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CONTENTS

Volume 331 Issue 6022

EDITORIAL 1242 Celebrating the Culture of Science John Durant and Alaa Ibrahim

R. Penrose, reviewed by R. Bousso

1267

NEWS OF THE WEEK 1246

BOOKS ET AL. 1266 Cycles of Time

A roundup of the week’s top stories

NEWS & ANALYSIS

EDUCATION FORUM 1269

1251

China Bets Big on Small Grants, Large Facilities

1252

Ten Months After Deepwater Horizon, Picking Up the Remnants of Health Data

1253

More Negative Data for Link Between Mouse Virus and Human Disease

1254

Price Tags for Planet Missions Force NASA to Lower Its Sights

PERSPECTIVES 1271 Listeria Unwinds Host DNA

1272

Rhodopsin as Thermosensor?

B. Minke and M. Peters >> Report p. 1333 1273

THE WAR IN AFGHANISTAN

Counting the Dead in Afghanistan War as a Laboratory for Trauma Research

LETTERS 1264 Projecting Human DNA Patent Numbers A. E. Mills and P. M. Tereskerz

Aerosol Chemistry and the Deepwater Horizon Spill H. Coe >> Report p. 1296

1275

Crise de Foie, Redux?

D. D. Moore >> Report p. 1315 1276

The Deep Social Structure of Humankind

B. Chapais >> Research Article p. 1286

China: Invest Wisely in Sustainable Water Use

1277

P. Gong et al.

Family Planning: Looking Beyond Access

Response

The Diamond Within a Silicon Analog of Cyclobutadiene Y. Apeloig >> Report p. 1306

W. N. Ryerson

1265

page 1254

J. R. Rohde >> Report p. 1319

>> Science Podcast 1261

Impact of Undergraduate Science Course Innovations on Learning M. A. Ruiz-Primo et al.

NEWS FOCUS 1256

The New Harvest

C. Juma, reviewed by D. Gollin

W. Cates Jr. et al.

REVIEW

TECHNICAL COMMENT ABSTRACTS

1279

How to Grow a Mind: Statistics, Structure, and Abstraction J. B. Tenenbaum et al.

page 1266

CONTENTS continued >>

COVER The North Atlantic Treaty Organization–led military coalition in Afghanistan has released its internal database of civilian casualties. In this visualization of the data, monthly casualties rise above the region of Afghanistan in which they occurred, from January 2009 (bottom, near map) to December 2010 (top). Over this period, 2537 civilians were killed (large dots), and 5594 were wounded (small dots), with weaponry and perpetrator coded by color. See News Focus story on p. 1256.

DEPARTMENTS 1239 1243 1244 1340 1341

This Week in Science Editors’ Choice Science Staff New Products Science Careers

Image: Created by George Michael Brower, based on a concept by John Bohannon

www.sciencemag.org

SCIENCE

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Published by AAAS

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CONTENTS

RESEARCH ARTICLES 1286

1315

Co-Residence Patterns in Hunter-Gatherer Societies Show Unique Human Social Structure

K. R. Hill et al. Individuals in residential groups in contemporary hunter-gatherer societies are unrelated to each other. >> Perspective p. 1276; Science Podcast 1289

D. Feng et al. Circadian control of liver metabolism is mediated by cycles of recruitment of the histone deacetylase HDAC3. >> Perspective p. 1275 1319

Ordered and Dynamic Assembly of Single Spliceosomes

1295

1322

Organic Aerosol Formation Downwind from the Deepwater Horizon Oil Spill

J. A. de Gouw et al. Organic compounds of intermediate volatility play an important role in the formation of secondary organic aerosols. >> Perspective p. 1273 1299

1302

Complex Multicolor Tilings and Critical Phenomena in Tetraphilic Liquid Crystals

1325

A Planar Rhombic Charge-Separated Tetrasilacyclobutadiene

1328

Tomography of Reaction-Diffusion Microemulsions Reveals Three-Dimensional Turing Patterns

T. Bánsági Jr. et al. Tomography reveals three-dimensional Turing patterns created by the Belousov-Zhabotinsky reaction running in a microemulsion.

1312

Creating Favorable Geometries for Directing Organic Photoreactions in Alkanethiolate Monolayers

Aging in the Natural World: Comparative Data Reveal Similar Mortality Patterns Across Primates

Positive Supercoiling of Mitotic DNA Drives Decatenation by Topoisomerase II in Eukaryotes

J. Baxter et al. Positive supercoiling of catenated DNA during cell division induces its enzymic decatenation to allow chromosome segregation. 1333

K. Suzuki et al. A stable silicon analog of a reactive, antiaromatic hydrocarbon has been synthesized and characterized. >> Perspective p. 1277 1309

Mechanistic Basis of Resistance to PCBs in Atlantic Tomcod from the Hudson River

A. M. Bronikowski et al. Aging patterns in humans fall within the parameters of other primates in natural populations. >> Science Podcast

X. Zeng et al. X-shaped molecules undergo reversible thermal transitions between phase-separated and mixed states. 1306

page 1309

I. Wirgin et al. Chronic pollution of the Hudson River, New York, results in rapid evolution of resistance to the pollutants.

Catastrophic Drought in the Afro-Asian Monsoon Region During Heinrich Event 1

J. C. Stager et al. An extreme megadrought occurred in the Afro-Asian monsoon region during an iceberg melting episode 50,000 years ago.

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response

A. Lebreton et al. A virulence factor secreted by Listeria monocytogenes induces expression of interferon-stimulated genes. >> Perspective p. 1271

A. A. Hoskins et al. Fluorescently labeled yeast spliceosome proteins reveal the events of intron splicing as it happens.

REPORTS

A Circadian Rhythm Orchestrated by Histone Deacetylase 3 Controls Hepatic Lipid Metabolism

Function of Rhodopsin in Temperature Discrimination in Drosophila

W. L. Shen et al. The light sensor rhodopsin appears to also function in temperature sensation in the fruit ﬂy. >> Perspective p. 1272 1336

page 1325

A Polarized Epithelium Organized by β- and α-Catenin Predates Cadherin and Metazoan Origins

D. J. Dickinson et al. An epithelium in Dictyostelium discoideum suggests convergence between multicellular animals and slime molds.

CONTENTS continued >>

M. Kim et al. Molecules align in molecular overlayers for photodimerization reactions that would be disfavored in solution.

www.sciencemag.org

SCIENCE

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CONTENTS

SCIENCEONLINE SCIENCENOW

SCIENCEXPRESS

www.sciencenow.org Highlights From Our Daily News Coverage

www.sciencexpress.org

Structure of an Agonist-Bound Human A2A Adenosine Receptor

F. Xu et al. Changes associated with conformationally selective agonist binding shed light on G protein–coupled receptor activation. 10.1126/science.1202793

The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inﬂammatory Arthritis in Mice

W. Tang et al. A growth factor protects against arthritis in mice by blocking tumor necrosis factor–dependent signaling. 10.1126/science.1199214

A Midzone-Based Ruler Adjusts Chromosome Compaction to Anaphase Spindle Length

G. Neurohr et al. The degree of chromosome condensation can be modulated within cells to ensure proper segregation. 10.1126/science.1201578

Enhanced Turbulence and Energy Dissipation at Ocean Fronts E. D’Asaro et al. Energy in surface ocean currents can dissipate into deep water via enhanced turbulence at the boundaries between water masses. 10.1126/science.1201515

Low-Power Switching of Phase-Change Materials with Carbon Nanotube Electrodes F. Xiong et al. The crystallinity and resistivity of a compound semiconductor was changed with current pulses delivered by nanoelectrodes. 10.1126/science.1201938

TECHNICALCOMMENTS Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” A. Graf et al. Full text at www.sciencemag.org/cgi/content/ full/331/6022/1265-c

Response to Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” M. D. Mahecha et al. Full text at www.sciencemag.org/cgi/content/ full/331/6022/1265-d

Crustaceans Crave a Little Quiet

Noisy reefs may lure some marine invertebrates, but they repel many others. http://scim.ag/crustaceans

SCIENCETRANSLATIONAL MEDICINE www.sciencetranslationalmedicine.org Integrating Medicine and Science 9 March issue: http://scim.ag/stm030911

Elephants Can Lend a Helping Trunk

RESEARCH ARTICLE: Neutrophils Activate Plasmacytoid Dendritic Cells by Releasing Self-DNA–Peptide Complexes in Systemic Lupus Erythematosus

Memristors Make Fast Work of Mazes

RESEARCH ARTICLE: Netting Neutrophils Are Major Inducers of Type I IFN Production in Pediatric Systemic Lupus Erythematosus

Pachyderms know when they need help— and are willing to wait for it. http://scim.ag/helping-trunk

R. Lande et al.

Electronic devices could test multiple potential escape routes at the same time. http://scim.ag/memristors

SCIENCESIGNALING

www.sciencesignaling.org The Signal Transduction Knowledge Environment 8 March issue: http://scim.ag/ss030811

RESEARCH ARTICLE: Vav1-Mediated Scaffolding Interactions Stabilize SLP-76 Microclusters and Contribute to Antigen-Dependent T Cell Responses

A. T. Sasaki et al.

C. M. Pﬂeger Cancers in which K-Ras activation drives tumor growth could be targeted by treatments blocking K-Ras ubiquitination.

PODCAST

SCIENCECAREERS

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

Reentering Academia: A Success Story

E. Pain University of Oxford chemist Carol Robinson returned to academia after an 8-year break and went on to win the FEBS/EMBO Women in Science Award. http://scim.ag/robinsonq_a I. S. Levine When a scientist is confronted with a colleague who is struggling with personal problems, there are no easy answers. http://scim.ag/mm_troubled

VOL 331

T. J. Merkel and J. M. DeSimone Nanodiamond-drug conjugates show antitumor activity in mouse models of chemoresistant breast and liver cancer.

SCIENCEPODCAST

On the 11 March Science Podcast: civilian casualties in Afghanistan, primate mortality rates, social structure in hunter-gatherer societies, and more.

SCIENCEINSIDER

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

M. González-Gaitán et al. Mathematical analysis reveals how a graded signal can induce a homogeneous response across a ﬁeld of cells.

Published by AAAS

RESEARCH ARTICLE: Nanodiamond Therapeutic Delivery Agents Mediate Enhanced Chemoresistant Tumor Treatment

www.sciencemag.org/multimedia/podcast Free Weekly Show

PERSPECTIVE: Ubiquitin on Ras— Warden or Partner-In-Crime?

SCIENCE

J. E. Craft New studies suggest that rational combinatorial targeting of the pathogenic mechanisms in lupus may be a winning therapeutic strategy.

PERSPECTIVE: Dodging Drug-Resistant Cancer with Diamonds

RESEARCH ARTICLE: Ubiquitination of K-Ras Enhances Activation and Facilitates Binding to Select Downstream Effectors

www.sciencemag.org

PERSPECTIVE: Dissecting the Immune Cell Mayhem that Drives Lupus Pathogenesis

E. K. Chow et al.

N. R. Sylvain et al. Scaffolding functions of Vav1 contribute to SLP-76 clustering and signaling downstream of the T cell receptor.

Mind Matters: Troubled Colleagues

G. S. Garcia-Romo et al.

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 ofﬁces. 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 20090–6178. 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 identiﬁcation code for Science is 0036-8075. Science is indexed in the Reader’s Guide to Periodical Literature and in several specialized indexes.

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EDITED BY CAROLINE ASH AND MARIA CRUZ

The spliceosome, which excises introns from precursors to messenger RNAs (pre-mRNAs), is a highly dynamic macromolecular machine consisting of ﬁve RNAs and scores of both core and accessory proteins. To overcome the difﬁculties of fully reconstituting the spliceosome from its puriﬁed components, Hoskins et al. (p. 1289) followed its assembly in yeast using colocalization single-molecule spectroscopy (CoSMoS) in unfractionated cell extracts. Their kinetic analysis reveals that the spliceosome assembles by ﬁrst binding U1 small nuclear RNA followed by U2, U4/U6.U5, and then the multiprotein Prp19-complex. No single subcomplex binding event limits the overall assembly rate, and the commitment of the pre-mRNA molecule to splicing progressively increases as the spliceosome assembles.

Prehistoric Social Networking A hunter-gatherer life-style is thought to have been the predominant social structure for most of human history. The common assumption is that hunter-gatherer groups consisted largely of related individuals: parents, siblings, offspring, and possibly extending to include spousal relations. Hill et al. (p. 1286; see the Perspective by Chapais) have analyzed the kin relationships of groups in 32 contemporary hunter-gatherer societies and surprisingly ﬁnd that most members of a group are not related to each other. Although siblings may often be co-resident, neither matrilineal nor patrilineal organization dominates. The occurrence of networks of interacting yet genetically unrelated individuals has implications for theories about the origins of prosocial behavior and of culture.

CREDIT (TOP TO BOTTOM): DIANA KATHARINE HUNT; SUZUKI ET AL.

Tomcod Toxicology From 1947 to 1976, two General Electric manufacturing facilities released around 600,000 kg of polychlorinated biphenyls (PCBs) into the Hudson River, NY, USA. Atlantic tomcod in the river accumulated some of the highest levels of PCBs ever seen in natural populations and became resistant to the poisonous effects of the contamination. The toxic effects of PCBs are usually mediated by cytochrome P4501A, which is regulated by the aryl hydrocarbon receptor (AHR), but the Hudson River tomcod population show poor inducibility of cytochrome P4501A. Wirgin et al. (p. 1322, published online 17 February) found that the Hudson River tomcod AHR2 gene had four distinct polymorphisms, but a two–amino acid deletion was found to reduce the ability of the AHR2-1

allele to bind PCBs and appears to be the basis of the ﬁsh’s resistance to poisoning. The presence of this allele at low frequencies in tomcod from nearby, cleaner, rivers indicates that AHR2-1 was also present at low frequency in the Hudson River tomcod before the contamination event and was very rapidly selected for after the PCB leak.

Add Water to Dry The intertropical convergence zone, the globecircling region where winds from the Northern and Southern hemispheres converge near the equator, is where most tropical rainfall occurs. The position of this region is variable on seasonal time scales, as well as on longer ones, being particularly susceptible to the effects of large-scale climate perturbations. Stager et al. (p. 1299, published online 24 February) connect one of these major perturbations with a concurrent widespread and severe drought in Africa. Large volumes of glacial meltwater and ice that entered the North Atlantic between 15,000 and 18,000 years ago shifted the convergence zone further south, thus depriving that region of Africa of the moisture that it received so abundantly both before and after that period.

Another Example of Frustration Geometry can be used to create frustrated systems (that is, those that do not pack in conﬁgurations of minimum energy), such as block copolymers, where two or more polymer molecules are forced to be near each other through

www.sciencemag.org SCIENCE VOL 331 Published by AAAS

a covalent chemical bond. Zeng et al. (p. 1302; see the cover) examine liquid crystalline rod-like molecules with two incompatible side groups that pack into honeycomb structures and form complex tiling patterns. Upon heating, a reversible phase transition is observed from a phaseseparated state to a mixed one, in a transition analogous to the Curie transition in simple and frustrated ferro- and antiferromagnets.

A Silicon Rhombus Unlike carbon atoms, silicon atoms tend not to engage in π-bonding in their natural compounds. By using bulky groups to force the silicon atoms into close proximity while inhibiting their reactivity toward other molecules nearby, Suzuki et al. (p. 1306; see the Perspective by Apeloig) have synthesized and structurally characterized a silicon analog of cyclobutadiene, the strained, π-bonded tetragonal hydrocarbon that exhibits anti-aromaticity. Whereas the carbon compound compensates for its electronic instability by lengthening and shortening its bonds in alternation, the four Si-Si bonds adopt essentially equal lengths. Instead, the silicon centers vary their angular disposition, and theoretical calculations support an accompanying variation in their charge polarizations.

Downloaded from www.sciencemag.org on March 10, 2011

<< Watching Splicing Live

Turing Patterns in 3D Turing patterns—self-organized structures created by systems that undergo reaction and diffusion—are a possible mechanism underlying

11 MARCH 2011

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This Week in Science

The 2011 AAAS Philip Hauge Abelson Advancing Science Symposium

Continued from page 1239

pattern formation in living organisms. Although there are many experimental studies of two-dimensional (2D) pattern formation—three-dimensional (3D) structures, the most relevant to biology, have been difﬁcult to observe and should, according to theoretical studies, show even richer patterns. Using tomographic methods, Bánsági et al. (p. 1309, published online 10 February) demonstrate the existence of patterns that are inherently 3D, as opposed to simple extensions of 2D patterns, in a reactiondiffusion system known as the Belousov-Zhabotinsky reaction, running in a microemulsion.

Jet travel and working the night shift uncouple patterns of eating and activity from the body’s internal circadian clock. Recent evidence indicates that such disruption of circadian rhythms has deleterious consequences for metabolic control and can increase the occurrence of diseases like obesity or diabetes. Feng et al. (p. 1315; see the Perspective by Moore) provide insight into the molecular mechanisms that couple circadian rhythms to metabolic control in the liver. The authors found that histone deacetylase 3 (HDAC3) bound to over 14,000 genes throughout the genome during the day when mice were inactive but only to about 100 genes during the night when the animals were active and feeding. Genes with products that function in liver metabolism tended to bind HDAC3, and the presence of HDAC3 was associated with histone deacetylation and decreased transcription. Thus, failure to coordinate expression of such genes with feeding and activity may contribute to the observed effects of circadian disruption on metabolism.

Tissues Simply The identiﬁcation of shared genes and derived characteristics among distantly related organisms help us to understand how cells and tissues are organized and how this organization evolved. Dickinson et al. (p. 1336) have discovered that unicellular organisms capable of social organization, such as the amoeba Dictyostelium discoideum, can differentiate cells into simple tissues. Dictyostelium epithelium is composed of a single layer of elongated polarized cells necessary for the formation of the fruiting body. The organization, morphology, and function of Dictyostelium epithelium are dependent on an ancestral signaling pathway that is comprised of the functional orthologs of α− and β-catenins, but that does not require Wnt signaling nor cadherins.

Bacterial-Host Exploitation Intracellular bacterial pathogens are responsible for a variety of diseases, including listeriosis and legionnaire’s disease. The bacterial pathogens subvert cellular functions through the interaction of bacterial effector proteins with host components. Lebreton et al. (p. 1319; published online 20 January; see the Perspective by Rohde) found that a virulence factor, LntA, of the bacterial pathogen Listeria monocytogenes targets the host cell nucleus to activate the type III–interferon signaling pathway. LntA counteracted the repression mediated by the chromatin regulator BAHD1 to subvert the heterochromatin machinery and reprogram the transcription of host genes involved in innate immunity. The pathogens thus manipulate epigenetic regulation to their advantage.

CREDIT: DANIEL J. DICKINSON

Feeling the Heat Given a choice, a fruit ﬂy will seek a temperature optimum of 18°C. This behavior requires a biochemical signaling pathway apparently initiated by a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor. Given the similarity of these signaling events to those initiated by rhodopsin, the G protein–coupled receptor that senses light, Shen et al. (p. 1333; see the Perspective by Minke and Peters) tested the effects of rhodopsin mutations on temperature selection in ﬂies and, surprisingly, found that it resulted in discrimination between temperatures within a few degrees of the the optimal 18°C, independent of light sensation. One or more accessory molecules may also be required, as the rhodopsin molecule itself does not seem to be the temperature sensor. www.sciencemag.org SCIENCE VOL 331

11 MARCH 2011 Published by AAAS

Breaching Barriers in Alzheimer’s Disease Overcoming the challenges of translating research into therapies Wednesday, 6 April 2011 8:30 A.M. to 6:00 P.M. AAAS Headquarters 1200 New York Ave., NW Washington, DC 20005 Organized by AAAS, Science Translational Medicine, and The Agouron Institute Free of charge, but advanced registration is required Register at www.aaas.org/go/abelson/ 202-326-6408 Speakers include: David Holtzman Washington University

H

Dennis Selkoe Harvard Medical School

H

Steve Paul Weill-Cornell Medical College

H

Don Cleveland UCSD

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Metabolic Lags

EDITORIAL

Celebrating the Culture of Science

10.1126/science.1204773

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11 MARCH 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

John Durant is director of the MIT Museum and executive director of the Cambridge Science Festival. E-mail: [email protected].

Alaa Ibrahim is an assistant professor in the Department of Physics at the American University in Cairo and director of the Cairo Science Festival. E-mail: [email protected].

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personal, social, economic, and political development. This has spurred many countries to increase their investments in science and technology. But funding research is not enough: Nations must also promote cultures that celebrate science and its values of reasoning, openness, tolerance, and respect for evidence, just as they celebrate the arts and humanities that enrich everyone’s lives. Not only formal education but informal outreach is critical in achieving this goal. Today, there is a great deal of creative experimentation with different methods of engaging the public with science. Our own experiences in organizing science festivals in the United States and Egypt illustrate the potential of one important form of public engagement. Science festivals are rich expressions of the cultural importance of science and technology. Typically, they give researchers the chance to interact directly with students and citizens from all walks of life. They also offer many different forms of engagement, from lectures, dialogues, panel discussions and debates; through hands-on demonstrations, shows, exhibitions, and workshops; to science-related theater, cafes, music, and stand-up comedy. The topics covered are equally diverse, but no matter what the ﬁeld, the goal is the same: to engage citizens with science in ways that are inspirational and empowering. Cambridge, Massachusetts, is a “science city,” with more science per square foot than most other places. But even there, local access to science is extremely uneven. An annual Cambridge Science Festival was launched 4 years ago as one approach to address this divide by offering everyone a chance to engage with the city’s scientists and engineers. Last spring, more than 40,000 people took up the opportunity to interact with scientists from all of Cambridge’s universities and dozens of its high-tech companies. Cairo is a much larger city than Cambridge, with very different needs and opportunities. Inspired by the U.S. Cambridge Science Festival and hosted by the American University in Cairo, the Cairo Science Festival was launched last year to increase awareness of, and opportunities in, science and its role in development, particularly for Egyptian youth, who represent the majority of Cairo’s population of 20 million. It was also an example of how festivals can forge bridges between nations. In 2010, audiences in Cambridge and Cairo were connected via videoconferencing in a series of daily sessions that allowed U.S. and Egyptian audiences to join in conversation with Nobel Prize winners about their work. This year, we plan to extend the collaboration to include, among other things, a transnational discussion about the role—and control—of the Internet during social and political revolutions, in particular the “January 25 Revolution” centered in Tahrir Square, literally on the doorstep of the Cairo Science Festival. To promote, support, and connect science festivals globally, a Science Festival Alliance was created in 2009 (www.ScienceFestivals.org). Last month, the alliance helped organize an International Public Science Events Conference in Washington, DC. This drew more than 200 participants (including scientists) who are involved in science festivals and science cafes in 16 different countries—from China, Japan, and Singapore to Egypt, Italy, the United Kingdom, and the United States. There we agreed to collaborate by, for example, sharing best practices and circulating successful programs. Increased support for such initiatives at the national and international levels will allow local cultures to develop a deeper connection to science, as well as connect otherwise distant communities and cultures in common encounters, inquiries, and understandings. We look forward to seeing science festivals – John Durant and Alaa Ibrahim proliferate across the globe.

CREDITS: (LEFT) THINKSTOCK; (RIGHT, TOP TO BOTTOM) MIT; AMERICAN UNIVERSITY IN CAIRO

THERE IS AN INCREASING WORLDWIDE CONSENSUS ON THE VITAL IMPORTANCE OF SCIENCE FOR

EDITORS’CHOICE EDITED BY KRISTEN MUELLER AND JAKE YESTON

PLANT SCIENCES

PHYSIOLOGY

What’s Bred in the Bone

Plastids, such as the chloroplasts of plants, use photosynthesis as an essential energy source for plant cell function. Plastids probably arose from bacterial endosymbionts, and over time, most ﬂowering plants, even those that no longer photosynthesize, have discarded many genes within their plastids. Delannoy et al. sequenced the plastid genome of the orchid, Rhizanthella gardneri. R. gardneri lives underground and is parasitic, meaning that it obtains its energy and nutrients indirectly by exploiting other plants. Its plastid, the smallest identiﬁed land-plant plastid to date, retained genes that are also found in other nonphotosynthetic parasitic plants that are not closely related. The genes appear to be transcribed and translated properly. These results suggest that all plant plastids contain key genes and transfer RNAs that function outside of photosynthesis, which explains their convergent retention among distant lineages of parasitic plants and may have implications beyond the plant kingdom. — LMZ Mol. Biol. Evol. 28, 10.1093/ molbev/msr028 (2011).

In females, the interaction between the reproductive system and bone physiology is well established: Loss of estrogen during aging is a causative factor in osteoporosis. Oury et al. now describe a bone– reproductive system connection in males, where osteocalcin, a hormone produced in bone cells, controls the production of testosterone in the testes. Mice deﬁcient in osteocalcin had smaller testes, decreased concentrations of blood testosterone, and reduced fertility. In the testes, osteocalcin increased expression of genes that participate in testosterone biosynthesis and spermatogenesis and inhibited stem cell death. Osteocalcin probably functions by binding the G protein–coupled receptor Gprc6a, which is speciﬁcally expressed in Leydig cells of the testes. Direct binding of osteocalcin to Gprc6a was not directly demonstrated, but mice with reduced expression of Gprc6a in Leydig cells showed impaired testicular function similar to that in osteocalicin-deﬁcient mice. Osteocalcin, which also regulates metabolism through effects on pancreatic beta cells and fat cells, thus appears to have important physiological roles in coordinating energy metabolism, bone remodeling, and reproductive function. — LBR

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CREDITS (LEFT TO RIGHT): DELANNOY ET AL., MOL. BIOL. EVOL. 28, 10.1093/MOLBEV/MSR028 (2011).; LI ET AL., PHYS. REV. LETT. 106, 84301 (2011)

Even Parasitic Plants Need Plastids

Cell 144, 10.1016/j.cell.2011.02.004 (2011). PHYSICS

A One-Way Wall of Silence The recent development of metamaterials and photonic crystals has provided a route to control the propagation of electromagnetic waves through the engineered structure of a material. Combined with transformation optics, such control is rewriting the expected rules of behavior governing the propagation of electromagnetic waves, and offers myriad possibilities ranging from imaging to communications and stealth applications. Sound is also a wave, and so the manipulation of acoustic waves may be expected to carry over by analogy to their electromagnetic counterparts. Li et al. present a sonic crystal composed of a periodic array of steel rods, the geometry of which was selected to give rise to a band gap, whereby the transmission of sound waves in a speciﬁc frequency range is prohibited in one direction but allowed in the opposite direction. The authors also show that by mechanically changing the spacing of the array (by rotating the square steel rods), the diode-like behavior can be switched on and off. A range of applications might be expected to follow, from acoustic isolation and ﬁltering to ultrasound imaging. — ISO Phys. Rev. Lett. 106, 84301 (2011). B I O M AT E R I A L S

Delivered on a Diamond Doxorubicin (Dox) is a powerful, broad-use anticancer drug, but its effectiveness is limited by toxic damage to healthy tissues and the development of drug resistance by the cancerous cells. One form of resistance arises through an increase in transporter proteins that will pump drugs out of a cell. Chow et al. explore the potential for small diamond particles, on the order of 2 to 8 nm in diameter, to increase the effectiveness of doxorubicin. The facets on the truncated octahedral architecture provide a large number of surfaces for drug conjugation, and charges on the surface enable water to bind, aiding dispersability and sustained therapeutic release. In comparison with unbound Dox at equal doses, conjugates of diamond-Dox showed slower in vivo clearance, lower toxicity, and a slower but more prolonged uptake of Dox by the cancerous cells, leading to greater apoptosis for mouse models of liver and mammary cancers. Of particular note was that the mammary tumor cells were known to show Dox resistance. The production of the diamond particles can be scaled up. They have furthermore shown wide biocompatibility, and they can be chemically modiﬁed for conjugation to a wide range of therapeutics, indicating that they could form a broad-based platform for the treatment of a range of conditions in which drug retention and delivery are limiting factors. — MSL Sci. Transl. Med. 3, 73ra21 (2011).

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See pages 784 and 785 of the 11 February 2011 issue or access www.sciencemag.org/about/authors

Cori Bargmann, The Rockefeller Univ. John I. Brauman, Chair, Stanford Univ. Richard Losick, Harvard Univ. Michael S. Turner, University of Chicago

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Robert H. Crabtree, Yale Univ. Wolfgang Cramer, Potsdam Inst. for Climate Impact Research F. Fleming Crim, Univ. of Wisconsin Jeff L. Dangl, Univ. of North Carolina Tom Daniel, Univ. of Washington Stanislas Dehaene, Collège de France Emmanouil T. Dermitzakis, Univ. of Geneva Medical School Robert Desimone, MIT Claude Desplan, New York Univ. Ap Dijksterhuis, Radboud Univ. of Nijmegen Dennis Discher, Univ. of Pennsylvania Scott C. Doney, Woods Hole Oceanographic Inst. Jennifer A. Doudna, Univ. of California, Berkeley Julian Downward, Cancer Research UK Bruce Dunn, Univ. of California, Los Angeles Christopher Dye, WHO Michael B. Elowitz, Calif. Inst. of Technology Tim Elston, Univ. of North Carolina at Chapel Hill Gerhard Ertl, Fritz-Haber-Institut, Berlin Barry Everitt, Univ. of Cambridge Paul G. Falkowski, Rutgers Univ. Ernst Fehr, Univ. of Zurich Tom Fenchel, Univ. of Copenhagen Alain Fischer, INSERM Wulfram Gerstner, EPFL Lausanne Karl-Heinz Glassmeier, Inst. for Geophysics & Extraterrestrial Physics Diane Grifﬁn, Johns Hopkins Bloomberg School of Public Health Taekjip Ha, Univ. of Illinois at Urbana-Champaign Christian Haass, Ludwig Maximilians Univ. Steven Hahn, Fred Hutchinson Cancer Research Center Gregory J. Hannon, Cold Spring Harbor Lab. Dennis L. Hartmann, Univ. of Washington Martin Heimann, Max Planck Inst., Jena James A. Hendler, Rensselaer Polytechnic Inst. Janet G. Hering, Swiss Fed. Inst. of Aquatic Science & Technology Ray Hilborn, Univ. of Washington Michael E. Himmel, National Renewable Energy Lab. Kei Hirose, Tokyo Inst. of Technology Ove Hoegh-Guldberg, Univ. of Queensland David Holden, Imperial College Lora Hooper, UT Southwestern Medical Ctr at Dallas Jeffrey A. Hubbell, EPFL Lausanne Steven Jacobsen, Univ. of California, Los Angeles Kai Johnsson, Ecole Polytechnique Federale de Lausanne

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Peter Jonas, Universität Freiburg Barbara B. Kahn, Harvard Medical School Daniel Kahne, Harvard Univ. Bernhard Keimer, Max Planck Inst., Stuttgart Robert Kingston, Harvard Medical School Hanna Kokko, Univ. of Helsinki Alberto R. Kornblihtt, Univ. of Buenos Aires Leonid Kruglyak, Princeton Univ. Lee Kump, Penn State Univ. Mitchell A. Lazar, Univ. of Pennsylvania David Lazer, Harvard Univ. Virginia Lee, Univ. of Pennsylvania Ottoline Leyser, Univ. of New York Olle Lindvall, Univ. Hospital, Lund Marcia C. Linn, Univ. of California, Berkeley John Lis, Cornell Univ. Richard Losick, Harvard Univ. Jonathan Losos, Harvard Univ. Ke Lu, Chinese Acad. of Sciences Laura Machesky, CRUK Beatson Inst. for Cancer Research Andrew P. MacKenzie, Univ. of St Andrews Anne Magurran, Univ. of St Andrews Oscar Marin, CSIC & Univ. Miguel Hernández Charles Marshall, Univ. of California, Berkeley Martin M. Matzuk, Baylor College of Medicine Grahma Medley, Univ. of Warwick Yasushi Miyashita, Univ. of Tokyo Richard Morris, Univ. of Edinburgh Edvard Moser, Norwegian Univ. of Science and Technology Sean Munro, MRC Lab. of Molecular Biology Naoto Nagaosa, Univ. of Tokyo James Nelson, Stanford Univ. School of Med. Timothy W. Nilsen, Case Western Reserve Univ. Pär Nordlund, Karolinska Inst. Helga Nowotny, European Research Advisory Board Stuart H. Orkin, Dana-Farber Cancer Inst. Christine Ortiz, MIT Elinor Ostrom, Indiana Univ. Andrew Oswald, Univ. of Warwick Jonathan T. Overpeck, Univ. of Arizona P. David Pearson, Univ. of California, Berkeley Reginald M. Penner, Univ. of California, Irvine John H. J. Petrini, Memorial Sloan-Kettering Cancer Center Simon Phillpot, Univ. of Florida Philippe Poulin, CNRS Colin Renfrew, Univ. of Cambridge Trevor Robbins, Univ. of Cambridge

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BOOK REVIEW BOARD

John Aldrich, Duke Univ. David Bloom, Harvard Univ. Angela Creager, Princeton Univ. Richard Shweder, Univ. of Chicago Ed Wasserman, DuPont Lewis Wolpert, Univ. College London

NEWS OF THE WEEK

AROUND THE WORLD 1, 4

Glory

6 2 7 5

London, U.K. 1

New Long-Term Study Of a Generation Announced

From their 25th week in the womb to their 21st birthday, 90,000 children born in the United Kingdom in 2012 will be tracked in a multimillion-pound longitudinal study, the U.K. government announced last week. Like the country’s 1946 National Survey of Health and Development, which followed just over 5000 people, the study aims to investigate how environmental and genetic factors during pregnancy and early infancy inﬂuence a child’s future. Parents of the 2012 cohort will provide blood samples and ﬁll in questionnaires during pregnancy, and later bring in their new family members to be weighed and assessed at 4 and 12 months. Monitoring will then continue through childhood and adolescence. Beijing, China 2

China Beefs Up Space Program In a speech to the annual National People’s Congress (NPC) last weekend, Premier Wen Jiabao hailed crewed space missions and lunar exploration as two major Chinese accomplishments over the past 5 years (see p. 1251). Now the country’s space ambitions are set on new heights. In an announcement timed with NPC, Ma Xingrui, general manager of China Aerospace Science and Technology Corp. (CASC)—the prime contractor for the country’s space program—revealed to Chinese media aggressive R&D spending plans for the next 5 years. China’s leaders tout the country’s space program as a shining example of indigenous innovation because it has developed much of the technology on its own. CASC says it will increase R&D spending to further boost own-

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ership of core space technology. Toward that end, a CASC subsidiary, the China Academy of Launch Vehicle Technology, is developing a launch vehicle capable of carrying 130 tons, equivalent in capacity to NASA’s retired Saturn V rocket, which launched the Skylab space station into low Earth orbit. Meanwhile, CASC has planned some 20 launches this year, including that of its ﬁrst orbiting “space lab,” Tiangong-1, in the second half of 2011. Tiangong-1 will orbit Earth for 2 years—ample time for Chinese spacecraft to practice docking maneuvers in anticipation of a future, full-ﬂedged space station. Vandenberg Air Force Base, California 3

Sic Transit Glory A rocket failure on 4 March sent NASA’s Earth-observing Glory satellite plunging into the South Paciﬁc. The loss of the satellite—launched to monitor subtle variations in the sun’s brightness and identify small particles called aerosols in Earth’s atmosphere—deprives climate scientists of key data needed to estimate future global warming. Problems arose about 3 minutes into the ﬂight, when a protective shroud called a fairing failed to peel away from the Taurus XL rocket as planned. Its weight dragged the rocket downward into the sea. An identical Taurus mishap doomed NASA’s Orbiting Carbon Observatory in February 2009, but engineers thought a new separation mechanism had solved the problem. The failure means Glory will miss out on continuing observations into the next 11-year sunspot cycle. “It’s a terrible loss,” says Glory team member Judith Lean, a solar physicist at the U.S. Naval Research Laboratory in Washington, D.C. “You don’t have many solar cycles in your lifetime.”

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The satellite also would have taken sophisticated readings of sunlight that aerosols in Earth’s atmosphere scatter back toward space. The information would have enabled scientists to identify the particles and gauge how much they offset global warming. http://scim.ag/glory-fail London, U.K. 4

Funding Bodies Decide Weight Of Research ‘Impact’

Last week, the United Kingdom’s higher education funding bodies revealed how much weight the country’s next nationwide assessment of science in its universities would give to the economic and social impact of research. Such impacts, which have not been part of previous assessments, will make up 20% of the overall scores in the U.K.’s 2014 Research Excellence Framework, which will determine how a large portion of the nation’s research funding is distributed. That weighting is less than the 25% the funding bodies originally proposed— they plan to ramp up the percentage in future assessments—but it’s still more than what was desired by critics who argue that the economic and social impacts of basic research can’t be meaningfully measured. Brasília, Brazil 5

Panel to Reassess R&D Brazil is putting the brakes on its rapidly growing budget for research and development while launching a broad review of its research priorities. Neuroscientist Miguel Nicolelis of Duke University in Durham,

NOTED

>In 1959, when computers were room-

sized behemoths with names like UNIVAC, the U.S. government launched a crash project to give them a common language. The result, COBOL, is now the subject of an exhibition opening 17 March at the Smithsonian National Museum of American History in Washington, D.C.

www.sciencemag.org

CREDIT: NASA

3

NEWS

http://scim.ag/brazil-RD

Washington, D.C. 6

Bioethics Panel Examines Global Clinical Trials

CREDITS (TOP TO BOTTOM): AMEL PAIN/EPA/LANDOV; ALEXANDER SAFONOV/GETTY IMAGES

Bioethics advisers to President Barack Obama have begun to assess whether U.S. rules adequately protect volunteers in global clinical trials. The review was prompted by the revela-

tion last fall that from 1946 to 1948, a U.S. federal researcher deliberately infected Guatemalan patients with syphilis to study treatments. In response, President Obama asked his Presidential Commission for the Study of Bioethical Issues to appoint an international panel to determine if “current rules for research participants protect people from harm or unethical treatment, domestically as well as internationally.” At a meeting last week, commission chair and University of Pennsylvania President Amy Gutmann announced the international panel: four experts from the bioethics commission and 10 others from outside the United States. The commission also heard from experts about how the number of trials abroad has burgeoned in the past decade as companies seek to cut costs. And as part of a fact-ﬁnding investigation requested by the president, commission staffers have begun digging through 477 boxes of documents on the Guatemala studies. The commission will report to the president by the end of the year. http://scim.ag/bio-ethics

NEWSMAKERS

Three Q’s Zahi Hawass made headlines last week when he announced that he intended to resign his post as minister of Egypt’s antiquities in the wake of the country’s revolution. Hawass is arguably the world’s most wellknown archaeologist, uncovering tombs on television and ruling Egypt’s ancient treasures with a ﬁrm hand. He had been a strong supporter of Egypt’s former president, Hosni Mubarak, and cited the lack of police control over archaeological sites during the past 6 weeks as the reason behind his planned departure. When he announced his intent to resign, Hawass acknowledged extensive looting at Saqqara, Giza, and other important ancient sites. Q: Why haven’t you actually resigned? I did not submit an ofﬁcial resignation because I have not been asked for one. I have announced my reasons and at the moment, I do not want to be part of the new government. >>

THEY SAID IT

Cocos Island, Costa Rica 7

More Room to Swim Costa Rica has expanded a national park in the Paciﬁc Ocean to nearly 1 million hectares, larger than Yellowstone National Park. Centered around Cocos Island, 550 kilometers from mainland Costa Rica, the new Seamounts Marine Management Area teems with endangered leatherback turtles and scalloped hammerhead sharks, as well as whitetip reef sharks, whale sharks, yellowﬁn tuna, and numerous endemic species—all now fully protected in the new nonﬁshing zone. It’s good news for Costa Rica’s population of leatherback turtles, which dropped 40% in the past 8 years owing to locals’ taste for leatherback eggs. The numbers of scalloped hammerhead sharks are also plummeting because of overﬁshing; they are prized for their large ﬁns, the key ingredient in shark ﬁn soup. The larger area should help protect the region’s unique underwater mountains and marine ecosystem, said biologist Marco Quesada of Conservation International, which helped Costa Rica create the park. www.sciencemag.org

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“I now know why you won the Nobel Prize. When they asked you a question, by God, you answered it.” —Representative Ralph Hall (R–TX), chair of the U.S. House of Representatives science committee, to Energy Secretary Steven Chu after one of many detailed and lengthy answers from Chu to questions posed by committee members at last week’s hearing on his department’s 2012 budget request.

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North Carolina, has been tapped to lead the “Commission of the Future,” a panel that will study the direction of Brazilian science. The group’s formation follows a $577 million reduction (a cut of about 11%) in the budget of the Ministry of Science and Technology this year. The nation’s research program was expanding until the fall (Science, 3 December 2010, p. 1306). But the new president, Dilma Rousseff, has ordered across-the-board government spending cuts in response to concerns about inﬂation. The 21-member R&D review group includes several U.S. and European scientists along with Brazilians.

NEWS OF THE WEEK

Q: What are your plans for the future? I will continue to write books, give lectures, and watch over Egyptian antiquities. I will report anything bad I hear about on my Web site and take a stand against anyone who threatens antiquities in the near future.

Hungarian Trio Shares €1 Million Prize A new €1 million award from a Danish nonproﬁt organization honors three Hungarian-born scientists for their contributions to European neuroscience. The inaugural prize from the Grete Lundbeck European Brain Research Foundation goes to Péter Somogyi of the University of Oxford in the United Kingdom, Tamás Freund of the Hungarian Academy of Sciences in Budapest, and Györgi Buzsáki of Rutgers University in Newark, New Jersey. The chair of the selection committee, Oxford neuroscientist Colin Blakemore, praised the men for “their wide-ranging, technically and conceptually brilliant research on the functional organization of neuronal circuits.” All three have focused largely on the hippocampus, a brain region central to memory formation. Somogyi has pioneered techniques for identifying different types of neurons and has applied them to map out circuits in that area, whereas Freund, his former student, has made important discoveries about the roles of inhibitory neurons there. Buzsáki, meanwhile, has specialized in recording neural activity in the hippocampus, often in freely moving animals, to investigate how neural circuits in this brain region contribute to memory and other cognitive functions.

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FINDINGS

Elephants Can Lend A Helping Trunk We humans readily understand when we need another person’s help to solve a problem. That kind of cooperative skill is an indication of our higher social cognition, psychologists say, and it’s rarely found in other species. But a new study, published online in the Proceedings of the National Academy of Sciences, shows that elephants, too, can easily see when a goal is only attainable with another elephant—and his or her trunk. To test elephants’ cooperative skills, Joshua Plotnik, then a psychology graduate student at Emory University in Atlanta and now at the University of Cambridge in the United Kingdom, used a sliding table bearing treats that could be moved only if two elephants pulled simultaneously on the ends of a single rope threaded through the table; otherwise, the rope would just come out. Twelve Asian elephants (Elephas maximus) were grouped in pairs and taught the pulling task. In a test of whether they understood that they needed help, the animals waited for their partners, sometimes for as long as 45 seconds, before pulling their end of the rope. “People might not be surprised that chimpanzees can solve this task,” says Satoshi Hirata, a primatologist at the Great Ape

Elephants cooperate to move a sliding table.

Research Institute in Okayama, Japan, who invented this dual rope-pulling exam. “But it is more surprising, even for researchers, that elephants can do it, too. It shows that they are highly socially intelligent.” http://scim.ag/helping-trunk

Bad for the Bone Last November, failure-weary HIV prevention researchers were energized by news that taking anti-HIV drugs could thwart transmission of the virus in transgender women and men who have sex with men. That success now comes with a new caveat. Two groups reported last week in Boston at the Conference on Retroviruses and Opportunistic Infections that the two drugs used together for what is known as Pre-Exposure Prophylaxis (PrEP) reduced bone density in trial participants. Researchers have long

Battle-Scarred Mars High-resolution images released last week by the European Space Agency reveal new details about an unusually elongated crater on Mars that may have been blasted by several objects striking the planet’s surface at a shallow angle. The unnamed crater, located in a heavily blemished portion of the Red Planet’s southern hemisphere, is about 78 kilometers long, approximately 25 kilometers across at its widest point, and about 2 kilometers deep. Two distinct blankets of material blasted from the impact zone suggest that at least two projectiles, possibly fragments of a once-intact body, gouged the crater. Three particularly deep spots in the crater (depicted in blue, inset) bolster the notion of multiple impactors, as does the presence of another elongated crater nearby that has a similar alignment.

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Q: Would you consider staying on if asked? For me to come back, the government has to give me full guarantees that the police will guard every site across Egypt and give me the funding to continue the work I have been doing for the last 8 years. There are 70 great projects that we currently have no money for, such as site-management plans; restoration and protection projects for pharaonic, Islamic, Coptic, and Jewish monuments; education programs for Egyptians; and training projects for young Egyptian archaeologists.

CREDITS (TOP TO BOTTOM): JOSHUA PLOTNIK AND RICHARD LAIR; ESA/DLR/FU BERLIN (G. NEUKUM)

>>NEWSMAKERS

NEWS $8600, $6200, $1000 The

scores of Representative Rush Holt (D–NJ), IBM’s supercomputer Watson, and Representative Bill Cassidy (R–LA) in an informal round of Jeopardy! played last week on Capitol Hill. Holt is a former physicist; Cassidy, a hepatologist.

281 February sales of General Motors’s new, electric Chevy Volt. That’s down from 321 in January. Meanwhile, Nissan moved just 67 units of its electric car, the Leaf; it sold 87 in January. $263 billion The cost of iden-

CREDITS (TOP TO BOTTOM): JAMES SOTIRAKIS/THINKSTOCK; SOURCE: DEISSEROTH LAB

tifying Earth’s estimated 5.4 million undiscovered animal species, according to a study in Trends in Ecology and Evolution. That’s far more than the $5 billion price tag famed ant biologist Edward O. Wilson placed on identifying all life forms in 2000.

known that one of the drugs, tenofovir, causes 2% to 4% bone density loss in HIVinfected people who use the compound as part of their treatment regimen. The PrEP data suggest the loss is only 1%, and no one has suffered bone fractures as a result. Still, concerns about side effects from PrEP run especially high because the anti-HIV drugs are being given to otherwise healthy people.

