar warming and salinization in the early 2000s was associated with weakening of wind-stress curl, hence weakened gyre circulation and decreased heat loss over the subpolar Atlantic (1). And indeed, the long decline of subpolar gyre surface currents from 1994 to 2000 revealed by satellite altimetry (15) corresponds with a weakening wind-curl field. Indices of wind-related climate variability have their basis in empirical orthogonal function [EOF; (14)] analysis of the winter (December to March) wind-stress curl, with use of the recently produced 20th century reanalysis [and the 60-year National Centers for Environmental Prediction/National Center for Atmospheric Research (NCEP/NCAR) reanalysis for comparison]. Both data sets give essentially the same spatial EOF patterns and amplitudes shown in Fig. 1A. EOF1 has an extremum west of Ireland, centered on the boundary between the subpolar and subtropical ocean gyres, and its time series (principal component, PC, in Fig. 1B) correlates highly with the NAO index. EOF2 (Fig. 1A) has extrema colocated with the ocean gyres, and its time series, PC2, is shown in Fig. 1B. EOF2 represents weakening and strengthening of the climatological-mean pattern and hence the strength of the gyre circulation. PC time series from NCEP/NCAR reanalysis data for the latter half of the century follow closely the 20th century reanalysis results. For the recent decades, 1960 to 2005, the PC2 time series of the wind-stress curl was found to be the key index associated with the saline and warm periods in the northern North Atlantic Ocean, because it controls expansion, contraction, and strength of both subpolar and subtropical gyres (1). With a longer atmospheric reanalysis, we can now address climate regimes associated with the SST variability such as the AMV, focusing on the wind-stress curl PC2 of the EOF analysis. To highlight the North Atlantic climate regimes, we computed sea level pressure (SLP), blocking, and turbulent heat-flux anomalies on the basis of composited differences between negative and positive events in wind-stress curl PC2 (>1 standard deviation) (14). All analysis results displayed have their basis in the 20th century reanalysis data. The number of wintertime blocking days (from December to March) was determined from the ensemble-mean daily 500-hPa height data. The mean annual blocking days over a decade (Fig. 2A) use the blocking definition (12) based on reversal north-south dynamic height gradient at 500 hPa within the latitude range 34N to 74N in the Atlantic sector and requiring reversal to persist 5 or more days (14). The 20th century reanalysis tends to overestimate blocking days in the latter half of the century compared with the NCEP/NCAR in the overlapping period (fig. S4). However, the temporal and spatial fluctuations are similar, such as widespread blocking activity in the 1960s and 2000s, weak blocking in the 1980s and 1990s, and two centers of activity in the 1950s. NCEP/NCAR reanalysis singles out the late 1960s as the most unusual period, with persistent blocking at every longitude between western Europe and Greenland (fig. S5). Despite the differences between the reanalyses, some decades display more blocking activity than others (Fig. 2A). The warmest periods occur in the North Atlantic Ocean when increased blocking occurs over the Western Europe and has an extension toward Greenland, that is, 1920 to 1970 and after 2000. This association is evident in the time series of blocked days in the subpolar Atlantic (Fig. 2B) and the AMO index of subpolar ocean surface temperature [as in (7, 14)], with and without the trend removed. The difference between full and detrended AMO index shows the importance of the global warming signal in the recent years (7). Wind-stress curl, EOF mode 2, has the same spatial pattern as the mean ocean gyres; we call EOF2/PC2 the gyre mode. It is distinct from the NAO, which more resembles EOF1. Wind-stress curl relates to the vorticity of the winds, hence to the Laplacian of SLP, which emphasizes smallerscale features than SLP-based analysis (1618): An SLP composite corresponding to negative minus positive curl PC2 events results in a pattern (Fig. 3A) resembling the eastern Atlantic pattern in SLP-EOF analysis (12, 19) and also the Atlantic Ridge pattern found with cluster analysis (16, 17). This SLP pattern has been identified as a blocking signature (12). This pattern was also recovered as an SLP difference field of warm years (19391968) minus cold years (19001929) (20). With different binning of the warm (19501964) and cold (19701984) years, the SLP pattern re-
Fig. 1. The winter (December through March, DJFM) wind-stress curl variability from the 20th century atmospheric reanalysis based on EOF analysis. (A) Spatial pattern of wind-stress curls EOF1 and EOF2. EOF1 (top) represents 22.3% of the wind-stress curl variability and has its centers of action displaced north-south relative to the subpolar ocean gyre. EOF2 (bottom), with 15.6% of the variance, has centers of action coinciding with the subpolar and northern subtropical ocean gyres. (B) Principal components of wind-stress curl EOFs from the 20th century reanalysis. PC1 (red), PC2 (blue), and from NCEP/NCAR reanalysis, PC1 (dashed black) and PC2 (dashed purple). The standard deviation of the PC2 from the 20th century reanalysis is 8.9 108 N m3 (includes interannual variability). Time series are smoothed by 10 binomial filters.
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sembles more the negative NAO pressure anomaly (21). The multidecadal depth-dependent AMV temperature mode found in the zonal average is also linked to the eastern Atlantic SLP pattern (8). There is no spatial similarity between the NAO pattern and the wind curl EOF2, and the PC2 is not strongly correlated with the NAO index, but the PC2 time series shows remarkably close relationship with the subpolar SLP anomaly (Fig. 3B) and, less significantly, with the subtropical Azores SLP center. The relation between the low-frequency Azores SLP and ocean gyre variability has been found from analysis of the sea level variability along the European coast (22). To establish the linkage between the windstress curl variability and blocking, we form composited difference fields by using the wind-stress curl PCs. The composite of anomalous blocking activity based on PC1 (fig. S6, as negative minus positive PC1 events) corresponds to Greenland blocking known to be associated with the negative NAO phase (18). On the other hand, the PC2 composites (again as negative minus positive PC2 events, Fig. 4A) show simultaneously increased activity of the western European blocking and a weaker Greenland blocking, flanked by decreased blocking over Scandinavia and southern Europe. This specific blocking anomaly reaching from Greenland to western Europe represents
Fig. 2. (A) Mean winter (DJFM) blocking days (color bar) by decade from the 20th century reanalysis. (B) DJFM blocking days in the region from 10E to 70W and 45N to 75N from the 20th century reanalysis (black) and from NCEP/NCAR reanalysis (blue). The two blocking time series have a correlation of 0.85 over the overlapping time period 19492008. The AMO index (dashed pink) is an area-averaged SST from 0N to 60N and 10E to 80W. The AMO index with global surface temperature evolution removed [as in (7)] is shown in solid red. The SST curves are smoothed by three binomial filters.
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fluctuations of the climatological-mean blocking pattern (fig. S3) with contributions from positive and negative NAO index, where the NAO-positive contributions occur over the eastern side of the North Atlantic and western Europe. Extending the classic NAO description of North Atlantic atmospheric variability by allowing east-west displacement of centers of action in this way is suggested in earlier work (9, 17). The active blocking band from Greenland to the western Europe was also seen in decadal variability of the extended early midcentury warm period (Fig. 2). With use of the same years as in (20) to define warm period (19391968) and cold period (19001929), the resulting difference in blocking days (Fig. 4B) has an anomaly of increased blocking from Greenland to Europe very similar to that corresponding to the curl PC2 (Fig. 4A). The areas of significance (95% or higher, 58 degrees of freedom) are stippled. Use of the AMO index (with no detrending) to form a composite (positive minus negative) of the blocking events (fig. S7) gives the same band of increased blocking as the wind stress curl PC2, although with somewhat less significance. In addition to the association of warm, saline ocean with weakening of the wind-stress curl and subpolar and subtropical gyre circulation, air-sea heat flux influences SST. The composite difference of turbulent (sensible and latent) heat flux corresponding to (negative minus positive) curl PC2 (Fig. 4C) shows the heat flux anomaly associated with the climate regimes, which have weak circulation gyres and high blocking activity. This heat flux anomaly favors heating in large part of the North Atlantic, including the equatorial region, with weak cooling in a narrow band in the midlatitudes. This heating anomaly pattern differs from that corresponding to the NAO index, where the subpolar heating or cooling is centered over the Labrador Sea (instead of the central subpolar gyre) and the cooling or heating over the Gulf Stream region is of the same order of magnitude as in the Labrador Sea. Figure 4 shows that a similar blocking activity can be recovered whether using the gyre index (wind-stress curl PC2) or the multidecadal SST variability as the compositing index. This is an implication that the particular blocking anomaly is a fundamental part of the forcing of the gyre variability at low frequencies. Moreover it is associated with surface heat exchange, which supports heat content variability, in turn amplifying the effects of expanding and contracting ocean gyres. In summary, variability of atmospheric blocking over years to several decades shows correlation with the ocean surface temperature and with significant changes in Atlantic Ocean circulation, mediated by wind-stress curl and air-sea heat exchange. The wind-stress curl variability of most importance for the subpolar gyre represents weakening and strengthening of the climatological-mean curl pattern (1). This same mode of variability is associated with a major shift in the upper ocean currents after year 2000 and warming and salinization of the subpolar gyre (1, 23). Wind-stress curl variability represents changes in the Atlantic storm track, frequency, and intensity; and particularly persistent events, that is, blocking, where storm tracks are shifted have a large impact on the curl. For example with negative NAO index, a block over southern Greenland forces the existence of a single upper-level midlatitude jet crossing the Atlantic (18). However, the index of our wind-stress curl pattern, the gyre mode, is not correlating strongly with NAO, nor projects purely on Greenland blocking but contains a large component of blocking centered on western Europe. Increased activity of both Greenland and western Europe blocking centers has occurred when the North Atlantic has been in a warm state. The gyre mode also supports atmosphere-ocean heat exchange, which sustains a warm state of the North Atlantic, from the subpolar region through most of the subtropics to the equator. This suggests that blocking activity is more fundamental than the NAO index in describing climatic changes in the Atlantic Ocean. The blocking events (lasting 5 days or more) provide an example of high-frequency atmospheric variability projecting on lower-frequency variability of the wind-stress curl. Winters with frequent blocking appear to persist for decades and are evidence of a highly disturbed hemispheric jet stream system. We speculate that the westerly wind stress directed along the Gulf Stream and North Atlantic Current system is less intense and less coherent in such conditions. Our analysis cannot separate cause and effect between high blocking activity and warm ocean surface, but the existing theory of the midlatitude atmosphere-ocean interaction supports increased persistence of atmospheric anomalies that created oceanic anomalies in the first place (24). The warm-ocean/cold-land anomaly pattern has been linked to a dynamical environment favorable for
Fig. 3. Relation between the gyre index and SLP. Analysis period is 19012008. (A) A composite difference of SLP based on negative minus positive wind-stress curl PC2 events stronger than one standard deviation shows the SLP anomaly associated with a weakened subpolar gyre. Color denotes SLP anomaly in hPa. Stippling denotes significance of difference at 95% level. (B) Subpolar SLP (blue) (average over 20W to 50W and 50N to 65N) and curl PC2 (red) from the 20th century reanalysis smoothed by 10 binomial filters. VOL 334 SCIENCE www.sciencemag.org
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blocking (25, 26). Blocking has also been shown to be sensitive to a warmer subpolar Atlantic in a reconstruction of the cold European winter of 2005/2006 (10). The possibility of coupled interaction of atmosphere with AMV seems likely, given the long-period variability of blocking reported here and in the even longer paleoclimate time series (27). AMV has been attributed to changes in the Atlantic Meridional Overturning Circulation (AMOC) in some numerical model experiments (28, 29). Also, on the basis of hydrographic data, AMV exhibits vertical structure, which could signal AMOC variability (8). Feedbacks between the hemispheric jet-stream waveguide, tropical and extra-tropical ocean temperatures, and the AMOC itself are possible and even likely.
A
1. S. Hkkinen, P. B. Rhines, D. L. Worthen, J. Geophys. Res. 116, C03006 (2011). 2. G. P. Compo, J. S. Whitaker, P. D. Sardeshmukh, Bull. Am. Meteorol. Soc. 87, 175 (2006). 3. G. P. Compo et al., Q. J. R. Meteorol. Soc. 137, 1 (2011). 4. D. B. Enfield, A. M. Mestaz-Nuez, P. J. Trimble, Geophys. Res. Lett. 28, 2077 (2001). 5. K. E. Trenberth, D. J. Shea, Geophys. Res. Lett. 33, L12704 (2006). 6. J. R. Knight, C. K. Folland, A. A. Scaife, Geophys. Res. Lett. 33, L17706 (2006). 7. M. Ting, Y. Kushnir, R. Seager, C. Li, J. Clim. 22, 1469 (2009). 8. I. V. Polyakov, V. A. Alexeev, U. S. Bhatt, E. I. Polyakova, X. Zhang, Clim. Dyn. 34, 439 (2010). 9. T. Woollings, B. J. Hoskins, M. Blackburn, P. Berrisford, J. Atmos. Sci. 65, 609 (2008). 10. M. Croci-Maspoli, H. C. Davies, Mon. Weather Rev. 137, 664 (2009). 11. J. Cattiaux et al., Geophys. Res. Lett. 37, L20704 (2010). 12. S. C. Scherrer, M. Croci-Maspoli, C. Schwierz, C. Appenzeller, Int. J. Climatol. 26, 233 (2006). 13. S. Tibaldi, F. Molteni, Tellus 42A, 343 (1990). 14. Supporting material on data sets and methods on Science Online. 15. S. Hkkinen, P. B. Rhines, Science 304, 555 (2004). 16. C. Cassou, L. Terray, J. W. Hurrell, C. Deser, J. Clim. 17, 1055 (2004). 17. J. W. Hurrell, C. Deser, J. Mar. Syst. 79, 231 (2010). 18. T. Woollings, A. Hannachi, B. Hoskins, A. Turner, J. Clim. 23, 1291 (2010). 19. M. Croci-Maspoli, C. Schwierz, H. C. Davies, Clim. Dyn. 29, 713 (2007). 20. C. Deser, M. L. Blackmon, J. Clim. 6, 1743 (1993). 21. Y. Kushnir, J. Clim. 7, 141 (1994). 22. L. Miller, B. C. Douglas, Geophys. Res. Lett. 34, L16602 (2007). 23. S. Hkkinen, P. B. Rhines, J. Geophys. Res. 114, C04005 (2009). 24. J. J. Barsugli, D. S. Battisti, J. Atmos. Sci. 55, 477 (1998). 25. A. Shabbar, J. Huang, K. Higuchi, Int. J. Climatol. 21, 355 (2001). 26. D. Barriopedro, R. Garcia-Herrera, A. R. Lupo, E. Hernandez, J. Clim. 19, 1042 (2006). 27. E. Rimbu, G. Lohmann, Clim. Past Discuss. 5, 2411 (2009). 28. T. L. Delworth, M. E. Mann, Clim. Dyn. 16, 661 (2000). 29. J. R. Knight, C. K. Folland, A. A. Scaife, Geophys. Res. Lett. 33, L17706 (2006). Acknowledgments: S.H. and D.L.W. were funded by NASA Headquarters Physical Oceanography Program and Ocean Surface Topography (OST) Science Team. P.B.R. is supported by NASA through the OST Science Team. We thank our reviewers for constructive criticism.
Fig. 4. Anomaly patterns of blocking activity. Analysis period is 19012008. (A) A composite difference of blocking days based on negative minus positive curl PC2 events stronger than one standard deviation shows the increased blocking occurring from Greenland to western Europe when the subpolar gyre is weak. Stippling denotes significance of difference at 95% level. (B) Warm (19391968) minus cold (19001929) years [as defined in (20)] difference in blocking days. Stippling denotes significance of difference at 95% level assuming 58 degrees of freedom. (C) Anomaly of the surface heat exchange associated with the gyre index. A composite difference of turbulent heat flux (positive upward; color denotes flux magnitude in units of W m2) based on negative minus positive curl PC2 events stronger than one standard deviation shows the widespread heat input to the ocean when the gyre circulation is also weak. Stippling denotes significance of difference at 95% level. www.sciencemag.org SCIENCE VOL 334

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The Influence of Late Quaternary Climate-Change Velocity on Species Endemism

B. Sandel,1,2* L. Arge,2 B. Dalsgaard,3 R. G. Davies,4 K. J. Gaston,5 W. J. Sutherland,3 J.-C. Svenning1 The effects of climate change on biodiversity should depend in part on climate displacement rate (climate-change velocity) and its interaction with species capacity to migrate. We estimated Late Quaternary glacial-interglacial climate-change velocity by integrating macroclimatic shifts since the Last Glacial Maximum with topoclimatic gradients. Globally, areas with high velocities were associated with marked absences of small-ranged amphibians, mammals, and birds. The association between endemism and velocity was weakest in the highly vagile birds and strongest in the weakly dispersing amphibians, linking dispersal ability to extinction risk due to climate change. High velocity was also associated with low endemism at regional scales, especially in wet and aseasonal regions. Overall, we show that low-velocity areas are essential refuges for Earths many small-ranged species. nthropogenic climate change is a major threat to Earths biodiversity and the ecosystem services it provides (1). As climate changes, the conditions suitable for local persistence of a particular species move across the surface of the Earth, driving species responses both to recent warming (24) and to long-term natural climate cycles (58). Species with strong dispersal abilities inhabiting relatively stable climates may track climate fairly closely. Conversely, species with weak dispersal abilities relative to the rate of climate change may fail fully to oc-
cupy climatically suitable areas (914) and may even go extinct, despite appropriate habitat being present elsewhere (1517). Climate-change velocity is a measure of the local rate of displacement of climatic conditions over Earths surface (18). It integrates macroclimatic shifts with local spatial topoclimate gradients and is calculated by dividing the rate of climate change through time by the local rate of climate change across space, yielding a local instantaneous velocity measure. By describing the local rate at which species must migrate to track
1 Ecoinformatics and Biodiversity Group, Department of Bioscience, Aarhus University, Aarhus 8000 C, Denmark. 2Center for Massive Data Algorithmics (MADALGO), Department of Computer Science, Aarhus University, Aarhus 8000 C, Denmark. 3 Conservation Science Group, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UK. 4School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. 5 Environment and Sustainability Institute, University of Exeter, Cornwall TR10 9EZ, UK.
*To whom correspondence should be addressed. E-mail: brody.sandel@biology.au.dk
Fig. 1. Global maps of (A) climate-change velocity since the Last Glacial Maximum (21,000 years ago); proportional endemism of (B) amphibians, (C) mammals, and (D) birds; and (E) relationships of past and predicted future climate-change velocity. Velocity is highest in northeastern North America and north-central Eurasia; these same areas display low or no endemism (the black line delimits areas that were glaciated at LGM). Mountain ranges and other low-velocity regions exhibit higher endemic richness of amphibians, mammals, and birds. Rank-transformed velocity since the LGM and rank-transformed velocities until 2080 show similar spatial patterns, but there are areas of important mismatch. Orange areas, where velocities in the past have been low but predicted future velocities are expected to be high, are a particular conservation concern.
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changing climate, climate-change velocity is more biologically relevant than are traditional macroclimatic anomalies (13, 16, 19, 20). Furthermore, because climate-change velocity incorporates finescale topoclimate gradients it captures the important buffering effect of topographic heterogeneity on climate change (21). For example, a given temperature change should have very different biological consequences depending on topographic context: In flat areas, considerable movement is required to track a 1C increase in temperature, whereas a short shift uphill could be sufficient in mountains. Thus, species distributed in topographically homogenous landscapes will experience higher climate-change velocities and therefore require stronger dispersal abilities to track climate change than those of species in heterogeneous landscapes. High climate-change velocities are likely to be associated with incomplete range filling and species extinctions (22). Not all species, however,
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are at equal risk from high velocity (20). Strong dispersers should be most able to maintain distributional equilibrium with climate conditions and are therefore likely to occupy more of their potential range and avoid extinction. Species with small ranges are at particular risk of extinction (20); they often have small population sizes and densities (23) and are less likely to occupy refuges that remain suitable during climate oscillations (15, 16, 24). Range size may also reflect other species characteristics that influence resilience to climate fluctuation, with widespread species tending to have broad climatic tolerances (25), generalist strategies (23), and strong dispersal capabilities (23). Hence, high-velocity regions should have fewer small-ranged species and fewer species with poor dispersal ability (16). To test these hypotheses, we used reconstructions of mean annual temperature at the Last Glacial Maximum (LGM; 21,000 years ago) to calculate a global map of climate-change velocity from the LGM to the present (26) and tested the effects of velocity on patterns of range size and species richness of amphibians, mammals, and birds (27). The difference between the LGM and the present is one of the strongest climatic shifts in all of the Quaternary (28), and its spatial pattern is probably similar to the spatial patterns of earlier climate cycles (16). Our analysis revealed that the LGM-to-present climate-change velocity exhibits marked geographic variation, with peaks in northeastern North America and north-central Eurasia (Fig. 1A). Velocities tended to be lower in the Southern Hemisphere and in mountainous areas. Globally, small-ranged (<250,000 km2; hereafter termed endemic) amphibians, mammals, and birds are concentrated where climate-change velocity is low (Fig. 1, B to D) [results were not sensitive to the particular definition of small ranges (27)]. In high-velocity northeastern North America and north-central Eurasia, endemic species are nearly absent, whereas low-velocity areas often harbor highly endemic faunas. For all species groups, low-velocity sites are more likely to harbor endemic species than are high-velocity sites (Fig. 2, insets; logistic regression with spatial filters: n = 2000 grid cells, all P < 0.0001). In addition, among regions (10 10 equivalents) containing at least one endemic species, velocity is strongly and negatively related to the proportion of amphibian [bivariate regression; n = 188 regions, coefficient of determination (r 2 ) = 0.283, P < 0.001], mammal (n = 231 regions, r 2 = 0.328, P < 0.001), and bird (n = 240 regions, r 2 = 0.385, P < 0.001) species that are endemic (Fig. 2). We excluded glaciated areas (Fig. 1A) from this analysis, but investigations showed that results were not sensitive to this decision. It is widely accepted that modern climate influences species distributions and diversity, whereas the role of historical determinants is less clear (23). We therefore examined models that incorporated descriptions of modern climate, the spatial pattern of modern climate conditions, and climate-change velocity. The spatial pattern of modern climate conditions was summarized by calculating, for each grid cell, the total area of land within a 1000-km radius that had a mean annual temperature (MAT) within 1C and mean annual precipitation (MAP) within 100 mm of that focal cell (24). The extent of analogous modern climate exerted strong influences on endemism, reflecting the fact that regionally rare climates are likely to contain small-ranged species (Table 1) (24). Endemism of all three groups was negatively related to productivity and to two measures of seasonality. In models that considered all variables together while controlling for spatial autocorrelation [simultaneous autoregressive models (SARs)], velocity was a highly significant negative predictor of endemism for all groups, and for the amphibians was second in importance only to the extent of analogous climate. We examined the effect of adding terms to this model to describe the interaction of velocity and modern climate descriptors. Including these terms generally had small effects on other coefficient estimates, and only one such interaction was significant (velocity precipitation seasonality for amphibians), so these interactions were not considered further. We examined all subsets of the full SAR models and compared them using Akaikes Information Criterion (AIC). For all groups, models incorporating velocity were always strongly preferred over equivalent models without velocity (mean AIC improvement: amphibians, 29.6; mammals, 38.5; and birds, 39.0). Across the full model set with and without climate-change velocity, there was high support for velocity as part of the best model (summed Akaike weights for velocity > 0.989 for all groups). These results show that past climate-change velocity and modern climate act together to determine global patterns of endemism. Because velocity incorporates fine-scale topographic effects on climate stability, it should also contribute to within-region variation in endemism. To test this, we divided the world into regions (10 10 equivalents) and asked whether velocity was correlated with endemism within these regions. For all three species groups, high-velocity areas within regions were associated with low endemism (global means of within-region correlation coefficients: amphibians, r = 0.160; mammals, r = 0.157; and birds, r = 0.091, all P < 0.01) (Fig. 3). This overall pattern is consistent with the loss of small-ranged species in high-velocity regions, but the local importance of velocity showed strong geographic variation. Velocity should have the strongest impact where the climate is sometimes highly suitable for a given group, potentially favoring the generation and maintenance of endemic diversity (29). In contrast, regions characterized by generally unfavorable conditions are expected to contain few endemics, whether or not velocity is low. In addition, the ability of species to cope with temperature fluctuations is thought to vary spatially, with species in highly seasonal
Fig. 2. The relationship between climate-change velocity and proportional endemism for (A) amphibians, (B) mammals, and (C) birds at a global scale within 10 10 regions, considering only unglaciated areas. Relationships are shown separately for the Northern (red) and Southern Hemispheres (blue) and for the global relationship (black line). Insets depict the relationship between velocity and the presence or absence of any small-ranged species. For each of 25 velocity quantiles, the blue bars indicate the proportion of locations with that velocity where small-ranged species occur. The black line displays a logistic regression fit to the relationship.
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areas tolerating a wider temperature range (30). We tested these hypotheses by identifying the modern climatic variables that most strongly control the within-region correlation of velocity and endemism patterns (table S9). For all three species groups, the models with the lowest AIC scores contained a single significant predictor and were consistent with the above predictions; velocity was particularly important for amphibians and mammals where precipitation was high [amphibians, n = 125 regions, standardized regression coefficient (b) = 0.185, P = 0.0465; mammals, n = 149 regions, b = 0.259, P = 0.0018) and for birds where temperature seasonality was low (n = 135 regions, b = 0.334, P = 0.0102) (Fig. 3). Although a comprehensive test has thus far been lacking, the importance of climate-change velocity for a group should depend on the dispersal abilities of its species. Hence, of the taxa analyzed, birds should be best at tracking highvelocity changes, whereas amphibians should be worst (9). Indeed, the importance of velocity in determining global patterns of endemism decreased from amphibians (standardized b = 0.288) to mammals (standardized b = 0.216) to birds (standardized b = 0.183) (Table 1). Furthermore, velocity was most tightly correlated with patterns of within-region endemism for amphibians and least correlated for birds. Lending additional support to the importance of dispersal ability, velocity was also more tightly associated with patterns of nonvolant mammal endemism (standardized global b = 0.166, P = 0.0058, mean local r = 0.165) than with bat endemism (standardized global b = 0.135, P = 0.0670, mean local r = 0.034). Global patterns of endemism were wellcorrelated among the three groups, suggesting that overall they respond to similar drivers. However, regions with low amphibian endemism relative to avian endemism (which are correlated, r = 0.681) tended to have high velocity, indicating that amphibians respond more strongly than do birds to climate change (multiple regression of amphibian endemism against velocity and avian endemism, n = 183 regions, standardized bvelocity = 0.281, P = 0.0010). Similar results were obtained for amphibian endemism relative to mammalian endemism (r = 0.693, n = 185 regions, bvelocity = 0.283, P = 0.0010) and mammalian endemism relative to avian endemism (r = 0.819, n = 212 regions, bvelocity = 0.175, P = 0.0028). Richness of the lowest three range-size quartiles in amphibians is low relative to the equivalent avian richness (n = 175 regions, bvelocity = 0.247, P = 0.0031) and mammal richness (n = 173 regions, bvelocity = 0.333, P < 0.0001) in high-velocity regions. As expected given their low dispersal ability, amphibian assemblages in high-velocity regions were thus particularly depauperate of endemic species relative to bird and mammal assemblages. The focal relationship between velocity and endemism is corroborated by other patterns in the distributions of all three groups. At both global and regional spatial scales, high velocity was associated with larger median range sizes, lower Table 1. Relationships of seven predictor variables to global patterns of proportional endemism for amphibians, mammals, and birds. GVI, generalized vegetation index; MAT, mean annual temperature; MAP, mean annual precipitation; TS, temperature seasonality; PS, precipitation seasonality; Extent, the regional extent of analogous climate; and Velocity, Late Quaternary climate-change velocity. Five comparative measures were used: the coefficient of determination from bivariate regressions (r 2), standardized regression coefficients estimated from ordinary least-squares multiple regression (OLS), simultaneous autoregressive models using all predictors (SAR), the reduced SAR model with the lowest AIC score (SARreduced), and Akaike weights based on SAR models. Blank cells indicate variables that were not included in the reduced model. Figures in text are based on the full SAR models, which were most consistently successful at removing residual spatial autocorrelation. The effect of the change in MAT between LGM and present (Anomaly) and topographic heterogeneity (TH) were tested by replacing velocity in all models with each of these variables. *P < 0.05, **P < 0.01, ***P < 0.001.
r2 Amphibians GVI MAT MAP TS PS Extent Velocity Anomaly TH Mammals GVI MAT MAP TS PS Extent Velocity Anomaly TH Birds GVI MAT MAP TS PS Extent Velocity Anomaly TH 0.227*** 0.002 0.090*** 0.068*** 0.073*** 0.415*** 0.283*** 0.082*** 0.134*** 0.253*** 0.158*** 0.203*** 0.313*** 0.039** 0.538*** 0.328*** 0.187*** 0.023* 0.297*** 0.102*** 0.137*** 0.348*** 0.036** 0.446*** 0.385*** 0.222*** 0.015 OLS 0.185** 0.010 0.125 0.018 0.233*** 0.439*** 0.252*** 0.203** 0.230** 0.169*** 0.228** 0.215** 0.180 0.194*** 0.490*** 0.199*** 0.155** 0.092 0.237*** 0.065 0.317*** 0.500*** 0.183*** 0.369*** 0.194*** 0.148*** 0.074 SAR 0.196** 0.008 0.115 0.001 0.244*** 0.414*** 0.288*** 0.213** 0.260*** 0.160*** 0.216* 0.190* 0.182 0.173*** 0.467*** 0.216*** 0.158** 0.106* 0.212*** 0.021 0.303*** 0.549*** 0.120* 0.323*** 0.183*** 0.121* 0.086* SARreduced 0.198** 0.11 0.241*** 0.411*** 0.289*** 0.207*** 0.285*** 0.160*** 0.216* 0.190* 0.182 0.173*** 0.467*** 0.216*** 0.158** 0.106* 0.213*** 0.302*** 0.565*** 0.114* 0.320*** 0.180*** 0.119* 0.081* WeightAIC 0.989 0.294 0.460 0.312 0.997 1.000 1.000 0.979 0.998 0.993 0.882 0.736 0.597 0.929 1.000 0.998 0.975 0.646 1.000 0.284 0.999 1.000 0.838 1.000 0.989 0.733 0.726
variation in range size within assemblages, reduced richness of species with range size below the median, and weaker, inconsistent, and sometimes positive relationships for richness of species with larger range sizes (figs. S1 to S6 and tables S1 to S6 and S9). High velocities have been proposed to be associated with incomplete range filling and species extinctions (10, 17, 22). Although we found no evidence for reduced range sizes with higher velocities, the decline in endemism and inferred importance of dispersal ability are consistent with the extinctions hypothesis. However, it may be that velocity per se is not driving endemism but simply correlates with other variables that do have a direct mechanistic link. Alternatively, a direct mechanistic link between velocity and endemism may not require species extinctions. We considered several such alternative hypotheses and show that none are consistent with the data. VOL 334 SCIENCE
High-velocity areas may coincide with those where analogous climate conditions have most expanded since the LGM; low endemism in these areas may occur because species in such areas have expanded their range size accordingly. Climate expansion since the LGM was highly correlated with the extent of modern analogous conditions (r = 0.794) but was a weaker predictor of endemism in all cases (compare Table 1 with table S12). Furthermore, the range expansion hypothesis predicts that the groups with strongest dispersal ability (birds) should have expanded their ranges most and therefore should show the strongest relationships to velocity, which is contrary to our results. It is also possible that velocity appears to be an important predictor, not because of a mechanistic link but only because it is derived from another variable that does have a direct link. However, climate-change velocity was a better predictor than either of its components
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Fig. 3. The global pattern of within-region fit of climate-change velocity to patterns of endemism. (A) Within each 10 10 region, circle size indicates the number of species groups for which velocity was significantly (P < 0.05) associated with endemism. The color of the circle corresponds to the mean correlation (topographic heterogeneity and macroclimate anomaly) and was the only one with consistent predictive power across scales (Table 1), which suggests that topographic heterogeneity and anomaly both contain important and complementary information. High endemism and low velocity occur not only in mountains but also in macroclimatically stable, relatively flat regions (such as portions of the Amazon basin and central Africa). At the same time, high-velocity mountain areas harbor low endemism (such as the Altai mountains of northern Mongolia and some mountains in the western United States). Does a direct role of velocity depend on extinctions, and are alternatives consistent with the data? Dynesius and Jansson (15) proposed three interrelated mechanisms linking climate stability and patterns of richness and range size: (i) Instability may select for increased dispersal ability and generalism, leading to large ranges; (ii) gradual speciation rates may be reduced in unstable areas, producing a lack of young, small-ranged species; and (iii) small-ranged species may have gone extinct in unstable areas. Underlying all of these hypotheses is the process of lineage extinction across a range of evolutionary scales, from selection acting on within-species lineages to the extinction of newly diverging or full species (31). In principal, reduced speciation rates [explana-
coefficient across all groups present within that region. (B to D) Globally, strong within-region correlations between velocity and endemism were associated with high precipitation (amphibians and mammals) and low temperature seasonality (birds). Envelopes around each line show the 90% confidence interval. Taken together, these results indicate that past climate changes have left important legacies in contemporary range size and species richness patterns, supplemented by the influences of modern climate and its spatial pattern. Small-ranged species constitute most of Earths species diversity (23); our findings show that these species, especially those from less vagile groups, are sensitive to climate movements and are concentrated in areas where possibilities for tracking past climate changes have been greatest. This conclusion also suggests that small-ranged, weakly dispersing species in previously stable regions experiencing high future climate-change velocities will be at greatest extinction risk from anthropogenic climate change.
1. C. Parmesan, G. Yohe, Nature 421, 37 (2003). 2. R. K. Colwell, G. Brehm, C. L. Cardels, A. C. Gilman, J. T. Longino, Science 322, 258 (2008). 3. J. Lenoir, J. C. Ggout, P. A. Marquet, P. de Ruffray, H. Brisse, Science 320, 1768 (2008). 4. C. Moritz et al., Science 322, 261 (2008). 5. B. Huntley, T. Webb III, J. Biogeogr. 16, 5 (1989). 6. P. K. Schoonmaker, D. R. Foster, Bot. Rev. 57, 204 (1991). 7. M. S. McGlone, Global Ecol. Biogeogr. Lett. 5, 309 (1996). 8. M. B. Davis, R. G. Shaw, Science 292, 673 (2001). 9. M. B. Arajo, R. G. Pearson, Ecography 28, 693 (2005). 10. J.-C. Svenning, F. Skov, Ecol. Lett. 7, 565 (2004). 11. J.-C. Svenning, F. Skov, Glob. Ecol. Biogeogr. 16, 234 (2007).
tion (ii)] might instead be due to high rates of gene flow among populations in unstable, shifting climates. However, this mixing should be most pronounced for strongly dispersing species. In contrast, lineage extinctions at all levels should have the strongest effects on weak dispersers, which is consistent with our results. Thus, elevated extinction rateslikely across a range of evolutionary scales (31)appear to best explain the association of low endemism with high velocity (17, 22). Our results have important implications for conservation in a world that is increasingly experiencing elevated climate-change velocities (18). Areas that have experienced high velocities in the past are on average also expected to experience high velocities over the next century (Fig. 1E and fig. S7). As we have shown, these areas are already missing small-ranged species, suggesting that most of the remaining species may cope well with future changes. However, there are important mismatches between the spatial patterns of past and future climate change; areas with low velocities in the past, high concentrations of endemic species, and high velocities into the future are a particular conservation concern. These areas include western Amazonia, where concentrations of endemic species that have experienced relatively low-velocity changes in the past may be faced with rapid climate shifts in the near future. SCIENCE VOL 334
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12. J.-C. Svenning, F. Skov, Ecol. Lett. 10, 453 (2007). 13. M. B. Arajo et al., Ecography 31, 8 (2008). 14. J.-C. Svenning, S. Normand, F. Skov, Ecography 31, 316 (2008). 15. M. Dynesius, R. Jansson, Proc. Natl. Acad. Sci. U.S.A. 97, 9115 (2000). 16. R. Jansson, Proc. Biol. Sci. 270, 583 (2003). 17. J.-C. Svenning, Ecol. Lett. 6, 646 (2003). 18. S. R. Loarie et al., Nature 462, 1052 (2009). 19. R. Jansson, T. J. Davies, Ecol. Lett. 11, 173 (2008). 20. T. J. Davies, A. Purvis, J. L. Gittleman, Am. Nat. 174, 297 (2009). 21. D. Scherrer, C. Krner, J. Biogeogr. 38, 406 (2010). 22. D. Nogus-Bravo, R. Ohlemller, P. Batra, M. B. Arajo, Evolution 64, 2442 (2010). 23. K. J. Gaston, The Structure and Dynamics of Geographic Ranges (Oxford Univ. Press, New York, 2003). 24. R. Ohlemller et al., Biol. Lett. 4, 568 (2008). 25. G. C. Stevens, Am. Nat. 133, 240 (1989). 26. This measure does not account for abrupt or transient changes within the time interval. Over periods of decades or centuries, relatively rapid changes may produce velocities considerably higher than those obtained by using just the LGM and present. 27. Materials and methods are available as supporting material on Science Online. 28. W. F. Ruddiman, Earths Climate: Past and Future (W.H. Freeman and Company, New York, 2001). 29. D. J. Currie et al., Ecol. Lett. 7, 1121 (2004). 30. C. K. Ghalambor, R. B. Huey, P. R. Martin, J. J. Tewksbury, G. Wang, Integr. Comp. Biol. 46, 5 (2006). 31. A. C. Carnaval, M. J. Hickerson, C. F. B. Haddad, M. T. Rodrigues, C. Moritz, Science 323, 785 (2009). Acknowledgments: We thank the Aarhus University Research Foundation for financial support. This study was also supported in part by MADALGOCenter for Massive Data Algorithmics, a Center of the Danish National Research Foundation. B.D. is supported by the Danish Council for Independent ResearchNatural Sciences, and W.J.S. is funded by Arcadia. We thank international climate modeling groups for providing their data for analysis, the Laboratoire des Sciences du Climat et de lEnvironnement for collecting and archiving the paleoclimate model data, the International Union for Conservation of Nature and Natural Resources for making the amphibian and mammal range data available, and the Natural Environment Research Councilfunded Avian Diversity Hotspots Consortium (NER/O/S/2001/01230) for the use of the bird range data. We thank four anonymous reviewers whose constructive comments improved this manuscript. Data are archived at Dryad (http://dx.doi.org/ 10.5061/dryad.b13j1).

www.sciencemag.org/cgi/content/full/science.1210173/DC1 Materials and Methods Figs. S1 to S12 Tables S1 to S12 References (3254) 22 June 2011; accepted 19 August 2011 Published online 6 October 2011; 10.1126/science.1210173
Long-Term Change in the Nitrogen Cycle of Tropical Forests