More Evidence That Chimps Die From AIDS Researchers at this year’s Conference on Retroviruses and Opportunistic Infections, held in Boston last week, have found ironclad evidence of AIDS in a wild chimp. At the 2009 conference, researchers who studied fecal samples from chimpanzees at Gombe Stream National Park in Tanzania— communities that have been followed since Jane Goodall started working there in 1960—showed that individuals infected with SIVcpz, a cousin of HIV, had shorter life spans. Necropsies of dead chimps revealed immune destruction, but for ethi-

Random Sample

Mixology Meets Materials Science Mixing business and pleasure, materials scientists in Japan say they can make the compound iron tellurium sulﬁde (FeTeS) conduct electricity without resistance if they ﬁrst soak the stuff in booze. Such superconductivity turns on when FeTeS is cooled below 8 K, but to achieve it Yoshihiko Takano of the National Institute for Materials Science in Tsukuba and his team usually expose a newly formed sample to air, water, or oxygen. Now they’ve found that a dip in warm wine, beer, or sake works just as well. “I enjoy the taste of wine, beer, and so on,” Takano says. Apparently, FeTeS does, too. Takano’s team made FeTeS samples consisting of many tiny crystalline grains, and the drinks induced superconductivity in more grains than did a tasteless mixture of water and ethanol, the team reports this week in Superconductor Science and Technology. So something else in the drinks may act on FeTeS, perhaps to catalyze reactions with oxygen, Takano says. Not everybody buys it. Single crystals of FeTeS are superconductive right out of the oven, which shows that intrinsically the compound requires no further processing, says physicist Cedomir Petrovic of Brookhaven National Laboratory in Upton, New York. Still, physicist Paul Canﬁeld of Ames Laboratory in Iowa says the preprint of the paper created a stir as scientists contemplated follow-up experiments: “A lot of people were thinking that they could score a lot of good hooch off their grants.”

cal reasons, the researchers could not prove their case by sampling chimp blood over time to track the decline of CD4 white blood cells, the virus’s main target. Now Lucie Etienne, who works with Martine Peeters at the Institute of Research for Development in Montpellier, France, has solid evidence from a wild-born male chimpanzee that tested positive for SIVcpz when he arrived at a chimpanzee sanctuary in Cameroon in November 2003. A test a few months later showed that he had 700 CD4 cells per microliter of blood. By August 2009, the count had dropped to 287; the average CD4 count of a healthy, agematched chimpanzee tested at the sanctuary was 1256. The SIVcpz-infected chimp has had many infections and lost 21% of his weight between August 2009 and January 2010, Etienne and her colleagues reported online 13 January in Retrovirology. Etienne says they may treat the chimpanzee with anti-HIV drugs.

Shedding Light on Anxiety Tucked deep inside the brain, the amygdala is the seat of fear and anxiety. Now researchers have used newly developed

www.sciencemag.org

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BY THE NUMBERS

“optogenetic” methods to indentify a subset of the amygdala’s neurons that seems to regulate anxiety levels. Mice are naturally anxious, says Karl Deisseroth, a neuroscientist at Stanford University and the senior author of the paper, which appears in Nature this week. In the lab, for example, mice hug the walls when placed in a big, open enclosure. To address these nervous tendencies, Deisseroth and colleagues focused on a speciﬁc group of neurons that originate in one part of the amygdala and terminate in another. After inserting a gene that made the neurons sensitive to light, they stimulated the neurons’ terminals via laser light pulsed through an optical ﬁber. Mice given this treatment were less afraid of open spaces. By clarifying the neural circuits affected by anxiety disorders, such studies could eventually lead to more targeted treatments, says neuroscientist Stephen Maren of the University of Michigan, Ann Arbor.

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NEWS OF THE WEEK

Q: What are your plans for the future? I will continue to write books, give lectures, and watch over Egyptian antiquities. I will report anything bad I hear about on my Web site and take a stand against anyone who threatens antiquities in the near future.

Hungarian Trio Shares €1 Million Prize A new €1 million award from a Danish nonproﬁt organization honors three Hungarian-born scientists for their contributions to European neuroscience. The inaugural prize from the Grete Lundbeck European Brain Research Foundation goes to Péter Somogyi of the University of Oxford in the United Kingdom, Tamás Freund of the Hungarian Academy of Sciences in Budapest, and Györgi Buzsáki of Rutgers University in Newark, New Jersey. The chair of the selection committee, Oxford neuroscientist Colin Blakemore, praised the men for “their wide-ranging, technically and conceptually brilliant research on the functional organization of neuronal circuits.” All three have focused largely on the hippocampus, a brain region central to memory formation. Somogyi has pioneered techniques for identifying different types of neurons and has applied them to map out circuits in that area, whereas Freund, his former student, has made important discoveries about the roles of inhibitory neurons there. Buzsáki, meanwhile, has specialized in recording neural activity in the hippocampus, often in freely moving animals, to investigate how neural circuits in this brain region contribute to memory and other cognitive functions.

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FINDINGS

Elephants Can Lend A Helping Trunk We humans readily understand when we need another person’s help to solve a problem. That kind of cooperative skill is an indication of our higher social cognition, psychologists say, and it’s rarely found in other species. But a new study, published online in the Proceedings of the National Academy of Sciences, shows that elephants, too, can easily see when a goal is only attainable with another elephant—and his or her trunk. To test elephants’ cooperative skills, Joshua Plotnik, then a psychology graduate student at Emory University in Atlanta and now at the University of Cambridge in the United Kingdom, used a sliding table bearing treats that could be moved only if two elephants pulled simultaneously on the ends of a single rope threaded through the table; otherwise, the rope would just come out. Twelve Asian elephants (Elephas maximus) were grouped in pairs and taught the pulling task. In a test of whether they understood that they needed help, the animals waited for their partners, sometimes for as long as 45 seconds, before pulling their end of the rope. “People might not be surprised that chimpanzees can solve this task,” says Satoshi Hirata, a primatologist at the Great Ape

Elephants cooperate to move a sliding table.

Research Institute in Okayama, Japan, who invented this dual rope-pulling exam. “But it is more surprising, even for researchers, that elephants can do it, too. It shows that they are highly socially intelligent.” http://scim.ag/helping-trunk

Bad for the Bone Last November, failure-weary HIV prevention researchers were energized by news that taking anti-HIV drugs could thwart transmission of the virus in transgender women and men who have sex with men. That success now comes with a new caveat. Two groups reported last week in Boston at the Conference on Retroviruses and Opportunistic Infections that the two drugs used together for what is known as Pre-Exposure Prophylaxis (PrEP) reduced bone density in trial participants. Researchers have long

Battle-Scarred Mars High-resolution images released last week by the European Space Agency reveal new details about an unusually elongated crater on Mars that may have been blasted by several objects striking the planet’s surface at a shallow angle. The unnamed crater, located in a heavily blemished portion of the Red Planet’s southern hemisphere, is about 78 kilometers long, approximately 25 kilometers across at its widest point, and about 2 kilometers deep. Two distinct blankets of material blasted from the impact zone suggest that at least two projectiles, possibly fragments of a once-intact body, gouged the crater. Three particularly deep spots in the crater (depicted in blue, inset) bolster the notion of multiple impactors, as does the presence of another elongated crater nearby that has a similar alignment.

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Q: Would you consider staying on if asked? For me to come back, the government has to give me full guarantees that the police will guard every site across Egypt and give me the funding to continue the work I have been doing for the last 8 years. There are 70 great projects that we currently have no money for, such as site-management plans; restoration and protection projects for pharaonic, Islamic, Coptic, and Jewish monuments; education programs for Egyptians; and training projects for young Egyptian archaeologists.

CREDITS (TOP TO BOTTOM): JOSHUA PLOTNIK AND RICHARD LAIR; ESA/DLR/FU BERLIN (G. NEUKUM)

>>NEWSMAKERS

NEWS $8600, $6200, $1000 The

scores of Representative Rush Holt (D–NJ), IBM’s supercomputer Watson, and Representative Bill Cassidy (R–LA) in an informal round of Jeopardy! played last week on Capitol Hill. Holt is a former physicist; Cassidy, a hepatologist.

281 February sales of General Motors’s new, electric Chevy Volt. That’s down from 321 in January. Meanwhile, Nissan moved just 67 units of its electric car, the Leaf; it sold 87 in January. $263 billion The cost of iden-

CREDITS (TOP TO BOTTOM): JAMES SOTIRAKIS/THINKSTOCK; SOURCE: DEISSEROTH LAB

tifying Earth’s estimated 5.4 million undiscovered animal species, according to a study in Trends in Ecology and Evolution. That’s far more than the $5 billion price tag famed ant biologist Edward O. Wilson placed on identifying all life forms in 2000.

known that one of the drugs, tenofovir, causes 2% to 4% bone density loss in HIVinfected people who use the compound as part of their treatment regimen. The PrEP data suggest the loss is only 1%, and no one has suffered bone fractures as a result. Still, concerns about side effects from PrEP run especially high because the anti-HIV drugs are being given to otherwise healthy people.

More Evidence That Chimps Die From AIDS Researchers at this year’s Conference on Retroviruses and Opportunistic Infections, held in Boston last week, have found ironclad evidence of AIDS in a wild chimp. At the 2009 conference, researchers who studied fecal samples from chimpanzees at Gombe Stream National Park in Tanzania— communities that have been followed since Jane Goodall started working there in 1960—showed that individuals infected with SIVcpz, a cousin of HIV, had shorter life spans. Necropsies of dead chimps revealed immune destruction, but for ethi-

Random Sample

Mixology Meets Materials Science Mixing business and pleasure, materials scientists in Japan say they can make the compound iron tellurium sulﬁde (FeTeS) conduct electricity without resistance if they ﬁrst soak the stuff in booze. Such superconductivity turns on when FeTeS is cooled below 8 K, but to achieve it Yoshihiko Takano of the National Institute for Materials Science in Tsukuba and his team usually expose a newly formed sample to air, water, or oxygen. Now they’ve found that a dip in warm wine, beer, or sake works just as well. “I enjoy the taste of wine, beer, and so on,” Takano says. Apparently, FeTeS does, too. Takano’s team made FeTeS samples consisting of many tiny crystalline grains, and the drinks induced superconductivity in more grains than did a tasteless mixture of water and ethanol, the team reports this week in Superconductor Science and Technology. So something else in the drinks may act on FeTeS, perhaps to catalyze reactions with oxygen, Takano says. Not everybody buys it. Single crystals of FeTeS are superconductive right out of the oven, which shows that intrinsically the compound requires no further processing, says physicist Cedomir Petrovic of Brookhaven National Laboratory in Upton, New York. Still, physicist Paul Canﬁeld of Ames Laboratory in Iowa says the preprint of the paper created a stir as scientists contemplated follow-up experiments: “A lot of people were thinking that they could score a lot of good hooch off their grants.”

cal reasons, the researchers could not prove their case by sampling chimp blood over time to track the decline of CD4 white blood cells, the virus’s main target. Now Lucie Etienne, who works with Martine Peeters at the Institute of Research for Development in Montpellier, France, has solid evidence from a wild-born male chimpanzee that tested positive for SIVcpz when he arrived at a chimpanzee sanctuary in Cameroon in November 2003. A test a few months later showed that he had 700 CD4 cells per microliter of blood. By August 2009, the count had dropped to 287; the average CD4 count of a healthy, agematched chimpanzee tested at the sanctuary was 1256. The SIVcpz-infected chimp has had many infections and lost 21% of his weight between August 2009 and January 2010, Etienne and her colleagues reported online 13 January in Retrovirology. Etienne says they may treat the chimpanzee with anti-HIV drugs.

Shedding Light on Anxiety Tucked deep inside the brain, the amygdala is the seat of fear and anxiety. Now researchers have used newly developed

www.sciencemag.org

SCIENCE

VOL 331

Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

BY THE NUMBERS

“optogenetic” methods to indentify a subset of the amygdala’s neurons that seems to regulate anxiety levels. Mice are naturally anxious, says Karl Deisseroth, a neuroscientist at Stanford University and the senior author of the paper, which appears in Nature this week. In the lab, for example, mice hug the walls when placed in a big, open enclosure. To address these nervous tendencies, Deisseroth and colleagues focused on a speciﬁc group of neurons that originate in one part of the amygdala and terminate in another. After inserting a gene that made the neurons sensitive to light, they stimulated the neurons’ terminals via laser light pulsed through an optical ﬁber. Mice given this treatment were less afraid of open spaces. By clarifying the neural circuits affected by anxiety disorders, such studies could eventually lead to more targeted treatments, says neuroscientist Stephen Maren of the University of Michigan, Ann Arbor.

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NEWS & ANALYSIS

2 011 B U D G E T

CREDIT: LI XUEREN/XINHUA/LANDOV

China Bets Big on Small Grants, Large Facilities BEIJING—Some nations talk about doubling the budgets of their basic research funding agencies. China’s doing it. The National Natural Sciences Foundation of China (NSFC), the country’s main agency for funding competitive, peer-reviewed research grants, will get 12 billion yuan ($1.83 billion) in 2011—a 17% increase over 2010 and twice its budget just 2 years ago. NSFC is not the only science winner in China’s 2011 budget, released here on 5 March. New spending plans promise massive investments in shared research facilities, such as new beamlines for structural biologists and materials scientists at the Shanghai Synchrotron Radiation Facility that opened in 2009. The rationale, science ofﬁcials say, is to erode barriers between scientists at universities and institutes. Still, some science policy experts and researchers in China decry the lack of a coherent strategy behind the ramp-up of science spending. China’s R&D programs, says one senior scientist who requested anonymity, are stacked “like a layer cake” with little holding them together. That cake was unveiled here at the opening session of the annual National People’s Congress. Delivering the equivalent of the U.S. president’s State of the Union address, Premier Wen Jiabao revealed in broad brushstrokes China’s priorities for 2011 and beyond. As in Europe and the United States, innovation is the watchword of the day. Wen said the central government is “keenly aware” of the country’s “insufﬁcient scientiﬁc and technological innovation capabilities” and later added that “China’s economy needs to be quickly put on the path of endogenous growth driven by innovation.” China’s central government hopes that plenty of cash will grease the wheels of its innovation juggernaut. During the 11th 5-year plan that ended in 2010, the central government spent 619.7 billion yuan ($94 billion at the current exchange rate) on science and technology, an average annual increase of 22%. Robust as that sounds, Wen cited the nation’s inability to raise total societal R&D spending to 2% of GDP as one of three failures of the 11th 5-year plan. The current ﬁgure

is 1.75%. This year’s target is 1.85%, which Wen says will rise to 2.2% by the end of the 12th 5-year plan in 2015. Toward that end, the central government has appropriated 194.41 billion yuan ($29.6 billion) for science and technology in 2011, a 12.5% increase over last year. Basic research funding will rise a modest 5.24% to 25.72 billion yuan ($3.9 billion). NSFC is getting the lion’s share: It is slated to receive 46% of China’s designated basic research funds. That will allow the agency to award more grants, extend general grants from 3 to 4 years, and double the average payout to 600,000 yuan ($91,000). The success rate for young scientist grants is expected to rise from 23% to 30%, says NSFC President Chen Yiyu. Other efforts classiﬁed as basic research may be pinched, notably the science ministry’s 973 grants program. That would be a minor setback for the science ministry, whose big science portfolio is about to be stuffed with cash. Science and technology megaprojects launched during the last 5-year plan and which will continue into the 12th are slated to receive 43.5 billion yuan ($6.62 billion) in 2011, a 30% increase over last year. Two megaprojects with substantial basic research components are “drug discovery and development” and “major infectious diseases.” These initiatives will build technology platforms and facilities for chemical biology and drug screening and for the quick identiﬁcation and discovery of new pathogens, says Chen Zhu, China’s health minister. Both projects involve “basic capacity building for research,” he says. “My personal analysis,” adds Luo Yongzhang, director of the National Engineering Laboratory for Antitumor Protein Therapeutics at Tsinghua University here, “is that the central government would like to build up S&T platforms to promote both basic and translational research.” Universities and the Chinese Academy of Sciences (CAS), which employ many of the nation’s scientists across several dozen institutes, are supposed to share these new

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resources, but they have a poor track record of working well together because of impediments to sharing data and authorship. In a nascent attempt to overcome such barriers, CAS, universities, and industry are being encouraged to form alliances on targeted projects, says Sun Song, director of CAS’s Institute of Oceanology in Qingdao, which has just launched an aquaculture alliance. Bridging the university-CAS gap is one challenge for the new CAS president, Bai Chunli. A physical chemist who introduced scanning tunneling microscopy to China in the early 1990s, Bai in recent years has overseen development of China’s nanoscience research. In a speech at CAS headquarters following his appointment last week, Bai vowed to liberate scientists at the academy’s institu-

Downloaded from www.sciencemag.org on March 10, 2011

NEWS & ANALYSIS

Innovation nation? Wen Jiabao vows that China will turn science achievements into “actual productive forces.”

tions from heavy administrative burdens so that they can spend more time on research. Many of China’s top leaders are engineers, and they want results. Early in his address, Wen lauded three “major breakthroughs” in S&T during the just-completed 5-year plan: human-occupied space missions, the lunar exploration program, and supercomputers. In the current 5-year plan, he said, China will “promote the conversion of advances in science and technology into actual productive forces.” In other words, basic research is ﬁne—as long as ﬁndings don’t sit around on the shelf.

–RICHARD STONE

With reporting by Hao Xin.

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Ten Months After Deepwater Horizon, Picking Up the Remnants of Health Data Almost as soon as the Macondo oil rig exploded in the Gulf of Mexico, cleanup workers began reporting to local health ofﬁces, complaining of ﬂulike symptoms, rashes, heat stroke, and stress. On 28 February, after 10 months of hearing such anecdotal stories—and still haunted by the government’s failure to collect health data following the Exxon Valdez disaster—the U.S. Department of Health and Human Services launched the long-awaited Gulf Longterm Follow-up (GuLF) Study through the National Institute of Environmental Health Sciences (NIEHS). The largest, most comprehensive study of long-term health effects from an oil spill, it will attempt to collect health data on cleanup workers by contacting 100,000 of them directly and tracking 55,000 for at least 5 years, looking at longterm problems such as cancer, birth defects, and psychosocial issues. But at this juncture, experts worry that they won’t know what to look for. Any shortterm physiological effects such as elevated levels of biomarkers or telltale rashes that could be deﬁnitively linked to the spill are long gone, as are toxicants in workers’ blood that could have provided information on exposure levels. What remains is an economically depressed community in which many suffer from stress-related illnesses that will be difﬁcult to pin on any particular cause. In June 2010, the Institute of Medicine (IOM) convened a workshop to recommend a research strategy. After hearing testimony from researchers such as toxicologist Blanca Laffon, who found DNA damage in cleanup workers from the 2002 Prestige tanker

1252

spill off the coast of Spain, and Lawrence Palinkas, who found psychological damage in Alaskans affected by the Exxon Valdez spill, the group recommended that the government undertake a massive, comprehensive health analysis—and fast. Throughout all the time it takes to develop a powerful long-term study, says epidemiologist Maureen Lichtveld of Tulane University in New Orleans, Louisiana, who served on the IOM committee, “at the other end of the table there are ﬁshermen who have worried for months about their health.” Lead investigator Dale Sandler of NIEHS says the agency moved as quickly as it could. Studies of this magnitude usually have a time lag of several years between conception and approval by review boards. “Miracles have happened to get this launched as quickly as it has,” she said. “People realize how important it is.” Even if the $19 million GuLF Study had been initiated immediately, it would still have been a difﬁcult undertaking. The problem with studying oil spills, Lichtveld says, is that unlike most occupational health studies in which a worker’s job responsibilities are clearly deﬁned, the duties—and thus the exposures—of cleanup workers are varied. Possible health effects from picking up tarballs on the beach, laying booms, or working on the rig itself will likely differ. Lichtveld and others predict that the largest health effects will be mental and psychosocial: depression and anxiety from losing jobs and livelihoods, for instance, and increased alcohol use, all of which can have physiological effects.

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CREDIT: THE TIMES-PICAYUNE/LANDOV

GULF OIL SPILL

The time gap only exacerbates the study’s challenges. Environmental health expert Bernard Goldstein of the University of Pittsburgh in Pennsylvania, who testiﬁed at the IOM workshop, says that by starting at this late date, “we’ve basically thrown out major tools for understanding the linkage between exposure and effect.” To further complicate the issue, industrial workers in the region are exposed to any number of hazards daily, so teasing apart effects of any single component is tough. Few data exist on the baseline health of the workers before the spill. One boon for the NIEHS study is that the National Institute for Occupational Safety and Health collected some baseline health data immediately after the accident when workers began safety training. Local health agencies that had been seeing patients are also working closely with NIEHS. These data will help immensely, says Sandler, but they’re insufﬁcient to establish the kind of baseline needed to discover any convincing links between exposures and effect. “This [study] should have been prepared to go immediately, and the very least we should learn from this is to be prepared for the next one, because there will be a next one,” says Goldstein. “That’s not a criticism of the investigators; it is a criticism of our federal government that does not have in place the kind of methodological approaches one would need to immediately start a study.” One of the most important lessons learned in developing the study, says Lichtveld, is the importance of working closely with the community, and she believes the GuLF Study is doing an excellent job. Enrollment and tracking of a cohort in an environmental epidemiology study is notoriously difﬁcult. But in this case, many Gulf Coast residents whose lives have been overturned by the spill seem eager to enroll. Alabama ﬁsher Chris Bryant, who sits on the study’s community advisory board, says that hundreds of people have turned up at each of the informational meetings he’s organized. “We’re looking at people’s health here, and people are pretty passionate about that,” he says. Perhaps as important as any actual data the GuLF Study may glean, Lichtveld says, is how it will inform the development of “offthe-shelf protocols” that would facilitate a quick response to future spills. “Hopefully, this is a lesson learned we never have to learn again,” she says. –SARA REARDON

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At risk. It won’t be easy to discover what health effects the gulf spill had on cleanup workers. But a new NIH study is going to try.

NEWS&ANALYSIS R E T R OV I R O LO G Y

injecting tissue from CWR22 into mice, growing tumors, injecting tissue from those “xenografts” into new mice, and repeating that passaging process until they could reliably grow large enough xenografts for study. In 1999, the same lab described a permanent BOSTON—A new finding presented at a The unusual life cycle of retroviruses cell line, 22Rv1, it had made from a CWR22 conference here last week throws cold water explains how such a recombination could xenograft. Because it was one of very few on the impassioned debate about the link occur, as Pathak described. Retroviruses cell lines available to study prostate cancer, between a novel mouse retrovirus and pros- contain RNA that, in addition to coding for it was widely used. Pathak’s group tracked tate cancer and chronic fatigue syndrome in viral proteins, carries instructions to make down samples from different passages of humans. Yet few believe it will end the con- the enzyme reverse transcriptase. After a CWR22, different versions of both 22Rv1 troversy, which began in 2006. retrovirus infects a cell, reverse transcrip- and a second cell line made later from In an extensive sleuthing expedition that tase converts the viral RNA into DNA, CWR22. Before 1996, no CWR22 samples looked back nearly 20 years, two collaborat- which is necessary for the virus to inte- contained XMRV DNA. Pathak’s lab found that some of the early ing research teams contend that they have grate with the host chromosomes. This is evidence that xenotropic murine leukemia the stage in which recombination between samples of xenografts did have a stretch of virus–related virus (XMRV) resulted from retroviral DNA from different genomes DNA that was nearly identical to about half of the XMRV genome. A group led by John the chance recombination of pieces of two can happen. mouse viruses in lab experiments and that Pathak explained how skepticism has Cofﬁn, who works at both NCI and Tufts the connections to human disease are spu- steadily built about the link between XMRV University here, made a similar discovery rious. “That nails it,” said retrovirologist and these diseases as several labs examined with different samples of xenografts. When the teams compared notes, they saw that the Nathaniel Landau of New York University. two sequences perfectly overlapped to form “Everyone working on this thing has this “It’s all contamination.” XMRV. “It was an amazing moment, the virus contaminating their stuff. It’s been —JOHN COFFIN, kind that happens once or twice in a career,” a tremendous waste of time and money.” TUFTS UNIVERSITY Coffin says. “It was like seeing a puzzle But even if XMRV is not a threat to human come together.” health, the fact that a retrovirus As Pathak emphasized in that can readily infect human his talk, the DNA sequences in cells was apparently generated what they dubbed preXMRV-1 by chance in the lab raises some and preXMRV-2 are nearly ideninteresting and potentially troutical to the XMRV sequences bling issues. reportedly found in humans but Vinay Pathak, a retrovirolosuspected to be a lab contamigist who works at the HIV Drug nant by some groups. Pathak’s Resistance Program run by the and Coffin’s teams both also U.S. National Cancer Instifound preXMRVs in some mice tute (NCI) in Frederick, Marystrains used in the experiments. land, presented the new data at But XMRV itself cannot infect the 18th Conference on Retro mouse cells, which means the viruses and Opportunistic InfecpreXMRVs could have recomtions, which focuses mainly on bined only after the mice received another retrovirus, HIV. (For prostate tumor transplants that additional coverage of the meetcontained human cells. Speciﬁing, see pages 1248 and 1249.) cally, RNA from both preXMRV The fact that the XMRV work garnered so much attention Confusing picture. New data say a lab accidentally created XMRV, shown here genomes must have been packhere reflects the high stakes. in an electron micrograph, and furthers doubt about its links to human disease. aged in a newly formed viral particle, or virion. When that virion The possibility that XMRV causes human disease has raised both patient samples and could not ﬁnd the virus infected a human cell derived from the hope and fear among patients and pub- or antibodies to it (Science, 17 September prostate tumor, the reverse transcriplic health ofﬁcials (Science, 2 July 2010, 2010, p. 1454). One 2009 study particularly tase enzyme accidentally mashed up the p. 18). For people who have prostate cancer piqued Pathak’s interest, as it showed how preXMRVs and created XMRV. “It’s a very or the bafﬂing chronic fatigue syndrome, a human prostate cancer cell line produced elegant study,” says phylogeneticist Stéphane Hué of University College London. “This is XMRV offered not only an explanation but high levels of the virus. The cell line was established at Case the birth date of the virus.” also a treatment: The virus is susceptible to Hammering the nail in further, Oya some anti-HIV drugs. Blood banks, on the Western Reserve University in Cleveland, other hand, have worried mightily that, as Ohio, from a human tumor called CWR22. Cingöz in Cofﬁn’s lab looked for XMRV in happened when the AIDS epidemic began, Prostate cancer tumors are difﬁcult to grow dozens of inbred and wild mice and reported they were unwittingly helping to spread a in lab experiments, but in 1993, research- that she found no evidence that the virus naters there reported that they had success by urally exists. dangerous retrovirus.

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CREDIT: ILA R. SINGH/UNIVERSITY OF UTAH

More Negative Data for Link Between Mouse Virus and Human Disease

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–JON COHEN

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Mars Sample Return

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Pricey. Collecting martian rocks for eventual return to Earth is the top large-mission recommendation, but the committee insists that the estimated cost of $3.5 billion be brought down.

P L A N E TA R Y S C I E N C E

Price Tags for Planet Missions Force NASA to Lower Its Sights HOUSTON, TEXAS—Scientists choosing the missions they want to send across the solar system in the next decade knew their recommended program wouldn’t come cheap. But “sticker shock hit us,” says Steven Squyres, chair of the U.S. National Research Council committee that has drawn up the Planetary Science Decadal Survey. For the ﬁrst time in a planetary science decadal survey, outside consultants estimated mission costs, and the process has produced “huge numbers” for the largest proposed missions, Squyres says. Those numbers have forced some painful and unprecedented recommendations in the committee’s report, which was released here Monday evening at the annual Lunar and Planetary Science Conference. The most expensive recommended missions—the ﬁrst step in returning samples from Mars and a close look at Jupiter’s moon Europa, which may bear life—must be scaled back if they are going to ﬂy. And that would only ﬁt the survey’s recommended missions into federal budget expectations last year, when the report was ﬁnalized. Then, NASA was assuming its planetary science budget would rise over the next decade. This year, planetary scientists are looking at a declining NASA budget for their ﬁeld. Cost loomed large for the committee from the start. The ﬁeld has long struggled with the “problem of ﬁrsts.” Soon after the ﬁrst quick ﬂyby of a planetary body like Mars, it’s on to the ﬁrst orbiting mission and then

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the ﬁrst lander. Firsts are the challenges that attract money from Congress and capture the taxpayers’ imaginations. But the next ﬁrst is always more complicated than the last. And greater complexity always means more expense. Consultants at Aerospace Corp. headquartered in El Segundo, California, produced a plot of past missions’ complexity versus cost of that mission, included in the survey. The plot dramatically shows how mission cost goes up exponentially with increasing complexity. It also shows there’s a price to pay for underestimating a mission’s appropriate cost. Failed missions always received less funding than successful missions of comparable complexity. Planetary scientists have not always done well anticipating the soaring costs that accompany rising complexity. Most infamously, the previous planetary science decadal survey—prepared without independent cost estimates—put the Mars Science Laboratory rover (now called Curiosity) in the medium, under-$650-million category. Now, several technical problems later and more ambitious than originally planned, Curiosity clocks in at a cost of $2.3 billion. “People who advocate missions are optimists,” Squyres says. But with Aerospace Corp. conducting independent cost estimates, he says, “the whole [survey] process avoids the optimism inherent in the cost-estimation process.” With more realistic prices in hand, the survey committee weighed in with four rec-

www.sciencemag.org

CREDIT: NASA

Cofﬁn believed earlier that studies linking XMRV to human disease deserved serious attention. He co-authored an article in the 23 October 2009 issue of Science, which included the ﬁrst report of XMRV in patients who had chronic fatigue syndrome. Led by Vincent Lombardi of the Whittemore Peterson Institute in Reno, Nevada, and NCI’s Francis Ruscetti, the study provided more evidence that, as Cofﬁn’s piece stated, “transmission happened in the outside world and was not a laboratory contaminant.” Now, Cofﬁn has changed his thinking. “It’s all contamination,” he says. At this point, Cofﬁn questions whether any human has been infected with the virus. “It remains a distant possibility,” he says. Hué, who works with Greg Towers in London, presented complementary 22Rv1 data at the conference that they published 20 December 2010 in Retrovirology (Science, 7 January, p. 17). They showed that XMRVs isolated from different 22Rv1 cell lines were more genetically diverse than sequences reportedly found in chronic fatigue and prostate cancer patients. If XMRV infected humans, copied itself, and spread to others, Hué says he would expect to see more diversity in the patients as it evolved to escape immune defenses. “I don’t think XMRV is a human pathogen,” Hué says. “It’s as simple as that.” Like Coffin, he doubts that XMRV has even infected a human but adds that “one can never say that something doesn’t exist.” “The evidence coming out at this meeting is incredibly impressive, and the weight of evidence is indicating that this is not a major human virus in terms of pathogenesis,” says Michael Busch, who heads the Blood Systems Research Institute in San Francisco, California, and is part of a working group convened by the U.S. Department of Health and Human Services to examine whether XMRV poses a threat to the country’s blood supply. But Busch said that before he concludes XMRV is simply a contaminant, he wants to see the results of studies they are coordinating between several labs with samples from agreed-upon patient and negative controls, as well as blood donors. If most of these fail to ﬁnd the virus, Busch says, “it’s going to eliminate concerns” that XMRV has caused these diseases. Even if XMRV has not harmed humans, Busch says we got lucky. This is the ﬁrst accidental generation of a retrovirus that can infect human cells. “It’s a warning shot,” Busch says. “We’ve created a highly infectious virus that may transmit to humans.”

NEWS&ANALYSIS

–JON COHEN

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Mars Sample Return

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Pricey. Collecting martian rocks for eventual return to Earth is the top large-mission recommendation, but the committee insists that the estimated cost of $3.5 billion be brought down.

P L A N E TA R Y S C I E N C E

Price Tags for Planet Missions Force NASA to Lower Its Sights HOUSTON, TEXAS—Scientists choosing the missions they want to send across the solar system in the next decade knew their recommended program wouldn’t come cheap. But “sticker shock hit us,” says Steven Squyres, chair of the U.S. National Research Council committee that has drawn up the Planetary Science Decadal Survey. For the ﬁrst time in a planetary science decadal survey, outside consultants estimated mission costs, and the process has produced “huge numbers” for the largest proposed missions, Squyres says. Those numbers have forced some painful and unprecedented recommendations in the committee’s report, which was released here Monday evening at the annual Lunar and Planetary Science Conference. The most expensive recommended missions—the ﬁrst step in returning samples from Mars and a close look at Jupiter’s moon Europa, which may bear life—must be scaled back if they are going to ﬂy. And that would only ﬁt the survey’s recommended missions into federal budget expectations last year, when the report was ﬁnalized. Then, NASA was assuming its planetary science budget would rise over the next decade. This year, planetary scientists are looking at a declining NASA budget for their ﬁeld. Cost loomed large for the committee from the start. The ﬁeld has long struggled with the “problem of ﬁrsts.” Soon after the ﬁrst quick ﬂyby of a planetary body like Mars, it’s on to the ﬁrst orbiting mission and then

11 MARCH 2011

VOL 331

SCIENCE

Published by AAAS

the ﬁrst lander. Firsts are the challenges that attract money from Congress and capture the taxpayers’ imaginations. But the next ﬁrst is always more complicated than the last. And greater complexity always means more expense. Consultants at Aerospace Corp. headquartered in El Segundo, California, produced a plot of past missions’ complexity versus cost of that mission, included in the survey. The plot dramatically shows how mission cost goes up exponentially with increasing complexity. It also shows there’s a price to pay for underestimating a mission’s appropriate cost. Failed missions always received less funding than successful missions of comparable complexity. Planetary scientists have not always done well anticipating the soaring costs that accompany rising complexity. Most infamously, the previous planetary science decadal survey—prepared without independent cost estimates—put the Mars Science Laboratory rover (now called Curiosity) in the medium, under-$650-million category. Now, several technical problems later and more ambitious than originally planned, Curiosity clocks in at a cost of $2.3 billion. “People who advocate missions are optimists,” Squyres says. But with Aerospace Corp. conducting independent cost estimates, he says, “the whole [survey] process avoids the optimism inherent in the cost-estimation process.” With more realistic prices in hand, the survey committee weighed in with four rec-

www.sciencemag.org

CREDIT: NASA

Cofﬁn believed earlier that studies linking XMRV to human disease deserved serious attention. He co-authored an article in the 23 October 2009 issue of Science, which included the ﬁrst report of XMRV in patients who had chronic fatigue syndrome. Led by Vincent Lombardi of the Whittemore Peterson Institute in Reno, Nevada, and NCI’s Francis Ruscetti, the study provided more evidence that, as Cofﬁn’s piece stated, “transmission happened in the outside world and was not a laboratory contaminant.” Now, Cofﬁn has changed his thinking. “It’s all contamination,” he says. At this point, Cofﬁn questions whether any human has been infected with the virus. “It remains a distant possibility,” he says. Hué, who works with Greg Towers in London, presented complementary 22Rv1 data at the conference that they published 20 December 2010 in Retrovirology (Science, 7 January, p. 17). They showed that XMRVs isolated from different 22Rv1 cell lines were more genetically diverse than sequences reportedly found in chronic fatigue and prostate cancer patients. If XMRV infected humans, copied itself, and spread to others, Hué says he would expect to see more diversity in the patients as it evolved to escape immune defenses. “I don’t think XMRV is a human pathogen,” Hué says. “It’s as simple as that.” Like Coffin, he doubts that XMRV has even infected a human but adds that “one can never say that something doesn’t exist.” “The evidence coming out at this meeting is incredibly impressive, and the weight of evidence is indicating that this is not a major human virus in terms of pathogenesis,” says Michael Busch, who heads the Blood Systems Research Institute in San Francisco, California, and is part of a working group convened by the U.S. Department of Health and Human Services to examine whether XMRV poses a threat to the country’s blood supply. But Busch said that before he concludes XMRV is simply a contaminant, he wants to see the results of studies they are coordinating between several labs with samples from agreed-upon patient and negative controls, as well as blood donors. If most of these fail to ﬁnd the virus, Busch says, “it’s going to eliminate concerns” that XMRV has caused these diseases. Even if XMRV has not harmed humans, Busch says we got lucky. This is the ﬁrst accidental generation of a retrovirus that can infect human cells. “It’s a warning shot,” Busch says. “We’ve created a highly infectious virus that may transmit to humans.”

NEWS&ANALYSIS Jupiter Europa Orbiter

Bringing it home. Robotically returning special rocks from the moon is a contender among $1 billion missions.

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Moonrise

CREDITS (LEFT TO RIGHT): JPL/NASA; NASA/ESA

Out there. A mission to Europa (foreground) could ﬂy only if its cost came down and NASA’s budget went up.

ommendations in the large or ﬂagship category, but each comes with a cost caveat. The committee’s highest priority for 2013 to 2022 is the Mars Astrobiology Explorer-Cacher (MAX-C), at an estimated cost of $3.5 billion in ﬁscal year 2015 dollars. (Advocates proposing the mission to the committee had pegged the cost at $2.2 billion.) The MAX-C rover would search for signs of life, past or present, and collect likely looking sedimentary rocks that could someday be lifted to orbit by the rocket of a second mission and carried to Earth in a third mission. But $3.5 billion was more than the committee could swallow. At that price, “MAX-C would take up a disproportionate share of NASA’s planetary budget,” the report says. So the mission should be ﬂown in the next decade only if the cost to NASA can be cut to about $2.5 billion. (The European Space Agency will share the costs of a two-rover mission.) “We think that’s doable,” Squyres says. If not, “the mission … should be deferred until a subsequent decade or cancelled,” according to the report. That’s not language often seen in decadal surveys. The survey’s second highest priority among flagship missions—also liferelated—is the Jupiter Europa Orbiter (JEO). From a tight orbit, JEO would get a long, close look at the ice-covered moon thought to harbor a deep ocean with “one of the most promising extraterrestrial habitable environments in our solar system,” according to the report. But at a projected cost of $4.7 billion, “it should ﬂy in the decade 2013-2022 only if changes to both the mission and the NASA planetary budget make it affordable.” In other words, the mission needs to shrink and the NASA planetary

budget needs to grow until other missions would be unaffected. The third large-mission priority is a dark horse, the Uranus Orbiter and Probe mission. The ice giant planets Uranus and Neptune are “the one class of planet we know least about,” Squyres says. Again, the committee warns that if costs grow signiﬁcantly above the projected cost of $2.7 billion, the mission should be downscaled or canceled. The ﬁnal recommendation is an either-or: a mission to study the climate of Venus pegged at $2.4 billion, or a spacecraft to orbit Saturn’s little moon Enceladus with its watery jets at $1.9 billion. Among proposed medium-size missions, the committee found a better match between ambition and budget. Its only recommendation on cost for these so-called New Frontiers Program missions was to keep their prices capped at $1 billion each but to start excluding the cost of the rocket from a mission’s budget. The committee did diverge from tradition when it declined to rank New Frontiers missions. It recommended that NASA— guided by peer review of missions—select two from a list of seven the committee whittled down from more than 20 proposed by planetary scientists. The seven selected missions span the solar system and, not incidentally, all the subgroups of the planetary community. The seven missions would return a sample from an icy comet, probe gas giant Saturn, explore the surface of ﬁery Venus, return a sample from a huge impact basin on the moon, visit asteroids orbiting the sun with Jupiter, repeatedly ﬂy by Jupiter’s radiation-soaked volcanic moon Io, and plant a network of geophysical observatories on the moon. “They’re all splendid concepts,” Squyres says.

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Here at the meeting, 1300 planetary scientists hearing Squyres present the report mostly commended him and the legion of committee members, panel members, contributors, and reviewers on the ﬁne document they had produced. Listeners also gasped at the costs attached to their hoped-for missions. And they knew the budget cutting going on in Washington would only make the outlook even worse. James Green, director of the planetary science division at NASA headquarters, showed his version of a budget graph from the report. The original has a “wedge” of funding for decadal missions that grows rapidly during the next 10 years under last year’s steadily rising version of the total planetary budget. Green had drawn a lower line representing the president’s 2012 budget request that he must use for planning. It cut the decadal funding wedge about in half. Squyres had two responses. The decadal survey “has decision rules to fit whatever budget wedge comes NASA’s way,” he told Science earlier. First the rules specify downscaling, delaying, or canceling large or “ﬂagship” missions, starting with the top priority, Mars sample return, and going down to the least expensive, Enceladus Orbiter. “If you can’t afford any of those, you’ve got no ﬂagship at all,” he said. A decade without a ﬂagship mission is quite possible, he later added. But Squyres, as a planetary scientist, had another response. The president’s budget is not carved in stone beyond 2012, he noted. If the community gets behind the decadal survey and takes their concerns to their representatives and senators, perhaps planetary science won’t have to live with a declining budget. Then perhaps the dire economic times will not mean the end of the next decade’s big ﬁrsts.

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–RICHARD A. KERR

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NEWSFOCUS

A military data set of civilian casualties, provided exclusively to Science, indicates that the war has become more lethal to the Afghan population, largely because of indiscriminate insurgent attacks KANDAHAR, AFGHANISTAN—By day, you don’t see the war. The wind throws up a red haze that obscures everything. Bearded Afghan men in colorful vests whip their donkeys, lugging vegetables and ﬁrewood. Unless you spot an armored convoy picking its way across the rugged landscape or hear a ﬁghter jet scream overhead, you couldn’t even guess the century. But once night falls, a brilliant cluster of lights appears, as if a new city has sprouted in the valley several kilometers south of the provincial capital. This is Kandahar Airﬁeld. What used to be a derelict airport littered with debris from the Soviet era has grown into a massive base for the International Security Assistance Force (ISAF).

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Inside its outer maze of concrete walls and machine gun nests is a world of surreal contrasts. Thousands of ISAF soldiers clog the streets, some hurrying to dinner at the end of a grueling workday, others just starting their night shifts. The conversations are in English, French, German, Dutch, and other languages from the 48 nations that have contributed troops to the ISAF coalition. There is no safety beyond the walls, so all recreation must happen here. The Americans brought in a string of restaurants, including T.G.I. Friday’s. The Canadians built a full-sized, openair hockey rink—the ﬂoor is concrete rather than ice—and they play in full gear under the glare of stadium lighting. Tonight, men and women in uniform amble along a wooden

11 MARCH 2011

promenade, licking ice cream cones and watching a volleyball game. The spell is broken as sirens blare and everyone dives for the ground. Once per day on average, insurgents manage to send small rockets ﬂying over the walls. The Canadians in the hockey rink famously play through these attacks, each team refusing to ﬂinch. But newcomers on the base can’t help but ﬂinch constantly—every jet overhead sounds like an incoming rocket. It turns out to be a drill this time. But an hour later, a dozen missiles erupt from the base’s launchers, breaking the speed of sound in seconds and fading over the horizon like cigarette embers. Somewhere to the west people are dying. What began as an invasion to capture Osama bin Laden in 2001 has become a fullblown occupation and counterinsurgency. As the war in Afghanistan grinds toward the decade mark—the longest in U.S. history— public support is waning. When he testiﬁes

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CREDIT: AFP/GETTY IMAGES

Counting the Dead in Afghanistan

Dangerous protectors. As the conﬂict in Afghani-

stan drags on, civilian casualties are increasing.

before the U.S. Congress next week, ISAF Commander Gen. David Petraeus will face some difﬁcult questions. Most contentious is the issue of civilian casualties. Media reports of surging violence give the impression of a country slipping out of control, but ISAF has reported steady progress. “There is so much rhetoric ﬂying around and none of it can be tested,” says Neil Johnson, a physicist at the University of Miami in Florida who studies the dynamics of warfare. “What we need is hard, reliable data.” A deluge of data arrived last year from WikiLeaks, the organization that has made public thousands of classiﬁed documents. It includes the raw observation of casualties by soldiers on the ground in Afghanistan, but the many unknowns surrounding those reports have left researchers puzzled about how to interpret them. A few independent organizations, including the United Nations, have published their own reports on civilian casualties in Afghanistan, but only for illustrating broad trends. The data underlying their reports have never been released. For the ﬁrst time, those data are now publicly available. In January, ISAF provided Science with a database of civilian casualties called CIVCAS. It is the military’s internal record of the death and injury of Afghan civilians, broken down by month, region, weaponry, and perpetrator. By its reckoning, 2537 civilians were killed and 5594 were wounded over the past 2 years, with 12% of those casualties attributed to ISAF forces and the rest to insurgents. The death toll is 93% identical to that in the WikiLeaks data, revealing those raw ﬁeld observations to be far more reliable than researchers had suspected. In February, after learning that the military was releasing these data, both the UN and an Afghan human rights organization agreed to release versions of their own civilian casualty data to Science. They show twice as many civilians killed over the same period, including 393 deaths by air strikes that were not counted in the military database. ISAF ofﬁcials acknowledge the gap. “The civilian casualties reported by the UN have always been higher than those reported by ISAF,” says U.S. Navy Rear Adm. Gregory Smith, the director of communications for NATO based in Kabul. “But the trends have been very consistent.” Science assembled a team of experts to analyze the released data sets. They conclude that while the war has grown deadlier for Afghan civilians over the past 2 years—

up to 20% more civilians were killed in 2010 compared with the year before—ISAF has become a safer ﬁghting force. The majority of deaths, and nearly all of the recent increase, are attributed to indiscriminate attacks by insurgents rather than ISAF soldiers. In spite of a troop surge and the launch of new operations against the Taliban last year, the data provided by the UN show a 26% drop in civilian deaths caused by military forces. And both the UN and ISAF data sets show a drop in deaths due to air strikes last year, by 50% and 10%, respectively. All of these data, as well as other information never before released, are now online at http://scim.ag/afghandata. Taken together, they provide the clearest picture yet of the human cost of the war.