Peter Hietz,1* Benjamin L. Turner,2 Wolfgang Wanek,3 Andreas Richter,4 Charles A. Nock,5 S. Joseph Wright2 Deposition of reactive nitrogen (N) from human activities has large effects on temperate forests where low natural N availability limits productivity but is not known to affect tropical forests where natural N availability is often much greater. Leaf N and the ratio of N isotopes (d15N) increased substantially in a moist forest in Panama between ~1968 and 2007, as did tree-ring d15N in a dry forest in Thailand over the past century. A decade of fertilization of a nearby Panamanian forest with N caused similar increases in leaf N and d15N. Therefore, our results indicate regional increases in N availability due to anthropogenic N deposition. Atmospheric nitrogen dioxide measurements and increased emissions of anthropogenic reactive N over tropical land areas suggest that these changes are widespread in tropical forests. nthropogenic N fixation has approximately doubled atmospheric deposition of reactive N in terrestrial ecosystems globally, with regional variation resulting from differences in the intensity of agriculture, the burning of fossil fuels, and biomass burning (1). Many temperate and boreal ecosystems are N limited; in these regions, atmospheric N deposition has caused the acidification of soils and waters, loss of soil cations, a switch from N to P limitation, a decline in the diversity of plant communities adapted to low N availability, and increases in carbon uptake and storage (2, 3). Natural N availability is much greater in many tropical forests than in most temperate forests due to high rates of N fixation by heterotrophic soil microbes
1 Institute of Botany, University of Natural Resources and Life Sciences, Gregor Mendel-Strae 33, 1180 Vienna, Austria. 2 Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancn, Republic of Panama. 3Department of Chemical Ecology and Ecosystem Research, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. 4Institute of Environmental Physics, University of Bremen, Otto-Hahn-Allee 1, D-28359 Bremen, Germany. 5Centre d tude de la Fort, Dpartement des Sciences Biologiques, Universit du Qubec Montral, Post Office Box 8888 Centre-ville Station, H3C 3P8 Montreal, Canada.
*To whom correspondence should be addressed. E-mail: peter.hietz@boku.ac.at
and rhizobia associated with legumes, which are abundant in many tropical forests (4). Nitrogen deposition is increasing in the tropics, and this region may see the most dramatic increases in the coming decades (1). It has been hypothesized that this will acidify soils, deplete soil nutrients, reduce tree growth and carbon storage, and negatively affect biodiversity in tropical forests (5, 6). Yet despite extensive speculation, there remains no direct evidence for changes in the N cycle in tropical forests. The ratio of stable N isotopes (d15N) reflects the nature of the N cycle in ecosystems, with higher values indicating greater N availability and a more open N cycle (7, 8). In temperate ecosystems where N deposition is low, leaf N concentrations and the d15N of leaves and wood decreased during the 20th century, indicating progressive N limitation in response to changes in land use (9) and increasing atmospheric CO2 concentrations (10). In contrast, wood d15N values have increased in temperate forests with high rates of N deposition or a history of recent disturbance, suggesting more open N cycles under such conditions (11, 12). We compared leaves from herbarium specimens (158 species) collected ~40 years ago (~1968) from a tropical moist forest on Barro VOL 334 SCIENCE
Colorado Island (BCI), Republic of Panama, with sun and shade leaves (340 species) collected in 2007. Over four decades, leaf d15N increases averaged 1.4 T 0.16 per mil () (SEM) and 2.6 T 0.1 when comparing 1960s leaves to conspecific 2007 shade and sun leaves, respectively. Based on their leaf mass per area, 1960s leaves included a mixture of both sun and shade leaves (13). The increase in leaf d15N occurred in both legumes (Fabaceae) and nonlegumes (Fig. 1, A and B). Foliar N concentrations in nonlegumes increased by 7.7 T 1.9% and 15.2 T 2.5% when comparing 1960s leaves to 2007 sun and shade leaves, respectively (Fig. 1C). Legumes had substantially greater foliar N concentrations than nonlegumes, and there was no overall change in their foliar N concentration between the 1960s and 2007 (Fig. 1D). To assess whether the changes detected on BCI are representative of tropical forests more broadly, we determined d15N in tree rings from three nonleguminous tree species in the Huai Kha Khaeng Reserve, a remote monsoon forest near the Thailand-Myanmar border. Significant increases in d15N during the past century were detected in all three species (Fig. 2). Similar changes were reported previously for tree rings in two Amazonian rainforest tree species (14). A forest N addition experiment conducted 1 km from BCI provides perspective on the changes in foliar N composition (15). Foliar d15N increased by 0.3 to 1.5 in four tree species and by ~0.5 to 1.2 in fine litter (15), and the N concentration in litterfall increased by 7% (16) after 8 to 9 years of fertilization with 125 kg N ha1year1. The observed increase in leaf d15N did not reflect the signal of the N fertilizer, which had a lower d15N (2.2) than leaves of nonfertilized trees in control plots (15) and therefore should have resulted in a decline rather than an increase in foliar d15N. Nitrogen fertilization also increased NO3 leaching (from 0.01 to 0.93 mg N liter1), NO flux (from 70 to 196 mg N m2 day1), and N2O flux (from 448 to 1498 mg N m2 day1) (15), confirming that the increase in leaf d15N after N fertilization was associated with a more
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open N cycle. Similar increased N losses and higher leaf d15N have been observed in temperate forests after N fertilization (17). Thus, the increases in foliar N and d15N between the 1960s and 2007 indicate a regional shift to a more open N cycle. The forests in Panama and Thailand are oldgrowth forests that have been intensively studied as part of a global network of long-term studies in very large forest plots (www.ctfs.si.edu). Using this information, we tested whether trends other than increased N deposition might plausibly explain the changes in N concentrations and d15N observed. On BCI, nothing in the forest structure or documented changes since 1982 suggest that it Fig. 1. Leaf d15N and N concentration in leaves collected ~1968 and 2007 for a tropical moist forest on Barro Colorado Island, Panama. (A and B) Foliar d15N and (C and D) foliar N concentration in leaves of nonlegumes [(A) and (C)] and legumes [(B) and (D)]. Probability (p) values (two-tailed paired Wilcoxon signed rank tests) and degrees of freedom (DF) are for comparisons between leaves collected ~1968 and conspecific sun or shade leaves collected in 2007. Leaf mass per area, which varies with light availability, suggests that herbarium leaves included a mixture of sun and shade leaves. is in a stage of natural breakdown or recovery. There were no significant changes in aboveground biomass, fast- or slow-growing trees, high or low wood density species, large or small seeded species, or large or understory trees (18). The most important events resulting in above-average forest disturbance were El Niorelated droughts, which occur approximately every 5 to 10 years and increase tree mortality. At the Thai site, major damage by fire is a rare but recurring event with a strong and widespread disturbance in the mid-1800s and, since then, several strong but localized disturbances of variable intensity in the 1910s, 1940s, and 1960s (19). Fire will result in a short peak in N availability, but the documented disturbance events cannot have resulted in the change in N availability we observe mainly in the second half of the 20th century. Precipitation can affect plant d15N (20) but did not change during recent decades on BCI or at the closest weather station to the Thai forest (fig. S2). Nitrogenfixing plants have a somewhat different d15N than nonN-fixing plants (as seen in the 0.5 to 1 lower d15N in legumes compared with nonlegumes), so a reduction in N fixation by legumes might result in an increase in ecosystem d15N. In this case, the difference in leaf d15N between legumes and nonlegumes should have decreased and leaf N concentrations decreased rather than increased between 1968 and 2007. Also, the proportion of legume trees did not change on BCI (7.25 to 7.52% of stems between 1982 and 2005, data available at www.ctfs.si.edu) and is low at Huai Kha Khaeng (3.17 and 3.05% in 1992 and 1999, respectively). The N deposited on BCI is probably from local sources as seen in regionally high tropospheric NO2 concentrations (fig. S3). Local sources are the increasing shipping traffic through the Panama Canal (fig. S4), which passes BCI, and Panama City, which is 40 km from BCI. The dry forest site in Southeast Asia is located in an area of moderate tropospheric NO2 concentrations similar to those above Panama, although concentrations are much higher nearby (fig. S5). Our results from Panama and Thailand are likely to apply broadly to tropical forests worldwide, because the largest tropospheric NO2 concentrations observed over our study sites (>1015 molecules cm2) are recorded over 21, 26, and 12% of tropical Africa, Asia, and America, respectively (fig. S6). Also, atmospheric N deposition recorded <1 km from BCI (9 kg ha1 year1) (15) is similar to model estimates (21, 22) and measurements (4) of N deposition in other tropical regions, and N emissions increased in large parts of the tropics (Fig. 3). Nitrogen deposition over tropical land area increased during the past decades and is projected to increase even further (1). This might alter the relative competitive ability of Fabaceae, many of which fix atmospheric N and are naturally N-rich (Fig. 1), leading to shifts in tree species composition. Where N deposition results in increased foliar N, as seen on BCI, the consequence should be increased photosynthetic carbon gain, at least on a leaf-area basis, because foliar N concentration scales with photosynthetic capacity (23). This is important for ecosystem models of tropical forests, where the consequences of N deposition are less well understood than for temperate forests (24). However, N deposition can also result in soil acidification and altered availability of other nutrients (5, 15), with a potentially negative effect on plant growth. Thus, regional differences in the deposition of N and possibly of other nutrients might contribute to the observed changes in tropical forests (18, 25, 26) and help to explain regional differences in forest response.
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Fig. 3. Anthropogenic N emissions in 2005 and 1970. NOx and NH3 emissions per 0.1 grid cell were obtained from European CommissionJoint Research Centre/ Netherlands Environmental Assessment Agency, EDGAR version 4.1, (http://edgar.jrc.ec.europa.eu/) 2010, and were converted to N emissions per surface area.
1. J. N. Galloway et al., Biogeochemistry 70, 153 (2004). 2. P. M. Vitousek et al., Ecol. Appl. 7, 737 (1997). 3. M. J. Wassen, H. O. Venterink, E. D. Lapshina, F. Tanneberger, Nature 437, 547 (2005). 4. L. O. Hedin, E. N. J. Brookshire, D. N. L. Menge, A. R. Barron, Annu. Rev. Ecol. Evol. Syst. 40, 613 (2009). 5. P. A. Matson, W. H. McDowell, A. R. Townsend, P. M. Vitousek, Biogeochemistry 46, 67 (1999). 6. G. K. Phoenix et al., Glob. Change Biol. 12, 470 (2006). 7. P. Hgberg, New Phytol. 137, 179 (1997). 8. L. Pardo et al., Biogeochemistry 80, 143 (2006). 9. K. K. McLauchlan, J. M. Craine, W. W. Oswald, P. R. Leavitt, G. E. Likens, Proc. Natl. Acad. Sci. U.S.A. 104, 7466 (2007). 10. K. K. McLauchlan, C. J. Ferguson, I. E. Wilson, T. W. Ocheltree, J. M. Craine, New Phytol. 187, 1135 (2010). 11. S. Elhani, J. M. Guehl, C. Nys, J. F. Picard, J. L. Dupouey, Tree Physiol. 25, 1437 (2005).
12. A. R. Bukata, T. K. Kyser, Environ. Sci. Technol. 39, 7777 (2005). 13. See supporting material on Science Online. 14. P. Hietz, O. Dnisch, W. Wanek, Environ. Sci. Technol. 44, 1191 (2010). 15. M. D. Corre, E. Veldkamp, J. Arnold, S. J. Wright, Ecology 91, 1715 (2010). 16. M. Kaspari et al., Ecol. Lett. 11, 35 (2008). 17. P. Hgberg, C. Johannisson, Plant Soil 157, 147 (1993). 18. J. Chave et al., PLoS Biol. 6, e45 (2008). 19. P. J. Baker, S. Bunyavejchewin, C. D. Oliver, P. S. Ashton, Ecol. Monogr. 75, 317 (2005). 20. B. Z. Houlton, D. M. Sigman, E. A. G. Schuur, L. O. Hedin, Proc. Natl. Acad. Sci. U.S.A. 104, 8902 (2007). 21. J. N. Galloway et al., Science 320, 889 (2008). 22. Y. Chen et al., Glob. Change Biol. 16, 2024 (2010). 23. I. J. Wright et al., Nature 428, 821 (2004). 24. G. Asner et al., Biogeochemistry 54, 1 (2001). 25. S. Lewis, J. Lloyd, S. Sitch, E. T. A. Mitchard, W. F. Laurance, Annu. Rev. Ecol. Evol. Syst. 40, 529 (2009). 26. S. J. Wright, Ann. N. Y. Acad. Sci. 1195, 1 (2010).
Acknowledgments: We thank D. Agudo, T. Romero, U. Hietz-Seifert, and M. Watzka for laboratory support and the F. H. Levinson Fund and the Austrian Science Fund (grant P19507-B17) for funding. P.H. took herbarium and wood samples and analyzed the leaf, wood, and N emission data; S.J.W. provided 2007 leaves; B.L.T. analyzed 2007 leaves; W.W. analyzed herbarium leaves and wood; C.A.N. synchronized wood cores; A.R. analyzed satellite data; and P.H., B.L.T., and S.J.W. wrote the manuscript. All authors contributed to the discussion of the data. Leaf and wood data are available from P.H.; NO2 satellite data are available from A.R.

www.sciencemag.org/cgi/content/full/334/6056/664/DC1 Materials and Methods Figs. S1 to S6 Table S1 References (2733) 1 August 2011; accepted 22 September 2011 10.1126/science.1211979
Neural Mechanisms for the Coordination of Duet Singing in Wrens

Eric S. Fortune,1,2* Carlos Rodrguez,2 David Li,1 Gregory F. Ball,1 Melissa J. Coleman3 Plain-tailed wrens (Pheugopedius euophrys) cooperate to produce a duet song in which males and females rapidly alternate singing syllables. We examined how sensory information from each wren is used to coordinate singing between individuals for the production of this cooperative behavior. Previous findings in nonduetting songbird species suggest that premotor circuits should encode each birds own contribution to the duet. In contrast, we find that both male and female wrens encode the combined cooperative output of the pair of birds. Further, behavior and neurophysiology show that both sexes coordinate the timing of their singing based on feedback from the partner and suggest that females may lead the duet.
from sensory cues produced by the partner or partners. We examined how sensory information from these two sources, autogenous and heterogenous respectively, is integrated in cortical (i.e., pallial) circuits. We used a model system, plaintailed wrens (Pheugopedius euophrys) (7), a species of neotropical birds that sing duets in which females and males rapidly alternate syllable production, sounding as if a single bird sang it (see movies S1 and S2) (8, 9).
1 Department of Psychological and Brain Sciences, Department of Neuroscience, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. 2Museo de Zoologa, Pontificia Universidad Catlica del Ecuador, Edificio de Ciencias Biolgicas, 12 de Octubre 1076 y Roca, Quito, Ecuador. 3W. M. Keck Science Department of Claremont McKenna College, Pitzer College, and Scripps College, 925 North Mills Avenue, Claremont, CA 91711, USA.
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ooperative behaviors are found across taxa and can be critical for survival and reproduction (16). To achieve cooperative
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Fig. 1. Changes in singing due to cooperative context. (A) Differences in the timing of syllable production by a female when singing in a duet (top, spectrogram) versus alone (bottom, spectrogram). Spectrograms show power (color: black, lowest, and light yellow, highest amplitudes) over the behaviorally relevant frequency range (ordinate) plotted as a function of time (seconds, abscissa). The syllables are labeled on the bottom of each spectrogram, with capital letters and magenta bars indicating the females syllables and lowercase letters and blue bars the males syllables. At the top of each spectrogram are colored bars (green, orange, yellow) that indicate the intervals between the peak frequencies of female syllables. The dotted lines and white arrows indicate the changes in timing that occur in solitary song. (B) Same presentation as (A), but a duet song with solitary syllables from the male. In this example, the bars at the top indicate the timings of the male syllables. Inset oscillogram shows the change in amplitudes from male solitary singing to subsequent duetting; male syllables are marked in blue. (C) Timing changes were also seen during bouts of duetting when the male failed to produce its What are the mechanisms that plain-tailed wrens use to cooperate for the production of their complex, learned duet songs? At one extreme, it is possible that the wrens each sing their sequence of syllables as a fixed action pattern (1014), following a common cue that initiates singing in both individuals. Alternatively, the wrens could respond on a syllable-by-syllable basis to the singing of the partner throughout the duration of the duet (15). These mechanisms will be reflected in both the behavioral performances of the wrens and in the neurophysiological activity that mediates the behavior. To assess the behavioral parameters that the wrens use for duetting, we monitored singing behavior of a population of plain-tailed wrens on the slopes of the Antisana volcano in Ecuador at the Yanayacu Biological Station and Center for Creative Studies (00.36S, 77.53W, altitude 2700 m) (fig. S1) between October 2009 and January 2011. We examined more than 1000 wren vocalizations captured in over 150 hours of acoustic recordings (see Materials and Methods) that were made in the Chusquea bamboo thickets where the wrens maintain territories. Duet singing is likely used in territorial defense (9, 15), although a complete description of the functional roles of duetting has not been achieved. We observed that wrens commonly produced duet songs, but both females and males also sang
syllables during a motif of duet song. Spectrogram (top) has the same format used in (A); the corresponding oscillogram below indicates female syllables in magenta and male syllables in blue. Audio files are available in the supporting online materials. spectrogram. Green bars indicate the (A) to (B) transition, orange (B) to (D), and yellow (D) to (A). The within-motif intervals, indicated by the green and orange bars, were either identical or longer in duration in the solitary song. In Fig. 1B, the mean intersyllable intervals between male syllables during duets are indicated with green bars, (b) to (d), and yellow bars (d) to (b). As in females, within-motif intervals, indicated by the green bars, are longer during solitary singing. These results suggest that heterogenous acoustic cues modulate the motor program for singing on a syllable-by-syllable basis (15) and that these sensory cues affect at least the duration and variability of intersyllable intervals. These behavioral data therefore also suggest that the nervous system is not using a fixed-action pattern to generate duet song but relies on a unique combination of sensory feedback from both autogenous and heterogenous sources. We also found more variability in male singing than in female singing. We commonly recorded solitary songs produced by females but infrequently recorded those by males. Solitary males produced low-amplitude songs, and therefore, the field recordings may have failed to capture many male songs because of lower amplitudes. The amplitude of male syllables, however, increased significantly by about 14.5 dB
alone. In general, the acoustic structure and sequence of syllables produced by each individual were identical both in duet and solitary singing (Fig. 1, A and B). Solitary songs are easily recognized by long intersyllable intervals (range 0.34 s to 1.6 s) that occur when the partner would normally sing its syllables during a duet. The presence of these intersyllable intervals in solitary songs suggests that the motor pattern generator for singing in the brain includes the appropriate timing of syllable production during the duet. Nevertheless, when either female or male wrens sing alone, they increase the durations of intersyllable intervals within each motif. These within-motif increases were on the order of tens of milliseconds (mode increase in duration from duet to solitary = 58 ms, n = 115 song samples; female intersyllable interval duration during a duet = 489 T 69 ms and alone = 524 T 99 ms; males during duet = 763 T 23 ms and alone 885 T 586 ms; mean T standard deviation). Also, the durations of intersyllable intervals were significantly more variable in solitary songs than in duet songs (F test, P < 0.05, df = 66). These changes can be seen in the examples of female and male wren singing shown in Fig. 1, A and B. In Fig. 1A, the mean intersyllable intervals between the female syllables sung during its duets are indicated by the colored bars at the top of each SCIENCE VOL 334
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from solitary to duet singing (Fig. 1B, inset) [t test, P < 0.01, df = 20; solitary syllable amplitude 17.4 T 11.7 (arbitrary units) and duet amplitude 534.75 T 52.85]. In contrast, females did not change syllable amplitudes [amplitude increase 0.13 dB, t test, P = 0.86, df = 20; solitary amplitude 565.9 T 203.3 (arbitrary units) and duet amplitude 582.1 T 219.1]. During duets, male syllable amplitudes were not significantly different from female syllables (t test, P = 0.46, df = 20), as can be seen in oscillograms in Figs. 1C and 2. Male variability was also evident in failures of the male wren to produce its syllables within motifs in the middle of longer duet sequences (Fig. 1C). During these omissions, females continued singing and lengthened intersyllable intervals (mode increase 61 ms, n = 44 song samples). These rapid modulations of singing in the middle of duets provide additional evidence that the birds are not relying on fixed-action patterns in the brain to generate duet song. Also, the sex differences observed in singing indicate that females and males share similar, but not identical, mechanisms for cooperative production of duets. Further, these data suggest an interesting hypothesis, that female plain-tailed wrens may provide the leading cues for duet singing (16, 17). Neurophysiological evidence may support this idea (below), but future experiments with manipulations of acoustic cues in playbacks are needed (17). Nevertheless, these data demonstrate moment-tomoment coordination of feedback arising from both partners during duet singing in plain-tailed wrens. To examine how cooperative duet singing is encoded in cortical circuits, we captured birds and performed neurophysiological experiments. We made extracellular recordings in HVC, a song control nucleus (18, 19), for up to 30 hours each in three female and three male wrens. Before capture, we recorded duets from each individual used in neurophysiological experiments, as HVC activity has been shown to be selective for the acoustic parameters of autogenous song elements (2024). We isolated 75 units from 42 recording sites (see Materials and Methods for details). Stimuli included duets and a series of manipulations of duet motifs, including isolated autogenous and heterogenous syllables from the duet, time reversals of the entire duet and subsets of syllables, a duet in which the syllables were presented in reverse order, and other ad hoc temporal manipulations. The majority of units in both females and males responded best to the duet song over all stimuli tested [85 and 87% for Z score and response strength measures (25), respectively, n = 61] (Fig. 2). The mean Z score for duets in females was 2.3 T 0.83 (range 0.8 to 3.9, n = 21) and in males 1.3 T 0.64 (range 0.2 to 3.0, n = 40). The distribution of response strengths (range 0.9 to 15.1 spikes/s) to duet songs for both females and males can be seen in Fig. 3A. The responses to duet stimuli were not simply a sum of responses to female and male syllables, because, in the majority of neurons, response strengths elicited by duet stimuli (7.9 T 6.4 spikes/s in females, n = 21; 6.1 T 3.7 in males, n = 40) were significantly greater (paired t tests: females, P < 0.01, n = 21; males, P < 0.01, n = 40) than the sum of the response strengths to female and male syllables presented alone (6.3 T 4.7 spikes/s in females, n = 21; and 4.4 T 2.8 males, n = 40) (Fig. 3, A and B). Further, we calculated d values (26) for each unit, comparing responses to duet stimuli with (i) the sum of the responses to the male and female syllables presented alone, (ii) the response to female syllables, and (iii) the responses to male syllables alone. The d values express the ability to discriminate between two stimuli, and in the comparisons shown here, d values greater than 0.5 indicate the selectivity of a neuron for the duet song over the other stimulus (26). Data for each unit in Fig. 3A are plotted with a symbol that represents the d measures that are greater than 0.5 for multiple comparisons. Most units (36 out of 61) responded selectively to the duet song when compared with the sum of the responses to the male and female syllables presented alone (filled circles), whereas only one unit did not respond preferentially to the duet song relative to each of the other stimuli (open circle). Eleven units preferred duet songs over male syllables only (horizontal bar), 3 units preferred duet over female syllables only (vertical bar), and 10 units preferred duet over both male and female syllables alone (cross) but not the sum (female + male). As female and male syllables were extracted unaltered from the duet, these stimuli had identical acoustic content, and therefore, it is the rapid alternation of female and male syllables in the duet that led to facilitated responses. In sum, both response strength and d measures indicate that a majority of HVC units exhibited facilitated responses to the combined duet performance, rather than responding best to each individuals own contribution to duets. We nevertheless found that both female and male syllables often elicited responses from HVC
Fig. 2. Example responses in HVC to song stimuli. Responses recorded in a female (A to D) and a male (E to H) wren. Bottom in each panel shows the stimulus oscillogram, top are raster plots showing the times of spikes for 20 stimulus repetitions, and middle is a histogram (50-ms bins) of the activity. Magenta indicates syllables produced by the female, blue by the male. Stimuli
were (A) and (E) duet song, (B) and (F) female syllables, (C) and (G) male syllables, and (D) and (H) reverse duet song. The female recording shown here had a strong preference for duet song over all other stimuli tested. The male recording shown here responded best to the duet song but, nonetheless, had a particularly strong response to the female syllables. SCIENCE www.sciencemag.org
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units in both female and male wrens. If these responses were related to the autogenous motor output, we would expect that female syllables would elicit stronger responses in female HVC than male syllables and vice versa for neurons in males. It is interesting that, in both females and males, the female syllables elicited significantly stronger responses than did the male syllables (Fig. 3) (paired t tests, females P < 0.01, n = 21 and males P < 0.01, n = 40). This is particularly surprising in males, because these responses are contrary to previous results obtained in several songbird species (22, 23, 27). Stronger responses to the conspecific partner in male wrens may be an adaptation for duet singing in these birds. The preference for female syllables in both female and male partners may be a neurophysiological correlate of the possible role of females in leading duet singing. That HVC neurons respond consistently to partner syllables is itself a surprising result, as previous studies showed that conspecific vocalizations generally elicit weak responses in HVC (20, 21). Indeed, such responses do not fit with current models of HVC function developed for nonduetting species, in which selective responses to autogenous stimuli in HVC are used to modulate motor programs (27, 28). The responses in both sexes to the partner syllables support behavioral observations that both sexes adhere to duet codes that are influenced by the partners previous syllables (15). The question remains, how are temporal interactions between females and males for the cooperative production of duets encoded? To examine this issue, we altered the timings between syllables. For example, we removed the intersyllable intervals in stimuli composed of only female or only male syllable sequences. Even though the syllables are identical in these stimuli, sequences with naturally occurring intersyllable intervals elicited significantly stronger responses than stimuli without these intervals (Fig. 4) ( paired t tests: females, P < 0.01, n = 21; males, P < 0.01, n = 40). This finding differs from previous results in which temporal combination sensitive HVC neurons in nonduetting species were not sensitive to durations of intersyllable intervals (29). This sensitivity to intersyllable intervals is likely another adaptation of the song circuit for the coordination of duet singing. The sensitivity to intersyllable intervals requires long-term, on the order of hundreds of milliseconds, integration of information. If these responses are produced by sensory information alone, a possible cellular strategy could use a combination of slow inhibition coupled with rapid excitation, as has been seen in counting neurons in amphibians (30). If these responses reflect underlying motor programs (10), it would suggest that sensory stimuli are activating and/or modulating a form of central pattern generator (CPG) (11, 13). Indeed, the activation of a CPG could explain the structure of solitary songs, in which appropriate syllable sequences and intervals are retained. The combination of mechanisms that we observed in plain-tailed wrens may be used to control cooperative behaviors across taxa. Consider, for example, two people cooperating to dance a tango. Certainly each person knows his or her own part of the dance and possibly the partners contribution, but these data suggest that premotor circuits in both individuals preferentially encode the combined cooperative behavior. Further,
Fig. 3. HVC neurons in both females and males exhibit facilitated responses to the duet song. (A) Response strength of 20 female units (magenta) and 40 male units (blue) to the duet song (ordinate) plotted against the sum of response strengths to the separate female and male components of the duet song (abscissa). Data above the identity line, in the yellow region, indicate superlinear responses to the duet than the sum of responses to female and male syllables presented alone. Filled circles () indicate that the d value (see text) for the comparison between the duet and the sum of the responses to the male and female syllables was > 0.5. Horizontal bar () indicates d values > 0.5 for the comparison between responses to the duet and male syllables and vertical bar ( ) for female syllables. A cross () indicates d >
0.5 for both female and male syllables, but not female + male. Open circles ( ) indicate d values of < 0.5 in all comparisons of the duet to other stimuli. (B) Responses for female and male units normalized to the strongest response from each unit. Duet songs elicited responses that were significantly stronger (paired t tests, P < 0.01; females, n = 21; males, n = 40) than responses to female, male, and summed (female + male) responses (the significant difference between duet and female + male responses are indicated by the green line labeled i). In addition, in both female and male wrens, the female syllables elicited significantly stronger responses (paired t tests, P < 0.01; females, n = 21; males, n = 40) than male syllables (indicated by the green line labeled ii). Bars indicate means, error bars indicate standard deviations.
Fig. 4. Neurons in HVC require appropriate intersyllable intervals. (A) Response strengths (spikes/s) to unaltered female syllables (ordinate) versus response strengths to the same syllables, but with the intersyllable intervals removed (abscissa). Blue dots are units recorded in males (n = 40) and magenta in females (n = 21). Data points in the yellow shaded region indicate that the unaltered syllable sequence elicited a greater response than the same www.sciencemag.org SCIENCE
syllables without intersyllable intervals. (B) Same as (A) but for male syllables. (C) Normalized responses as in Fig. 2B. Removal of intersyllable intervals from either female or male syllable sequences resulted in significantly reduced response strengths in both male and female units (green lines, paired t tests, P < 0.01; female syllables, green line i; male syllables, ii). Bars indicate means, error bars indicate standard deviations. VOL 334 4 NOVEMBER 2011
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information from the leader may be preferentially represented in the brains of both individuals. Finally, coordination of timing during cooperation is likely mediated by interactions between CPGs and both autogenous and heterogenous sensory information.
1. R. No, Anim. Behav. 71, 1 (2006). 2. M. P. Crawford, Comp. Psychol. Monogr. 14, 1 (1937). 3. N. S. Clayton, J. M. Dally, N. J. Emery, Philos. Trans. R. Soc. London B Biol. Sci. 362, 507 (2007). 4. F. Pron, L. Rat-Fischer, M. Lalot, L. Nagle, D. Bovet, Anim. Cogn. 14, 545 (2011). 5. D. J. Wheatcroft, T. D. Price, Trends Ecol. Evol. 23, 416 (2008). 6. D. J. Hoare, I. D. Couzin, J. G. J. Godin, J. Krause, Anim. Behav. 67, 155 (2004). 7. N. I. Mann, F. K. Barker, J. A. Graves, K. A. Dingess-Mann, P. J. Slater, Mol. Phylogenet. Evol. 40, 750 (2006). 8. N. I. Mann, K. A. Dingess, K. F. Barker, J. A. Graves, P. J. Slater, Behaviour 146, 1 (2009). 9. N. I. Mann, K. A. Dingess, P. J. Slater, Biol. Lett. 2, 1 (2006). 10. M. A. Long, D. Z. Jin, M. S. Fee, Nature 468, 394 (2010). 11. E. T. Vu, M. E. Mazurek, Y. C. Kuo, J. Neurosci. 14, 6924 (1994). 12. M. A. Long, M. S. Fee, Nature 456, 189 (2008). 13. D. Margoliash et al., Brain Behav. Evol. 44, 247 (1994). 14. N. Tinbergen, The Study of Instinct (Oxford Univ. Press, New York, 1951). 15. D. M. Logue, C. Chalmers, H. A. Gowland, Anim. Behav. 75, 1803 (2008). 16. D. M. Logue, Anim. Behav. 73, 105 (2007). 17. R. N. Levin, Anim. Behav. 52, 1107 (1996). 18. E. S. Fortune, D. Margoliash, J. Comp. Neurol. 360, 413 (1995). 19. F. Nottebohm, T. M. Stokes, C. M. Leonard, J. Comp. Neurol. 165, 457 (1976). 20. R. Mooney, J. Neurosci. 20, 5420 (2000). 21. D. Margoliash, J. Neurosci. 6, 1643 (1986). 22. D. Margoliash, J. Neurosci. 3, 1039 (1983). 23. R. Mooney, W. Hoese, S. Nowicki, Proc. Natl. Acad. Sci. U.S.A. 98, 12778 (2001). 24. T. A. Nick, M. Konishi, J. Neurobiol. 62, 469 (2005). 25. M. J. Coleman, R. Mooney, J. Neurosci. 24, 7251 (2004). 26. M. M. Solis, A. J. Doupe, J. Neurosci. 17, 6447 (1997). 27. J. F. Prather, S. Peters, S. Nowicki, R. Mooney, Nature 451, 305 (2008). 28. A. S. Dave, D. Margoliash, Science 290, 812 (2000). 29. D. Margoliash, E. S. Fortune, J. Neurosci. 12, 4309 (1992). 30. C. J. Edwards, T. B. Alder, G. J. Rose, Nat. Neurosci. 5, 934 (2002). Acknowledgments: This research would not have been possible without the help and support of S. Burneo, curator, Museum of Zoology (QCAZ), Pontificia Universidad Catlica del Ecuador in Quito. This project used equipment previously purchased with funds from NSF award IOS-0817918 to E.S.F. Support was also provided by the Johns Hopkins University and Claremont McKenna, Pitzer, Scripps Colleges. We thank H. Greeney, J. Simbaa, R. Jarrn, A. Saa, and K. Cisneros for logistical support; N. Cowan, J. Knierim, R. Krahe, and H. Zakon for helpful discussions and feedback; and D. Margoliash for his advice and insights. Behavioral and neurophysiological data are available upon request. Animal tissues are archived in the QCAZ in Quito, Ecuador, and are available in accordance with the relevant laws and regulations of the Republic of Ecuador.

www.sciencemag.org/cgi/content/full/334/6056/666/DC1 Materials and Methods Fig. S1 References Audio Clips S1 to S5 Movies S1 and S2 15 June 2011; accepted 6 September 2011 10.1126/science.1209867
Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling
Seung Chul Shin,1,3* Sung-Hee Kim,1 Hyejin You,1,2 Boram Kim,1,2 Aeri C. Kim,1,2 Kyung-Ah Lee,1 Joo-Heon Yoon,3 Ji-Hwan Ryu,3 Won-Jae Lee1 The symbiotic microbiota profoundly affect many aspects of host physiology; however, the molecular mechanisms underlying host-microbe cross-talk are largely unknown. Here, we show that the pyrroloquinoline quinonedependent alcohol dehydrogenase (PQQ-ADH) activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signaling (IIS) in Drosophila to regulate host homeostatic programs controlling developmental rate, body size, energy metabolism, and intestinal stem cell activity. Germ-free animals monoassociated with PQQ-ADH mutant bacteria displayed severe deregulation of developmental and metabolic homeostasis. Importantly, these defects were reversed by enhancing host IIS or by supplementing the diet with acetic acid, the metabolic product of PQQ-ADH. ll metazoans harbor substantial numbers of commensal microorganisms in the gut. It has been well established that commensal bacteria have positive impacts across a wide range of host physiology, including regulation of immunity and metabolism (13). Recent progress toward understanding gut-microbe interactions using Drosophila revealed that a fine-
1 School of Biological Science, Seoul National University and National Creative Research Initiative Center for Symbiosystem, Seoul 151-742, South Korea. 2Department of Bioinspired Science and Division of Life and Pharmaceutical Science, Ewha Womans University, Seoul 120-750, South Korea. 3Research Center for Human Natural Defense System, Yonsei University College of Medicine, CPO Box 8044 Seoul, South Korea.
*Present address: Korea Polar Research Institute, Incheon 406-840, South Korea. These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: lwj@snu.ac.kr
tuned regulation of gut immunity is required for the preservation of a healthy commensal community structure to promote host fitness and ensure normal host survival rates (4). Furthermore, the indigenous gut microbiota also controls the normal turnover rate of gut epithelial cells by regulating intestinal stem cell activity (5). Recently, it has been shown that the normal microflora is deeply involved in the energy balance and metabolic homeostasis of host animals (69). However, our current understanding of the impact of gut microbiota on host physiology is descriptive, due in part to technical difficulties associated with in-depth integrated genetic analysis of both the microbes and the host. To overcome these limitations, we used the combination of Drosophila and its commensal Acetobacter as a model of host-microbe interaction to enable us to perform a simultaneous genetic analysis of both host and microbe in vivo. VOL 334 SCIENCE
To observe the systemic effects of the symbiotic commensal community on the host, we first examined host growth rate and body size in the presence and absence of the commensal microflora by generating conventionally reared and germ-free animals (10). In conventionally reared fly larvae, the time to develop into a puparium was <7 days; it lengthened to ~9 days in germfree larvae when they fed on the axenic standard cornmeal diet (Fig. 1A). Interestingly, the effect of commensal bacteria on host development was more pronounced when the amount of yeast in the diet was reduced (Fig. 1A and fig. S1). Most notably, conventionally reared larvae developed into puparia in ~9 days, whereas germ-free larvae died at first instar if fed a diet containing <0.1% yeast or if yeast was substituted by casamino acids (Fig. 1A and fig. S1). Casamino acids were found to be essential nutrients for host growth in the absence of yeast. Under these conditions, germ-free larvae had a body size <10% of corresponding conventionally reared larvae 120 hours after egg laying (Fig. 1A and fig. S1). At this time point, the effect of the microbiota on host growth was most pronounced. These results indicate that commensal microbiota is able to influence the systemic development of Drosophila by affecting both growth rate and body size. All metazoan guts harbor complex commensal communities: hundreds of species are present in humans (11). In Drosophila, the adult midgut is typically in stable contact with a symbiotic commensal community composed of 5 to 20 different microbial species that consist primarily of members of the Acetobacter and Lactobacillus genera (1214). We found that the midgut of laboratory-reared Drosophila harbors five major commensal bacterial species, Commensalibacter intestini, Acetobacter pomorum, Gluconobacter morbifer, Lactobacillus plantarum, and Lactobacillus brevis (12, 15). Taking advantage of
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this simple commensal community, we examined whether the beneficial role of commensal microbiota is attributed to the combined effects of whole commensal community or whether it can be attributed to the activities of a subset of the microbiota. We exposed germ-free embryos fed on medium containing casamino acids instead of yeast to each of the five species of commensal bacteria and found that all were able to colonize the host gut independently. Colonization of A. pomorum alone in the germ-free gut was sufficient for restoring the developmental rate and body size of casamino acidfed larvae such that the developmental rate and body size of the host animals were comparable to those of conventionally reared larvae or germ-free larvae colonized by all five bacteria species (Fig. 1B). The other four bacteria species had a minor effect on larval growth and development (Fig. 1B). Acetic acid bacteria such as Asaia sp. colonize gut epithelial cells of mosquito (Anopheles sp.) through a specific association between the insect epithelial cells and extracellular polysaccharide matrix that surrounds bacterial cells (16, 17). Because our A. pomorum strain also produced large quantities of extracellular polysaccharides (when cultured in vitro, we observed a thick layer of extracellular polysaccharides), contact between Drosophila gut epithelial cells and A. pomorum may be similar to that observed between Asaia and Anopheles gut cells. To understand the beneficial role of A. pomorum on host developmental homeostasis at the molecular level, we established a draft genome sequence of A. pomorum with a whole-genome shotgun strategy and subsequently performed a transposon Tn5-mediated random mutagenesis to generate an A. pomorum mutant library. The draft genome sequence of A. pomorum consisting of 67 contigs in 19 scaffolds contains 2696 predicted genes with a total genome size of ~2.8 Mb. To screen for bacterial genes conferring benefits on the host, we measured growth rate and body size for each cohort of fly larvae colonized by each mutant (N ~ 3000 each) 120 hours after egg laying (fig. S2). We performed two replicates of the genome-wide screening. We found that 23 cohorts of mutant A. pomorum monoassociated larvae were consistently smaller compared with the wild-type (WT) A. pomorum monoassociated larvae. Analysis of the molecular lesions in the 23 mutant strains identified 14 genes, including 11 genes involved in the periplasmic pyrroloquinoline quinonedependent alcohol dehydrogenase (PQQ-ADH)dependent oxidative respiratory chain and three genes encoding proteins with other functions (figs. S3 and S4 and table S1). Larvae colonized with any of the 14 mutant strains showed developmental defects (Fig. 1C), although the bacteria colonized the larval gut as efficiently as WT A. pomorum (fig. S5). Given that the majority of the bacterial genes we identified as having a growth-promoting effect on their host are known to be involved in the PQQ-ADHdependent oxidative respiratory chain (fig. S3) and that five different mutations in the PQQ-ADH-I gene were independently identified (fig. S4 and table S1), we focused on the role of bacterial PQQ-ADH-I on host physiology. Among the five mutants, we subsequently used P3G5 for in-depth analyses. The P3G5 strain itself exhibited a similar in vitro growth rate to that of the parental WT A. pomorum and colonized gut epithelia as efficiently as WT A. pomorum (fig. S6). Under this condition, we observed that the development time to reach puparium formation extended to ~14 days in P3G5-monoassociated larvae compared with <10 days in WT A. pomorummonoassociated larvae (Fig. 2A). In low yeast medium, we observed similar results to those from larvae fed on casamino acids (Fig. 2A). Control experiments showed that the feeding rate between WT A. pomorum and P3G5-monoassociated larvae did not differ (fig. S7). In addition to a slower larval developmental rate, we found that P3G5-monoassociated adults were significantly
Fig. 1. Genome-wide screening of the Acetobacter genes essential for host growth. (A) The time to reach puparium formation of conventionally reared w 1118 larvae and germ-free w 1118 larvae fed a standard laboratory diet containing different yeast concentrations (2, 1, 0.5, 0.25, or 0.1%) or the casamino acid diet (casamino acid). The sizes of larvae at the 120th hour of development after egg laying are shown. Data were analyzed using an analysis of variance (ANOVA) followed by Tamhanes T2 post hoc test; values represent mean SEM (***P < 0.001). (B) A. pomorum is sufficient for full larval development. Germ-free embryos were associated with each of the five species of commensal bacteria or all five commensal bacteria (+A. pomorum/C. intestini/G. morbifer/L. plantarum/L. brevis). Germ-free embryos and conventionally reared embryos were also used as control. The time to reach puparium formation was measured in the casamino acid diet. The sizes of larvae at the 120th hour of development after egg laying are shown. Data were analyzed using an ANOVA followed by Tamhanes T2 post hoc test; values represent mean SEM (***P < 0.001). (C) All 14 screened mutant strains were defective in promoting host development. Germ-free embryos were colonized with each of the 14 mutant A. pomorum strains and the fly larvae fed the casamino acid diet. Mutated gene names and clone identification numbers are shown. Germ-free embryos associated with WT A. pomorum (wild-type) were used as the control. The time to reach puparium formation of WT A. pomorummonoassociated embryos was arbitrarily designated as 1, and the results are shown as relative time to puparium formation. Data were analyzed using the KruskalWallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (**P < 0.01, ***P < 0.001). In box-plot diagrams, black lines and boxes represent the median and first and third quartiles of the values; whiskers extend to minimum and maximum values. In an independent experiment, the time to reach puparium formation of each group of monoassociated larvae (n = ~20) was measured. www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011
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Fig. 3. Deregulation of developmental and metabolic homeostasis in P3G5monoassociated animals is rescued by enhancing host IIS. In all experiments, animals maintained in the casamino acid media were compared. The animals used in this study were: wild-type + Hs-GAL4, WT A. pomorummonoassociated control flies carrying Hs-GAL4 alone; P3G5 + Hs-GAL4, P3G5-monoassociated control flies carrying Hs-GAL4 alone; and P3G5 + Hs-GAL4>UAS-DILP2, P3G5-monoassociated flies carrying Hs-GAL4>UASDILP2. (A) P3G5-induced dFOXO nuclear localization is abolished upon ectopic DILP2 expression leading to cytoplasmic retention of dFOXO. Fat body tissues of the early third instar larvae were used. (B) Time to puparium formation and the sizes of the larvae at the 120th hour of development after egg laying. Data are analyzed using an ANOVA followed by Tamhanes T2
post hoc test; values represent mean SEM (***P < 0.001). (C) Adult body size. Body weights of female adults (5 days old) were measured. Data are analyzed using an ANOVA followed by Tamhanes T2 post hoc test; values represent mean SEM (***P < 0.001). (D) Wing area, cell number, and size. Wings of female adult flies (5 days old) were used in this study. Data were analyzed using Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (*P < 0.05, **P < 0.01, ***P < 0.001). (E) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (**P < 0.01). SCIENCE www.sciencemag.org
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Fig. 2. Commensal PQQ-ADH activity is required for diverse ranges of host homeostatic programs controlling developmental rate, body size, and metabolism. In all experiments, germ-free embryos monoassociated with the WT A. pomorum (+wild-type) and P3G5 mutant strain (+P3G5) maintained in the casamino acid media were compared. (A) Time to puparium formation and sizes of larvae 120 hours after egg laying. A standard cornmeal-agar medium containing 0.1% yeast was also used. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (B) Adult body sizes. Body weights of adult flies (5 days old) were measured. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (C) Wing area, cell number, and size. Female adult flies (5 days old) were used. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (D) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using the Mann-Whitney U test (*P < 0.05 and **P < 0.01).
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smaller than WT A. pomorummonoassociated adults (Fig. 2B). Consistent with smaller body size, P3G5-monoassociated adults had smaller wings, with reduced cell size and number, compared with those of control adults (Fig. 2C), and also small intestines (fig. S7). Furthermore, we found that PQQ-ADH activity of A. pomorum ensured the basal number of intestine stem cells (ISCs) and the epithelial cell renewal rate via induction of Unpaired-3 (Upd3) expression for Janus kinasesignal transducers and activators of transcription signaling activation (fig. S8). The overall body and tissue size reduction observed in the P3G5-monoassociated animals is reminiscent of animals with defective insulin/ insulin-like growth factor signaling (IIS) (1822). It is known that IIS mutant animals show diabetic phenotypes, including an increase in circulating sugars and stored lipid levels (20, 23, 24). When we examined the levels of sugars and lipids in P3G5-monoassociated larvae, we found elevated levels of total sugars [glucose and trehalose (the major disaccharide in insects)] and triacylglycerides (the main form of stored lipids) compared with those seen in control larvae (Fig. 2D). Consistent with the IIS mutant-like phenotype, we found that WT A. pomorum, but not P3G5, was able to induce IIS activation as evidenced by phosphoinositide 3-kinase (PI3K) activation and cytoplasmic retention of the forkhead transcription factor (dFOXO) (fig. S9). Furthermore, bacterial PQQ-ADH activity is required for full expression of Drosophila insulin-like peptides (DILPs) in the larval brain, suggesting that PQQ-ADH activity contributes to IIS activation in part through DILP induction (fig. S9). Importantly, we did not observe all the effects of WT A. pomorum on host developmental and metabolic homeostasis in the absence of host IIS activation (fig. S10), indicating that A. pomorum affects host physiological homeostasis through IIS activation. To further investigate whether metabolic and developmental defects found in a P3G5monoassociated animal were caused by low IIS pathway activity, we attempted to restore host homeostasis by enhancing IIS activity through ectopic overexpression of DILP2 (Hs-GAL4>UASDILP2) in P3G5-monoassociated animals. The results (Fig. 3A) show that dFOXO nuclear localization in the fat body, seen in the P3G5monoassociated control larvae (carrying the HsGAL4 driver alone), was abolished upon ectopic DILP2 expression (that is, P3G5-monoassociated Hs-GAL4>UAS-DILP2 larvae), leading to cytoplasmic retention of dFOXO. Importantly, all metabolic and developmental defects (Fig. 3, B to E), as well as ISC deregulation (fig. S11) caused by P3G5 bacteria were largely rescued in P3G5monoassociated Hs-GAL4>UAS-DILP2 animals. Interestingly, DILP overexpression could not rescue developmental defects found in germ-free larvae monoassociated with other nonA. pomorum commensal bacteria (fig. S12), indicating that the DILP effect is specific for P3G5-monoassociated animals. PQQ-ADH is the primary dehydrogenase in the ethanol oxidative respiratory chain of Acetobacter involved in acetic acid production (25). We found that all screened mutant bacteria including the P3G5 strain showed impaired or severely reduced acetic acid production (fig. S13),
Fig. 4. Deregulation of developmental and metabolic homeostasis in P3G5-monoassociated animals is rescued by acetic acid supplementation. In all experiments, animals maintained in the casamino acid media were compared. The animals used in this study were: wild-type, WT A. pomorum-monoassociated control flies; P3G5, P3G5-monoassociated control flies; P3G5 + acetic acid, P3G5-monoassociated flies in the presence of 0.2% acetic acid; and Germ-free + acetic acid, germ-free control flies in the presence of 0.2% acetic acid. (A) Acetic acid supplementation in casamino acid media induces IIS activation in P3G5-monoassociated larvae. PI3K activation state was evaluated by examining membrane targeting of pleckstrin homologygreen fluorescent protein (PHGFP), and localization of dFOXO was examined by immunostaining with an antibody to dFOXO. Fat body tissues of the early third instar larvae were used. DAPI, 4,6-diamidino-2-phenylindole. (B) Time to puparium formation, and sizes of larvae at the 120th hour of development after egg laying. Data were analyzed using an ANOVA followed by Tamhanes T2 post hoc test. Values represent mean SEM (***P < 0.001). (C) Adult body size. Body weights of adult flies (5 days old) were measured. Data were analyzed using an ANOVA followed by Tamhanes T2 post hoc test; values represent mean SEM (***P < 0.001). (D) www.sciencemag.org SCIENCE
Wing area, cell number, and size. Wings of female adult flies (5 days old) were used in this study. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (***P < 0.001). (E) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (*P < 0.05, **P < 0.01). VOL 334 4 NOVEMBER 2011
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suggesting that acetic acidproducing ability is an important factor that affects host physiology. Although acetic acid supplementation in casamino acid media did not result in any appreciable effect on host development in the absence of bacteria (i.e., germ-free animals) or in the presence of commensal bacterium other than A. pomorum (i.e., germ-free animals monoassociated with C. intestini, G. morbifer, L. plantarum, or L. brevis), all disease phenotypes (defects in IIS, development, metabolism and ISCs) found in P3G5-monoassociated animals could be effectively reversed by acetic acid supplementation (Fig. 4 and fig. S13). Although the exact mechanism of acetic acid action remains to be elucidated, it is known to affect blood glucose level and insulin signaling in mammals by reducing the digestion rate of complex carbohydrates in the diet (26). However, the aforementioned P3G5induced deregulation found when the fly larvae were fed a complex carbohydrate diet (i.e., containing cornmeal) was also observed in fly larvae fed a diet containing simple carbohydrates, such as sucrose or glucose (fig. S13). Furthermore, these defects were reversed by supplementing the simple sugar diet with acetic acid (fig. S13), indicating that acetic acid may influence host IIS and development through a mechanism other than by reducing the digestion rate of complex carbohydrates from the diet. Given that acetic acid can rescue host physiology only in the presence of P3G5 bacterial metabolic activity, we can conclude that both PQQ-ADHdependent acetic acid generation and PQQ-ADHindependent acetic acid metabolism are required to promote the effect of A. pomorum on host IIS. Further dissection of the A. pomorumcontrolled gut factor(s) that mediates the effect of acetic acidproducing and using bacterial metabolic activity on host IIS will provide an important link between gut microbiome activity and host metabolic homeostasis. In summary, the present study showed that the PQQ-ADH respiratory chain of the A. pomorum and IIS of the host interact to maintain the gutmicrobe mutualism. Bacterial PQQ-ADH is required, but not sufficient, to explain all of the A. pomorummediated effects on host physiology, and host signaling pathways, other than IIS, may also be modulated by gut bacteria. Our Drosophila-Acetobacter interaction system is a useful genetic model for understanding the mechanistic links between microbiome-modulated host signaling pathways and host physiology.
1. F. Bckhed, R. E. Ley, J. L. Sonnenburg, D. A. Peterson, J. I. Gordon, Science 307, 1915 (2005). 2. T. A. Koropatnick et al., Science 306, 1186 (2004). 3. J. L. Round et al., Science 332, 974 (2011); 10.1126/science.1206095. 4. Y. S. Bae, M. K. Choi, W. J. Lee, Trends Immunol. 31, 278 (2010). 5. N. Buchon, N. A. Broderick, S. Chakrabarti, B. Lemaitre, Genes Dev. 23, 2333 (2009). 6. M. Vijay-Kumar et al., Science 328, 228 (2010); 10.1126/science.1179721. 7. P. J. Turnbaugh et al., Nature 444, 1027 (2006). 8. A. M. OHara, F. Shanahan, EMBO Rep. 7, 688 (2006). 9. P. J. Turnbaugh et al., Nature 457, 480 (2009). 10. Materials and methods are available as supporting material on Science Online. 11. J. Qin et al.; MetaHIT Consortium, Nature 464, 59 (2010). 12. J.-H. Ryu et al., Science 319, 777 (2008); 10.1126/science.1149357. 13. C. Ren, P. Webster, S. E. Finkel, J. Tower, Cell Metab. 6, 144 (2007). 14. C. N. Wong, P. Ng, A. E. Douglas, Environ. Microbiol. 13, 1889 (2011). 15. S. W. Roh et al., Appl. Environ. Microbiol. 74, 6171 (2008). 16. E. Crotti et al., Environ. Microbiol. 11, 3252 (2009). 17. G. Favia et al., Proc. Natl. Acad. Sci. U.S.A. 104, 9047 (2007). 18. B. A. Edgar, Nat. Rev. Genet. 7, 907 (2006). 19. W. Brogiolo et al., Curr. Biol. 11, 213 (2001). 20. E. J. Rulifson, S. K. Kim, R. Nusse, Science 296, 1118 (2002). 21. M. Tatar et al., Science 292, 107 (2001). 22. K. D. Baker, C. S. Thummel, Cell Metab. 6, 257 (2007). 23. R. Bhni et al., Cell 97, 865 (1999). 24. S. J. Broughton et al., Proc. Natl. Acad. Sci. U.S.A. 102, 3105 (2005). 25. T. Yakushi, K. Matsushita, Appl. Microbiol. Biotechnol. 86, 1257 (2010). 26. C. S. Johnston, I. Steplewska, C. A. Long, L. N. Harris, R. H. Ryals, Ann. Nutr. Metab. 56, 74 (2010). Acknowledgments: This work was supported by the National Creative Research Initiative Program (to W.-J.L.), the BK 21 program, the Basic Science Research Program (2011-0001168, to J.-H.R. and J.-H.Y.), and a Gwangju Institute of Science and TechnologySystems Biology grant (2011) of the Ministry of Science and Technology Korea. We thank D. Daffonchio and K. Matsushita for helpful comments on Acetobacter. The A. pomorum draft genome sequence is deposited in DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank with accession no. AEUP01000000.