South Vietnamese allies. “We were told that we were over there only as advisers,” he says. Even the ofﬁcial number of troops deployed to Vietnam looked fishy. “We were being manipulated by the government through the media,” says Sutherland, now a statistician at the University of Massachusetts, Amherst. Forty years later, with wars raging in Iraq and Afghanistan, the military has become far more cautious with casualty statistics. The new approach was described by U.S. Army Gen. Tommy Franks. “We don’t do body counts,” he told reporters in Afghanistan in 2002. Although the deaths of coalition soldiers in Iraq and Afghanistan are available— for example, at www.icasualties.org—an “information vacuum” has surrounded civilian casualties, says Michael Spagat, an economist at Royal Holloway, University of LonCounting bodies don. So researchers trying to measure the Millions of people have died in modern wars, human cost of the wars have had to turn to but the exact number is anyone’s guess. His- other data sources. torians estimate that at least 10 million peoOne source is the media. The Iraq Body ple were killed in World War I and at least Count (IBC) Web site has tallied Iraqi civil50 million in World War II. Although records ian casualties—over 100,000 and counting— exist for the number of soldiers who went to from media reports since the U.S.-led invawar and never came home, most sion in March 2003. However, civilian deaths went uncounted. this method can provide only a The military did not systemlower limit to the true number. sciencemag.org atically track casualties beyond Another source is the civilOfﬁcial data their own troops. ian population itself. Several sets, resources That changed in the late research teams have used housefor exploring the infor1960s, when Americans opened hold surveys to estimate casualmation, and podcast their newspapers to ﬁnd an ofﬁties in Iraq. A 2006 survey pubinterview with John Bohannon at http://scim. lished in The Lancet claiming a cial count of the people dying in ag/afghanspecial. the Vietnam War. Each week, the civilian death toll of 600,000 has U.S. military released those numbeen widely criticized (Science, 6 bers to the media, dividing the weekly casu- March 2009, p. 1278). A larger survey in Iraq alties between U.S. soldiers, U.S. allies, and led by the World Health Organization came to “Communists killed.” One of the readers was a ﬁgure close to 150,000 for the same period. Michael Sutherland, then a statistics Ph.D. But in Afghanistan, these methods may student at Harvard University. “The military be impossible. The country’s size and popuhad a goal of achieving a 10-to-1 kill ratio,” lation are close to those of Iraq—both have he says. “The idea was that if we were kill- 30 million people in an area comparable to the ing 10 of their guys for every one of ours, we size of France—but that is where the similariwere winning.” Sutherland, who was eligible ties end. Whereas Iraq is a ﬂat country with a for the draft and had friends already serving well-educated and mostly urban population, in Vietnam, started collecting and analyzing Afghanistan is a nightmare for ﬁeld research. those weekly numbers. Most Afghans live in small villages nestled The kill ratio was indeed approaching within rugged and poorly connected river 10-to-1, but he discovered other patterns. valleys. Their inaccessibility thwarts surIt was clear that at least some of the data veys, and a lack of journalists on the ground were fabricated. The frequency of the num- makes rigorous media-based casualty counts bers’ last digits was skewed, with far too few “extremely difﬁcult,” says John Sloboda, the zeros and ﬁves compared with chance. The director of IBC based in London. reason? “If you’re making up numbers, you These difﬁculties have not stopped some never say that 150 Communists were killed,” organizations from publishing estimates of Sutherland says. “Instead, you use 147 or the death toll in Afghanistan. The Red Cross 152.” By comparing the trends in deaths, he has monitored the flow of casualties into also found that U.S. soldiers were clearly hospitals, for example, and the UN has colthe ones ﬁghting the battles rather than the lected statistics through its regional ofﬁces.

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THE WAR IN AFGHANISTAN | NEWSFOCUS

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From WikiLeaks to CIVCAS release Starting in July 2010, the largest leak of secret military information in history went public. WikiLeaks gave media outlets 92,000 internal military documents related to the war in Afghanistan and 400,000 from Iraq, allegedly provided by a low-ranking U.S. soldier who is now in custody. The documents include the raw operational reports from troops on the ground between January 2004 and December 2009. In Iraq over that period, soldiers reported a total of 79,000 civilian deaths, 15,000 of which the media missed (Science, 29 October 2010, p. 575). Afghanistan has been spared Iraq’s sectarian violence. Over the same 6-year period, the leaked documents note 4024 Afghan civilian deaths. The information vacuum was breached, but researchers have been wary of using the data. The military’s raw operational reporting was not intended for research, so any errors, biases, and inconsistencies they might contain are unknown. Did investigations conﬁrm those casualties? How many bodies identiﬁed as “combatants” later turned out to be civilians, and how many of the wounded subsequently died from their injuries? Without those answers, the data do little to dispel the fog of uncertainty. Late last summer, a confidential source within ISAF informed me that the military was curating a database of civilian casualties. He described a dedicated military team that investigates civilian casualties and analyzes trends in the ﬁnal tally to help ISAF reduce the number. In a series of e-mail exchanges with Science, ISAF ofﬁcials conﬁrmed that such a tracking system does exist and that its output is an internal database of civilian casualties called CIVCAS. In October 2010, ISAF hosted me in Kabul and Kandahar as an embedded reporter. I was given access to military personnel at every level of the civilian casualty–tracking system, from the collection and quality-checking of CIVCAS data to the analysis that leads to new combat directives. I was also able to tour medical facilities and interview medical personnel

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(see p. 1261). What I was not allowed to do was take the data with me. ISAF ofﬁcials were concerned that sensitive information associated with civilian casualty data—such as the tactics and movements of troops—could be revealed. But after 3 months of negotiation, ISAF agreed to give the entire CIVCAS database to Science for public release. “Our database is 100% transparent,” says Smith. “Ultimately, this is a war being fought here in Afghanistan on behalf of the Afghan people.” Along with the death rate of soldiers, he says, this is “the most signiﬁcant data set in identifying whether or not you’re making progress.” In a series of conﬁdential meetings over the past year, ISAF has provided the UN and

“Ultimately, this is a war being fought … on behalf of the Afghan people.” The information compiled by ISAF is “the most signiﬁcant data set in identifying whether or not you’re making progress.” —REAR ADM. GREGORY SMITH, NATO

some human rights organizations full access to CIVCAS. “The other organizations will share their compiled data but not their raw data,” says Smith. “That’s really to protect their own access, freedom, and independence, which is certainly something we recognize and respect.” After learning that ISAF was releasing CIVCAS, some of those organizations provided Science with data as well. The United Nations Assistance Mission in Afghanistan (UNAMA) provided 3 years of their monthly casualty statistics, including a detailed accounting of deaths by air strikes since January 2009. The Kabul-based organization Afghanistan Rights Monitor (ARM) provided Science with the highest-resolution data of all, describing individual incidents during the ﬁrst half of 2010. “Providing these data is a great public service,” says Sutherland, who is undertaking the ﬁrst statistical analysis. Science is making all these available at http://scim.ag/afghandata. ISAF has released additional sets of information to help researchers analyze the casualty data, including precise monthly troop deployment numbers over the past 3 years. Perhaps the most important is

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a document describing the standard operating procedures of ISAF’s civilian casualty–tracking system. “This is crucial,” says Johnson. “It’s not enough to see the numbers; you need to know how they are collected.” From deaths to data Nestled deep inside Kandahar Airfield is an inner bastion, like the nucleus of a cell. Stacked white trailers have been converted into a village of temporary ofﬁces. At the center is the Combined Joint Operations Center. Dozens of ofﬁcers sit in an auditorium facing a wall of giant screens. Some show maps of the combat space, and others show live video feeds from unmanned drones. Since operations were launched last year to push the Taliban out of their original homeland around Kandahar, this has become the nerve center of the war. “We have a casualty,” says an Australian ofﬁcer sitting in the top row. (She requested that her name not be used because she is not an authorized ISAF spokesperson.) On her computer is a window of spooling text called JChat—it looks like an Internet chat session—providing a real-time view of the events of the war. One line of text is red, indicating a request for medical evacuation. It is one of many in this region, with about a dozen evacuations requested per day. “It’s an Afghan girl wounded by shrapnel,” she says, deciphering the string of acronyms in JChat. The ofﬁcer is not concerned about who caused the injury. Her job is to save the girl’s life by identifying the most efficient path to a doctor. She plots the grid references on a map, and a series of overlapping circles appear around the nearest helicopters. The path is computed and the orders are given. Help is on the way. Meanwhile, a parallel stream of data has already started to ﬂow. “This is the start of a process that we call Consequence Management,” says Col. Martin Bricknell, a senior U.K. military doctor and the medical director for southern Afghanistan. The soldiers on the ground with the injured girl have already radioed in a First Impression Report, describing the deaths and injuries that occurred. Once they return to base, they will give their commander a more detailed account. Within 9 days, ISAF Headquarters is expecting a CIVCAS assessment report. Besides reviewing the known facts of the case, the purpose is to improve future operations, Bricknell says. “Is there anything that we can learn from that so we can reduce casualties next time?” At that point, the ISAF leadership will decide whether to investigate further. “We are absolutely determined to [track] the con-

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Although these organizations have published estimates periodically, none has given researchers access to their data to make an independent assessment. Of course, the organization in the best position to directly record civilian casualties is the military itself, with nearly 150,000 observers on the ground witnessing the violence every day. But it seemed that the military kept no record of those observations— that is, until last year when WikiLeaks showed otherwise.

THE WAR IN AFGHANISTAN | NEWSFOCUS Civilian Casualties in Afghanistan 2009 – 10

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Regional Command (Southwest)

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Direct Fire Indirect Fire

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Improvised Explosive Device Complex

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Wounded

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Casualties in time and space. The seasonal rhythms and shifting battleﬁelds of the war emerge in this view of the 8131 Afghan civilians killed or injured over the past 2 years, recorded in a military database called CIVCAS. (No data were available for the ﬁrst 5 months of 2010 in the Southwest region.)

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Road Traffic Accidents JUL

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CREDIT: GEORGE MICHAEL BROWER

MAR FEB 2009

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Kabul Kandahar

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sequences of the conﬂict to the civilian population,” Bricknell says. “Therefore we take every allegation of a civilian casualty very seriously.” If local government ofﬁcials give a different account of the event, for example, an incident assessment team will be assembled and sent into the ﬁeld. For the most serious incidents, ofﬁcials from the UN or other organizations join the team. The CIVCAS database tracks all of these deaths and injuries. Between January 2009 and December 2010, it logged a total of 2537 civilians killed and 5594 wounded. About 80% of the deaths and injuries are attributed to insurgents. (The CIVCAS data go back to January 2008, but insurgent-caused casualties were not tracked until 2009.) Throughout the war, critics have accused ISAF of undercounting civilian casualties, particularly those caused by their own soldiers. Just last month, a battle in Kunar province on Afghanistan’s eastern border with Pakistan generated conﬂicting accounts. According to villagers, ISAF killed 65 civilians, including 50 women and children. According to ISAF, only insurgents were killed. The data provided by UNAMA do show far more casualties than those from ISAF. For 2009 and 2010, its data include 5191 civilian deaths, over 70% of them caused by “antigovernment elements,” 20% by “pro-government forces,” and the rest undetermined. Compared with CIVCAS, they attribute nearly three times the number of civilian deaths caused by military forces, only a small portion of which are Afghan national rather than ISAF forces. One of the most significant discrepancies comes from the 529 civilians that UNAMA claims were killed by “air attacks” in 2009 and 2010. CIVCAS shows only 136 civilians killed by jets and helicopters over that period. Asked whether casualty reporting by soldiers might be biased, British Army Lt. Col. George Wilson, who oversees ISAF Consequence Management, demurs. “No, I think the converse,” he says. “I genuinely think we get honest reporting from the ground.” According to Wilson and other ISAF sources, the cause of the disparity in body counts is methodology. “Raw numbers will never be the same, and there’s a good reason for that,” Smith says. “We do not have a presence in all 34 provinces,” he says, and therefore CIVCAS does not track all casualties. “We only count that which we see.” In some cases, such as alleged casualties from air strikes, “we can do a tremendous amount of forensics, … [but] seldom do we see the actual bodies. Sometimes we have access to someone who was wounded, but not always,” he says. “You can

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do overflights of cemeteries to determine any fresh grave sites and conﬁrm or deny the numbers involved.” He adds that other organizations trying to track civilian casualties face the same challenges. Smith does not question the accuracy of UNAMA’s body count. “The UN has a much broader mandate” to track civilian casualties, he says, “and the resources to do that.” Rather than creating a deﬁnitive record for history, the purpose of CIVCAS, he says, is “marking progress.” A safer force Over the past month, Sutherland, Spagat, Johnson, and other experts have analyzed CIVCAS and the other civilian casualty data sets at Science’s request. We have also built a timeline of the past 2 years of the war that compares those data with the casualties reported in the media, available at http:// scim.ag/afghandata.

“Counting seems like such a simple thing, but it is the only way to see the actual effect of the war.” —MICHAEL SUTHERLAND, UNIVERSITY OF MASSACHUSETTS, AMHERST

By all accounts, the war has grown deadlier for Afghan civilians. The CIVCAS data show a 19% increase in the total number of civilians killed in 2010 compared with the year before, and the UN data show a 15% jump. But at the same time, there are signs that ISAF has become a safer ﬁghting force, treading more lightly on local populations. Although the overall death toll in Afghanistan has risen, the increase was not wrought by soldiers. Over 90% of last year’s spike in CIVCAS, and the entirety of that in the UN data, is attributed to insurgents. IED explosions have continued to cause the majority of civilian deaths. By contrast, 2010 saw a 26% drop in the number of civilians killed by soldiers in the UN data. In their own data, ISAF admitted to killing 12% more civilians compared with the year before, while wounding 20% fewer. This happened in the context of the largest military offensive in years and a surge that doubled the number of troops in Afghanistan to 140,000 last year. The lack of a corresponding spike in military-caused casualties is surprising. This is especially true of air strikes, by far the most dangerous military activity for civilians. In the data provided by the UN, air strikes killed 171 civilians in 2010, half as

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many as in 2009. CIVCAS, which separately deﬁnes casualties caused by jets and helicopters, shows 11% fewer deaths. There are also subtler signs of progress in the data. In April last year, ISAF units received a new directive concerning “escalation of force”: the shooting of civilians due to communication breakdowns. Most of these incidents occur at the hundreds of checkpoints that ISAF operates across the country. In the course of analyzing their casualty data, ISAF commanders noticed a trend. “What became very clear to me is that all the [civilian] fatalities occurred between the 100-meter point and the 0-meter point” approaching a checkpoint, Wilson says. Beyond that range, a ﬂare usually sufﬁces to warn drivers to slow down. But if a vehicle has not slowed down yet, Wilson says, “this is where as a soldier you suddenly think, ‘I’m about to die because this vehicle is going to drive in here and detonate.’ ” The problem, he says, is that soldiers in that situation had no nonlethal options. The new directive gave soldiers more options for warning drivers at a distance, Wilson says. These include laser dazzlers, paint ball guns, and even chalk bullets. “If you ﬁre them at a vehicle,” he says, “they will ping off and make such a loud noise that, if they’re a genuinely innocent person, they’ll get the message.” The CIVCAS data put numbers to that narrative. Deaths due to escalation of force dropped by 50% in the 8 months after the April 2010 directive went out, compared with the same 8 months in 2009. While they applaud the release of these data sets, researchers are grappling with their limitations. “One problem is that organizations are all using different deﬁnitions in their data,” Johnson says. For example, whereas the UN data have separate categories for casualties caused by “mortar and rocket ﬁre” and “shooting,” ISAF pools all ground-based battle casualties into “direct ﬁre” and “indirect ﬁre.” The different organizations also divide the country along slightly different lines for coding the location of casualties. “They should be trading notes,” Johnson says, “at least so their data can be easily compared.” Such limitations would be solved if all data for civilian casualties were released at the level of individual events rather than aggregated monthly. “Ultimately, that is the only way you can verify them,” says IBC’s Sloboda. The data provided to Science by ARM come closest to this level of resolution, but so far, they only cover the ﬁrst half of last year. “Counting seems like such a simple thing,” Sutherland says. “But it is the only way to see the actual effect of the war.”

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THE WAR IN AFGHANISTAN | NEWSFOCUS From soldier to subject. Researchers are collecting

War as a Laboratory For Trauma Research

CREDIT: AP

The military is sending scientists onto the battleﬁeld to ﬁnd ways to improve emergency medicine, but the research faces a practical and ethical mineﬁeld KANDAHAR AIRFIELD, AFGHANISTAN— Easing back into a borrowed chair in a tiny trailer office here on the base, Cmdr. Lisa Osborne should be enjoying a moment of calm. But all she can talk about is work. “I saw a guy show up here in trauma. You wouldn’t believe the condition he was in,” she says, recalling the victim of an improvised explosive device (IED). The extent of the injuries was shocking, even to the hardened medics working here in Afghanistan. “He was missing both legs and his whole backside. But he was alive, breathing on his own.” For most people, a scene like that would fill them with nothing but horror. But for Osborne, a U.S. Navy anesthesiologist and medical researcher, it was a reminder of hard-earned progress. “Years ago, that kind of patient would be dead,” she says. “No

question.” What saved him was a series of extremely rapid interventions, including new techniques for applying tourniquets, drugs, and blood products. Those insights came from medical research conducted in Iraq and Afghanistan, orchestrated through a U.S. military program called the Joint Combat Casualty Research Team (JC2RT). As its deputy director, Osborne runs the show. In many ways, war is the perfect laboratory for trauma medicine research. On any given day, dozens or even hundreds of casualties arrive by helicopter to military hospitals across Afghanistan. IEDs are the number one risk, often combining burns, deep lacerations from shrapnel, and brain trauma from blast waves. Injuries like these are too rare to study in peacetime. And because all the patients in these studies are military

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personnel, they come with exhaustive data relating to preinjury health and postinjury outcome. Many of the insights gained from battleﬁeld studies have found their way into civilian emergency medicine. But war is also the most chaotic and stressful environment imaginable for doing science. “Someone has to take down all this data,” Osborne says. “I saw this poor girl trying to turn this bloodsmeared page, trying to get data off of it. … That’s the reality. Until you’ve lived that, you don’t realize how difﬁcult it is.” Adding to the difﬁculty, controversy has dogged JC2RT projects, including charges by journalists that researchers rushed experimental treatments onto the battleﬁeld without proper ethical review or sufﬁcient safety testing, needlessly risking the lives of soldiers. Science investigated these issues with the help of two bioethicists and several sources from both civilian and military trauma medicine.

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data from casualties like this one in Afghanistan.

Exploring the golden hour From the moment a bullet or piece of shrapnel hits the body, the clock is ticking. Trauma medics call it the “golden hour,” the small window of time in which the patient’s life can be saved. Death can come in a mat-

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a single patient in Afghanistan show the 1-to-1 ratio of blood products now used.

Once a research project passes this prescreening, it must be approved by an independent U.S. Army institutional review board (IRB) based at Fort Detrick, Maryland. That review process is identical to those at any research institution, Osborne says, sourcing outside experts as needed. Osborne and her colleagues identify issues likely to cause problems with reviewers, giving researchers a chance to address them early “so that when [the proposal] goes to IRB it has a chance,” Osborne says. At least one of the investigators must be present to undertake the study in the ﬁeld. “Most of these folks, they’re only here for a short amount of time. If their stuff gets held up in IRB for 6 months, they miss the boat. We try to prevent that from happening. But it does happen.” To date, about 100 projects have made it through this gauntlet and into the ﬁeld, so far producing dozens of peer-reviewed research papers (see table on p. 1263). All of them take advantage of data from the U.S. military’s Joint Theater Trauma Registry (JTTR), a continuously updated record of trauma cases in Iraq and Afghanistan. JTTR currently contains the case histories of 40,000 patients, including medical observations, treatments, and outcomes in minute detail. Most of the research has yielded incremental improvements to existing treatments. But some have overturned paradigms of trauma care, says Osborne. One example is blood transfusion. Some battlefield injuries are so severe that patients require massive transfusions, sometimes as much as ﬁve times the volume of blood in the body. The standard transfusion practice begins with concentrated red blood cells in a saline solution, with the other blood

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Serious charges Wartime medical research has a troubled history. The most notorious examples are the experiments performed on prisoners by German and Japanese doctors during World War II. As a 2002 directive by the U.S. Department of Defense states, “The involvement of prisoners of war as human subjects of research is prohibited.” So even harmless experiments on detainees are forbidden by JC2RT, says Osborne. Nonetheless, its research projects have attracted their share of controversy.

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Whole blood. These empty transfusion bags from

components—plasma containing platelets and clotting factors—added sparingly later. “What we realized,” says Osborne, “is that if you wait on those clotting factors, then you’re always behind. You can keep putting in red blood cells and they just keep pouring right out [of the wounds].” The transfusion protocol for severe injuries has now been revamped based on JC2RT research. For injuries that require massive transfusions, the equivalent of whole blood is now given immediately, including a full complement of clotting factors. The researchers found that it reduced mortality rates in these patients from 65% to 17%. Another dramatic change from JC2RT research is how “damage control” surgery is performed. “We found that it’s critical that we not close some injuries when they come in here,” Osborne says, “because the outcomes are massive infections and sepsis.” Instead, blood vessels are tied off and the wound is left open, sometimes for several days, vigorously cleaning it with saline. “We used to close that wound 7 years ago,” she says. Other problems have yet to be solved. One of the most urgent is compartment syndrome, the accumulation of ﬂuid between tissue layers that can result in amputation or even death if the pressure cuts off blood to organs. One JC2RT project is testing whether the pressure in an injured limb can be diagnosed earlier by detecting a drop in oxygen in the tissue. Another unsolved problem explored by JC2RT projects is how to diagnose brain trauma, for example, using ultrasound to measure the blood pressure of vessels within the eye. Because of the streamlined process, insights from those projects will move quickly onto the battleﬁeld. In medical research, says Osborne, “you normally have a 10-year pipeline” between the ﬁrst experiment and a new treatment for patients. “For us, it can be half a year,” she says. The speedy turnaround has doubtless saved lives. But at the same time, it has invited intense scrutiny.

CREDITS: ISAF

ter of minutes, hours, or days, but the most crucial interventions must be made immediately. War has been “an amazing learning environment,” says Osborne, with each conﬂict pushing trauma medicine forward. New motorized ambulances in World War I saved wounded soldiers by getting them from the front lines to the hospitals quickly. World War II saw the ﬁrst large-scale use of antibiotics. Medics in the Korean War pioneered repair and grafting techniques for vascular surgery. In the Vietnam War, portable radiology equipment and ventilators were tested in the ﬁeld. With the wars in Afghanistan and Iraq, combat medicine has faced a new problem. “The difﬁculty for the current generation is getting approval for research,” says Col. Martin Bricknell, a senior doctor in the U.K. Royal Army Medical Corps who hosted this reporter’s visit to military facilities in Kandahar (see p. 1256). “It is considerably more rigorous than it used to be, and therefore the lag between good idea to outcomes is much greater.” JC2RT was created in 2006 to streamline that process. “We’re a team of eight,” says Osborne, a petite, blonde 42-year-old in the middle of her 6-month tour of duty in Afghanistan. When she is not at war, Osborne is the director of anesthesiology research at the Uniformed Services University of the Health Sciences in Bethesda, Maryland. “Most of us have Ph.D.s,” she says, “but we’re not data collectors.” Instead, JC2RT is a gatekeeper and overseer. Ideas for projects are put forward by researchers from all branches of the U.S. military, often with university-based scientiﬁc partners. As the proposals roll in, Osborne says, “I’m the one who has to give people the bad news that their research project isn’t going to work.” A typical problem is feasibility. “The people who are writing these [proposals] are just coming into theater,” Osborne says. “They don’t realize that there’s no way they’re going to be able to do it.” As an example, she describes a project that would have required infrared photography of injured soldiers’ limbs. “You often have a whole sea of people working on the patient. You can’t tell them, ‘Okay, everybody step back. I’m going to snap some photos.’ ” Another fatal ﬂaw is the use of experimental medical devices. “It’s a deal-breaker,” Osborne says, because of U.S. federal regulations. “If you use them, you must have informed consent from the patient. Period.” Most wounded soldiers plucked from the battleﬁeld are in no state to grant consent for a new device, even if they are conscious.

SOURCE: JC2RT

Trauma Research on the Battlefield Research topic

Summary

Example of output or project status

Damage control resuscitation

Methods for resuscitating patients with massive blood loss

M. A. Borgman et al., The ratio of blood products transfused affects mortality in patients receiving massive transfusions at a combat support hospital. J Trauma 63, 805 (2007).

Compartment syndrome

Using oxygen concentration and other markers to detect dangerous fluid buildup in injured limbs

Ongoing

Prehospital lifesaving interventions

Assessing the performance of immediate interventions in trauma care

Ongoing

Vascular surgery

Comparing the effectiveness of vascular injury treatment for wounded soldiers

S. M. Gifford et al., Effect of temporary shunting on extremity vascular injury: an outcome analysis from the Global War on Terror vascular injury initiative. J Vasc Surg 50, 549 (2009).

Traumatic brain injury

Biomarkers and quantitative EEG for detecting brain injury and characterizing immunodeficiency

Ongoing

Critical Care Air Transport Team (CCATT)

Analysis of resuscitation and early clinical outcomes as a function of aeromedical platform

M. D. Goodman et al., Traumatic brain injury and aeromedical evacuation: when is the brain fit to fly? J Surg Res 164, 286 (2010).

Combat medicine training

Assessing the effectiveness of medical training in the context of Iraq and Afghanistan

J. A. Tyler et al., Current US military operations and implications for military surgical training. J Am Coll Surg 211, 658 (2010).

Tourniquets

Optimizing the use of tourniquets for improving survival and reducing amputations

J. F. Kragh Jr. et al., Survival with emergency tourniquet use to stop bleeding in major limb trauma. Annals of Surgery 249, 1 (2009).

Soft tissue injury

Assessing the effectiveness of the vacuum assisted device and other innovative treatments for soft tissue damage

R. Fang et al., Feasibility of negative pressure wound therapy during intercontinental aeromedical evacuation of combat casualties. J Trauma 69, S140 (2010).

Explosive mass casualty

Developing guidelines for intensive care for multiple casualties caused by explosions

B. W. Propper et al., Surgical response to multiple casualty incidents following single explosive events. Annals of Surgery 250, 311 (2009).

A 2009 investigative report by The Baltimore Sun, which has been widely circulated among civilian and military doctors, alleged that researchers sidestepped standard ethical practices in Iraq and Afghanistan. “The military exposed hundreds of soldiers and Marines to the risks of unproven treatments that were unlikely to do much good,” the report claims. U.S. Air Force Lt. Col. Todd Rasmussen, deputy commander of the U.S. Army Institute of Surgical Research in San Antonio, Texas, who oversees JC2RT, declined to comment, saying only that the article reﬂected “vigorous but appropriate academic deliberations” within the medical research community. Science asked two bioethicists, Daniel Wikler of Harvard Medical School in Boston and Norman Fost of the University of Wis-

consin, Madison, to assess the issues raised by the article and, more generally, JC2RT’s procedures for ensuring that battlefield research is carried out ethically. Fost, who is the architect of the current standard ethical procedures for trauma medicine research, pulled in several other experts. “I talked at length with four experienced surgeons, three of whom specialize in trauma and have military experience, two of whom have served multiple tours in Iraq and/or Afghanistan,” he says. He also reviewed numerous articles in peer-reviewed journals and U.S. Food and Drug Administration documents. Both bioethicists posed questions about JC2RT research directly to Osborne through a series of e-mail exchanges. Fost’s conclusion: None of the examples of unethical research held up under scrutiny.

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But the criticism is not surprising. “There is scarcely a disease or treatment in the world without strong disagreement among knowledgeable people about the best approach,” Fost says. As for the charge of lax ethical review, Fost found the opposite to be true. “There was a continuous dedication to evaluating practices in a way that many civilian medical centers would envy,” he says. Beyond the standard IRB approval process, new treatments are evaluated in real time with weekly “morbidity and mortality conferences” that include military physicians from around the world. They are part of a “serious quality-improvement program,” Fost says, which is “beyond anything I’m familiar with in civilian medicine.” Wikler agrees that there is no clear evidence of wrongdoing but adds that the extra scrutiny is justiﬁed. “There is a long tradition of using soldiers as guinea pigs for research,” he says. “And trauma medicine research in general has the problem of a lack of consent from unconscious patients,” which raises the bar on what types of experiments are permissible. JC2RT research “falls into this double shadow,” Wikler says. “But that doesn’t mean it was wrong.” “The moral of this story is that continuous research is not only desirable but ought to be seen as obligatory, barring insuperable ethical barriers,” Wikler says. “All of us hope that if we are injured in a trafﬁc accident and are brought to a hospital, the ER docs won’t be using primitive medical techniques because research on banged-up patients was forbidden.” This is also the view of Peggy Knudson, a trauma surgeon and researcher at the University of California, San Francisco, who has trained U.S. military doctors in Iraq and has advised the military on JC2RT research. Knudson says some of the treatments tested in Iraq and Afghanistan are already being applied in civilian hospitals in the United States. “I copied the military burn protocol and brought it back to my [hospital],” she says. She was also impressed by the new methods for applying tourniquets and temporarily shunting blood vessels. “I use them both.” She says that the military’s massive blood-transfusion protocol is being tested at 11 civilian trauma centers across the United States. Here in Afghanistan, Osborne has to get back to work supervising the research projects. “I can’t wait for tomorrow,” she says. Once per week, she stops being a scientist and becomes a doctor, waiting for the injured to arrive from all directions.

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THE WAR IN AFGHANISTAN | NEWSFOCUS

–JOHN BOHANNON

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COMMENTARY At the end, start again?

Sensing light and heat

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LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES

LETTERS edited by Jennifer Sills

Projecting Human DNA Patent Numbers IN THE NEWS FOCUS STORY “THE HUMAN genome (patent) project” (4 February, p. 530), S. Kean focuses on the concern that large numbers of human gene patents and assignees holding these patents can block or have a chilling effect on commercialization. We believe that Kean may have overstated these issues. First, the graph in the story includes the numbers of all gene and DNA patents, which include plants and animals as well as human DNA patents. Narrowing to the subset of human DNA patents would produce a smaller number. Second, the story does not take into account patent renewal rates. Studies have shown that 66% of all patents expire due to failure to maintain (1). If this holds true in the ﬁeld of human DNA, there may be many fewer patents in the future as current patents

SINCE 2006, CHINA HAS SUFFERED SEVERE droughts every year, including one in the humid southwest in the spring of 2010. The current severe drought in the North China Plain is threatening more than 6.5 million hectares of winter wheat. Public health has been heavily affected by poor water quality. From 2006 to 2010, an additional 200,000 to 500,000 water-related diseases were reported each year, compared with those reported in 2004 to 2005 (1). On 29 January 2011, the Chinese government announced a plan to invest US$60 billion per year for 10 years to achieve sustainable water use. However, the increased investment mainly targets improving infrastructure “hardware,” such as building new reservoirs, drilling wells, increasing water supply for key

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ANN E. MILLS* AND PATTI M. TERESKERZ

Center for Biomedical Ethics and Humanities, University of Virginia, Charlottesville, VA 22908, USA. *To whom correspondence should be addressed. E-mail: [email protected]

References and Notes

1. M. Lemley, N.W. U.L. Rev. 95, 1495 (2001). 2. V. Konde, “Consolidation strategies of the biotechnology companies,” Nat. Network (30 September 2010); http:// network.nature.com/groups/biotechmanage/forum/ topics/8279. 3. Conﬂict of interest: The authors have previously been consultants for the Biotechnology Industry Organization through a contract with the University of Virginia.

irrigation districts, and implementing interbasin water transfer projects. The plan largely ignores information sharing, good planning, and governance “software,” which should be given priority (2, 3). Since the 1950s, China has invested huge amounts of money and labor into developing irrigation and water conservancy facilities. This has helped to feed the increasingly large population of China, but current water use is not sustainable (2, 4). The transition from farmer-initiated development of rural water facilities to the central government–dominated construction of major water infrastructures starting around 1978 has led to an imbalance in the irrigation network. Its main arteries are often well constructed, but small arterioles and capillaries to connect farmlands are underfunded. The end-pipe irrigation system at the farm level is critical in preventing disasters. In addition,

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policy implementation based on administrative units rather than watersheds hampers effective rural water management. Therefore, in addition to water conservation and efﬁciency options (3, 5), two urgent issues must be addressed. The increased government funding must be directed to the improvement, construction, and management of rural water systems. Integrated water resource management must be adopted to

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 disqualiﬁed. To submit a Letter, go to www.submit2science.org.

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CREDIT: COMSTOCK/THINKSTOCK

China: Invest Wisely in Sustainable Water Use

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expire. Third, if the industry has consolidated (2), then perhaps fewer entities hold these patents than indicated by the story. There is a lack of empirical evidence about the numbers of human DNA patents, including adjustments for mergers, acquisitions activity, subsidiaries, and spelling variations. Our hope is that further study will give decision-makers a more complete and accurate picture of the genetics industry.

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provide a holistic solution of water problems at watershed scales, with goals including environmental sustainability, human health, and economic and social well-being, in addition to the traditional agricultural proﬁtability and yields (6).

PENG GONG,1,2* YONGYUAN YIN,3 CHAOQING YU1

Center for Earth System Science, Tsinghua University, Beijing, China. 2Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA. 3State Key Laboratory of Remote Sensing Science, Jointly Sponsored by Institute of Remote Sensing Applications, Chinese Academy of Sciences and Beijing Normal University, China. 1

*To whom correspondence should be addressed: E-mail: [email protected]

References

1. Ministry of Health of the People’s Republic of China, December 2010 Survey of Outbreak of Nationally Enlisted Infectious Diseases (www.moh.gov.cn/publicﬁles/ business/htmlﬁles/mohjbyfkzj/s3578/201101/50310. htm) [in Chinese]. 2. P. H. Gleick, Science 302, 1524 (2003). 3. C. Q. Yu, Nature 470, 307 (2011). 4. Y. Y. Yin, N. Clinton, B. Luo, L. C. Song, in Climate Change and Vulnerability, N. Leary et al., Eds. (Earthscan, London, 2008), chap. 5. 5. P. H. Gleick, in The World’s Water 2008–2009, P. H. Gleick et al., Eds. (Island Press, Washington, DC, 2009), chap. 5. 6. J. Sachs et al., Nature 466, 558 (2010).

Family Planning: Looking Beyond Access IN THEIR POLICY FORUM “FAMILY PLANNING and the millennium development goals” (24 September 2010, p. 1603), W. Cates Jr. et al. write, “Over 215 million women who desire safe, effective family planning lack access.” This represents a common misunderstanding of the data from demographic and health surveys. Many people think that the term “unmet need,” which is used to describe the estimated 215 million women in marriages or unions who don’t want to be pregnant and are not using contraception, is actually the phenomenon of unmet demand for contraception. It is not. In most countries, lack of access to family planning is a very minor reason for nonuse. For example, in Nigeria, lack of access is cited by 0.2% of nonusers who don’t want to be pregnant (1). Many of these women don’t want or intend to use modern fam-

Response

ily planning methods because (i) they have heard it is dangerous, (ii) their male partners are opposed, (iii) their religion is opposed, or (iv) they are leaving decisions on the number of children “up to God” (2, 3). Of the 215 million women in the “unmet need” category, about 78 million women use traditional, less reliable methods of family planning, while 137 million use no method at all (4). Bringing about a major increase in contraceptive use can only be accomplished through the use of communication to overcome the informational barriers. Improved communication may also beneﬁt the 578 million men and women in marriages or unions worldwide who do not practice family planning because they have no desire to prevent additional pregnancies (5). Some of this group may choose to use family planning once they better understand the health and economic benefits of delayed, spaced, and limited childbearing. It is important to have an ample supply of low-cost contraceptive choices, delivered in a consumer-friendly way in the context of broad reproductive health care. There is a shortage of contraceptives in some countries and a need for new contraceptive technologies. However, the need goes well beyond providing access to contraception.

WILLIAM N. RYERSON

Population Media Center and Population Institute, 145 Pine Haven Shores Road, Shelburne, VT 05482, USA. E-mail: [email protected]

References and Notes

1. National Population Commission (NPC) [Nigeria] and ICF Macro, “Nigeria demographic and health survey 2008” (NPC and ICF Macro, Abuja, Nigeria, 2009). 2. E. van de Walle, Demography 27, 487 (1992). 3. W. N. Ryerson, FOCUS 5, 22 (1995). 4. J. Bongaarts, S. W. Sinding, Int. Perspect. Sex. Reprod. Health 35, 39 (2009). 5. The ﬁgure of 578 million was derived as follows: 1.146 billion women of reproductive age worldwide are in a union or marriage (6), indicating about 2.292 billion people of reproductive age are in a union. Of these, 44% (6), or 1.008 billion, do not use modern contraceptive methods. After subtracting the 430 million (215 million women and their partners) in the unmet need category (those that do not want a pregnancy in the next 2 years), 578 million remain. 6. United Nations, Department of Economic and Social Affairs, Population Division, “World contraceptive use 2009” (2009). 7. I thank C. Haub for help with demographic data.

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WE APPRECIATE RYERSON’S OBSERVATIONS and analysis. As is common practice in family planning literature, the word “access” here is meant to include the spectrum of knowledge, information, services, and opportunities necessary for women and men to use in making informed choices about preventing unintended pregnancies. We agree with Ryerson’s suggested revision to the sentence “Over 215 million women who desire safe, effective family planning lack access.” To be precise, we should have written, “Over 200 million women who do not wish to be pregnant are not using contraception because of lack of family planning information or services.” Such attention to semantic accuracy is important in advocacy broadly, and especially so for international family planning.

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When livers get fatty

WILLARD CATES JR.,* E. MEGAN DAVIDSON AVERILL, TRICIA PETRUNEY

FHI, Durham, NC 27713, USA. *To whom correspondence should be addressed. E-mail: [email protected]

TECHNICAL COMMENT ABSTRACTS

Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” Alexander Graf, Lutz Weihermüller, Johan A. Huisman, Michael Herbst, Harry Vereecken Mahecha et al. (Reports, 13 August 2010, p. 838) estimated the temperature sensitivity of ecosystem respiration (Q10) and showed that temperature sensitivity and its site-to-site variability are lower than previously reported. We demonstrate that their Q10 value of 1.4 is an underestimate if interpreted as the averaged sensitivities of all ecosystem components, because fast temperature ﬂuctuations penetrate poorly into the soil. Full text at www.sciencemag.org/cgi/content/full/331/ 6022/1265-c

Response to Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” Miguel D. Mahecha, Markus Reichstein, Nuno Carvalhais, Gitta Lasslop, Holger Lange, Sonia I. Seneviratne, Rodrigo Vargas, Christof Ammann, M. Altaf Arain, Alessandro Cescatti, Ivan A. Janssens, Mirco Migliavacca, Leonardo Montagnani, Andrew D. Richardson We estimated the sensitivity of terrestrial ecosystem respiration to air temperature across 60 FLUXNET sites by minimizing the effect of seasonally confounding factors. Graf et al. now offer a theoretical perspective for an extension of our methodology. However, their critique does not change our main ﬁndings and, given the currently available observational techniques, may even impede a comparison across ecosystems. Full text at www.sciencemag.org/cgi/content/full/331/ 6022/1265-d

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Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” Alexander Graf,* Lutz Weihermüller, Johan A. Huisman, Michael Herbst, Harry Vereecken Mahecha et al. (Reports, 13 August 2010, p. 838) estimated the temperature sensitivity of ecosystem respiration (Q10) and showed that temperature sensitivity and its site-to-site variability are lower than previously reported. We demonstrate that their Q10 value of 1.4 is an underestimate if interpreted as the averaged sensitivities of all ecosystem components, because fast temperature fluctuations penetrate poorly into the soil. espiration is an important feedback mechanism in Earth’s climate system because of its strong response to temperature. The factor by which respiration multiplies in response to a 10° temperature increase (Q10) was reported to vary considerably not only between ecosystem components but also between ecosystems. Recently, Mahecha et al. (1) determined a convergent Q10 value of 1.4 (T0.1) for a wide range of mean annual temperatures and biomes using FLUXNET (2) ecosystem respiration data and the scale-dependent parameter estimation (SCAPE) methodology to exclude confounding effects. This value is considerably lower than previous estimates, and the authors pointed out that it has to be interpreted carefully, in particular with respect to soil respiration. They recommended further evaluation using soil respiration and soil temperature measurements. We commend the authors for their valuable contribution to the analysis of both aboveground and soil respiration. By extending their synthetic validation experiment with soil respiration, we could indeed reproduce their finding of overestimated Q10 values by the conventional methodology. However, we also found that the SCAPE methodology leads to an underestimation of Q10 whenever the following criteria apply: (i) Soil respiration at depths where shortterm temperature fluctuations are dampened contributes substantially to ecosystem respiration and (ii) Ecosystem-level Q10 is defined as the contribution-weighted average of the Q10s of all single ecosystem components, and thus as the sensitivity that applies when all parts of the system warm equally. Our study may in part explain the low Q10 estimate of 1.4 that was critically discussed by the authors themselves and in a related commentary article (3). The SCAPE methodology (1) aims at excluding confounding effects from the temperature-

R

Forschungszentrum Juelich, Agrosphere Institute (IBG-3), Institute for Bio- and Geosciences, 52425 Juelich, Germany. *To whom correspondence should be addressed. E-mail: [email protected]

respiration relation by focusing on less affected temporal scales. It is based on the observation that confounding effects on temperature sensitivity act on different time scales. For example, biomass and litter production change on seasonal and longer time scales. As a consequence, basal respiration Rb exhibits an annual cycle. If Rb and the annual temperature cycle are correlated, this will affect the apparent temperature sensitivity of respiration. Therefore, Mahecha et al. decomposed temperature and log-transformed ecosystem respiration time series from a wide range of sites into frequency bins, with the first bin containing all fluctuations with a period longer than 3 months. For each frequency bin, they calculated Q10 values separately. Using synthetic experiments, Mahecha et al. demonstrated that the Q10 values for intermediate and high frequencies converged toward the prescribed value. The conventional Q10 and the Q10 of the lowest frequency bins, in contrast, were subject to large errors. The validation experiments assumed that the temperature time series reflects the conditions at the location that acts as a source for respiration. For soil respiration, this is not necessarily the case because of the damping of temperature fluctuations as a function of soil depth. This effect has already been recognized as a major source of uncertainty in the determination of the temperature sensitivity of soil respiration (4–12). In addition, it can be shown that highfrequency temperature variations are dampened more than low-frequency temperature variations (13). In an artificial experiment with diurnal and annual temperature waves, it was shown that the sensitivity of apparent Q10 values to the choice of soil temperature measurement depth can be reduced by daily averaging of the soil respiration and soil temperature time series (8). Cycles between the diurnal and annual cycle, however, were not considered in (8). The analysis of Mahecha et al. (1) was based on daily (nighttime) averages of temperature and respiration. Annual variations were also excluded by the SCAPE methodology, and the remaining intermediate frequencies were used to determine

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Q10. At these frequencies, soil temperature may still differ considerably from air temperature. When air temperature is statistically related to a respiration flux that is composed of contributions from various sources in a range of depths with varying soil temperature, the resulting Q10 cannot be considered as the weighted average of the intrinsic temperature sensitivity of these sources. To illustrate this, we present a synthetic experiment similar to that described in (1). We used six soil temperature time series at different depths in addition to the air temperature time series to generate the artificial respiration signal (see supporting online material for details). The results obtained in (1) are reproduced in the upper row of Fig. 1. The Q10 values converge toward the prescribed Q10 for high frequencies, whereas the conventional Q10 matches the prescribed value of 2 only if Rb is constant. For the case in which 33% of respiration is belowground (second row), all binned Q10 values underestimate the prescribed value except for the value corresponding to the lowest-frequency bin. When the belowground contribution increases further (third and fourth row), the high-frequency Q10 approaches unity. The magnitude of the contribution of soil respiration to ecosystem respiration can vary between a few percent and more than 50%, depending on the ecosystem and on whether or not root respiration is included in the belowground respiration (14, 15). We therefore suggest extending the SCAPE methodology to account for the vertical source distribution of soil respiration as affected by varying soil temperature, even if it aims at ecosystemlevel sensitivity only. A global convergence may still be found. However, the Q10 value obtained by such an extended methodology may vary between 1.4 and previously reported estimates, thus bridging the gap between these disparate results. For application in models that are driven by air temperature and aim at short-term changes, such as gap-filling models, values determined with the SCAPE methodology may even be the more appropriate ones, as it implicitly contains the damping of fluctuations with soil depth. On the decadal temporal scale covered by global climate modeling, however, trends in soil temperature almost equal trends in air temperature because of the nature of heat transfer (13). In such a model, the same value would lead to a systematic underestimation of long-term respiration.

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TECHNICAL COMMENT

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

M. D. Mahecha et al., Science 329, 838 (2010). D. Baldocchi, Aust. J. Bot. 56, 1 (2008). P. B. Reich, Science 329, 774 (2010). J. Lloyd, J. A. Taylor, Funct. Ecol. 8, 315 (1994). M. Pavelka, M. Acosta, M. V. Marek, W. Kutsch, D. Janous, Plant Soil 292, 171 (2007). M. Reichstein, C. Beer, J. Plant Nutr. Soil Sci. 171, 344 (2008). M. Bahn et al., Ecosystems 11, 1352 (2008). A. Graf et al., Biogeosciences 5, 1175 (2008). M. J. I. Briones, NATO Science for Peace and Security C, 317 (2009).