www.sciencemag.org/cgi/content/full/334/6056/670/DC1 Materials and Methods Figs. S1 to S13 Table S1 References (2737) 16 August 2011; accepted 8 September 2011 10.1126/science.1212782
N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

Daniel C. Scott,1,2 Julie K. Monda,1 Eric J. Bennett,3* J. Wade Harper,3 Brenda A. Schulman1,2 Although many eukaryotic proteins are amino (N)terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12s N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12s interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition. any eukaryotic proteins are N-terminally acetylated (14). Genetic data underscore the importance of N-terminal methionine acetylation (1, 510), although specif-
E1E2E3 ubiquitin-like protein (UBL) conjugation cascades. First, an E2 transiently binds E1 for generation of a thioester-linked E2~UBL intermediate, which then interacts with an E3. For RING E3s, the UBL is transferred from E2 to an E3-associated targets lysine, producing an isopeptide-bonded target~UBL complex. E2 core domains are sufficient for binding E1s and RING E3s (11). Contacts beyond E2 cores often mediate pathway-specific interactions. A unique N-terminal extension on Nedd8s E2, Ubc12, binds both E1 and E3 (1216). Nedd8 transfer from Ubc12 to cullins involves a dual E3 mechanism (16): A RING E3, Rbx1, is essential for cullin neddylation; a co-E3, Dcn1, contains a potentiating neddylation domain (Dcn1P)
1 Structural Biology Department, St. Jude Childrens Research Hospital, Memphis, TN 38105, USA. 2Howard Hughes Medical Institute, St. Jude Childrens Research Hospital, Memphis, TN 38105, USA. 3Cell Biology Department, Harvard Medical School, Boston, MA 02115, USA.
ic interactions mediated by N-acetyl-methionine are largely unknown. We examined how N-acetylmethionine can direct protein interactions by studying an E2 enzyme. E2s play central roles in VOL 334 SCIENCE
*Present address: Division of Biological Sciences, University of CaliforniaSan Diego, La Jolla, CA 92093, USA. To whom correspondence should be addressed. E-mail: brenda.schulman@stjude.org
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thought to bind different Ubc12 and cullin surfaces (1518). Notably, human Dcn1 acts as an oncogene (19). Because Dcn1s E3 activity was elusive with bacterially expressed Ubc12 (14, 17), we asked whether in eukaryotes Ubc12 might be modified. Tandem mass spectrometry (MS/MS) identified exogenous yeast (y) and human (h), and endogenous human Ubc12 as being N-terminally acetylated on Met1 (Fig. 1A and fig. S1). Although mammalian N-terminal acetyltransferases (Nats) appear partially redundant (3), yNat specificities are well defined (20). yUbc12s N-terminal MetLeu sequence was predicted to retain Met1 and be acetylated by the Mak3p-Mak10p-Mak31p complex comprising yNatC (21). Indeed, Mak3 gene deletion prevented yUbc12 N-terminal acetylation in yeast, and bacterially expressed yNatC catalyzed yUbc12 N-terminal acetylation (Fig. 1). Thus, yNatC performs yUbc12 N-terminal acetylation. To address whether Ubc12 N-terminal acetylation influences Nedd8 ligation, we examined yNedd8~yCul1 steady-state levels in yeast with Nat gene deletions. Only yeast lacking NatC components displayed decreased yNedd8~yCul1 (Fig. 1D). Furthermore, loss of NatC activity was synthetically lethal in combination with the cdc34-2 temperature-sensitive allele (Fig. 1E)a hallmark for Nedd8 pathway components due to roles in yCul1/SCF-regulated cell division (14, 22). Thus, yUbc12 N-terminal acetylation is important for yCul1 neddylation and function in vivo. In vitro, Ubc12 N-terminal acetylation dictated Dcn1P-mediated Nedd8 transfer to Cul1 in pulse-chase assays comparing N-terminally acetylated Ubc12AcMet, Ubc12Met (identical sequence but not N-terminally acetylated), and Ubc12GSMet (unacetylated with Gly-Ser-Met at the N terminus) (16). yDcn1P E3 activity was substantially increased for yUbc12AcMet, with lower yDcn1P enhancement of yNedd8 transfer to yCul1 from yUbc12Met or yUbc12GSMet consistent with the residual neddylation in NatC null yeast (Figs. 1D and 2A). hUbc12 N-terminal acetylation was
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XCorr 3.7106 Cn 0.3964
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Substrate: yUbc12Met yUbc12AcMet + - + - + NatC - - - + + yUbc12AcMet - + + - - yUbc12Met

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22,068.88 yUbc12-His WT Yeast
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22,069.29 yUbc12-His E. coli +yMak10 +yMak31 Predicted mass +yMak3 Met- 22,026.20 Ac- 22,068.21
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0 21 22
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mass *10 3
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at 1 t3
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ak 31
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ak
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yCul1~yNedd8 yCul1 NatA NatB NatC cdc34-2 cdc34-2:mak10 WT mak10 22C 30C 34C 37C
Fig. 1. Ubc12 is N-terminally acetylated in eukaryotic cells. (A) Liquid chromatography (LC)MS/MS spectrum from endogenous hUbc12s N-terminal peptide after Glu-C digestion and desalting, indicating XCorr and DCN values, y ions (red), and b ions (blue) used to match peptide sequence. (B) In vitro bacterially expressed yNatC reactions with [14C]-acetylcoenzyme A and www.sciencemag.org SCIENCE
Ubc12Met (free N terminus) or Ubc12AcMet (preacetylated negative control). (C) MaxEnt LC-TOF (time-of-flight) spectra of yUbc12-His6 purified from wild-type (WT) or Dmak3 yeast, or from coexpression with yNatC in Escherichia coli. (D) Immunoblot of yCul1 (Cdc53p) from indicated yeast strains. (E) Genetic interactions between NatC subunit mak10 and cdc34-2. VOL 334 4 NOVEMBER 2011
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absolutely required for hDcn1P-mediated potentiation of neddylation (Fig. 2B and fig. S2). In all cases, Ubc12 N-terminal acetylation was specific for Dcn1P E3 activity, because Dcn1P-independent Rbx1-mediated transfer of Nedd8 to Cul1 was independent of the state of Ubc12s N terminus (Fig. 2 and fig. S2). Nonetheless, even in the presence of Dcn1P and Ubc12 N-terminal acetylation, Cul1 neddylation required Rbx1s RING E3 activity and was blocked by the CAND1 inhibitor (fig. S2). Thus, in addition to roles of the acetylated Ubc12-Dcn1 E2-E3 complex, in vivo steady-state Cul1~Nedd8 levels may also reflect Dcn1- and acetylation-independent regulation. To obtain mechanistic insights, we quantified Ubc12-Dcn1P interactions by isothermal titration calorimetry (ITC). N-terminal acetylation increased Ubc12s affinity for Dcn1P by about two orders of magnitude, and this was recapitulated by synthetic Ubc12 N-terminal peptides, which also inhibited neddylation reactions (Fig. 2C and figs. S3 and S4). In contrast, N-terminal acetylation had little effect on Ubc12 peptide binding to E1 (Fig. 2C and fig. S5). To understand how N-acetyl-methionine mediates interactions, we determined crystal structures of yeast and human Dcn1P bound to acetylated Ubc12 peptides (table S1 and fig. S6). As in prior structures, Dcn1P forms a helical domain containing two EF-handlike folds (14, 16, 23). The Ubc12 N-terminal peptides are a-helical, as in full-length yUbc12GSMet (16). A Dcn1P groove at the junction between the two EF-handlike subdomains cradles Ubc12s helix, culminating in Ubc12s N-acetyl-methionine burial in a conserved, deep, hydrophobic pocket in Dcn1P (Fig. 3 and figs. S7 and S8). N-acetyl-methionine recognition consists of three major components (Fig. 3). First, the methyl portion of the acetyl group fits snuggly in a hydrophobic pocket. Second, the amide makes a hydrogen bond with a structurally conserved carbonyl oxygen from Dcn1. Third, the Met1 side chain is also fully enwrapped by the hydrophobic pocket. The structures suggest two mechanisms by which Ubc12s N-terminal acetylation dictates binding to Dcn1P. First, the acetyl group interacts directly with Dcn1P. Second, acetylation eliminates an N-terminal positive charge, which would impede burial in the hydrophobic pocket. These concepts were substantiated by assaying Dcn1P binding to N-terminally formylated Ubc12 peptides, which lack the acetyl methyl but retain the amide and are uncharged. N-terminal capping via formylation did improve binding ~10-fold in comparison to unacetylated peptides, although the dissociation constants (Kds) were decreased ~9- and ~17-fold compared with the acetylated human and yeast peptides, respectively, highlighting the importance of the acetyl methyl (Fig. 3E). Further agreeing with the structures, Ubc12s N-terminal Met1 was also required to bind Dcn1P (Fig. 3E).
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Titrant yDcn1P yDcn1 yUbc12

P
Protein
K d (M) >36 0.375 >40 0.444 NB 0.970 >130 1.11 0.070 0.016 0.585 0.943
H (cal/mol) 6,005 3,840 3,777 4,145 NB -6,931 1,259 -6,107 -8,571 -11,260 -8,881 -9,425
S (cal/mol/deg) 41.4 42.9 33.4 43.6 NB 3.21 22.2 5.84 2.65 -3.80 -1.99 -4.79
N 0.35 0.70 1.16 0.99 NB 0.76 0.75 0.92 1.05 0.99 1.29 1.14
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yE1 yE1 hE1 hE1
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Ac-hUbc121-26 :
Fig. 2. Dependence of Dcn1 co-E3 activity on Ubc12 N-acetylation. (A) Pulse-chase [32P]~yNedd8 transfer from yUbc12 variants to yCul1 C-terminal domain (yCul1C) complexed with Rbx1 with or without yDcn1P. (B) Same as (A), but with human proteins. (C) Thermodynamic parameters for Ubc12 or its peptides binding to Dcn1P and E1 by ITC. NB, no binding. Ubc12s N-acetyl-methionine is sealed into Dcn1Ps hydrophobic pocket by Ubc12s N-terminal helix positioning hydrophobic residues 2 and 4 (Fig. 3). On one side, yLeu2/hIle2 buries the acetyl. On the other side, Leu4 seals the Met1 side chain into place. Residues downstream from Ubc12s helix also interact with Dcn1P (fig. S9). Although yUbc12GSMets N-terminal extension is helical (16), we wished to test the structurally observed role for the helix with human proteins because hUbc12Mets N-terminal region forms an extended structure in complex with E1 (12, 13). A 2.0 resolution structure with a stapled-helix (24) peptide superimposed with unstapled hUbc12AcMet-hDcn1P, confirming solvent exposure of the staple (Fig. 3F). Helical stapling improved binding to hDcn1P ~14-fold, largely due to decreasing the entropic cost (Fig. 3E). Moreover, helical stapling eliminated E1 binding. Thus, locking the flexible hUbc12 VOL 334 SCIENCE N-terminal region into a helix contributes to the hDcn1P interaction. Additional Dcn1P elements secure Ubc12s N-acetyl-methionine in place. First, yDcn1s Tyr190/ hDcn1-Tyr181 clamps between Ubc12s N-acetylMet1 and yLeu2/hIle2, pressing Ubc12s N-acetylmethionine into Dcn1Ps hydrophobic pocket. Second, the loop between Dcn1Ps E and F a helices closes down on Ubc12s N-acetylmethionine. In prior yDcn1P structures lacking Ubc12 (14, 16, 23), these elements are repositioned to occlude the hydrophobic pocket (fig. S10). yDcn1P apparently initially engages Ubc12s acetylated N terminus and subsequently clamps it down. Conformational flexibility may account for yDcn1Ps low-level activity toward yUbc12 even without N-terminal acetylation. Given yDcn1Ps structural malleability, we reasoned that mutations alleviating repulsion of an N-terminal charge might enhance yDcn1Ps
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Fig. 3. Dcn1P recognition of Ubc12s N-acetyl-methionine. (A) hCul1WHB (not shown)-hDcn1P-AcetylhUbc121-15 structure, with hDcn1P surface colored by conservation among human and yeast orthologs and AcetylhUbc121-15 peptide in cyan. (B) Close-up of hUbc12s acetylated N terminus (cyan) binding hDcn1P (salmon) in cartoon (left) or hUbc12s N-acetylMet1 and residues 2 and 4 as spheres in a mesh view of hDcn1P (right). (C) Close-up of hUbc12s acetylated N terminus (cyan) binding hDcn1P surface colored by electrostatic potential. (D) Same as (B), but with yeast proteins. (E) Thermodynamic parameters for Ubc12 peptide binding to Dcn1P by ITC. 5:9S refers to helical staple. *Reference from Fig. 2C. NB, no binding. (F) Solvent exposure of helical staple in hUbc12 peptide (cyan) bound to hDcn1P (surface, colored by electrostatic potential).
Fig. 4. Structure-based Dcn1 mutant compensation for lack of Ubc12 N-terminal acetylation. (A) Pulse-chase [32P]-yNedd8 transfer from yUbc12Met (top) or yUbc12AcMet (bottom) to yCul1C-yRbx1 with structurebased yDcn1P mutants (note different time courses). (B) Thermodynamic parameters for binding between yDcn1P or the Tyr190Ala (Y190A) mutant to unacetylated and acetylated yUbc12. *Reference from Fig. 2C. (C) Immunoblots for yCul1 (Cdc53p, top) or HA-tag (bottom) from midlog whole-cell extracts from Ddcn1 or Ddcn1/Dmak10 yeast harboring empty, WT Dcn1-HA, or Y190A Dcn1-HA expression vectors.
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low-level E3 activity toward yUbc12Met. The structure indicated that Asp substitutions for yDcn1P Leu110 or Leu173 would approach yUbc12Mets N terminus to balance the positive charge. Also, an Ala replacement for the Tyr190 clamp would not force a charged yUbc12Mets N terminus directly into the hydrophobic pocket. Indeed, the three Dcn1P mutants showed enhanced E3 activity specifically toward unacetylated yUbc12 (Fig. 4A). We asked whether a structure-based mutation could compensate for in vivo defects in cullin neddylation resulting from loss of NatC-mediated yUbc12 acetylation. Thus, we expressed hemagglutinin (HA)tagged Dcn1 or the Tyr190Ala mutant (we could not express comparable levels of the other mutants in yeast), in strains deleted for Dcn1 alone, or both Dcn1 and the NatC subunit Mak10. As with the in vitro enzymology and improved binding, the Tyr190Ala mutant rescued the defect in yCul1~yNedd8 conjugate formation that resulted from lack of NatC activity (Fig. 4). We showed N-terminal acetylation of Ubc12 to be an avidity enhancer, contributing a critical interaction within a highly interconnected neddylation complex. As only part of molecular recognition within large multicomponent complexes, many interactions depending on N-terminal acetylation likely remain unknown and may be auxiliary (2, 20, 25). Our study raises the question of whether rules dictating N-terminal acetylation determined evolution of interactions controlling functions of N-terminally acetylated proteins. Specificity may also involve proximal elements, such as Ubc12s N-terminal helix. Because N-acetylmethionine can be completely enwrapped in a hydrophobic environment where it would be unfavorable to bury the positive charge masked by acetylation, we propose that N-acetyl-methionine can serve as a distinctive residue type allowing burial of protein N-termini into hydrophobic pockets of interacting proteins. Such N-acetyl-methionine binding sites may serve as targets for small molecules disrupting these critical interactions.
19. I. Sarkaria et al., Cancer Res. 66, 9437 (2006). 20. B. Polevoda, F. Sherman, J. Mol. Biol. 325, 595 (2003). 21. B. Polevoda, F. Sherman, J. Biol. Chem. 276, 20154 (2001). 22. D. Lammer et al., Genes Dev. 12, 914 (1998). 23. X. Yang et al., J. Biol. Chem. 282, 24490 (2007). 24. G. H. Bird, F. Bernal, K. Pitter, L. D. Walensky, Methods Enzymol. 446, 369 (2008). 25. B. Polevoda, F. Sherman, Biochem. Biophys. Res. Commun. 308, 1 (2003). Acknowledgments: This was supported by American Lebanese Syrian Associated CharitiesSt. Jude, NIH, and Howard Hughes Medical Institute (B.A.S.), grants from NIH and Millennium Pharmaceuticals ( J.W.H.), and Damon Runyon Cancer Research Foundation (E.J.B.). We thank S. Gygi, I. Kurinov, C. Ralston, R. Cassell, P. Rodrigues, K. Kodali, V. Pagala, R. Schekman, D. W. Miller, S. Bozeman, D. J. Miller, J. Bollinger, and C. Rock for assistance, reagents, and/or discussions. D.C.S., J.K.M., B.A.S., and St. Jude Childrens Research Hospital have applied for a patent on uses of Ubc12 N-terminal acetylation for inhibiting neddylation. Research Collaboratory for Structural Bioinformatics structural accession codes: 3TDI, 3TDU, 3TDZ. Author contributions: D.C.S., J.K.M., and E.J.B. designed, performed, and analyzed experiments; D.C.S. and B.A.S. wrote the manuscript, with all authors contributing; J.W.H. and B.A.S. advised and assisted on all aspects.

www.sciencemag.org/cgi/content/full/science.1209307/DC1 Materials and Methods Figs. S1 to S10 Table S1 References (2644) 3 June 2011; accepted 9 September 2011 Published online 22 September 2011; 10.1126/science.1209307
mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase
Roberto Zoncu,1,2,3,4 Liron Bar-Peled,1,2,3 Alejo Efeyan,1,2,3 Shuyu Wang,1,2,3 Yasemin Sancak,1,2,3 David M. Sabatini1,2,3,4,5* The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H+adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acidsensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing. mino acids are the building blocks of proteins and intermediates in lipid and adenosine triphosphate (ATP) synthesis. They also initiate a signaling cascade that leads to activation of the master growth regulator mTOR complex 1 (mTORC1). This multicomponent proVOL 334 SCIENCE
tein kinase integrates inputs from growth factors as well as nutrient and energy supplies to control many biosynthetic and catabolic processes (1). Most signals upstream of mTORC1 converge on TSC1-TSC2, a heterodimeric tumor suppressor that negatively regulates the Rheb guanosine triphosphatase (GTPase), which is an essential activator of mTORC1 protein kinase activity (2, 3). In contrast, amino acids signal to mTORC1 by promoting its binding to a distinct family of GTPases, the Rag GTPases (4, 5). The Rags form heterodimers consisting of RagA or RagB, which are highly similar to each other, bound to RagC or RagD, which are also highly related. In an amino acidsensitive fashion, the Rag GTPases recruit mTORC1 to the surface of lysosomes, which also contain Rheb (5). The
1 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA. 2Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA. 3David H. Koch Institute for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. 4Broad Institute, Seven Cambridge Center, Cambridge, MA 02142, USA. 5Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA.
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*To whom correspondence should be addressed. E-mail: sabatini@wi.mit.edu
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1. T. Arnesen, PLoS Biol. 9, e1001074 (2011). 2. T. Arnesen et al., Proc. Natl. Acad. Sci. U.S.A. 106, 8157 (2009). 3. P. Van Damme et al., PLoS Genet. 7, e1002169 (2011). 4. C. H. Yi et al., Cell 146, 607 (2011). 5. S. H. Askree et al., Proc. Natl. Acad. Sci. U.S.A. 101, 8658 (2004). 6. T. Kanki et al., Mol. Biol. Cell 20, 4730 (2009). 7. S. J. Dixon et al., Proc. Natl. Acad. Sci. U.S.A. 105, 16653 (2008). 8. R. Behnia, B. Panic, J. R. Whyte, S. Munro, Nat. Cell Biol. 6, 405 (2004). 9. S. R. Setty, T. I. Strochlic, A. H. Tong, C. Boone, C. G. Burd, Nat. Cell Biol. 6, 414 (2004). 10. C. S. Hwang, A. Shemorry, A. Varshavsky, Science 327, 973 (2010). 11. Y. Ye, M. Rape, Nat. Rev. Mol. Cell Biol. 10, 755 (2009). 12. D. T. Huang et al., Nat. Struct. Mol. Biol. 11, 927 (2004). 13. D. T. Huang et al., Nature 445, 394 (2007). 14. T. Kurz et al., Mol. Cell 29, 23 (2008). 15. A. Y. Kim et al., J. Biol. Chem. 283, 33211 (2008). 16. D. C. Scott et al., Mol. Cell 39, 784 (2010). 17. T. Kurz et al., Nature 435, 1257 (2005). 18. G. Huang, A. J. Kaufman, Y. Ramanathan, B. Singh, J. Biol. Chem. 286, 10297 (2011).
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trimeric Ragulator complex, which comprises the p18, p14, and MP1 proteins, anchors the Rag GTPases to the lysosome and, like the Rags, is necessary for mTORC1 activation by amino acids (6). Together, the Ragulator and Rag heterodimer form an amino acidregulated docking site for mTORC1 on the lysosomal surface. Amino acid signaling has been proposed to begin, alternatively, at the plasma membrane or inside the cell, but this key issue remains unsettled (710). The localization of the Rag GTPases on lysosomes, but not on other Rheb-containing endomembranes, suggests that this organelle has an important role in amino acid signaling to mTORC1. To determine whether lysosome-associated processes and proteins participate in the activation of mTORC1 by amino acids, we used RNA interference (RNAi) in Drosophila S2 cells to reduce the expression of a number of genes with roles in lysosomal biogenesis and function (table S1). Double-stranded RNAs (dsRNAs) targeting most of the genes did not affect the amino acidinduced phosphorylation of the ribosomal protein S6 kinase (dS6K) on T398, a readout of dTORC1 activity (table S1). In contrast, dsRNAs targeting vhaAC39, vha16, vha100-1, and vha100-2, all encoding components of the vacuolar H+-ATPase (v-ATPase), suppressed dS6K phosphorylation to degrees similar to that of a dsRNA targeting dRagC (table S1, Fig. 1, A and B, and fig. S1A). The dsRNAs to vhaAC39 also decreased the size of S2 cells (Fig. 1C). Consistent with the results in Drosophila cells, lentiviral short-hairpin RNAs (shRNAs) targeting human ATP6V0c, the ortholog of Drosophila vha16, suppressed amino acid induced phosphorylation of S6K1 in human embryonic kidney (HEK) 293T cells (Fig. 1D and fig. S1B). These results implicate the v-ATPase in the activation of mTORC1 by amino acids. The v-ATPase consists of multicomponent V0 and V1 domains and operates through an incompletely understood mechanism in which each cycle of ATP hydrolysis by the V1 sector generates torque that rotates the membrane domain of V0, known as the rotor. In turn, this movement enables the transfer of protons into the lysosomal lumen, causing its acidification (11). The macrolides concanamycin A (ConA) and salicylihalamide A (SalA) are structurally diverse inhibitors of the v-ATPase that do not have other known targets (1114). In 293T cells, both ConA and SalA inhibited amino acidinduced phosphorylation of S6K1 in a concentration-dependent manner (Fig. 1, E and F). The inhibition of S6K1 phosphorylation occurred after short (15- to 60-min) treatment times (fig. S2A) and without concomitant alterations in lysosomal morphology (fig. S2, B and C) or inhibition of Akt phosphorylation, a readout of growth factor signaling (Fig. 1, E and F). The finding that the v-ATPase and its activity are necessary for mTORC1 activation by amino acids led us to consider potential roles for it in the pathway. One possibility is that the v-ATPase functions downstream of amino acids and is part of the amino acidinduced signaling pathway that culminates in mTORC1 activation. Another conceivable function is that the proton gradient generated by the v-ATPase is required for amino acids to be transported into the cellular compartment where an amino acid sensor is located. To bypass the transport function, we tested whether the v-ATPase is required for alcohol ester derivatives of amino acids to activate mTORC1. These esters freely diffuse across membranes and, within the cytoplasm and lysosomes, are hydrolyzed by esterases into native amino acids (15). A mixture of amino acid esters or leucine methyl ester activated mTORC1 with efficiencies comparable to those of their respective native amino acids (fig. S1, C and D). Moreover, ConA also inhibited the S6K1 phosphorylation induced by amino acid esters (Fig. 1G and fig. S1E). Consistent with these findings, SalA also suppressed mTORC1 activation induced by cycloheximide, which, by inhibiting translation, boosts concentrations of intracellular amino acids (7, 16) (Fig. 1H). Thus,
Fig. 1. Requirement of the v-ATPase for mTORC1 activation by amino acids. (A) dsRNA-mediated depletion of vhaAC39 in Drosophila S2 cells. Cells were deprived for amino acids for 1.5 hours and then stimulated with complete medium for 30 min. Proteins from cell lysates were analyzed for phosphorylation of dS6K at threonine 398 (T398). Depletion of vhaAC39 by two distinct dsRNAs is compared to that of dRagC. (B) dsRNA-mediated depletion of both vha100-1 and vha100-2 in S2 cells suppresses amino acidinduced T398 phosphorylation of dS6K. (C) Cell size measurement after depletion of vhaAC39 in S2 cells with two dsRNAs (red and blue) compared to a control dsRNA (black). (D) S6K1 phosphorylation at T389 in HEK-293T cells treated with shRNA targeting GFP, RagC and RagD, and V0c. Cells were deprived of amino acids for 50 min and, where indicated, stimulated for 10 min. Immunoblotting was used to www.sciencemag.org SCIENCE
detect the indicated proteins. (E) S6K1 phosphorylation in HEK-293T cells deprived of amino acids for 50 min in the presence of the indicated concentrations of ConA and then stimulated for 10 min with amino acids. (F) S6K1 phosphorylation in HEK-293T cells deprived of amino acids for 50 min in the presence of the indicated concentrations of SalA and restimulated for 10 min with amino acids. (G) ConA blocks mTORC1 activation by alcohol esters of amino acids. HEK-293T cells were deprived of amino acids for 50 min and then stimulated for 10 min with amino acids or alcohol esters of amino acids in the presence of 2 mM ConA where indicated. (H) Activation of mTORC1 by intracellular amino acids. HEK-293T cells were deprived of amino acids for 50 min and stimulated with amino acids or cycloheximide in dimethyl sulfoxide (DMSO) or 2 mM SalA. Immunoblotting was used to detect the indicated proteins. VOL 334 4 NOVEMBER 2011
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these results are consistent with the v-ATPase having a role downstream of intracellular amino acids in the initiation or propagation of the amino acidinduced signal to mTORC1. A key event in amino acid signaling is the Rag GTPasemediated translocation of mTORC1 to the surface of lysosomes (5, 6). In cells treated with ConA or SalA or depleted of ATP6V0c, mTOR failed to cluster onto lysosomes in response to amino acids and instead was found in a diffuse staining pattern (Fig. 2, A and B, and fig. S3, A to C). Unlike the Ragulator (6), the v-ATPase appears not to be necessary for anchoring the Rag GTPases to the lysosomal surface, because pharmacological or RNAi-mediated inhibition of the v-ATPase did not affect lysosomal localization of RagC (Fig. 2C and fig. S3, C and D). To test whether the v-ATPase might function upstream of the Rag GTPases, we used a RagB mutant that is constitutively active (RagBGTP) and renders mTORC1 signaling insensitive to amino acid starvation (4, 5). If the v-ATPase is required for the activation of the Rag GTPases, expression of RagBGTP should rescue the defects in mTOR lysosomal recruitment and S6K1 phosphorylation caused by ConA and SalA. Indeed, in cells stably expressing RagBGTP, the lysosomal localization of mTOR and the phosphorylation of S6K1 were insensitive not only to amino acid starvation but also to ConA and SalA treatment (Fig. 2, D and E, and fig. S3F). Consistent with these results, in knockin mouse embryonic fibroblasts (MEFs) that express active RagAGTP from the endogenous RagA locus, SalA (Fig. 2F) and ConA (fig. S3G) did not block the constitutive S6K1 phosphorylation caused by RagAGTP. Collectively, these results place the v-ATPase downstream of amino acids but upstream of the activation of the Rag GTPases; they also exclude its involvement in other regulatory inputs to mTORC1, such as controlling Rheb activity (6). We tested whether a physical interaction exists between the v-ATPase and the Rags or Ragulator or both. Semi-quantitative mass spectrometric analyses of anti-FLAG immunoprecipitates prepared from 293T cells expressing FLAG-tagged Ragulator components ( p18 or p14) or RagB revealed the presence of many subunits of the v-ATPase (Fig. 3A). Immunoblot assays with antibodies to endogenous V0 (c and d1) and V1 (A, B2, and D) subunits confirmed that Ragulator coimmunoprecipitates with the V0 and V1 domains (Fig. 3, B and C), whereas the Rags coimmunoprecipitate with V1 subunits only (fig S4, A and B). Although easily detected in immunoblot assays as coimmunoprecipitating with Ragulator, the c subunit of V0 was not detected by mass spectrometry, probably because of its highly hydrophobic nature. The v-ATPase did not coimmunoprecipitate with lysosomal (LAMP1) or cytoplasmic (Metap2) control proteins (Fig. 3, B and C). In vitro assays with purified recombinant proteins verified a direct interaction between the V0 component d1 and p18, but not p14, and between the V1 component D with p18 and, to a lesser degree, with p14 (Fig. 3D). No direct interactions were detected between the Rag GTPases and purified v-ATPase subunits (fig. S4C), which is consistent with the relatively low abundance of v-ATPase subunits
Fig. 2. Requirement of the v-ATPase for lysosomal recruitment of mTORC1 by the Rag GTPases. (A) Immunofluorescence images of mTOR and LAMP2 in HEK-293T cells deprived of amino acids (a.a.) (top) or deprived and then stimulated (bottom) in the presence of DMSO (left) or 2.5 mM SalA (right). (B) HEK-293T cells expressing a lentivirally encoded shRNA targeting GFP (left) or V0c (right) were deprived of amino acids (top) or deprived and then stimulated (bottom). (C) Staining for RagC and LAMP2 in HEK-293T cells deprived of amino acids (top) or deprived and then stimulated (bottom) in the presence of DMSO (left) or 2.5 mM SalA (right). (D) HEK-293T cells stably expressing the constitutively active RagBQ99L mutant (293T RagBGTP) were deprived of amino acids (top) or deprived and stimulated (bottom) in the presence of DMSO (left) or 2.5 mM ConA (right). (E) S6K1 phosphorylation in HEK-293T cells and HEK-293T RagBGTP cells deprived of amino acids for 50 min in the presence of DMSO or 2 mM SalA and stimulated for 10 min with amino acids. (F) S6K1 phos-
phorylation in wild-type MEFs (RagA+/+) or in MEFs homozygous for the constitutive active RagA Q66L mutant (RagAGTP/GTP); cells were deprived of amino acids for 50 min in the presence of DMSO or 2.5 mM SalA and stimulated for 10 min with amino acids. In all images, insets show selected fields that were magnified five times and their overlays. Scale bars represent 10 mm. SCIENCE www.sciencemag.org
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in the RagB immunoprecipitates analyzed by mass spectrometry (Fig. 3A). Thus, in addition to scaffolding the Rag GTPases to the lysosomal surface, Ragulator provides a physical and functional link between the v-ATPase and the Rag GTPases. Consistent with amino acids acting upstream of the v-ATPase, amino acids regulated the interaction between the V1 domain of v-ATPase and Ragulator and Rag GTPases. Amino acid starvation and stimulation strengthened and weakened, respectively, the interaction (Fig. 3E and fig. S4D). In contrast, amino acids did not affect the binding of Ragulator with the V0 domain of the v-ATPase (Fig. 3E) or of the V1 and V0 subunits with each other [as does glucose starvation (17)]
Fig. 3. Interaction of the v-ATPase with the Ragulator-Rag GTPases. (A) Cartoon summarizing mass spectrometry analyses of immunoprecipitates from HEK-293T cells expressing FLAG-p18 (left), FLAG-p14 (center), and FLAG-RagB (right). v-ATPase subunits are color-coded according to their peptide representation (scale at the far right). (B) Binding of Ragulator to the V0 domain. HEK-293T cells stably expressing FLAGtagged p18 and p14 were lysed and subjected to FLAG immunoprecipitation (IP) followed by immunoblotting for V0c and V0d1. FLAG-LAMP1 and FLAG-Metap2 served as negative controls. (C) Binding of Ragulator to the V1 domain. HEK-293T cells stably expressing FLAG-tagged p18, p14, LAMP1, and Metap2 were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for V1A, V1B2, and V1D. (D) (Top) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated with recombinant V0d1 fused to glutathione S-transferase (HA-GST-V0d1), immobilized on glutathione agarose beads. Samples were subjected to immunoblotting for FLAG to detect bound Ragulator components. HA-GST-Rap2A served as a negative control. (Bottom) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated with recombinant V1D fused to glutathione S-transferase (HA-GST-V1D). HA-GST-metap2 served as a negative control. (E) The Ragulator-V1 interaction, but not the Ragulator-V0 interaction, is regulated by amino acids. HEK-293T cells stably expressing FLAG-tagged p18, p14, and Metap2 were deprived of amino acids for 90 min or deprived and then stimulated with amino acids for 15 min. After lysis, samples were subjected to FLAG immunoprecipitation and immunoblotting for the indicated v-ATPase subunits. (F) SalA blocks regulation of the Ragulator-V1 interaction by amino acids. HEK-293T cells stably expressing FLAG-p14 were deprived of amino acids for 90 min, or deprived and then stimulated with amino acids for 15 min, in the presence of DMSO or 2 mM SalA. Samples were lysed, FLAG-immunoprecipitated, and immunoblotted for the indicated proteins. (G) Cartoon summarizing the Ragulatorv-ATPase interactions identified in (A) to (F). Orange denotes regulation by amino acids; blue indicates lack of regulation. www.sciencemag.org SCIENCE VOL 334 4 NOVEMBER 2011
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(fig. S4D). Treatment of cells with SalA, like amino acid deprivation, strengthened the interaction between Ragulator and V1 domain subunits and, moreover, largely prevented amino acids from weakening the interaction (Fig. 3F and fig. S4E). Thus, amino acids induce a structural rearrangement of the v-ATPaseRagulator Rag GTPase complex that is blocked by SalA (Fig. 3G). The v-ATPase is large and complex and has many functions that cannot easily be teased apart in live cells (11, 1822). Thus, to better understand its role in amino acid signaling to mTORC1, we developed a cell-free system that recapitulates the amino acidinduced binding of mTORC1 to the Rag GTPases on the lysosomal surface [see methods in the supporting online material (SOM)]. We prepared a light organelle fraction from 293T cells expressing FLAG-RagB that had been deprived of amino acids. The organelles were briefly stimulated with amino acids or amino acid esters and then incubated with cytosol containing Myc-tagged Raptor (Fig. 4A and fig. S5A). In this system, amino acids, and especially amino acid esters, increased binding of Myc-Raptor to FLAG-RagBcontaining vesicles but not to control vesicles (Fig. 4A and fig. S5B). As expected, in preparations containing the FLAGRagBGTP mutant, the binding of Myc-Raptor was constitutively high and largely insensitive to amino acids (fig. S5C and S5D). We also prepared purified lysosomes by immunoisolating them from 293T cells (see methods in SOM) (fig. S6, A to C). Again, amino acid esters induced binding of Myc-tagged Raptor to isolated lysosomes (fig. S6D). Cytosol appeared to be dispensable, because highly purified, FLAG-tagged Raptor showed amino acidinduced binding to organelles containing GST-tagged Rag heterodimers (fig. S6E). Thus, lysosomes contain all the machinery required for sensing amino acids and activating the Rag GTPases. In the in vitro system, the alcohol esters of amino acids were more effective than native amino acids in inducing the RagB-Raptor interaction (Fig. 4A). A possible reason for this is that amino acids must enter and accumulate in lysosomes to initiate signaling, and that the amino acid esters do so more easily in the in vitro preparation (15). To test the requirement for an intralysosomal accumulation, we used treatments that permeabilize the lysosomal membrane and thus allow amino acids to leak out. Treatment of the organelle preparation with Streptolysin O, which introduces nanometer-sized holes into the lysosomal membrane, or Triton X-100, which dissolves the membranes without disrupting the v-ATPase RagulatorRag interaction, completely suppressed the effect of amino acids or their esters to promote the binding of Raptor to RagB (Fig. 4B). The PAT1 (SLC36A1) transporter is a proton-coupled amino acid transporter that localizes specifically to lysosomes (fig. S7A) and exports amino acids
cytosol input: DMSO: FCCP: AMP-PNP (1mM): AMP-PNP (10mM): amino acid esters:
Fig. 4. In vitro analysis of mTORC1 activation by amino acids. (A) Cell-free binding of Myc-Raptor to FLAG-RagB but not to FLAG-Rap2Acontaining vesicles. Organelle preparations were left unstimulated or were stimulated with amino acids or amino acid esters and incubated with Myc-Raptorcontaining cytosol. After FLAG immunoprecipitation, bound Myc-Raptor was detected by immunoblotting. (B) Intact FLAG-RagB lysosomes, FLAG-RagB lysosomes permeabilized with streptolysin O, and FLAG-RagB lysosomes permeabilized with Triton X-100 were left unstimulated, stimulated with amino acids, or stimulated with amino acid esters. Myc-Raptor was detected by immunoblotting. (C) S6K1 phosphorylation at T389 in HEK-293T cells transiently expressing FLAG-S6K1, FLAG-S6K1 + Myc-PAT1, FLAG S6K1 + HAGST-tagged active Rag mutants, or FLAG-S6K1 + Myc-PAT1 + HAGST-active Rags. Cells were deprived of amino acids for 50 min or starved and then stimulated for 10 min (see methods in SOM). The indicated proteins were detected by immunoblotting. The band pattern of Myc-PAT1 is probably due to glycosylation. (Right) Immunofluorescence images of lysosomes from HEK-293T cells transiently expressing Myc-PAT1 and stained for Myc tag (top, red in the merge) and for LAMP2 (center, green in the merge).
(D) Accumulation of 14C-labeled amino acids into lysosomes immunopurified from HEK-293T cells expressing LAMP1-mRFP-FLAGX2. Lysosomes were either left intact or permeabilized with Triton X-100 or streptolysin O before measurement. Overexpression of PAT1 largely abolished amino acid accumulation inside lysosomes. Each value represents the mean T SD of three independent samples. (E) FLAG-RagB lysosomes were treated with DMSO or 2 mM SalA, activated with amino acid esters, and then incubated with Myc-Raptor. An organellar fraction from FLAG-metap2expressing cells served as a negative control. (F) FLAG-RagB lysosomes were stimulated with amino acid esters in the presence of the proton ionophore FCCP or the nonhydrolyzable ATP analog AMP-PNP at 1 mM or 10 mM. Organelle samples were then incubated with Myc-Raptor cytosol, followed by FLAG immunoprecipitation and immunoblotting for Myc-Raptor and endogenous mTOR. (G) Model for inside-out activation of mTORC1 by lysosomal amino acids. The accumulation of amino acids inside the lysosomal lumen generates an activating signal that is transmitted to the Rag GTPases via the v-ATPaseRagulator interaction. In turn, the Rags physically recruit mTORC1 to the lysosomal surface. SCIENCE www.sciencemag.org
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out of the lysosomal lumen (23). In intact cells, overexpression of PAT1 completely suppressed mTORC1 activation by amino acids, and this effect was fully rescued by coexpression of constitutively active RagBGTP (Fig. 4C). In contrast, overexpression of PAT4 (SLC36A4), an amino acid transporter that does not localize to the lysosome, had no effect on mTORC1 activation by amino acids (fig. S7, B and C). These results strongly suggest that amino acid signaling begins inside the lysosome. Consistent with this possibility, stimulation of amino acidstarved 293T cells with 14C-amino acids led to the rapid appearance of labeled amino acids within lysosomes immunoisolated through a FLAG-tagged lysosomal protein (Fig. 4D and fig. S6, A and B). Amino acid accumulation was reverted by lysosome permeabilization and largely prevented by PAT1 overexpression (Fig. 4D). In the in vitro system, disruption of the v-ATPase by SalA or by shRNA against V0c blocked the amino acidinduced interaction of Raptor with RagB (Fig. 4E and fig. S5E). SalA causes structural rearrangements in the v-ATPase that inhibit both its capacity to hydrolyze ATP and to establish the lysosomal proton gradient (13, 24). Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), an ionophore that dissipates the lysosomal proton gradient without interfering with the v-ATPase (25), did not prevent the amino acidinduced binding of Raptor to RagB (Fig. 4F). Moreover, amino acids or their esters did not alter the lysosomal pH of intact cells, nor did they perturb lysosomal acidification in vitro (fig. S8, A to C). In contrast, the nonhydrolyzable ATP analog adenosine 5-(b,g-imido)triphosphate (AMP-PNP), which inhibits the ATPase activity of V1 and the consequent rotation of the stalk and the V0 proteolipid subunits, blocked the amino acidinduced interaction between Raptor and RagB in a concentrationdependent manner (Fig. 4F and fig. S8D). Thus, ATP hydrolysis and the associated rotation of the v-ATPase appear to be essential to relay an amino acid signal from the lysosomal lumen to the Rag GTPases, whereas the capacity of the v-ATPase to set up the lysosomal proton gradient is dispensable in the in vitro system. We propose a lysosome-centric inside-out model of amino acid sensing by mTORC1 in which amino acids must accumulate in the lysosomal lumen to initiate signaling (Fig. 4G). The v-ATPase is required for amino acid signaling to mTORC1 and functions between amino acids and the nucleotide loading of the Rag GTPases. Its placement in the pathway and its amino acid sensitive interactions with the Rag-Ragulator complex implicate it as an essential component of the amino acid sensing mechanism.
1. R. Zoncu, A. Efeyan, D. M. Sabatini, Nat. Rev. Mol. Cell Biol. 12, 21 (2011). 2. K. Inoki, Y. Li, T. Xu, K. L. Guan, Genes Dev. 17, 1829 (2003). 3. A. R. Tee, B. D. Manning, P. P. Roux, L. C. Cantley, J. Blenis, Curr. Biol. 13, 1259 (2003). 4. E. Kim, P. Goraksha-Hicks, L. Li, T. P. Neufeld, K. L. Guan, Nat. Cell Biol. 10, 935 (2008). 5. Y. Sancak et al., Science 320, 1496 (2008). 6. Y. Sancak et al., Cell 141, 290 (2010). 7. A. Beugnet, A. R. Tee, P. M. Taylor, C. G. Proud, Biochem. J. 372, 555 (2003). 8. J. Boh, A. Low, R. R. Wolfe, M. J. Rennie, J. Physiol. 552, 315 (2003). 9. G. R. Christie, E. Hajduch, H. S. Hundal, C. G. Proud, P. M. Taylor, J. Biol. Chem. 277, 9952 (2002). 10. B. Wu et al., J. Cell Biol. 173, 327 (2006). 11. M. Forgac, Nat. Rev. Mol. Cell Biol. 8, 917 (2007). 12. B. J. Bowman, M. E. McCall, R. Baertsch, E. J. Bowman, J. Biol. Chem. 281, 31885 (2006). 13. M. R. Boyd et al., J. Pharmacol. Exp. Ther. 297, 114 (2001). 14. M. Huss et al., J. Biol. Chem. 277, 40544 (2002). 15. J. P. Reeves, J. Biol. Chem. 254, 8914 (1979). 16. D. J. Price, R. A. Nemenoff, J. Avruch, J. Biol. Chem. 264, 13825 (1989). 17. S. Bond, M. Forgac, J. Biol. Chem. 283, 36513 (2008). 18. C. M. Cruciat et al., Science 327, 459 (2010). 19. P. R. Hiesinger et al., Cell 121, 607 (2005). 20. A. Hurtado-Lorenzo et al., Nat. Cell Biol. 8, 124 (2006). 21. C. Peters et al., Nature 409, 581 (2001). 22. Y. Yan, N. Denef, T. Schpbach, Dev. Cell 17, 387 (2009). 23. C. Sagn et al., Proc. Natl. Acad. Sci. U.S.A. 98, 7206 (2001). 24. X. S. Xie et al., J. Biol. Chem. 279, 19755 (2004). 25. B. E. Steinberg et al., J. Cell Biol. 189, 1171 (2010). Acknowledgments: We thank members of the Sabatini Lab as well as R. Perera for helpful suggestions, E. Spooner for the mass spectrometric analysis, and J. De Brabander (University of Texas Southwestern) for salicylihalamide A. Supported by grants from the National Institutes of Health (CA103866 and AI47389) and Department of Defense (W81XWH-07-0448) to D.M.S., awards from the W.M. Keck Foundation and LAM Foundation to D.M.S., and fellowship support from the Jane Coffin Childs Memorial Fund for Medical Research and the LAM Foundation to R.Z., from the Human Frontier Science Program to A.E., and from the Medical Scientist Training Program to S.W. D.M.S. is an investigator of the Howard Hughes Medical Institute.

www.sciencemag.org/cgi/content/full/334/6056/678/DC1 Materials and Methods Figs. S1 to S8 Table S1 References 15 April 2011; accepted 13 September 2011 10.1126/science.1207056
RNAP II CTD Phosphorylated on Threonine-4 Is Required for Histone mRNA 3 End Processing
Jing-Ping Hsin, Amit Sheth, James L. Manley* The RNA polymerase II (RNAP II) largest subunit contains a C-terminal domain (CTD) with up to 52 Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 consensus repeats. Serines 2, 5, and 7 are known to be phosphorylated, and these modifications help to orchestrate the interplay between transcription and processing of messenger RNA (mRNA) precursors. Here, we provide evidence that phosphorylation of CTD Thr4 residues is required specifically for histone mRNA 3 end processing, functioning to facilitate recruitment of 3 processing factors to histone genes. Like Ser2, Thr4 phosphorylation requires the CTD kinase CDK9 and is evolutionarily conserved from yeast to human. Our data thus illustrate how a CTD modification can play a highly specific role in facilitating efficient gene expression. he carboxyl-terminal domain (CTD) of the RNA polymerase II (RNAP II) largest subunit (Rpb1) consists of Tyr1-Ser2-Pro34 Thr -Ser5-Pro6-Ser7 (YSPTSPS) consensus tandem repeats, which are conserved from yeast to human. The CTD, through phosphorylation on serine residues, links transcription to mRNA pro-
cessing events (13). Ser5 is phosphorylated by cyclin-dependent kinase 7, CDK7, a subunit of the general transcription factor TFIIH, and this modification functions to facilitate capping (4, 5). During transcriptional elongation, Ser2 is phosphorylated by CDK9/P-TEFb, which helps to coordinate RNA 3 end processing and transcription SCIENCE VOL 334
termination (6, 7). Ser7 phosphorylation has been implicated in transcription and 3 end processing of genes encoding certain small noncoding RNAs (8, 9). Tyr1 can also be phosphorylated by the c-Abl kinase (10). Although Thr4 has been reported to be phosphorylated in fission yeast (11), there is no evidence that this residue is modified in other species or what might the function of Thr4 be. To investigate CTD function in a genetically tractable vertebrate cell system, we created an Rpb1 conditional knockout chicken cell line, DT40-Rpb1, by using methods developed previously to study other conserved proteins (12, 13) (fig. S1). These cells express, as the only source of Rpb1, a tetracycline (tet)-repressible cDNA encoding hemagglutinin (HA)tagged human Rpb1 (human and chicken Rpb1 are 97% identical, and the CTD is very highly conserved among vertebrates; fig. S2). After addition of tet, Rpb1 became undetectable between 12 and 18 hours (Fig. 1A and fig. S3), and DT40-Rpb1 cells stopped
Department of Biological Sciences, Columbia University, New York, NY 10027, USA. *To whom correspondence should be addressed. E-mail: jlm2@columbia.edu
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growing and began to die by 24 hours (Fig. 1B). An important use of DT40 derivatives such as DT40-Rpb1 is for genetic complementation [e.g., (14)]. Here, a plasmid expressing FLAG-tagged wild-type (WT) Rpb1 was found to restore viability to DT40-Rpb1 cells in the presence of tet. Expression of an Rpb1 derivative (26r) containing 26 all-consensus repeats plus the 10 C-terminal most residues that confer stability (15), also restored viability. However, a comparable derivative containing 30 repeats with all Thr4 residues mutated to Val (T4V) was inviable. All three Rpb1 derivatives were expressed equivalently (Fig. 1C), and WT and 26r-expressing cells grew similarly after addition of tet, whereas T4V cells stopped growing and began dying by 24 hours (Fig. 1D). We next investigated the possible basis for T4V cell inviability. Comparison of overall transcription (by 3H pulse labeling) in WT, 26r, and T4V cells treated with tet for 40 hours (Fig. 2A) revealed only a modest decrease (20%) in polyadenylated [poly (A)+ ] and poly (A) RNA fractions in T4V compared with 26r cells, indicating that total transcription and synthesis of poly (A)+ mRNA was not significantly affected by the T4V substitutions. A 20-min induction of the inducible Egr1 gene (16) in T4V and 26r cells, grown in tet for 24 hours, resulted in an equivalent ~300-fold mRNA increase in both (Fig. 2B). Additionally, no differences were observed in unspliced versus spliced and uncleaved versus total (a measure of 3 processing) Egr1 mRNA (fig. S4), and 3 processing of several additional poly (A)+ mRNAs was similar in T4V and 26r cells (fig. S5). Thus, transcription and processing of poly (A)+ mRNAs was not significantly affected in T4V cells. We next measured accumulation of several types of RNAP II products in T4V and 26r cells treated with tet for 40 hours (Fig. 2C). Levels of U1 snRNA were unaffected, as were levels of two poly (A)+ mRNAs, RplPl and glyceraldehyde-3phosphate dehydrogenase (GAPDH). However, levels of all four nonpoly (A)+ replication-dependent histone mRNAs were significantly reduced in two independent T4V cell lines, to ~15% the levels observed in 26r cells. Consistent with this, histone H3 protein levels were reduced in T4V cells ~50% after 30 or 40 hours of tet treatment (Fig. 2D). Levels of other proteins tested were not significantly affected (Fig. 2D and fig. S6). Decreased histone gene transcription could explain the defect in histone mRNA accumulation in T4V cells. However, nuclear run-on analysis showed no differences, or even a slight increase, with three histone genes in 26r and T4V cells after 24 or 40 hours of tet treatment (fig. S7, A and B). We also performed chromatin immunoprecipitation (ChIP) with antibodies against Rpb1 (N20) in T4V and 26r cells treated with tet for 30 hours. RNAP II levels at the transcription start site (TSS) and 3 end of H2A, H2B, and H3 genes were not reduced, and indeed slightly elevated at the TSS, in T4V cells (Fig. 3A, left). Similar results were observed along the length
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Fig. 2. T4V cells are defective in histone expression. (A) WT, 26r, and T4V cells were treated with tet for 40 hours; RNA was labeled for 30 min and purified; and poly (A)+ and poly (A) fractions quantitated by scintillation counting. 26r T4V Cells per minute (CPM) counts relative to 26r are shown. Cell line N = 3. (B) T4V and 26r cells were treated with tet for 24 hours before induction with ionomycin and phorbol 12-myristate 13-acetate (PMA) for 20 min. EGR1 mRNA levels were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) following normalization to 18S RNA levels. N = 3. (C) T4V (two independent lines) and 26r cells were treated with tet for 40 hours. RNA was isolated, and indicated RNAs quantitated by RT-qPCR, normalized to 5S RNA levels, and displayed relative to 26r. N = 3. (D) T4V and 26r cells were treated with tet for 30 or 40 hours. Cell lysates were analyzed by Western blotting (top). Ratios of H3/actin and H3/GAPDH were determined and are displayed relative to values obtained with 26r cells. N = 3. Error bars display standard deviation. VOL 334 SCIENCE www.sciencemag.org
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of the RPLP1 gene (Fig. 3A, right). We conclude that histone gene transcription was unaffected in T4V mutant cells. The role of the CTD in 3 end formation suggested that Thr4 might be required specifically in histone mRNA 3 formation, which uses a distinct mechanism from poly (A)+ mRNAs (17). When histone 3 processing is defective, cryptic downstream polyadenylation sites can be used,
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Fig. 3. T4V cells are defective in histone mRNA 3 end processing. (A) Rpb1 ChIP. Diagrams depict genes analyzed. Thick lines represent genes, and dashed lines, transcripts. Triangles denote 3 cleavage sites. Positions of amplicons are indicated. T4V and 26r cells were grown in the presence of tet for 30 hours then used for ChIP. DNA samples were analyzed by qPCR. Signals were normalized as described in Materials and Methods. N = 3. Fig. 4. Thr4 is phosphorylated throughout eukaryotes and requires CDK 9. (A) T4V and 26r cells were treated with tet for 30 hours. Western blotting of input and Flag IP samples was done with P-Thr4 (right) and N20 (left) antibodies. (B) Total cell lysates from yeast, KC, 26r, and HeLa cells were prepared and used for Western blotting. Total Rpb1 was detected by 8WG16 (left), and Thr4-phosphorylated Rpb1, with P-Thr4 (right). Band indicated with an asterisk is a cross-reacting protein. (C) DT40 cells were treated with tet for 24 hours, the indicated concentrations of flavopiridol were added, and cells grown for an additional 6 hours. Cell lysates were analyzed by Western blotting. (D) Lysates from 26r and S2A cells treated with tet for 30 hours were analyzed by Western blotting.
(B) T4V and 26r cells were treated with tet for 40 hours, RNA was isolated, and RT was performed by using oligo-dT or random hexamer oligos. qPCR was performed; values were normalized to Rplp1 mRNA levels and are shown relative to 26r (top). The ratio of poly (A)+/total histone transcripts was calculated and plotted (bottom). N = 3. (C) CPSF100 ChIP. N = 5. (D) SLBP ChIP. N = 3. Error bars indicate standard deviation.
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resulting in formation of poly (A)+ histone mRNAs (18). We grew 26r and T4V cells in the presence of tet for 40 hours, and, for all four histone genes, ~twofold more poly (A)+ mRNA was recovered from T4V cells than from 26r cells (Fig. 3B, top), and the fraction of histone mRNA in these cells that was poly (A)+ increased 10- to 50-fold (Fig. 3B, bottom). Histone pre-mRNA 3 processing requires a complex set of evolutionarily conserved factors, only some of which are shared with the poly (A)+ mRNA 3 processing machinery (17). Using ChIP, we examined recruitment of one common [cleavage polyadenylation specificity factor 100 (CPSF100)] and one histone-specific [stem loop binding protein (SLBP)] factor to several histone genes and to the RPLP1 gene. CPSF-100 levels on the RPLP1 gene peaked at the promoter, consistent with previous observations [e.g., (19)], and were equivalent in 26r and T4V cells treated with tet for 30 hours (Fig. 3C, right). On the three histone genes tested, CPSF-100 levels were slightly reduced at the TSS in T4V cells but significantly lower at the 3 end (Fig. 3C, left), consistent with the defect in 3 processing. As expected in 26r cells, SLBP was detected at background levels on the RPLP1 gene (Fig. 3D), whereas significant levels were found associated with the two histone genes tested. However, SLBP levels were significantly reduced both at the TSS and at the 3 processing site in T4V cells (Fig. 3D). Total levels of SLBP, CPSF-100, and other 3 processing factors tested were equivalent in the two cell lines (Fig. 2D and fig. S6). The Thr to Val mutation is a conservative change, and our results thus suggest that modification (e.g., phosphorylation) of Thr4 is important for its function. To investigate whether Thr4 is phosphorylated, we used an antibody raised against and specific for a Thr4-phosphorylated CTD heptad repeat (P-Thr4; fig. S8). Immunoprecipitation (IP)-Westerns with extracts from 26r and T4V cells, using anti-FLAG for IP and P-Thr4 for Western (Fig. 4A), detected a band corresponding to a protein of the expected size in both input and IP from the 26r cells, but this species was detected only at very low levels in the T4V samples, indicating that Thr4 is phosphorylated in the 26r CTD. Thr4 is highly conserved throughout eukaryotes, and Western analysis with P-Thr4 of lysates from yeast, fly (KC), 26r, and human (HeLa) cells revealed a band that is the size expected for Thr4-phosphorylated Rpb1 in all samples (Fig. 4B). To identify the possible kinase responsible for Thr4 phosphorylation, we examined whether Thr4 phosphorylation in DT40 cells was sensitive to the CDK9/P-TEFb inhibitors DRB and flavopiridol (20) and found that it was strongly inhibited by both (Fig. 4C and fig. S9). This did not reflect a requirement for prior Ser2 phosphorylation, because Thr4 phosphorylation was detected in DT40-Rpb1 cells expressing an Rpb1 derivative with all Ser2 residues mutated to Ala2 (S2A) (Fig. 4D). Linking this to the defect in histone mRNA processing in T4V cells, histone mRNA levels in flavopiridol-treated 26r cells were reduced to levels found in T4V cells (fig. S10). Consistent with this, knockdown of CDK9 in human embryonic kidney 293 cells was previously shown to impair SLBP recruitment to histone genes and lead to accumulation of poly (A)+ histone mRNA (21). Our experiments have provided insight into the intricate mechanisms used by cells to couple transcription by RNAP II to subsequent RNA processing. Our data provide evidence that histone mRNA 3 end formation specifically requires Thr4 phosphorylation, but it is likely that this modification is important for other CTD functions. Although our experiments have not uncovered evidence for this, the fact that Thr4 is phosphorylated in yeast, which produces histone mRNA 3 ends by the same mechanism as other mRNAs, suggests the existence of additional functions in mRNA synthesis and/or processing.
Y. Hirose, J. L. Manley, Genes Dev. 14, 1415 (2000). T. Maniatis, R. Reed, Nature 416, 499 (2002). M. J. Moore, N. J. Proudfoot, Cell 136, 688 (2009). P. Komarnitsky, E. J. Cho, S. Buratowski, Genes Dev. 14, 2452 (2000). 5. S. C. Schroeder, B. Schwer, S. Shuman, D. L. Bentley, Genes Dev. 14, 2435 (2000). 6. S. H. Ahn, M. Kim, S. Buratowski, Mol. Cell 13, 67 (2004). 7. Z. Ni, B. E. Schwartz, J. Werner, J.-R. Suarez, J. T. Lis, Mol. Cell 13, 55 (2004). 8. R. D. Chapman et al., Science 318, 1780 (2007). 9. S. Egloff et al., Science 318, 1777 (2007). 10. R. Baskaran, M. E. Dahmus, J. Y. J. Wang, Proc. Natl. Acad. Sci. U.S.A. 90, 11167 (1993). 11. H. Sakurai, A. Ishihama, Genes Cells 7, 273 (2002). 12. J. Wang, Y. Takagaki, J. L. Manley, Genes Dev. 10, 2588 (1996). 13. Y. Takagaki, J. L. Manley, Mol. Cell 2, 761 (1998). 14. J. Wang, S. H. Xiao, J. L. Manley, Genes Dev. 12, 2222 (1998). 15. R. D. Chapman, M. Conrad, D. Eick, Mol. Cell. Biol. 25, 7665 (2005). 16. C. Y. Chen, L. W. Forman, D. V. Faller, Mol. Cell. Biol. 16, 6582 (1996). 17. W. F. Marzluff, E. J. Wagner, R. J. Duronio, Nat. Rev. Genet. 9, 843 (2008). 18. K. D. Sullivan, T. E. Mullen, W. F. Marzluff, E. J. Wagner, RNA 15, 459 (2009). 19. O. Rozenblatt-Rosen et al., Proc. Natl. Acad. Sci. U.S.A. 106, 755 (2009). 20. S. H. Chao, D. H. Price, J. Biol. Chem. 276, 31793 (2001). 21. J. Pirngruber et al., EMBO Rep. 10, 894 (2009). Acknowledgments: We thank T. Kashima for plasmids and help with construction of the DT40-Rpb1 cell line, W. Marzluff for the SLBP antibody, Z. Dominski for the Lsm11 antibody, and Novus Biologicals for the P-Thr4 antibody. This work was supported by NIH grant R01 GM28983. 1. 2. 3. 4.

www.sciencemag.org/cgi/content/full/334/6056/683/DC1 Materials and Methods Figs. S1 to S10 References 24 March 2011; accepted 29 August 2011 10.1126/science.1206034
Drosophila CENH3 Is Sufficient for Centromere Formation

Mara Jos Mendiburo,1,2 Jan Padeken,1,2 Stefanie Flp,3 Aloys Schepers,3 Patrick Heun1,4* CENH3 is a centromere-specific histone H3 variant essential for kinetochore assembly. Despite its central role in centromere function, there has been no conclusive evidence supporting CENH3 as sufficient to determine centromere identity. To address this question, we artificially targeted Drosophila CENH3 (CENP-A/CID) as a CID-GFP-LacI fusion protein to stably integrated lac operator (lacO) arrays. This ectopic CID focus assembles a functional kinetochore and directs incorporation of CID molecules without the LacI-anchor, providing evidence for the self-propagation of the epigenetic mark. CID-GFP-LacIbound extrachromosomal lacO plasmids can assemble kinetochore proteins and bind microtubules, resulting in their stable transmission for several cell generations even after eliminating CID-GFP-LacI. We conclude that CID is both necessary and sufficient to serve as an epigenetic centromere mark and nucleate heritable centromere function.
faithfully propagated through each cell cycle. With the exception of Saccharomyces cerevisiae, DNA sequence is considered neither necessary nor sufficient to mark centromeres in most eukaryotes, suggesting that centromere identity in many organisms is determined epigenetically (1). CENH3 (CENP-A in mammals, CID in Drosophila) is a centromere-specific histone H3 variant that replaces canonical histone H3 in centromeric nucleosomes. It is essential for centromere function and kinetochore assembly (25)
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and microtubule attachment. Most eukaryotic organisms contain only one centromere per chromosome, which is specifically positioned and VOL 334 SCIENCE
1 Max Planck Institute of Immunobiology, Stbeweg 51, 79108 Freiburg, Germany. 2Albert-Ludwigs-Universitt, Freiburg, Germany. 3Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Marchioninistrasse 25, 81377 Munich, Germany. 4BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universitt, Freiburg, Germany.
*To whom correspondence should be addressed. E-mail: heun@ie-freiburg.mpg.de
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and thus a prime candidate epigenetic mark for determining centromere identity. Global misincorporation of CID into chromosome arms leads to the formation of functional ectopic kinetochores only in a small subset of sites (6, 7), hindering a direct correlation between CID presence and kinetochore formation. To determine whether CID is sufficient for directing kinetochore assembly and nucleate centromere identity, we targeted a CID-GFP-Lac Repressor (LacI) fusion protein to an array of lac operator (lacO) sequences stably integrated in Drosophila Schneider S2 cells (Fig. 1A and fig. S1B). Inducible CID-GFP-LacI or GFP-LacI fusion proteins are efficiently targeted to lacO sequences only upon pulse induction (Fig. 1B), whereas low leaky expression of CID-GFP-LacI in uninduced cells correlates with an exclusive centromere localization (Fig. 1B and fig. S1A). CENH3 has been shown to adopt a specialized nucleosomal structure, which is proposed to mark centromeric chromatin (814). To determine whether CID-GFP-LacI is incorporated in nucleosomes at the lacO, mononucleosomes from GFP-LacI and CID-GFP-LacI cells were separated on a sucrose gradient. Analysis of the fractions from Fig. 1. CID-GFP-LacI is efficiently targeted to lacO sequences and induces kinetochore assembly. (A) Schematic representation of the experimental setup. (B) Examples of the lacO-containing chromosome 3 from fixed mitotic CID-GFP-LacI and GFP-LacI cells before and after induction with CuSO4 are shown. (C) Fixed mitotic chromosomes of CID-GFPLacI cells were processed 1 day after pulse induction for immunofluorescence (IF) with antibodies to GFP and CENP-C or NDC80. The lacO-containing chromosome is indicated by an asterisk and shown magnified in the right panels. (D) Same as on (B), with GFP-LacI cells 1 day after the pulse induction. (E) Quantification of the percentage of cells showing colocalization between GFP and different kinetochore proteins in the lacO for the CID-GFP-LacI and GFP-LacI cells similar to (A), (B), and fig. S2, A and B. Values are average T SD of at least three independent experiments. CENP-C, nCID-GFP-LacI = 33, nGFP-LacI = 43. MAD2, nCID-GFP-LacI = 38, nGFP-LacI = 37. POLO, nCID-GFP-LacI = 36, nGFP-LacI = 56. NDC80, nCID-GFP-LacI = 33, nGFP-LacI = 52. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 (two-sided Fishers exact test). White arrows indicate endogenous centromeres and green arrows the lacO integration in all panels. Scale bars, 3 mm. both cell lines revealed that lacO DNA comigrates with fractions containing H3-nucleosomes, suggesting that they are chromatinized (fig. S2, A and B). GFP-LacI did not comigrate with nucleosomes, likely due to its release from lacO binding after micrococcal nuclease treatment. In contrast, CID-GFP-LacI was found in nucleosomecontaining fractions, indicating that it can interact with chromatin independently of the LacI binding. Nucleosome incorporation was confirmed by coimmunoprecipitation of CID-GFP-LacI with histone H2A in the nucleosome fraction (fig. S2C). To exclude that CID-GFP-LacI migration pattern is only due to the contribution of protein localized to endogenous centromeres, we created a mutant CID fused to GFP-LacI that does not target to centromeres and is therefore present only at the lacO array (fig. S2E). In this mutated protein, three amino acids [G175S, L177V, and L178M (CIDsvm)] in the CENP-A targeting domain (CATD) of CID are replaced by the corresponding residues of histone H3.1, as shown previously for human CENP-A (15) (fig. S2D). The migration pattern of CIDsvmGFP-LacI in sucrose gradients resembles that of CID-GFP-LacI (fig. S2A), supporting the notion that CID-GFP-LacI is in nucleosomes also at lacO regions. Targeting of CID-GFP-LacI, but not GFPLacI, to the lacO recruited kinetochore proteins, such as CENP-C and the microtubule-binding protein NDC80/HEC1, 1 day after pulse induction (Fig. 1, C to E) (13, 14, 16). Probing for the spindle assembly checkpoint proteins POLO kinase and MAD2 revealed the same pattern, suggesting that ectopically targeted CID is sufficient to direct kinetochore assembly (Fig. 1E and fig. S1, C and D). The formation of a functional ectopic kinetochore at the lacO is expected to generate a dicentric chromosome causing chromosome breakage and anaphase defects (12). Indeed, we observe a higher number of aberrant mitotic chromosome configurations in cells expressing CID-GFP-LacI compared with control GFP-lacI cells and containing the lacO array (Fig. 2). The epigenetic model for centromere identity predicts that a functional centromere self-directs the loading of new centromeric marks after each cell division (1). If CID-GFP-LacI identifies the lacO as a centromere, CID molecules without a LacI fusion should also be recruited to lacO
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regions. To investigate this, we created a stable cell line containing both an inducible CID-GFP-LacI and a constitutively expressed CID-hemagglutinin (HA) HA construct. Upon pulse induction and targeting of CID-GFP-LacI, we found a low initial CID-HA recruitment to the lacO (9.7%) (Fig. 3A), increasing by two-fold 7 days later (24%) (Fig. 3B). Higher-resolution analysis using stretched chromatin fibers revealed that at initial time points CID-HA localization is restricted to the lacO region (fig. S3A), whereas 7 days Fig. 2. The lacO-containing chromosome is involved in mitotic defects and fragmentation. (A) IF fluorescence in situ hybridization (IF-FISH) on fixed mitotic chromosomes of CID-GFP-LacI cells 1 day after induction reveals a GFP-positive lacO-containing fragment (indicated by an asterisk and magnified in the insets). (B) Example of a mitotic chromosome breaking within the lacO sequence upon CID-GFPLacI targeting (green arrow). The white arrow indicates the endogenous centromere. (C) Lagging chromosome containing CID-GFP-LacI colocalizing with lacO sequences (white arrow). Grayscale images of individual channels are shown below. Scale bars, 3 mm. (D) Frequency of anaphase figures with defects involving lacO sequences as shown in (C). Horizontal bars are average of two independent experiments. nCID-GFP-LacI = 48, nGFP-LacI = 109. (E) Fragmented lacO-containing chromosomes in CID-GFP-LacI and GFP-LacI cells as shown in (A) and (B). Values are average T SD of at least two independent experiments. nCID-GFP-LacI = 42, nGFP-LacI = 47. *, P < 0.05; **, P < 0.005 (two-sided Fishers exact test). after pulse induction, CID-HA (Fig. 3C) or CID (fig. S3B) is also found spreading into adjacent regions (Fig. 3D). CID-HA or CID were never found in lacO sequences in the presence of GFP-LacI (fig. S3C). CID-GFP-LacI targeting to an ectopic chromosomal locus results in mitotically unstable dicentric chromosomes, hindering the analysis of long-term inheritance of the centromere function. To directly test de novo centromere heritability, we analyzed whether CID-GFP-LacI targeting confers mitotic stability to a centromere-devoid episomal DNA element carrying lacO sequences and a G418-resistance cassette. This plasmid was transfected into S2 cell lines constitutively expressing low levels of either CID-GFP-LacI or GFP-LacI and kept under selection pressure for 28 days (Fig. 4A) (17, 18). The initial transfection efficiency was comparable in both cell lines (Fig. 4B, DpnI). However, we observed a decline in the amount of replicated plasmids (+DpnI) in GFP-LacIexpressing cells, whereas CID-GFP-
Fig. 3. CID tethered to lacO sites creates a selfpropagating epigenetic mark. CID-GFP-LacI expression was induced in the presence of constitutively low levels of CID-HA. Representative images of mitotic chromosomes 1 day (A) and 1 week (B) after pulse induction are shown. White arrows indicate endogenous centromeres, green arrows the lacO site. The percentages of cells where CID-GFP-LacI sites colocalize with CID-HA are shown. n1day = 143, n7days = 29. (C) IF-FISH on a stretched chromatin fiber 1 week after pulse induction. White arrows indicate spreading CID-HA. Dashed white lines delimit the lacO sequence. Scale bars, 3 mm. (D) The distance between the most distal HA signal relative to the lacO was measured and normalized to the size of the lacO FISH signal to correct for differential stretching. n1day = 11, n7days = 19. *, P < 0.05 (two-tailed Students t test).
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LacI cells maintained the plasmid until the end of the experiment (Fig. 4B, +DpnI). Efficient replication and proper segregation are the two major factors defining the stability of plasmids (18). For the first 7 days, both cell lines replicated the plasmid with similar efficiency (Fig. 4B and fig. S5, F and G), indicating that correct segregation is the main factor contributing to its maintenance in CID-GFP-LacI cells. This was also observed when selection was removed after 16 days or never applied (fig. S5E). Furthermore, we find CENP-C, NDC80, and microtubule connections localizing to CID-GFP-LacIbound lacO plasmids in mitosis (Fig. 4, C and D, and fig. S4, A and C). These plasmids separate earlier than endogenous chromosomes and generally display a symmetric distribution on the spindle poles (fig. S4, B and C). GFP-LacI cells grew poorly after transfection, and the few mitotic cells detected showed no colocalization of GFP-positive lacO plasmids with either CENP-C or NDC80 (fig. S4D). Further characterization of recovered plasmids showed that plasmid size remained stable except for variability in the repetitive lacO array and did not acquire endogenous Drosophila sequences (fig. S5, A to C). A minor fraction of concatenated plasmids did not correlate with efficient recruitment of kinetochore proteins (fig. S5D). The centromeric mark can self-propagate in the lacO plasmid, as shown by the recruitment of CID-HA to targeted CID-GFP-LacI plasmids and the spreading of both into backbone sequences (Fig. 4E and figs. S5C and S6A). To determine whether centromeric chromatin is inherited even after removal of CID-GFP-LacI targeting, we transiently cotransfected the lacO plasmid and a CID-GFP-LacI expression construct to deliver an initial expression pulse that is progressively lost. Episomal lacO plasmids were detected as nonchromosomal CID/CENP-C foci, with or without a GFP signal. Early after transfection, we found cells that contained both GFP-positive and -negative foci (green bars in Fig. 4F) and a smaller cell population that had already lost CID-GFPLacI expression but contained nonchromosomal CID/CENP-C foci (black bars in Fig. 4F). The percentage of these GFP-negative plasmids increased until day 27, when all detected CID/CENP-C foci were devoid of any GFP signal (Fig. 4F and fig. S6, B to E). We conclude that an initial targeting of CID-GFP-LacI to the lacO plasmid enables the nucleation of centromere function, which is maintained independently of CID-GFP-LacI expression and targeting. In recent years, functional biosynthetic kinetochores have been constructed by targeting kinetochore components (1921) and chromatin modifiers (22, 23) to minichromosomes. Even though the possibility of bypassing CENH3 chromatin for the assembly of a kinetochore suggested that CENH3 is dispensable for this function (21), no evidence of centromere heritability was provided by these studies. Recent reports showing CENP-Adependent kinetochore formation in vitro (24) or by artificially targeting the CENP-A loading factor in humans (HJURP) (25) further stress the importance of CENH3 in kinetochore assembly. The results presented here show that CENH3 behaves as a true centromeric epigenetic mark not only by being sufficient for the recruitment of kinetochore proteins during mitosis but also for directing its own incorporation and maintaining centromere function through each cell division.
1. R. C. Allshire, G. H. Karpen, Nat. Rev. Genet. 9, 923 (2008). 2. M. R. Przewloka et al., PLoS ONE 2, e478 (2007). 3. V. Rgnier et al., Mol. Cell. Biol. 25, 3967 (2005). 4. M. D. Blower, G. H. Karpen, Nat. Cell Biol. 3, 730 (2001). 5. L. E. Jansen, B. E. Black, D. R. Foltz, D. W. Cleveland, J. Cell Biol. 176, 795 (2007). 6. P. Heun et al., Dev. Cell 10, 303 (2006). 7. A. M. Olszak et al., Nat. Cell Biol. 13, 799 (2011). 8. T. Furuyama, S. Henikoff, Cell 138, 104 (2009). 9. Y. Dalal, H. Wang, S. Lindsay, S. Henikoff, PLoS Biol. 5, e218 (2007). 10. N. Sekulic, E. A. Bassett, D. J. Rogers, B. E. Black, Nature 467, 347 (2010). 11. B. E. Black et al., Nature 430, 578 (2004). 12. B. McClintock, Genetics 26, 234 (1941). 13. I. M. Cheeseman, A. Desai, Nat. Rev. Mol. Cell Biol. 9, 33 (2008). 14. D. R. Foltz et al., Nat. Cell Biol. 8, 458 (2006). 15. B. E. Black et al., Mol. Cell 25, 309 (2007). 16. R. D. Vierstra, J. Callis, Plant Mol. Biol. 41, 435 (1999). 17. B. Hirt, J. Mol. Biol. 26, 365 (1967). 18. D. Pich, S. Humme, M. P. Spindler, A. Schepers, W. Hammerschmidt, Nucleic Acids Res. 36, e83 (2008). 19. S. Lacefield, D. T. Lau, A. W. Murray, Nat. Cell Biol. 11, 1116 (2009). 20. E. Kiermaier, S. Woehrer, Y. Peng, K. Mechtler, S. Westermann, Nat. Cell Biol. 11, 1109 (2009). 21. K. E. Gascoigne et al., Cell 145, 410 (2011). 22. M. Nakano et al., Dev. Cell 14, 507 (2008). 23. A. Kagansky et al., Science 324, 1716 (2009). 24. A. Guse, C. W. Carroll, B. Moree, C. J. Fuller, A. F. Straight, Nature 477, 354 (2011). 25. M. C. Barnhart et al., J. Cell Biol. 194, 229 (2011).
Fig. 4. CID-GFP-LacI confers stability to episomal lacO plasmids and allows epigenetic inheritance of centric chromatin after its elimination. (A) Schematic representation of the plasmid rescue assay. (B) The average of three independent plasmid rescue experiments is shown T SEM. tc, transfection control; utc, untransfected control. K = 1000. (C) Immunostainings of CID-GFP-LacI cells in metaphase 9 days after transfection with a lacO plasmid. (D) Similar to (C), showing microtubule-plasmid attachments. (E) Cells described in (A) were cotransfected with a CID-HA expression construct and the lacO plasmid and subjected to aHA and aGFPchromatin immunoprecipitation experiments 6 days later. (F) Quantification of the percentage of extrachromosomal CID/CENP-C foci in mitotic cells after transient cotransfection of lacO and CID-GFP-LacI constructs. Black: GFP-negative CID/CENP-C foci coming from GFP-negative cells. Green: Foci from cells that contain at least one GFP-positive focus (GFP-positive cells). Scale bars, 3 mm. www.sciencemag.org SCIENCE VOL 334
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Acknowledgments: We thank G. H. Karpen, A. Straight, T. Maresca, E. Salmon, C. Sunkel, S. Heidmann, and C. Lehner for reagents. We also thank L. Jansen for critical reading of the manuscript and the members of the Grosschedl Department, particularly S. Chlamydas for pioneering experiments and A. Olszak, H.-J. Schwarz, D. Venegas, S. Daujat, S. Saccani, and D. van Essen for reagents and helpful discussions. A.S. is supported by the Sonderforschungsbereich (SFB) 646, SFB/TR05, and Schwerpunktprogramm 1230 of the Deutsche Forschungsgemeinschaft. P.H. is funded by the Max Planck Society, the Human Frontier Science Program (Career Development Award; CDA), and the Excellence Initiative of the German Federal and State Governments (EXC 294). M.-J.M. and J.P. are grateful for an International Max Planck Research School (IMPRS) fellowship provided by the Max Planck Institute of Immunobiology and Epigenetics.