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Fig. 1. Results of synthetic experiments. Realworld daily time series of air temperature and soil temperature at various depths were used for generating artificial respiration signals from various layers of the ecosystem. Total ecosystem respiration was calculated as the sum of weighted contributions from these layers for different cases of vertical source distribution (left column). The uppermost case corresponds to the assumption implicit in the artificial experiments in (1), namely that all respiration sources are subject to air temperature; the lowermost case corresponds to the extreme where all sources are belowground. The prescribed Q10 value was 2, and the prescribed amplitude of Rb for different cases is given in the bottom row. The case shown on the right corresponds to the example given in figure S2 in (1). Each subpanel gives the conventional Q10 and the frequency-dependent Q10 values estimated from the artificial respiration data and the air temperature time series. The upper right subpanel is similar to figure S3 in (1), but without artificial noise. 10. N. Pingintha, M. Y. Leclerc, J. P. Beasley Jr., G. Zhang, C. Senthong, Tellus B Chem. Phys. Meterol. 62, 47 (2010). 11. J. A. Subke, M. Bahn, Soil Biol. Biochem. 42, 1653 (2010). 12. C. L. Phillips, Glob. Change Biol. 17, 515 (2011). 13. W. R. van Wijk, Ed., Physics of Plant Environment (North Holland Publishing, Amsterdam, 1963).

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14. M. Aubinet et al., Agric. For. Meteorol. 149, 407 (2009). 15. T. Zha, Z. Xing, K.-Y. Wang, S. Kellomaki, A. G. Barr, Ann. Bot. 99, 345 (2007).

Fig. S1 Table S1 References

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1265-c/DC1 Materials and Methods

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25 August 2010; accepted 11 February 2011 10.1126/science.1196948

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TECHNICAL COMMENT

Response to Comment on “Global Convergence in the Temperature Sensitivity of Respiration at Ecosystem Level” Miguel D. Mahecha,1,2* Markus Reichstein,1 Nuno Carvalhais,1,3 Gitta Lasslop,1 Holger Lange,4 Sonia I. Seneviratne,2 Rodrigo Vargas,5,6 Christof Ammann,7 M. Altaf Arain,8 Alessandro Cescatti,9 Ivan A. Janssens,10 Mirco Migliavacca,9,11 Leonardo Montagnani,12,13 Andrew D. Richardson14 We estimated the sensitivity of terrestrial ecosystem respiration to air temperature across 60 FLUXNET sites by minimizing the effect of seasonally confounding factors. Graf et al. now offer a theoretical perspective for an extension of our methodology. However, their critique does not change our main findings and, given the currently available observational techniques, may even impede a comparison across ecosystems. n a recent paper, we applied a methodology to estimate the temperature sensitivity of ecosystem respiration (the total CO2 release by plants and soils) from long-term monitoring data (1). One central finding was that the overall short-term temperature sensitivity of ecosystem respiration is relatively uniform across biomes and different climate zones. Our approach is based on the assumption that ecosystem-atmosphere interactions vary on multiple time scales, as shown in a series of site-level studies (2, 3). The conceptual advantage of the proposed scale-dependent parameter estimation (SCAPE) methodology compared with conventional estimates has now been confirmed by the synthetic experiments reported by Graf et al. (4): SCAPE approximates the shortterm temperature sensitivity of ecosystem respi-

I

1

Max Planck Institute for Biogeochemistry, 07745 Jena, Germany. Institute for Atmospheric and Climate Science, Eidgenössische Technische Hochschule, Zurich Universitätsstrasse 16, 8092 Zurich, Switzerland. 3Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, 2829–516 Caparica, Portugal. 4 Norsk Institutt for Skog og Landskap, N–1431 Ås, Norway. 5 Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA. 6Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE, Carretera Ensenada–Tijuana No. 3918, Zona Playitas, C.P. 22860, Ensenada, Mexico. 7Agroscope Reckenholz-Tänikon, Federal Research Station, Reckenholzstrasse 191, CH–8046 Zurich, Switzerland. 8School of Geography and Earth Sciences and McMaster Centre for Climate Change, McMaster University, Hamilton, Ontario, Canada. 9European Commission, Joint Research Center, Institute for Environment and Sustainability, Ispra, Italy. 10Department of Biology, University of Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium. 11Remote Sensing of Environmental Dynamics Laboratory, Dipartimento di Scienze dell’Ambiente e del Territorio, University of Milano– Bicocca, Milano, Italy. 12Servizi Forestali, Agenzia per l’Ambiente, Provincia Autonoma di Bolzano, Bolzano, Italy. 13Faculty of Sciences and Technologies, Free University of Bozen–Bolzano, Piazza Università 1, 39100, Bolzano, Italy. 14Harvard University Department of Organismic and Evolutionary Biology, Harvard University Herbaria, 22 Divinity Avenue, Cambridge, MA 02138, USA. 2

*To whom correspondence should be addressed. E-mail: [email protected].

ration in the presence of seasonally confounding effects quite well. However, Graf et al. (4) correctly point out that the reported Q10;sc could be an underestimate if substantial fractions of the released CO2 are originating from deeper soil layers where temperature dynamics are delayed and dampened compared with air temperature. This valid point (5, 6) has already been anticipated in our original paper (1). Graf et al. (4) emphasize the difference between an intrinsic temperature sensitivity of respiration and the findings in (1). This objection illustrates a possible misunderstanding. Hence, we use this opportunity to further clarify a few conceptual aspects of our study. First, although our study was motivated by the search for an intrinsic temperature sensitivity of ecosystem respiratory processes [in the sense of (7), referring to kinetic properties], we did recognize that such an intrinsic value does not exist at the ecosystem level and can only be approximated. As illustrated by Reich (8), a series of individual processes contribute to the overall release of CO2, via different mechanisms, and each of these processes may obey different metabolic relations (9). Therefore, we suggest using the term “SCAPE Q10” (denoted as Q10;sc) in forthcoming analyses. In fact, by definition no intrinsic Q10 exists at the ecosystem level (6). Second, the main virtue of the presented Q10;sc values is that they are not influenced by the seasonal confounders (6, 10). We interpret Q10;sc as a parametric diagnostic of the shortterm temperature sensitivity of respiration at ecosystem level. Such an integrated measure is essential for scrutinizing the validity of biosphere models. An ecosystem-level Q10;sc can form the basis for evaluating current and future coupled climate–carbon cycle simulations, similar to the climate sensitivity of the global carbon cycle, which is a diagnostic at Earth-system level (11).

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Third, Graf et al. (4) conclude that we should have included temperature dampening effects to obtain more representative Q10;sc estimates. We agree with this idea and see its value for future applications. However, the prerequisite would be the availability of continuous soil temperature and flux data at different depths allowing us to pin-point the exact location of the sources of CO2 in the soil column. At present, however, such an approach is not feasible based on available FLUXNET data archives (12). It would also require taking delays in respiration responses into account, for instance, due to delayed belowground allocation and exudation rates (13). Furthermore, fluctuations in soil water content may play an important role, acting as a direct constraint for soil respiration (14) but also by determining heat conduction (and storage) in the soil (15). This latter effect is itself dependent on the soil type, peat soils being far less conductive than sandy or clay soils, even with higher water content (15). Hence, the question of how to disentangle the temperature sensitivity of plant respiration remains unclear in this context. Besides the methodological advances, our reported Q10;sc values are practicable because they have been derived based on a minimum of a priori assumptions. Using air temperature instead of soil temperature profiles guarantees a comparability of the integrated response of soils and vegetation. The consistency of the analysis across so many sites is a key prerequisite for comparing different biomes and provides reference temperature sensitivities for terrestrial biosphere models that generally operate on very large spatial scales. The analysis by Graf et al. (4) would instead lead directly to the formulation of an ecosystemspecific empirical model, which was not the goal of our analysis. As a final remark, we would like to highlight that the importance of our paper lies in the homogeneity of the temperature sensitivity observed across biomes and plant functional types. This emergent behavior at ecosystem level deserves more attention in future studies, where a modeldata integration approach built on the ideas described by Graf et al. (4) may considerably advance our understanding of ecosystem-atmosphere exchange processes.

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TECHNICAL COMMENT

References 1. M. D. Mahecha et al., Science 329, 838 (2010). 2. P. C. Stoy et al., Biogeosciences 6, 2297 (2009). 3. R. Vargas, M. Detto, D. D. Baldocchi, M. F. Allen, Glob. Change Biol. 16, 1589 (2010). 4. A. Graf, L. Weihermüller, J. A. Huisman, M. Herbst, H. Vereecken, Science 331, 1265 (2011); www.sciencemag.org/cgi/content/full/331/6022/1265-c. 5. M. Pavelka, M. Acosta, M. V. Marek, W. Kutsch, D. Janouš, Plant Soil 292, 171 (2007). 6. M. Reichstein, C. Beer, J. Plant Nutr. Soil Sci. 171, 344 (2008). 7. E. A. Davidson, I. A. Janssens, Nature 440, 165 (2006). 8. P. B. Reich, Science 329, 774 (2010). 9. M. Acosta, M. Pavelka, R. Pokornŷ, D. Janouš, M. V. Marek, Ann. Bot. 101, 469 (2008).

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TECHNICAL COMMENT 12. Fluxnet Synthesis data set; www.fluxdata.org. 13. Z. Kuzyakov, O. Gavrichovka, Glob. Change Biol. 16, 3386 (2010). 14. M. Almagro, J. López, J. I. Querejeta, M. Martínez-Mena, Soil Biol. Biochem. 41, 594 (2009).

15. D. Hillel, Introduction to Environmental Soil Physics (Elsevier, Amsterdam, 2003). 1 October 2010; accepted 11 February 2011 10.1126/science.1197033

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10. D. A. Sampson, I. A. Janssens, J. Curiel Yuste, R. Ceulemans, Glob. Change Biol. 13, 2008 (2007). 11. D. C. Frank et al., Nature 463, 527 (2010).

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The End of the World Is Flat Raphael Bousso

T

he universe began as kind of distortion, a “conforCycles of Time a horizontal line: this mal transformation,” is permuch we knew. More mitted when passing from the An Extraordinary New recently, in a breathtaking real space-time to its depicView of the Universe experimental effort, we distion. This restriction ensures by Roger Penrose covered that the world will that the paths of light rays Bodley Head, London, 2010. end in a horizontal line. But if are faithfully represented. 300 pp. £25, C$37.95. ISBN the end looks like the beginNothing can travel faster than 9780224080361. Forthcoming ning, could it be that they are light, so no event can inﬂu(May) from Knopf, New York. $28.95. ISBN 9780307265906. the same thing? Was our Big ence parts of the universe that Bang the last gasp of a previare too remote for light rays to ous world? And will the death reach. We see only a portion of our universe mark the birth of a new one? of our own universe, a few billion light-years Such speculations are at the heart of Cycles of across, because light has only traveled for a Time, the latest book by mathematical physi- few billion years since the universe began. cist Roger Penrose. Because a Penrose diagram has the same In 1905, Einstein created the special the- light rays, it accurately captures the causal ory of relativity, fusing space and time into structure of the original, undistorted unia single four-dimensional entity. A decade verse. By discarding an overwhelming later, he formulated a theory of gravity, wealth of detail, it allows the called general relativity. It posits that the most important geometric feageometry of four-dimensional space-time is tures to become apparent. The curved by massive objects such as the Sun. presence of a black hole can be The resulting shape, in turn, tells matter par- obscured by heaps of equations, ticles how to move. As long as the warping but in a Penrose diagram, it is is small, this scheme provides the illusion of obvious. One glance sufﬁces to a gravitational “force” pulling Earth toward recognize that the black hole sinthe Sun, whereas in fact our planet simply gularity is not a point in space follows the straightest possible path through (a common misconception), but a curved geometry. rather a moment of time that is In many popular accounts, one ﬁnds this eventually reached by all objects notion illustrated by a rubber sheet that is that enter the black hole. The Big indented by the Sun, while Earth rolls around Bang, too, is seen to be a moment in the mold. But analogies of this kind are of time; but it marks the begininadequate for visualizing many fascinating ning, not the end, of the classical phenomena predicted by Einstein’s theory. space-time geometry. (Such iniHow are we to picture the Big Bang—the tial or ﬁnal moments of time can dense, hot initial state of our universe? How be represented by the horizontal should one imagine the singularity at the cen- lines alluded to at the beginning ter of a black hole? Black holes and cosmol- of this review.) ogy are the settings in which general relativPenrose diagrams changed ity truly comes into its own. But for half a the way we think about curved century, physicists lacked powerful tools for space-time. The emphasis on visualizing them. global and causal structure liberEnter the Penrose diagram. To picture ated a stagnant ﬁeld, ushering in the universe on a sheet of paper, it shrinks a renaissance that stretched from inﬁnite regions of space and time to ﬁnite Penrose’s and Stephen Hawksize. It zooms in on space-time singulari- ing’s celebrated singularity theoties, such as the Big Bang, by stretching out rems (1), through the discovery the very early universe. But only a particular that black holes have thermodynamic properties (2, 3), to The reviewer is at the Center for Theoretical Physics, Departthe recognition of a completely ment of Physics, University of California, Berkeley, CA general relation between classi94720–7300, USA, and Lawrence Berkeley National Labocal space-time geometry and its ratory, Berkeley, CA 94720–8162, USA. E-mail: bousso@ lbl.gov quantum information content (4).

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Key signposts guiding our search for a quantum theory of gravity—Bekenstein entropy, Hawking radiation, and the holographic principle—owe a large debt to the methods pioneered by Penrose in the 1960s. Our own universe exhibits a superﬁcial similarity between the beginning of time and the inﬁnite future: both look like horizontal lines in the Penrose diagram. As a result, we could stack one copy of the diagram on top of another, like so many wood blocks. A horizontal line separating two copies would seem to play a dual role as the future boundary of one universe and as the big bang of another. In Cycles of Time, Penrose asks us to take this whimsical picture seriously. In his viewpoint, the entire history of our universe is reduced to an “aeon,” a mere segment in an inﬁnite chain of beginnings and ends. Penrose’s proposal ascribes to his diagrams a universal status that they do not possess. Our world, in its rich phenomenology, is far more than a Penrose diagram. In nearly every respect, the beginning and the end of the observable universe are totally different.

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CREDIT: FRY DESIGN/GETTY IMAGES

BOOKS ET AL.

BOOKS ET AL. generalized second law of thermodynamics” ECONOMICS (2). Proposed by Jacob Bekenstein, this law maintains that disorder does not decrease even when disordered matter is lost into black holes. This is surprising at ﬁrst, but no counterexample has ever been found. In fact, the law turned out to hold even in a situation that Douglas Gollin Bekenstein did not anticipate and that is relevant for Penrose’s proposal. In 1974, Hawkfrica is a continent of farmers. ing discovered that black holes emit radiaSouth of the Sahara, about 60% of tion (3). Over enormous time scales, a black the economically active population hole will dissolve into a cloud of Hawking makes a living from agriculture. Most of the radiation and disappear. Careful calculations continent’s farms are extremely small, and showed that the disorder of the Hawking productivity levels are very low. cloud is always much greater For example, a 2003 surthan that of the matter that was vey of households in MozamThe New Harvest destroyed when it fell into the bique found about 3.2 million Agricultural Innovation black hole (5, 6). farm households, cultivating in Africa This is an example of an average of 1.4 hectares. A by Calestous Juma the generalized second law typical farm household (literOxford University Press, at work. The processing of ally) scratches out a meager New York, 2010. 296 pp. matter by black holes, far living from the soil: over 80% $99, £60. ISBN 9780199783205. from imposing order, vastly of the farms used no source of Paper, $19.95, £12.99. increases the disorder. This power other than hand tools ISBN 9780199783199. result is hardly new or conand human labor; more than troversial, and it invalidates 90% used neither chemical one of the central tenets of Cycles of Time. fertilizers nor pesticides. Crop output is corIt is all the more puzzling that the result respondingly low: Yields of the major food goes unmentioned in the book, especially grains (maize, sorghum, and rice) are below as in places Penrose goes to great lengths to one metric ton per hectare, leaving farm defend implausible assumptions—includ- households with little surplus for sale to the ing some that fail to help his case. He sup- market. For the grain crops, 75 to 95% of the poses that information is lost in black holes, output is consumed on the farms where it is apparently in the belief that this would imply produced (1). that black holes destroy disorder. But if this To use a different metric of comparison assumption were true, it would only make (and a different country), a typical maize his task more hopeless. Information, in a farmer in Kenya produces just under 1 kg of very precise sense, is the opposite of dis- maize for every hour spent working on her order. Penrose’s assumed information loss farm (2). By contrast, a farmer in Iowa— would introduce fundamental ignorance working with far more capital and much betabout the microscopic state of the Hawk- ter technology—can expect to produce over ing cloud (thus increasing its “ﬁne-grained 1000 kg of maize per hour of labor. entropy”) while having no effect on the In short, sub-Saharan Africa faces vast macroscopic disorder (the “coarse-grained and daunting productivity deficits with entropy”), which increases in any case. respect to the rest of the world. Millions of In the end, then, it is difﬁcult to recom- people in the region are living and working in mend Cycles of Time. Experts will already be low-productivity, quasi-subsistence agriculfamiliar with the topics discussed in its stron- ture, with desperately low standards of living. ger passages, such as the thermodynamic Moreover, the data from recent years suggest arrow of time and Penrose diagrams. And that agriculture in Africa may actually be fallcasual readers will have a hard time separat- ing further behind rather than catching up. ing the wheat from the chaff. In the face of this situation, it is easy to feel discouraged and pessimistic. Yet in The New References Harvest, Calestous Juma offers a remarkably 1. S. W. Hawking, R. Penrose, Proc. R. Soc. London Ser. A optimistic outlook for agriculture in Africa. 314, 529 (1970). Juma (a Kenyan scholar of sustainable devel2. J. D. Bekenstein, Lett. Nuovo Cimento 4, 737 (1972). 3. S. W. Hawking, Nature 248, 30 (1974). opment at Harvard University) argues that

A Hopeful Vision of Food in Africa

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4. R. Bousso, J. High Energy Phys. 1999, 004 (1999). 5. J. D. Bekenstein, Phys. Rev. D 9, 3292 (1974). 6. J. D. Bekenstein, Phys. Rev. D 12, 3077 (1975).

10.1126/science.1203148

The reviewer is at the Department of Economics, Williams College, Williamstown, MA 01267, USA. E-mail: douglas. [email protected]

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Near the Big Bang, particles were densely packed and very hot; but the universe will be empty and cold in the far future. The laws of physics contain many absolute scales that Penrose diagrams—designed as they are to distill out the causal structure—know nothing about. All hydrogen atoms, say, have the same ﬁxed size in the real world; in a Penrose diagram, their size is arbitrary and changing. Could it be that all absolute scales dissolve in the very early and late universe? Penrose suggests as much, in an attempt to lend the diagram a more fundamental role at least in these limits. But nucleosynthesis, say, occurs at a particular temperature in the early universe, and the cosmological constant leads to a particular expansion rate at late times. Many more examples teach us that the universe has always contained, and always will contain, information that its Penrose diagram lacks. The cyclic proposal is motivated by a legitimate concern. The second law of thermodynamics tells us that entropy, a measure of disorder, is extremely unlikely to decrease. An egg may fall, break, and leave a mess on the ﬂoor, but chances are that the mess will not collect itself into an intact egg, which then jumps back on the table. Left to its own devices, matter will become more disordered over time until it reaches a state of maximum entropy, in which it spends the overwhelming fraction of its time. But the universe is not in such a state. The disorder in the universe has been increasing since the very earliest times, and it continues to grow today. We must understand why the universe began in an incredibly special state, so well ordered that 14 billion years later, the universe still has not reached maximum disorder. Penrose is at his best when he explains this deep and beautiful mystery, and the book may be worth reading for this chapter alone. However, he compounds the shortcomings of his cyclic universe model when he argues that it can solve the low-entropy problem. At this point, another idea is introduced: like vacuum cleaners, black holes appear to reduce disorder by swallowing matter. By the end of one “aeon,” Penrose argues, most matter has ended up in giant black holes. Very little entropy remains, and the next aeon can commence in perfect order. The second law guarantees that a vacuum cleaner does not actually decrease the overall disorder; at best, it just shifts it around. In fact, the machine creates far more entropy than it destroys (for example, by heating up the air in the room). A black hole, it turns out, is not different. Penrose’s assertion that black holes destroy entropy is ﬂatly contradicted by “the

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BOOKS ET AL. generalized second law of thermodynamics” ECONOMICS (2). Proposed by Jacob Bekenstein, this law maintains that disorder does not decrease even when disordered matter is lost into black holes. This is surprising at ﬁrst, but no counterexample has ever been found. In fact, the law turned out to hold even in a situation that Douglas Gollin Bekenstein did not anticipate and that is relevant for Penrose’s proposal. In 1974, Hawkfrica is a continent of farmers. ing discovered that black holes emit radiaSouth of the Sahara, about 60% of tion (3). Over enormous time scales, a black the economically active population hole will dissolve into a cloud of Hawking makes a living from agriculture. Most of the radiation and disappear. Careful calculations continent’s farms are extremely small, and showed that the disorder of the Hawking productivity levels are very low. cloud is always much greater For example, a 2003 surthan that of the matter that was vey of households in MozamThe New Harvest destroyed when it fell into the bique found about 3.2 million Agricultural Innovation black hole (5, 6). farm households, cultivating in Africa This is an example of an average of 1.4 hectares. A by Calestous Juma the generalized second law typical farm household (literOxford University Press, at work. The processing of ally) scratches out a meager New York, 2010. 296 pp. matter by black holes, far living from the soil: over 80% $99, £60. ISBN 9780199783205. from imposing order, vastly of the farms used no source of Paper, $19.95, £12.99. increases the disorder. This power other than hand tools ISBN 9780199783199. result is hardly new or conand human labor; more than troversial, and it invalidates 90% used neither chemical one of the central tenets of Cycles of Time. fertilizers nor pesticides. Crop output is corIt is all the more puzzling that the result respondingly low: Yields of the major food goes unmentioned in the book, especially grains (maize, sorghum, and rice) are below as in places Penrose goes to great lengths to one metric ton per hectare, leaving farm defend implausible assumptions—includ- households with little surplus for sale to the ing some that fail to help his case. He sup- market. For the grain crops, 75 to 95% of the poses that information is lost in black holes, output is consumed on the farms where it is apparently in the belief that this would imply produced (1). that black holes destroy disorder. But if this To use a different metric of comparison assumption were true, it would only make (and a different country), a typical maize his task more hopeless. Information, in a farmer in Kenya produces just under 1 kg of very precise sense, is the opposite of dis- maize for every hour spent working on her order. Penrose’s assumed information loss farm (2). By contrast, a farmer in Iowa— would introduce fundamental ignorance working with far more capital and much betabout the microscopic state of the Hawk- ter technology—can expect to produce over ing cloud (thus increasing its “ﬁne-grained 1000 kg of maize per hour of labor. entropy”) while having no effect on the In short, sub-Saharan Africa faces vast macroscopic disorder (the “coarse-grained and daunting productivity deficits with entropy”), which increases in any case. respect to the rest of the world. Millions of In the end, then, it is difﬁcult to recom- people in the region are living and working in mend Cycles of Time. Experts will already be low-productivity, quasi-subsistence agriculfamiliar with the topics discussed in its stron- ture, with desperately low standards of living. ger passages, such as the thermodynamic Moreover, the data from recent years suggest arrow of time and Penrose diagrams. And that agriculture in Africa may actually be fallcasual readers will have a hard time separat- ing further behind rather than catching up. ing the wheat from the chaff. In the face of this situation, it is easy to feel discouraged and pessimistic. Yet in The New References Harvest, Calestous Juma offers a remarkably 1. S. W. Hawking, R. Penrose, Proc. R. Soc. London Ser. A optimistic outlook for agriculture in Africa. 314, 529 (1970). Juma (a Kenyan scholar of sustainable devel2. J. D. Bekenstein, Lett. Nuovo Cimento 4, 737 (1972). 3. S. W. Hawking, Nature 248, 30 (1974). opment at Harvard University) argues that

A Hopeful Vision of Food in Africa

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4. R. Bousso, J. High Energy Phys. 1999, 004 (1999). 5. J. D. Bekenstein, Phys. Rev. D 9, 3292 (1974). 6. J. D. Bekenstein, Phys. Rev. D 12, 3077 (1975).

10.1126/science.1203148

The reviewer is at the Department of Economics, Williams College, Williamstown, MA 01267, USA. E-mail: douglas. [email protected]

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Near the Big Bang, particles were densely packed and very hot; but the universe will be empty and cold in the far future. The laws of physics contain many absolute scales that Penrose diagrams—designed as they are to distill out the causal structure—know nothing about. All hydrogen atoms, say, have the same ﬁxed size in the real world; in a Penrose diagram, their size is arbitrary and changing. Could it be that all absolute scales dissolve in the very early and late universe? Penrose suggests as much, in an attempt to lend the diagram a more fundamental role at least in these limits. But nucleosynthesis, say, occurs at a particular temperature in the early universe, and the cosmological constant leads to a particular expansion rate at late times. Many more examples teach us that the universe has always contained, and always will contain, information that its Penrose diagram lacks. The cyclic proposal is motivated by a legitimate concern. The second law of thermodynamics tells us that entropy, a measure of disorder, is extremely unlikely to decrease. An egg may fall, break, and leave a mess on the ﬂoor, but chances are that the mess will not collect itself into an intact egg, which then jumps back on the table. Left to its own devices, matter will become more disordered over time until it reaches a state of maximum entropy, in which it spends the overwhelming fraction of its time. But the universe is not in such a state. The disorder in the universe has been increasing since the very earliest times, and it continues to grow today. We must understand why the universe began in an incredibly special state, so well ordered that 14 billion years later, the universe still has not reached maximum disorder. Penrose is at his best when he explains this deep and beautiful mystery, and the book may be worth reading for this chapter alone. However, he compounds the shortcomings of his cyclic universe model when he argues that it can solve the low-entropy problem. At this point, another idea is introduced: like vacuum cleaners, black holes appear to reduce disorder by swallowing matter. By the end of one “aeon,” Penrose argues, most matter has ended up in giant black holes. Very little entropy remains, and the next aeon can commence in perfect order. The second law guarantees that a vacuum cleaner does not actually decrease the overall disorder; at best, it just shifts it around. In fact, the machine creates far more entropy than it destroys (for example, by heating up the air in the room). A black hole, it turns out, is not different. Penrose’s assertion that black holes destroy entropy is ﬂatly contradicted by “the

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efﬁcient water puriﬁcation systems. How seriously should we take such techno-optimism? At times, a reader may be skeptical. For example, the author suggests that with greater investments in human capacity, “[b]ioinformatics could do for Africa what computer software did for India.” Perhaps this is true, as a statement of remote possibility. But given the current state of biological research in subSaharan Africa—and the distance by which the region lags behind China, India, and other countries—it seems farfetched. Beyond his techno-optimism, Juma is also upbeat about new institutional models that will lend themselves to agricultural innovation in Africa. These include intergovernmental agreements, public-private partnerships for research, “innovation clusters,” and a range of other mechanisms. He views such institutional changes as playing a central role in agricultural innovation. The New Harvest presents hundreds of illustrations and examples of technological and social innovations that have exciting potential for Africa. These often come from the creative engineering efforts of small startup ﬁrms or the problem-solving efforts of nongovernmental organizations. Although these examples do indeed convey a sense of the possibilities, readers may feel that the author is uncritically repeating the hype that these organizations use to woo investors and donors. While Juma wants his readers to grasp the possibilities that innovation offers, it would be useful to point out that many of these ventures are in early phases and may never go much further. Juma acknowledges the vast need for costly investments in physical infrastructure—such as roads and transport networks, power generation and energy delivery, and irrigation systems. He also recognizes the

References

1. World Bank, Agriculture, Environment, and Social Development Unit, “Mozambique agricultural development strategy: Stimulating smallholder agricultural growth” (Report 32416-MZ, World Bank, Washington, DC, 2006); http://siteresources.worldbank.org/MOZAMBIQUEEXTN/ Resources/Moz_AG_Strategy.pdf. 2. T. Suri, Econometrica 79, 159 (2011).

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Africa stands on the cusp of a new wave of technological progress that will boost agriculture and contribute to broad-based economic development. Juma writes that the necessary technologies already exist. Many of them, he argues, come from outside the traditional areas of agricultural science. For example, Juma suggests that advances in information and communications technologies have the potential to transform African agriculture. He is also bullish about biotechnology, nanotechnology, and other frontiers of innovation. For many of these technologies, he notes, diffusion can occur quickly and easily through the efforts of entrepreneurs and businesses. Mobile phones offer an example of the kind of technological diffusion that Juma has in mind. By some estimates, mobile phone coverage in Africa has now reached over 60% of the continent’s population. The spread of mobile phones has required little “push” effort from governments or donor agencies; instead, it has been driven by a vast demand. The required investments in infrastructure were modest: cell towers operated by solar power sources are now ubiquitous. And the market is robust, supported by legions of informal entrepreneurs selling phones, airtime, and even, in areas without electricity, charging services. Juma suggests that similar models will shape the diffusion of computers and Internet services in Africa—with rapid innovation in turn leading to unexpected solutions to the problems of low-productivity agriculture. He comments, “few technologies have the potential to revolutionize the African agricultural sector as much as the Internet.” Similarly, nanotechnologies can lead to rapid diagnosis of plant and animal diseases and can allow the development of cheap and

need for investments in human capital: health care and education. Nonetheless, he glosses over the costs and difﬁculties of providing this physical and social infrastructure. Road building is an unavoidably “old technology” that requires a lot of boots on the ground and a lot of earth-moving equipment. Education similarly requires time, teachers, and at least modest facilities. These are not insurmountable problems, but Juma offers few insights into how African countries can meet the challenges they present. He invokes “regional cooperation” as the solution to many problems of physical infrastructure, and his discussion of education focuses mostly on ways to include agriculture in the curriculum. He suggests that the dismal state of educational systems in many African countries should be viewed as “an opportunity for governments to adopt more community-driven models that prioritize education in a holistic way that improves community involvement, child achievement, agricultural production, and the standard of living for rural populations.” Language like this offers little concrete guidance. In summary, Juma’s account succeeds in offering a glimpse of the possible. The book provides a welcome relief from the gloom and despair in popular narratives about African agriculture. But some of the optimistic anecdotes seem to come uncritically from the pitches and prospectuses of ambitious researchers and nongovernmental organizations. The book’s sense of optimism appears to be driven less by the changes taking place in rural Africa than by the conversations taking place around Boston, Massachusetts, where the author is based. Overall, The New Harvest would be more compelling if Juma had matched his discussion of possibilities with a frank assessment of the political, technical, and economic difﬁculties that face African countries and institutions. History teaches us never to doubt the power of science and innovation to bring about change. In the long run, this lesson should apply no less in African agriculture than in other areas of human endeavor. But in the short run, many challenges and obstacles remain.

10.1126/science.1203125

CREDIT: RACHEL KRAMER

Subsistence rice farming in Madagascar.

EDUCATIONFORUM SCIENCE EDUCATION

Impact of Undergraduate Science Course Innovations on Learning

Despite revealing some positive impacts, studies too often suffer from weak design and inadequate reporting.

Maria Araceli Ruiz-Primo*, Derek Briggs, Heidi Iverson, Robert Talbot, Lorrie A. Shepard

Innovations: Concepts and Methods

Five inclusion criteria were used to screen articles during the literature search: (i) focus on undergraduate education in biology, chemistry, engineering, or physics; (ii) School of Education and Human Development, University of Colorado Denver, Denver, CO 80217, USA. *Author for correspondence. E-mail: maria.ruiz-primo@ ucdenver.edu

inclusion of one or more student-centered innovations; (iii) set in a “real-world” regular classroom and/or laboratory environment, as opposed to conducted in an education laboratory; (iv) published in a peer-reviewed journal between 1990 and 2007; and (v) results communicated in English. [See supporting online material (SOM) for details about study methods.] Of the 868 articles on course innovations gathered, 82, 18, 23, and 74 described at least one comparative study in biology, chemistry,

engineering, or physics, respectively. Comparative studies involve a contrast between students who have and have not received a given instructional innovation (i.e., treatment versus control), making it possible to evaluate the effect of course innovations on student learning. The unit of analysis was “study,” a unique set of data collected under a single research plan from a sample of respondents (9, 10). An article that reports results of multiple innovations, comparison groups, or outcome measures could have multiple studies. The ﬁnal pool included 98, 26, 38, and 148 studies in biology, FOUR TYPES OF INNOVATIONS STUDIED chemistry, engineering, and physics, respectively. Conceptually Oriented Tasks (COTs): Studies were coded on two •elicit students’ level of understanding of key science concepts; dimensions: conceptual char•identify students’ misconceptions, “common sense” knowledge at acteristics of the innovations, odds with actual concepts that are known to affect students’ and methodological characterlearning; istics of the study designs. This •engage students in conceptual schemes within a topic rather than approach permitted us to cluster isolated facts; help students focus on methods of problem conceptually and/or methodrepresentation and approaches to think about the problem ologically similar studies (10). components (solving strategies); Periodic agreement checks •engage students with real-world problems in creative ways that revealed acceptable intercoder reflect a conceptually integrated understanding of the content agreement: 89% on average. Collaborative Learning (CL) Activities: Analysis of conceptual char•engage students with peers (groups as small as pairs) as a acteristics indicated that most component of the learning process; studies (69%) involved more •provide students the opportunity to engage in explanations and than one innovation. The most discussions as they describe their reasoning, interpretations, and frequent combination included solutions to problems. COTs and CL (26%). Examples of specific, well-known Technology (TECH): programs that were given this •helps students visualize processes and/or concepts; combination of codes included •helps students manipulate variables by collecting and/or analyzing Tutorials in Introductory Physdata that can help them understand a concept or a process or ics (11) and Powerful Ideas in solve real-world problems; Physical Science (12, 13), in •helps students test theories and models with simulated data; which students work in small •provides feedback (online homework, such as problem sets). groups collaboratively on conInquiry-Based Projects (IBPs): ceptual questions designed to elicit and resolve students’ com•provide students the opportunity to undertake research projects mon misconceptions. The com(may or may not be in a real-world setting) that require more than one class period to complete; bination of COT, CL, and TECH codes was also quite common •require students to develop a procedure and/or plan to complete (27%) for established prothe project; grams of instruction in physics. •require students to follow a structured procedure and/or plan For example, in Peer Instrucprovided in advance as a framework to complete the project. tion (3), students use electronic Four types of innovation studied. clickers to respond to conceptu-

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t many colleges and universities, the traditional model of science instruction—a professor lecturing a large group of students—is being transformed into one in which students play a more active role in learning. This has been attributed to mounting evidence that traditional lectures, recitations, and laboratory sessions do not guarantee that students develop deep understanding of critical concepts (1–5). Since 1991, the National Science Foundation (NSF) increased its support to research projects that reconceptualize undergraduate science instruction. The resulting increase of “student-centered” instructional innovations studies raises questions, for example: How do these undergraduate course innovations vary? Do student-centered innovations in undergraduate science courses have a positive impact on student learning? These types of questions motivated a 3-year, NSF-funded research study that intended to characterize undergraduate course innovations described in published journal articles and to quantify their impact on student learning in biology, chemistry, engineering, and physics courses. We expand upon prior studies (6–8) to consider more types of innovations from more disciplines. Unlike other syntheses, we did not rely on terms used by authors to refer to their innovations, as names may reﬂect a general term (e.g., technology) with different meanings. Instead, we classiﬁed studies based on four, non–mutually exclusive innovations: conceptually oriented tasks (COTs), collaborative learning (CL), technology (TECH), and inquiry-based projects (IBPs) (see the ﬁrst table). Our categories allowed characterization of innovations in more detail than in previous syntheses (8).

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ally oriented questions before and after discussing the questions with their peers. Peer Instruction is also used in the discipline of biology with (14) and without (15, 16) the use of electronic clickers. In engineering, the most common innovations involve technology alone (32%), e.g., computer-based simulations from which “virtual experiments” can be conducted as they typically would be in a laboratory (17). Effect Sizes: Positive, but Variable

sible threats to the internal validity of most studies, because there are few examples in which students were randomly assigned to treatment and control conditions. To make matters worse, a substantial number of studies fail to administer pretests, making it impossible to rule out preexisting differences in achievement between groups (selection bias) that could artiﬁcially inﬂate or obscure the effect of the innovations. Effect sizes for comparative studies with random assignment are lower than those without random assignment, which indicates that the latter designs are likely to produce inflated estimates of effectiveness. Another important methodological threat to the validity of these studies is a lack of attention to technical characteristics of the instruments used to measure learning outcomes. For example, of the 71 physics studies included, the vast majority (92%) do not provide information about the validity and reliability of the instruments used. Only 3% of studies overall pay attention to these properties that enable a reader to conclude that a given test truly measures what it is claimed to measure. In the spirit of improving scientific research of instructional innovations, we make the following recommendations, which we view as a joint responsibility of funding agencies, researchers, journal editors, and reviewers. First, all studies need to include descriptive statistics (sample sizes, means, standard deviations) for all treatment and control groups on all testing occasions. Second, whenever possible, researchers should attempt to randomly assign students to treatment and control conditions. When this is not possible, efforts should be made to demonstrate that the groups are comparable

An effect size statistic was used to compare instructional innovations on student learning, expressing different outcome measures on a common scale, i.e., the mean effect as a proportion of the standard deviation of the outcome variable (18) (see the second table). Many of the studies did not report summary statistics needed to compute an effect size (means and standard deviations); thus, 46% of 310 studies had to be excluded from the synthesis (the highest percentage in physics, 52%). Only 18 (11%) of the remaining 166 studies involved random assignment of students to treatment or control conditions (experimental design). The most frequent design (89%) can be classiﬁed as a “quasiexperiment” (19), in that students were compared across treatment and control conditions, but were not assigned to these conditions at random. Further, 64% of quasi-experimental studies did not include a pretest to establish baseline conditions before the intervention. The variability of effect sizes within and across disciplines was substantial. The average effect sizes (20) found in biology (0.54) and physics (0.59) were considerably larger than those found in chemistry (0.27) and engineering (0.08). Quality of research design played a clear role, with the mean effect size for randomized experiments EFFECT SIZES OF INNOVATIONS AND (0.26) considerably smaller than for COMBINATIONS OF INNOVATIONS quasi-experiments (0.50). Caveats and Recommendations

This evidence suggests that undergraduate course innovations in biology, chemistry, engineering, and physics have positive effects on student learning. However, some caveats are in order. First, as mentioned, almost half of the comparative studies collected for review had to be excluded because they lacked the simple descriptive statistics needed to compute an effect-size estimate. It is unknown whether these results generalize to what would have been found from the excluded studies. Second, it is difﬁcult to rule out plau-

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Innovations

n

Mean

Median

SD

COTs

9

0.47

0.22

0.70

CL

5

0.68

0.69

0.16

TECH

30

0.37

0.38

0.38

COTs+CL

41

0.54

0.54

0.43

COTs+TECH

18

0.41

0.44

0.61

TECH+IBP

9

–0.11

–0.25

0.67

COTs+CL+TECH

39

0.46

0.50

0.50

Other

15

0.84

0.74

0.65

Total

166

0.47

0.47

0.54

before the treatment with respect to variables (e.g., prior academic achievement). Finally, researchers should be attentive to the quality of their outcome measures; if measures are not valid and reliable, subsequent interpretations can become equivocal. Although the poor quality of some research in this ﬁeld, and the speciﬁc shortcomings that commonly undermine studies, have been discussed before, journals continue publishing these types of papers. We are hopeful that our new analyses provide more simple and straightforward emphasis on these critical issues. Experts in experimental research and methodology in education and experts in educational assessment can contribute a great deal to improve research on instructional innovations in science. References and Notes

1. I. Halloun, D. Hestenes, Am. J. Phys. 53, 1043 (1985). 2. D. Hestenes, Am. J. Phys. 66, 465 (1998). 3. E. Mazur, Peer Instruction (Series in Educational Innovation, Prentice Hall, Upper Saddle River, NJ, 1997). 4. L. C. McDermott, E. F. Redish, Am. J. Phys. 67, 755 (1999). 5. L. C. McDermott, P. S. Shaffer, M. D. Somers, Am. J. Phys. 62, 46 (1994). 6. R. J. Beichner et al., in Research-Based Reform of University Physics, edited by E. F. Redish and P. J. Cooney [Reviews in Physics Education Research (PER), American Association of Physics Teachers, College Park, MD, 2007]; www.per-central.org/document/ServeFile.cfm?ID=4517. 7. L. Springer, M. E. Stanne, S. S. Donovan, Rev. Educ. Res. 69, 21 (1999). 8. R. R. Hake, Am. J. Phys. 66, 64 (1998). 9. G. V. Glass, Educ. Res. 5, 3 (1976). 10. M. W. Lipsey, D. B. Wilson, Practical Meta-Analysis (SAGE Publications, Thousand Oaks, CA, 2001). 11. L. C. McDermott, P. S. Shaffer, Tutorials in Introductory Physics (Prentice Hall, Upper Saddle River, NJ, 1998). 12. D. P. Casavant et al., Powerful Ideas in Physical Science (American Association of Physics Teachers, College Park, MD, ed. 3, 2001); www.aapt.org/Publications/pips_ samples/cover.pdf. 13. L. Liang, D. Gabel, Int. J. Sci. Educ. 27, 1143 (2005). 14. S. Freeman, et al.., Educ. Life Sci. Educ. 6, 132 (2007). 15. M. J. Giuliodori, H. L. Lujan, S. E. DiCarlo, Adv. Physiol. Educ. 30, 168 (2006). 16. S. P. Rao, S. E. DiCarlo, Adv. Physiol. Educ. 24, 51 (2000). 17. T. F. Wiesner, Q. Lan, J. Eng. Educ. 93, 195 (2004). 18. Given two studies with the same effect, an effect-size statistic will be larger if one study has more homogenous students (smaller standard deviation) than the other. 19. W. R. Shadish, T. D. Cook, D. T. Campbell, Experimental and Quasi-Experimental Designs for Generalized Causal Inference (Houghton Mifﬂin, Boston, MA, 2002). 20. We report the mean effect size without weighting by sample size of each study, despite the inferential argument in favor of weighting the effect sizes of studies as a function of their “precision,” which will be highest for those studies involving the largest samples of students. However, such an approach rests on the assumption that each study represents a “sample” from some larger study “population.” We do not feel such an assumption is warranted here, so we avoid the computation of standard errors for inferential purposes. Instead, we prefer to view our effect sizes as descriptive statistics.

Supporting Online Material

www.sciencemag.org/cgi/content/full/331/6022/1269/DC1

“Other” refers to innovation combinations wherein fewer than ﬁve studies were combined. See SOM for details.

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EDUCATIONFORUM

PERSPECTIVES MICROBIOLOGY

Listeria Unwinds Host DNA

A bacterium uses a viral strategy to manipulate the host cell immune response and optimize conditions for infection.

CREDIT: Y. HAMMOND/SCIENCE

I

nterferons (IFNs) are released by mammalian cells upon attack by microbial pathogens, alerting neighboring cells to prepare a defense that includes the activation of so-called IFN-stimulated genes. Although this response nearly always limits viral replication, its role during bacterial infection has not been clear. In some cases, the IFN response accelerates bacteria clearance, but in other cases, it results in a more severe disease (1, 2). The latter is true for Listeria monocytogenes, the bacterium that causes a range of human illnesses, from gastroenteritis to fatal meningitis. On page 1319 of this issue, Lebreton et al. (3) identify a new virulence factor, LntA, secreted by L. monocytogenes, that controls the expression of IFN-stimulated genes. The mechanism allows the bacterium to govern both the induction and repression of the host cell immune response, perhaps to optimize conditions for speciﬁc stages of infection or colonization of speciﬁc tissues. The expression of IFN-stimulated genes during infection occurs in response to cell signaling cascades that activate two families of transcription factors: nuclear factor κB and the interferon response factors (IRFs), in particular IRF-3/7 and IRF-9. These genes are also subject to repression by protein complexes that promote the formation of heterochromatin (which is transcriptionally inactive) (4). Lebreton et al. show that L. monocytogenes triggers the assembly of protein complexes that silence the expression of IFNstimulated genes in infected epithelial cells (see the figure). These complexes contain BAHD1, a protein that promotes the formation of heterochromatin (5). The complexes also contain HP1 and TRIM28 (or KAP-1), proteins that recruit and coordinate the structural elements of heterochromatin. Lebreton et al. show that infection of epithelial cells induces expression of type III IFNs (or IFN-λ). These cytokines comprise IL-28A, IL-28B, and IL-29, and are structurally distinct from type I and type II IFNs (6, 7). Type III IFN production would normally trigger expression of downstream IFN-stimulated genes; however, these are the genes subject to BAHD1-mediated represDepartment of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia 75015, Canada. E-mail: [email protected]

sion. In addition to suppressing this host immune response, Lebreton et al. also discovered that L. monocytogenes produces LntA, which relieves the repression of IFNstimulated genes by blocking the recruitment of BAHD1-containing silencing complexes. Thus, the bacterium secretes a factor that promotes the host cell immune response. Until now, direct evidence that bacterial pathogens produce factors that modify chromatin has been lacking (8). The ﬁndings of Lebreton et al. provide a strong foundation for making inroads into the role of type III IFNs and their importance in infection. Why has Listeria evolved a mechanism to induce a strong IFN response? How does this result in more severe disease? One effect of IFN signaling is to induce arrest of the cell division cycle (9). This may be important to L. monocytogenes, given that other gastrointestinal pathogens produce factors that block cell-cycle progression to favor condi-

tions for colonization (10) and dissemination. Other pathogens take advantage of proinﬂammatory conditions in the gut to favor colonization (11). Many viruses govern chromatin in a spatiotemporal manner in accordance with their infection strategy. Some establish long-term latent infections in host cells in which viral genomes are silenced, allowing evasion of the host’s immune surveillance system. Conversely, reactivation of latent viral genomes involves dissociation of silencing complexes. For example, TRIM28-containing complexes silence viral genes required for the lytic cycle of some viruses (12). Speciﬁc viral factors target such silencing complexes and enable a latent-to-lytic switch (13). Lebreton et al. deduced that L. monocytogenes also couples chromatin control with speciﬁc stages of its infection from the surprising observation that infection of mice with strains that either lacked lntA or constitutively expressed lntA Virus

Bacteria Early infection BAHD1 TRIM28

Viral latency Host gene expression off

HP1

? TRIM28

Viral gene expression off

HP1

IFNstimulated genes

Lytic genes

Late infection

Lytic phase

?

LntA

BAHD1 TRIM28

HP1

Downloaded from www.sciencemag.org on March 10, 2011

John R. Rohde

Bacterial protein relieves repression

? TRIM28

HP1

Viral protein relieves repression

Host gene expression on

Host immune response

Viral gene expression on

Entry into lytic phase

Microbial control of heterochromatin. In response to infection by the bacterium L. monocytogenes, host cell immune response genes (IFN-stimulated genes) are repressed by host proteins that form a silencing complex. The bacterial protein LntA relieves this repression. For some viral infections, lytic viral genes are silenced by host cell complexes similar to those controlled by L. monocytogenes. Viral factors that promote either latency or entry to lytic phase must directly or indirectly control the activity of these silencing complexes. In both cases, the spatial and temporal signals that govern these important decisions remain unclear.

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PERSPECTIVES lence factors into the host is an oversimpliﬁed view. The delivery of virulence factors by bacteria that use a type III secretion system is subject to temporal delivery rather than an all-ornone response. Salmonella, for example, control the timing and hierarchy of effector delivery through its syringe-like apparatus into host cells (15). Similarly, Yersinia inject an effector (YopK) into host cells where it then acts as a throttle to regulate the delivery of subsequent effectors (16). Unveiling the mechanisms that control when and where bacteria deliver their virulence factors may help develop strategies to combat them.

3. A. Lebreton et al., Science 331, 1319 (2011). 4. S. Kamitani et al., Biochem. Bios. Res. Commun. 370, 366 (2008). 5. H. Bierne et al., Proc. Natl. Acad. Sci. U.S.A. 106, 13826 (2009). 6. S. V. Kotenko et al., Nat. Immunol. 4, 69 (2003). 7. P. Sheppard et al., Nat. Immunol. 4, 63 (2003). 8. M. A. Hamon, P. Cossart, Cell Host Microbe 4, 100 (2008). 9. L. Dumoutier et al., J. Biol. Chem. 279, 32269 (2004). 10. J. Cui et al., Science 329, 1215 (2010). 11. S. E. Winter et al., Nature 467, 426 (2010). 12. D. Wolf, S. P. Goff, Cell 131, 46 (2007). 13. P. C. Chang et al., Cancer Res. 69, 5681 (2009). 14. A. P. Bhavsar et al., Nature 449, 827 (2007). 15. M. Lara-Tejero et al., Science 331, 1188 (2011); 10.1126/science.1201476. 16. R. Dewoody, P. M. Merritt, A. S. Houppert, M. M. Marketon, Mol. Microbiol. 10.1111/ j.1365-2958.2011.07534.x (2011).

References

1. R. M. O’Connell et al., J. Exp. Med. 200, 437 (2004). 2. S. Stockinger, T. Decker, Immunobiology 213, 889 (2008).

10.1126/science.1203271

CELL BIOLOGY

Rhodopsin as Thermosensor?

A molecule that senses light is also important for temperature discrimination.

Baruch Minke and Maximilian Peters

H

ow does an organism whose internal temperature varies with ambient temperature—a poikilotherm— sense temperature and choose the right habitat? On page 1333 in this issue, Shen et al. (1) report that temperature discrimination by ﬂy larvae involves the molecule rhodopsin, a molecule known to function in visual sensation. The ﬁnding raises questions about different roles for rhodopsin that depend on the cellular context. Transient receptor potential (TRP) ionchannel subfamilies (TRPV, TRPM, and TRPA) function as thermosensors in vertebrates and invertebrates, including worms, ﬂies, and mammals (2). In ﬂy larvae (Drosophila melanogaster), which survive (and grow) in a limited range of 18° to 24°C, thermosensation involves TRPA1 (3). To search for a receptor that activates the channel through a thermosensation cascade, Shen et al. used a behavioral assay in which Drosophila larvae choose between an ideal temperature of 18°C and a comfortable temperature range of 19° to 24°C. Surprisingly, these experiments showed that the visual pigment rhodopsin (found primarily in photoreceptor cells) functions in thermosensation because Department of Medical Neurobiology, The Institute of Medical Research Israel-Canada (IMRIC) and the Kühne Minerva Center for Studies of Visual Transduction, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem 91120, Israel. E-mail: [email protected]

1272

GDP

Gαq

AF

AF

R

GTP

GTP

γ β

γ β

GDP Na+ Gαq

Ca2+

PLC-β

R TRPA1

Heat

Not exclusive to light. In the proposed thermosensation cascade of Drosophila larvae, a change in temperature activates rhodopsin (R), which interacts with a still-unidentiﬁed accessory factor (AF) that accelerates its intrinsic thermal activity. Rhodopsin can also absorb photons of light, but this has no effect on its thermal activation. Heat-activated rhodopsin (RHA) then activates a heterotrimeric G protein (Gq) by promoting GDP (guanosine 5′-diphosphate) to GTP (guanosine 5′-triphosphate) exchange. This then activates PLC-β, and opens TRPA1 channels in a yet unknown manner. [The ﬁgure is derived from (4) with permission from B. Katz and B. Minke]

mutant ﬂies lacking rhodopsin lost their thermosensitivity in the comfortable temperature range (preference for 18°C). In addition, wild-type larvae preferred 18°C even in the dark, where photoactivation of rhodopsin does not occur. Illumination also did not affect wild-type larva thermosensation even though rhodopsin is activated under this condition. This suggests that, in this particular assay, rhodopsin functions as a thermosensor and not in its known role as a light sensor. The authors also found that thermal sensitivity is

not restricted to ﬂy rhodopsin. Indeed, the phenotype of rhodopsin-deﬁcient ﬂies was rescued by expression of murine melanopsin, the photopigment involved in regulating circadian rhythm in mice. The ﬁndings of Shen et al. raise several questions about rhodopsin. One issue pertains to rhodopsin’s extreme thermostability. In Drosophila photoreceptor cells, absorption of a single photon of light by rhodopsin isomerizes the retinoid chromophore from the 11-cis to the all-trans form, inducing a

11 MARCH 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

had a similar effect—a decrease in bacterial burden in the animals. Thus, tight control of LntA expression during the infection may be necessary for the bacteria to control the host immune response and ensure their survival. The authors did identify two bacterial factors required for LntA production, but the spatiotemporal cues for this regulation remain unknown. Likewise, the signaling events that result in the BAHD1-mediated repression of INF-stimulated genes in response to L. monocytogenes infection are unknown. Invading microbes are sensed by surveillance mechanisms of the host’s innate immune system; successful pathogens deploy factors that interrupt this process (14). Tight control of lntA expression would be in line with growing evidence that constitutive delivery of viru-

PERSPECTIVES lence factors into the host is an oversimpliﬁed view. The delivery of virulence factors by bacteria that use a type III secretion system is subject to temporal delivery rather than an all-ornone response. Salmonella, for example, control the timing and hierarchy of effector delivery through its syringe-like apparatus into host cells (15). Similarly, Yersinia inject an effector (YopK) into host cells where it then acts as a throttle to regulate the delivery of subsequent effectors (16). Unveiling the mechanisms that control when and where bacteria deliver their virulence factors may help develop strategies to combat them.

3. A. Lebreton et al., Science 331, 1319 (2011). 4. S. Kamitani et al., Biochem. Bios. Res. Commun. 370, 366 (2008). 5. H. Bierne et al., Proc. Natl. Acad. Sci. U.S.A. 106, 13826 (2009). 6. S. V. Kotenko et al., Nat. Immunol. 4, 69 (2003). 7. P. Sheppard et al., Nat. Immunol. 4, 63 (2003). 8. M. A. Hamon, P. Cossart, Cell Host Microbe 4, 100 (2008). 9. L. Dumoutier et al., J. Biol. Chem. 279, 32269 (2004). 10. J. Cui et al., Science 329, 1215 (2010). 11. S. E. Winter et al., Nature 467, 426 (2010). 12. D. Wolf, S. P. Goff, Cell 131, 46 (2007). 13. P. C. Chang et al., Cancer Res. 69, 5681 (2009). 14. A. P. Bhavsar et al., Nature 449, 827 (2007). 15. M. Lara-Tejero et al., Science 331, 1188 (2011); 10.1126/science.1201476. 16. R. Dewoody, P. M. Merritt, A. S. Houppert, M. M. Marketon, Mol. Microbiol. 10.1111/ j.1365-2958.2011.07534.x (2011).

References

1. R. M. O’Connell et al., J. Exp. Med. 200, 437 (2004). 2. S. Stockinger, T. Decker, Immunobiology 213, 889 (2008).

10.1126/science.1203271

CELL BIOLOGY

Rhodopsin as Thermosensor?

A molecule that senses light is also important for temperature discrimination.

Baruch Minke and Maximilian Peters

H

ow does an organism whose internal temperature varies with ambient temperature—a poikilotherm— sense temperature and choose the right habitat? On page 1333 in this issue, Shen et al. (1) report that temperature discrimination by ﬂy larvae involves the molecule rhodopsin, a molecule known to function in visual sensation. The ﬁnding raises questions about different roles for rhodopsin that depend on the cellular context. Transient receptor potential (TRP) ionchannel subfamilies (TRPV, TRPM, and TRPA) function as thermosensors in vertebrates and invertebrates, including worms, ﬂies, and mammals (2). In ﬂy larvae (Drosophila melanogaster), which survive (and grow) in a limited range of 18° to 24°C, thermosensation involves TRPA1 (3). To search for a receptor that activates the channel through a thermosensation cascade, Shen et al. used a behavioral assay in which Drosophila larvae choose between an ideal temperature of 18°C and a comfortable temperature range of 19° to 24°C. Surprisingly, these experiments showed that the visual pigment rhodopsin (found primarily in photoreceptor cells) functions in thermosensation because Department of Medical Neurobiology, The Institute of Medical Research Israel-Canada (IMRIC) and the Kühne Minerva Center for Studies of Visual Transduction, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem 91120, Israel. E-mail: [email protected]

1272

GDP

Gαq

AF

AF

R

GTP

GTP

γ β

γ β

GDP Na+ Gαq

Ca2+

PLC-β

R TRPA1

Heat

Not exclusive to light. In the proposed thermosensation cascade of Drosophila larvae, a change in temperature activates rhodopsin (R), which interacts with a still-unidentiﬁed accessory factor (AF) that accelerates its intrinsic thermal activity. Rhodopsin can also absorb photons of light, but this has no effect on its thermal activation. Heat-activated rhodopsin (RHA) then activates a heterotrimeric G protein (Gq) by promoting GDP (guanosine 5′-diphosphate) to GTP (guanosine 5′-triphosphate) exchange. This then activates PLC-β, and opens TRPA1 channels in a yet unknown manner. [The ﬁgure is derived from (4) with permission from B. Katz and B. Minke]

mutant ﬂies lacking rhodopsin lost their thermosensitivity in the comfortable temperature range (preference for 18°C). In addition, wild-type larvae preferred 18°C even in the dark, where photoactivation of rhodopsin does not occur. Illumination also did not affect wild-type larva thermosensation even though rhodopsin is activated under this condition. This suggests that, in this particular assay, rhodopsin functions as a thermosensor and not in its known role as a light sensor. The authors also found that thermal sensitivity is

not restricted to ﬂy rhodopsin. Indeed, the phenotype of rhodopsin-deﬁcient ﬂies was rescued by expression of murine melanopsin, the photopigment involved in regulating circadian rhythm in mice. The ﬁndings of Shen et al. raise several questions about rhodopsin. One issue pertains to rhodopsin’s extreme thermostability. In Drosophila photoreceptor cells, absorption of a single photon of light by rhodopsin isomerizes the retinoid chromophore from the 11-cis to the all-trans form, inducing a

11 MARCH 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

had a similar effect—a decrease in bacterial burden in the animals. Thus, tight control of LntA expression during the infection may be necessary for the bacteria to control the host immune response and ensure their survival. The authors did identify two bacterial factors required for LntA production, but the spatiotemporal cues for this regulation remain unknown. Likewise, the signaling events that result in the BAHD1-mediated repression of INF-stimulated genes in response to L. monocytogenes infection are unknown. Invading microbes are sensed by surveillance mechanisms of the host’s innate immune system; successful pathogens deploy factors that interrupt this process (14). Tight control of lntA expression would be in line with growing evidence that constitutive delivery of viru-

PERSPECTIVES rhodopsin chromophore is required for both light absorption as well as proper trafﬁcking of the full-length rhodopsin protein to the cell surface (7). Therefore, without a chromophore, opsin is not functionally expressed and temperature discrimination is lost. Future experiments will be required to differentiate between these two roles of the rhodopsin chromophore and determine whether the nascent opsin is the thermosensor. Shen et al. also emphasize that TRPA1 channel activation via the rhodopsin-initiated enzymatic cascade (as opposed to direct temperature-induced activation of the channel) is advantageous because small temperature differences are ampliﬁed. Rhodopsin has supreme sensitivity to light. A single photon activates rhodopsin and triggers activation of many downstream signaling molecules, thus amplifying the response to the stimulus. However, it is not clear that there is ampliﬁcation triggered by rhodopsin in a thermosensation cascade. In the photoreceptor system, all signaling proteins are expressed at extremely high levels (millions of copies per cell), which is necessary for signal ampliﬁcation (4). However, the amount of rhodopsin in the thermosensitive larva cells is minuscule (close to the detection limit), and the amounts of Gq and PLC-β were not measured. Yet, given that the abundance of signaling proteins is key to signal ampliﬁcation, measuring the expression level of each of these components is essential to explain how they enable

larvae to discriminate a temperature difference of only a few degrees Celsius. Another crucial question arising from the ﬁndings of Shen et al. is where the temperature-sensitive cells or organ resides in ﬂy larvae. Clearly it is not in the eye, because eliminating rhodopsin from the photoreceptor cells made the ﬂies blind but not temperature insensitive. Recently, it was discovered that Drosophila larvae sense and avoid light through photoreceptors in their body wall (8). Do these or other neurons in this organ also mediate temperature sensation? The ﬁndings of Shen et al. open a new avenue for understanding temperature sensation. Previously, temperature-sensitive transcription factors and TRP ion channels were shown to participate in animal thermosensation. Perhaps the class of temperature-sensitive proteins will expand to include light-sensitive G protein–coupled receptors like rhodopsin.

Downloaded from www.sciencemag.org on March 10, 2011

major conformational change in rhodopsin. Rhodopsin is a G protein–coupled receptor whose isomerization ultimately leads to the opening of TRP channels through the activation of the enzyme phospholipase C–β (PLC-β) via a heterotrimeric G protein (Gq) (4). Nevertheless, the probability that either vertebrate (5) or ﬂy (6) rhodopsins undergo thermal isomerization has been estimated at once in ~1000 years at 24°C. If this were not the case, both the human visual system and that of Drosophila would suffer from the same thermal noise that causes false photon detection seen in digital cameras. To account for this thermostability, Shen et al. propose that an accessory factor might interact with rhodopsin and accelerate its intrinsic thermal activity (see the ﬁgure). Yet, despite numerous multidisciplinary studies on rhodopsin, no such factor has been detected. Identiﬁcation of this factor, however, is crucial for understanding how rhodopsin functions as a thermosensor. A solution to this dilemma could be the use of the opsin moiety of rhodopsin without the associated chromophore (retinoid) as a thermosensor. However, Shen et al. found that larvae lacking either vitamin A (a precursor for retinoids), or a protein required for generating the chromophore, also lost their thermal preference. Therefore, both the chromophore and the opsin are required for rhodopsin-mediated thermosensation. Complicating matters, in ﬂy photoreceptor cells, the

References

1. W. L. Shen et al., Science 331, 1333 (2011). 2. A. Dhaka, V. Viswanath, A. Patapoutian, Annu. Rev. Neurosci. 29, 135 (2006). 3. Y. Kwon, H. S. Shim, X. Wang, C. Montell, Nat. Neurosci. 11, 871 (2008). 4. B. Katz, B. Minke, Front. Cell. Neurosci. 3, 2 (2009). 5. K. W. Yau, G. Matthews, D. A. Baylor, Nature 279, 806 (1979). 6. R. C. Hardie et al., Neuron 36, 689 (2002). 7. K. Ozaki, H. Nagatani, M. Ozaki, F. Tokunaga, Neuron 10, 1113 (1993). 8. Y. Xiang et al., Nature 468, 921 (2010). 10.1126/science.1203482

ATMOSPHERIC SCIENCE

Aerosol Chemistry and the Deepwater Horizon Spill

Airborne observations reveal the role of less volatile organic compounds in forming atmospheric aerosols.

Hugh Coe

I

n Earth’s atmosphere, particles smaller than 1 µm, known as aerosols, scatter incoming solar radiation and act as sites for condensation of water during cloud formation. Human activities can alter this population of particles, thereby affecting climate and air quality (1, 2). Our inability to accurately predict the composition and mass of atmospheric aerosols, however, is inhibiting progress in both areas. Understanding the formation of organic aerosols, a large class of submicrometer particles (3), has Centre for Atmospheric Science, School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Manchester M13 9PL, UK. E-mail: hugh.coe@ manchester.ac.uk

proven to be a challenge; laboratory experiments have previously not been reconciled with field measurements ( 4, 5). Recent theoretical frameworks point to the importance of semivolatile organic compounds (SVOCs) and organic compounds of intermediate volatility (IVOCs) as precursors (6, 7), but investigators have lacked observational evidence. On page 1295 of this issue, de Gouw et al. (8) help to clarify the role of SVOCs and IVOCs in forming organic aerosols. Using airborne measurements taken downwind of the oil slick resulting from the 2010 Deepwater Horizon (DWH) accident in the Gulf of Mexico, they reveal that the oxidation of IVOCs and SVOCs in the

atmosphere plays a dominant role in forming organic particles. The DWH offshore drilling platform exploded on 20 April 2010, allowing crude oil to ﬂow from the ocean bed (9) until 15 July 2010. A substantial proportion of the oil reached the surface, and the more volatile fraction evaporated (see the figure). Researchers conducted two ﬂights over the spill during June 2010 to document its impact on regional air quality. De Gouw et al. identiﬁed a narrow plume of gaseous VOCs from the spill, but they observed a much broader plume of organic particles. Observations showed that the particle mass ﬂux and particle size increased with

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PERSPECTIVES rhodopsin chromophore is required for both light absorption as well as proper trafﬁcking of the full-length rhodopsin protein to the cell surface (7). Therefore, without a chromophore, opsin is not functionally expressed and temperature discrimination is lost. Future experiments will be required to differentiate between these two roles of the rhodopsin chromophore and determine whether the nascent opsin is the thermosensor. Shen et al. also emphasize that TRPA1 channel activation via the rhodopsin-initiated enzymatic cascade (as opposed to direct temperature-induced activation of the channel) is advantageous because small temperature differences are ampliﬁed. Rhodopsin has supreme sensitivity to light. A single photon activates rhodopsin and triggers activation of many downstream signaling molecules, thus amplifying the response to the stimulus. However, it is not clear that there is ampliﬁcation triggered by rhodopsin in a thermosensation cascade. In the photoreceptor system, all signaling proteins are expressed at extremely high levels (millions of copies per cell), which is necessary for signal ampliﬁcation (4). However, the amount of rhodopsin in the thermosensitive larva cells is minuscule (close to the detection limit), and the amounts of Gq and PLC-β were not measured. Yet, given that the abundance of signaling proteins is key to signal ampliﬁcation, measuring the expression level of each of these components is essential to explain how they enable

larvae to discriminate a temperature difference of only a few degrees Celsius. Another crucial question arising from the ﬁndings of Shen et al. is where the temperature-sensitive cells or organ resides in ﬂy larvae. Clearly it is not in the eye, because eliminating rhodopsin from the photoreceptor cells made the ﬂies blind but not temperature insensitive. Recently, it was discovered that Drosophila larvae sense and avoid light through photoreceptors in their body wall (8). Do these or other neurons in this organ also mediate temperature sensation? The ﬁndings of Shen et al. open a new avenue for understanding temperature sensation. Previously, temperature-sensitive transcription factors and TRP ion channels were shown to participate in animal thermosensation. Perhaps the class of temperature-sensitive proteins will expand to include light-sensitive G protein–coupled receptors like rhodopsin.

Downloaded from www.sciencemag.org on March 10, 2011

major conformational change in rhodopsin. Rhodopsin is a G protein–coupled receptor whose isomerization ultimately leads to the opening of TRP channels through the activation of the enzyme phospholipase C–β (PLC-β) via a heterotrimeric G protein (Gq) (4). Nevertheless, the probability that either vertebrate (5) or ﬂy (6) rhodopsins undergo thermal isomerization has been estimated at once in ~1000 years at 24°C. If this were not the case, both the human visual system and that of Drosophila would suffer from the same thermal noise that causes false photon detection seen in digital cameras. To account for this thermostability, Shen et al. propose that an accessory factor might interact with rhodopsin and accelerate its intrinsic thermal activity (see the ﬁgure). Yet, despite numerous multidisciplinary studies on rhodopsin, no such factor has been detected. Identiﬁcation of this factor, however, is crucial for understanding how rhodopsin functions as a thermosensor. A solution to this dilemma could be the use of the opsin moiety of rhodopsin without the associated chromophore (retinoid) as a thermosensor. However, Shen et al. found that larvae lacking either vitamin A (a precursor for retinoids), or a protein required for generating the chromophore, also lost their thermal preference. Therefore, both the chromophore and the opsin are required for rhodopsin-mediated thermosensation. Complicating matters, in ﬂy photoreceptor cells, the

References

1. W. L. Shen et al., Science 331, 1333 (2011). 2. A. Dhaka, V. Viswanath, A. Patapoutian, Annu. Rev. Neurosci. 29, 135 (2006). 3. Y. Kwon, H. S. Shim, X. Wang, C. Montell, Nat. Neurosci. 11, 871 (2008). 4. B. Katz, B. Minke, Front. Cell. Neurosci. 3, 2 (2009). 5. K. W. Yau, G. Matthews, D. A. Baylor, Nature 279, 806 (1979). 6. R. C. Hardie et al., Neuron 36, 689 (2002). 7. K. Ozaki, H. Nagatani, M. Ozaki, F. Tokunaga, Neuron 10, 1113 (1993). 8. Y. Xiang et al., Nature 468, 921 (2010). 10.1126/science.1203482

ATMOSPHERIC SCIENCE

Aerosol Chemistry and the Deepwater Horizon Spill

Airborne observations reveal the role of less volatile organic compounds in forming atmospheric aerosols.

Hugh Coe

I

n Earth’s atmosphere, particles smaller than 1 µm, known as aerosols, scatter incoming solar radiation and act as sites for condensation of water during cloud formation. Human activities can alter this population of particles, thereby affecting climate and air quality (1, 2). Our inability to accurately predict the composition and mass of atmospheric aerosols, however, is inhibiting progress in both areas. Understanding the formation of organic aerosols, a large class of submicrometer particles (3), has Centre for Atmospheric Science, School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Manchester M13 9PL, UK. E-mail: hugh.coe@ manchester.ac.uk

proven to be a challenge; laboratory experiments have previously not been reconciled with field measurements ( 4, 5). Recent theoretical frameworks point to the importance of semivolatile organic compounds (SVOCs) and organic compounds of intermediate volatility (IVOCs) as precursors (6, 7), but investigators have lacked observational evidence. On page 1295 of this issue, de Gouw et al. (8) help to clarify the role of SVOCs and IVOCs in forming organic aerosols. Using airborne measurements taken downwind of the oil slick resulting from the 2010 Deepwater Horizon (DWH) accident in the Gulf of Mexico, they reveal that the oxidation of IVOCs and SVOCs in the

atmosphere plays a dominant role in forming organic particles. The DWH offshore drilling platform exploded on 20 April 2010, allowing crude oil to ﬂow from the ocean bed (9) until 15 July 2010. A substantial proportion of the oil reached the surface, and the more volatile fraction evaporated (see the figure). Researchers conducted two ﬂights over the spill during June 2010 to document its impact on regional air quality. De Gouw et al. identiﬁed a narrow plume of gaseous VOCs from the spill, but they observed a much broader plume of organic particles. Observations showed that the particle mass ﬂux and particle size increased with

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PERSPECTIVES precursors of the organic particles. The saturation ratio (C*) of an organic compound is the concentration at which the compound will partition equally between the gas and condensed phases. Recently, researchers have used the saturation ratios of a range of compounds known to arise from the oxidation of precursor compounds in the atmosphere to sort these compounds by their volatility. This distribution is known as a volatility basis set, and investigators have used it to create models that have accurately represented measured organic aerosol mass (7, 10). This approach offers considerable potential for understanding aerosol formation, but it needs to be tuned to local conditions based on measurements. It has been used in chamber studies to show that less than 15% of the atmospheric particulate from the photo-oxidation of diesel (11) and wood smoke (12) arises from known precursor compounds of organic aerosol. These studies suggested that the bulk of the ﬁnal particulate was formed from IVOC (C* ≈ 103 to 106 µg m–3) and

SVOC (C* ≈ 10−1 to 103 µg m–3) precursors. These precursors already have low volatility, and their subsequent oxidation readily forms compounds with saturation concentrations that are lower than typical ambient particle concentrations (1 to 10 µg m–3) (3), driving particle formation by condensation. Perhaps the only positive outcome of the DWH disaster is that it provided de Gouw et al. with an ideal experiment to probe the contribution of IVOCs and SVOCs to organic aerosol formation. In particular, it provided a point source of a complex mixture of organic compounds with a wide range of volatilities, and a mobile sea surface that allowed the precursors to spread as a function of their volatilities. The C* value most consistent with the observations of the slick was 105 µg m–3. This value is much higher than the ambient particulate mass concentrations of around 10 µg m–3, implying that substantial processing of the precursor material takes place in the atmosphere on time scales of 2 hours or less. De Gouw et al. also point out that there is no enhancement of particulate organic matter in the narrow plume containing the most volatile material; this ﬁnding, which suggests that it is the IVOCs and SVOCs that are forming the particulate, is consistent with the results of earlier chamber studies. These direct observations are an important step in our understanding of organic aerosol. They highlight the urgent need to measure IVOCs and SVOCs and to identify their key components. This is a major challenge facing atmospheric measurement science. The work also indicates that the organic compounds responsible for particle formation are not the same compounds that control ozone formation, which are generally of higher volatility. This result may inﬂuence the future regulation of ozone and particulates.

Downloaded from www.sciencemag.org on March 10, 2011

distance from the source; this demonstrated that the organic particulate was formed in the atmosphere from the oxidation of precursor compounds emitted from the surface oil and subsequent condensation, and did not arise from other potential sources. The narrow plume of VOCs resulted from rapid evaporation of the most volatile components of freshly surfaced oil. However, using a simulation of oil spread over the ocean surface and a description of evaporation rate based on volatility, de Gouw et al. showed that different compounds evaporated over very different spatial scales, with less volatile compounds spreading over a wider area before evaporating. They used this approach to estimate the range of volatilities of the precursor compounds that contributed most to the formation of organic particulate. They showed that the size and persistence of the slick was consistent with the width of the atmospheric plume, and with the evaporation of hydrocarbons composed of 14 to 16 carbon atoms. These hydrocarbons were the most likely

Organic process. (A) Oil from the DWH well spread across the ocean surface in a narrow plume, from which the most volatile organic carbon compounds (VOCs) quickly evaporated. Semivolatile and intermediate-volatility compounds (SVOCs and IVOCs) spread over a wider area because they took longer to evaporate. Oxidation of IVOCs and SVOCs in the atmosphere contributed heavily to the formation of organic aerosols. (B) The distribution of mass fraction of the hydrocarbons as a function of their volatility in the oil, represented by the saturation concentration C* (8). At the DWH spill, the size of the oil slick that best matched the downstream plume of organic particulate was consistent with precursor compounds to aerosol formation with C* ≈ 105 µg m–3, which equates to hydrocarbons composed of 14 to 16 carbon atoms.

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10.1126/science.1203019

CREDIT: ADAPTED BY P. HUEY/SCIENCE

References

1. P. Forster 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). 2. B. Brunekreef, S. T. Holgate, Lancet 360, 1233 (2002). 3. Q. Zhang et al., Geophys. Res. Lett. 34, L13801 (2007). 4. R. Volkamer et al., Geophys. Res. Lett. 33, L17811 (2006). 5. C. L. Heald et al., Geophys. Res. Lett. 32, L18809 (2005). 6. N. M. Donahue et al., Atmos. Environ. 43, 94 (2009). 7. J. L. Jimenez et al., Science 326, 1525 (2009). 8. J. A. de Gouw et al., Science 331, 1295 (2011). 9. Final Report, National Commission on the BP Deepwater Horizon Oil Spill and Offshore Drilling (www.oilspillcommission.gov/ﬁnal-report). 10. A. L. Robinson et al., Science 315, 1259 (2007). 11. E. A. Weitkamp, A. M. Sage, J. R. Pierce, N. M. Donahue, A. L. Robinson, Environ. Sci. Technol. 41, 6969 (2007). 12. A. P. Grieshop, N. M. Donahue, A. L. Robinson, Atmos. Chem. Phys. Discuss. 8, 17095 (2008).

PERSPECTIVES PHYSIOLOGY

Crise de Foie, Redux?

Two nuclear factors work in concert to regulate circadian expression of metabolic genes.

David D. Moore

CREDIT: Y. HAMMOND/SCIENCE

D

www.sciencemag.org SCIENCE VOL 331 11 MARCH 2011 Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

isruption of circadian a rapid and high accumulation rhythms due to rotatof liver triglycerides and florid ing shift work affects hepatic steatosis. Similar, but less up to 20% of the workforce in marked results were observed industrialized countries, resultwith loss of Rev-erbα, potening in a nearly twofold greater tially as a consequence of comrisk of developing the metapensatory effects of the hepatic bolic syndrome in this populaexpression of its close relative, tion (1). The role of the circaRev-erbβ. However, increased dian clock in maintaining metliver fat was not observed in an abolic balance is conﬁrmed by earlier study of the loss of Revthe obesity, insulin resistance, erbα in a different genetic backand fatty liver observed in mice ground (7). with mutations in clock compoThe study by Feng et al. pronents (2). Fatty liver (steatosis, vides an example of an unexpector nonalcoholic fatty liver disedly speciﬁc metabolic function ease) is a major driver of metfor what might have been considabolic dysregulation (3). On ered a nonspeciﬁc transcriptional HDAC3 HDAC3 page 1315 of this issue, Feng cofactor. Particularly through its NCoR NCoR et al. (4) identify a molecular interaction with the general coAc RevLipogenic erbα mechanism that links circadian repressor NCoR, HDAC3 could genes off Ac disruption and fatty liver. potentially associate with many Histone deacetylase 3 transcription factors. In the liver, (HDAC3) removes acetyl however, this study suggests a groups from modiﬁed lysines near-exclusive relationship with in histone proteins. It is also a Rev-erbα (and Rev-erbβ). This is component of transcriptional reminiscent of the phenotype that repressor complexes associ- Metabolic power trio. Diurnal variation in recruitment of HDAC3 to the mouse liver results from loss of the steroid ated with the compaction of genome via interaction with Rev-erbα and NCoR results in a circadian rhythm of epi- receptor coactivator 2 (SRC-2) in DNA and proteins into chroma- genomic modiﬁcation and gene expression. Lack of HDAC3 in the liver results in unre- the mouse liver, which is similar tin. Focusing on the genome- strained lipogenesis and rapid development of fatty liver. to that of the glycogen-storage wide localization of HDAC3 disorder von Gierke’s disease, binding sites in mouse liver, Feng et al. recruitment to target sites. Indeed, decreased caused by decreased glucose-6-phosphatase observed a huge diurnal variation: More than HDAC3 recruitment followed genetic dele- expression (8). One apparent difference is that 99.9% of the 14,000 speciﬁc DNA binding tion of Rev-erbα in mouse liver. in the case of SRC-2, only a single gene tarsites identiﬁed in the daytime, when the mice What do Rev-erbα and HDAC3 do when get is involved, whereas HDAC3 affects more are inactive, vanish 12 hours later at night, they inﬁltrate into the liver chromatin of sleep- than 10,000 overlapping HDAC3 and Revwhen the mice are active and eating. Remark- ing mice? Both histone acetylation around erbα binding sites. However, this discrepably, the total amount of HDAC3 protein in the HDAC3–Rev-erbα sites and RNA poly- ancy is less relevant than it appears, because the liver did not vary. Binding of HDAC3 merase II occupancy of nearby target genes further studies with SRC-2 have revealed correlated with that of its nuclear receptor co- decline when the deacetylase is recruited, a more coordinated gene expression prorepressor (NCoR) partner, and also of Rev- and these responses are lost in liver lack- gram to control overall energy accretion (9), erbα, a constitutive transcriptional repressor. ing HDAC3. Thus, the diurnal recruitment and it is apparent that the 10,000 HDAC3– Rev-erbα activity has direct metabolic regu- of HDAC3 results in genome-wide rhythmic Rev-erbα sites do more than regulate triglyclatory effects in the liver (5). It is a core com- epigenomic modification (histone acetyla- eride accumulation. Thus, both co-regulaponent of the circadian clock (6), which func- tion) that modulates gene expression. tors direct highly coordinated metabolic protions in every cell of the mammalian body. The nearby targets include abundant genes grams, and it remains an intriguing question The congruence of the circadian oscillation involved in lipid biosynthesis, suggesting how a huge range of potential co-regulator– of Rev-erbα expression with HDAC3 occu- that HDAC3 and Rev-erbα shut down inap- co-regulator and co-regulator–transcription pancy of DNA suggested that the deacetylase propriate anabolic pathways such as lipogen- factor interactions are somehow ignored, and depends on the transcriptional repressor for esis during the normal fasting period (see the only those that drive their speciﬁc metabolic ﬁgure). Indeed, mice genetically engineered outputs are allowed. to lack HDAC3 expression in liver displayed Another issue relates to the vexing probDepartment of Molecular and Cellular Biology, Baylor Colnot just a loss of the normal diurnal repression lem of comparing circadian and metabolic lege of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA. of genes such as fatty acid synthase, but also cycles. It will be a challenge to understand E-mail: [email protected]

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PERSPECTIVES the physiological integration of the HDAC3– Rev-erbα network with the presumably separate, but reinforcing, food-dependent effects of insulin, for example. Another important issue will be elucidating the speciﬁc effect of the HDAC3–Rev-erbα network on the metabolically enriched 10% of the liver transcriptome that shows circadian oscillation. So, is it time to disinter the French “crise de foie”? This mythical malady was considered a consequence of overindulgence, which certainly links it to fatty liver. Severe jet lag

decreases hepatic Rev-erbα mRNA expression (10), potentially producing the same effects seen in the Rev-erbα–deﬁcient mice studied by Feng et al. Moreover, acute circadian disruption can cause transient liver damage in mice, which is associated with disruption of bile acid homeostasis (11). Considering the links between shift workers and not only the metabolic syndrome, but potentially also liver damage (12, 13), it is possible that repeated circadian disruption may induce at least a modest liver crisis.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

References

D. De Bacquer et al., Int. J. Epidemiol. 38, 848 (2009). J. Bass, J. S. Takahashi, Science 330, 1349 (2010). V. T. Samuel et al., Lancet 375, 2267 (2010). D. Feng et al., Science 331, 1315 (2011). L. Yin et al., Science 318, 1786 (2007). N. Preitner et al., Cell 110, 251 (2002). G. Le Martelot et al., PLoS Biol. 7, e1000181 (2009). A. R. Chopra et al., Science 322, 1395 (2008). A. R. Chopra et al., Cell Metab. 13, 35 (2011). E. Filipski et al., J. Natl. Cancer Inst. 97, 507 (2005). K. Ma et al., PLoS ONE 4, e6843 (2009). K. Nakamura et al., Ind. Health 36, 218 (1998). M. Mukai et al., Dig. Dis. Sci. 47, 549 (2002). 10.1126/science.1203194

The Deep Social Structure of Humankind

Primatology and anthropology converge on the uniqueness of human society.

Bernard Chapais

D

espite the variability of chimpanzee groups, it is possible to identify some common features that characterize a unique “chimpanzee society.” The same is possible with other social species except, apparently, our own. Given the extreme diversity of human societies, deﬁning a common denominator—or “deep structure” (1)—has appeared unrealistic. The problem lies in cumulative cultural evolution. Culture has generated so many elaborations of that deep structure that, now, it is hardly discernible, as if heavily embellished versions of the same alphabet letter now mask the letter itself. Two sources of data, however, can help circumvent this problem: comparing hunter-gatherer societies, which are the most informative groups for understanding the social evolution of humans; and comparing human huntergatherer societies to nonhuman primate societies. Together, these analyses enable us to identify the unique attributes of human society. Hill et al. (2) nicely illustrate this process on page 1286 of this issue by providing quantitative data on the social structure of huntergatherers that support a model of humankind’s deep social structure that was derived from a comparison with primate societies (1). Hill et al. present the ﬁrst data on actual residence and biological and afﬁnal (in-law) kinship patterns among more than 5000 members of 32 present-day foraging societies. Their ﬁndings validate and enrich an existing model (1) that highlights the disDepartment of Anthropology, University of Montreal, Post Ofﬁce Box 6128, Station Centre-ville, Montréal Quebec H3C 3J7, Canada. E-mail: [email protected]

1276

In contrast, human residence patterns are much more flexible, with both Group A Group B sexes staying or leaving. Hill et al. conﬁrm this pattern for hunter-gatherers. Fourth, because of dispersal in primates, relatives of different sexes, for example, brothers and sisters, stop interacting with each other around puberty. The human pattern is strikingly different: Dispersed Males Females Sibling-bonds Pair bonds kin maintain lifetime Creating human society. The unique multigroup structure of human bonds (3). In particular, societies may have originated with the linkage of kinship bonds and brother-sister bonds are spousal bonds after the evolution of pair bonding in the human lineage. manifest in several ethnoAccording to that model, the elementary unit of between-group alliances graphical classics, such was a pair bond (red) connecting the spouses’ kin living in distinct groups as marriages between (A and B) and linking the two sets of in-laws. cross-cousins (children tinctive features of human society compared of a brother and a sister) and special avuncuwith other primate societies. First, this model lar (uncle-nephew) bonds. In hunter-gatherer sees human societies as multilevel, nested bands, Hill et al. show that cross-sex siblings structures of alliances. Human groups are often reside together. Fifth, in large mixed-sex always part of more inclusive social entities, primate groups, individuals have preferential which themselves belong to even more inclu- bonds with their matrilineal (mother’s line) sive structures. In contrast, the vast majority kin, but patrilineal kin recognition is either of primate societies are independent, single- absent or limited and fragmentary (5, 6). In group structures. Second, the vast majority marked contrast, kin recognition in humans of human groups are communities of fami- is bilineal and of unparalleled extent. Sixth, lies formed by conjugal partners (3), most of humans maintain preferential bonds with whom are monogamous, a remarkably unique their afﬁnes, or in-laws (spouses’ relatives pattern in the primate order (4). Third, in large and relatives’ spouses). This is a uniquely mixed-sex primate groups, either the males human feature (3, 7), and Hill et al. show that or the females move to another group around close and distant afﬁnes account for a large puberty, losing contact with their natal group proportion of coresident group members in permanently; dispersal is strongly sex-biased. hunter-gatherers.

11 MARCH 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

ANTHROPOLOGY

PERSPECTIVES the physiological integration of the HDAC3– Rev-erbα network with the presumably separate, but reinforcing, food-dependent effects of insulin, for example. Another important issue will be elucidating the speciﬁc effect of the HDAC3–Rev-erbα network on the metabolically enriched 10% of the liver transcriptome that shows circadian oscillation. So, is it time to disinter the French “crise de foie”? This mythical malady was considered a consequence of overindulgence, which certainly links it to fatty liver. Severe jet lag

decreases hepatic Rev-erbα mRNA expression (10), potentially producing the same effects seen in the Rev-erbα–deﬁcient mice studied by Feng et al. Moreover, acute circadian disruption can cause transient liver damage in mice, which is associated with disruption of bile acid homeostasis (11). Considering the links between shift workers and not only the metabolic syndrome, but potentially also liver damage (12, 13), it is possible that repeated circadian disruption may induce at least a modest liver crisis.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

References

D. De Bacquer et al., Int. J. Epidemiol. 38, 848 (2009). J. Bass, J. S. Takahashi, Science 330, 1349 (2010). V. T. Samuel et al., Lancet 375, 2267 (2010). D. Feng et al., Science 331, 1315 (2011). L. Yin et al., Science 318, 1786 (2007). N. Preitner et al., Cell 110, 251 (2002). G. Le Martelot et al., PLoS Biol. 7, e1000181 (2009). A. R. Chopra et al., Science 322, 1395 (2008). A. R. Chopra et al., Cell Metab. 13, 35 (2011). E. Filipski et al., J. Natl. Cancer Inst. 97, 507 (2005). K. Ma et al., PLoS ONE 4, e6843 (2009). K. Nakamura et al., Ind. Health 36, 218 (1998). M. Mukai et al., Dig. Dis. Sci. 47, 549 (2002). 10.1126/science.1203194

The Deep Social Structure of Humankind

Primatology and anthropology converge on the uniqueness of human society.

Bernard Chapais

D

espite the variability of chimpanzee groups, it is possible to identify some common features that characterize a unique “chimpanzee society.” The same is possible with other social species except, apparently, our own. Given the extreme diversity of human societies, deﬁning a common denominator—or “deep structure” (1)—has appeared unrealistic. The problem lies in cumulative cultural evolution. Culture has generated so many elaborations of that deep structure that, now, it is hardly discernible, as if heavily embellished versions of the same alphabet letter now mask the letter itself. Two sources of data, however, can help circumvent this problem: comparing hunter-gatherer societies, which are the most informative groups for understanding the social evolution of humans; and comparing human huntergatherer societies to nonhuman primate societies. Together, these analyses enable us to identify the unique attributes of human society. Hill et al. (2) nicely illustrate this process on page 1286 of this issue by providing quantitative data on the social structure of huntergatherers that support a model of humankind’s deep social structure that was derived from a comparison with primate societies (1). Hill et al. present the ﬁrst data on actual residence and biological and afﬁnal (in-law) kinship patterns among more than 5000 members of 32 present-day foraging societies. Their ﬁndings validate and enrich an existing model (1) that highlights the disDepartment of Anthropology, University of Montreal, Post Ofﬁce Box 6128, Station Centre-ville, Montréal Quebec H3C 3J7, Canada. E-mail: [email protected]

1276

In contrast, human residence patterns are much more flexible, with both Group A Group B sexes staying or leaving. Hill et al. conﬁrm this pattern for hunter-gatherers. Fourth, because of dispersal in primates, relatives of different sexes, for example, brothers and sisters, stop interacting with each other around puberty. The human pattern is strikingly different: Dispersed Males Females Sibling-bonds Pair bonds kin maintain lifetime Creating human society. The unique multigroup structure of human bonds (3). In particular, societies may have originated with the linkage of kinship bonds and brother-sister bonds are spousal bonds after the evolution of pair bonding in the human lineage. manifest in several ethnoAccording to that model, the elementary unit of between-group alliances graphical classics, such was a pair bond (red) connecting the spouses’ kin living in distinct groups as marriages between (A and B) and linking the two sets of in-laws. cross-cousins (children tinctive features of human society compared of a brother and a sister) and special avuncuwith other primate societies. First, this model lar (uncle-nephew) bonds. In hunter-gatherer sees human societies as multilevel, nested bands, Hill et al. show that cross-sex siblings structures of alliances. Human groups are often reside together. Fifth, in large mixed-sex always part of more inclusive social entities, primate groups, individuals have preferential which themselves belong to even more inclu- bonds with their matrilineal (mother’s line) sive structures. In contrast, the vast majority kin, but patrilineal kin recognition is either of primate societies are independent, single- absent or limited and fragmentary (5, 6). In group structures. Second, the vast majority marked contrast, kin recognition in humans of human groups are communities of fami- is bilineal and of unparalleled extent. Sixth, lies formed by conjugal partners (3), most of humans maintain preferential bonds with whom are monogamous, a remarkably unique their afﬁnes, or in-laws (spouses’ relatives pattern in the primate order (4). Third, in large and relatives’ spouses). This is a uniquely mixed-sex primate groups, either the males human feature (3, 7), and Hill et al. show that or the females move to another group around close and distant afﬁnes account for a large puberty, losing contact with their natal group proportion of coresident group members in permanently; dispersal is strongly sex-biased. hunter-gatherers.

11 MARCH 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on March 10, 2011

ANTHROPOLOGY

PERSPECTIVES erated pace. Cooperation in other primates is limited to the coordination of individuals belonging to the same group. The advent of the primitive tribe moved cooperation to substantially higher levels of complexity. It paved the way for the coordination of whole social groups, hence creating the nested character of human social structure. Such multilevel alliance structures raise challenging questions. For instance, what “cognitive prerequisites” were necessary for social groups to act as individual units and coordinate their actions in relation to other units? Did hominins, for example, require a theory of mind (the attribution of mental states to others) and shared intentionality (the recognition that I and others act as a collective working toward the same goal) (10) to achieve that level of cooperation?