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12 April 2011; accepted 19 September 2011 10.1126/science.1206880
Exercise and Genetic Rescue of SCA1 via the Transcriptional Repressor Capicua
John D. Fryer,1* Peng Yu,1 Hyojin Kang,1 Caleigh Mandel-Brehm,1 Angela N. Carter,2 Juan Crespo-Barreto,1 Yan Gao,1 Adriano Flora,1 Chad Shaw,1,3 Harry T. Orr,4 Huda Y. Zoghbi1,2,5,6,7 Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by up-regulation of epidermal growth factor and consequential down-regulation of Capicua, which is an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of all disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reduction of Capicua levelseither genetically or by exercisemitigated the disease phenotypes by dampening the toxic gain of function. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases. pinocerebellar ataxia type 1 (SCA1) is characterized by a progressive loss of motor skills, usually beginning with impaired gait and balance (1). As with other neurodegenerative diseases, the disease protein Ataxin-1 (ATXN1) is abundantly expressed in most neurons, yet some neuronal populations are more vulnerable than others. In SCA1, cerebellar Purkinje cells
are first to show dysfunction; eventually, other neuronal populationsincluding deep cerebellar and brainstem nucleiare affected, leading to premature death (2). Although exercise has beneficial effects on many brain functions (3), it is not clear whether it would be protective in SCA1 or would accelerate neuronal demise by increasing the activity and metabolic demands on these
1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. 2Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA. 3Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX 77030, USA. 4Institute of Translational Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA. 5Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA. 6Jan and Dan Duncan Neurological Research Institute, Texas Childrens Hospital, Houston, TX 77030, USA. 7Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
*Present address: Department of Neuroscience, Mayo Clinic, FL 32224, USA. Present address: Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. To whom correspondence should be addressed. E-mail: hzoghbi@bcm.edu
Fig. 1. Exercise extends life span in Atxn1154Q mice through up-regulation of EGF and consequential downregulation of Cic. (A) Median survival was 272 days for nonexercised Atxn1154Q mice versus 317 days for exercised Atxn1154Q mice (45-day extension, n = 6 mice per group, P < 0.01 by log-rank test). (B) Exercise caused a 50% increase in brainstem EGF levels in WT mice and a 74% increase in Atxn1154Q mice measured with enzymelinked immunosorbent assay (mean T SEM, n = 5 mice per group). (C) Exercise caused a 24 and 22% decrease in brainstem Cic levels in WT and Atxn1154Q mice, respectively, measured with quantitative reverse transcription polymerase chain reaction RT-PCR (mean T SEM, n = 5 mice per group). (D) Western blotting for Cic demonstrates an exercise-induced decrease in Cic levels in the brainstem,
whereas Atxn1 or Atxn1154Q remain unaffected. (E) Primary brainstem neuronal cultures treated with recombinant EGF for 72 hours show a dosedependent decrease in the level of Cic but not Atxn1. SCIENCE www.sciencemag.org
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already vulnerable neuronal populations, as has been suggested for other neurodegenerative diseases (4, 5). To determine the effects of exercise in SCA1, we implemented a mild exercise regimen in Atxn1154Q knock-in mice, which bear 154 CAG repeats targeted into the endogenous mouse locus so as to create a model that recapitulates all aspects of SCA1 (6). From 4 to 8 weeks of age, wild-type (WT) or Atxn1154Q mice were placed on a fixed-speed rotarod apparatus five times per week, whereas control mice were placed on an
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immobile rotarod apparatus. At 10 weeks of age, we found no significant improvements in motor performance (fig. S1, A and B), but Atxn1154Q mice that were exercised showed a remarkable and highly significant extension of life span (Fig. 1A). To determine the molecular mechanism of this rescue, we measured the expression of several growth factors in vulnerable tissues (cerebellum and brainstem) 1 week after the last exercise session. We found a sustained increase in the level of epidermal growth factor (EGF) in the brainstem but not the cerebellum (Fig. 1B and fig. S1). Because Drosophila data indicate that Capicua (Cic) lies downstream of EGF signaling (7), and we previously showed that Cic interacts with Atxn1 (8), we measured Cic levels after exercise and found a significant decrease in the brainstem (Fig. 1, C and D) but not the cerebellum (fig. S1B). Exercise did not affect brainstem Atxn1154Q protein levels (fig. S1F). Additionally, primary brainstem neuronal cultures treated with recombinant EGF for 72 hours showed a dosedependent decrease in Cic levels (Fig. 1E). Thus, EGF regulates Cic levels in vivo and in vitro, and reduction of Cic might modulate the survival of Atxn1154Q mice. To formally test the effect of reduced Cic levels on SCA1 phenotypes, we generated a loss-of-function allele of Cic and back-crossed these mice onto a C57BL/6 strain (fig. S2A). Two isoforms of Cic, long (Cic-L) and short (Cic-S), are transcribed from alternative promoters. Cic-L/ mice completely lack the Cic-L isoform, with ~10% of Cic-S remaining (fig. S2B). Cic-L+/ mice had a ~50% reduction of both Cic-L and Cic-S. Cic protein is reduced in the Atxn1/ cerebellum (8), and we also found a Cic dose-dependent reduction of Atxn1 and its paralog Ataxin-1Like (Atxn1L) in WT, Cic-L+/, and Cic-L/ cerebella (fig. S2B), confirming the interdependency of Cic and Atxn1 paralog proteins in vivo. To determine whether reducing the level of Cic would affect disease course in SCA1, we bred Atxn1154Q male mice to Cic-L+/ female mice in order to generate WT, Cic-L+/, Atxn1154Q, and Atxn1154Q;Cic-L+/ mice, all on a pure C57BL/6 background. Because of their motor impairments, Atxn1154Q mice showed less total locomotor activity in the open-field assay than did WT mice, but Atxn1154Q;Cic-L+/ mice performed significantly better than did their Atxn1154Q littermates (Fig. 2A). Reduced Cic levels also significantly improved the motor coordination defects normally seen in Atxn1154Q mice (Fig. 2, B and C). Reduced Cic levels also improved the learning and memory deficits that Atxn1154Q mice normally exhibit in the conditioned fear assay (Fig. 2D). These improved phenotypes were accompanied by reduction in neuropathology: Atxn1154Q;Cic-L+/ mice had significantly more Purkinje cells than did Atxn1154Q mice at 40 weeks of age (Fig. 3, A and B). Thus, a 50% reduction of Cic levels is enough to mitigate the behavioral defects and Purkinje cell loss in Atxn1154Q mice. Reduction of Cic levels also improved phenotypes that could be attributed to other brain regions. After weaning, Atxn1154Q mice lost weight progressively, but loss of one Cic allele was enough to significantly rescue this weight loss (Fig. 3C).
Fig. 2. Reduction of Cic rescues multiple behaviors in SCA1 mice. Reduction of Cic by 50% in Atxn1154Q mice resulted in motor improvements in the (A) open-field assay, (B) dowel test, (C) rotarod, and (D) conditioned fear assay of learning and memory. Legend for each panel is indicated in (B). Values represent mean T SEM with n = 9 to 10 mice per genotype. Analysis of variance (ANOVA) with post-hoc t tests were performed. #P < 0.05, ##P < 0.01 between WT and Atxn1154Q mice; *P < 0.05, **P < 0.01 between Atxn1154Q and Atxn1154Q;Cic-L+/ mice.
Fig. 3. Reduction of Cic rescues multiple phenotypes in SCA1 mice. A 50% reduction of Cic in Atxn1154Q mice rescued (A) and (B) Purkinje cell integrity (n = 4 mice per genotype), (C) body weight, and (D) premature lethality (P < 0.05 by log-rank test). The median life span of Atxn1154Q mice in this cohort is in close agreement with the median life span from the unexercised Atxn1154Q cohort (Fig. 1), with some increased variability due to greater numbers of litters being used in this larger cohort. For body weight and lethality, n = 11 WT mice, n = 12 Cic-L+/ mice, n = 14 Atxn1154Q mice, and n = 16 Atxn1154Q;Cic-L+/ mice. www.sciencemag.org SCIENCE VOL 334
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As with the exercised Atxn1154Q mice, the median age of survival of the Atxn1154Q;Cic-L+/ mice was significantly older than that of Atxn1154Q mice (Fig. 3D). These data probably explain why exercise improved the longevity of Atxn1154Q mice but not the impaired coordination: Exercise reduced Cic levels only in the brainstem and not in the cerebellum. To determine the mechanism by which constitutive reduction of Cic rescues SCA1, we focused on the cerebellum, the primary site of dysfunction. We examined how the Atxn1154Q protein influenced the transcriptional repressor function of Cic by comparing microarrays of Cic-L/ cerebella (table S1) with previous microarrays from Atxn1154Q cerebellum (9). We identified many hyper-repressed genes (up-regulated in Cic-L/ but down-regulated in Atxn1154Q ), with >50% containing a Cic motif (table S2) (10). We selected several of these hyper-repressed target genes and found that genes that were down-regulated in Atxn1154Q cerebellum were in fact restored to near WT levels in Atxn1154Q; Cic-L+/ cerebellum, with significantly more Cic bound to their promoters in Atxn1154Q versus WT mice (Fig. 4A). This suggests that the mechanism of disease rescue is relief of Cic hyperrepression conferred by polyglutamine-expanded Atxn1. In Drosophila, overexpression of Atxn182Q is rescued by overexpression of Cic and is exacerbated by Cic reduction (8, 11). Although this is likely due to a titration of the endogenous Drosophila Cic away from its normal transcriptional targets, it suggests that polyglutamine-expanded Atxn1 could cause a loss of Cic transcriptional function (derepression). We identified many derepressed genes [up-regulated in both Cic-L/ and Atxn1154Q cerebella (table S3)] that were in fact even further up-regulated in Atxn1154Q;Cic-L+/ cerebellum, with significantly less Cic bound to their promoters in Atxn1154Q versus WT mice (Fig. 4B). This suggests that polyglutamineexpanded Atxn1 does indeed cause Cic to lose some transcriptional repressor activity, but this could not explain the genetic rescue; if partial loss of Cic activity was the driving factor in pathogenesis, the disease phenotype would be exacerbated in Atxn1154Q;Cic-L+/ mice. We propose that the polyglutamine-expanded Atxn1 causes Cic to bind more tightly to certain transcriptional targets (hyper-repressing them) while concomitantly causing Cic to bind less toand thus upregulate (derepress)other transcriptional targets. Genetic or exercise-induced reduction of Cic relieves the toxic hyper-repressive activity despite the concomitant loss of normal repressive function (fig. S3), which is consistent with the fact that the gain-of-function mechanism is the driving mechanism for toxicity in SCA1. Whereas other polyglutamine diseases are caused by a gain-of-function mechanism despite partial loss of function of the involved protein mediated through different protein partners (9, 1113), here we have demonstrated that polyglutamineexpanded protein can cause concomitant gain- and loss-of-function effects on the same native protein partner. The level of polyglutamine-expanded Atxn1 protein was reduced by ~9% in Atxn1154Q; Cic-L+/ mice as compared with Atxn1154Q mice (fig. S2, C and D), and although this could possibly contribute to the phenotypic rescue, we suggest that the rescue is more likely caused by relief of Cic-dependent hyper-repression. Therapeutics aimed at lowering Cic levels or disrupting the Cic-Atxn1154Q protein interaction could potentially ameliorate the disease. The effect of exercise on life span was long lasting, well after its discontinuation, underscoring the potential value of exercise beyond motor improvements. Thus, SCA1 individuals might benefit from an exercise program early in disease course (14, 15). Genetic reduction of Cic also increased the life span of Atxn1154Q mice (by 3.5 weeks), but the magnitude of the survival effect was greater (~6 weeks) in exercised Atxn1154Q mice. Thus, other pathways such as enhanced growth factor signaling are likely to contribute to the effect. We cannot rule out the possibility that more intense or longer-duration exercise could cause a sustained EGF increase and Cic decrease in the cerebellum that could lead to motor improvements. The exercise regimen we chose was quite gentle; a more rigorous or sustained exercise paradigm that engages the cerebellum more intensely might reduce cerebellar Cic levels and
Fig. 4. Atxn1154Q causes concomitant gain and loss of Cic transcriptional function. (A) Atxn1154Q hyper-repressed cerebellar transcripts containing a Cic motif, but these were normalized to near WT levels in Atxn1154Q;Cic-L+/ mice as measured with quantitative RT-PCR (mean T SEM, n = 6 mice per genotype), with more Cic bound to their promoters in Atxn1154Q versus WT littermates as measured with chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR, mean T SEM, n = 4 mice per genotype). (B)
Atxn1154Q also derepressed cerebellar transcripts containing a Cic motif, but these transcripts were further up-regulated in Atxn1154Q;Cic-L+/ mice through quantitative RT-PCR (mean T SEM, n = 6 mice per genotype), with less Cic bound to their promoters in Atxn1154Q versus WT littermates as measured with ChIP-qPCR (mean T SEM, n = 4 mice per genotype). Data analyzed by means of ANOVA with post-hoc t test. *P < 0.05 between WT and Atxn1154Q; #P < 0.05 between Atxn1154Q and Atxn1154Q;Cic-L+/ mice. SCIENCE www.sciencemag.org
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improve cerebellar phenotypes. It is encouraging that exercise and the accompanying increase in neuronal activity and metabolic demands do not seem to exacerbate the disease process in vulnerable neuronal populations, which may be important in a variety of neurodegenerative disorders.
1. H. T. Orr et al., Nat. Genet. 4, 221 (1993). 2. H. Y. Zoghbi, H. T. Orr, J. Biol. Chem. 284, 7425 (2009). 3. C. W. Cotman, N. C. Berchtold, L.-A. Christie, Trends Neurosci. 30, 464 (2007). 4. A. Chi, G. Benzi, M. Dossena, R. Mutani, G. Mora, Brain 128, 472 (2005). 5. D. J. Mahoney, C. Rodriguez, M. Devries, N. Yasuda, M. A. Tarnopolsky, Muscle Nerve 29, 656 (2004). 6. K. Watase et al., Neuron 34, 905 (2002). 7. S. Astigarraga et al., EMBO J. 26, 668 (2007). 8. Y. C. Lam et al., Cell 127, 1335 (2006). 9. J. Crespo-Barreto, J. D. Fryer, C. A. Shaw, H. T. Orr, H. Y. Zoghbi, PLoS Genet. 6, e1001021 (2010). 10. M. Kawamura-Saito et al., Hum. Mol. Genet. 15, 2125 (2006). 11. J. Lim et al., Nature 452, 713 (2008). 12. P. S. Thomas Jr. et al., Hum. Mol. Genet. 15, 2225 (2006). 13. J. M. Van Raamsdonk et al., Hum. Mol. Genet. 14, 1379 (2005). 14. D. V. Vaz et al., Clin. Rehabil. 22, 234 (2008). 15. W. Ilg et al., Neurology 73, 1823 (2009). Acknowledgments: J.D.F. and H.Y.Z. conceived of the study and designed experiments. J.D.F., C.M.B., A.N.C., and Y.G. performed behavioral assays and provided input on analysis. J.D.F. and J.C.-B. performed molecular work and analysis. P.Y., H.K., and C.S. analyzed microarray data. J.D.F. and H.Y.Z. wrote the manuscript with input from J.C.-.B, A.F., and H.T.O. We are grateful to G. Schuster for the generation of mutant mice and the members of the H.Y.Z. laboratory for comments and discussions on the manuscript. This research was supported by NIH grants NS27699, NS27699-20S1ARRA, and HD24064 (Baylor College of MedicineIntellectual and Developmental Disabilities Research Center) to H.Y.Z.; 1F32NS055545 to J.D.F.; and NS022920 and NS045667 to H.T.O. H.Y.Z. is an investigator with the Howard Hughes Medical Institute, holds a patent on SCA1 diagnostic testing, and is on the scientific advisory board of Pfizer Neuroscience Program.

www.sciencemag.org/cgi/content/full/334/6056/690/DC1 Materials and Methods Figs. S1 to S3 Tables S1 to S4 References (1618) 15 August 2011; accepted 21 September 2011 10.1126/science.1212673
Fei Wang,1,2 Jun Zhu,1 Hong Zhu,1,2 Qi Zhang,1 Zhanmin Lin,1 Hailan Hu1* Dominance hierarchy has a profound impact on animals survival, health, and reproductive success, but its neural circuit mechanism is virtually unknown. We found that dominance ranking in mice is transitive, relatively stable, and highly correlates among multiple behavior measures. Recording from layer V pyramidal neurons of the medial prefrontal cortex (mPFC) showed higher strength of excitatory synaptic inputs in mice with higher ranking, as compared with their subordinate cage mates. Furthermore, molecular manipulations that resulted in an increase and decrease in the synaptic efficacy in dorsal mPFC neurons caused an upward and downward movement in the social rank, respectively. These results provide direct evidence for mPFCs involvement in social hierarchy and suggest that social rank is plastic and can be tuned by altering synaptic strength in mPFC pyramidal cells. ominance hierarchy is a fundamental organizing mechanism for most animal societies (1). The dominance status determines access to resources and profoundly affects survival, health, reproductive success, and multiple behaviors (24). Once established, the hierarchy rank is relatively stable and can minimize intense fights among group members (5). Dominance hierarchy emerges early in development and is present in children as young as 2 years old (6). Moreover, dominance has been linked to heritable traits in animals (7, 8) and humans (9). The ubiquitous, inheritable nature and the early developmental emergence suggest that dominance hierarchy is encoded by innate neural mechanism (10). Yet, current understanding of the neural mechanisms associated with social hierarchythe neural circuits underlying status-related behavioral differences and the mechanism for initiating changes in social statusis surprisingly limited.
1 Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 2Graduate School of Chinese Academy of Sciences, Shanghai 200031, China.
*To whom correspondence should be addressed. E-mail: hailan@ion.ac.cn
To investigate the neural mechanisms underlying social hierarchy, a robust and reliable behavioral assay is essential. The tube test, in which one mouse forces its opponent out backward from a narrow tubedeveloped to measure the dominance tendency of mice (11) (Fig. 1A)was previously used mostly for comparison between mice of different strains (11) or genotypes (12). To measure the hierarchical relation of animals within the same social group, we applied the tube test to cage groups of four male C57/BL6 mice, living together for at least 2 weeks. Mice were tested pair-wise using a round robin design, and the social rank was assessed on the basis of winning against the other three cage mates (Fig. 1, A and D). We validated the tube test to be a reliable measure of dominance ranking by the following three criteria: 1) Transitivitywhen mouse A is more dominant over B and B more dominant over C, then A should be dominant over C (Fig. 1B). We analyzed the relation of any three mice from a cage group; rank was transitive in 95% (251 out of 264) cases (Fig. 1B). Consistent with this high transitivity, in 89% (78 out of 88) of the trials, a four-mouse group adopted a linear social diagram among the four possible combinations (Fig. 1C). SCIENCE VOL 334
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2) Stability over time (2). For stability evaluation, rank was measured daily for 7 days (Fig. 1, D and E). Mice adopted the same rank position as the previous day in 59% (127 out of 216) of the comparisons. Having a reward for taking the tube test (see Methods) did not affect the linearity or stability of the rank (Fig. 1C and fig. S1A). It was noteworthy that the time spent in the tube was significantly shorter when the mouse of the lowest rank (rank-4) was involved or as rank distance increased (Fig. 1F). Moreover, rank did not correlate with either the weight (when weight difference is below 15%) (P = 0.96, one-way analysis of variance) (fig. S1B) or the locomotive activity in the open field (P = 0.41) (fig. S1C). 3) Consistency with results of other dominance measures. To rule out factors unrelated to dominance, e.g., sensorimotor capacity, learning ability, and persistence, we compared the tube test results with those obtained by five additional dominance assays. First, in the visible burrow system (VBS), where food and water are relatively difficult to access and weight change reflects dominance (13), the tube-test ranks correlated linearly with weight changes (Fig. 2A and fig. S2A). Second, tube-test ranks also linearly correlated with the ranks by the agonistic behavior test, in which dominance is determined by scoring agonistic behaviors in a novel context (Fig. 2B and fig. S2B). Third, in the barber assay where the most dominant mouse barbers the whiskers of its cage mates (Dalila effect) (12, 14), the barber mouse was ranked first by the tube test in six of seven cages (Fig. 2C). Fourth, in the urinemarking assay, where dominant mice mark larger territories than subordinate ones (15), we found 71% (30 out of 42) consistency with the tube-test result (Fig. 2D). Finally, in the test of ultrasonic vocalization toward females in which dominant males emit more 50- to 70-kHz ultrasonic courtship songs (16), we found rank-1 mice in the tube test emitted markedly more ultrasounds toward a female than mice of lower ranks in 9 out of 10 cages (Fig. 2E and fig. S2C). In addition, dominance ranking significantly correlated among these five tests themselves (fig. S2). Although the reliability and validity of each assay in measuring the dominance behavior may be debatable
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or context-dependent (5, 17), the high consistency of ranking results from these multiple assays strongly supports the notion that dominance is a common dependable variable underlying a variety of behaviors that use different sensorimotor skills (fig. S3). Where and how is the hierarchical information encoded in the brain? Functional brain imaging studies in humans have implicated the dorsolateral prefrontal cortex (dlPFC) and medial prefrontal cortex (mPFC) in dominance hierarchy related behaviors (18, 19). The functional homo100
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the rank positions of one cage of mice tested daily over 6 days. (E) Summary graph for 21 cages measured. The average rank positions of animals belonging to each rank group from the previous day were calculated. (F) Normalized time spent in the tube encountering for the six pairing conditions (n = 10 cages), e.g., 12 stands for rank-1 against rank-2. Wilson rank-sum test (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars, SEM.
Fig. 2. Comparison of the tube test A B Visible burrow system with five other dominance tests. (A Agonistic behavior assay (VBS) to E) (Left) schematics of each test. 4 4 (A) (Right) Average weight-change 3 3 ranks in VBS plotted against the tube-test ranks. P = 0.0003, Fishers 2 2 exact test, same for (B) to (D). For R2 = 0.97 R2 = 0.91 1 1 n = 10 n = 16 (A), (B), and (E), n = number of cages. (B) The agonistic behavior score was Water Food 1 2 3 4 1 2 3 4 calculated by subtracting the number Rank in tube test Rank in tube test of offensive behaviors from that of E Ultrasonic test C Barber test defensive behaviors during a 20-min Barber Non-barber 4 Unfamiliar Barber Non-barber period in a novel cage. (Right) AvMale 7 female 3 erage ranks in agonistic behavior assay plotted against the tube-test 2 ranks. P = 0.003. (C) (Right) NumR2 = 0.93 1 ber of animals at each tube-test rank n = 10 position from all the barber and 0 1 2 3 4 2 3 4 1 nonbarber mice. P = 0.02. (D) (Left) Rank in tube test Rank in tube test Representative picture of the urine20 200 Ultrasound events marking patterns of a rank-1 to -4 D Urine marking assay Number Duration mice pair as revealed by ultraviolet Rank-1 Dominant Subordinate n = 10 12 30 light. (Right) Contingency table 100 10 showing number of animals in each Rank-4 category. n = 42 pairs from eight ** *** ** *** 30 12 cage groups. P = 0.003. (E) (Top right) *** *** 0 0 1 2 3 4 Average ranks in the ultrasound 10 sec Dominant Subordinate Rank in tube test test against the tube-test ranks. In tube test P = 2 107. (Bottom left) Example traces of the ultrasound emitted by a pair of rank-1 and rank-4 male The P value is obtained by two-tailed Students t test on natural log mice when encountering a female. (Bottom right) Summary graph of the transformed data in comparison with rank-1. **P < 0.01; ***P < 0.001. Error number and duration of ultrasounds evoked by mice of each rank position. bars, SEM.
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log of these regions in rodents is the dorsal mPFC, including the anterior cingulate cortex (AC) and the prelimbic cortex (PL) (Fig. 3A) (20). Moreover, mPFC lesion lowered the social rank in rats (21). Therefore, we investigated potential differences in the synaptic properties in this region between dominant and subordinate mice. Acute brain slices containing mPFC were made from rank-1 and rank-4 mice, and miniature excitatory synaptic currents (mEPSCs) mediated by the a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors were measured by whole-cell recording from layer V pyramidal neurons, the primary mPFC output neurons (Fig. 3B). The amplitudes, but not the frequency, of mEPSCs were significantly larger in rank-1 than rank-4 mice of the same cage group (P < 0.0001, KolmogorovSmirnov two-sample test) (Fig. 3, C and D). The difference was significant for each of the five pairs of mice recorded (Fig. 3E). These results indicate dominant mice have a higher excitatory synaptic strength in layer V pyramidal neurons than their subordinates, which could result in stronger output of these excitatory neurons to other brain regions. Consistently, dominant mice exhibited significantly higher number of c-Fos positive neurons in the PL region of mPFC after the tube test (Fig. 3, F and G). To determine whether the difference in mPFC synaptic efficacy could contribute to the rank status, we manipulated the function of excitatory synapses in dorsal mPFC using a Sindbis virusbased in vivo recombinant DNAdelivery technique (2224) (Fig. 4A). Small guanosine triphosphatases Ras and Rap potentiate and depress AMPA receptormediated transmission, respectively, in the hippocampus by regulating synaptic trafficking of AMPA receptors (25). We verified these effects in the mPFC neurons in vivo by localized mPFC injection of Ras- or Rap-expressing Sindbis viruses and whole-cell recording of injected neurons (Fig. 4B). Sindbis virus preferentially infects pyramidal neurons (fig. S4), and the basic electrophysiological properties of infected neurons, including the input resistance and leak current, were indistinguishable from those of uninfected neurons within 48 hours of infection (fig. S4, see also SOM text note 1). At 12 to 24 hours postinfection, neurons infected with Ras virus had higher amplitudes of AMPA receptormediated EPSCs (179 T 24% of controls, P = 0.0093, Wilcoxon signed rank test), in comparison with neighboring uninfected control cells (Fig. 4, B and E, and fig. S5A), whereas Rap-expressing neurons had lower EPSC amplitudes (Fig. 4E and fig. S5B, 71 T 9% of controls, P = 0.025). The behavioral consequences of these synaptic manipulations were further assessed by bilateral high-titer viral injection into the dorsal mPFC of one mouse from each cage group with stable ranks (persisting for at least four continuous daily trials before the injection) (fig. S7). Mice infected with Ras virus moved upward in the rank, starting as early as 12 hours after viral injection (Fig. 4, F and G, and fig. S8A). In contrast, mice infected with Rap virus moved downward in the rank (Fig. 4, F and G, and fig. S8B). Infection of virus expressing green fluorescent protein (GFP) alone did not result in any rank shift (Fig. 4, F and G, and fig. S8E). As the synaptic effect of Ras may involve both pre- and postsynaptic changes (25, 26) and Ras-induced synaptic potentiation was relatively transient (<3 days) (fig. S6), we then manipulated the AMPA receptormediated transmission more specifically by using constructs that express either the AMPA receptor subunit GluR4, which undergoes synaptic delivery and increases synaptic transmission under basal conditions (27, 28) (see also SOM text note 2), or the C terminus of GluR4 (R4Ct), which can block synaptic trafficking of AMPA receptors (27) (see also SOM text note 3). Recording in acute mPFC slices from virus-infected mice revealed that viral expression of GluR4 potentiated (173 T 22% of controls, P = 0.01), whereas R4Ct depressed (24 T 2% of controls, P = 0.0006), AMPA-EPSCs in mPFC (Fig. 4, C to E, and fig. S5). Correspondingly, GluR4 increased, whereas R4Ct decreased the tube-test rank of injected animals (Fig. 4, F to G, and fig. S8). Analysis of the viral infection sites reveals that the rank change correlated best with the infection rates in the PL region (fig. S7). Injection of the same amount of R4Ct virus into the M1 motor cortex had no effect on the rank (Fig. 4G
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and fig. S9), which indicated that the effect is mPFC-specific. To confirm that viral manipulations in mPFC affected dominance instead of other variables that could alter tube-test behavior (fig. S3), we performed an additional dominance measure, the ultrasonic vocalization test. When tested for ultrasound vocalizations toward a female, low-rank mice injected with GluR4-expressing virus displayed a significant increase, whereas high-rank mice injected with R4Ct-expressing virus showed a marked decrease in both the frequency and duration of ultrasound production at 36 to 60 hours after viral injection (Fig. 4H and fig. S10), resulting in a bidirectional shift in ultrasonic test rank (Fig. 4I and fig. S10). Here, we established that the tube test is a simple and reliable method that provides hierarchical ranking of social groups in mice. Using this and other dominance measures, we identified the mPFC circuitry, in particular the dorsal mPFC, as a neural substrate for dominance hierarchy. The bidirectional modulation suggests that the effect of mPFC synaptic efficacy on dominance is specific and unlikely to be due to secondary effects. How could mPFC contribute to the determination of social status? Social hierarchy behavior depends on a collection of cognitive traits involving recognition of social status, learning of social norms, and detection of violation of social norms (10). mPFC has been explicitly implicated in social cognition (29). Through its projections
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Fig. 4. Modulations of mPFC synaptic efficacy caused bidirectional shift of hierarchical rank. (A) Example of GFP-GluR4 virus injection sites (counterstained with Hoechst). White dashed lines outline the mPFC. (B) Simultaneous whole-cell recording of EPSCs from a pair of layer V pyramidal neurons while layer II/III was stimulated. (Top left) Illustration of the recording configuration. (Top right) Patching of a pair of infected (by GFP-Ras) and uninfected mPFC neurons under transmitted and fluorescent light microscopy. (Bottom) Example of evoked EPSC recorded at 60 and +40 mV from a neuron pair. Stimulation artifacts are marked by filled circles. (C and D) Effects of virus expressing GFP-GluR4 (C) and red FP (RFP)R4Ct (D) on the AMPA receptor and Nmethyl-D-aspartate (NMDA) receptormediated EPSCs (left) and the tube-test rank dynamics of a four-mouse group (right). Arrowhead indicates the injection at 0 hours time point. (E and F) Summary of effects on AMPA receptor
mediated EPSCs (E) and tube-test rank (F) as induced by injection of each viral construct. n in (F) is the same as in (G). (G) Summary of percentage of animals showing rank increase (left upward column) or decrease (right downward column) in the tube test. (H) Natural logtransformed number of ultrasonic vocalization events toward a female before and after viral injection. Natural log transformation was taken to normalize the data and reduce the data span. Each line represents data from one mouse. (I) Summary of percentage of mice showing rank increase or decrease in ultrasound test induced by GluR4 and R4Ct viruses. (C) to (F) and (I) Wilcoxon signed rank test, in comparison with neighboring noninjected control neurons (C) to (E) or with rank change of animals injected with GFP-expressing virus (F) or noninjected animals (I). (G) Fishers exact test, compared with GFP-injected animals. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars, SEM. SCIENCE www.sciencemag.org
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to regions such as dorsal raphe, ventral tegmental area, hypothalamus, and amygdala, mPFC exerts top-down controls on serotonin and dopamine release, endocrine function, and fear response. All of these could contribute to key features of the dominance behaviors, including aggressiveness, stress responsiveness, and fearfulness. It will be of interest to determine which of these mPFC downstream circuits are specifically involved in setting the dominance hierarchy and to investigate how the dominance rank is initiated and maintained by differential neuronal activities in these circuits. Likewise, it will also be important to understand how the behavioral specificity of mPFC is generated by distinct upstream inputs, given the multiple functions that mPFC has been implicated in [reviewed by (30) and, recently, (3134)]. The identification of a neural substrate of dominance hierarchy should provide new insights into the coding of a fundamental social behavior in the mammalian nervous system.
1. E. O. Wilson, Sociobiology: The New Synthesis (Harvard Univ. Press, Cambridge, MA, 1975). 2. I. S. Bernstein, Behav. Brain Sci. 4, 419 (1981). 3. R. M. Sapolsky, Science 308, 648 (2005). 4. S. R. Yeh, R. A. Fricke, D. H. Edwards, Science 271, 366 (1996). 5. C. Drews, Behaviour 125, 283 (1993). 6. D. G. Frankel, T. Arbel, Int. J. Behav. Dev. 3, 287 (1980). 7. J. Masur, M. A. Benedito, Nature 249, 284 (1974). 8. D. A. Dewsbury, Anim. Behav. 39, 284 (1990). 9. A. Mehrabian, Curr. Psychol. 14, 261 (1996). 10. D. D. Cummins, Synthese 122, 3 (2000). 11. G. Lindzey, H. Winston, M. Manosevitz, Nature 191, 474 (1961). 12. A. S. Garfield et al., Nature 469, 534 (2011). 13. H. Arakawa, D. C. Blanchard, R. J. Blanchard, Behav. Brain Res. 176, 27 (2007). 14. S. Y. Long, Anim. Behav. 20, 10 (1972). 15. L. C. Drickamer, Behav. Processes 53, 113 (2001). 16. J. Nyby, G. A. Dizinno, G. Whitney, Behav. Biol. 18, 285 (1976). 17. D. Benton, J. C. Dalrymplealford, P. F. Brain, Anim. Behav. 28, 1274 (1980). 18. C. F. Zink et al., Neuron 58, 273 (2008). 19. J. Y. Chiao, Curr. Opin. Neurobiol. 20, 803 (2010). 20. H. B. Uylings, H. J. Groenewegen, B. Kolb, Behav. Brain Res. 146, 3 (2003). 21. R. R. Holson, Physiol. Behav. 37, 239 (1986). 22. H. Marie, W. Morishita, X. Yu, N. Calakos, R. C. Malenka, Neuron 45, 741 (2005). 23. S. G. McCormack, R. L. Stornetta, J. J. Zhu, Neuron 50, 75 (2006). 24. H. L. Hu et al., J. Neurosci. 28, 7847 (2008). 25. J. J. Zhu, Y. Qin, M. Zhao, L. Van Aelst, R. Malinow, Cell 110, 443 (2002). 26. S. A. Kushner et al., J. Neurosci. 25, 9721 (2005). 27. J. J. Zhu, J. A. Esteban, Y. Hayashi, R. Malinow, Nat. Neurosci. 3, 1098 (2000). 28. J. J. Zhu, J. Neurosci. 29, 6320 (2009). 29. D. M. Amodio, C. D. Frith, Nat. Rev. Neurosci. 7, 268 (2006). 30. R. P. Vertes, Synapse 51, 32 (2004). 31. H. E. Covington 3rd et al., J. Neurosci. 30, 16082 (2010). 32. O. Yizhar et al., Nature 477, 171 (2011). 33. K. Guillem et al., Science 333, 888 (2011). 34. D. Tse et al., Science 333, 891 (2011). Acknowledgments: We thank J. Zhu for the gifts of pSindbis GluR4 and R4Ct constructs and discussion on GluR4; R. Malinow for support of the initial exploration of the tube test in his laboratory; M. Poo, H. Kessels, J. Feldman, and R. Sapolsky for discussion and critical reading of the manuscript; J. Yan for help with statistics; B. Lu for the idea of agonistic behavior test; B. Zhang, K. Zhang, and M. Streets for technical help; Y. Yanagawa for the Gad67-GFP mouse; and X. Yu for ultrasound equipment. The work was supported by Chinese 973 Program (2011CBA00400), Projects of the Scientific Research Foundation, the Hundreds of Talents Program, and the Shanghai Pujiang Talent Program to H.H. Author contributions: F.W. performed most experiments. J.Z. participated in the behavioral and viral injection experiments. F.W., J.Z., and H.H. analyzed data and prepared the figures. H.Z, Q.Z., and Z.L. assisted in experiments. H.H. designed the study and wrote the manuscript.

www.sciencemag.org/cgi/content/full/science.1209951/DC1 Materials and Methods SOM Text Figs. S1 to S10 References 17 June 2011; accepted 9 September 2011 Published online 29 September 2011; 10.1126/science.1209951
Social Network Size Affects Neural Circuits in Macaques

J. Sallet,1,2* R. B. Mars,1,2* M. P. Noonan,1,2* J. L. Andersson,2 J. X. OReilly,2 S. Jbabdi,2 P. L. Croxson,1,3 M. Jenkinson,2 K. L. Miller,2 M. F. S. Rushworth1,2 It has been suggested that variation in brain structure correlates with the sizes of individuals social networks. Whether variation in social network size causes variation in brain structure, however, is unknown. To address this question, we neuroimaged 23 monkeys that had been living in social groups set to different sizes. Subject comparison revealed that living in larger groups caused increases in gray matter in mid-superior temporal sulcus and rostral prefrontal cortex and increased coupling of activity in frontal and temporal cortex. Social network size, therefore, contributes to changes both in brain structure and function. The changes have potential implications for an animals success in a social context; gray matter differences in similar areas were also correlated with each animals dominance within its social network. he evolution of primate brains is thought to be associated with the demands of living in a complex social environment (1). Recent evidence shows that differences in brain structure correlate with variation in individuals social network size (2); some brain structures are
1 Department of Experimental Psychology, University of Oxford, Oxford OX1 3UD, UK. 2Oxford Centre for Functional Magnetic Resonance Imaging of the Brain (FMRIB), University of Oxford, Oxford OX1 3UD, UK. 3Icahn Medical Institute, Mount Sinai School of Medicine, New York, NY 10029, USA.
*These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: jerome.sallet@psy.ox.ac.uk
larger in people in regular contact with a larger number of relatives, friends, and colleagues. However, the direction of cause and effect underlying this phenomenon is unknown. Although this issue has not been directly investigated, sensorimotor experience is known to lead to brain structural changes even during adulthood (3, 4). For instance, learning to use a tool increases gray matter density in the intraparietal sulcus (IPS), caudal superior temporal sulcus (STS), and somatosensory cortex in the rhesus macaque (Macaca mulatta) (3). Here, we exploit the pseudo-randomized assignment of individual animals to social groups SCIENCE VOL 334
in a research colony (5) to demonstrate that variation in young adult rhesus macaques social environments changes structure and function in a distributed neural circuit centered on mid-STS, anterior cingulate cortex (ACC), and rostral prefrontal cortex (rPFC). First, we conducted a deformation-based morphometric (DBM) analysis (6) of magnetic resonance imaging (MRI) scans of brain structure from 23 young adult [4.33 T 0.52 years (mean T SD)] monkeys (14 males) (5). Scanned animals were drawn from 34 animals from different groups within a research colony. The animals were housed in groups of between one and seven individuals. We considered the number of housemates of each monkey as a measure of social network size. The organization of monkeys into groups was not randomized in a conventional sense but instead depended on factors that were independent of social characteristics; these included the requirements of independent programs of neuroscientific research and veterinary considerations [full details of housing arrangements are provided (5)]. A true randomization of animals would have been virtually impossible given numerous considerations, including the constraints imposed by the licensing of experimental procedures, the cost of such a project, and the potential for disruption to other research programs. While some unobserved variables might have contributed to the outcomes we report, group assignment was not carried out on the basis of social character-
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istics, and it was not the case that less sociable animals were housed together in smaller groups. Although one might imagine that a very unsociable animal might be identified and isolated, this does not imply that the next most unsociable animals would be housed together in a duo and the next most in a trio and so on. The one singly housed animal in the current experiment was an individual that was left after other members of the group had been used in other procedures. [Results after data from the singleton and even duos are omitted are also shown (5).] In summary, the assignment of group size in the current experiment is as close to randomized assignment as is currently feasible. Because the social network size is a parameter that is not controlled by the animals, then, in this context, any interindividual brain differences that are correlated with social network size will be a consequence of the imposed social network size. The constitutions of the groups studied were defined 0.53 T 0.81 year after the animals arrival in the facilities. In all cases, animals had been in a group of the stated size for some time (1.22 T 0.6 years) before scanning. In addition to social network size, the age, weight, sex, and the number of MRI structural scans contributing to each individuals average structural MRI scan were included as regressors in a nonparametric general linear model (GLM) analysis. The dependent variable analyzed was the determinant of the Jacobian matrix from the nonlinear registration for each individuals structural scan, a scalar value that represents the amount each voxel in an individuals brain would need to be expanded or compressed to match the group-average brain. This method has proven suitable for studying brain plasticity (6). We identified gray matter areas in which the Jacobian determinant was significantly related to a regressor (P < 0.005, volume > 5 mm3). Positive linear correlations were found between social network size and Jacobian determinants in several regions, most notably temporal cortex in mid-STS, adjacent inferior temporal (IT) gyrus, rostral superior temporal gyrus (STG), and temporal pole (Fig. 1, A and B, and table S3). No negative correlation was found. In the areas in which effects were found, and over the social network sizes we investigated here, average gray matter density increased by 5.42% T 2.73 per
Fig. 1. Gray matter positively correlated (P < 0.005, cluster size > 5 mm3) with network size in the temporal lobe and rPFC (n = 23). (A) Illustrations of the relationship between the determinant of the Jacobian matrix of the MRI warp fields applied during registration and network size at the centers of these regions. The x and y values correspond to coordinates (expressed in millimeters) within the Montreal Neurological Institute macaque rhesus template space. Red arrows indicate regions where network size effects
were observed. White arrows indicate anatomical landmarks. PS, principal sulcus; STS, superior temporal sulcus; STG, superior temporal gyrus; IT, inferotemporal cortex; rPFC, rostral prefrontal cortex; TP temporal pole; L, left. (B) Illustration of relationship between network size and Jacobian determinants in the mid-STS/IT, rostral STS, and rPFC. Jacobian determinants greater than 1 indicate that voxels in an individuals MRI scan must be compressed to match the group template in MNI space. SCIENCE www.sciencemag.org
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member of the social network. The STS clusters were in regions that are, in monkeys, responsive to visually presented faces and body movements (79). Some of these areas have been shown to respond to such stimuli with an activity profile that resembles that of areas in the human caudal STS at the temporo-parietal junction (TPJ) (8). The rostral STG has been implicated in the encoding of vocalization in macaques and in the holding of semantic knowledge in humans (10, 11). In macaques, lesions of the temporal pole disrupt emotional responsiveness (12). The gray matter increases in these areas could, therefore, reflect an increasing need to decode the significance of the facial expressions, gestures, and vocalizations of a greater number of individuals and combinations of individuals as network size increased. A correlation was also found between social network and Jacobian determinants in the amygdala (Fig. 1A). Lesions of the amygdala also disrupt emotional responsiveness in both humans and macaques (13, 14). Bickart and colleagues (2) have reported a similar correlation in human subjects but, unlike here, they were unable to determine whether differences in amygdala size were the consequence of differences in experienced social network size. Our initial analysis also identified clusters in rPFC, in the rostral principal sulcus, at the border between dorsolateral prefrontal cortex (area 46) and the frontal pole (area 10) (Fig. 1, A and B, and table S3). In human subjects, an rPFC area rostral to the paracingulate sulcus, together with the STS, is active when predictions are made and updated about the intentions of others (15, 16). Such predictions will have to be made about more individuals, and combinations of individuals, as social network size increases. It is possible that the rPFC region that we have identified in the macaques in the present study is similar to the human rPFC area (17, 18). In humans, rPFC, together with STS and TPJ, is associated with theory of mind and prediction of another individuals overt behavior as well as their intentions (15, 16). It is not clear if macaques have a theory of mind and are able to represent others intentions (15, 16), but they can make decisions based on inferences about what others can see (19), which might be a precursor of such an ability. There is also evidence that the activity of dorsolateral prefrontal neurons is modulated when choices are made in the context of an interactive game (20) and by the relative dominance of different individuals during interactions (21). We searched for an effect of network size on other areas sometimes called social brain areas without success (14, 16, 2224). For example, we did not observe significant correlation between network size and Jacobian determinants in the ventral premotor cortex, inferior parietal lobule, or anterior, lateral, and ventral IPS. These areas of the social brain have been described, in both humans and macaques, as part of the mirror neuron system (16). Neurons in these areas are responsive when macaques make movements and when they perceive the movements of others. It has been suggested that they are part of a system for understanding others actions and intentions. Inclusion in a larger social network did not entail that other, nonsocial aspects of the environment were enriched, but it did entail that each individual spent more time engaged in social interactions with other individuals (Pearsons r = 0.9735, n = 11, P < 0.0001; fig. S1). It therefore seems, regardless of whatever role it may have in action perception, that the mirror neuron system
Fig. 2. Gray matter in rPFC correlated positively (P < 0.005, cluster size > 5 mm3) with dominance (blue, n = 11), network size (red, n = 23), and conjunction of dominance and network size (yellow). (A) Illustrations of the relationship between the Jacobian determinant and relative dominance status at the centers of the overlapping regions. Blue arrows indicate regions where dominance status effects were observed. White arrows indicate anatomical landmarks. The x and y values correspond to coordinates (expressed in mm) within the MNI macaque rhesus template space. Abbreviations as for Fig. 1. (B) Illustration of relationship between relative dominance status and the Jacobian determinants in the rPFC and IT.
Fig. 3. (A) ACC gyrus regions in which BOLD signal indices of brain activity in the resting state were correlated with STS activity. (B) Illustration of correlation. www.sciencemag.org SCIENCE VOL 334
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may not be challenged by the need to make more frequent inferences about a greater number of other individuals when social group sizes are larger. It might be expected that increases in gray matter in a network of brain areas concerned with social cognition would lead to more successful social behavior. To test this proposition, we examined whether variation in the same brain structures was correlated with social rank after controlling for social network size. Social rank is an index of success in social settings and in macaques it is correlated with access to valued resources (25); social rank in male macaques is dependent on the ability to form coalitions, which in turn is dependent on the ability to form pair bonds (25). It is therefore plausible that social rank might be dependent on brain networks for social cognition. We measured the social rank of 11 male macaques relative to other members of their social groups, in four groups, on the basis of observations of agonistic relationships (table S2). Each individual was assigned a cardinal index of social dominance (26). Not only did we ensure that individuals investigated in this more detailed analysis of behavior were all of the same sex, but we also controlled for the effect of social network size by selecting individuals from four groups of similar social network sizes (9 of 11 animals were taken from groups with either four or five members). This GLM analysis included the cardinal index of social dominance and additional regressors of age, weight, and social network size (despite the little variation in this factor for the individuals included in this analysis), and number of structural MRI scans contributing to each individuals average MRI scan. Gray matter in rPFC, in a region adjacent to the area where an rPFC effect of social network size was observed, increased with increasing social dominance (Fig. 2 and table S3). Average gray matter density increased by 0.31% T 0.14 each time the relative dominance status was increased by 1%. In an additional test, we sought voxels in which there was an overlap of both social network and dominance effects. Smaller regions of IT and rPFC showed a conjunction effect (Fig. 2). That is, even after taking into account age, weight, and social network size, increased IT and rPFC size correlated with higher social rank. In summary, larger social networks cause changes in cortex in or adjacent to regions where gray matter is correlated with social dominance. Finally, we investigated whether social network size affects brain activity as well as brain structure. We tested whether the coupling of activity, assessed using resting state functional MRI available from 21 of the same animals, increased with social network size. For each monkey a 1.5 mm by 1.5 mm by 1.5 mm region of interest seed area was positioned within the STS cluster, and the time series of the blood oxygen leveldependent (BOLD) signal was extracted. We then identified areas of functional connectivity with STS by regressing the STS functional time series against activity in every voxel in the brain, using a GLM in which age, weight, sex, and whole-brain mean time series were also included as co-regressors of no interest (5). First, we conducted region-of-interest analyses testing whether social network size increased functional coupling between the mid-STS seed and the rPFC regions identified in the previous structural analyses. The functional coupling between midSTS activity and activity within a 2.5 mm by 2.5 mm by 1.5 mm region of interest covering the rPFC increased with increasing social network size (Pearsons r = 0.386, n = 21, P = 0.042). Similarly, the functional coupling between the temporal and frontal areas, IT and rPFC, that were identified in the DBM analysis of dominance increased with increasing dominance (Pearsons r = 0.526, n = 11, P = 0.049). The areas are monosynaptically interconnected (27). More pronounced, however, were the results from the whole-brain analysis of functional coupling with STS, which identified increased coupling between activity in mid-STS and the ACC gyrus (z > 2.3, P < 0.05; Fig. 3, A and B, and table S5). An identical part of the macaque ACC gyrus has been implicated in the valuation of social information from conspecifics (28), and a related area has a similar function in humans (22). In both humans and macaques, it is specifically the ACC gyrus, rather than the adjacent ACC sulcus, that plays this role in social cognition. The ACC gyrus and mid-STS are also monosynaptically interconnected (29). Functional coupling between the STS and several extrastriate visual areas was also found (Fig. 3A and table S5). Several of the regions are part of the ventral visual processing stream that provides mid-STS with visual input. In summary, we have identified a distributed neural circuit in which changes in structure and functional coupling occur as a function of social network size. The findings inform accounts of brain evolution that emphasize the pressure exerted by complexity of the social environment (1). One prediction of such accounts is that individual variation in brain anatomy should have implications for an individuals success within the social group, and we have demonstrated that brain structure correlates with measures of social dominance that remained constant over at least 4 months when group constitution did not change. The pattern of results is especially concordant with suggestions that complexity of social environments may have had a greater impact on specific brain circuits rather than the brain as a whole (22, 30). These results may also have implications for understanding changes seen in neural circuits for social cognition in clinical conditions associated with alterations in social interaction; changes in brain areas may be partly a consequence, and not just the cause, of alterations in social interactions. Finally, the results raise the possibility that individual differences in patterns of inter-regional coupling of huVOL 334 SCIENCE man brain activity measured in the resting state are a function of variation in social network size.
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www.sciencemag.org/cgi/content/full/334/6056/697/DC1 Materials and Methods SOM Text Fig. S1 Tables S1 to S5 References and Notes 20 June 2011; accepted 27 September 2011 10.1126/science.1210027
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Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.dtl for more information. Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are featured in this space. Emphasis is given to purpose, chief characteristics, and availability of products and materials. Endorsement by Science or AAAS of any products or materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.
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POSITIONS OPEN
POSITIONS OPEN
TENURE-TRACK FACULTY POSITIONS in Stem Cell Columbia Stem Cell Initiative Tapping the Potential of Stem Cells for Human Health Research at Columbia University Medical Center The Columbia Stem Cell Initiative (CSCI; website http://www.ColumbiaStemCell.org) brings together more than 120 groups working to tap the potential of stem cells for human health. Their research covers all aspects of stem cell research, from basic mechanisms of self-renewal and differentiation in model organisms to clinical and preclinical applications in disease contexts. To further strengthen this initiative and to create strategic overarching projects from basic science to regenerative medicine, we seek to recruit three new faculty members at the ASSISTANT PROFESSOR and ASSOCIATE PROFESSOR level. Applicants_ research may focus directly on stem cell biology or therapy or may use stem cell-derived cells as a central experimental tool for studying disease or physiological mechanisms. There is no restriction as to tissue or disease area. Successful candidates will be members of the Department of Rehabilitation and Regenerative Medicine, and will also generally hold a joint appointment in a basic science department relevant to their research field. Faculty will be provided with an attractive startup package and a salary commensurate with experience. Modern laboratory space in proximity to other stem cell groups and other translational centers will be provided. The new recruits will have access to all the facilities and potential for interactions and collaboration provided by CSCI at both the Medical School and Morningside Heights campuses of Columbia University. Applications should be sent to Dola Sengupta (Program Coordinator, CSCI; e-mail: ds2865@columbia. edu). Candidates should submit a cover letter, curriculum vitae, research statement, up to three publications, and contact information for three people who will write letters of reference. Applications should be received by December 15, 2011. Inquiries may be addressed to Christopher Henderson (Director, CSCI; e-mail: ch2331@columbia.edu). Columbia University is an Equal Opportunity/Affirmative Action Employer. FACULTY POSITION in Chemical Biology Department of Chemistry and the Carolina Center for Genome Sciences University of North Carolina at Chapel Hill The Department of Chemistry and the Carolina Center for Genome Sciences at the University of North Carolina (UNC) at Chapel Hill invite applications for a tenure-track faculty position at the ASSISTANT PROFESSOR level. Areas of interest include chemical biology and chemical genomics. However, all areas of chemistry, broadly defined, will be considered. Successful candidates are expected to develop an exceptional research program and to teach at both the graduate and undergraduate levels. Research space will be allocated in the new Genome Sciences Building, which will contain faculty from the Departments of Biology, Chemistry, Statistics and Operations Research, and Computer Science. Applications will only be accepted electronically. Applicants should submit a single PDF containing (in this order) a cover letter, curriculum vitae, research plan, and teaching statement, and up to three reprints/preprints to website: http://jobs.unc.edu/2501931. Applicants should also arrange to have three letters of recommendation sent to e-mail: chemsearch@unc.edu. Review of applications will begin December 1, 2011, and will continue until the position is filled. Questions should be directed to: Chair, Faculty Search Committee, Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, e-mail: chemsearch@unc.edu. UNC is an Equal Opportunity/Affirmative Action Employer, and strongly encourages applications from women and minorities.
FACULTY POSITION in Developmental Integrative Biology The University of North Texas (UNT) seeks candidates at the Associate or Full Professor level to join our growing Developmental Integrative Biology Research Cluster (DIB; website: developmentalbiology. unt.edu) comprising nine faculty and more than 50 graduate students and postdoctoral fellows. Areas of research should focus on developmental aspects of functional/comparative genomics, physiology, neuroscience, cell biology, endocrinology, or genetics, with expertise in the cardiovascular system, respiration, stress response, and/or metabolism. The candidate must have an established international reputation and an externally funded research program. Candidates will have a Ph.D. in Physiology or related field, have previous teaching experience, and are expected to support the instructional and research goals of the university at both the undergraduate and graduate levels. Competitive startup funding and salary will be provided. Laboratory space is available in the newly constructed state-of the-art Life Sciences Complex. For further information, see websites: http://www. unt.edu, http://www.biol.unt.edu, and http:// research.unt.edu/clusters. All applicants must apply online at website: http:// facultyjobs.unt.edu/applicants/Central?quickFind0 51506. For questions, contact Michael Hedrick, Search Committee Chair, at e-mail: michael.hedrick@unt. edu. Applications will be reviewed beginning October 30, 2011, and will continue until the search is closed. UNT is an Affirmative Action/ADA/Equal Opportunity Employer. ASSISTANT PROFESSOR OF BIOLOGY Tenure-track position beginning Fall 2012 in neurophysiology to join the Biology department and contribute to our growing Neuroscience program. Responsibilities will be to teach introductory molecular/ cellular biology and upper level courses in biology and neuroscience, and to direct undergraduate research in the College_s required Independent Study Program. Preference will be given to applicants who can advise student research projects focused on electrophysiology, microscopy, histology, and/or developmental biology. Applicants should have a Ph.D.; postdoctoral research and/or teaching experience preferred. Send curriculum vitae, statements of research and teaching philosophy, graduate and undergraduate transcripts, and three letters of recommendation to Dr. D. Fraga, Chair of Biology, the College of Wooster, Wooster, Ohio (e-mail: dfraga@wooster.edu). Electronic applications are preferred, and should be received by December 15, 2011 for full consideration. The College of Wooster is an independent college of the liberal arts and sciences with a commitment to excellence in undergraduate education. The College values diversity, strives to attract qualified women and minority candidates, and encourages individuals belonging to these groups to apply. Wooster seeks to ensure diversity by its policy of employing persons without regard to age, sex, color, race, creed, religion, national origin, disability, veteran status, sexual orientation, gender identity and expression, or political affiliation. The College of Wooster is an Equal Opportunity/Affirmative Action Employer. Employment is subject to federal laws requiring verification of identity and legal right to work in the United States as required by the Immigration Reform and Control Act. Drug-free workplace. ASSISTANT PROFESSOR OF BIOLOGY Penn State Brandywine invites applications for a tenure-track Assistant Professor of Biology; start August 2012. Teach undergraduate biology. Publication of research in referred journals and service activities expected. Ph.D. in biology or closely related area required; prefer expertise in molecular or cell biology. To learn about the campus and Penn State University, visit website: http://www.psu.edu/ur/ cmpcoll.html. To learn more about the position and how to apply, visit website: http://www.psu.jobs/ Search/Opportunities.html and follow the BFaculty[ link. Affirmative Action/Equal Opportunity Employer.
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www.sciencecareers.org
Postdoctoral Fellowships in Genomic Biology