References and Notes

1. B. Chapais, Primeval Kinship: How Pair-Bonding Gave Birth to Human Society (Harvard University Press, Cambridge, 2008). 2. K. R. Hill et al., Science 331, 1286 (2011). 3. L. Rodseth et al., Curr. Anthropol. 32, 221 (1991). 4. B. Chapais, in Oxford Handbook of Evolutionary Family Psychology, C. Salmon, T. Shackelford, Eds. (Oxford University Press, New York, 2011), pp. 33–50. 5. D. Rendall, in Kinship and Behavior in Primates, B. Chapais, C. Berman, Eds. (New York, Oxford University Press, 2004), pp. 295–316. 6. A. Widdig, Biol. Rev. Camb. Philos. Soc. 82, 319 (2007). 7. R. Fox, The Red Lamp of Incest (Dutton, New York, 1980). 8. K. E. Langergraber, J. C. Mitani, L. Vigilant, Proc. Natl. Acad. Sci. U.S.A. 104, 7786 (2007). 9. R. W. Wrangham, in The Evolution of Human Behavior: Primate Models, W. G. Kinzey, Ed. (SUNY Press, Albany, 1987), pp. 51–71. 10. M. Tomasello, Origins of Human Communication (MIT Press, Cambridge MA, 2008). 11. I thank R. Crépeau, D. Pérusse, and S. Teijeiro for helpful comments on the manuscript. 10.1126/science.1203281

CHEMISTRY

The Diamond Within a Silicon Analog of Cyclobutadiene Yitzhak Apeloig The distinctive rhombic structure of the central ring of a stable silicon analog of cyclobutadiene provides new insights into antiaromaticity.

O

rganic chemists recognized the distinctive stability and reactivity of aromatic molecules long before chemical bonding was explained in terms of quantum mechanical interactions of electrons. Early quantum mechanics studies by Hückel showed that molecules containing rings with 4n + 2 delocalized π-electrons (such as benzene, an n = 1 case) will gain extra stability [see, e.g., (1)]. Later studies showed that rings with 4n π-electrons, such as cyclobutadiene (C4H4), will be destabilized and highly reactive and they were termed antiaromatic (2). Understanding antiaromaticity in C4H4, an apparently simple molecule, has presented a challenge. The consensus view of its groundstate structure (1, 3) was upset by recent studies of 1,3-dimethylcyclobutadiene (4–7). Additional insights can be gained from studies of silicon (Si) analogs of C4H4, which are challenging to prepare because of the weakness and high reactivity of Si π-bonds [see Schulich Faculty of Chemistry and Lise Meitner Minerva Center for Computational Quantum Chemistry, Technion– Israel Institute of Technology, Haifa 32000, Israel. E-mail: [email protected]

(8) and references therein]. On page 1306 of this issue, Suzuki et al. (9) describe the synthesis and characterization of a derivative of the silicon analog of C4H4 (compound 1 in the ﬁrst ﬁgure, panel A) that has two conjugated Si=Si double bonds within a four-member ring. Their study provides an understanding of how the structure of 1 accommodates antiaromatic π-interactions. The reactivity of C4H4 is so high that it can be isolated only at low temperatures in frozen glasses of organic solvents (10, 11), or at ambient temperature by sequestering it in an unreactive molecular host that inhibits its dimerization (12). There are three possible models for the shape and ground-state electronic conﬁguration of C4H4. Theoretical and experimental studies have shown that the simplest model, the square-shaped molecule 2a (see the ﬁrst ﬁgure, panel B), is not the ground state. This structure unpairs the electrons in the highest occupied level and places them into two degenerate singly occupied molecular orbitals (SOMOs). As is often seen for highly symmetric structures where the degenerate highest occupied molecular orbitals (HOMOs) are not fully occupied, a symme-

www.sciencemag.org SCIENCE VOL 331 11 MARCH 2011 Published by AAAS

1277

Downloaded from www.sciencemag.org on March 10, 2011

That unique social structure leads to questions of how it evolved. A key event might have been the advent of pair bonding in the human lineage (see the ﬁgure). Our closest relatives, chimpanzees and bonobos, live in large mixed-sex groups. They are territorial, with male chimpanzees attacking strangers. They mate promiscuously, with both sexes having multiple short-term partners. They exhibit male residence and female transfer (the females move to new groups and the males stay), a pattern that translates into extensive patrilines of males that include their fathers, grandfathers, uncles, and other male kin. However, patrilineal kin do not recognize each other (8), so this genealogical structure is to a large extent “socially silent.” Now, suppose that pair bonding evolved in this type of social structure. This brought about the multifamily composition of human groups, with enduring associations between mothers and fathers enabling children to recognize their fathers. This, in turn, made it possible for children to recognize their father’s relatives; that is, pair bonding would reveal the underlying genealogical structure and create bilineal kinship. From then on, a father, upon meeting another group, would recognize his daughter (if she transferred to it), and his grand-offspring, and refrain from attacking them. He would also recognize his daughter’s preferential bond with her sexual mate (his “son-in-law”), a factor alleviating conﬂicts between male afﬁnes. Similarly, grandfathers, brothers and uncles would recognize their transferred kin and their afﬁnes, instigating a state of mutual tolerance. According to this model (1), the ﬁrst multigroup social entities evolved after sexual promiscuity gave way to pair bonding in mixed-sex hominin groups exhibiting male residence and female transfer, a residence pattern presumed to be homologous (similar through common descent) in humans and chimpanzees (9). In the nascent “tribe,” males were now able to circulate freely between groups in which they had kin and in-laws, cross-sex kin maintained lifetime bonds, and betweengroup alliances were ensured by kinship bonds, “marital” ties, and the ensuing extensive network of bonds between in-laws. Hill et al.’s ﬁndings offer empirical support to many aspects of this model. Somewhat paradoxically, then, the dramatic and fortuitous extension of kin recognition brought about by pair bonding would have launched the evolution of supragroup social structures in which a large proportion of individuals were now distantly related—just the pattern observed by Hill et al. At that point, hominin social evolution would have taken place at a new, accel-

PERSPECTIVES erated pace. Cooperation in other primates is limited to the coordination of individuals belonging to the same group. The advent of the primitive tribe moved cooperation to substantially higher levels of complexity. It paved the way for the coordination of whole social groups, hence creating the nested character of human social structure. Such multilevel alliance structures raise challenging questions. For instance, what “cognitive prerequisites” were necessary for social groups to act as individual units and coordinate their actions in relation to other units? Did hominins, for example, require a theory of mind (the attribution of mental states to others) and shared intentionality (the recognition that I and others act as a collective working toward the same goal) (10) to achieve that level of cooperation?

References and Notes

1. B. Chapais, Primeval Kinship: How Pair-Bonding Gave Birth to Human Society (Harvard University Press, Cambridge, 2008). 2. K. R. Hill et al., Science 331, 1286 (2011). 3. L. Rodseth et al., Curr. Anthropol. 32, 221 (1991). 4. B. Chapais, in Oxford Handbook of Evolutionary Family Psychology, C. Salmon, T. Shackelford, Eds. (Oxford University Press, New York, 2011), pp. 33–50. 5. D. Rendall, in Kinship and Behavior in Primates, B. Chapais, C. Berman, Eds. (New York, Oxford University Press, 2004), pp. 295–316. 6. A. Widdig, Biol. Rev. Camb. Philos. Soc. 82, 319 (2007). 7. R. Fox, The Red Lamp of Incest (Dutton, New York, 1980). 8. K. E. Langergraber, J. C. Mitani, L. Vigilant, Proc. Natl. Acad. Sci. U.S.A. 104, 7786 (2007). 9. R. W. Wrangham, in The Evolution of Human Behavior: Primate Models, W. G. Kinzey, Ed. (SUNY Press, Albany, 1987), pp. 51–71. 10. M. Tomasello, Origins of Human Communication (MIT Press, Cambridge MA, 2008). 11. I thank R. Crépeau, D. Pérusse, and S. Teijeiro for helpful comments on the manuscript. 10.1126/science.1203281

CHEMISTRY

The Diamond Within a Silicon Analog of Cyclobutadiene Yitzhak Apeloig The distinctive rhombic structure of the central ring of a stable silicon analog of cyclobutadiene provides new insights into antiaromaticity.

O

rganic chemists recognized the distinctive stability and reactivity of aromatic molecules long before chemical bonding was explained in terms of quantum mechanical interactions of electrons. Early quantum mechanics studies by Hückel showed that molecules containing rings with 4n + 2 delocalized π-electrons (such as benzene, an n = 1 case) will gain extra stability [see, e.g., (1)]. Later studies showed that rings with 4n π-electrons, such as cyclobutadiene (C4H4), will be destabilized and highly reactive and they were termed antiaromatic (2). Understanding antiaromaticity in C4H4, an apparently simple molecule, has presented a challenge. The consensus view of its groundstate structure (1, 3) was upset by recent studies of 1,3-dimethylcyclobutadiene (4–7). Additional insights can be gained from studies of silicon (Si) analogs of C4H4, which are challenging to prepare because of the weakness and high reactivity of Si π-bonds [see Schulich Faculty of Chemistry and Lise Meitner Minerva Center for Computational Quantum Chemistry, Technion– Israel Institute of Technology, Haifa 32000, Israel. E-mail: [email protected]

(8) and references therein]. On page 1306 of this issue, Suzuki et al. (9) describe the synthesis and characterization of a derivative of the silicon analog of C4H4 (compound 1 in the ﬁrst ﬁgure, panel A) that has two conjugated Si=Si double bonds within a four-member ring. Their study provides an understanding of how the structure of 1 accommodates antiaromatic π-interactions. The reactivity of C4H4 is so high that it can be isolated only at low temperatures in frozen glasses of organic solvents (10, 11), or at ambient temperature by sequestering it in an unreactive molecular host that inhibits its dimerization (12). There are three possible models for the shape and ground-state electronic conﬁguration of C4H4. Theoretical and experimental studies have shown that the simplest model, the square-shaped molecule 2a (see the ﬁrst ﬁgure, panel B), is not the ground state. This structure unpairs the electrons in the highest occupied level and places them into two degenerate singly occupied molecular orbitals (SOMOs). As is often seen for highly symmetric structures where the degenerate highest occupied molecular orbitals (HOMOs) are not fully occupied, a symme-

www.sciencemag.org SCIENCE VOL 331 11 MARCH 2011 Published by AAAS

1277

Downloaded from www.sciencemag.org on March 10, 2011

That unique social structure leads to questions of how it evolved. A key event might have been the advent of pair bonding in the human lineage (see the ﬁgure). Our closest relatives, chimpanzees and bonobos, live in large mixed-sex groups. They are territorial, with male chimpanzees attacking strangers. They mate promiscuously, with both sexes having multiple short-term partners. They exhibit male residence and female transfer (the females move to new groups and the males stay), a pattern that translates into extensive patrilines of males that include their fathers, grandfathers, uncles, and other male kin. However, patrilineal kin do not recognize each other (8), so this genealogical structure is to a large extent “socially silent.” Now, suppose that pair bonding evolved in this type of social structure. This brought about the multifamily composition of human groups, with enduring associations between mothers and fathers enabling children to recognize their fathers. This, in turn, made it possible for children to recognize their father’s relatives; that is, pair bonding would reveal the underlying genealogical structure and create bilineal kinship. From then on, a father, upon meeting another group, would recognize his daughter (if she transferred to it), and his grand-offspring, and refrain from attacking them. He would also recognize his daughter’s preferential bond with her sexual mate (his “son-in-law”), a factor alleviating conﬂicts between male afﬁnes. Similarly, grandfathers, brothers and uncles would recognize their transferred kin and their afﬁnes, instigating a state of mutual tolerance. According to this model (1), the ﬁrst multigroup social entities evolved after sexual promiscuity gave way to pair bonding in mixed-sex hominin groups exhibiting male residence and female transfer, a residence pattern presumed to be homologous (similar through common descent) in humans and chimpanzees (9). In the nascent “tribe,” males were now able to circulate freely between groups in which they had kin and in-laws, cross-sex kin maintained lifetime bonds, and betweengroup alliances were ensured by kinship bonds, “marital” ties, and the ensuing extensive network of bonds between in-laws. Hill et al.’s ﬁndings offer empirical support to many aspects of this model. Somewhat paradoxically, then, the dramatic and fortuitous extension of kin recognition brought about by pair bonding would have launched the evolution of supragroup social structures in which a large proportion of individuals were now distantly related—just the pattern observed by Hill et al. At that point, hominin social evolution would have taken place at a new, accel-

try-breaking process, called a but adopts an unprecedented rhomA Jahn-Teller (J-T) distortion lowbic or diamond-like structure—a Et Me R R Et Me ers the molecule’s energy. The structure predicted but not previ2 + Si Si 1 – ground-state structure of C4H4 ously observed in C4H4 derivatives. – Si3 R R Si R Si Si R results from a covalent J-T disAll four Si-Si distances are essen4 + Si Si Et Me 1,R = EMind tortion that leads to the rectantially the same and are intermediate Et Me R R gular molecule 2b (see the ﬁrst between typical Si–Si and Si=Si bond EMind ﬁgure, panel B). In this struclengths, so there is no bond alternaB ture, the two nonbonding election. The silicon atoms with acute Carbon Silicon trons in the SOMOs are paired inner-ring angles (76°), Si1 and Si3, Covalent J-T distortion Polar J-T distortion and occupy the HOMO, which have a pyramidal geometry, but the is lower in energy. This orbital silicon atoms with wide inner-ring SOMO can be viewed as two π(C=C) angles (103°), Si2 and Si4, are planar bonds with an antibonding (see the second ﬁgure). Suzuki et al. HOMO HOMO interaction between them. attribute the unusual structure of 1 to This understanding of the a charge-separated structure, as repHOMO-1 HOMO ground state of C4H4 was the resented by the electronic resonance HOMO-1 Si consensus view ( 1, 3) until structures (see the ﬁrst ﬁgure, panel 4e– Si 4e– Si 4e– 2010, when a debate erupted A), that becomes competitive with Si regarding the coexistence of other structures because of the rela2b 2c 3c 2a both the square 2a and the rectively weak π(Si=Si) bond and the tangular 2b structures and in ﬂexible silicon σ-bond framework. Antiaromaticity, silicon style. (A) Two resonance structures of compound 1,3-dimethylcyclobutadiene The study of Suzuki et al. is one 1, a tetrasilicon analog of the antiaromatic compound C4H4 synthesized by (4–7). A third structure of C4H4 Suzuki et al., are shown; the charge-separated resonance is shown on the of many that address the concepts 2c, could form through a polar right. The bulky substituent EMind is 1,1,7,7-tetraethyl-3,3,5,5-tetramethyl- of aromaticity and antiaromaticity, J-T distortion (see the ﬁrst ﬁgs-hydrindacen-4-yl. (B) Different models of the electronic structure of C4H4 which continue to generate wide and its Si analog are depicted by their occupied molecular orbitals. ure, panel B). In the HOMO, two interest and controversy [see (17, electrons are localized to only 18)]. An intriguing question that the atoms in positions 1 and 3, and they have a same type of compound. For example, C=C their results pose is whether EMind4C4 and nonbonding interaction. This planar rhombic bonds have a planar geometry, whereas Si=Si EMind4Ge4—the lighter and heavier conge(or “diamond”-like) structure was considered bonds are bent with pyramidal Si centers and ners of silicon, respectively—also adopt a theoretically (3) but has never been observed sometimes are twisted. Allene (C3H4) is lin- rhombic diamond-like structure like that of experimentally. ear, but its silicon analog is strongly bent (8). 1. Such studies may provide further insights Aromatic molecules containing silicon Previously reported cyclic Si4R4 deriva- into the consequences of antiaromaticity in have been synthesized and they exhibit aro- tives (where R is an organic group) were sta- small rings. maticity [see (13) and references therein], but bilized by complexation to a transition metal References formally antiaromatic systems containing sili- and have a central ring that is nearly square 1. F. A. Carey, R. J. Sundberg, Advanced Organic Chemistry con have only now been reported (9). The syn- planar [see (16) and references therein]. The (Springer, New York, ed. 5, 2007), part A. thesis of a Si analog of C4H4 1 by Suzuki et al. structure of 1 is surprising in that it is planar 2. R. Breslow, Acc. Chem. Res. 6, 393 (1973). 3. C. Gellini, P. R. Salvi, Symmetry 2, 1846 (2010). is remarkable in that Si=Si (and 4. Y.-M. Legrand, A. van der Lee, M. Barboiu, Science 329, Si=C) bonds are much less stable 299 (2010). than C=C bonds, and the Si4 ring 5. D. Scheschkewitz, Science 330, 1047-c (2010). 6. I. V. Alabugin, B. Gold, M. Shatruk, K. Kovnir, Science would be further destabilized by 330, 1047-d (2010). antiaromaticity. Large substitu7. Y.-M. Legrand, A. van der Lee, M. Barboiu, Science 330, ents such as the EMind group (see 1047-e (2010). 8. V. Y. Lee, A. Sekiguchi, Organic Compounds of Low Coorthe ﬁrst ﬁgure, panel A) help block dinate Si, Ge, Sn and Pb Compounds, from Phantom Spedimerization or the approach of cies to Stable Compounds (Wiley, Chichester, UK, 2010). reagents to the otherwise highly 9. K. Suzuki et al., Science 331, 1306 (2011). Si 2 10. O. L. Chapman, C. L. McIntosh, J. Pacansky, J. Am. Chem. reactive molecules (8, 14, 15). Soc. 95, 244 (1973). Suzuki et al. report a 9% yield 11. G. Maier, Angew. Chem. Int. Ed. Engl. 13, 425 (1974). 1 3 Si Si of 1 for a one-step reduction of 12. D. J. Cram, M. E. Tanner, R. Thomas, Angew. Chem. Int. 4 Ed. Engl. 30, 1024 (1991). Si (EMind)SiBr3. The very large 13. V. Y. Lee, A. Sekiguchi, Angew. Chem. Int. Ed. 46, 6596 EMind groups are effective at (2007). shielding the Si=Si bonds; the 14. R. West, M. J. Fink, J. Michl, Science 214, 1343 (1981). 15. A. G. Brook et al., J. Chem. Soc. Chem. Commun. 1981, half-life of 1 is about 3 days 191 (1981). in benzene solution at room 16. V. Y. Lee et al., Dalton Trans. 39, 9229 (2010). 17. Thematic issue on aromaticity, P. v. R. Schleyer, Ed., temperature. Chem. Rev. 101 (5), 7–1566 (2001). Comparisons of the structure 18. Thematic issue on aromaticity, P. v. R. Schleyer, Ed., of the central ring of 1 to C4H4 Chem. Rev. 105 (10), 3433–3947 (2005). Diamond ring setting. The x-ray crystal structure of 1 shows must consider the differences in how the four silicon atoms form a rhombus and are protected by 10.1126/science.1202157 how C and Si form bonds in the the large EMind groups.

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REVIEW moment when all these fields converged on a common paradigm for understanding the mind), the labels “Bayesian” or “probabilistic” are merely placeholders for a set of interrelated principles and theoretical claims. The key ideas can be thought of as proposals for how to answer three central questions:

How to Grow a Mind: Statistics, Structure, and Abstraction Joshua B. Tenenbaum,1* Charles Kemp,2 Thomas L. Griffiths,3 Noah D. Goodman4

The Challenge: How Does the Mind Get So Much from So Little? or scientists studying how humans come to understand their world, the central challenge is this: How do our minds get so much from so little? We build rich causal models, make strong generalizations, and construct powerful abstractions, whereas the input data are sparse, noisy, and ambiguous—in every way far too limited. A massive mismatch looms between the information coming in through our senses and the ouputs of cognition. Consider the situation of a child learning the meanings of words. Any parent knows, and scientists have confirmed (1, 2), that typical 2-yearolds can learn how to use a new word such as “horse” or “hairbrush” from seeing just a few examples. We know they grasp the meaning, not just the sound, because they generalize: They use the word appropriately (if not always perfectly) in new situations. Viewed as a computation on sensory input data, this is a remarkable feat. Within the infinite landscape of all possible objects, there is an infinite but still highly constrained subset that can be called “horses” and another for “hairbrushes.” How does a child grasp the boundaries of these subsets from seeing just one or a few examples of each? Adults face the challenge of learning entirely novel object concepts less often, but they can be just as good at it (Fig. 1). Generalization from sparse data is central in learning many aspects of language, such as syntactic constructions or morphological rules (3). It presents most starkly in causal learning: Every statistics class teaches that correlation does

F

1 Department of Brain and Cognitive Sciences, Computer Science and Artificial Intelligence Laboratory (CSAIL), Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. 2Department of Psychology, Carnegie Mellon University, Pittsburgh, PA 15213, USA. 3Department of Psychology, University of California, Berkeley, Berkeley, CA 94720, USA. 4Department of Psychology, Stanford University, Stanford, CA 94305, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

not imply causation, yet children routinely infer causal links from just a handful of events (4), far too small a sample to compute even a reliable correlation! Perhaps the deepest accomplishment of cognitive development is the construction of larger-scale systems of knowledge: intuitive theories of physics, psychology, or biology or rule systems for social structure or moral judgment. Building these systems takes years, much longer than learning a single new word or concept, but on this scale too the final product of learning far outstrips the data observed (5–7). Philosophers have inquired into these puzzles for over two thousand years, most famously as “the problem of induction,” from Plato and Aristotle through Hume, Whewell, and Mill to Carnap, Quine, Goodman, and others in the 20th century (8). Only recently have these questions become accessible to science and engineering by viewing inductive learning as a species of computational problems and the human mind as a natural computer evolved for solving them. The proposed solutions are, in broad strokes, just what philosophers since Plato have suggested. If the mind goes beyond the data given, another source of information must make up the difference. Some more abstract background knowledge must generate and delimit the hypotheses learners consider, or meaningful generalization would be impossible (9, 10). Psychologists and linguists speak of “constraints;” machine learning and artificial intelligence researchers, “inductive bias;” statisticians, “priors.” This article reviews recent models of human learning and cognitive development arising at the intersection of these fields. What has come to be known as the “Bayesian” or “probabilistic” approach to reverse-engineering the mind has been heavily influenced by the engineering successes of Bayesian artificial intelligence and machine learning over the past two decades (9, 11) and, in return, has begun to inspire more powerful and more humanlike approaches to machine learning. As with “connectionist” or “neural network” models of cognition (12) in the 1980s (the last

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1) How does abstract knowledge guide learning and inference from sparse data? 2) What forms does abstract knowledge take, across different domains and tasks? 3) How is abstract knowledge itself acquired? We will illustrate the approach with a focus on two archetypal inductive problems: learning concepts and learning causal relations. We then briefly discuss open challenges for a theory of human cognitive development and conclude with a summary of the approach’s contributions. We will also draw contrasts with two earlier approaches to the origins of knowledge: nativism and associationism (or connectionism. These approaches differ in whether they propose stronger or weaker capacities as the basis for answering the questions above. Bayesian models typically combine richly structured, expressive knowledge representations (question 2) with powerful statistical inference engines (questions 1 and 3), arguing that only a synthesis of sophisticated approaches to both knowledge representation and inductive inference can account for human intelligence. Until recently it was not understood how this fusion could work computationally. Cognitive modelers were forced to choose between two alternatives (13): powerful statistical learning operating over the simplest, unstructured forms of knowledge, such as matrices of associative weights in connectionist accounts of semantic cognition (12, 14), or richly structured symbolic knowledge equipped with only the simplest, nonstatistical forms of learning, checks for logical inconsistency between hypotheses and observed data, as in nativist accounts of language acquisition (15). It appeared necessary to accept either that people’s abstract knowledge is not learned or induced in a nontrivial sense from experience (hence essentially innate) or that human knowledge is not nearly as abstract or structured (as “knowledge-like”) as it seems (hence simply associations). Many developmental researchers rejected this choice altogether and pursued less formal approaches to describing the growing minds of children, under the headings of “constructivism” or the “theory theory” (5). The potential to explain how people can genuinely learn with abstract structured knowledge may be the most distinctive feature of Bayesian models: the biggest reason for their recent popularity (16) and the biggest target of skepticism from their critics (17).

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In coming to understand the world—in learning concepts, acquiring language, and grasping causal relations—our minds make inferences that appear to go far beyond the data available. How do we do it? This review describes recent approaches to reverse-engineering human learning and cognitive development and, in parallel, engineering more humanlike machine learning systems. Computational models that perform probabilistic inference over hierarchies of flexibly structured representations can address some of the deepest questions about the nature and origins of human thought: How does abstract knowledge guide learning and reasoning from sparse data? What forms does our knowledge take, across different domains and tasks? And how is that abstract knowledge itself acquired?

The Role of Abstract Knowledge Over the past decade, many aspects of higherlevel cognition have been illuminated by the

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mathematics of Bayesian statistics: our sense of similarity (18), representativeness (19), and randomness (20); coincidences as a cue to hidden causes (21); judgments of causal strength (22) and evidential support (23); diagnostic and conditional reasoning (24, 25); and predictions about the future of everyday events (26).

The claim that human minds learn and reason according to Bayesian principles is not a claim that the mind can implement any Bayesian inference. Only those inductive computations that the mind is designed to perform well, where biology has had time and cause to engineer effective and efficient mechanisms, are likely to

be understood in Bayesian terms. In addition to the general cognitive abilities just mentioned, Bayesian analyses have shed light on many specific cognitive capacities and modules that result from rapid, reliable, unconscious processing, including perception (27), language (28), memory (29, 30), and sensorimotor systems (31). In contrast, in tasks that require explicit conscious manipulations of probabilities as numerical quantities—a recent cultural invention that few people become fluent with, and only then after sophisticated training—judgments can be notoriously biased away from Bayesian norms (32). At heart, Bayes’s rule is simply a tool for answering question 1: How does abstract knowledge guide inference from incomplete data? Abstract knowledge is encoded in a probabilistic generative model, a kind of mental model that describes the causal processes in the world giving rise to the learner’s observations as well as unobserved or latent variables that support effective prediction and action if the learner can infer their hidden state. Generative models must be probabilistic to handle the learner’s uncertainty about the true states of latent variables and the true causal processes at work. A generative model is abstract in two senses: It describes not only the specific situation at hand, but also a broader class of situations over which learning should generalize, and it captures in parsimonious form the essential world structure that causes learners’ observations and makes generalization possible. Bayesian inference gives a rational framework for updating beliefs about latent variables in generative models given observed data (33, 34). Background knowledge is encoded through a constrained space of hypotheses H about possible values for the latent variables, candidate world structures that could explain the observed data. Finer-grained knowledge comes in the “prior probability” P(h), the learner’s degree of belief in a specific hypothesis h prior to (or independent of) the observations. Bayes’s rule updates priors to “posterior probabilities” P(h|d) conditional on the observed data d: P(hjd) ¼

Fig. 1. Human children learning names for object concepts routinely make strong generalizations from just a few examples. The same processes of rapid generalization can be studied in adults learning names for novel objects created with computer graphics. (A) Given these alien objects and three examples (boxed in red) of “tufas” (a word in the alien language), which other objects are tufas? Almost everyone selects just the objects boxed in gray (75). (B) Learning names for categories can be modeled as Bayesian inference over a tree-structured domain representation (2). Objects are placed at the leaves of the tree, and hypotheses about categories that words could label correspond to different branches. Branches at different depths pick out hypotheses at different levels of generality (e.g., Clydesdales, draft horses, horses, animals, or living things). Priors are defined on the basis of branch length, reflecting the distinctiveness of categories. Likelihoods assume that examples are drawn randomly from the branch that the word labels, favoring lower branches that cover the examples tightly; this captures the sense of suspicious coincidence when all examples of a word cluster in the same part of the tree. Combining priors and likelihoods yields posterior probabilities that favor generalizing across the lowest distinctive branch that spans all the observed examples (boxed in gray).

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P(djh)P(h) ºP(djh)P(h) ∑h′ ∈H P(djh′ )P(h′ ) ð1Þ

The posterior probability is proportional to the product of the prior probability and the likelihood P(d|h), measuring how expected the data are under hypothesis h, relative to all other hypotheses h′ in H. To illustrate Bayes’s rule in action, suppose we observe John coughing (d), and we consider three hypotheses as explanations: John has h1, a cold; h2, lung disease; or h3, heartburn. Intuitively only h1 seems compelling. Bayes’s rule explains why. The likelihood favors h1 and h2 over h3: only colds and lung disease cause coughing and thus elevate the probability of the data above baseline. The prior, in contrast, favors h1 and h3 over h2: Colds and heartburn are much more common than lung disease. Bayes’s rule weighs

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REVIEW

hypotheses according to the product of priors and likelihoods and so yields only explanations like h1 that score highly on both terms. The same principles can explain how people learn from sparse data. In concept learning, the data might correspond to several example objects (Fig. 1) and the hypotheses to possible extensions of the concept. Why, given three examples of different kinds of horses, would a child generalize the word “horse” to all and only horses (h1)? Why not h2, “all horses except Clydesdales”; h3, “all animals”; or any other rule consistent with the data? Likelihoods favor the more specific patterns, h1 and h2; it would be a highly suspicious coincidence to draw three random examples that all fall within the smaller sets h1 or h2 if they were actually drawn from the much larger h3 (18). The prior favors h1 and h3, because as more coherent and distinctive categories, they are more likely to be the referents of common words in language (1). Only h1 scores highly on both terms. Likewise, in causal learning, the data could be co-occurences between events; the hypotheses, possible causal relations linking the events. Likelihoods favor causal links that make the co-occurence more probable, whereas priors favor links that fit with our background knowledge of what kinds of events are likely to cause which others; for example, a disease (e.g., cold) is more likely to cause a symptom (e.g., coughing) than the other way around. The Form of Abstract Knowledge Abstract knowledge provides essential constraints for learning, but in what form? This is just question 2. For complex cognitive tasks such as concept learning or causal reasoning, it is impossible to simply list every logically possible hypothesis along with its prior and likelihood. Some more sophisticated forms of knowledge representation must underlie the probabilistic generative models needed for Bayesian cognition. In traditional associative or connectionist approaches, statistical models of learning were defined over large numerical vectors. Learning was seen as estimating strengths in an associative memory, weights in a neural network, or parameters of a high-dimensional nonlinear function (12, 14). Bayesian cognitive models, in contrast, have had most success defining probabilities over more structured symbolic forms of knowledge representations used in computer science and artificial intelligence, such as graphs, grammars, predicate logic, relational schemas, and functional programs. Different forms of representation are used to capture people’s knowledge in different domains and tasks and at different levels of abstraction. In learning words and concepts from examples, the knowledge that guides both children’s and adults’ generalizations has been well described using probabilistic models defined over tree-structured representations (Fig. 1B) (2, 35). Reasoning about other biological concepts for natural kinds (e.g., given that cows and rhinos have protein X in their muscles, how likely is it

that horses or squirrels do?) is also well described by Bayesian models that assume nearby objects in the tree are likely to share properties (36). However, trees are by no means a universal representation. Inferences about other kinds of categories or properties are best captured by using probabilistic models with different forms (Fig. 2): twodimensional spaces or grids for reasoning about geographic properties of cities, one-dimensional orders for reasoning about values or abilities, or directed networks for causally transmitted properties of species (e.g., diseases) (36). Knowledge about causes and effects more generally can be expressed in a directed graphical model (9, 11): a graph structure where nodes represent variables and directed edges between nodes represent probabilistic causal links. In a medical setting, for instance (Fig. 3A), nodes might represent whether a patient has a cold, a cough, a fever or other conditions, and the presence or absence of edges indicates that colds tend to cause coughing and fever but not chest pain; lung disease tends to cause coughing and chest pain but not fever; and so on. Such a causal map represents a simple kind of intuitive theory (4), but learning causal networks from limited data depends on the constraints of more abstract knowledge. For example, learning causal dependencies between medical conditions is enabled by a higher-level framework theory (37) specifying two classes of variables (or nodes), diseases and symptoms, and the tendency for causal relations (or graph edges) to run from diseases to symptoms, rather than within these classes or from symptoms to diseases (Fig. 3, A to C). This abstract framework can be represented by using probabilistic models defined over relational data structures such as graph schemas (9, 38), templates for graphs based on types of nodes, or probabilistic graph grammars (39), similar in spirit to the probabilistic grammars for strings that have become standard for representing linguistic knowledge (28). At the most abstract level, the very concept of causality itself, in the sense of a directed relationship that supports intervention or manipulation by an external agent (40), can be formulated as a set of logical laws expressing constraints on the structure of directed graphs relating actions and observable events (Fig. 3D). Each of these forms of knowledge makes different kinds of prior distributions natural to define and therefore imposes different constraints on induction. Successful generalization depends on getting these constraints right. Although inductive constraints are often graded, it is easiest to appreciate the effects of qualitative constraints that simply restrict the hypotheses learners can consider (i.e., setting priors for many logical possible hypotheses to zero). For instance, in learning concepts over a domain of n objects, there are 2n subsets and hence 2n logically possible hypotheses for the extension of a novel concept. Assuming concepts correspond to the branches of a specific binary tree over the objects, as in Fig. 1B, restricts this space to only

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n − 1 hypotheses. In learning a causal network over 16 variables, there are roughly 1046 logically possible hypotheses (directed acyclic graphs), but a framework theory restricting hypotheses to bipartite disease-symptom graphs reduces this to roughly 1023 hypotheses. Knowing which variables belong to the disease and symptom classes further restricts this to roughly 1018 networks. The smaller the hypothesis space, the more accurately a learner can be expected to generalize, but only as long as the true structure to be learned remains within or near (in a probabilistic sense) the learner’s hypothesis space (10). It is no coincidence then that our best accounts of people’s mental representations often resemble simpler versions of how scientists represent the same domains, such as tree structures for biological species. A compact description that approximates how the grain of the world actually runs offers the most useful form of constraint on inductive learning. The Origins of Abstract Knowledge The need for abstract knowledge and the need to get it right bring us to question 3: How do learners learn what they need to know to make learning possible? How does a child know which tree structure is the right way to organize hypotheses for word learning? At a deeper level, how can a learner know that a given domain of entities and concepts should be represented by using a tree at all, as opposed to a low-dimensional space or some other form? Or, in causal learning, how do people come to correct framework theories such as knowledge of abstract disease and symptom classes of variables with causal links from diseases to symptoms? The acquisition of abstract knowledge or new inductive constraints is primarily the province of cognitive development (5, 7). For instance, children learning words initially assume a flat, mutually exclusive division of objects into nameable clusters; only later do they discover that categories should be organized into tree-structured hierarchies (Fig. 1B) (41). Such discoveries are also pivotal in scientific progress: Mendeleev launched modern chemistry with his proposal of a periodic structure for the elements. Linnaeus famously proposed that relationships between biological species are best explained by a tree structure, rather than a simpler linear order (premodern Europe’s “great chain of being”) or some other form. Such structural insights have long been viewed by psychologists and philosophers of science as deeply mysterious in their mechanisms, more magical than computational. Conventional algorithms for unsupervised structure discovery in statistics and machine learning— hierarchical clustering, principal components analysis, multidimensional scaling, clique detection— assume a single fixed form of structure (42). Unlike human children or scientists, they cannot learn multiple forms of structure or discover new forms in novel data. Neither traditional approach to cognitive development has a fully satisfying response: Nativists have assumed that,

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if different domains of cognition are represented in qualitatively different ways, those forms must be innate (43, 44); connectionists have suggested these representations may be learned but in a generic system of associative weights that at best only approximates trees, causal networks, and other forms of structure people appear to know explicitly (14). Recently cognitive modelers have begun to answer these challenges by combining the structured knowledge representations described above with state-of-the-art tools from Bayesian statis-

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tics. Hierarchical Bayesian models (HBMs) (45) address the origins of hypothesis spaces and priors by positing not just a single level of hypotheses to explain the data but multiple levels: hypothesis spaces of hypothesis spaces, with priors on priors. Each level of a HBM generates a probability distribution on variables at the level below. Bayesian inference across all levels allows hypotheses and priors needed for a specific learning task to themselves be learned at larger or longer time scales, at the same time as they constrain lower-level learn-

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Fig. 2. Kemp and Tenenbaum (47) showed how the form of structure in a domain can be discovered by using a HBM defined over graph grammars. At the bottom level of the model is a data matrix D of objects and their properties, or similarities between pairs of objects. Each square of the matrix represents whether a given feature (column) is observed for a given object (row). One level up is the structure S, a graph of relations between objects that describes how the features in D are distributed. Intuitively, objects nearby in the graph are expected to share similar feature values; technically, the graph Laplacian parameterizes the inverse covariance of a gaussian distribution with one dimension per object, and each feature is drawn independently from that distribution. The highest level of abstract principles specifies the form F of structure in the domain, in terms of grammatical rules for growing a graph S of a constrained form out of an initial seed node. Red arrows represent P(S|F) and P(D|S), the conditional probabilities that each level specifies for the level below. A search algorithm attempts to find both the form F and the structure S of that form that jointly maximize the posterior probability P(S,F|D), a function of the product of P(D|S) and P(S|F). (A) Given as data the features of animals, the algorithm finds a tree structure with intuitively sensible categories at multiple scales. (B) The same algorithm discovers that the voting patterns of U.S. Supreme Court judges are best explained by a linear “left-right” spectrum. (C) Subjective similarities among colors are best explained by a circular ring. (D) Given proximities between cities on the globe, the algorithm discovers a cylindrical representation analogous to latitude and longitude: the cross product of a ring and a ring. (E) Given images of realistically synthesized faces varying in two dimensions, race and masculinity, the algorithm successfully recovers the underlying two-dimensional grid structure: a cross product of two chains.

ing. In machine learning and artificial intelligence (AI), HBMs have primarily been used for transfer learning: the acquisition of inductive constraints from experience in previous related tasks (46). Transfer learning is critical for humans as well (SOM text and figs. S1 and S2), but here we focus on the role of HBMs in explaining how people acquire the right forms of abstract knowledge. Kemp and Tenenbaum (36, 47) showed how HBMs defined over graph- and grammar-based representations can discover the form of structure

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Fig. 3. HBMs defined over graph schemas can explain how intuitive theories are acquired and used to learn about specific causal relations from limited data (38). (A) A simple medical reasoning domain might be described by relations among 16 variables: The first six encode presence or absence of “diseases” (top row), with causal links to the next 10 “symptoms” (bottom row). This network can also be visualized as a matrix (top right, links shown in black). The causal learning task is to reconstruct this network based on observing data D on the states of these 16 variables in a set of patients. (B) A two-level HBM formalizes bottom-up causal learning or learning with an uninformative prior on networks. The bottom level is the data matrix D. The second level (structure) encodes hypothesized causal networks: a grayscale matrix visualizes the posterior probability that each pairwise causal link exists, conditioned on observing n patients; compare this matrix with the black-and-white ground truth matrix shown in (A). The true causal network can be recovered perfectly only from observing very many patients (n = 1000; not shown). With n = 80, spurious links (gray squares) are inferred, and with n = 20 almost none of the true structure is detected. (C) A threelevel nonparametric HBM (48) adds a level of abstract principles, represented by a graph schema. The schema encodes a prior on the level below (causal network structure) that constrains and thereby accelerates causal learning. Both schema and network structure are learned from the same data observed in (B). The governing similarity in a domain. Structures of different forms—trees, clusters, spaces, rings, orders, and so on—can all be represented as graphs, whereas the abstract principles underlying each form are expressed as simple grammatical rules for growing graphs of that form. Embedded in a hierarchical Bayesian framework, this approach can discover the correct forms of structure (the grammars) for many real-world domains, along with the best struc-

schema discovers the disease-symptom framework theory by assigning variables 1 to 6 to class C1, variables 7 to 16 to class C2, and a prior favoring only C1 → C2 links. These assignments, along with the effective number of classes (here, two), are inferred automatically via the Bayesian Occam's razor. Although this three-level model has many more degrees of freedom than the model in (B), learning is faster and more accurate. With n = 80 patients, the causal network is identified near perfectly. Even n = 20 patients are sufficient to learn the high-level C1 → C2 schema and thereby to limit uncertainty at the network level to just the question of which diseases cause which symptoms. (D) A HBM for learning an abstract theory of causality (62). At the highest level are laws expressed in first-order logic representing the abstract properties of causal relationships, the role of exogenous interventions in defining the direction of causality, and features that may mark an event as an exogenous intervention. These laws place constraints on possible directed graphical models at the level below, which in turn are used to explain patterns of observed events over variables. Given observed events from several different causal systems, each encoded in a distinct data matrix, and a hypothesis space of possible laws at the highest level, the model converges quickly on a correct theory of intervention-based causality and uses that theory to constrain inferences about the specific causal networks underlying the different systems at the level below.

ture (the graph) of the appropriate form (Fig. 2). In particular, it can infer that a hierarchical organization for the novel objects in Fig. 1A (such as Fig. 1B) better fits the similarities people see in these objects, compared to alternative representations such as a two-dimensional space. Hierarchical Bayesian models can also be used to learn abstract causal knowledge, such as the framework theory of diseases and symptoms (Fig. 3), and other simple forms of intui-

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tive theories (38). Mansinghka et al. (48) showed how a graph schema representing two classes of variables, diseases and symptoms, and a preference for causal links running from disease to symptom variables can be learned from the same data that support learning causal links between specific diseases and symptoms and be learned just as fast or faster (Fig. 3, B and C). The learned schema in turn dramatically accelerates learning of specific causal relations (the

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directed graph structure) at the level below. Getting the big picture first—discovering that diseases cause symptoms before pinning down any specific disease-symptom links—and then using that framework to fill in the gaps of specific knowledge is a distinctively human mode of learning. It figures prominently in children’s development and scientific progress but has not previously fit into the landscape of rational or statistical learning models. Although this HBM imposes strong and valuable constraints on the hypothesis space of causal networks, it is also extremely flexible: It can discover framework theories defined by any number of variable classes and any pattern of pairwise regularities on how variables in these classes tend to be connected. Not even the number of variable classes (two for the diseasesymptom theory) need be known in advance. This is enabled by another state-of-the-art Bayesian tool, known as “infinite” or nonparametric hierarchical modeling. These models posit an unbounded amount of structure, but only finitely many degrees of freedom are actively engaged for a given data set (49). An automatic Occam’s razor embodied in Bayesian inference trades off model complexity and fit to ensure that new structure (in this case, a new class of variables) is introduced only when the data truly require it. The specific nonparametric distribution on node classes in Fig. 3C is a Chinese restaurant process (CRP), which has been particularly influential in recent machine learning and cognitive modeling. CRP models have given the first principled account of how people form new categories without direct supervision (50, 51): As each stimulus is observed, CRP models (guided by the Bayesian Occam’s razor) infer whether that object is best explained by assimilation to an existing category or by positing a previously unseen category (fig. S3). The CrossCat model extends CRPs to carve domains of objects and their properties into different subdomains or “views,” subsets of properties that can all be explained by a distinct way of organizing the objects (52) (fig. S4). CRPs can be embedded in probabilistic models for language to explain how children discover words in unsegmented speech (53), learn morphological rules (54), and organize word meanings into hierarchical semantic networks (55, 56) (fig. S5). A related but novel nonparametric construction, the Indian buffet process (IBP), explains how new perceptual features can be constructed during object categorization (57, 58). More generally, nonparametric hierarchical models address the principal challenge human learners face as knowledge grows over a lifetime: balancing constraint and flexibility, or the need to restrict hypotheses available for generalization at any moment with the capacity to expand one’s hypothesis spaces, to learn new ways that the world could work. Placing nonparametric distributions at higher levels of the HBM yields flexible inductive biases for lower

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levels, whereas the Bayesian Occam’s razor ensures the proper balance of constraint and flexibility as knowledge grows. Across several case studies of learning abstract knowledge—discovering structural forms, causal framework theories, and other inductive constraints acquired through transfer learning—it has been found that abstractions in HBMs can be learned remarkably fast from relatively little data compared with what is needed for learning at lower levels. This is because each degree of freedom at a higher level of the HBM influences and pools evidence from many variables at levels below. We call this property of HBMs “the blessing of abstraction.” It offers a top-down route to the origins of knowledge that contrasts sharply with the two classic approaches: nativism (59, 60), in which abstract concepts are assumed to be present from birth, and empiricism or associationism (14), in which abstractions are constructed but only approximately, and only slowly in a bottom-up fashion, by layering many experiences on top of each other and filtering out their common elements. Only HBMs thus seem suited to explaining the two most striking features of abstract knowledge in humans: that it can be learned from experience, and that it can be engaged remarkably early in life, serving to constrain more specific learning tasks. Open Questions HBMs may answer some questions about the origins of knowledge, but they still leave us wondering: How does it all start? Developmentalists have argued that not everything can be learned, that learning can only get off the ground with some innate stock of abstract concepts such as “agent,” “object,” and “cause” to provide the basic ontology for carving up experience (7, 61). Surely some aspects of mental representation are innate, but without disputing this Bayesian modelers have recently argued that even the most abstract concepts may in principle be learned. For instance, an abstract concept of causality expressed as logical constraints on the structure of directed graphs can be learned from experience in a HBM that generalizes across the network structures of many specific causal systems (Fig. 3D). Following the “blessing of abstraction,” these constraints can be induced from only small samples of each network’s behavior and in turn enable more efficient causal learning for new systems (62). How this analysis extends to other abstract concepts such as agent or object and whether children actually acquire these concepts in such a manner remain open questions. Although HBMs have addressed the acquisition of simple forms of abstract knowledge, they have only touched on the hardest subjects of cognitive development: framework theories for core common-sense domains such as intuitive physics, psychology, and biology (5, 6, 7). First steps have come in explaining developing theories of mind, how children come to understand explicit false beliefs (63) and in-

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dividual differences in preferences (64), as well as the origins of essentialist theories in intuitive biology and early beliefs about magnetism in intuitive physics (39, 38). The most daunting challenge is that formalizing the full content of intuitive theories appears to require Turingcomplete compositional representations, such as probabilistic first-order logic (65, 66) and probabilistic programming languages (67). How to effectively constrain learning with such flexible representations is not at all clear. Lastly, the project of reverse-engineering the mind must unfold over multiple levels of analysis, only one of which has been our focus here. Marr (68) famously argued for analyses that integrate across three levels: The computational level characterizes the problem that a cognitive system solves and the principles by which its solution can be computed from the available inputs in natural environments; the algorithmic level describes the procedures executed to produce this solution and the representations or data structures over which the algorithms operate; and the implementation level specifies how these algorithms and data structures are instantiated in the circuits of a brain or machine. Many early Bayesian models addressed only the computational level, characterizing cognition in purely functional terms as approximately optimal statistical inference in a given environment, without reference to how the computations are carried out (25, 39, 69). The HBMs of learning and development discussed here target a view between the computational and algorithmic levels: cognition as approximately optimal inference in probabilistic models defined over a learner’s subjective and dynamically growing mental representations of the world’s structure, rather than some objective and fixed world statistics. Much ongoing work is devoted to pushing Bayesian models down through the algorithmic and implementation levels. The complexity of exact inference in large-scale models implies that these levels can at best approximate Bayesian computations, just as in any working Bayesian AI system (9). The key research questions are as follows: What approximate algorithms does the mind use, how do they relate to engineering approximations in probabilistic AI, and how are they implemented in neural circuits? Much recent work points to Monte Carlo or stochastic sampling– based approximations as a unifying framework for understanding how Bayesian inference may work practically across all these levels, in minds, brains, and machines (70–74). Monte Carlo inference in richly structured models is possible (9, 67) but very slow; constructing more efficient samplers is a major focus of current work. The biggest remaining obstacle is to understand how structured symbolic knowledge can be represented in neural circuits. Connectionist models sidestep these challenges by denying that brains actually encode such rich knowledge, but this runs counter to the strong consensus in cognitive science and artificial intelligence that symbols and structures are essential for thought. Uncovering their neural

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basis is arguably the greatest computational challenge in cognitive neuroscience more generally—our modern mind-body problem. Conclusions We have outlined an approach to understanding cognition and its origins in terms of Bayesian inference over richly structured, hierarchical generative models. Although we are far from a complete understanding of how human minds work and develop, the Bayesian approach brings us closer in several ways. First is the promise of a unifying mathematical language for framing cognition as the solution to inductive problems and building principled quantitative models of thought with a minimum of free parameters and ad hoc assumptions. Deeper is a framework for understanding why the mind works the way it does, in terms of rational inference adapted to the structure of realworld environments, and what the mind knows about the world, in terms of abstract schemas and intuitive theories revealed only indirectly through how they constrain generalizations. Most importantly, the Bayesian approach lets us move beyond classic either-or dichotomies that have long shaped and limited debates in cognitive science: “empiricism versus nativism,” “domain-general versus domain-specific,” “logic versus probability,” “symbols versus statistics.” Instead we can ask harder questions of reverseengineering, with answers potentially rich enough to help us build more humanlike AI systems. How can domain-general mechanisms of learning and representation build domain-specific systems of knowledge? How can structured symbolic knowledge be acquired through statistical learning? The answers emerging suggest new ways to think about the development of a cognitive system. Powerful abstractions can be learned surprisingly quickly, together with or prior to learning the more concrete knowledge they constrain. Structured symbolic representations need not be rigid, static, hard-wired, or brittle. Embedded in a probabilistic framework, they can grow dynamically and robustly in response to the sparse, noisy data of experience. References and Notes 1. P. Bloom, How Children Learn the Meanings of Words (MIT Press, Cambridge, MA, 2000). 2. F. Xu, J. B. Tenenbaum, Psychol. Rev. 114, 245 (2007). 3. S. Pinker, Words and Rules: The Ingredients of Language (Basic, New York, 1999). 4. A. Gopnik et al., Psychol. Rev. 111, 3 (2004). 5. A. Gopnik, A. N. Meltzoff, Words, Thoughts, and Theories (MIT Press, Cambridge, MA, 1997). 6. S. Carey, Conceptual Change in Childhood (MIT Press, Cambridge, MA, 1985). 7. S. Carey, The Origin of Concepts (Oxford Univ. Press, New York, 2009). 8. P. Godfrey-Smith, Theory and Reality (Univ. of Chicago Press, Chicago, 2003). 9. S. Russell, P. Norvig, Artificial Intelligence: A Modern Approach (Prentice Hall, Upper Saddle River, NJ, 2009). 10. D. McAllester, in Proceedings of the Eleventh Annual Conference on Computational Learning Theory [Association for Computing Machinery (ACM), New York, 1998], p. 234. 11. J. Pearl, Probabilistic Reasoning in Intelligent Systems (Morgan Kaufmann, San Francisco, CA, 1988).