at the University of Illinois at Urbana-Champaign
The Institute for Genomic Biology at the University of Illinois at Urbana-Champaign offers a number of fellowships for truly exceptional young scholars who have completed their Ph.D. within the last several years, and are looking for a stimulating and supportive interdisciplinary environment to carry out independent and collaborative research in the eld of genomic biology. IGB Fellows will spend up to three years conducting research in one of several research themes in the Institute, and ideally this research will also overlap with two or more of these thematic areas. Visit www.igb.uiuc.edu/content/fellows-application for more information about the Institute, the research themes and the application procedures. The closing date for all positions is December 15, 2011. Fellows will be announced on or about January 15, 2012.
Biocomplexity
We seek a scientist with interests that include the evolutionary process, systems biology, and ecosystem dynamics. e successful candidate will join a multidisciplinary group exploring collective eects in biology from a strongly quantitative perspective. Ongoing research addresses the evolution of translation, the role of horizontal gene transfer in shaping communities of microbes and phages, geomicrobiology, and the systems biology of cells and ecosystems. (Nigel Goldenfeld, eme Leader)
Mining Microbial Genomes

e Fellow will be involved in one of several multidisciplinary projects focused on (1) the discovery, design, and development of novel antibiotics, or (2) the assignment of function to novel enzymes discovered in genome projects. e ideal candidate will have a proven record of expertise in microbially produced natural products and/or enzyme evolution. We are interested in candidates with previous experience in bacterial metabolism, bacterial genetics, molecular biology, biochemistry, enzyme evolution, metabolic engineering, organic synthesis, mass spectroscopy, bioinformatics and/or metagenomics. (Bill Metcalf, eme Leader)
Regenerative Biology & Tissue Engineering

e Fellow will be involved in one or more of our multidisciplinary projects related to regenerative biology and harnessing the potential of adult/embryonic stem cells for tissue engineering applications. Of particular interest is leveraging theme expertise in biomaterials fabrication, drug delivery systems, microuidics-based in vitro experimental platforms, and in vivo evolutionary biology and regeneration medicine studies. e ideal candidate will have experience in one or more areas of (stem) cell biology, induced pluripotent cell technology, biomaterials, microuidics, and/or tissue engineering. (Paul Kenis, eme Leader)
Genomics of Neural and Behavioral Plasticity

We seek a biologist with strong bioinformatics skills and training in one or more of the following areas: evolutionary biology, neuroscience, animal behavior, molecular biology, genomics or systems biology. Applicants with expertise in both biology and bioinformatics will be strongly preferred. e successful candidate will join a multidisciplinary team that is using genomics to identify both conserved and novel mechanisms of neural and behavioral plasticity in diverse animal systems. Fellows are expected to conduct research that contributes to the development of the themes goals by integrating components from theme members individual research programs. (David Clayton, Interim eme Leader)
Genomic Ecology of Global Change

e Fellow will be involved in a cross-disciplinary project investigating how changes in networks of genes aect plant and ecosystem function when challenged by elements of global change, including greater carbon dioxide, ozone, drought, temperature, disease and herbivory. e ideal candidate will have a strong background in plant biology and a record of expertise in molecular biology, genomic ecology, physiology or modeling of gene networks or ecosystem function. e ability to work creatively and productively in a highly interdisciplinary and collaborative environment is essential. (Don Ort, eme Leader)
Host-Microbe Systems
e Fellow will be responsible for developing DNA isolation, microbial isolation, 16S rRNA gene sequencing and other metagenomic analysis techniques for surveying microbial content of the human and nonhuman primate vaginal and intestinal microbiomes. Additional responsibilities will include the culture isolation and genome sequencing and other molecular biology techniques to examine microbial, metabolic, and immunologic contents, phylogenetic comparisons, and performing analyses using bioinformatics and other computational and analytical methods. e ideal candidate will have a strong background in microbiology, biochemistry, or a related eld with experience and expertise in molecular microbial ecology and bioinformatics and/or biostatisitcs. (Brenda Wilson, eme Leader)
Energy Biosciences Institute

e Energy Biosciences Institute (EBI) is an externally funded theme within the IGB. It is the largest academia collaboration to date, currently receiving $500 million over ten years and focusing on the development of secondgeneration biofuels intended to signicantly slow the rate of global climate change. Its research ranges from systems biology of fermentative organisms to quantication of ecosystem services provided by new sustainable biofuel crops. e full range of research can be seen at www.energybiosciencesinstitute.org. We seek an outstanding candidate across these areas interested in applying genomic biology to understanding and developing opportunities for improving sustainable biofuel production. Research can be at any point in the supply chain from improving feedstocks and their environmental sustainability to producing fuel. e appointee will work in an interdisciplinary laboratory of over 100 exceptional colleagues focused on this challenge. e appointment would also involve collaboration with our partners: UC Berkeley and BP. (Steve Long, eme Leader)
Business, Economics, and Law of Genomic Biology

We seek an individual with training in economics, business, law, or strategy and with an interest in technology entrepreneurship, technology industries, and biotechnology. e Fellow will join a multidisciplinary group that includes business, law, and technology experts; agricultural economics faculty; and personnel from the campus Oce of Technology Management. Our theme is exploring issues in university-industry technology transfer, industry evolution, intellectual property protection, the competitive and cooperative dynamics for both entrepreneurial start-ups and existing corporations, the impact that globalization of biotechnology has on the evolution of industry, and the position of U.S. rms in the global marketplace. (Jay Kesan, eme Leader)
Cellular Decision Making in Cancer

We seek an individual with interest in quantitative biology. Our theme faculty members have expertise in single molecule biophysics, genomics and chemical biology. Building on the current strengths on cell death, antiviral signaling, stem cell dierentiation, live cell probing of decision making and genome instability modeling, we aim to develop a multiple-scale narrative on how single molecule events in the cell are integrated into the protein networks to determine the cell fate. Cancer is a major focus area of research. (Taekjip Ha, eme Leader)
The University of Illinois is an Afrmative Action/ Equal Opportunity Employer

www.igb.illinois.edu
featured employer
FOCUS ON CAREERS
FOCUS ON NEUROSCIENCE
The Many Fields of Neuroscience
Shifting from Synapses to Society

Neuroscience has come a long way since the staining and identication of the neuron by Camillo Golgi and Ramn y Cajal over a century ago. Now the eld has joined forces with other disciplines such as chemistry, computer science, engineering, and psychology, creating areas of focus that range from individual cells to social communities. Combining specialties has helped progress the understanding of social behavior as well as various psychological disorders, which some say are the nal frontiers in biological science. By Jacqueline Ruttimann Oberst Story Landis
By turning on or off specic neurons, one can identify their place in particular neurocircuits and how these circuits function in normal behavior or go haywire in disease. We can use this
Feng Zhang
sk neuroscientists to dene the area that they are studying and one is bound to get a different answer every time. No longer tting into one niche, the eld can delve into the microcosm of molecules and cells but also expand out into the macrocosm of mankind itself. The complexity of issues were addressing now is at a completely different level than what we did 15 years ago, says Nora Volkow, director of the U.S. National Institute on Drug Abuse at the National Institutes of Health (NIH). In the past, it used to be one receptor in one area of the brain. Now we have the tools to monitor the complete system at any given point in timethat is, the entire brain and how it changes at short- and long-term intervals. We now can start to study all the proteins in the cell and their interactions, how cells communicate with one another to create networks, and how these relate to behaviors. Within the past 1020 years, three areas have come on the scene: transdifferentiation, optogenetics, and social neuroscience. From the Lilliputian to the large-scale, these subelds all aid in puzzling together the various pieces that comprise the brain.
technology to identify molecular targets and develop better drugs.Feng Zhang
CELL FATE REALIZED: TRANSDIFFERENTIATION

Stem cells and regenerative medicine have worked their way into the eld of neuroscience in the form of transdifferentiation. In this process, either a tissue-specic stem or precursor cell begets cells that it normally was not destined to produce. Transdifferentiation occurs in nature, albeit rarely. For example, when the lens of the eye is removed in salamanders, iris cells fashion themselves into lens cells. Fifty years ago, cell fate conversion consisted of a cloning technique called somatic cell nuclear transfer, which allows somatic cell DNA to be inserted into an enucleated egg cell. The technology has spawned such animals as Dolly the sheep but has fallen short of cloning non-human primates. In 2005, the eld became checkered when a South Korean research team, led by Woo Suk Hwang, claimed to have derived human embryonic stem cells using this technology, only to subsequently admit that they fabricated the data. However, the eld was redeemed one year later when Shinya Yamanakas group at Kyoto University in Japan utilized four embryonic stem cell genesOct3/4, Sox2, c-Myc, and Klf4to convert mouse and human skin broblasts into embryonic stem celllike cells called induced pluripotent stem (iPS) cells. Since then other
John Cacioppo
CREDITS: (STORY LANDIS) NINDS, NIH; (BACKGROUND) ISTOCKPHOTO.COM/ERAXION
researchers have used different gene or chemical concoctions to turn broblasts into iPS cells. One uses recipes kind of like in The Joy of Cooking, explains Story Landis, director of the U.S. National Institute of Neurological Disorders and Stroke at the NIH. Now you dont have to start at an embryonic stem cell or induce a pluripotent stem cell. Instead, you can take a broblast and treat it in a special way to directly turn it into various cell types. continued on p. 710
UPCOMING FEATURES
Focus on ChinaDecember 9 BS/MS Scientists (online only)January 13 Faculty: Lab CultureFebruary 3
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THE LUNDBECK FOUNDATION JUNIOR GROUP LEADER FELLOWSHIPS

The Lundbeck Foundation hereby invites applications for ve fellowships within biomedicine and two within natural sciences which will be granted to especially promising young researchers and their research groups. Priority will be given to at least three biomedical fellowships within the Foundationss focus areas: neurology, psychiatry and allergology/immune modulation.
The fellowships are awarded for ve years and each fellowship amounts to DKK 10 million (approx. Euro 1,3 million). The subject area should be frontline basic- or applied research within the scope of the Foundations grant strategy, which can be seen at www.lundbeckfonden.dk The fellowships may well attract Danish or foreign researchers from abroad who wish to move to Denmark and continue their research here. The call is also open for applicants at Danish universities and university hospitals. The fellowships are intended for seven researchers who are qualied to establish or develop their own research groups within the health- or natural sciences and who have received their Ph.D. degree within the last 5-7 years.
The application should include an account of the projects research plan, collaborators, budget and how the research group is envisioned to be placed within a Danish research institution. In addition, it should include a letter of intent from a resident researcher at the host institution, who makes him- or herself available as a mentor to facilitate the applicants establishment of the research group as an integral part of the host institution. Further guidance is provided in the application form. The application, written in English, should be sent via the Foundations Electronic Application System for Junior Group Leader Fellowships 2012 at www.lundbeckfonden.dk no later than December 15., 2011. For further information please contact Ulla Jakobsen, Science Manager at the Lundbeck Foundation, phone: (+45) 39 12 80 11 or at mail@lundbeckfonden.dk
The Lundbeck Foundation has controlling shareholdings in its subsidiaries H. Lundbeck, ALK-Abell and Falck. In addition, the Foundation manages nancial investments of approx. 1.3 billion. The Foundation supports research within the medical and natural sciences. In 2010, the Foundation had a prot after tax of approx. 400 million and made research grants of approx. 50 million.
Lundbeckfonden Vestagervej 17, DK-2900 Hellerup Tel. +45 39 12 80 00 www.lundbeckfonden.dk
HARVARD UNIVERSITY CENTER FOR BRAIN SCIENCE FACULTY POSITION
Faculty Positions in Neuroscience

The Department of Neuroscience (www.neurosci.tufts.edu) at Tufts University School of Medicine is expanding by adding tenure-track faculty positions. Positions are available at Assistant, Associate and Full Professor levels. The department will build on its core strengths and focus on the study of synapses, disorders of the nervous system and neuron-glial interactions. We are particularly interested in building on our research that is relevant to epilepsy, depression, neurodegenerative disorders and the neurobiology of obesity. We are seeking candidates who use innovative approaches to investigate problems that cross levels of investigation from molecular and cellular to systems and/or behavioral neuroscience. Candidates using molecular, genetic, electrophysiological and/or imaging methodologies to study neurons, synapses and networks are particularly encouraged to apply. We offer generous start-up packages, newly renovated laboratory space and a highly collaborative environment offering opportunities for both basic and translational research. Applicants should hold a Ph.D. and/or M.D. degree and have several years of productive postdoctoral experience. Successful candidates will be expected to develop thriving, well-funded research programs and to contribute to graduate and medical education. Please submit electronic applications including a CV, a statement of research interests and the names and email addresses of at least three references to: neuroscifacultyrecruitment@tufts.edu. TUSM is an Equal Opportunity Afrmative Action Employer. Women and minorities are encouraged to apply.
The Center for Brain Science is seeking an Assistant Professor who works in the area of systems neuroscience, broadly construed. We are particularly interested in candidates whose research programs apply novel experimental and computational approaches to the study of the structure and function of neural circuits and their relationship to behavior. Successful candidates will hold an academic appointment in a participating department in the Faculty of Arts and Sciences at Harvard University. The Center for Brain Science brings together scientists involved in research on systems neuroscience. Our aims include discovering and understanding the neural circuits that underlie behavior in diverse animal species. CBS fosters interactions across disciplinary boundariesfaculty from several academic departments share neighboring research and meeting space and common research facilities; its connections reach out across the University. Further information about the Center for Brain Science is available at http://cbs.fas.harvard.edu/. Candidates should have demonstrated excellence in both research and teaching. A strong doctoral record is required and postdoctoral experience preferred. Teaching opportunities will include offerings at both undergraduate and graduate levels. To apply, please submit a cover letter, curriculum vitae, research statement, statement of teaching philosophy, up to three publications, and contact information for three people who will write letters of reference on your behalf to http://academicpositions.harvard.edu/postings/3718. Your application, including letters of reference, should be complete by 28 November 2011. Applications from, or nominations of, women and minority candidates are strongly encouraged. Harvard University is an Afrmative Action/ Equal Opportunity Employer.
focus on neuroscience
FOCUS ON CAREERS
We hope to readily reprogram easily accessible somatic cells from a patient with one of these neurological diseases into iPS cells or directly into neurons to model the disease and develop personalized treatments. Sheng Ding
FEATURED PARTICIPANTS
Center for Cognitive and Social Neuroscience, University of Chicago ccsn.uchicago.edu Gladstone Institute of Cardiovascular Disease www.gladstone.ucsf.edu Massachusetts Institute of Technology www.mit.edu National Institute of Neurological Disorders and Stroke www.ninds.nih.gov National Institute on Drug Abuse www.nida.nih.gov Society for Neuroscience www.sfn.org Society for Social Neuroscience www.s4sn.org Stanford University www.stanford.edu University of Oxford www.ox.ac.uk
The technique has various applications in neuroscience. Transdifferentiation gives you an unprecedented opportunity to study neurological diseases such as autism, schizophrenia, Alzheimers disease, and Parkinsons disease, explains Sheng Ding, senior investigator of the Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco. We hope to readily reprogram easily accessible somatic cells from a patient with one of these neurological diseases into iPS cells or directly into neurons to model the disease and develop personalized treatments. Postdoctoral researchers and graduate students interested in the eld should enter now, according to Ding. He proposes that new therapeutics developed through iPS cell technology will be available in 10 years.
SHEDDING LIGHT: OPTOGENETICS

The brain consists of approximately 100 billion neuronsabout the same number as stars in a galaxy. Each neuron can also have anywhere from 1,000 to 10,000 synapses. Depending on the type of information that a neuron is sending, the signaling speeds can vary from 0.6 m/s (in the case of transmitting pain) to upwards of 120 m/s (in the case of muscle stimulation). Hence neuronal mass and speed make studying brain functions daunting. Scientists have used a variety of techniques to elucidate neuronal function, but each has its own shortcomings. Electrophysiological techniques that physically delve electrodes into brain tissue are restricted by the depth to which probes can be placed and have limited ability to distinguish a single cell type amongst the myriad of cells interspersed throughout the brain. Pharmacological or genetic manipulations can help isolate signals from specic cell types; however, the results are often slow to take effect, from hours or days to months. Enter optogenetics or the merging of optics and genetics to allow control of very well-dened events within a particular cell, explains Karl Deisseroth, associate professor of psychiatry and bioengineering at Stanford University, who coined the term. Gero Miesenbck, a physiology professor at the University of Oxford describes the technique as using two avors of light-responsive proteins: sensors that light up when a neuron becomes active and actuators that absorb light and turn activity on or off. Deemed Method of the Year by Nature Methods in 2010 and
highlighted in the Insights of the Decade special section by Science that same year, optogenetics is a newcomer in the neuroscience realm, emerging less than 10 years ago. This eld however, borrows from observations and discoveries made 3040 years ago. In 1979, Francis Crick pointed out the difculty of using electrodes to pinpoint specic neurons in the brain and later speculated that light might be able to hone in on one type of cell and leave others unaltered. At the time though, no neuroscientist knew how to make neurons responsive to light. Over the years, biologists discovered many different kinds of lightresponsive proteins, or opsins. Among these, ion channels that open when a chemical co-factor, all-trans-retinal, absorbs photons were found in algae. However, the genes encoding these opsins were not identied until 2003, and neurobiologists focused rather on cell-directed tools that used combinations of custom-made chemicals and genes to alter neuronal function. Until 2005, when Deisseroths group discovered that these microbial opsins could precisely control neurons in response to light and, in 2006, showed that even adult vertebral tissues, including the brain, express natural alltrans-retinal. Prior to these studies, Miesenbcks lab had developed other strategies for optogenetic control of nerve cells by reassembling fruit y (Drosophila) opsin signaling pathways in neurons or combining light-activated chemicals with introduced genes. In 2005, they remote-controlled y behavior with light. His group also developed a genetic means to visualize nerve cell activity by creating synapto-pHluorin, a pH-sensitive form of green uorescent protein. Deisseroths group subsequently demonstrated the use of microbial opsins for neuronal control in freely moving mammals. They described beroptic interfaces that can be implanted in the brain to provide the light needed to activate these channels and target specic neurons in the recesses of the brain. Now, optogenetics is ubiquitous in neuroscience, and a variety of tools can be used to either activate or inhibit a neuron. It offers the best of all worlds: You can manipulate a specic cell type within a specic brain region, and you can do so with millisecond precision. This means that we can start to tease apart the functions of different cell types, activating or inactivating them to causally test their roles in brain continued on p. 712
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CREDIT: PHOTO BY CHRIS GOODFELLOW
Platform Managers in Neuroscience

Linkping University is one of Swedens six large universities, currently enrolling 27,000 students. e university recently launched a Centre for Neurobiology, involving some 20 independent research groups from two faculties: the Faculty of Medicine and the Faculty of Science and Engineering. With the goal of developing advanced common research platforms, the Centre for Neurobiology is now announcing three platform manager positions. In addition to running each platform and working together with the di erent research groups, there is also excellent opportunity for the successful candidate for conducting independent technology-oriented research. We are currently seeking experts in the following areas: Rodent transgenesis, Computer-based imaging, and Behavioural phenotyping. For more details regarding the Centre, the positions, and how to submit your application please go to: http:// www.hu.liu.se/neuro?l=en. For information regarding Linkping University, please go to: http://www.hu.liu. se/?l=en, http://www.liu.se/?l=en.
The Norwegian University of Science and Technology (NTNU) in Trondheim represents academic eminence in technology and the natural sciences as well as in other academic disciplines ranging from the social sciences, the arts, medicine, architecture to ne art. Cross-disciplinary cooperation results in innovative breakthroughs and creative solutions with far-reaching social and economic impact.
Faculty of Medicine
Professorship/ Qualication Fellowship in Systems Neuroscience

The Kavli Institute for Systems Neuroscience of the Faculty of Medicine at the Norwegian University of Science and Technology (NTNU) invites applications for a faculty position in systems neuroscience. The new position is part of NTNUs strategic effort in the eld of neuroscience. We seek applicants with experience and interest in using state-ofthe-art molecular and cellular technologies to understand neural networks and behaviour. The successful candidate holds a Ph.D. and has experience leading a research team and attracting international funding. The candidate has a track record in both molecular-cellular and systems neuroscience, with outstanding publications, and she/he demonstrates ability to develop an internationally competitive research programme. Participation in teaching activities at masters and PhD level is required. The applicant will benet from the strong infrastructure at the Kavli Institute for Systems Neuroscience at NTNU (www.ntnu.no/cbm). Start-up funding, including scientic equipment and PhD/post doctoral fellows, is negotiable. The position is advertised at the rank of Professor but may alternatively be dened as a qualication fellowship for a period of no longer than 3 years in case of sufcient future potential.Young applicants not yet qualied for full professorship are thus encouraged to apply. Applicants should submit a cover letter, a CV with a complete publication list, copies of 5 selected papers, a summary of research achievements, a research plan, and 3 letters of reference (referees should send letters directly to Edvard Moser, Director of the Kavli Institute, edvard.moser@ntnu.no). The complete advertisement is available at www.jobbnorge.no. Submit applications through www.jobbnorge.no within 1 December, 2011. For further information about the position contact Edvard Moser, edvard.moser@ntnu.no, tel. +47 73598278; information about the application process contact Brit Lbeck Fladvad, HR- section, Faculty of Medicine, brit.advad@ntnu.no. See also http://www.medisin.ntnu.no/eng/
Neuroscience postdoc programme

Linkping University is one of Swedens six large universities, currently enrolling 27,000 students. e university recently launched a Centre for Neurobiology, involving some 20 independent research groups from two faculties: the Faculty of Medicine and the Faculty of Science and Engineering. e Centre for Neurobiology is now seeking Postdoctoral Fellows (2+2 years) within several neuroscience research areas: Addiction, Animal Behaviour, Electrophysiology and Circuits, Neuroimaging and Neuroengineering, Neurodegeneration, Neuroendocrinology, Neurodevelopment, Pain, and Psychiatric Functional Brain Imaging. For more details regarding the Centre, the di erent research labs involved in the program, and to submit a letter-of-intent please go to: http://www.hu.liu.se/ neuro?l=en. For information regarding Linkping University, please go to: http://www.hu.liu.se/?l=en, http://www.liu.se/?l=en.
Jobbnnorge.no
focus on neuroscience
FOCUS ON CAREERS
[Enter optogenetics or] the merging of optics and genetics to allow control of very well-dened events within a particular cell. Karl Deisseroth
function and behavior, comments Joanna Mattis, a graduate stu dent in Deisseroths lab. Feng Zhang, a former graduate student of Deisseroth who is now an assistant professor of neuroscience at the Massachusetts Institute of Technology, adds: By turning on or off specic neurons, one can identify their place in particular neurocircuits and how these circuits function in normal behavior or go haywire in disease. We can use this technology to identify molecular targets and develop better drugs. Optogenetics studies from Miesenbck and Deisseroths groups, as well as others, have literally and metaphorically shed light on neural codes relevant to Parkinsons disease, autism, schizophrenia, drug abuse, anxiety, and depression. For future and current graduate students in neuroscience, Deisseroth advises that they follow their passion. Students should pursue the things that interest them, comments Deisseroth. The history of optogenetics is a parable for maintenance of basic science research. These days everybody is trying to justify their biology work in terms of disease relevance. Whereas deep insights into neurology and psychological diseases have been provided using optogenetics, the essential tools for this work were taken from algae and archaebacteria, remote and odd forms of life that were studied for many decades by people who had no consciousness of disease relevance and studied them just for their beauty. Because of the breadth and depth of data that is now coming into the eld, Zhang advises students to get multidisciplinary training. Things are becoming high throughput and experiments are done on a shorter timescale. Before, it took a year to test a hypothesis, now one can do it in a couple of months, he says. Dont just get training in biology, but also in computational biology and physics. The more versatile a person is, the more contributions this person will be able to make.
now director of the Center for Cognitive and Social Neuroscience at the University of Chicago. Biologists thought social processes had little relevance to the basic structure and function of human biology. Social scientists thought we were centuries away from biology being able to contribute to solutions to world wars, great depressions, and social injustices. There have been a lot of changes since then. These changes came from the convergence of data from psychology and biology studies using traditional animal models. For example, knowledge about social bonding (attachment, altruism, trust) advanced from the discovery that oxytocin and vasopressin receptors are localized in different brains regions of the more social prairie vole compared to the more solitary montane and meadow voles. Because of this research, clinical studies are emerging investigating intranasal oxytocin as a treatment for autism. Social neuroscience has an application for various mental disorders, for example, depression and autism, since these all have a social component, states Cacioppo. The eld draws upon numerous neurobiological techniques such as functional magnetic resonance imaging (fMRI), transcranial magnetic stimulation, electrocardiograms, and studies of patients with focal brain lesions. Social neuroscience is becoming more of a heavyweight in science now that we have tools, theories, and a common language to communicate with one another, says Greg Norman, a post doc in Cacioppos lab. This is the science of the mindnot just psychology, not just biologybut the integration of the human condition. It encompasses many elds. You can be a geneticist or a sociologist and still be a social neuroscientist. This multidisciplinary approach can be both a strength and a weakness. Although more data is generated from collaboration, each discipline has its own jargon, often obfuscating each level of analysis. There is a challenge in trying to get people to use a common language instead of just talking past each other. And trying to understand how all the pieces t into a whole is really difcult, adds Norman. Our eld encompasses genetics all the way to the study of societies. You can be in this eld for 100 years and still not comprehend its breadth. To avoid competition among the disciplines and bring them together, the Society for Social Neuroscience, for which Cacioppo is president, has separate awardsone for animal science and one for human science. But he hopes that in time, we wont have this distinction.
STATE OF (NEURO)SCIENCE
These additional facets of neurosciencetransdifferentiation, optogenetics, social neurosciencereect the overall state of science. Fifty years ago a solitary genius was doing the work, now the geniuses are working in teams, says Cacioppo. Its not only how science is performed that has changed, but also budgets. Its the best of times and its the worst of times, says Landis. There are wonderful opportunities to use all of this technology but not enough funding for all of the possible projects. Choosing the most promising areas to pursue will require difcult choices.
Jacqueline Ruttimann Oberst is a freelance writer living in Chevy Chase, Maryland. DOI: 10.1126/science.opms.r1100111
STRENGTH IN NUMBERS: SOCIAL NEUROSCIENCE

The brain does not work in isolation and neither do humans. We are, after all, social creatures. A new eld has arisen from this ideasocial neurosciencethe study of the neural, hormonal, cellular, and genetic mechanisms that dene social species. For 40 years, traditional neuroscience considered the nervous system as an isolated entity devoid of any signicant inuences from the social environment. Biology and social sciences, at best, were at odds, explains John Cacioppo, who is one of the founders of the eld and is
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CREDIT: CHRISTOPHER KROEGER
Chair and Professor Department of Neurosciences
POSTDOCTORAL FELLOW
Neuroscience
A Postdoctoral fellow position is available in the lab of Michael J. Friedlander, Ph.D. to work on effects of temporal patterns of synaptic activation on long term plasticity in neocortex in normal and injured brains. Interest and skills in synaptic physiology, optical imaging and synaptic plasticity are preferred. Position includes competitive salary and benefits, state of the art facilities, and dynamic neuroscience research community. There is the opportunity to join a major new neuroscience program and group, and to develop one's own projects, as well. The University Research Institute is located in picturesque Roanoke Valley in Roanoke, VA, midway between Washington, DC and Charlotte, NC. Please send letter of interest, CV and list of references to: Michael J. Friedlander, Ph.D., Executive Director and Professor, Virginia Tech Carilion Research Institute at friedlan@vtc.vt.edu. For additional information please visit:
The School of Medicine at Case Western Reserve University invites applications and nominations for the position of Chair of the Department of Neurosciences. This distinguished department was established in 1989 and has been led by two eminent Neuroscientists, Story Landis and Lynn Landmesser. It includes 16 primary faculty members who span broad areas of neuroscience, including developmental and molecular neurobiology, cellular neurobiology and systems level studies of neural circuits and their relation to behavior. In addition to strong basic science, research programs are addressing causes and therapies related to spinal cord injury, stroke, Alzheimers disease, Rett syndrome, and multiple sclerosis. The department has an outstanding graduate program, continuously supported by NIH since its founding. Broad interdisciplinary training is provided by more than 40 participating faculty from 15 departments. Further growth and enhancement of the department under the new chair will be supported by an attractive recruitment package. Thirty secondary faculty members from both basic and clinical departments participate in collaborative research and training programs. To promote collaborative research, the Center for Translational Neuroscience occupies space contiguous with the Department and houses faculty from other departments engaged in neuroscience research. The unied campus at CWRU facilitates collaborative interactions between neuroscientists in the Schools of Medicine, Engineering and Arts and Sciences, and with researchers in nearby afliated hospitals. Research missions are supported by excellent core facilities (behavioral testing, whole animal imaging, confocal/multiphoton imaging, structural biology, proteomics and others) and a recently expanded and renovated animal facility, including a state-of-the-art transgenic facility. Applicants for this position must have a Ph.D. and/or an M.D. degree with a distinguished record of scientic achievement, demonstrated leadership skills, dedication to the research mission, and a commitment to education and mentorship of students and faculty. Appointment as Professor of Neurosciences with tenure is anticipated. Please submit a CV and letter of interest addressing research, educational, administrative and leadership goals and vision to Neuroscience Chair Search Committee, c/o Denise Davis (Denise.Davis@case.edu). In employment, as in education, Case Western Reserve University is committed to Equal Opportunity and Diversity. Women, veterans, members of underrepresented minority groups, and individuals with disabilities are encouraged to apply. Case Western Reserve University provides reasonable accommodations to applicants with disabilities. Applicants requiring a reasonable accommodation for any part of the application and hiring process should contact the Ofce of Inclusion, Diversity and Equal Opportunity at 216-368-8877 to request a reasonable accommodation. Determinations as to granting reasonable accommodations for any applicant will be made on a case-by-case basis. http://neurosciences.case.edu
http://research.vtc.vt.edu/
and
http://research.vtc.vt.edu/employees/ michael-j-friedlander/
Virginia Tech has a strong commitment to the principle of diversity and, in that spirit, seeks a broad spectrum of candidates including women, minorities, and people with disabilities.
Faculty Position in Systems Neuroscience Department of Physiology Northwestern University Feinberg School of Medicine
A tenure-track position is open for a full-time faculty researcher (PhD, MD/PhD or MD) in systems neuroscience. Applicants should have an outstanding record of research in systems neuroscience that adds to existing departmental strengths in sensorimotor integration, pain perception, and learning and memory. Individuals using animal models and innovative experimental techniques including multi-electrode recordings or optical imaging, as well as advanced computational methods are particularly encouraged to apply, but the scientic excellence of the candidate is more important than the particular area of research. Rank is open. All applicants should have substantial peer-reviewed publications that demonstrate research productivity and the ability to perform cutting edge research. Candidates for an Assistant Professor position should have postdoctoral research experience. Candidates seeking appointment at the Associate Professor or Full Professor level should have substantial research productivity and a history of grant support and academic service. The successful applicant will also have the opportunity to participate in graduate and medical education. Starting date is negotiable. Applicants should send (single PDF) a cover letter, curriculum vitae, a two-page description of research accomplishments and plans, and the name and contact information for three references by e-mail to neuroscience@northwestern.edu Northwestern University is an Afrmative Action, Equal Opportunity Employer. Women and minorities are encouraged to apply. Hiring is contingent upon eligibility to work in the United States.
POSITIONS OPEN
Director of Basic Sciences Temasek Life Sciences Laboratory, Singapore

Temasek Life Sciences Laboratory (TLL) invites applications for appointment as Director of Basic Sciences. Temasek Life Sciences Laboratory is a non-pro t research Ins tute located on the Campus of the Na onal University of Singapore (www.tll.org.sg). Temasek Life Sciences Laboratory is a liated to the Na onal University of Singapore and Nanyang Technological University and enjoys close links with other research organiza ons based in Singapore and elsewhere. Research in Temasek Life Sciences Laboratory falls into two broad categories - Basic Sciences and Strategic Research. Research work within Basic Sciences has strengths in Cell Biology, Developmental Biology, Molecular Pathogenesis, Genomics, and Structural Biology and uses a variety of bacterial, fungal, plant and animal models. The Temasek Life Sciences Laboratory has excellent infrastructure and facili es for research. The Director of Basic Sciences will report to the Execu ve Director and work closely with the other members of the TLL Management team. The candidate will oversee the Basic Sciences Research Program to maintain high standards and interna onal visibility. The candidate will also coordinate the recruitment and assessment of basic sciences principal inves gators. The candidate will be expected to forge new research ini a ves with local and interna onal research organiza ons. The candidate should be an outstanding scholar who will be able to provide strong leadership in research and training. The candidate should have an excellent track record and an interna onal recogni on in research in any area of research represented at the Temasek Life Sciences Laboratory. Administra ve experience would be an added advantage. Research support and laboratory facili es are available. Remunera on will be commensurate with the candidates quali ca ons and experience. Interested par es should submit their applica ons, supported by a detailed resume and names of at least six referees to: Prof. Chan Soh Ha, Execu ve Director Temasek Life Sciences Laboratory, The Na onal University of Singapore,1 Research Link, Singapore 117604 chansh@tll.org.sg (Closing date for applica on: 31 January 2012)
Faculty Position Molecular and Cellular Cancer Biology University of Pittsburgh Cancer Institute
The University of Pittsburgh Cancer Institute (UPCI) (www.upci.upmc.edu ) at the University of Pittsburgh has a strong program in molecular and cellular cancer biology (www.upci.upmc.edu/mccbp ) and seeks to recruit faculty at the Assistant Professor level to develop outstanding research programs that bring approaches complementing our existing strengths in three broad areas. These include: genome stability (DNA damage and repair, aging and cancer, telomere biology and maintenance, and cell cycle control); cellular responses to stress (gene expression, signal transduction and ER stress); mitochondrial physiology (apoptosis, metabolism and cancer). There are opportunities for extensive collaborations within the University of Pittsburgh Medical Center and Carnegie Mellon University, as well as opportunities for participation in related graduate programs. We seek candidates who will establish competitive research programs in the elds of DNA repair and chromatin/nuclear structure, DNA replication, protein trafcking, signal transduction or transcription. We would particularly welcome applications from individuals who use state-of the art biochemical, imaging and molecular biology tools working in an array of biological systems that can translate bench science to clinical cancer applications. Candidates with a track record of independent funding and publications in high impact journals will be given the highest consideration. Successful candidates will be expected to run a vibrant collaborative program supported by external funding. A competitive salary and research start-up package will be provided. The University of Pittsburgh School of Medicine is consistently among the top ten in NIH-funded medical schools in the U.S. and is located in one of Americas most livable cities. Positions will be coordinated with Departments in the University of Pittsburgh and are tenure track. To apply, please send your curriculum vitae, a one-page summary of your research plans, and three letters of recommendation to: Bennett Van Houten, PhD, UPCI Research Pavilion, Hillman Cancer Center Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213-1863, e-mail: derussoj@upmc.edu. Applications will be reviewed and evaluated upon receipt of full applications on an ongoing basis. The University of Pittsburgh is an Afrmative Action, Equal Opportunity Employer.
TENURE-TRACK FACULTY POSITION
DEPARTMENT OF BIOCHEMISTRY
The Department of Biochemistry invites applications for a tenure-track position at the Assistant or Associate Professor level. We are seeking individuals to establish internationally leading programs that apply the tools of structural and chemical biology to topics of biomedical relevance. The Medical College of Wisconsin (www.mcw.edu) is the largest private research institution in Wisconsin, conducting nearly $150 million annually in funded research. The Department of Biochemistry (www.mcw.edu/ biochemistry) is equipped with state-of-the-art X-ray and NMR facilities, and MCW is home to NIH-designated technology development centers for EPR spectroscopy and mass spectrometry. Institutional disease-focused initiatives provide translational research opportunities for candidates with an interest in structure-based drug discovery in many areas including cancer, cardiovascular disease and diabetes. Applicants must have a doctoral degree, at least 2 years of postdoctoral training, a strong publication record, and potential to obtain extramural funding. Competitive salary support, start-up funds and new laboratory space will be provided. Applications should include a cover letter, curriculum vitae, statement of research interests, copies of key publications, and three reference letters. Send application materials and reference letters electronically in pdf format to: Structural Biology Search Committee Department of Biochemistry Medical College of Wisconsin 8701 Watertown Plank Road Milwaukee, WI 53226 E-mail: akear@mcw.edu EOE M/F/D/V (www.mcw.edu/hr)
Your assignment
KTH Royal Institute of Technology has research activities in six strategic areas, thereby deepening our impact on the worlds great challenges. We are now looking for international calibre research talent to join us, and help us create a brighter tomorrow.
grand challenges in research
Our strategic research areas are: Information and communication technology e-Science Transportation Energy Molecular biosciences Production engineering Please visit www.kth.se/sra for more information about the available positions, our research and KTH.
KTH, founded in 1827, is Swedens leading technical university. We account for one-third of Swedens technical research and engineering education capacity at a university level. Education and research cover a broad spectrum from the natural sciences through all the branches of engineering, as well as architecture, industrial engineering and management, urban planning, work science and environmental engineering.
IST AUSTRIA IS LOOKING FOR
Professors and Assistant Professors

IST Austria (Institute of Science and Technology Austria) invites applications for Professors and Assistant Professors in all elds of the natural and mathematical sciences and related disciplines. Outstanding scientists in physics, chemistry, and mathematics are especially encouraged to apply. The Institute, which is situated on the outskirts of Vienna, was established by the Austrian government with a focus on basic research. The campus opened in 2009 and is expected to grow to 45 research groups and over 500 employees by 2016. IST Austria is entitled to award PhD degrees and includes an English-language graduate school. It aims to achieve an international mix of scientists and chooses them solely on the basis of their individual excellence and potential contribution to research. The Institute recruits tenured and tenure-track leaders of independent research groups. The successful candidates will receive a substantial annual research budget but are expected to also apply for external research grants.
For further information and access to the online application material, please consult: www.ist.ac.at/professor-applications Deadline for receiving Assistant Professor applications: January 15, 2012
IST Austria values diversity and is committed to equality. Female researchers are encouraged to apply.
The MAX DELBRCK CENTER FOR MOLECULAR MEDICINE (MDC) BERLIN-BUCH and the CHARIT UNIVERSITTSMEDIZIN BERLIN (CHARIT) invite applications for the following position:
Full University Professorship of Experimental Cardiovascular Research

Salary Group W3 BBesG (Code number: Prof. 388/2011)
The MDC is committed to expanding its impact in the eld of Experimental Cardiovascular Research and is seeking applications from outstanding individuals with international reputation in relevant areas of research including genetics, genomics, (patho)physiology of the heart, vascular biology or metabolic diseases. Successful candidates will conduct visionary independent research, obtain extramural funding and engage in collaborative projects with groups at the MDC. They will also integrate with and substantially contribute to Cardio Berlin, an institutionalized cooperation of the MDC, the CHARIT, and the Deutsches Herzzentrum Berlin with focus on the prevention of all stages of the cardiovascular continuum. The successful applicant will be scienti c member of the MDC Berlin-Buch and of the Medical Faculty of the CHARIT. The position is af liated with the MDC Berlin-Buch. Quali cations: Junior Professorship or postdoctoral thesis (Habilitation) or equivalent scienti c achievements and teaching quali cations (as per 100 Berlin Higher Education Act - BerlHG). The MDC Berlin-Buch and the CHARIT are equal opportunity employers. The applications of women are explicitly encouraged. For further information about the MDC Berlin-Buch and the CHARIT please visit our web sites http://www.mdc-berlin.de or http://www.charite.de. For enquiries about the position please contact Thomas Willnow (willnow@mdc-berlin.de). Applications should be sent by December 16, 2011 including a curriculum vitae, list of publications, an outline of present and planned research and other relevant material (see: http://www.charite.de/en/charite/organization/careers/employment_market/internship/) either to or to Prof. Dr. Annette Grters-Kieslich Dean Charit Universittsmedizin Berlin 10098 Berlin (Germany) Prof. Dr. Walter Rosenthal Scienti c Director Max Delbrck Center for Molecular Medicine (MDC) Berlin-Buch 13092 Berlin (Germany)
The MDC Berlin-Buch is a member of the Helmholtz Association of National Research Centers, supported by the Federal Government of Germany and the Land Berlin. The CHARIT is the Medical School of the Humboldt-Universitt zu Berlin and the Freie Universitt Berlin. For electronic applications please use the e-mail: jobs@mdc-berlin.de and professur-bewerbung@charite.de. Please send your application as one le, pdf-format, maximum size 1 Mb.
MICROBIOLOGY/IMMUNOLOGY FACULTY POSITIONS

The Department of Biological Sciences, College of Sciences, Old Dominion University, invites applications for several tenure track/tenured Faculty positions (beginning July 2012) at the Assistant Professor, Associate Professor, or Professor level as part of a major recruitment initiative. We are particularly interested in: (1) investigators in host-pathogen interactions and molecular pathogenesis; (2) molecular or cellular immunologists employing contemporary molecular biology approaches. A successful candidate must establish and maintain a vigorous research program that will attract peer-reviewed funding, and provide excellent education to our graduate and undergraduate students. All applicants must have a Ph.D., M.D. or an equivalent degree in an appropriate field. Applicants at the Associate Professor or Professor level must demonstrate substantial research accomplishments, a consistent record of independent peer-reviewed funding, and have active competitive grants. Interactions are encouraged with other departments/units of the College and the University, as well as the Eastern Virginia Medical School. A cross-appointment with the Center for Molecular Medicine (Chris D. Platsoucas, Ph.D., Center Director) is available. State salary support and competitive start-up packages are available. The Department of Biological Sciences receives substantial support from state funds, as well as from research grants from federal and other granting agencies. The department has strong Ph.D. and M.S. graduate programs that currently enroll over 125 students. The College of Sciences is undergoing a major research expansion. Over the last three years research grant awards to the College have increased by 78% to $20.6 million in FY2010. Old Dominion University (www.odu.edu) is a state supported, Carnegie doctoral research extensive institution enrolling more than 24,000 students including 6,000 graduate students. Interested individuals should submit curriculum vitae, a statement of research achievements and research plans, and the names, addresses, e-mail addresses and phone numbers of three references to Wayne Hynes, Ph.D., Professor and Chairman, Department of Biological Sciences, Old Dominion University at MICB@odu.edu. Review of applicants will begin immediately and continue until the positions are filled.
Old Dominion University is an affirmative action, equal opportunity institution and requires compliance with the Immigration Reform and Control Act of 1986.
Faculty Position in Microbiology

The University of Georgia invites applications for a full time, tenure track Assistant Professor position in the areas of microbial physiology and/or microbial ecology. The successful candidate will be expected to establish a nationally recognized research program involving the role of microorganisms in complex environments, in biogeochemically important processes, and/or in biotechnological applications. Topics of interest include microbial diversity, energy-generating pathways, symbioses, bioprocessing and bioremediation, extremophiles, and the molecular basis of unusual lifestyles or physiology. Applicants should have a Ph.D. in Microbiology, Biotechnology, Ecology or a related discipline and at least two years of postdoctoral research experience. Successful applicants will be expected to establish a vigorous externally-funded research program and to instruct and mentor undergraduate and graduate students. To apply, the following should be submitted at: http://recruitment.frankli n.uga.edu (1) a single PDF containing cover letter, curriculum vitae, and 1-2 page statements of research interests and teaching philosophy; (2) a single PDF containing reprints of three research papers; and (3) three letters of recommendation submitted directly by the references. For questions, please contact Nancy Perkins at micro@uga.edu or 706-542-2677. The University of Georgia is located 70 miles northeast of Atlanta in Athens, Georgia. Founded in 1785, the University of Georgia is the nations rst state-chartered university. More information on Athens, Georgia can be found at: http://www.visitathensga.com/. To learn more about the Department of Microbiology and the University of Georgia, visit our website at www.uga.edu/mib. Review of applications will begin January 15, 2012, and the search will remain open until the position is lled. The start date for the position is August 13, 2012. The Department of Microbiology of the Franklin College of Arts and Sciences and the University of Georgia are committed to increasing the diversity of faculty and students and sustaining a work and learning environment that is inclusive. Women, minorities and people with disabilities are encouraged to apply. The University of Georgia is an EEO/AA Institution.
The National Socio-Environmental Synthesis Center in Annapolis, MD Announces its opening and rst call for research applications
SESYNC is the newest NSF-funded national synthesis center, hosting researchers to integrate diverse forms of information, data, and models to inspire novel research and provide innovative solutions to socio-environmental problems. Through its diverse programs, the Center invites researchers from across the globe to work on-site at the SESYNC headquarters in Annapolis. Applications are invited from social, natural and computational scientists for support through one of SESYNCs programs: Thematic Pursuits, Ventures, Workshops, Socio-environmental Education, Short-courses, Policy & Practice Fellows, Postdoctoral Fellows, and Sabbatical Fellows
Founding Theme: Ecological Wealth and Changing Human Populations

SESYNCs Founding Theme focuses on understanding how shifts in the distribution of human populations across the landscape have inuenced, and been inuenced by, natural systems and the goods and services they provide. We invite applications to support Pursuits by individuals or teams focused on major scientic questions central to this Theme. The center will have 2-3 Themes ongoing at any time and all future Themes will be identied through a community-driven process beginning early Spring 2011. Applications due January 31, 2012.
Digital Information Research Specialist

Position Opening for a scholar-programmer to join SESYNCs cyberinfrastructure team. Advanced degree in environmental science with computational focus or advanced degree in computational science, library and information science, or computer science (Ph.D. preferred but not required). For best consideration, apply by December 31, 2011.
Postdoctoral and Sabbatical/Research Fellowships

Researchers with expertise and ideas to employ integrative, analytical, computational, or informatic approaches focused on the structure, functioning, and sustainability of socio-environmental systems are invited to apply. Applicants may have received their PhD in ANY relevant area of research including anthropology, ecology, economics, education, geography, mathematics, statistics, computer science, political science, public policy, planning, etc. Postdoctoral positions are for 2 years and sabbatical/research fellows 2-12 months. Applications due January 31, 2012.
For details on SESYNC programs and careers please see www.SESYNC.org.
Baylor College of Medicine FACULTY POSITION Verna and Marrs McLean Department of Biochemistry and Molecular Biology Applications are being accepted for a tenure-track or tenured faculty position at any rank in the Verna and Marrs McLean Department of Biochemistry and Molecular Biology, known for its innovative and diverse research programs. Applicants should have a strong record of research productivity and creativity and should show promise for, or demonstrated success in, securing research funding. Applications will be considered from candidates with research interests in any areas relevant to molecular biomedical research. Areas of particular interest include chemical biology and drug discovery, structural biology of membrane proteins, signaling, metabolomics, proteomics and systems biology. We offer highly competitive start-up packages and salaries, outstanding research facilities, including many specialized cores and centers, as well as an unusually large and interactive research community within the Texas Medical Center and nearby institutions of the Gulf Coast Consortium. An endowed chair may be available for an appropriate senior candidate. Applications will be reviewed as they are received; priority will be given to completed applications received by December 15, 2011. Applicants should submit a statement of research interests, curriculum vitae, and contact information for three individuals who have agreed to provide letters of reference, as a single PDF le via e-mail to Theodore G. Wensel, Ph.D., twensel@bcm.edu, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Baylor College of Medicine is an Equal Opportunity/Afrmative Action/Equal Access Employer.
NATIONAL SPACE BIOMEDICAL RESEARCH INSTITUTE VICE PRESIDENT AND INSTITUTE ASSOCIATE DIRECTOR
The National Space Biomedical Research Institute (NSBRI) is an academic consortium competitively engaging more than 60 institutions to conduct integrated biomedical research and technology development, to ensure safe long-duration human spaceight and to apply the advances to improve health on Earth. NSBRI invites applications for the position of Vice President and Associate Institute Director. The position oversees the Institutes diverse Science and Technology Program, Education and Outreach Program, and carries major responsibilities in the partnership with NASA. The Vice President and Associate Institute Director serves as a key interface with outstanding investigators, team leadership, students, institutions, NASA, industry, and national and international collaborators to coordinate and implement a scientic program of national stature and importance. Effective utilization of NSBRIs new demonstration laboratories, located in the BioScience Research Collaborative, Houston, TX, provides an opportunity to help shape the Institutes expanding capabilities on behalf of our nations space program. Reporting to the NSBRI President and Institute Director, the Vice President and Institute Associate Director is a corporate ofcer of NSBRI and a member of the senior management team. He or she is expected to hold a full-time academic appointment at Baylor College of Medicine, at the rank of Associate Professor or full Professor, in a department appropriate to the eld of expertise. In addition to a Ph.D., M.D. or other doctorate degree, applicants for the position must have at least 10 years of scientic leadership experience, with evidence of excellence in scientic and/or technical achievement, competitive research support, and productive interactions involving distinguished researchers in diverse disciplines. Desired characteristics include effective leadership and management skills, knowledge of space life sciences, and integrity and credibility deserving of NSBRIs multiple stakeholders. Interested candidates should submit a curriculum vitae in condence before December 15, 2011, to: Search Committee National Space Biomedical Research Institute BioScience Research Collaborative 6500 Main Street, Suite 910 Houston, TX 77030-1402 Applicants are also required to apply directly to vacancy #214122 at www.medschooljobs.org. For more information on the NSBRIs research and education programs, please visit www.nsbri.org. Baylor College of Medicine is an Equal Opportunity, Afrmative Action and Equal Access Employer.
Department of Physics and Astronomy Experimental/Theoretical Biomedical/ Biophysics Tenure-Track Faculty Position
The Department of Physics and Astronomy at Wayne State University invites applications for a faculty appointment, subject to administrative approval, in the area of experimental or theoretical biomedical/biophysics. We are seeking individuals with exceptional promise for a tenure-track assistant professor position beginning in the Fall 2012 semester. Senior appointments may be considered for highly qualied applicants. We are particularly looking for researchers who can collaborate in or complement ongoing research activities in the department. Active departmental research in biomedical/biophysics includes: Fluorescence tracking of single molecular motors, Raman spectroscopy of biological tissues, single molecule force measurements, uorescence correlation spectroscopy, nanoparticle synthesis for drug delivery and cancer treatments, and nonlinear dynamics of cell injury. The department also has strong research programs in condensed matter physics, atomic physics, high-energy particle physics, astrophysics, and relativistic heavy ions physics. In addition, there are opportunities for collaborating with researchers in other areas of the university, including the nationally recognized medical school. The successful applicant is expected to become an effective teacher at both the physics undergraduate and graduate levels, to supervise M.S./Ph.D. dissertations, to lead an internationally competitive, fundable research program in biomedical/biophysics, and to take an active role in the undergraduate biomedical physics program. All applicants should send a curriculum vitae and a detailed statement of research interests through the Academic Application on the WSU Online Hiring System at jobs.wayne.edu (Posting #038155). More information about the application process, including on-line application instructions and position number, is available on the Department website at http:/www.physics.wayne.edu. Applicants should also arrange to have at least three letters of reference sent by December 15, 2011 either through email or by regular mail to: Peter Hoffmann, Chair, Physics Search Committee, Department of Physics and Astronomy, 666 W. Hancock, Wayne State University Detroit, MI 48201 [Email address: BMPSearch@physics.wayne.edu]. We will begin reviewing applications December 15, 2011. Wayne State is an Afrmative Action/Equal Opportunity Employer. Women and members of minority groups are encouraged to apply.
Two Professors in Medical Image Analysis

Two full time positions are available, both placed at the Research and development platform MedTech West. Administratively placed at Chalmers University of Technology and University of Gothenburg. The duties of the positions include research, teaching and supervision. For further information about the positions and how to apply, please visit Vacancies at
www.chalmers.se
The Region Vstra Gtaland, Sweden, has initiated the Regional Imaging and Intervention Centre, to be opened in 2015 at Sahlgrenska University Hospital, Gothenburg. The advertised positions should be viewed in this context as a centre of this kind depends on advanced and cutting edge imaging technologies and techniques.
Director, Center for Immunology

The Director will lead the new Center for Immunology with responsibility to expand the existing Immunology research program in the Department of Pathology and lead interdepartmental research and education programs in Immunology. A substantial start-up package will be provided for the Directors laboratory, and the Director will have responsibility to lead recruitment of 3-4 additional faculty positions supported by start-up packages. The current Immunology programs strengths include fundamental immunology (innate immunity, signaling, MHC molecules, APCs, T cell biology), immunology of infectious diseases (particularly tuberculosis and HIV), autoimmunity (e.g. IBD) and other topics. Annual research funding in areas related to infectious disease immunology, pathogenesis and biology exceeds $100M at CWRU and afliated institutions. Research training is focused on the Immunology Training Program (http: //www.case.edu/med/pathology/training/itp.html). Candidates should be established scientists with international stature and successful and rigorous research programs. Leadership experience and a successful track record as a scientic mentor are desired. Appointment as Professor with tenure is anticipated.
The Immune Disease Institute, Childrens Hospital Boston and Harvard Medical School are affirmative action/equal opportunity employers. Women and minority candidates are strongly encouraged to apply.
Please send a cover letter, CV, and contact information for three references to Clifford V. Harding, Chair, Department of Pathology, c/o Denise Davis (dmd10@case.edu). In employment and education, CWRU is committed to equal opportunity and diversity. Women, veterans, members of underrepresented minority groups, and individuals with disabilities are encouraged to apply. Accommodations for application and hiring are described at http://www.case.edu/diversity/faculty/writinganad.html.
HARVARD MEDICAL SCHOOL
Department of Health and Human Services National Institutes of Health / National Eye Institute International Programs Manager, GS-601-15 (Organizational Title: Associate Director for International Programs)
POSITION: The National Eye Institute (NEI), a major component of the National Institutes of Health (NIH) and the Department of Health and Human Services (DHHS), is seeking exceptional candidates for the position of Associate Director for International Program Activities. The NEI is responsible for a national and international program to conduct and support research, training, health information dissemination, and other programs with respect to blinding eye diseases, visual disorders, mechanisms of visual function, preservation of sight, and the special health problems and requirements of individuals who are visually impaired. The incumbent reports directly to the Director, NEI and is the chief technical and policy advisor on global vision health and international programs. The Associate Director will conduct planning, management, and evaluation of the international cooperative research activities in vision disorders and will (1) serve as the NEI focal point with the Fogarty International Center and the State Department, and other Federal organizations involved in international health activities; (2) coordinate vision research activities under bilateral agreements between the United States and other countries; and manage international contracts and budgets; (3) maintain liaison with international organizations involved in the prevention of blindness and vision disorders; (4) plan and implement programs for international exchange of scientists; (5) assume a leadership role in fostering communications between the NEI researchers and the international scientic community; and (6) travel to national and international meetings, workshops, and other forums to foster global vision research. The position includes opportunities for the pursuit of specic international agendas and collaborations with global vision health and research partners. Applicants should be known and respected within their profession, both nationally and internationally. QUALIFICATIONS REQUIRED: Applicants must possess a degree in an academic eld related to the health sciences or allied sciences appropriate to the work of the position. Experience in academic, professional society, and/or industry settings is important for the level of liaison and collaboration required with constituents from sectors such as ophthalmic and optometric health service organizations, government agencies, and international research groups. SALARY AND BENEFITS: GS-15 salary range is $123,758 - $155,500. Full Federal benets are available including leave, health and life insurance, long-term care insurance, retirement, and retirement savings plan (401K equivalent). HOW TO APPLY: Interested candidates may apply at www.usajobs.com to vacancy announcement NIH-NEI-DE-12-499213, beginning 11/28/2011. Applications must be received by midnight December 27, 2011. For questions, contact Thomascene White, Human Resources Specialist, Ofce of Human Resources at whitet1@mail.nih.gov. The NIH encourages the application and nomination of qualied women, minorities, and individuals with disabilities. DHHS, NIH and NEI ARE EQUAL OPPORTUNITY EMPLOYERS.
Department of Health and Human Services National Institutes of Health National Institute of Neurological Disorders and Stroke
The University of Campinas (UNICAMP), one of the leading research universi es in Brazil and South America, is seeking highly quali ed facul es for tenured posi ons to carry out teaching and research in Bioenergy. The Bioenergy Laboratory at UNICAMP will o er lab and o ce space for research in basic and applied science. Also, FAPESP, a leading science and technology funding agency in Brazil, has created a speci c program for funding proposals in this area of research. Applicants should be PhDs with high academic achievements and are expected to carry out teaching and research ac vi es in one of the following areas: Sensors development for applica on in agriculture Photosynthesis biomolecules or biomime c structures Plant produc vity and photosynthesis Process engineering related to the u liza on of microorganisms for bioenergy produc on Sustainability in bioenergy produc on The annual star ng salary of a tenured faculty at UNICAMP is approximately R$ 109,200 (around US$ 65,900). In case of interest, please send your CV (English or Portuguese) to: carreiras@reitoria.unicamp.br (Subject: Bioenergy + area of interest). Applicants whose CVs best suit the posi ons requirements will be invited to par cipate in an open selec on examina on. For further informa on, please contact: info.carreiras@reitoria.unicamp.br.
Facility Manager Protein/Peptide Sequencing Facility The Division of Intramural Research of the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH in Bethesda, MD is searching for outstanding applicants for a Facility Manager for its Protein/Peptide Sequencing Facility. The successful incumbent will oversee a mix of service-based and project-based activities while developing new collaborations both in biophysical protein analysis and proteomics. Though this is primarily a service facility, there are ample opportunities for collaboration and activities to explore and extend the facilitys work in new directions. The successful candidate will work closely with leadership in NINDS to make recommendations for improvement of resources for protein analysis and proteomics to serve the NINDS and NIH neuroscience communities, with the possibility of hiring further support staff in the future if needed. Opportunities for developing training curricula or new courses are available. Presently the facility is equipped with the following: Thermo LTQ ion trap LC/MS, AB/SCIEX MALDI TOF/TOF MS, Waters Q-TOF Micro LC/MS, Agilent Model 1100 HPLC, ABI Protein Sequencer, and a Digilab In-Gel Digestion Robot. Approximately 10-15 different groups within NINDS and the broader NIH neuroscience community make use of the facility. Based on current usage, the primary requirement for the position is skill in analysis of puried proteins and complexes, with a smaller component of proteomics. This is a Staff Scientist position effective early 2012. A Ph.D. in Chemistry or Biological Sciences with an emphasis in biological protein analysis using mass spectrometry, and two or more years of postdoctoral experience are required. The salary is commensurate with experience. Applicants should send a cover letter describing qualications and career plans, a curriculum vitae with bibliography, and a single page document describing his/her Philosophy of Core Facility Management, and have three letters of reference sent to: Peggy Rollins, Ofce of the Scientic Director, Division of Intramural Research, NINDS, NIH, Building 35 Room GA908, Bethesda, MD 20892 or Peggy.Rollins@ninds.nih.gov. Review of applications will begin on November 30, 2011, but applications will be received until the position is lled. HHS and NIH are Equal Opportunity Employers.
FACULTY POSITIONS
Biology NYU ABU DHABI
NYU Abu Dhabi is currently inviting applications for two positions (open rank, tenure or tenure-track) for appointments as faculty in its Division of Science and Mathematics. We encourage applications from candidates who have research interests in neurobiology or molecular and cell biology. Candidates will be expected to have or develop active research programs and to participate in the divisions teaching activities at the undergraduate level. New York University (NYU) has established itself as a Global Network University, a multisite, organically connected network encompassing key global cities and idea capitals. The network has three foundational, degree-granting campuses: New York, Abu Dhabi and Shanghai, complemented by a network of over fifteen research and study-away sites across five continents. Faculty and students will circulate within this global network in pursuit of common research interests, the promotion of cross-cultural understanding and solutions for problems, both local and global. Entering its second year, NYU Abu Dhabi has already recruited a cohort of faculty who are at once distinguished in their research and teaching. Our first two classes of students are drawn from around the world and surpass all traditional recruitment benchmarks, both US and global. NYU Abu Dhabis highly selective liberal arts enterprise is complemented by an institute for advanced research, sponsoring cutting-edge projects across the arts, humanities, social sciences, natural sciences and engineering. The terms of employment are very competitive and include housing and educational subsidies for children. Faculty may also spend time at NYU New York and other sites of the global network, engaging in both research and teaching opportunities. The appointment might begin as soon as September 1, 2012, but candidates may elect to start as late as September 1, 2013. Applications are due by November 30, 2011; applications received later will be reviewed until the positions are filled. We will consider senior candidates on a rolling basis. Candidates should submit a curriculum vitae, statements of research and teaching interests (not to exceed three pages each), no more than three representative publications and three letters of reference in PDF format to be considered. Please visit our website at http://nyuad. nyu.edu/human.resources/open.positions.html for instructions and other information on how to apply. If you have any questions, please e-mail nyuad.science@nyu.edu.
Cellular Biology of Parasitism Assistant/Associate Professor

The Department of Cellular Biology at the University of Georgia invites applications for a tenure track Assistant/Associate Professor position. We seek a highly qualied individual with a research program in host-parasite interactions at the cellular, metabolic or immunologic level, with particular interest in programs focusing on malaria. The Department has strong representation in the Center for Tropical and Emerging Global Diseases, one of the worlds leading centers for parasite research. The position includes a very competitive salary, excellent laboratory space and a generous start-up package that can be enhanced by an endowment. Applicants should send a cover letter, curriculum vitae, research statement and teaching philosophy and arrange to have 3 letters of reference sent to: cbsearch@uga.edu. Applicants must hold a PhD or related graduate degree and have postdoctoral research training. Appointment will be commensurate with experience. Applications received by January 10, 2012 are assured of full consideration. The Franklin College of Arts and Sciences, its many units, and the University of Georgia are committed to increasing the diversity of its faculty and students, and sustaining a work and learning environment that is inclusive. Women, minorities and people with disabilities are encouraged to apply. The University of Georgia is an Afrmative Action/Equal Opportunity Institution.
NYU Abu Dhabi is an Equal Opportunity/Affirmative Action Employer.
Faculty Position in Metabolism Research Faculty Position in Cancer Research

The Childrens Research Institute (CRI) at the University of Texas Southwestern Medical Center seeks applications for a tenure-track faculty position in Dallas, TX at the Instructor, Assistant Professor, or Professor level in the area of cancer biology. Outstanding investigators at any academic rank will be considered. Candidates must have a Ph.D., M.D. or equivalent degrees, a track record of outstanding research, and the ability to direct an independently-funded research program. The UT Southwestern Medical Center has a distinguished history of excellence in disease-related basic science research. The CRI is a new institute dedicated to recruiting outstanding scientists dedicated to solving fundamental problems in human disease and to providing a dynamic, stimulating and highly collaborative scientic environment. Major areas of focus within the CRI will include stem cell biology and cancer in addition to metabolism. Please submit a CV, a 2-page summary of past accomplishments and research plans, and ask three references to submit letters to Beth Morris at beth.morris@utsouthwestern.edu. CRI is a collaborative venture with Childrens Medical Center of Dallas
The Childrens Research Institute (CRI) at the University of Texas Southwestern Medical Center seeks applications for a tenure-track faculty position in Dallas, TX at the Instructor, Assistant Professor, or Professor level in the area of metabolism and disease. Outstanding investigators at any academic rank will be considered. Candidates must have a Ph.D., M.D. or equivalent degrees, a track record of outstanding research, and the ability to direct an independently-funded research program. Areas of specic interest for this position include metabolomics, metabolic ux analysis, mitochondrial biology and other areas of metabolism relevant to human health and development. In addition to analytical equipment dedicated to the investigators studies, CRI members will also have access to a metabolomics core housing instrumentation for ultra-high pressure liquid chromatography, triple-quadrupole mass spectrometry and gas chromatography/mass spectrometry. NMR spectroscopy, 13C dynamic nuclear polarization, a human 7-Tesla MRI and a state-of-the-art mouse metabolic phenotyping facility are also available on campus to provide an unparalleled breadth of metabolic analysis. The UT-Southwestern Medical Center has a distinguished history of excellence in disease-related basic science research. The CRI is a new institute dedicated to recruiting outstanding scientists dedicated to solving fundamental problems in human disease and to providing a dynamic, stimulating and highly collaborative scientic environment. Major areas of focus within the CRI will include stem cell biology and cancer in addition to metabolism. Please submit a CV, a 2-page summary of past accomplishments and research plans, and ask three references to submit letters to Beth Morris at beth.morris@utsouthwestern.edu. CRI is a collaborative venture with Childrens Medical Center of Dallas
UT Southwestern is an Equal Opportunity/Afrmative Action Employer.
UT Southwestern is an Equal Opportunity/Afrmative Action Employer.
Donald Hedberg Distinguished Professor of Entrepreneurial Studies in the Natural Sciences

Carthage College seeks an experienced and accomplished candidate to become the next Donald Hedberg Distinguished Professor of Entrepreneurial Studies in the Natural Sciences and to lead the Entrepreneurship in the Natural Sciences Program (Science Works). Founded in 1994, the Science Works program has propelled hundreds of Carthage undergraduates beyond traditional post-graduate options and into areas that are entrepreneurial or innovative in nature. The Hedberg Chair oversees the Science Works Program, which includes the minor in Entrepreneurial Studies in the Natural Sciences. The successful candidate will engage in teaching and mentoring of undergraduate students in research and the development of technical business plans. The Hedberg Chair is expected to strengthen the program by seeking extramural funding and partnerships across the Milwaukee-Chicago corridor. The Chair will work collaboratively with departments throughout the College to expand entrepreneurship opportunities for Carthage students. The Chair is housed in the Natural Science Division of the College and works with a faculty that has a long history of collaboration and interdisciplinary work. The College welcomes applications from scholars and professionals with a history of successful innovation and leadership in a technical field. The successful candidate should hold a Ph.D. at the time of appointment, but candidates with a significant combination of academic and professional accomplishment will be considered. Carthage was founded in 1847 and is a private college of the liberal arts and sciences affiliated with the ELCA Lutheran Church with approximately 2,500 full-time and 720 part-time students. The College awards degrees in more than 40 subject areas and has a long-standing commitment to educating the whole person by nourishing the intellectual, spiritual, emotional, social, and physical dimensions of students lives. The Carthage faculty is composed of over 150 scholars invested in teaching. The College is located on the picturesque shore of Lake Michigan midway between Chicago and Milwaukee and is a focal point in the Kenosha community. The search committee will begin the review of credentials and continue until the position is filled. For best consideration, candidates should submit a curriculum vitae and a letter of interest in the position outlining a teaching philosophy and a vision for technical entrepreneurship in the 21st Century by December 7, 2011. All application materials should be submitted as a single pdf document online through www.carthage.edu/careers. Nominations, inquiries, or expressions of interests should be directed to (preferably electronically): Julio C. Rivera, Jr. Provost and Search Committee Chair Carthage College 2001 Alford Park Drive Kenosha, WI 53140 e-mail: hedberg-search@carthage.edu voice: 262-551-5850 fax: 262-551-5843
U.S. Environmental Protection Agency Ofce of Research and Development HIGH-LEVEL CAREER OPPORTUNITIES
EPAs Ofce of Research and Development (ORD) is seeking internationally recognized scientists/engineers to ll two positions in the National Risk Management Research Laboratory (NRMRL). One position is located in Ada, Oklahoma and one position is located in Cincinnati, Ohio. NRMRLs leadership in science and engineering is recognized throughout EPA and the environmental community as the source of responsive, objective solutions to complex, multidisciplinary environmental problems. More information about NRMRL can be found at www.epa.gov/nrmrl/. ORD plans to ll these positions using EPAs Title 42 Authority, which offers up to 5-year renewable appointments at highly competitive, market-based salaries. The positions are part of a larger EPA effort to use state-of-the-science approaches and technologies in its mission of protecting human health and the environment. The ideal candidates will have a degree in a relevant eld, extensive specialized experience and accomplishments, and an international reputation for technical excellence. Positions and major duties include: RTP-ORD-42-2011-0003, Director, Ground Water and Ecosystems Restoration Division (Ada, Oklahoma). Interdisciplinary -Biologist, Engineer, Physical Scientist, Chemist Leading the nations only research program focused on groundwater protection and restoration. The high visibility science program currently encompasses studies of the relationship between groundwater resources and hydraulic fracturing for extraction of natural gas and carbon sequestration. The responsibilities include advocacy, management of a large and highly skilled staff and supervision of a state-of-the-art research facility. The goal of the program is to bring innovation to achieve sustainable solutions to complex environmental problems with a focus on groundwater resources. RTP-ORD-42-2011-0004, Director, Water Supply and Water Resources Division (Cincinnati, Ohio). Interdisciplinary - Biologist, Engineer, Physical Scientist, Chemist Leading a research program focused primarily on protection of water resources and drinking water treatment. The mission of the Division is to achieve sustainable solutions to complex water issues facing the nation and the globe. Current research areas in the Division are associated with solving critical issues on sustainable water resources and include aging water infrastructure, adaptation to climate change, adoption of new treatment technologies and management of emerging microbial and chemical contaminants in drinking water and distribution systems. Responsibilities for all positions include providing hands-on leadership of ORDs research programs, serving as a senior spokesperson/ representative, identifying collaborative opportunities with outside organizations, and playing a vital leadership role within NRMRL. Salary and Benets: Salary is up to $200,000 per annum, depending upon qualications, experience, and other factors. The selected applicant will be eligible for full benets including health and life insurance, retirement, vacation and sick leave. For more information about the organization and the job announcements, including contacts for questions about the position, please refer to www.epa.gov/ORD/NRMRL/jobs/index.html.
www.carthage.edu
The U.S. Environmental Protection Agency is an Equal Opportunity Employer.
Endowed Chair Professorship in Biochemistry or the Department of Molecular, Cellular and Developmental Biology
The University of Colorado Boulder is seeking applications and nominations for the Jennie Smoly Caruthers Endowed Chair in Biochemistry or Molecular Biology. The scientist will hold the rank of Professor in the Department of Chemistry and Biochemistry or the Department of Molecular, Cellular and Developmental Biology. It is expected that the selected candidate will be an internationally recognized, outstanding scientist with a track record of well-funded research, broadly cited publications and will have demonstrated success in teaching, graduate training, and service to the academic community. The successful candidate will be located in research space that will be shared with other world-class scientists conducting multidisciplinary research in systems biology, biochemistry, bioorganic chemistry, computational biology, and biomedical engineering. It is expected that the individual occupying this endowed chair will conduct collaborative research with colleagues in order to solve complex biomedical problems, requiring the expertise from several disciplines. Applications are accepted electronically at https: //www.jobsatcu.com, posting #814500. Applications must include a cover letter, a statement of current and future research interests, a curriculum vitae including previous and current funding, and letters from at least four references. Review of applications will begin on September 1, 2011. Applications will be accepted until the position is lled. The University of Colorado does not discriminate in any condition of employment on the basis of race, color, national origin, sex, age, disability, creed, religion, sexual orientation, or veteran status. Equal Employment Opportunity (EEO) shall apply to all terms, conditions, and privileges of employment, including hiring, probation, testing, training and development, promotion, transfer, compensation, benets, educational assistance, discipline, termination, layoffs, social, cultural and recreational programs, and retirement. See www.colorado.edu/ArtsSciences/Jobs/ for full job description. The University of Colorado at Boulder is committed to diversity and equality in education and employment.
TENURE-LINE FACULTY POSITION IN COMPUTATIONAL AND SYSTEMS IMMUNOLOGY The Stanford Institute for Immunity, Transplantation and Infection invites applications for a tenure line faculty position at the level of Assistant Professor in the area of computational and systems immunology. The predominant criterion for appointment in the University Tenure Line is a major commitment to research and teaching. Candidates must have a doctoral degree and formal advanced training in Biomedical Informatics or Computer Science as well as a strong background in Immunology, and must have excellent teaching and communications skills. The search committee encourages applications from candidates who have research interests in computational analyses of immunology, especially using systems methods as they pertain to human data and diseases. The successful candidate will conduct a vigorous, independent research program that includes the development and application of bioinformatics and informatics solutions to immunological research, particularly as it relates to the human immune system and disease. Additionally, the candidate will be expected to advise graduate students and should possess creativity and strong interpersonal skills in order to work in a collaborative and diverse faculty. Applicants should send curriculum vitae, a brief statement of their research interests, and the names of three referees to: Mark M. Davis, Ph.D., Director Institute for Immunity, Transplantation and Infection c/o Michele King, ITI Program Manager bioinformatics-search@stanford.edu We will begin reviewing applicants on December 15, 2011. Stanford University is an Equal Opportunity Employer and is committed to increasing the diversity of its faculty. It welcomes nominations of and applications from women and members of minority groups, as well as others who would bring additional dimensions to the Universitys research, teaching and clinical missions.
FACULTY POSITION STRUCTURAL BIOLOGY, BIOCHEMISTRY AND BIOPHYSICS University of Connecticut - Storrs
The Department of Molecular and Cell Biology at the University of Connecticut invites applications for a 9-month ASSISTANT OR ASSOCIATE PROFESSOR TENURE TRACK position to complement and expand upon existing strengths in the areas of Structural Biology, Biochemistry and Biophysics, starting August 23, 2012 (Search #2012118). Rank will be commensurate with experience. The successful candidate will be expected to have or develop a productive, independent research program, teach at the undergraduate and graduate levels, and employ structural or biophysical approaches to address important biological problems. Minimum qualications include a Ph.D. in Biophysics, Biochemistry or a closely related eld, postdoctoral experience and an outstanding record of research accomplishments in structural biology or biophysics. Equivalent foreign degrees are acceptable. Preferred qualications include the ability to contribute through research, teaching, and/or public engagement to the diversity and excellence of the learning experience. This position is at the main campus in Storrs; however, incumbents may also work at the University of Connecticuts regional campuses in Avery Point, Hartford, Stamford, Torrington, Waterbury and West Hartford. Applicants should login at http://www.jobs.uconn.edu to submit a CV and concise statements of research and teaching interests. In addition, applicants should arrange to have at least three letters of reference sent to sb3@uconn.edu as a PDF document on letterhead with signature. To ensure full consideration, applications should be received by December 15, 2011. The University of Connecticut is an EEO/AA Employer.
ASSOCIATE PROFESSOR/PROFESSOR Cancer Biology

The Leroy T. Canoles Jr. Cancer Research Center and the Department of Microbiology and Molecular Cell Biology invite applications for the position of Associate Professor/Professor. We seek outstanding candidates with demonstrated excellence in cancer research as evidenced through peer-reviewed publication and external funding. The LTCJCRC has existing strength in cancer translational research and capitalizes upon both clinical and basic science infrastructure. The center is located on the 4th oor of the new Education and Research Building providing modern laboratory space, state-of-the-art research resources and a centralized meeting and ofce footprint. Candidates with expertise in cancer biology, cancer metastasis, treatment, molecular signaling or biomarker discovery using either proteomics/genomics and/or animal models are sought. However, all outstanding candidates in cancer research are encouraged to apply. A competitive start-up package will be provided to the successful candidate with appointment to the Leroy T. Canoles Jr. Cancer Research Center and the Department of Microbiology and Molecular Cell Biology. Interested individuals should send a complete curriculum vitae, a statement of current and future research interests, and the contact information for three references to: O. John Semmes, Ph.D., Director, Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, ERB 425, 651 Colley Avenue, Norfolk, VA 23507 or via e-mail: conyerbl@evms.edu. Review of applications will begin January 1, 2012, and will continue until the position is lled. EVMS was founded by the community to improve the health of the community through teaching, discovering and caring. A collaborative culture at EVMS draws like-minded students, physicians and scientists from all over the country and encourages a multidisciplinary research approach with an emphasis on translational research. EVMS is an Afrmative Action/Equal Opportunity Employer and Drug and Tobacco Free Work Place.
Science Scholarships and Fellowships
UNCF/MERCK SCIENCE INITIATIVE

The UNCF/Merck Science Initiative is an innovative approach that creates opportunities in the biological, chemical and engineering sciences for African American students throughout the country.
Assistant/Associate/Full Professor/Endowed Chair Positions Molecular Virology and Prokaryotic Systems Biology Department of Biochemistry and Molecular Biology
The department seeks to add highly qualied tenure-track or tenured faculty at any academic rank with demonstrated excellence in broad areas of molecular virology or systems biology of prokaryotes. These additions to our faculty represent the commitment of the Eberly College of Science and the Huck Institutes of the Life Sciences to expand collaborative research in infectious diseases, genomics, biofuels and the environment. Molecular virology candidates are sought with expertise in broad areas including, but not limited to, mechanisms of viral replication, host responses to viral infection and viral antagonism of host responses. Investigators combining molecular approaches with cell-culture and animal models are encouraged to apply. Prokaryotic systems biology candidates are sought with expertise in broad areas engaging the domains Bacteria or Archaea including infectious disease, biofuels, ecology and the environment. Candidates aspiring to combine genomics and bioinformatics with quantitative approaches towards understanding global metabolism and host or environmental interactions are encouraged to apply. Successful candidates will have the opportunity to participate in several centers including Infectious Disease Dynamics, Molecular Immunology and Infectious Disease, Comparative Genomics and Bioinformatics, Astrobiology Research and the Institutes for Energy and the Environment. These positions feature outstanding research space in the recently completed, state-of-the-art, Millennium Science Complex, competitive start-up packages and access to university-subsidized research support facilities including genomics, DNA core, X-ray crystallography, high-throughput calorimetry, high-eld NMR, proteomics mass spectrometry, electron and confocal microscopy, ow cytometry and fermentation (http://www.huck.psu.edu/). The successful candidate will be expected to direct an innovative, well-funded research program, to contribute to the teaching mission of the department and to participate actively in service. Applications should be submitted as a single pdf document via e-mail to Traci Shimmel (tshimmel@psu.edu). Document should include curriculum vitae, description of research interests and teaching goals (2-3 pages). PLEASE REFERENCE JOB NUMBER 35399B. Review of applications will begin in December and continue until positions are lled.
Penn State is committed to Afrmative Action, Equal Opportunity and the diversity of its workforce.
UNDERGRADUATE
Science Research Scholarship Awards
n Scholarships up to $25,000 n A paid summer internship at a Merck facility with stipend totaling more than $5,000 n Mentoring and networking opportunities n Eligibility: College juniors, science or engineering majors, 3.3 GPA
GRADUATE
Science Research Dissertation Fellowships
n Fellowships up to $53,500 n Mentoring and networking opportunities n Eligibility: Ph.D. or equivalent degree candidates engaged in dissertation research in the biological, chemical or engineering fields
Careers with Mass Appeal
Tenure-Track Faculty Positions