12. J. McClelland, D. Rumelhart, Eds., Parallel Distributed Processing: Explorations in the Microstructure of Cognition (MIT Press, Cambridge, MA, 1986). 13. S. Pinker, How the Mind Works (Norton, New York, 1997). 14. T. Rogers, J. McClelland, Semantic Cognition: A Parallel Distributed Processing Approach (MIT Press, Cambridge, MA, 2004). 15. P. Niyogi, The Computational Nature of Language Learning and Evolution (MIT Press, Cambridge, MA, 2006). 16. T. L. Griffiths, N. Chater, C. Kemp, A. Perfors, J. B. Tenenbaum, Trends Cogn. Sci. 14, 357 (2010). 17. J. L. McClelland et al., Trends Cogn. Sci. 14, 348 (2010). 18. J. B. Tenenbaum, T. L. Griffiths, Behav. Brain Sci. 24, 629 (2001). 19. J. Tenenbaum, T. Griffiths, in Proceedings of the 23rd Annual Conference of the Cognitive Science Society, J. D. Moore, K. Stenning, Eds. (Erlbaum, Mahwah, NJ, 2001), pp. 1036–1041. 20. T. Griffiths, J. Tenenbaum, in Proceedings of the 23rd Annual Conference of the Cognitive Science Society, J. D. Moore, K. Stenning, Eds. (Erlbaum, Mahwah, NJ, 2001), pp. 370–375. 21. T. L. Griffiths, J. B. Tenenbaum, Cognition 103, 180 (2007). 22. H. Lu, A. L. Yuille, M. Liljeholm, P. W. Cheng, K. J. Holyoak, Psychol. Rev. 115, 955 (2008). 23. T. L. Griffiths, J. B. Tenenbaum, Cognit. Psychol. 51, 334 (2005). 24. T. R. Krynski, J. B. Tenenbaum, J. Exp. Psychol. Gen. 136, 430 (2007). 25. M. Oaksford, N. Chater, Trends Cogn. Sci. 5, 349 (2001). 26. T. L. Griffiths, J. B. Tenenbaum, Psychol. Sci. 17, 767 (2006). 27. A. Yuille, D. Kersten, Trends Cogn. Sci. 10, 301 (2006). 28. N. Chater, C. D. Manning, Trends Cogn. Sci. 10, 335 (2006). 29. R. M. Shiffrin, M. Steyvers, Psychon. Bull. Rev. 4, 145 (1997). 30. M. Steyvers, T. L. Griffiths, S. Dennis, Trends Cogn. Sci. 10, 327 (2006). 31. K. P. Körding, D. M. Wolpert, Nature 427, 244 (2004). 32. A. Tversky, D. Kahneman, Science 185, 1124 (1974). 33. E. T. Jaynes, Probability Theory: The Logic of Science (Cambridge Univ. Press, Cambridge, 2003). 34. D. J. C. Mackay, Information Theory, Inference, and Learning Algorithms (Cambridge Univ. Press, Cambridge, 2003). 35. F. Xu, J. B. Tenenbaum, Dev. Sci. 10, 288 (2007). 36. C. Kemp, J. B. Tenenbaum, Psychol. Rev. 116, 20 (2009). 37. H. M. Wellman, S. A. Gelman, Annu. Rev. Psychol. 43, 337 (1992). 38. C. Kemp, J. B. Tenenbaum, S. Niyogi, T. L. Griffiths, Cognition 114, 165 (2010). 39. T. L. Griffiths, J. B. Tenenbaum, in Causal Learning: Psychology, Philosophy, and Computation, A. Gopnik, L. Schulz, Eds. (Oxford University Press, Oxford, 2007), pp. 323–345. 40. J. Woodward, Making Things Happen: A Theory of Causal Explanation (Oxford Univ. Press, Oxford, 2003). 41. E. S. Markman, Categorization and Naming in Children (MIT Press, Cambridge, MA, 1989). 42. R. N. Shepard, Science 210, 390 (1980). 43. N. Chomsky, Rules and Representations (Basil Blackwell, Oxford, 1980). 44. S. Atran, Behav. Brain Sci. 21, 547, (1998). 45. A. Gelman, J. B. Carlin, H. S. Stern, D. B. Rubin, Bayesian Data Analysis (Chapman and Hall, New York, 1995). 46. C. Kemp, A. Perfors, J. B. Tenenbaum, Dev. Sci. 10, 307 (2007). 47. C. Kemp, J. B. Tenenbaum, Proc. Natl. Acad. Sci. U.S.A. 105, 10687 (2008). 48. V. K. Mansinghka, C. Kemp, J. B. Tenenbaum, T. L. Griffiths, in Proceedings of the 22nd Conference on Uncertainty in Artificial Intelligence, R. Dechter, T. Richardson, Eds. (AUAI Press, Arlington, VA, 2006), pp. 324–331. 49. C. Rasmussen, in Advances in Neural Information Processing Systems (MIT Press, Cambridge, MA, 2000), vol. 12, pp. 554–560. 50. J. R. Anderson, Psychol. Rev. 98, 409 (1991). 51. T. L. Griffiths, A. N. Sanborn, K. R. Canini, D. J. Navarro, in The Probabilistic Mind, N. Chater, M. Oaksford, Eds. (Oxford Univ. Press, Oxford, 2008). 52. P. Shafto, C. Kemp, V. Mansinghka, M. Gordon, J. B. Tenenbaum, in Proceedings of the 28th Annual Conference of the Cognitive Science Society (Erlbaum, Mahwah, NJ, 2006), pp. 2146–2151.

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53. S. Goldwater, T. L. Griffiths, M. Johnson, Cognition 112, 21 (2009). 54. M. Johnson, T. L. Griffiths, S. Goldwater, in Advances in Neural Information Processing Systems (MIT Press, Cambridge, MA, 2007), vol. 19, pp. 641–648. 55. T. L. Griffiths, M. Steyvers, J. B. Tenenbaum, Psychol. Rev. 114, 211 (2007). 56. D. Blei, T. Griffiths, M. Jordan, J. Assoc. Comput. Mach. 57, 1 (2010). 57. T. L. Griffiths, Z. Ghahramani, in Advances in Neural Information Processing Systems (MIT Press, Cambridge, MA, 2006), vol. 18, pp. 475–482. 58. J. Austerweil, T. L. Griffiths, in Advances in Neural Information Processing Systems (MIT Press, Cambridge, MA, 2009), vol. 21, pp. 97–104. 59. N. Chomsky, Language and Problems of Knowledge: The Managua Lectures (MIT Press, Cambridge, MA, 1986). 60. E. S. Spelke, K. Breinlinger, J. Macomber, K. Jacobson, Psychol. Rev. 99, 605 (1992). 61. S. Pinker, The Stuff of Thought: Language as a Window into Human Nature (Viking, New York, 2007). 62. N. D. Goodman, T. D. Ullman, J. B. Tenenbaum, Psychol. Rev. 118, 110 (2011). 63. N. Goodman et al., in Proceedings of the 28th Annual Conference of the Cognitive Science Society (Erlbaum, Mahwah, NJ, 2006), pp. 1382–1387. 64. C. Lucas, T. Griffiths, F. Xu, C. Fawcett, in Advances in Neural Information Processing Systems (MIT Press, Cambridge, MA, 2009), vol. 21, pp. 985–992. 65. B. Milch, B. Marthi, S. Russell, in ICML 2004 Workshop on Statistical Relational Learning and Its Connections to Other Fields, T. Dietterich, L. Getoor, K. Murphy, Eds. (Omnipress, Banff, Canada, 2004), pp. 67–73. 66. C. Kemp, N. Goodman, J. Tenenbaum, in Proceedings of the 30th Annual Meeting of the Cognitive Science Society (Publisher, City, Country, 2008), pp. 1606–1611. 67. N. Goodman, V. Mansinghka, D. Roy, K. Bonawitz, J. Tenenbaum, in Proceedings of the 24th Conference on Uncertainty in Artificial Intelligence (AUAI Press, Corvallis, OR, 2008), vol. 22, p. 23. 68. D. Marr, Vision (W. H. Freeman, San Francisco, CA, 1982). 69. J. B. Tenenbaum, T. L. Griffiths, in Advances in Neural Information Processing Systems, T. Leen, T. Dietterich, V. Tresp, Eds. (MIT Press, Cambridge, MA, 2001), vol. 13, pp. 59–65. 70. A. N. Sanborn, T. L. Griffiths, D. J. Navarro, in Proceedings of the 28th Annual Conference of the Cognitive Science Society (Erlbaum, Mahwah, NJ, 2006), pp. 726–731. 71. S. D. Brown, M. Steyvers, Cognit. Psychol. 58, 49 (2009). 72. R. Levy, F. Reali, T. L. Griffiths, in Advances in Neural Information Processing Systems, D. Koller, D. Schuurmans, Y. Bengio, L. Bottou, Eds. (MIT Press, Cambridge, MA, 2009), vol. 21, pp. 937–944. 73. J. Fiser, P. Berkes, G. Orbán, M. Lengyel, Trends Cogn. Sci. 14, 119 (2010). 74. E. Vul, N. D. Goodman, T. L. Griffiths, J. B. Tenenbaum, in Proceedings of the 31st Annual Conference of the Cognitive Science Society (Erlbaum, Mahwah, NJ, 2009), pp. 148–153. 75. L. Schmidt, thesis, Massachusetts Institute of Technology, Cambridge, MA (2009). 76. We gratefully acknowledge the suggestions of R. R. Saxe, M. Bernstein, and J. M. Tenenbaum on this manuscript and the collaboration of N. Chater and A. Yuille on a forthcoming joint book expanding on the methods and perspectives reviewed here. Grant support was provided by Air Force Office of Scientific Research, Office of Naval Research, Army Research Office, NSF, Defense Advanced Research Projects Agency, Nippon Telephone and Telegraph Communication Sciences Laboratories, Qualcomm, Google, Schlumberger, and the James S. McDonnell Foundation.

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Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1279/DC1 SOM Text Figs. S1 to S5 References 10.1126/science.1192788

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Co-Residence Patterns in Hunter-Gatherer Societies Show Unique Human Social Structure Kim R. Hill,1* Robert S. Walker,2* Miran Božičević,1 James Eder,1 Thomas Headland,3,4 Barry Hewlett,5,6 A. Magdalena Hurtado,1 Frank Marlowe,7 Polly Wiessner,8 Brian Wood9 Contemporary humans exhibit spectacular biological success derived from cumulative culture and cooperation. The origins of these traits may be related to our ancestral group structure. Because humans lived as foragers for 95% of our species’ history, we analyzed co-residence patterns among 32 present-day foraging societies (total n = 5067 individuals, mean experienced band size = 28.2 adults). We found that hunter-gatherers display a unique social structure where (i) either sex may disperse or remain in their natal group, (ii) adult brothers and sisters often co-reside, and (iii) most individuals in residential groups are genetically unrelated. These patterns produce large interaction networks of unrelated adults and suggest that inclusive fitness cannot explain extensive cooperation in hunter-gatherer bands. However, large social networks may help to explain why humans evolved capacities for social learning that resulted in cumulative culture. ur ancestors lived as hunter-gatherers until the beginning of the Holocene; during that time, they spread across the globe and developed features that distinguish us as an outlier among life forms on this planet (1). Our biological success appears to be based on both cooperation with non-kin and exceptional reliance on cultural transmission, yet critical questions remain about why these traits emerged in humans but not other animals. Contemporary huntergatherers often show extensive cooperation among members of a residential unit (referred to here as a “band”) in ways not paralleled by any other primate. This includes band-wide food sharing (2); high levels of allomaternal child care (3); daily cooperative food acquisition, construction, and maintenance of living spaces and transportation of children and possessions (4); and provisioning of public goods on a daily basis. The widespread flow of goods and services within hunter-gatherer bands coevolved with a life history that included slow juvenile growth, late sexual maturity, high fertility, high adult survivorship, and a long postreproductive life span (1, 5, 6). The combination of juvenile allomaternal provisioning, adult shared energy budgets, and helper-dependent life history

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1 School of Human Evolution & Social Change, Arizona State University, Tempe, AZ 85287, USA. 2Department of Anthropology, University of Missouri, Columbia, MO 65211, USA. 3 Department of Anthropology, SIL International, Dallas, TX 75236, USA. 4Department of Anthropology, Southern Methodist University, Dallas, TX 75275, USA. 5Department of Anthropology, Hawassa University, Hawassa, Ethiopia. 6Department of Anthropology, Washington State University, Vancouver, WA 98686, USA. 7Department of Anthropology, Durham University, Durham DH1 3LE, UK. 8Department of Anthropology, University of Utah, Salt Lake City, UT 84112, USA. 9Department of Anthropology, Stanford University, Stanford, CA 94305, USA.

*To whom correspondence should be addressed. E-mail: [email protected] (K.R.H.); [email protected] (R.W.)

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traits has led some to label hunter-gatherers as cooperative breeders (7, 8). Evolutionary behavioral scientists have attempted to understand how cooperative temperament might spread in small-scale human societies and how it might account for “prosocial” and “otherregarding” behaviors (9), exceptional social cognition and shared intentionality (10), orientation toward teaching and learning (11), a taste for equitable distribution (12), and a widespread willingness to punish norm violators, even when not directly affected by the noncooperative behavior (13). These traits appear to be derived in Homo because they are rare or absent in other apes (14). If hunter-gatherers engage in extensive cooperation within residential bands, we must determine the composition of these units to assess how such behavior could have evolved. Traditionally, anthropologists have suggested that huntergatherer co-residence is almost entirely based on kinship [e.g., (15, 16)], and evolutionary psychologists have embraced this idea in order to develop “mismatch hypotheses” about cooperation among non-kin in modern societies (17). Evolutionary researchers have also argued both that female philopatry and maternal grandmother provisioning is ancestral (5) and that male philopatry, typical of other African hominoids (18, 19) and leading to adult male provisioning (8), is the ancestral human pattern. If either of these is correct, and if foraging bands are mainly collections of close kin, inclusive fitness gains might be the primary motivator of ancestral human cooperation. Chapais (18) recently developed an alternative model of ancestral human social structure derived from pair-bonding and paternal investment within chimpanzee-like social groups. He suggested that the affiliation of several unrelated males to the same female (related as daughter, sister, wife, son’s wife, or brother’s wife) could ameliorate

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hostile relations across ancestral male philopatric groups, and could allow for cross-group visiting and opportunistic co-residence by kin of either sex and frequent interactions with affines. This model predicts lifetime cooperation and frequent co-residence of adult brothers and sisters as a unique feature of human society [(18), p. 129]. These features result in metagroups (“tribes”) composed of multiple residential bands that exchange spouses [(18), pp. 216–228]. Most important, the resultant social structure would lead to a high number of co-resident adults who are not genetically related [(18), pp. 235–243]. If this model is correct, and if most band members are not closely linked by either kinship or shared genetic descendants, a variety of mechanisms that promote dyadic and indirect reciprocity (19, 20) or favor social sanctioning of noncooperators (21, 22) may be required to explain the emergence of extensive cooperation in ancestral human societies. Hunter-gatherer residential patterns are also critical for assessing models of cultural evolution. If friendly visiting between groups is common and social network sizes are large, frequent interaction allows for increased observation of rare innovations that are unlikely to be discovered by individual learning. Under these conditions, costly social learning mechanisms are more likely to evolve (21), and cultural traits are more likely to accumulate complexity and efficiency (23). Thus, the emergence of metagroup social structure might explain why humans, but not other social-learning animals, evolved the cognitive mechanisms that produce cumulative culture, and why H. sapiens were able to replace other hominins as they spread out of Africa (24). Cross-cultural analysis. We analyzed data on band composition collected from published literature and unpublished field notes for a worldwide sample of 32 hunter-gatherer societies (25). Tabulations of the mean number of primary adult kin (i.e., parents, siblings, and offspring) that co-reside in bands clearly support the bisexual philopatry and dispersal model of co-residence. We found no significant difference in the mean number of parents living with adult sons versus daughters in 19 societies (Table 1). In a minority of societies, parents were statistically more likely to co-reside with adult offspring of one sex (seven societies with sons, six societies with daughters), but even in these societies many parents lived with adult offspring of the other sex. Likewise, adult brothers and sisters frequently co-reside in all societies; thus, men often co-reside with male in-laws (fig. S1). In about half of the societies, men and women lived with no more same-sex adult siblings than opposite-sex siblings (table S1, 18 of 32 societies). The pattern of frequent brother-sister co-residence is not just due to bride service (in which new husbands live with parents-in-law and work for them) but is evident across the entire adult life span, even in groups that show preferential clustering of male kin, and

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even when controlling for mortality effects by considering only living siblings (Fig. 1). Despite the common occurrence of brothersister co-residence, statistical analysis also illustrates a greater tendency for close kin of men to co-reside than for close female kin to live together. For example, the mean number of adult brothers residing with each other is significantly higher than the mean number of adult sisters coresiding in 17 societies, but vice versa in only three societies (Table 1), and the total number of co-residing primary kin of males is typically higher than the number of co-residing primary kin of females (Table 1 and fig. S1). The data demonstrate that primary kin associations are typical, but that most adult band members are not close kin in any of the societies in our sample. The mean total number of co-resident adult primary kin per band is only 1.8 for these 32 societies (range 0.45 to 5.27, Table 1). This represents only a small proportion (7%) of the total co-resident adults in the censused bands

(mean experienced band size = 28.2). To determine exactly who the remaining band members are, we require complete genealogical and marital information on all adults in the population (available only for the Ache and Ju/’hoansi). We analyzed the complete composition of 58 precontact Ache bands (n = 980 adults) using demographic and marital databases (26), and one of us (P.W.) tabulated relationships for all adult dyads in six bands of Ju/’hoansi foragers (n = 89 adults). Even though Ache bands are larger than Ju/’hoansi bands (21 versus 15 adults), typical band composition from a randomly chosen adult ego’s point of view shows a common pattern: First, ego and his/her primary and distant kin connected by up to five meiotic links (genetic coefficient of relatedness r = 1 to 0.03125) make up only about 25% of the adults who co-reside in Ache and Ju/’hoansi bands (Fig. 2 and table S2). Smaller Ju/’hoansi bands contain a greater proportion of primary kin, whereas larger Ache bands

contain a higher proportion of distant kin. Second, spouse and spouse’s kin constitute about 16% of the adults in these bands. Thus, primary and distant kin of a family unit make up only about 40% of the co-resident adult members of a band. Among the individuals genetically unrelated to either spouse or ego, the spouses of ego’s primary kin often carry the same kinship term as spouse’s primary kin (e.g., brother-in-law, sister-in-law, etc.) and may be treated equally. These make up about 6% of adult band members. Other adults connected through a multiple-linked chain of distant genetic and marriage ties (i.e., “distant affines” such as cousin’s spouse or “affines of affines” such as spouse’s brother’s wife’s brother) make up about half the co-resident adults members of a band. Finally, about one-quarter of the individuals in a band are not linked directly to ego by any known genealogical or marriage tie. It is noteworthy that flexible bilocal residence leads to neither sex being surrounded mainly by kin (table S3).

Table 1. Mean number of co-residing adult primary kin for men and women, with societies sorted from most to least virilocal (male-biased) co-residence. Significant differences were determined by resampling. The mean experienced band size is the mean of all band sizes weighted by the number of individuals who lived in bands of that size. Men’s parents

Women’s parents

Men’s brothers

Men’s sisters

Women’s brothers

Women’s sisters

Men’s primary kin

Women’s primary kin

Mean experienced band size

Gunwinggu Labrador Inuit Semang Iglulik Belcher Island Mbuti Hiwi Angmagsalik Ainu Ache Paliyan Nunamuit Aka Chenchu Netsilik Agta Slavey Ojibwa Wanindiljaugwa Copper Dogrib Ju/ ’hoansi Shoshoni Batak Alyawarra Vedda Paiute Apache Takamiut Hadza Hill Pandaram Miwuyt

1.26* 0.24 0.76* 0.60* 0.15 0.57* 0.54 0.75* 0.15 0.50* 0.28 0.37 0.56 0.44* 0.39 0.35 0.50 0.52 0.07 0.20 0.89 0.71 0.11 0.53 0.57 0.40 0.16 0.49 0.47 0.38 0.16 0.06

0.30 0.14 0.21 0.23 0.10 0.21 0.44 0.20 0.24 0.36 0.16 0.38 0.49 0.25 0.32 0.27 0.46 0.43 0.33† 0.40† 0.94 0.62 0.11 0.53 0.60 0.88† 0.52† 0.56 0.76 0.40 0.50† 0.30†

2.50* 0.36* 0.72* 0.56* 0.50* 0.59* 1.58* 0.25 0.89* 0.59* 0.48* 0.88* 0.51* 0.45* 0.27* 0.42* 1.00 0.78* 1.56 0.33* 1.23 0.37 0.37 0.69 0.65 0.60 0.32 0.29 0.67 0.32 0.16 0.11

0.75 0.18 0.44 0.19 0.45 0.37 0.58 0.25 0.57 0.33 0.57 0.79 0.27 0.40 0.19 0.37 0.29 0.75 1.54 0.13 1.23 0.50 0.24 0.58 0.44 0.30 0.21 0.32 0.33 0.42 0.24 0.22

0.60 0.11 0.46 0.18 0.43 0.33 0.55 0.20 0.53 0.34 0.63 0.75 0.21 0.37 0.18 0.35 0.27 0.71 1.37 0.13 1.16 0.46 0.21 0.73 0.33 0.33 0.21 0.33 0.31 0.38 0.27 0.17

0.60 0.00 0.00 0.13 0.10 0.27 0.51 0.20 0.34 0.16 0.21 0.52 0.33 0.21 0.06 0.27 0.53 0.44 1.46 0.07 1.04 0.31 0.33 0.53 0.88 0.44 0.21 0.48† 0.88 0.68† 0.18 0.42†

5.27* 0.93* 2.40* 1.74* 1.25* 1.88* 3.13* 1.63 1.78* 1.77* 1.57 2.44* 1.86 1.52 1.15 1.42 2.14 2.45* 3.61 0.96 4.20 2.15 0.78 2.25 2.21 1.90 1.03 1.55 2.07 1.43 0.84 0.56

2.20 0.45 1.18 0.97 0.73 1.21 2.05 1.10 1.33 1.36 1.21 2.00 1.55 1.28 0.97 1.22 1.86 2.13 3.16 0.90 4.12 2.14 0.81 2.42 2.43 2.31 1.28 1.96† 2.64 1.93† 1.31† 1.10†

18.0 24.1 19.3 25.5 11.9 32.9 36.7 17.0 14.9 20.7 25.0 64.9 16.6 17.6 17.0 12.8 22.3 56.6 21.4 16.3 81.6 14.5 7.3 42.6 39.2 11.6 11.7 18.6 15.9 35.1 5.8 11.9

Average

0.44

0.39

0.66*

0.45

0.42

0.40

1.93

1.67

28.2

Society

*Significantly more kin co-residing with men (P < 0.05).

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†Significantly more kin co-residing with women (P < 0.05).

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RESEARCH ARTICLES Proportion of siblings co-residing

0.5

A

Women’s brothers Women’s sisters Baseline

0.4 0.3 0.2

0.1 0.0 15-21

22-29

30-44

B

45+

Men’s brothers Men’s sisters Baseline

0.4 0.3

Fig. 2. Mean band composition from adult ego’s point of view for 58 Ache bands and 6 Ju/’hoansi bands (data in table S2). The category “distant affines” includes spouses of distant kin, distant kin of spouse, and affines of affines. The category “No relation” includes all adult dyads that cannot be connected in five or fewer total steps of kinship and marriage with no more than two marriage links in the chain of connection. The shaded region shows all band members genetically related to ego.

0.2

0.1 0.0 15-21

22-29

30-44

45+

Age

Fig. 1. Comparison of same-sex and cross-sex sibling co-residence contingent on survival by age for precontact Ache women (A) and men (B). Data points show mean proportions per age group of surviving siblings who co-reside. Bars indicate 95% confidence intervals; triangles mark the baseline proportion of co-residence for all adults in the population who are more than three genealogical steps removed (and not married to each other). Brotherbrother and brother-sister co-residence is common throughout the life span, but after their 30s, women are no more likely to co-reside with another living sister than with any randomly selected, unrelated adult. Conclusion. Formerly, anthropologists believed that hunter-gatherers lived in patrilocal bands composed mainly of close kin (15). Here, we present a statistical analysis of hunter-gatherer band composition based on actual residence rather than cultural rules. We show that bisexual philopatry and dispersal are typical and result in frequent adult brother-sister co-residence. This social pattern is not reported for any other primate or vertebrate, as far as we know. We hypothesize that monogamous pair bonding, paternal recognition within cooperatively breeding social units (8), and bisexual dispersal facilitate frequent and friendly intergroup relations (18) and migration and low group genetic relatedness of band co-residents. The hunter-gatherer social structure we describe has important implications for theories about the evolution of cooperation and cultural capacity. First, bands are mainly composed of individuals either distantly related by kinship and/or marriage or unrelated altogether. In our sample of 32 societies, primary kin generally make up less than 10% of a residential band. For example, in the Ache we estimate the mean genetic coefficient of relatedness (Hamilton’s r) between

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adults in 58 precontact bands to be only 0.054 (n = 19,634 dyads, SE = 0.0001). This agrees with Ache informants who reported that during the precontact period they often lived with people described as “friends, not relatives.” The Ju/’hoansi results in Fig. 2 suggest that mean relatedness in other groups is not too different from the Ache. Thus, we cannot necessarily assume that cognitive features such as inequality aversion and enhanced prosocial emotions evolved in ancestral environments composed mainly of close kin. Given the constant flow of individuals between groups, genetic group selection at the level of the band also seems improbable. Instead, cultural group selection (27) may lead to the spread of cooperative institutions within ethnic groups, which might then create a context favoring the genetic evolution of prosocial cognitive mechanisms through individual-level selection. Second, mathematical models suggest that large interaction networks may be required for culture to accumulate (21, 23). In small populations, cultural innovations can be lost because of infrequent interaction between potential models and imitators and/or stochastic events that eliminate models with particular cultural knowledge. For example, one of the authors (K.R.H.) observed that the Northern Ache, isolated from their ancestral core territory in the 19th century, were unable to make fire by the time they were contacted in the 1970s. However, older informants stated that their parents and grandparents had told them that their ancestors could make fire, and had partially described to them the technique, even though none had ever observed it directly. In contrast, the Southern Ache groups did maintain firemaking knowledge until their first contact in the late 20th century. Likewise, Tasmanians failed to maintain previously known methods for fishing when their island was cut off from mainland Australia in the early Holocene (23), and fishing technology in Polynesia shows reduced complexity

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on islands with smaller populations (28). In contrast, Wiessner [(29) and references therein] conducted studies on style and social information in material culture among Kalahari Bushman bands from four language families connected by intermarriage, exchange partnerships, and visiting. She found that tapered bone points were rapidly replaced everywhere by iron tips when fence wire became available in the 20th century, and that new point styles emerged and became relatively homogeneous within language groups over a period of 40 years. When people reside together, they have frequent opportunities to observe innovations, evaluate their success, and imitate traits judged most successful or most common. Our analyses suggest that the increased network size that follows a unique shift in ancestral human residential structure may have led to greater exposure to novel ideas worth copying, and may explain why humans, but not other animals, evolved costly social learning mechanisms (such as high-fidelity overimitation or conformity-biased transmission) that may have resulted in cumulative cultural evolution (21). This unique expansion of network size in our hominin ancestors can be detected archaeologically by the emergence of long-distance flows of tools and raw materials that appear at least as early as the middle Pleistocene (30). The extent to which modern hunter-gatherer patterns represent ancestral patterns is a complex question. For example, most of our sample societies were censused after the elimination of warfare, and many had been geographically displaced or lived in environments that had been substantially depleted of large game. All modern hunter-gatherers use projectile weapons that were not available to our distant ancestors. Without causal models of residential association that consider the impacts of technology, warfare, cooperative hunting, territorial inheritance, depletion, and demographic crashes, we should be cautious about

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Proportion of siblings co-residing

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References and Notes 1. K. Hill, M. Barton, A. M. Hurtado, Evol. Anthropol. 18, 187 (2009). 2. M. Gurven, Behav. Brain Sci. 27, 543 (2004). 3. P. K. Ivey, G. A. Morelli, E. Z. Tronick, in Hunter-Gatherer Childhoods: Evolutionary, Developmental and Cultural Perspectives, B. Hewlett, M. Lamb, Eds. (Aldine de Gruyter, New York, 2005), pp. 191–213. 4. K. Hill, Hum. Nat. 13, 105 (2002). 5. K. Hawkes, J. F. O’Connell, N. G. Jones, H. Alvarez, E. L. Charnov, Proc. Natl. Acad. Sci. U.S.A. 95, 1336 (1998).

6. H. S. Kaplan, K. R. Hill, J. B. Lancaster, A. M. Hurtado, Evol. Anthropol. 9, 156 (2000). 7. S. B. Hrdy, Mothers and Others (Belknap, Cambridge, MA, 2009). 8. K. R. Hill, A. M. Hurtado, Proc. Biol. Sci. 276, 3863 (2009). 9. C. F. Camerer, E. Fehr, Science 311, 47 (2006). 10. E. Herrmann, J. Call, M. V. Hernàndez-Lloreda, B. Hare, M. Tomasello, Science 317, 1360 (2007). 11. G. Csibra, Trends Cogn. Sci. 11, 95 (2007). 12. E. Fehr, H. Bernhard, B. Rockenbach, Nature 454, 1079 (2008). 13. J. Henrich et al., Science 312, 1767 (2006). 14. J. B. Silk, Science 311, 1248 (2006). 15. E. Service, Primitive Social Organization (Random House, New York, 1962). 16. J. Helm, in Man the Hunter, R. B. Lee, I. DeVore, Eds. (Aldine, Chicago, 1968), pp. 118–139. 17. M. Chudek, W. Zhao, J. Henrich, in Signaling, Commitment, and Emotion, R. Joyce, K. Sterelny, B. Calcott, Eds. (MIT Press, Cambridge, MA, 2010), pp. 1–24. 18. B. Chapais, Primeval Kinship: How Pair-Bonding Gave Birth to Human Society (Harvard Univ. Press, Cambridge, MA, 2008). 19. W. Allen-Arave, M. Gurven, K. R. Hill, Evol. Hum. Behav. 29, 305 (2008). 20. K. Panchanathan, R. Boyd, Nature 432, 499 (2004).

Ordered and Dynamic Assembly of Single Spliceosomes Aaron A. Hoskins,1,2 Larry J. Friedman,2 Sarah S. Gallagher,3* Daniel J. Crawford,1,2 Eric G. Anderson,1 Richard Wombacher,3† Nicholas Ramirez,1‡ Virginia W. Cornish,3 Jeff Gelles,2§ Melissa J. Moore1§ The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells. he spliceosome is a complex macromolecular machine responsible for removing introns from nascent transcripts via pre-mRNA (precursor to mRNA) splicing (1). The spliceosome is composed of five small nuclear RNAs (snRNAs) and ~100 core proteins minimally required for activity in vitro (2). The snRNAs

T

1 Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. 2Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA. 3 Department of Chemistry, Columbia University, New York, NY 10027, USA.

*Present address: Environmental Protection Agency, Washington, DC 20004, USA. †Present address: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg D-69120, Germany. ‡Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. §To whom correspondence should be addressed. E-mail: [email protected] (J.G.); [email protected] (M.J.M.)

and many core proteins are arranged into stable subcomplexes constituting small nuclear ribonucleoprotein particles [U1 and U2 small nuclear ribonucleoproteins (snRNPs) and the U4/U6.U5 tri-snRNP] and the multiprotein Prp19-complex (NTC). Although association of U1 with premRNA can occur in the absence of adenosine triphosphate (ATP), stable association of all other subcomplexes requires ATP hydrolysis. Intron excision occurs after the spliceosome has been fully assembled and activated by additional structural rearrangements (3). Current models of spliceosome assembly, activation, and catalysis generally depict it as an ordered (U1 → U2 → tri-snRNP → NTC → activation → catalysis), one-way process (3). Yet deviations from the ordered assembly model have been reported (4–6), with some studies suggesting that both spliceosome assembly and catalysis are dynamic and reversible (7–9). None of these studies, however, directly examined the ki-

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21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

R. Boyd, P. J. Richerson, Proc. Br. Acad. 88, 77 (1996). R. Boyd, S. Mathew, Science 316, 1858 (2007). J. Henrich, Am. Antiq. 69, 197 (2004). A. Powell, S. Shennan, M. G. Thomas, Science 324, 1298 (2009). See supporting material on Science Online. K. Hill, A. M. Hurtado, Ache Life History: The Ecology and Demography of a Foraging People (Aldine, New York, 1996). R. Boyd, P. J. Richerson, J. Theor. Biol. 215, 287 (2002). M. A. Klein, R. Boyd, Proc. Biol. Sci. 277, 2559 (2010). P. Wiessner, Am. Antiq. 48, 253 (1983). S. Mcbrearty, A. S. Brooks, J. Hum. Evol. 39, 453 (2000). We thank each of our study populations for cooperation with data collection, W. Denham for compiling the GCBS database, and B. Chapais, M. Flinn, P. Gardner, M. Gurven, and N. Peterson for discussions.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1286/DC1 Materials and Methods SOM Text Figs. S1 to S12 Figs. S1 to S3 References 14 October 2010; accepted 18 January 2011 10.1126/science.1199071

netics of subcomplex association with pre-mRNA. We monitored subcomplex dynamics during spliceosome assembly in real time by combining yeast genetic engineering, chemical biology, and a multiwavelength fluorescence technique, colocalization single-molecule spectroscopy (CoSMoS) (10). Labeling spliceosome subcomplexes. We previously established that splicing of single premRNA molecules can be monitored by multiwavelength total internal reflection fluorescence (TIRF) microscopy in the complex environment of Saccharomyces cerevisiae whole-cell extract (yeast WCE) (11). To enable kinetic analysis of spliceosome assembly, we have now developed methods to introduce fluorophores into individual spliceosomal subcomplexes in WCE. Protein labeling was accomplished using homologous recombination to fuse either a SNAP (an alkylguanine S-transferase) (12) or an Escherichia coli DHFR (dihydrofolate reductase) tag (13) onto the C terminus of numerous spliceosomal proteins. These tags enabled us to incorporate bright, photostable organic dyes into the subcomplexes and to avoid the poor photon output and blinking behavior of single fluorescent proteins (14). Integration of two orthogonal tags allows for simultaneous monitoring of two different subcomplexes by CoSMoS (Fig. 1). To ensure functionality of the tagged species, we tagged only essential proteins and verified that the resultant strains (table S1) had growth rates and in vitro splicing activities comparable to the parental strain (figs. S1 to S3). By using several selectable markers, we were able to incorporate up to three tags into a single strain. Multiple tags present in the same subcomplex minimized artifacts due to incomplete labeling, photobleaching, and/or long-lived dark-state formation of single fluorophores (15). DHFR tags were labeled by adding excess (20 nM) fluorophore-trimethoprim (TMP) con-

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Downloaded from www.sciencemag.org on March 10, 2011

the use of specific modern groups as analogs for past patterns. Nonetheless, the robustness of our main result suggests that our foraging ancestors evolved a novel social structure that emphasized bilateral kin associations, frequent brother-sister affiliation, important affinal alliances, and coresidence with many unrelated individuals. How this social structure evolved, and how it in turn affected cooperation and cultural capacity—and the role of language in all these features—are key to understanding the emergence of human uniqueness.

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References and Notes 1. K. Hill, M. Barton, A. M. Hurtado, Evol. Anthropol. 18, 187 (2009). 2. M. Gurven, Behav. Brain Sci. 27, 543 (2004). 3. P. K. Ivey, G. A. Morelli, E. Z. Tronick, in Hunter-Gatherer Childhoods: Evolutionary, Developmental and Cultural Perspectives, B. Hewlett, M. Lamb, Eds. (Aldine de Gruyter, New York, 2005), pp. 191–213. 4. K. Hill, Hum. Nat. 13, 105 (2002). 5. K. Hawkes, J. F. O’Connell, N. G. Jones, H. Alvarez, E. L. Charnov, Proc. Natl. Acad. Sci. U.S.A. 95, 1336 (1998).

6. H. S. Kaplan, K. R. Hill, J. B. Lancaster, A. M. Hurtado, Evol. Anthropol. 9, 156 (2000). 7. S. B. Hrdy, Mothers and Others (Belknap, Cambridge, MA, 2009). 8. K. R. Hill, A. M. Hurtado, Proc. Biol. Sci. 276, 3863 (2009). 9. C. F. Camerer, E. Fehr, Science 311, 47 (2006). 10. E. Herrmann, J. Call, M. V. Hernàndez-Lloreda, B. Hare, M. Tomasello, Science 317, 1360 (2007). 11. G. Csibra, Trends Cogn. Sci. 11, 95 (2007). 12. E. Fehr, H. Bernhard, B. Rockenbach, Nature 454, 1079 (2008). 13. J. Henrich et al., Science 312, 1767 (2006). 14. J. B. Silk, Science 311, 1248 (2006). 15. E. Service, Primitive Social Organization (Random House, New York, 1962). 16. J. Helm, in Man the Hunter, R. B. Lee, I. DeVore, Eds. (Aldine, Chicago, 1968), pp. 118–139. 17. M. Chudek, W. Zhao, J. Henrich, in Signaling, Commitment, and Emotion, R. Joyce, K. Sterelny, B. Calcott, Eds. (MIT Press, Cambridge, MA, 2010), pp. 1–24. 18. B. Chapais, Primeval Kinship: How Pair-Bonding Gave Birth to Human Society (Harvard Univ. Press, Cambridge, MA, 2008). 19. W. Allen-Arave, M. Gurven, K. R. Hill, Evol. Hum. Behav. 29, 305 (2008). 20. K. Panchanathan, R. Boyd, Nature 432, 499 (2004).

Ordered and Dynamic Assembly of Single Spliceosomes Aaron A. Hoskins,1,2 Larry J. Friedman,2 Sarah S. Gallagher,3* Daniel J. Crawford,1,2 Eric G. Anderson,1 Richard Wombacher,3† Nicholas Ramirez,1‡ Virginia W. Cornish,3 Jeff Gelles,2§ Melissa J. Moore1§ The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells. he spliceosome is a complex macromolecular machine responsible for removing introns from nascent transcripts via pre-mRNA (precursor to mRNA) splicing (1). The spliceosome is composed of five small nuclear RNAs (snRNAs) and ~100 core proteins minimally required for activity in vitro (2). The snRNAs

T

1 Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. 2Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA. 3 Department of Chemistry, Columbia University, New York, NY 10027, USA.

*Present address: Environmental Protection Agency, Washington, DC 20004, USA. †Present address: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg D-69120, Germany. ‡Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. §To whom correspondence should be addressed. E-mail: [email protected] (J.G.); [email protected] (M.J.M.)

and many core proteins are arranged into stable subcomplexes constituting small nuclear ribonucleoprotein particles [U1 and U2 small nuclear ribonucleoproteins (snRNPs) and the U4/U6.U5 tri-snRNP] and the multiprotein Prp19-complex (NTC). Although association of U1 with premRNA can occur in the absence of adenosine triphosphate (ATP), stable association of all other subcomplexes requires ATP hydrolysis. Intron excision occurs after the spliceosome has been fully assembled and activated by additional structural rearrangements (3). Current models of spliceosome assembly, activation, and catalysis generally depict it as an ordered (U1 → U2 → tri-snRNP → NTC → activation → catalysis), one-way process (3). Yet deviations from the ordered assembly model have been reported (4–6), with some studies suggesting that both spliceosome assembly and catalysis are dynamic and reversible (7–9). None of these studies, however, directly examined the ki-

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21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

R. Boyd, P. J. Richerson, Proc. Br. Acad. 88, 77 (1996). R. Boyd, S. Mathew, Science 316, 1858 (2007). J. Henrich, Am. Antiq. 69, 197 (2004). A. Powell, S. Shennan, M. G. Thomas, Science 324, 1298 (2009). See supporting material on Science Online. K. Hill, A. M. Hurtado, Ache Life History: The Ecology and Demography of a Foraging People (Aldine, New York, 1996). R. Boyd, P. J. Richerson, J. Theor. Biol. 215, 287 (2002). M. A. Klein, R. Boyd, Proc. Biol. Sci. 277, 2559 (2010). P. Wiessner, Am. Antiq. 48, 253 (1983). S. Mcbrearty, A. S. Brooks, J. Hum. Evol. 39, 453 (2000). We thank each of our study populations for cooperation with data collection, W. Denham for compiling the GCBS database, and B. Chapais, M. Flinn, P. Gardner, M. Gurven, and N. Peterson for discussions.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1286/DC1 Materials and Methods SOM Text Figs. S1 to S12 Figs. S1 to S3 References 14 October 2010; accepted 18 January 2011 10.1126/science.1199071

netics of subcomplex association with pre-mRNA. We monitored subcomplex dynamics during spliceosome assembly in real time by combining yeast genetic engineering, chemical biology, and a multiwavelength fluorescence technique, colocalization single-molecule spectroscopy (CoSMoS) (10). Labeling spliceosome subcomplexes. We previously established that splicing of single premRNA molecules can be monitored by multiwavelength total internal reflection fluorescence (TIRF) microscopy in the complex environment of Saccharomyces cerevisiae whole-cell extract (yeast WCE) (11). To enable kinetic analysis of spliceosome assembly, we have now developed methods to introduce fluorophores into individual spliceosomal subcomplexes in WCE. Protein labeling was accomplished using homologous recombination to fuse either a SNAP (an alkylguanine S-transferase) (12) or an Escherichia coli DHFR (dihydrofolate reductase) tag (13) onto the C terminus of numerous spliceosomal proteins. These tags enabled us to incorporate bright, photostable organic dyes into the subcomplexes and to avoid the poor photon output and blinking behavior of single fluorescent proteins (14). Integration of two orthogonal tags allows for simultaneous monitoring of two different subcomplexes by CoSMoS (Fig. 1). To ensure functionality of the tagged species, we tagged only essential proteins and verified that the resultant strains (table S1) had growth rates and in vitro splicing activities comparable to the parental strain (figs. S1 to S3). By using several selectable markers, we were able to incorporate up to three tags into a single strain. Multiple tags present in the same subcomplex minimized artifacts due to incomplete labeling, photobleaching, and/or long-lived dark-state formation of single fluorophores (15). DHFR tags were labeled by adding excess (20 nM) fluorophore-trimethoprim (TMP) con-

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Downloaded from www.sciencemag.org on March 10, 2011

the use of specific modern groups as analogs for past patterns. Nonetheless, the robustness of our main result suggests that our foraging ancestors evolved a novel social structure that emphasized bilateral kin associations, frequent brother-sister affiliation, important affinal alliances, and coresidence with many unrelated individuals. How this social structure evolved, and how it in turn affected cooperation and cultural capacity—and the role of language in all these features—are key to understanding the emergence of human uniqueness.