Department of Chemistry
The Department of Chemistry of the University of Massachusetts Lowell invites applications for two tenured or tenure-track faculty positions. The Department consists of 16 full-time faculty and offers BS, MS, and PhD degrees. Successful candidates will be expected to teach Chemistry at the undergraduate and graduate levels and develop/sustain active, high quality, externally funded and nationally and internationally recognized research programs. A PhD in chemistry or very closely related discipline is required; postdoctoral experience is preferred. Applicants at all ranks will be considered. However, preference may be given to those with a record of excellence in teaching, scholarship, and funded research. Applicants having expertise in the following areas are especially encouraged to apply. 1) Analytical Chemistry, with applications in areas such as sensor development, rapid screening methods, diagnostic testing, method development for testing the purity, stability and efficacy of new drugs related to pharmaceutical research and high throughput monitoring for pharmaceutical, homeland security and environmental applications. 2) Biochemistry, with research interests in biomolecule-drug interactions, metabolism, molecular basis of drug design, drug pharmacokinetics and pharmacodynamics, enzymatic pro-drug processing, biological therapeutics, nucleic acid-based drugs, establishing and validating biomarkers. 3) Inorganic Chemistry, with research that would fit into the Analytical, Biochemistry, Organic, or Polymer Science programs. Examples include chemical and biological sensing, mechanistic biochemistry, drug design/delivery, and inorganic polymers with energy storage/conversion/ distribution applications. 4) Polymer Science, with expertise in synthesis and applications of polymeric biomaterials, such as polymers for controlled drug release, polymer-drug conjugates, polymers for medical devices and medical device coatings.
POSTDOCTORAL
Science Research Fellowships
n Fellowships up to $92,000 n Mentoring and networking opportunities n Eligibility: Ph.D. or equivalent degree recipients in the biological or chemical research fields
APPLY ON-LINE
http://umsi.uncf.org Submit by December 1, 2011 T 703 205 3400 F 703 205 3550 E uncfmerck@uncf.org
GENERAL ELIGIBILITY REQUIREMENTS: Must be African American and a U.S. citizen or a permanent resident
Review of applications will commence immediately and the search will continue until suitable candidates are found. For complete posting details, application deadlines, and to apply, please visit http://jobs.uml.edu. Only online applications will be accepted.
The University of Massachusetts is an Equal Opportunity/Affirmative Action Title IX, H/V, ADA 1990 employer.
ANNOUNCEMENTS
Stanford University Dean of the School of Medicine

Stanford University invites nominations and applications for the position of Dean of the School of Medicine, beginning with the 2012-2013 academic year. The Dean is responsible for the direction of the training, research, and clinical missions of the school. We seek individuals with outstanding scholarly and professional careers who have demonstrated potential for academic and administrative leadership and present a salient vision of unied excellence across the School of Medicine. Located in close proximity to the six other schools in the University, and the adult, pediatric, and VA hospitals, the School of Medicine provides an educational environment that encourages intellectual freedom and unparalleled opportunities for cross-disciplinary research to solve major human health problems. Stanford School of Medicine offers a stimulating setting for students and faculty who are interested in developing a scholarly, investigative approach to problems in the practice, economics, and socio-political facets of medicine and creating new knowledge through basic science research. Accordingly, Stanford has designed its M.D., Ph.D., and Medical Scientist Training (M.D./Ph.D.) programs to achieve two goals: to develop in all medical students the capacity for leadership in the scientic practice of clinical medicine; and to provide all trainees with outstanding opportunities to prepare themselves for careers in basic, clinical, or the social aspects of medicine. Stanfords basic science and clinical faculty, who are among the national leaders in their respective elds, create and support a culture of innovation and creativity that is essential for excellence in research, education and clinical care. The Dean of the School of Medicine must have vision and leadership to sustain and enhance excellence in all parts of the schools mission, including fostering innovative educational, entrepreneurial, and research interactions with Stanfords other schools and partnering with hospital leadership for delivery of outstanding patient care. The Dean is responsible for administration of the School, participation in University governance and representing the School to outside constituencies. In addition, the Dean will represent the School of Medicine in working with the leadership of Stanford Hospital and Clinics and the Lucile Packard Childrens Hospital to establish clinical strategies and long-term goals. Consideration of nominations and applications for the position of Dean of the School of Medicine will begin immediately. Please address inquiries, nominations, and applications by January 3, 2012 to: deansearch@stanford.edu
Welcomes applications for its departments below at ranks of Instructor, Assistant & Associate Professor, or Professor.
Stanford University is an Equal Opportunity, Afrmative Action Employer and actively works to ensure that the search identies qualied candidates who are women and/or members of ethnic minorities. The University has a strong institutional commitment to the principle of diversity. AA/EOE.
FACULTY POSITION CRYO-ELECTRON MICROSCOPY VANDERBILT UNIVERSITY

The Department of Molecular Physiology and Biophysics and the Center for Structural Biology at Vanderbilt University invite applications for a tenure-track/tenured appointment in the area of biophysics/structural biology with an emphasis on cryo-electron microscopy approaches. Appointment is possible at the Full, Associate or Assistant Professor level. Applicants must have a Ph.D. and/or M.D. degree, and an outstanding record of research achievement that demonstrates the potential or ability to direct a world-class independent research program. The appointee will nd a rich multi-disciplinary research environment and opportunities for close collaborations within the School of Medicine, the School of Engineering, and the College of Arts & Science. Vanderbilt University is located in Nashville Tennessee, a cosmopolitan city rich in cultural activities. Please send a curriculum vitae, statement of research and teaching interests, and three letters of recommendation to: Hassane Mchaourab, Ph.D., Chair, Search Committee, e-mail: mpbfaculty .search@vanderbilt.edu. Review of applications will begin in December 2011 and continue until the position is lled. Vanderbilt University is an Equal Employment Opportunity/Afrmative Action Employer. Women and minority candidates are encouraged to apply.
TRANSLATIONAL BIOMEDICAL RESEARCH OPPORTUNITY Genomic Medicine/Bioinformatics

Sigfried and Janet Weis Center for Research is seeking outstanding independent scientists for full-time research positions at ranks equivalent to Assistant, Associate or Full Professor in the areas of Genomics and Bioinformatics. The Weis Center is a basic and translational research facility of Geisinger Clinic located at Geisinger Medical Center (GMC) in Danville, PA. Genomic Medicine is a strategic focus for translational research at Geisinger. Genomic Medicine is a strategic focus for translational research at Geisinger. About the position: Expertise in laboratory, computational, or statistical genetic approaches Expand ongoing research on the genetic basis of disease Proven records of innovative research with relevance to human disease Collegial environment with collaborative research opportunities Geisinger Health Systems advanced electronic medical record system and health information technology infrastructure allows for electronic capture of clinical data and large biorepository of patient specimens. Technical resources include instrumentation for confocal,TIRF, and single cell fluorescence imaging, microarray analysis, genotyping, DNA sequencing, and flow cytometry, and an AAALAC-accredited animal facility. Substantial resources are available for start-up, ongoing research support and salary. Qualified individuals should submit curriculum vitae, statement of research interests and three reference letters to Ms. Kristin Gaul,Weis Center for Research, Geisinger Clinic, via email (kgaul@geisinger.edu). Please refer to position WCR-3638 in the subject line. Applications will be accepted until the positions are filled. For more information on research programs at Geisinger visit our website at http://www.geisinger.org/professionals/research/wcr.
Geisinger Health System is an Affirmative Action/Equal Opportunity Employer
H E A LT H S Y S T E M
REDEFINING THE BOUNDARIES OF MEDICINE
THE 2012 LOUISA GROSS HORWITZ PRIZE FOR BIOLOGY OR BIOCHEMISTRY

The Louisa Gross Horwitz Prize was established under the will of the late S. Gross Horwitz through a bequest to Columbia University and is named to honor the donors mother. Louisa Gross Horwitz was the daughter of Dr. Samuel David Gross (1805-1889), a prominent surgeon of Philadelphia and author of the outstanding Systems of Surgery who served as President of the American Medical Association. Each year since its inception in 1967, the Louisa Gross Horwitz Prize has been awarded by Columbia University for outstanding basic research in the fields of biology or biochemistry. The purpose of this award is to honor a scientific investigator or group of investigators whose contributions to knowledge in either of these fields are deemed worthy of special recognition. The Prize consists of an honorarium and a citation which are awarded at a special presentation event. Unless otherwise recommended by the Prize Committee, the Prize is awarded annually. Dr. Jeffrey C. Hall, Brandeis University, Waltham, MA, Dr. Michael Rosbash, Brandeis University, Waltham, MA and Dr. Michael W. Young, The Rockefeller University, New York, NY were the 2011 awardees.
QUALIFICATIONS FOR THE AWARD

The Prize Committee recognizes no geographical limitations. The prize may be awarded to an individual or a group. When the prize is awarded to a group, the honorarium will be divided among the recipients, but each member will receive a citation. Preference will be given to work done in the recent past. Nominations must be submitted electronically at: http://www.cumc.columbia.edu/horwitz/ Nominations should include: 2. A summary, preferably less than 500 words, of the significance of this research in the fields of biology or biochemistry. 3. A brief biographical sketch of the nominee, including positions held and awards received by the nominee. 4. A listing of up to ten of the nominees most significant publications relating to the research noted under item 1. 5. A copy of the nominees curriculum vitae.
Deadline date: January 31, 2012
C A L L F O R N O M I N AT I O N S 2012 Vanderbilt Prize in Biomedical Science

e Vanderbilt Prize in Biomedical Science was created to honor and recognize a woman scientist of national reputation who has a stellar record of research accomplishments and is known for her mentorship of women. e winner nurtures the career, research, and studies of a promising woman beginning her Ph.D. studies at Vanderbilt. e Vanderbilt Prize in Biomedical Science includes a $25,000 cash award to the recipient, who will visit Vanderbilt to give a lecture and serve as a mentor to the Vanderbilt Prize Scholar. NOMINATIONS DEADLINE IS JANUARY 31, 2012 Previous winners: 2011: Dr. Titia de Lange, Rockefeller University 2010: Dr. Nancy Andrews, Duke University 2009: Dr. Susan Taylor, Univ. of California, San Diego 2008: Dr. Ann Graybiel, MIT 2007: Dr. Elizabeth Blackburn, UCSF & 2009 Nobel laureate 2006: Dr. Nancy Andreasen of the University of Iowa
Contact Danielle Certa for a nomination form: danielle.certa@vanderbilt.edu | (615) 936-6228 Or visit https://medschool.vanderbilt.edu/dean/ and click Vanderbilt Prize in Biomedical Science.
Call for Nomina ons & Scholarships | 2012

The Dan David Prize is an interna onal enterprise which annually awards three prizes of US$ 1M each in elds chosen within the three me dimensions to individuals with proven, excep onal excellence and contribu on to humanity in the sciences, arts, humani es and public service. Ten percent of the prize is donated by the laureates as scholarships to outstanding young researchers, doctoral and postdoctoral students studying topics related to the chosen elds.
Selected Fields for 2012

History/Biography Plas c Arts Genome Research
Past Time Dimension Present Time Dimension Future Time Dimension
Nomina ons deadline: November 30th, 2011 Scholarships deadline: March 15th, 2012 Further details at www.dandavidprize.org
prizes
1. A summary, preferably less than 500 words, of the research on which this nomination is based.
POSITIONS OPEN
POSITIONS OPEN
POSITIONS OPEN
NEUROLOGIST POSITION Columbia University The Department of Neurology at Columbia University and NewYork-Presbyterian Hospital is seeking neurologists to join its faculty. We are looking for neurologists with general or subspecialty training who are interested in an academic neurology practice. Subspecialty training in headache and pain, multiple sclerosis, neuromuscular, or epilepsy highly desirable. The majority of clinical time will be spent in an outpatient practice based primarily at Columbia University Medical Center in New York City. Teaching residents, medical students, and fellows is an integral part of the position. Opportunities to participate in clinical research are available. Protected research time and appropriate resource support will be provided. Candidates should be board certified or board eligible in neurology. Subspecialty fellowship training desirable but not mandatory. Faculty rank expected to be at ASSISTANT or ASSOCIATE PROFESSOR level, commensurate with experience and achievement. Assistant Professor level candidates must provide evidence of research accomplishments and have some teaching experience. External research funding preferred. Qualified candidates should send a letter of interest and curriculum vitae in Word format to: Richard Mayeux, M.D., M.Sc. Department of Neurology 622 West 168th Street, P&S Box 16 New York, NY 10032 E-mail: rpm2@columbia.edu Apply electronically through website: https:// academicjobs.columbia.edu/applicants/Central? quickFind=55516. Columbia University is an Equal Opportunity/Affirmative Action Employer. TWO TENURE/TENURE-TRACK FACULTY POSITIONS North Dakota State University The Department of Biological Sciences seeks to fill two faculty positions in the areas ecology and regulatory biology to begin Fall 2012. Candidates are expected to develop an extramurally funded research program that complements existing research, supervise graduate students, and teach courses in their area at the undergraduate/graduate levels. Appointments at the ASSISTANT PROFESSOR level are anticipated, but more senior levels are possible with suitable experience. See website: http://www.ndsu. edu/biology/ for a complete description and application. Review of applications will begin December 15, 2011. North Dakota State University is an Equal Opportunity/ Affirmative Action Employer. BIOLOGICAL ANTHROPOLOGIST The University of California, Santa Barbara, Department of Anthropology, Integrative Anthropological Sciences Unit (IAS), invites applications for a tenure-track position at the ASSISTANT PROFESSOR level specializing in human biology with an active field and laboratory research program focusing on areas such as human physiology, reproduction, endocrinology, growth and development, health and disease, life history, population biology, immunology, epidemiology, genetics, or neurobiology. Applications are due by December 20, 2011 for primary consideration but the position will remain open until filled. See website: http://www.anth.ucsb.edu for more details.
TWO POSITIONS in Biological Sciences Terrestrial Vertebrate Biologist Animal Physiologist The Department of Biological Sciences at California State University, Long Beach (CSULB) invites applications for two tenure-track positions; a terrestrial vertebrate biologist at the ASSISTANT PROFESSOR level, and an animal physiologist at the ASSISTANT/ASSOCIATE PROFESSOR level. We seek broadly trained individuals who address fundamental research questions in these areas and have a strong interest in teaching at a research-oriented, comprehensive university. For further information, please see the position descriptions at website: http:// www.csulb.edu/divisions/aa/personnel/ jobs/cnsm/. CSULB is an Equal Opportunity Employer.
ASSISTANT PROFESSOR University of Washington The Department of Biochemistry at the University of Washington (UW) School of Medicine in Seattle invites applications for a tenure-track Assistant Professor position. We are seeking a creative scientist using innovative molecular approaches to understand cellular, developmental, or synthetic biology. Our department is also strongly committed to teaching at the undergraduate, graduate, and medical school levels. UW faculty engage in teaching, research, and service. Applicants should have a Ph.D. in the biosciences or an M.D. Submit a curriculum vitae, research prospectus, and reprints or preprints as PDFs to e-mail: bcsearch@ uw.edu by December 15, 2011. Three letters of recommendation (PDFs preferred) should be electronically sent separately to Trisha Davis, Chair, Search Committee, also at e-mail: bcsearch@uw.edu. The University of Washington is an Affirmative Action/Equal Opportunity Employer. CONSERVATION BIOLOGIST Assistant Professor SIU Carbondale The Department of Zoology at SIU Carbondale invites applications for a tenure-track position in animal conservation biology at the Assistant Professor level. The successful candidate will be expected to enhance departmental research and graduate program capabilities by developing an externally funded research program. Teaching duties will be in the areas of conservation biology, ecology, and general zoology or biology. Minimum Qualifications: (1) Ph.D. in Zoology/Biology with demonstrated research experience and graduate training in area of specialization; (2) demonstrated grant success or evidence of strong potential to obtain external funding; and (3) a publication record and scholarly activities commensurate with experience. Preference will be given to individuals with postdoctoral experience who are working at the community level, using modern analytical techniques, and have strong quantitative skills. SIU Carbondale is a large, research-oriented institution situated in a pleasant small-town setting southeast of St. Louis. The Department of Zoology has 24 fulltime faculty members, about 240 undergraduate majors, and some 90 M.S. and Ph.D. students. Its research emphases include ecology and ecosystem studies, ecotoxicology, evolutionary genetics, fisheries biology, and wildlife biology. For further information, visit the department website: http://www.zoology.siu.edu. To apply, send a cover letter highlighting qualifications for the position described, a detailed statement of research interests and plans, and a statement of teaching interests and capabilities; curriculum vitae; names, e-mails, addresses, and telephone numbers of three professional references. Closing date for applications is 30 November 2011 or until filled. Stating date is 16 August 2012. Mail Applications To: Dr. Matt Whiles, Conservation Biology Search Committee, Department of Zoology, Mail code 6501, SIU Carbondale, 1125 Lincoln Drive, Carbondale, IL 62901. E-mail: mwhiles@zoology.siu.edu. SIU Carbondale is an Affirmative Action/Equal Opportunity Employer that strives to enhance its ability to develop a diverse faculty and staff, and to increase its potential to serve a diverse student population. All applications are welcomed and encouraged and will receive consideration. POSTDOCTORAL POSITION Germline Stem Cells Studies include culture, differentiation, and gene activity of male germline stem cells. See Science 316:404, 2007 and PNAS 106:21672, 2009. Send curriculum vitae, names of three references, and a letter describing research experience to: R. L. Brinster, School of Veterinary Medicine, University of Pennsylvania. E-mail: cpope@vet.upenn.edu.
FACULTY POSITION University of Wisconsin, Madison ASSISTANT PROFESSOR (tenure-track) position in Exercise Physiology available starting August 27, 2012. Earned Doctorate is required and postdoctoral research experience is preferred. Area of research interest is open within the broad confines of biological aspects of exercise. We are seeking individuals capable of developing and maintaining a high quality, externally funded, independent research program. We are also committed to hiring an individual who can provide quality instruction in Exercise Physiology to undergraduate and graduate students. Experience working with diverse populations is a plus. Information about the department is available at website: http://www. education.wisc.edu/kinesiology/. Applicants should send a letter of application, curriculum vitae, statement of research interests, copies of up to three published articles in refereed journals, and names, mailing addresses, e-mail addresses, and telephone numbers of three references to: Professor Gary Diffee, University of Wisconsin, Department of Kinesiology, 2000 Observatory Drive, Madison, WI 53706. We encourage the submission of all materials electronically in PDF or Word format to e-mail: diffee@education. wisc.edu. To ensure full consideration, application should be received by December 15, 2011, but applications will be accepted until the position is filled. UW-Madison is an Equal Opportunity/Affirmative Action Employer. We promote excellence through diversity and encourage all qualified individuals to apply. Unless confidentiality is requested in writing, information regarding the applicants must be released upon request. Finalists cannot be guaranteed confidentiality. FACULTY POSITION in Biomolecular Structure Determination The University of Houston (UH) invites applications for a faculty position in Biomolecular Structure Determination. Preference will be given to a scientist with an interest in NMR or X-ray diffraction, which complements current strengths at UH. The University of Houston has outstanding facilities in high field NMR and X-ray diffraction. Applicants for the position should have a Ph.D. in a related field, an outstanding record of research, and a strong commitment to education. The tenure-track position could be in any one of the departments of Biology-Biochemistry, Chemistry, or Physics and may be filled at any rank. Please send curriculum vitae, an outline or research interests and three letters of recommendation to: Professor Robert Fox, Department of Biology and Biochemistry, University of Houston, Houston, TX 772045001. Consideration of applications will begin in November 15, 2011 and will continue until the position is filled. The University of Houston is an Affirmative Action/Equal Opportunity Employer. Minorities, women, veterans, and person with disabilities are encouraged to apply.
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The BD Annexin V FITC assay shown illustrates the detection of camptothecininduced apoptosis in Jurkat cells using the BD Pharmingen Annexin V FITC Apoptosis Detection Kit. Representative Annexin V FITC-A vs Propidium Iodide-A contour plots using untreated and camptothecin-treated cells show an increase in the Annexin V+ PI population and a decrease in the Annexin V PI population.
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GENOMICS
Gene-Expression Analysis
In This Issue
To quantify the expression of specic genes, researchers can use a variety of techniques, including arrays, PCR, and high throughput sequencing. However, getting accurate results still depends on precisely carrying out these methods, even with increasingly user-friendly technologies. In fact, as more scientists study gene expression, the standards for analysis are growing more rigorous to ensure that only accurate data are published. Likewise, software has been keeping pace, helping researchers follow protocols and analyze their results.
See full story on page 702.
Upcoming Features
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And the 2011 winner is...

Tiago Branco, M.D., Ph.D.
Postdoctoral Research Fellow University College London
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US$ 25,000 Prize
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Congratulations to Tiago Branco on winning the 2011 Eppendorf & Science Prize for his studies on how dendrites discriminate temporal input sequences and apply different integration rules depending on input location. The results of his research provide insight on how the brain performs computations, and suggest that even single neurons can solve complex computational tasks. The annual US$ 25,000 Eppendorf & Science Prize for Neurobiology honors scientists, like Dr. Branco, for their outstanding contributions to neurobiology research. Dr. Branco is the tenth recipient of this international award. He will be honored at a ceremony held during the week of the 2011 Annual Meeting of the Society for Neuroscience in Washington, DC. You Could Be Next. If you are 35 years of age or younger and currently performing neurobiology research, you could be next to win the 2012 Prize. Deadline for entries is June 15, 2012.
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FOCUS ON CAREERS
The Many Fields of Neuroscience

Shifting from Synapses to Society
In This Issue
Neuroscience has come a long way since the staining and identication of the neuron over a century ago. Now the eld has joined forces with other disciplines such as chemistry, computer science, engineering, and psychology, creating areas of focus that range from individual cells to social communities. Combining specialties has helped progress the understanding of social behavior as well as various psychological disorders, which some say are the nal frontiers in biological science.
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GENOMICS
Gene-Expression Analysis s Exploits More Technologies es

To quantify the expression of specic genes, researchers can use a variety of techniques, including arrays, PCR, and high throughput t sequencing. However, getting accurate results still depends on precisely carrying out these methods, even with increasingly user-friendly technologies. In fact, as more scientists study gene expression, the standards for analysis are growing more rigorous to ensure that only accurate data are published. Likewise, software has been keeping pace, helping researchers follow protocols and analyze their results. By Mike May
A scientist needs to decide which technology to use qPCR [real-time polymerase chain reaction], microarrays, or something like RNA sequencing. Those are the big ones.
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CREDIT: ISTOCKPHOTO.COM/RED_MOON_RISE
ne of the biggest challenges for researchers in todays gene-expression analysis arises from choice. A scientist needs to decide which technology to useqPCR [real-time polymerase chain reaction], microarrays, or something like RNA sequencing. Those are the big ones, says Heidi Kijenski, microarray marketing director at Agilent Technologies in Santa Clara, California. Even the now old-school approach to analyzing gene expression by way of arrays keeps offering new twists. For example, the higher density (four-fold higher than the companys previous technology) SurePrint G3 Gene Expression Microarrays from Agilent include one million oligonucleotide probes, which can be arranged to study two samples of 400,000 features each, four samples of 180,000 features apiece, or eight samples with 60,000 features for each. These higher-density arrays come in catalogue or custom formats, Kijenski says. This allows a researcher the option of customizing the content for any geneexpression project. She adds, This includes any organism where the customer has the sequence. Moreover, it takes only weeks to get a custom array, and it doesnt cost any more than a catalogue one. Even the catalogue versions offer considerable variety. There are 31 organisms in all, Kijenski says. To enable even better performance, Agilent developed its new, compact SureScan Microarray Scanner. Its a smaller footprint and provides better sensitivity and image resolution, Kijenski says. Other companies are also pushing array technology to new capabilities. As an example, Kevin Cannon, vice president of gene expression at Affymetrix in Santa Clara, California, points out the companys new Human Transcriptome and Splice Junction Array. It includes almost 7 million transcripts99 percent coverage of the human genes, he says. A comparative study by Stanford University researchers showed that this array was as sensitive as RNA sequencing in gene-expression proling studies, and its more reproducible, faster, and more cost effective. In addition, Dara Wright, vice president of clinical applications at Affymetrix, notes that gene-expression signatures are becoming increasingly useful for clinical molecular pathology applications. The Powered by Affymetrix Program, for example, enables companies to license Affymetrixs GeneChip technology to develop and clinically validate devices based on multiplex
gene-expression signatures. Wright explains that the programs current focus is primarily on developing prognostic cancer applications, and after years of applied research and the anticipation of clinical utility, we are now seeing tests emerge for routine [clinical] use, she says. One such clinical application, developed on the Affymetrix gene-expression platform, is the Pathwork Tissue of Origin Test, which uses RNA extracted from a tumor to assess the expression of more than 2,000 genes. That gene-expression pattern can reveal features of the tumor, such as its metastatic potential.
MULTIPLEXING PCR
A lot of researchers have been focusing on subsets of gene markers, or biomarkers, says Handy Yowanto, global product manager at Beckman Coulter in Brea, California. The companys XP-PCR Process for gene expression makes biomarker multiplexing easy, according to Yowanto. This technology consists of four steps: total RNA isolation, reverse transcription to form cDNA, multiplex PCR amplication, and fragment separation. During the PCR step, says Yowanto, The universal tags attached to gene-specic primers are used to amplify all of the target genes collectively. He adds, This minimizes the primer bias typically associated with traditional multiplexed applications.

GENOMICS
The specicity of this technology stems from the target-specic primers along with PCR-product separation using capillary electrophoresis. This enables down to one-base fragment resolution, explains Yowanto. In addition, he points out that researchers can include multiple housekeeping genes as controls within a single reaction, so they dont have to worry about well-to-well or run-to-run variations. The technology is beginning to gain traction, Yowanto notes and its being used in a range of applications. As examples, he mentions cancer research and microbial detection. For primer design, researchers can use Beckman Coulters eXpress Designer software. In addition, Yowanto points out a few primer-design tips: You should make sure that the primer is intron-spanning to minimize the interference with genomic DNA. Also, make sure primers are not designed where SNPs could occur because that might reduce the quantication. He adds, Designing primers will get easier over time as the users get more experience with this technology in different experiments.
FEATURED PARTICIPANTS
Agilent Technologies www.agilent.com Affymetrix www.affymetrix.com Beckman Coulter www.beckmancoulter.com Biogazelle www.biogazelle.com Bio-Rad www.bio-rad.com Fluidigm www.uidigm.com Illumina www.illumina.com Life Technologies www.lifetechnologies.com Nanostring Technologies www.nanostring.com Polytechnic Institute of LaSalle Beauvais www.lasalle-beauvais.fr/ spip.php?lang=en Queen Mary, University of London www.qmul.ac.uk SAS www.sas.com Technical University of Munich www.tum.de Thermo Fisher Scientic www.thermosher.com
CPR FOR PCR

Getting accurate results from PCR demands considerable attention to detail. As early as 1997 Stephen Bustin, professor , of molecular science at Queen Mary, University of London, realized this constraint. It became clear to me early on that the way things were being done was not sufcient if you want to quantify the results, he says. Specically, Bustin says, researchers often do not include enough information in their publications about how they perform quantitative PCR. When there is information, he adds, its likely to be wrong. So theres a real problem with peerreviewed literature. In 2009, Bustin and his colleagues published the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Some companies quickly joined this ght. Bio-Rad was certainly the pioneer, says Bustin. It has put a huge amount of effort in publicizing and implementing the guidelines with road shows and so on. Some other large vendorsincluding Agilent Technologies, Life Technologies, and Thermo Fisher Scienticalso provide MIQE-compliant assays. Bustin also points out that qbasePLUS from Biogazelle in Zwijnaarde, Belgium, is the only qPCR analysis software that complies with MIQE. According to Frank Bizouarn, eld application specialist in the Bio-Rad gene expression division, qPCR is the tool to analyze gene expression, but you need diligence in how you set up and run your experiments. For sample preparation, he notes Bio Rads Experion automated electrophoresis station determines if the RNA quality is good or bad for gene-expression analysis. He adds that Bio-Rad sponsored the rst MIQE qPCR iPad app, developed by Michael W. Pfaf of the Technical University of Munich and Af Abdel Nour of the Polytechnic Institute of LaSalle Beauvais. With this, says Bizouarn, you can check off MIQE guidelines as you go along.
groups of cells. The averagein the middlewould not represent the expression in any of the cells. Instead of assessing the expression at a population level, some answers can only be found in single cells. In lots of biological processes, single cells may be driving or dominating the behavior, says Unger. As an example, he mentions cancer stem cells. In looking at single cells, though, Unger encourages researchers to study multiple genes. If youre looking at only a few genes at a time, he says, then you will miss patterns in the ex pression. He points out that the Fluidigm BioMark HD System for real-time PCR plus its Dynamic Array chips can simultaneously track 96 genes in 96 cells. Not just any technology provides enough resolution to work with single cells. By their very nature, single cells are tiny, which means theres not much material to work with, says Ken Livak, senior scientic fellow at Fluidigm. The microuidic technology underlying the BioMark HD can actually control cells one by one. To see how different cells vary in expression, though, a researcher must analyze many cells. In just three hours, according to Livak, the BioMark HD can analyze 96 cells, which can produce as many as 9,216 data points (from 96 genes in 96 cells).
EXPRESSION BY COUNTING
All of the methods in the past that looked at gene expression were relatively indirectmeasuring expression levels by labeling transcripts and hybridizing them to an array, says Gary Schroth, distinguished scientist at Illumina, headquartered in San Diego, California. The trend is very clear that the world is moving more and more towards sequencing or counting assays. He adds, RNA sequencing is more about counting and directly characterizing the molecules in your biological sample. In January 2007 according to Schroth, his group continued ,
EXAMINING SINGLE-CELL EXPRESSION

The population average is a lie, says Marc Unger, chief scien tic ofcer at Fluidigm in South San Francisco, California. For gene expression, the population average of any given gene is not necessarily characteristic of any of the cells in the population. For example, the histogram of gene expression of a partic ular genes intensity might show two peaks from two different
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GENOMICS
probe to count the target molecules. The workow is simple and results are so reproducible that researchers choose not to run replicates, say Grimley. This performance combined with the fact that the technology is compatible with a variety of sample input types, such as [formalin-xed, parafn-embedded], is resulting in strong adoption by the oncology research community.
COUNTING NONCODING RNA

Theres lots of interest in noncoding RNAs, microRNAs, says Dennis Fantin, product management leader for qPCR at Life Technologies in Carlsbad, California. In analyzing gene expression, results show that miRNA plays a regulatory role. They dont work through proteins, but interact directly with mRNA to inhibit translation, says Fantin. You cant explain everything about gene expression with transcription alone. To help researchers study the gene-expression impact of miRNA, Life Technologies developed its TaqMan OpenArray MicroRNA Panels. Fantin adds that the company offers predened TaqMan, noncoding-RNA assays, which work just like TaqMan gene-expression assays. For researchers who nd new miR NAs and long noncoding RNAs and need an assay, Life Technologies offers a custom service. Scientists can also use the nCounter miRNA Expression Assay Kits. The human miRNA product includes more than 700 miRNAs, says Grimley. The company also makes panels for mouse and rat miRNA. The study of miRNAs is becoming so popular that the company just launched its new nCounter miRGE assays, which enable the simultaneous detection and quantication of mRNAs and miRNAs in a single tube.
Theres lots of interest in noncoding RNAs, microRNAs.
Mike May is a freelance writer and editor for science and technology. DOI: 10.1126/science.opms.p1100059
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was the rst to do what researchers now call RNA-seq, which is sequencing full-length cDNA with short-read technology. In just a few years, this technology contributed to many publications. As Schroth says, Now, there are more than 300 peerreviewed publications using RNA-seq methods. He also notes the increasing throughput, going from 4060 million reads per run in 2007 to 3 billion with todays Illumina HiSeq 2000 system. The length of the reads has also tripled in that timeincreasing from 35 to 100 base pairs. In addition most of todays studies use paired-end sequencing, which was not available in 2007 . Ongoing releases of kits also make RNA sequencing more appealing. At the end of 2010, for example, Illumina released its TruSeq RNA Sample Prep Kits. Currently, these kits allow 24 samples to be indexed per sequencing lane, which enables researchers to multiplex samples and capitalize on the increased throughput of the sequencing systems. These prep kits allow labs to process dozens or even hundreds of samples a week, says Schroth, adding that labs with very high throughput needs can even automate the sample preparation using the kits. Other technologies also use counting. For example, the nCounter platform from NanoString Technologies in Seattle, Washington, detects up to 800 target molecules in a single-tube reaction for analyzing gene expression, micro RNA (miRNA), and copy-number variation. Its a direct digital counting method, says Chris Grimley, vice president of marketing at NanoString. Barcode-labeled probes hybridize directly to a target molecule in solution. The reporter probe carries the signal, and the capture probe allows the complex to be immobilized for data collection. Researchers can select from a range of existing panels, including the recently launched leukemia panel, which simultaneously detects and quanties 25 fusion-gene isoforms and 23 additional mRNAs related to leukemia. Alternatively scientists can design custom panels using their specic targets of interest. The nCounter Prep Station processes the samples and the nCounter Digital Analyzer uses the uorescent reporter on the
DEALING WITH EXPRESSION DATA

Datasets generated in gene-expression experiments keep getting bigger. This is challenging when trying to analyze a whole dataset, says Shannon Conners, JMP life sciences product manager at SAS in Cary, North Carolina. Beyond dataset size, researchers face other analytical challenges. People want to look at several factors at once, and they want to remove effects that might be caused by samples being taken at different sites or times, says Conners. She adds that researchers often want to search for correlations in gene-expression data acquired on different platforms. SASs JMP Genomics software provides a wide range of analytical tools. For example, Conners says, It includes tools for the analysis of continuous intensities from array data that work with very big datasets. It also includes tools for count data from high throughputsequencing studies. She adds that SAS tai lors these analytical tools for specic data types. For example, there are simplied workows for things like RNA-sequencing analysis, she says. These tools and more come in the new JMP Genomics 5.1. The analytical tools from SAS and the other technologies discussed here reveal the growing power that comes from combining approaches to analyze gene expression. Todays technological combinations make it faster and easier to dig deeper into gene expression under a wider range of circumstances.

NEW PRODUCTS: GENOMICS
RNA/DNA ISOLATION KITS

The MagMAX FFPE Isolation Kits are two new magnetic bead-based kits designed specically for the isolation of both total RNA and DNA from formalin-xed parafn-embedded (FFPE) samples. The MagMAX FFPE kits offer the yield, purity, and downstream performance characteristics that scientists expect, while also delivering faster workow protocols, higher throughput capacity, and eliminating user-exposure to toxic organic solvents typically associated with the FFPE purication process. To enhance user productivity, MagMAX FFPE Kits incorporate magnetic bead-based technology, yielding a workow amenable to high throughput processing. Offered in a 96-well format, MagMAX FFPE Kits enable an automated-assist protocol, which can effectively reduce hands-on processing time by 50% or more. By substituting xylene-like solvents with a novel solubilization reagent, MagMAX FFPE kits combine protease enzymes and digestion additives. This combo-reagent allows users to eliminate the deparanization step of the protocol, and the toxicity associated with traditional FFPE processing. Life Technologies
For info: 760- 603-7200 www.lifetech.com
ChIP-Seq PREPARATION KIT

The Magna ChIP-Seq preparation kit is designed to simplify genome-wide proling of DNA-protein interactions by chromatin immunoprecipitation and next generation sequencing (ChIPSeq). In contrast to other products that support only part of the ChIP-Seq workow, the Magna ChIP-Seq kit is a complete solution that provides a set of validated reagents for both chromatin immunoprecipitation and next generation library construction. These reagents, in combination with a detailed protocol and quality control guidelines, simplify the ChIP-Seq process to allow researchers to map interactions of histones, transcription factors, and other chromatin-associated proteins on a genome-wide scale. As successful immunoprecipitation is critical to construction of the sequencing library, the kit includes positive and negative control antibodies and a set of polymerase chain reaction primers that can be used as in-process controls or to verify technique. The kit has been used to construct ChIP-Seq libraries from as little as 1 ng of puried ChIP DNA, meaning that DNA segments associated with low abundance proteins can be sequenced. EMD Millipore
For info: 800-645-5476 www.millipore.com
cDNA SYNTHESIS KIT The new iScript Advanced cDNA synthesis kit for RT-qPCR allows researchers to generate more real-time polymerase chain reaction (PCR) data from a single 20 L reverse transcription reaction for their gene expression analyses. With a maximum capacity of 7 g of .5 input RNA, the iScript Advanced cDNA synthesis kit for RT-qPCR surpasses the capacity of its nearest rival by 2 g. The iScript Advanced cDNA synthesis kit for RT-qPCR offers additional advantages over competitor kits in terms of simplicity and speed. The kit has only two essential components: an iScript Advanced reaction mix and an iScript Advanced reverse transcriptase. The protocol takes only 35 minutes. The iScript Advanced cDNA synthesis kit for RTqPCR offers excellent sensitivity and efciency in both real-time PCR (using SYBR Green, EvaGreen, or probes) and conventional RT-PCR. Bio-Rad
For info: 800-424-6723 www.bio-rad.com
FISH PROBES
Stellaris FISH (uorescence in situ hybridization) is an RNA visualization method that allows simultaneous detection, localization, and quantication of individual mRNA molecules at the sub-cellular level in xed samples. Stellaris FISH probes are manufactured on a custom basis, including software for optimum probe design. In addition, premade probe sets for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human and mouse applications are available. This novel RNA FISH technology represents a fast and easy-to-use method to achieve conclusive results through compelling images of RNA expression. By enabling scientists to localize and count discrete molecules of mRNA using wideeld uorescence microscopy, Stellaris FISH probes can lead to streamlined studies in stem cell, cancer, pathology, developmental biology, transcription regulation, and neuroscience research. Stellaris FISH can also be combined with existing technologies such as real-time polymerase chain reaction, DNA FISH, immunohistochemistry, and Western blotting to provide complementary information. Biosearch Technologies
For info: 800-436-6631 www.biosearchtech.com
ORGANIC RNA EXTRACTION

The new Direct-zol RNA MiniPrep facilitates efcient and consistent broad range purication of high quality (DNA-free) total RNA directly from samples stored in Tri-Reagent or similar acid-guanidinium-phenol reagents. This innovative procedure bypasses phase separation and precipitation with a spin column and eliminates problems with phenol carryover, all traits typically associated with organic extraction-based methods. The Direct-zol RNA MiniPrep is part of the next gen of RNA prep that will meet the demands of todays scientists requiring RNA that is ideal for stringent analytical methods like miRNA proling, RNA-seq, and viral identication. The procedure couples the effectiveness of infectious agent inactivation and sample preservation with a convenient no hassle, no-mess procedure for DNA-free RNA. Zymo Research Corporation
For info: 888-882-9682 www.zymoresearch.com
Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.dtl for more information. Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are featured in this space. Emphasis is given to purpose, chief characteristics, and availability of products and materials. Endorsement by Science or AAAS of any products or materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.
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TOP IMAGE: Heterochromatin is characterized by a repressive, tight packaging of nucleosomes, which impedes transcription factors from gaining access to regulatory sites on the DNA. Methylation of cytosine bases in regions called CpG islands, found in many gene promoters, leads to formation of transcriptionally repressed heterochromatin. Methylation of cytosine bases by DNA methyltransferases (DNMTs) facilitates recruitment of Methyl-CpG-binding Protein 2 (MeCP2), which brings along other associated proteins, including histone deacetylases (HDACs), histone methyltransferases (HMTs), and Heterochromatin Protein 1 (HP1). These proteins then facilitate the deacetylation and methylation of histone proteins, resulting in the formation and maintenance of the repressive state of heterochromatin. To view our epigenetics digital animation movie and for more information, please visit www.cellsignal.com.
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CONTENTS

EDITORIAL 567 Understanding the Human Brain NEWS OF THE WEEK

Volume 334 Issue 6056

Sydney Brenner and Terrence J. Sejnowski

NEWS & ANALYSIS

A. D. Gitler >> Report p. 690

Another Reason to Exercise

Paradigm Shifts in Dye-Sensitized Solar Cells

M. Maroteaux and M. Mameli >> Report p. 693

D. Basko >> Report p. 648

H. Abrahamsen and H. Stenmark >> Report p. 678

NEWS FOCUS 584 Vigil at North Koreas Mount Doom

T. Woollings >> Report p. 655

Sharp Insights and a Sharp Tongue

LETTERS 593 Conservation: Limits of Land Sparing

R. Ohlemller >> Reports pp. 652 and 660

Conservation: Model Management Intensity

ESSAY 615 Eppendorf Winner: The Language of Dendrites

TECHNICAL COMMENT ABSTRACTS CORRECTIONS AND CLARIFICATIONS

BOOKS ET AL. 598 The Copernican Question

The Cell Biology of Synaptic Plasticity CONTENTS continued >>

R. S. Westman, reviewed by P. Dear C. C. Mann, reviewed by J. Farmer

The Inuence of Late Quaternary ClimateChange Velocity on Species Endemism

Long-Term Change in the Nitrogen Cycle of Tropical Forests

Structural Dynamics of a Catalytic Monolayer Probed by Ultrafast 2D IR Vibrational Echoes

Neural Mechanisms for the Coordination of Duet Singing in Wrens

pages 610 & 648

A Fluoride-Derived Electrophilic Late-Stage Fluorination Reagent for PET Imaging

Ionic LiquidMediated Selective Conversion of CO2 to CO at Low Overpotentials

N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

Hot CarrierAssisted Intrinsic Photoresponse in Graphene

The Pace of Shifting Climate in Marine and Terrestrial Ecosystems

Bidirectional Control of Social Hierarchy by Synaptic Efcacy in Medial Prefrontal Cortex

Drosophila CENH3 Is Sufcient for Centromere Formation

Atmospheric Blocking and Atlantic Multidecadal Ocean Variability

Social Network Size Affects Neural Circuits in Macaques

CONTENTS continued >>

Fermi Detection of a Luminous -Ray Pulsar in a Globular Cluster

www.sciencenow.org Highlights From Our Daily News Coverage

Happiness Associated With Longer Life

Cover Your Ears!

Musicologists learn what makes chalkboard screeches so painful. http://scim.ag/_chalkboard

The Case of the Haunted Golf Club

www.sciencesignaling.org The Signal Transduction Knowledge Environment 1 November issue: http://scim.ag/ss110111

www.sciencecareers.org/career_magazine Free Career Resources for Scientists

Taken for Granted: Getting to Aaahhh!

Conquering Information Overload

In Person: At Summer Davos

Deep Human Genealogies Reveal a Selective Advantage to Be on an Expanding Wave Front

Editorial Expression of Concern on Stapel and Lindenberg Report

www.sciencetranslationalmedicine.org Integrating Medicine and Science 2 November issue: http://scim.ag/stm110211

news.sciencemag.org/scienceinsider Science Policy News and Analysis

FOCUS: Epstein-Barr VirusAn Important Vaccine Target for Cancer Prevention

PERSPECTIVE: Epigenetics of Nicotine Another Nail in the Coughing

EDITED BY STELLA HURTLEY

Electrons Staying Hot

The Domineering Brain and Its Synapses

Echoes from the Surface

Splitting CO2 and Water

The Warm and the Cold of It

www.sciencemag.org SCIENCE VOL 334

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