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competent pre-mRNA. Similar results were obtained with the analogous SNAP-tagged strains (fig. S5, B to E). Although the above results indicated that we could observe subcomplex association with surfacetethered pre-mRNA molecules, they did not reveal what fraction of those pre-mRNAs ultimately spliced. To address this, we combined our previously described Cy3/Alexa647 splicing reporter pre-mRNA (11) with extracts in which either U1 or NTC was labeled with SNAP-Atto488 (Fig. 3). In these experiments, disappearance of fluorescence from the Alexa647-labeled intron without loss of the Cy3-labeled exon demonstrates that either the pre-mRNA was spliced and the spliceosome/lariat intron complex departed from the surface-tethered mRNA or that the Alexa647 was photobleached (11).

with pre-mRNA during assembly and then be expelled before catalytic activation (2, 19). The tri-snRNP and NTC were individually labeled via Brr2 and Snu114 (core U5 components) and Cef1 and Ntc90 (core NTC components). Both U5 and NTC are thought to remain spliceosomeassociated throughout activation and catalysis, departing only upon mRNA product release. As expected, only U1 spots accumulated on WT pre-mRNA in the absence of ATP (Fig. 2E and movie S1). In contrast, all subcomplexes accumulated in the presence of ATP, albeit at different rates (Fig. 2F and movie S2). These rates were consistent with an apparent order of assembly: U1 → U2 → tri-snRNP → NTC. Like U1, accumulation of U2, U5, and NTC was also dependent on an intact 5′ SS (fig. S5A), confirming the specificity of the interactions for a splicing-

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jugates (e.g., Cy3-TMP) to WCE. TMP binding to DHFR is noncovalent, but the ternary complex formed between DHFR, TMP, and endogenous NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) (20-30 mM in WCE) is extremely long-lived (16). SNAP tags were covalently labeled by incubating WCE with benzylguanine dye conjugates (e.g., SNAP-DY549) and then removing excess dye by gel filtration. SDS–polyacrylamide gel electrophoresis (SDSPAGE) confirmed labeling specificity (fig. S4A) and efficiency (70 to 90% labeling of functional SNAP tags) (fig. S4B). None of the dye adducts or labeling procedures employed here greatly inhibit splicing in vitro (figs. S2 and S3). Single-molecule experiments were carried out in WCE containing fluorescently tagged proteins, an O2 scavenging system, and triplet-state quenchers (17). Data were acquired using a TIRF microscope with laser excitation at 488, 532, and 633 nm. Such TIRF experiments detect surfacebound molecules as discrete spots, while fluorescent components in solution remain as diffuse background. To monitor spliceosome assembly, a model pre-mRNA derived from the rp51a transcript (11, 18) containing a single fluorophore and 3′ biotin was tethered to a streptavidinderivatized glass surface at densities of 100 to 250 pre-mRNA molecules per 314 mm2 field of view (FOV) (Fig. 1). Arrivals and departures of individual spliceosomal subcomplexes were visualized as the appearance and disappearance of fluorescent spots that colocalized with surfacetethered pre-mRNAs. Subcomplexes accumulate on surface-tethered pre-mRNAs and form functional spliceosomes. The first subcomplex to bind during spliceosome assembly is thought to be U1 snRNP, which interacts with the 5′ splice site (SS). To validate our approach, we monitored U1 association (with DHFR/Cy3-TMP tags on U1 components Snp1 and Prp40) in the presence of ATP with either wildtype (WT) pre-mRNA or a mutant version in which the 5′ SS had been mutated (G/GUAUGU → c/aUAccU). No stable association was observed in the absence of tethered RNA or with the 5′ SS mutant pre-mRNA (Fig. 2, A and B). As expected, U1 spots were present on a surface containing WT pre-mRNA (Fig. 2C). Monitoring of U1 association with WT pre-mRNA over time revealed rapid surface accumulation of U1 signals during the first 5 min (Fig. 2D and movies S1 and S2). In contrast, no time-dependent signal accumulation was observed in the absence of pre-mRNA or with the 5′ SS mutant. Thus, the long-lived signals are dependent both on the presence of premRNA and an intact 5′ SS. We next compared the kinetics of U1 association with those of U2, tri-snRNP, and the NTC. Binding events for individual subcomplexes were monitored in separate experiments using WCEs containing two DHFR/Cy3-TMP tags on a given subcomplex (table S1). U2 was labeled via the U2-SF3b components Cus1 and Hsh155. Both U1 and U2-SF3b are thought to stably associate

RESEARCH ARTICLES (53 T 5%) of pre-mRNAs that acquired NTC lost intron fluorescence, suggesting that commitment increases as assembly proceeds. Analysis of individual U1 and NTC binding event lifetimes indicated that pre-mRNAs that ultimately lost their intron signals tended to have U1 lifetimes about twice as long and NTC lifetimes about half as long (fig. S6). One possible explanation is that productive U1 association is stabilized by binding of additional spliceosome assembly factors. Conversely, the shorter NTC lifetime may indicate that properly assembled spliceosomes proceed rapidly through activation, catalysis, and mRNA product release soon after NTC binding. Order and kinetics of spliceosome assembly. Although the experiments in Fig. 2 can define the

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population-averaged timing with which different subcomplexes arrive at the pre-mRNA, they do not directly assess the order of subcomplex addition on individual pre-mRNA molecules. Further, the data in Fig. 2 and fig. S5 are composites of subcomplex association and dissociation events, photobleaching, and TMP dye exchange and are additionally complicated by variations in WCE splicing activity. These issues can be resolved by using CoSMoS to simultaneously follow the pre-mRNA association of two spliceosomal subcomplexes in the same WCE. To do so, we used two DHFR/Cy5-TMP tags and a single SNAP DY549 tag to label two subcomplexes (e.g., U1DHFR and U2-SNAP) with different fluorophores in the same extract (triple-label extracts,

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Similar to our previous observations (11), the extent of intron fluorescence loss was 15 T 2% (SE) and 18 T 2% for ATP-containing U1-SNAP and NTC-SNAP extracts, respectively, compared with 4 T 1% for an inactive no-ATP control (where loss measures photobleaching). These results indicate that active spliceosomes are formed on the surface-linked pre-mRNAs in our labeled extracts. For both U1 and NTC, we could observe numerous pre-mRNAs that both gained the labeled subcomplex and lost intron fluorescence (table S2). Interestingly, only 21 T 3% of premRNAs that had at least one U1 binding event also lost intron fluorescence. This indicates that interaction with U1 does not absolutely commit a pre-mRNA to splicing. In contrast, roughly half

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indicated. For WT pre-mRNA, the decrease in spot number after 10 min is due to Cy3-TMP photobleaching in this experiment. Experiments in (A) to (D) contained ATP. (E and F) Smoothed (9-point moving block averaged) curves of indicated subcomplex spots per pre-mRNA versus time, minus (E) or plus (F) ATP. Each subcomplex was monitored in a different WCE. Data in (F) are the average of n = 4 replicates. VOL 331

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Fig. 1), and visualized them binding to individual Alexa488-labeled pre-mRNA molecules (Fig. 4, A and B). As was observed with the individually labeled extracts, when both U1 and U2 were labeled in the same WCE, only U1 colocalized with pre-mRNAs in the absence of ATP, whereas both U1 and U2 colocalized with pre-mRNAs in the presence of ATP (Fig. 4, B and C, and movie S3). When individual pre-mRNA molecules were followed over time, the largest class (49%) (table S3) exhibited at least one discrete onset of U1 fluorescence and at least one discrete onset of U2 fluorescence (Fig. 4D and fig. S7). Other classes exhibited only U1 binding (18%), only U2 binding (6%), or no binding events (27%). These latter subpopulations may arise from the presence of some nonfluorescent subcomplexes in the extract and/or from alternative conformations of the pre-mRNA (7, 15) that prevent spliceosome assembly. U1 and U2 spots persisted for seconds to minutes before disappearing due to either dye photobleaching or subcomplex dissociation. For U1, which was labeled with two DHFR tags, fluorescence typically vanished in one or two discrete steps (96% of events) (table S4). Analogously, for U2, which was labeled with one SNAP tag, fluorescence most often vanished in a single step (88% of events). Thus, only one copy each of U1 and U2 is present at any given time on the majority of pre-mRNAs. To quantitatively evaluate the U1 and U2 binding order on individual pre-mRNA molecules (Fig. 4D), we calculated tU2-tU1, the difference between the arrival times of the two subcomplexes (20). A histogram (Fig. 4E) shows that the overwhelming majority (90%) of these delay times were positive, indicating that U2 binding nearly always followed U1 binding. This conclusion was confirmed by correlation analysis

of the absolute binding times (fig. S8), which revealed that even U1 binding events occurring late in the experiment were soon followed by U2 binding. Although U1 and U2 appeared to arrive simultaneously on a small minority (9 out of 223 events) of pre-mRNAs, some of these are likely cases of U1 and U2 arriving in rapid succession separated by a delay that the experimental time resolution (5 to 6 s) was insufficient to resolve (20) (table S5). Thus, assembly is highly ordered, with U1 always or almost always binding before U2. Further, >95% of pre-mRNAs that acquire both U1 and U2 acquire them separately rather than as a preformed U1/U2 complex. Consequently, formation of a U1/U2 complex before association with pre-mRNA cannot be a requirement for splicing because the fraction of premRNAs that splice is greater than 5% (table S2). To examine the ordering of later assembly steps, we used the same methodologies with other triply labeled yeast strains. U2 fluorescence almost always preceded onset of U5 fluorescence (Fig. 4F, fig. S9, and table S6); 97% of the tU5-tU2 values were positive (Fig. 4G). Similarly, U5 fluorescence almost always preceded onset of NTC fluorescence (Fig. 4H, fig. S10, and table S7); 91% of the tNTC-tU5 delay values were positive (Fig. 4I). In both the U2/U5 and U5/NTC data sets, very few traces (table S5) exhibited apparent simultaneous binding of the subcomplexes, and analysis of all traces suggested that at most one copy each of U5 and NTC were present on the majority of pre-mRNAs (table S4). In sum, our data indicate that when spliceosome assembly is followed on individual RP51A pre-mRNA molecules, the predominant reaction pathway is highly ordered (U1 → U2 → tri-snRNP → NTC). Further, the experiments indicate little or no preassociation for any pair of subcomplexes studied (table S5). As with U1/U2, these data demon-

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strate that no preassociation of these subcomplexes is required for splicing. On top of providing information about binding order, the CoSMoS methodology permits measurement of defined kinetic parameters. The arrival times of the first U1 subcomplex on each pre-mRNA and all three time-delay data sets (tU2-tU1, tU5-tU2, and tNTC-tU5) are well fit by single exponential distributions (fig. S11), allowing determination of apparent first-order rate constants (Fig. 5). All four rate constants fall in a narrow range (0.1 to 0.4 min−1), suggesting that no single subcomplex association step predominantly limits the rate of spliceosome assembly on RP51A pre-mRNA. In addition to arrival times, the triple-label experiments also allowed us to examine the order of subcomplex loss from pre-mRNA. Preliminary analysis revealed that U1 fluorescence tended to be lost before U2 fluorescence, and U2 fluorescence tended to be lost before U5 fluorescence. Only with U5 and NTC did a significant number of pre-mRNAs lose fluorescence from both subcomplexes simultaneously (table S8). These results are consistent with known postassembly events, including ordered loss of U1 and the SF3b component of U2 during spliceosome activation and subsequent simultaneous loss of U5 and NTC coincident with spliced mRNA release (2, 19). Although additional analyses of photobleaching and Cy5-TMP dye exchange rates will be required to fully interpret these results, they do indicate that subcomplex dissociation coupled to activation and spliceosome disassembly is detectable using this methodology. Definitive analysis of subcomplex dissociation relative to catalysis and intron release awaits future development of more photostable splicing reporters. We also examined dissociation kinetics of each subcomplex (20). In all cases, good fits of

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these mechanisms may be possible by combining CoSMoS with appropriate mutants and inhibitors of assembly. Pre-mRNAs can engage subcomplexes multiple times. Subsequent to dissociation of a

more than one species from which each subcomplex can dissociate. Thus, subcomplex dissociation is more complex than some current models suggest, and there are multiple mechanisms consistent with our data (fig. S13). Elucidation of

dwell-time distributions required a function containing more than one exponential term (fig. S12 and table S9). This presence of both short(t1 < 1 min) and long-lived (t2 > 1 min) characteristic dwell times indicates that there is

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the absence of ATP, U1 had a dwell-time distribution nearly identical to that observed in the presence of ATP (fig. S17 and table S9). This suggests that ATP hydrolysis by RNA helicases or other snRNP remodeling enzymes in WCE is not required for U1 dissociation. A previous study using native PAGE reported two different ATP-independent U1:pre-mRNA complexes: dun and dcommit (21). The more abundant dun (unstable and uncommitted) did not survive challenge from competitor RNAs, whereas the minor dcommit represented a more stable, challenge-resistant species. Because it could be chased into subsequent steps of the splicing pathway, dcommit is likely the same species as U1-containing commitment complexes (CC1 and/or CC2) (22). Our analysis of U1 snRNP

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particular subcomplex, many pre-mRNA molecules reacquired a copy of the same subcomplex. On individual pre-mRNAs, U1 often appeared to bind and dissociate repeatedly (Fig. 5A and fig. S14). Use of two covalent SNAP labels on U1 allowed us to verify by photobleaching that the majority of reoccurring U1-SNAP signals resulted from association and dissociation of different U1 molecules (20) (fig. S15 and table S10) rather than the blinking of a single molecule (15). Further, using the splicing reporter pre-mRNA (Fig. 3), we could observe multiple U1 binding events on pre-mRNAs that spliced (20 T 7% of pre-mRNAs that lost intron fluorescence acquired multiple U1 signals) (fig. S16). Thus premRNAs that have multiple encounters with U1 are not irreversibly trapped in an inactive state. In

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pair of splice sites to splicing, then alternative splice site choice can potentially be regulated at any stage of assembly. This hypothesis is bolstered by observations that some regulation of alternative splicing apparently occurs at late stages of assembly (23, 24). By making possible kinetic analysis of spliceosome assembly in whole-cell extracts, this work opens the door to answering fundamental questions concerning the mechanisms of pre-mRNA splicing. The combination of CoSMoS with chemical and genetic tools is a powerful approach for elucidating the mechanisms of complex biological processes, even when those processes can only be studied in cell extracts. These methods should prove broadly useful for analyzing many other complex macromolecular machines. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

T. W. Nilsen, Bioessays 25, 1147 (2003). P. Fabrizio et al., Mol. Cell 36, 593 (2009). M. C. Wahl, C. L. Will, R. Lührmann, Cell 136, 701 (2009). S. W. Stevens et al., Mol. Cell 9, 31 (2002). Y. Z. Xu et al., EMBO J. 23, 376 (2004). M. Schneider et al., Mol. Cell 38, 223 (2010). J. Abelson et al., Nat. Struct. Mol. Biol. 17, 504 (2010). C. K. Tseng, S. C. Cheng, Science 320, 1782 (2008). L. Liu, C. C. Query, M. M. Konarska, Nat. Struct. Mol. Biol. 14, 519 (2007). L. J. Friedman, J. Chung, J. Gelles, Biophys. J. 91, 1023 (2006). D. J. Crawford, A. A. Hoskins, L. J. Friedman, J. Gelles, M. J. Moore, RNA 14, 170 (2008). A. Juillerat et al., Chem. Biol. 10, 313 (2003). L. W. Miller, Y. Cai, M. P. Sheetz, V. W. Cornish, Nat. Methods 2, 255 (2005). R. M. Dickson, A. B. Cubitt, R. Y. Tsien, W. E. Moerner, Nature 388, 355 (1997).

Organic Aerosol Formation Downwind from the Deepwater Horizon Oil Spill J. A. de Gouw,1,2* A. M. Middlebrook,1 C. Warneke,1,2 R. Ahmadov,1,2 E. L. Atlas,3 R. Bahreini,1,2 D. R. Blake,4 C. A. Brock,1 J. Brioude,1,2 D. W. Fahey,1 F. C. Fehsenfeld,1,2 J. S. Holloway,1,2 M. Le Henaff,3 R. A. Lueb,5 S. A. McKeen,1,2 J. F. Meagher,1 D. M. Murphy,1 C. Paris,3 D. D. Parrish,1 A. E. Perring,1,2 I. B. Pollack,1,2 A. R. Ravishankara,1 A. L. Robinson,6 T. B. Ryerson,1 J. P. Schwarz,1,2 J. R. Spackman,1,2 A. Srinivasan,3 L. A. Watts1,2 A large fraction of atmospheric aerosols are derived from organic compounds with various volatilities. A National Oceanic and Atmospheric Administration (NOAA) WP-3D research aircraft made airborne measurements of the gaseous and aerosol composition of air over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico that occurred from April to August 2010. A narrow plume of hydrocarbons was observed downwind of DWH that is attributed to the evaporation of fresh oil on the sea surface. A much wider plume with high concentrations of organic aerosol (>25 micrograms per cubic meter) was attributed to the formation of secondary organic aerosol (SOA) from unmeasured, less volatile hydrocarbons that were emitted from a wider area around DWH. These observations provide direct and compelling evidence for the importance of formation of SOA from less volatile hydrocarbons.

O

n 20 April 2010, the Deepwater Horizon (DWH) offshore drilling unit exploded, causing the riser pipe to rupture and

crude oil to flow into the Gulf of Mexico from a depth of ~1500 m. The oil leak rate was estimated to be 68,000 barrels per day (1), and much of that

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15. I. Rasnik, S. A. McKinney, T. Ha, Nat. Methods 3, 891 (2006). 16. S. M. Dunn, R. W. King, Biochemistry 19, 766 (1980). 17. R. Dave, D. S. Terry, J. B. Munro, S. C. Blanchard, Biophys. J. 96, 2371 (2009). 18. B. Séraphin, M. Rosbash, EMBO J. 10, 1209 (1991). 19. R. M. Lardelli, J. X. Thompson, J. R. Yates 3rd, S. W. Stevens, RNA 16, 516 (2010). 20. Materials and methods are available as supporting material on Science Online. 21. S. W. Ruby, J. Biol. Chem. 272, 17333 (1997). 22. P. Legrain, B. Seraphin, M. Rosbash, Mol. Cell. Biol. 8, 3755 (1988). 23. M. Chen, J. L. Manley, Nat. Rev. Mol. Cell Biol. 10, 741 (2009). 24. S. Bonnal et al., Mol. Cell 32, 81 (2008). 25. We thank J. Chung, A. Okonechnikov, J. Yan, J. Haber, S. Lovett, I. Correa, M.-Q. Xu, Z. Chen, and B. Smith for helpful discussions and assistance. This work was supported by NIH RO1s GM043369 (J.G.), GM81648 (J.G.), GM053007 (M.J.M), GM54469 (V.W.C.), RC1 GM091804 (V.W.C), National Research Service Award fellowship GM079971 (A.A.H.), and K99/R00 GM086471 (A.A.H.). D.J.C., S.S.G., and R.W were supported by NIH training grant GM759628, a National Defense Science and Engineering Graduate fellowship, and a Deutscher Akademischer Austausch Dienst fellowship, respectively. M.J.M. is a Howard Hughes Medical Institute investigator. V.W.C. holds patents on the TMP-tag technology, and the technology is licensed and commercialized by Active Motif.

Supporting Online Information www.sciencemag.org/cgi/content/full/331/6022/1289/DC1 Materials and Methods Figs. S1 to S21 Scheme S1 Tables S1 to S12 Movies S1 to S3 References 7 October 2010; accepted 28 January 2011 10.1126/science.1198830

REPORTS oil accumulated on the sea surface. A NOAA WP-3D research aircraft equipped with a large number of instruments to characterize trace gases and aerosols (2) performed two flights near DWH on 8 and 10 June to explore the atmospheric impacts of the spilled oil and of the cleanup activities near DWH. This report discusses one of those impacts: the formation of large concentrations of secondary organic aerosol (SOA) observed downwind from the oil spill. These findings have implications for our general understanding of organic aerosol, which is a large but poorly understood class of atmospheric aerosol

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pre-mRNAs (Fig. 5, B to D, and figs. S14 and S19). As seen with U1, multiple NTC binding events could be detected on the splicing reporter pre-mRNA (4 T 2% of pre-mRNAs both lost their intron signal and acquired NTC more than once) (fig. S16). The number of binding events observed per pre-mRNA molecule was dependent on the subcomplex being studied. U1 exhibited by far the largest number of binding events, with the number of events systematically decreasing for each successive subcomplex in the pathway (fig. S20). This suggests that at each step of subcomplex addition, some fraction of the pre-mRNA molecules are lost to side pathways that do not lead to productive splicing (Fig. 5E). Discussion. Taken together, the data from this real-time kinetic analysis of spliceosome assembly are consistent with existing models and lead to new insights. Spliceosome assembly on the RP51A substrate is highly ordered (U1 → U2 → tri-snRNP → NTC), and pre-association of the subcomplexes is not required for splicing. Although no single step appears to irreversibly commit this pre-mRNA to splicing, commitment increases as spliceosome assembly proceeds. Further, spliceosome assembly on this pre-mRNA is kinetically efficient, with no single subcomplex binding step predominantly restricting the overall rate. Finally, we have directly observed multiple binding events for all subcomplexes, demonstrating that subcomplex binding is reversible. Together, these findings have important implications for the regulation of alternative splicing. If spliceosome assembly is reversible and no single assembly step irreversibly commits a particular

1 Chemical Sciences Division, Earth System Research Laboratory, National Oceanic and Atmospheric Administration, Boulder, CO 80305, USA. 2Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80309, USA. 3Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA. 4Department of Chemistry, University of California, Irvine, CA 92697, USA. 5Atmospheric Chemistry Division, Earth System Laboratory, National Center for Atmospheric Research, Boulder, CO 80301, USA. 6Center for Atmospheric Particle Studies, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

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pair of splice sites to splicing, then alternative splice site choice can potentially be regulated at any stage of assembly. This hypothesis is bolstered by observations that some regulation of alternative splicing apparently occurs at late stages of assembly (23, 24). By making possible kinetic analysis of spliceosome assembly in whole-cell extracts, this work opens the door to answering fundamental questions concerning the mechanisms of pre-mRNA splicing. The combination of CoSMoS with chemical and genetic tools is a powerful approach for elucidating the mechanisms of complex biological processes, even when those processes can only be studied in cell extracts. These methods should prove broadly useful for analyzing many other complex macromolecular machines. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

T. W. Nilsen, Bioessays 25, 1147 (2003). P. Fabrizio et al., Mol. Cell 36, 593 (2009). M. C. Wahl, C. L. Will, R. Lührmann, Cell 136, 701 (2009). S. W. Stevens et al., Mol. Cell 9, 31 (2002). Y. Z. Xu et al., EMBO J. 23, 376 (2004). M. Schneider et al., Mol. Cell 38, 223 (2010). J. Abelson et al., Nat. Struct. Mol. Biol. 17, 504 (2010). C. K. Tseng, S. C. Cheng, Science 320, 1782 (2008). L. Liu, C. C. Query, M. M. Konarska, Nat. Struct. Mol. Biol. 14, 519 (2007). L. J. Friedman, J. Chung, J. Gelles, Biophys. J. 91, 1023 (2006). D. J. Crawford, A. A. Hoskins, L. J. Friedman, J. Gelles, M. J. Moore, RNA 14, 170 (2008). A. Juillerat et al., Chem. Biol. 10, 313 (2003). L. W. Miller, Y. Cai, M. P. Sheetz, V. W. Cornish, Nat. Methods 2, 255 (2005). R. M. Dickson, A. B. Cubitt, R. Y. Tsien, W. E. Moerner, Nature 388, 355 (1997).

Organic Aerosol Formation Downwind from the Deepwater Horizon Oil Spill J. A. de Gouw,1,2* A. M. Middlebrook,1 C. Warneke,1,2 R. Ahmadov,1,2 E. L. Atlas,3 R. Bahreini,1,2 D. R. Blake,4 C. A. Brock,1 J. Brioude,1,2 D. W. Fahey,1 F. C. Fehsenfeld,1,2 J. S. Holloway,1,2 M. Le Henaff,3 R. A. Lueb,5 S. A. McKeen,1,2 J. F. Meagher,1 D. M. Murphy,1 C. Paris,3 D. D. Parrish,1 A. E. Perring,1,2 I. B. Pollack,1,2 A. R. Ravishankara,1 A. L. Robinson,6 T. B. Ryerson,1 J. P. Schwarz,1,2 J. R. Spackman,1,2 A. Srinivasan,3 L. A. Watts1,2 A large fraction of atmospheric aerosols are derived from organic compounds with various volatilities. A National Oceanic and Atmospheric Administration (NOAA) WP-3D research aircraft made airborne measurements of the gaseous and aerosol composition of air over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico that occurred from April to August 2010. A narrow plume of hydrocarbons was observed downwind of DWH that is attributed to the evaporation of fresh oil on the sea surface. A much wider plume with high concentrations of organic aerosol (>25 micrograms per cubic meter) was attributed to the formation of secondary organic aerosol (SOA) from unmeasured, less volatile hydrocarbons that were emitted from a wider area around DWH. These observations provide direct and compelling evidence for the importance of formation of SOA from less volatile hydrocarbons.

O

n 20 April 2010, the Deepwater Horizon (DWH) offshore drilling unit exploded, causing the riser pipe to rupture and

crude oil to flow into the Gulf of Mexico from a depth of ~1500 m. The oil leak rate was estimated to be 68,000 barrels per day (1), and much of that

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15. I. Rasnik, S. A. McKinney, T. Ha, Nat. Methods 3, 891 (2006). 16. S. M. Dunn, R. W. King, Biochemistry 19, 766 (1980). 17. R. Dave, D. S. Terry, J. B. Munro, S. C. Blanchard, Biophys. J. 96, 2371 (2009). 18. B. Séraphin, M. Rosbash, EMBO J. 10, 1209 (1991). 19. R. M. Lardelli, J. X. Thompson, J. R. Yates 3rd, S. W. Stevens, RNA 16, 516 (2010). 20. Materials and methods are available as supporting material on Science Online. 21. S. W. Ruby, J. Biol. Chem. 272, 17333 (1997). 22. P. Legrain, B. Seraphin, M. Rosbash, Mol. Cell. Biol. 8, 3755 (1988). 23. M. Chen, J. L. Manley, Nat. Rev. Mol. Cell Biol. 10, 741 (2009). 24. S. Bonnal et al., Mol. Cell 32, 81 (2008). 25. We thank J. Chung, A. Okonechnikov, J. Yan, J. Haber, S. Lovett, I. Correa, M.-Q. Xu, Z. Chen, and B. Smith for helpful discussions and assistance. This work was supported by NIH RO1s GM043369 (J.G.), GM81648 (J.G.), GM053007 (M.J.M), GM54469 (V.W.C.), RC1 GM091804 (V.W.C), National Research Service Award fellowship GM079971 (A.A.H.), and K99/R00 GM086471 (A.A.H.). D.J.C., S.S.G., and R.W were supported by NIH training grant GM759628, a National Defense Science and Engineering Graduate fellowship, and a Deutscher Akademischer Austausch Dienst fellowship, respectively. M.J.M. is a Howard Hughes Medical Institute investigator. V.W.C. holds patents on the TMP-tag technology, and the technology is licensed and commercialized by Active Motif.

Supporting Online Information www.sciencemag.org/cgi/content/full/331/6022/1289/DC1 Materials and Methods Figs. S1 to S21 Scheme S1 Tables S1 to S12 Movies S1 to S3 References 7 October 2010; accepted 28 January 2011 10.1126/science.1198830

REPORTS oil accumulated on the sea surface. A NOAA WP-3D research aircraft equipped with a large number of instruments to characterize trace gases and aerosols (2) performed two flights near DWH on 8 and 10 June to explore the atmospheric impacts of the spilled oil and of the cleanup activities near DWH. This report discusses one of those impacts: the formation of large concentrations of secondary organic aerosol (SOA) observed downwind from the oil spill. These findings have implications for our general understanding of organic aerosol, which is a large but poorly understood class of atmospheric aerosol

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pre-mRNAs (Fig. 5, B to D, and figs. S14 and S19). As seen with U1, multiple NTC binding events could be detected on the splicing reporter pre-mRNA (4 T 2% of pre-mRNAs both lost their intron signal and acquired NTC more than once) (fig. S16). The number of binding events observed per pre-mRNA molecule was dependent on the subcomplex being studied. U1 exhibited by far the largest number of binding events, with the number of events systematically decreasing for each successive subcomplex in the pathway (fig. S20). This suggests that at each step of subcomplex addition, some fraction of the pre-mRNA molecules are lost to side pathways that do not lead to productive splicing (Fig. 5E). Discussion. Taken together, the data from this real-time kinetic analysis of spliceosome assembly are consistent with existing models and lead to new insights. Spliceosome assembly on the RP51A substrate is highly ordered (U1 → U2 → tri-snRNP → NTC), and pre-association of the subcomplexes is not required for splicing. Although no single step appears to irreversibly commit this pre-mRNA to splicing, commitment increases as spliceosome assembly proceeds. Further, spliceosome assembly on this pre-mRNA is kinetically efficient, with no single subcomplex binding step predominantly restricting the overall rate. Finally, we have directly observed multiple binding events for all subcomplexes, demonstrating that subcomplex binding is reversible. Together, these findings have important implications for the regulation of alternative splicing. If spliceosome assembly is reversible and no single assembly step irreversibly commits a particular

1 Chemical Sciences Division, Earth System Research Laboratory, National Oceanic and Atmospheric Administration, Boulder, CO 80305, USA. 2Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO 80309, USA. 3Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA. 4Department of Chemistry, University of California, Irvine, CA 92697, USA. 5Atmospheric Chemistry Division, Earth System Laboratory, National Center for Atmospheric Research, Boulder, CO 80301, USA. 6Center for Atmospheric Particle Studies, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

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that affects air quality and climate change (3). A parallel analysis of data from the flights over the oil spill focused on the quantification of atmospheric emissions in general, on the air-water partitioning of volatile organic compounds (VOCs), and on an estimate of the oil leak rate (4). A large fraction of aerosol in the atmosphere consists of organic material (5). In the polluted atmosphere, the dominant fraction of this organic aerosol (OA) is secondary (6): It is formed in the atmosphere from gas-phase species. Recent research has indicated that SOA formation in polluted air is much more efficient than expected from the measured VOCs [volatile is defined here as having an effective saturation concentration, C*, of >106 mg m−3 (7)] and from their particulate mass yields as determined in the laboratory (8–11). One potential explanation for this discrepancy is the formation of SOA from semivolatile organic compounds (SVOCs; C* = 10−1 to 103 mg m−3) or organic compounds of intermediate volatility (IVOCs; C* = 103 to 106 mg m−3) (7, 12). Because SVOCs and IVOCs are typically co-emitted with VOCs, this mechanism has not been unambiguously observed in the atmosphere. As a result, it is currently unknown how much of the discrepancy between measured and expected SOA can be attributed to formation from SVOCs and IVOCs. The oil spill provided a unique environment to study SOA formation from VOCs and IVOCs separately, because organic compounds were released from different parts of the oil slick depending on their volatility. During both flights over the oil slick, a narrow plume of VOCs and a much wider plume of OA were observed downwind of DWH (Fig. 1). Results from the 10 June 2010 flight are discussed here in detail, as higher and more constant wind speeds on this day led to a more easily interpretable data set. The extent of the surface oil slick on 10 June (Fig. 1) is estimated from a composite of multiple satellite instruments (13). VOCs and OA were not enhanced everywhere over the oil but were instead confined to much narrower plumes downwind of DWH. Data from two periods (defined in Fig. 1C) are further examined in Fig. 2. Period P1 represents the measurements made closest to DWH; period P2 was farthest from DWH. The evaporation of freshly surfaced oil was the dominant source of the VOCs measured downwind of DWH (4). In laboratory analyses of oil spilled at DWH (14), the first 23% of the mass evaporated within 2 hours (fig. S1). During a 2-hour period, the sea surface transport of oil is a few kilometers at most, which explains why the VOCs were emitted from only a small area and confined to a narrow plume (Fig. 1C). As described elsewhere in detail (4), the composition of VOCs measured in canister samples collected in the plumes was dominated by alkanes and aromatics. The relative abundance of the VOCs measured in the atmosphere reflected the composition of the spilled oil itself, with the exception of a subset of lighter VOCs that dissolved

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either completely or partially during transport from the sea bottom to the surface. Other sources of VOCs (e.g., from ships and flares) were much smaller and had a different VOC composition; burning of surface oil did not occur on 10 June. A plume of OA was observed downwind of DWH (Fig. 1B), with mass loadings peaking at >25 mg m−3 on 8 June and >10 mg m−3 on 10 June; these values are in the range of OA observed in U.S. urban atmospheres (5). Because the measured OA was not correlated with tracers of incomplete combustion, such as black carbon aerosol and carbon monoxide (Fig. 2), we can rule out a combustion-related source. For reasons outlined below, we argue that the OA was largely formed from vapors released from the oil and the condensation of their atmospheric oxidation products onto existing particles. First, the mass flux of OA increased with distance downwind of DWH. Second, measured particle number size dis-

tributions indicated a growth in particle size downwind of DWH (Fig. 2B). This rules out wave-generated emission of aerosol from the sea surface—a known source of aerosol in marine air (15)—as the dominant source of OA in this case, because it would have led to the same particle sizes at all downwind distances. Total particle number concentrations, not shown here, remained relatively constant between periods P1 and P2, which, together with the observed increase in aerosol mass (Fig. 2A), rules out coagulation as the primary cause of the growth in particle size. One further observation regarding the OA is noted here but not explored in detail. Mass spectral analysis of the aerosol (5) indicates that both oxygenated organic aerosol (OOA) and hydrocarbon-like organic aerosol (HOA) increased in the aerosol downwind, with a larger contribution from HOA than from OOA (Fig. 2A). Although HOA and OOA are typically

Fig. 1. (A) Flight track on 10 June 2010, with data points below 900 m color- and size-coded by the measured concentration of organic aerosol. The gray area underlying the flight track represents the extent of the oil slick derived from multiple satellite observations. (B and C) The area indicated by the white square in (A) is shown in more detail in (B) and color- and size-coded by the measured C9-aromatics in (C). Data from the periods P1 and P2, indicated in red in (C) with arrows indicating the flight direction, are shown in more detail in Fig. 2. Degrees latitude and longitude are indicated along the y and x axes, respectively.

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attributed to direct emission from combustion sources and to secondary formation, respectively, our results show that HOA can also be formed in the atmosphere. Aromatics and C8 to C11 alkanes are known to be SOA precursors (16, 17) and were measured at very high mixing ratios downwind of DWH (Fig. 1C), higher than typically observed in urban areas (18, 19). However, the SOA was not primarily formed from these VOCs, because the observed OA plume was much wider than the VOC plume and because VOCs were not enhanced upwind from the OA plume. Instead, we argue here that IVOCs evaporating from the oil were the SOA precursors. As their evaporation is

slower, these species were transported on the sea surface away from the area where the spilled oil surfaced and were released to the atmosphere from a wider area (Fig. 3A). Further evidence for this conceptual model is obtained from the plume widths of the measured VOCs, whose vapor pressures span about two orders of magnitude. Figure 3B shows that close to DWH, the width of the VOC plume increased with the molecular weight of the VOC. SOA was not additionally enhanced in the narrow VOC plume despite very high mixing ratios of precursors. Calculations with the Weather Research and Forecasting model coupled with Chemistry (WRF-Chem) indicates that the very

Fig. 2. Time series of selected data from periods P1 and P2 (Fig. 1): (A) organic aerosol and its contributions from hydrocarbon-like organic aerosol (HOA) and oxygenated organic aerosol (OOA), (B) particle number size distributions, (C) black carbon (BC) aerosol, (D) aromatic volatile organic compounds (VOCs), and (E) carbon monoxide (CO). www.sciencemag.org

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high VOC mixing ratios combined with moderately enhanced NOx (1 to 5 parts per billion by volume) resulted in low concentrations of hydroxyl radicals in the narrow VOC plume. Therefore, only a small fraction of the measured VOCs reacted to form SOA in the downwind distance sampled by the aircraft. What were the precursors of the SOA formed downwind of DWH? We constructed a volatility distribution of the oil (Fig. 4A)—that is, the mass fraction as a function of the effective saturation concentration C* (7)—using the hydrocarbon composition of the spilled oil (4) and assuming that the value of C* for a hydrocarbon is the same as that for an n-alkane with the same number of carbon atoms (fig. S2). Assuming that the evaporation rate of a compound is proportional to its vapor pressure, we can fit the measured evaporation curve for the spilled oil (fig. S1) using the volatility distribution (Fig. 4A) and one free parameter, A, that equates the evaporation rate of each volatility class to A × C*. The best fit, shown by the envelope of contributions from all volatility classes in Fig. 4B, suggests that for C* ≥ 108 mg m−3 (≤C8 hydrocarbons), evaporation takes <1 hour. For C* = 104 to 107 mg m−3 (C9 to C18 hydrocarbons), evaporation takes place on time scales varying from 1 to 1000 hours. For C* ≤ 103 mg m−3 (≥C19 hydrocarbons), evaporation takes >1000 hours. To relate the time scales for evaporation of the precursor VOCs to spatial scales, we used an offline Lagrangian particle transport model to simulate surface oil trajectories on the basis of hourly seawater velocity data from the Naval Research Laboratory’s HYCOM-based 0.04° Gulf of Mexico Ocean Prediction system (20, 21). In the model, 1000 to 5000 particles representing freshly surfaced oil were released within an area of 4 km by 4 km, and their spreading on the sea surface due to advection, wind drift, and parameterized subgrid scale diffusion was described. From the time since surfacing, the model calculates the area where emissions to the atmosphere occur for species with evaporation lifetimes of 1 to 1000 hours, respectively (Fig. 4C and fig. S3). In the model, compounds that evaporate in <10 hours are released to the atmosphere from a small area near DWH, whereas compounds that evaporate in >100 hours are released from an area that is much larger than the extent of the OA plume. We conclude that the compounds responsible for SOA formation were most likely released on evaporation time scales of 10 to 100 hours. The best fit to the evaporation curve in Fig. 4B suggests that on these time scales, evaporation is dominated by C* = 105 mg m−3 compounds (C14 to C16 hydrocarbons), and we conclude that these species were the most likely precursors of the observed SOA. The effective saturation concentration (C*) equals the ambient mass loading at which partitioning of a compound shifts between the gas and condensed phases; a C* value of 105 mg m−3 is much higher than ambient mass loading (~10 mg m−3),

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Fig. 3. (A) Conceptual model describing the observations of VOCs and organic aerosol downwind from the oil spill. Oil from the leaking riser pipe surfaces in a relatively small area. The most volatile fraction of the oil evaporates within hours, leading to a narrow atmospheric plume of VOCs downwind from the spill site. The less volatile fraction takes longer to evaporate, during which time the oil spreads over a larger area. Organic aerosol is formed from the less volatile fraction and is observed in a wider plume downwind. (B) Measurements of different aromatic species shortly downwind from the oil spill demonstrate that the plume broadens as a function of decreasing volatility, in accordance with the conceptual model. Mixing ratios were normalized to their maximum value during this transect to facilitate the comparison of plume shapes.

which suggests that a substantial chemical transformation of these hydrocarbon precursors must take place to lower their volatility and produce SOA. On the basis of the distance of the downwind transect P2 (Fig. 1C) to the DWH spill site (45 km) and the average wind speed (5 m s−1), we estimate that this chemical transformation occurred in <3 hours. How efficient was SOA formation from C* = 105 mg m−3 compounds over the oil? The total leak rate of oil from DWH on 10 June was estimated to be 2.03 × 106 kg day−1 (4). Of this total, 11% (2.2 × 105 kg day−1) is estimated to be in the C* = 105 mg m−3 class (Fig. 4A). The flux of OA farthest downwind of DWH (estimated from the integral of the measured concentration times orthogonal wind speed and multiplied by the depth of the boundary layer) (4, 22) was 8 × 104 kg day−1, which may be a lower estimate because the aircraft did not sample across the full width of the SOA plume (Fig. 1B). From these numbers, we estimate the SOAyield for C* = 105 mg m−3 compounds to be ~36%, in approximate agreement with laboratory studies (12). Previous work has suggested that SOA formation from SVOCs and IVOCs could be an important source of aerosol in the United States (12). However, field verification of this chemistry has been difficult because SVOCs and IVOCs are typically co-emitted with VOCs; for that

Fig. 4. (A) Volatility distribution of oil spilled at DWH derived from the oil composition. The numbers on the top axis indicate the number of carbon atoms of the corresponding hydrocarbons. (B) Evaporation of oil as a function of time (blue circles) (14) fit using the volatility distribution from (A). (C) Modeled distribution of a compound that surfaces at the DWH spill site and evaporates with a mean lifetime of 1, 10, and 100 hours, respectively. The blue curves outline the areas where concentrations are ≥10% of the maximum value. The flight track on 10 June 2010, color-coded by the measured concentration of organic aerosol, is added for comparison.

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References and Notes 1. T. J. Crone, M. Tolstoy, Science 330, 634 (2010); 10.1126/science.1195840. 2. See supporting material on Science Online. 3. M. Kanakidou et al., Atmos. Chem. Phys. 5, 1053 (2005). 4. T. B. Ryerson et al., Geophys. Res. Lett. 10.1029/ 2011GL046726 (2011).

5. Q. Zhang et al., Geophys. Res. Lett. 34, L13801 (2007). 6. J. de Gouw, J. L. Jimenez, Environ. Sci. Technol. 43, 7614 (2009). 7. A. L. Robinson et al., Science 315, 1259 (2007). 8. J. A. de Gouw et al., J. Geophys. Res. 110, D16305 (2005). 9. R. Volkamer et al., Geophys. Res. Lett. 33, L17811 (2006). 10. H. Matsui et al., J. Geophys. Res. 114, D04201 (2009). 11. D. Johnson et al., Atmos. Chem. Phys. 6, 403 (2006). 12. N. M. Donahue, A. L. Robinson, S. N. Pandis, Atmos. Environ. 43, 94 (2009). 13. NOAA-NESDIS, Satellite Derived Surface Oil Analysis Products—Deepwater Horizon (www.ssd.noaa.gov/PS/ MPS/deepwater.html). 14. Spill Related Properties of MC 252 Crude Oil Sample ENT-052210-178, SL Ross Environmental Research Ltd. for British Petroleum (July 2010); see www.restorethegulf. gov/sites/default/files/documents/pdf/OilBudgetCalc_ Full_HQ-Print_111110.pdf, Appendix 8. 15. D. V. Spracklen, S. R. Arnold, J. Sciare, K. S. Carslaw, C. Pio, Geophys. Res. Lett. 35, L12811 (2008). 16. N. L. Ng et al., Atmos. Chem. Phys. 7, 3909 (2007). 17. C. E. Jordan et al., Atmos. Environ. 42, 8015 (2008).

Catastrophic Drought in the Afro-Asian Monsoon Region During Heinrich Event 1 J. Curt Stager,1,2* David B. Ryves,3 Brian M. Chase,4,5 Francesco S. R. Pausata6,7,8 Between 15,000 and 18,000 years ago, large amounts of ice and meltwater entered the North Atlantic during Heinrich stadial 1. This caused substantial regional cooling, but major climatic impacts also occurred in the tropics. Here, we demonstrate that the height of this stadial, about 16,000 to 17,000 years ago (Heinrich event 1), coincided with one of the most extreme and widespread megadroughts of the past 50,000 years or more in the Afro-Asian monsoon region, with potentially serious consequences for Paleolithic cultures. Late Quaternary tropical drying commonly is attributed to southward drift of the intertropical convergence zone, but the broad geographic range of the Heinrich event 1 megadrought suggests that severe, systemic weakening of Afro-Asian rainfall systems also occurred, probably in response to sea surface cooling. eridional repositioning of the intertropical convergence zone (ITCZ), the primary source of rainfall in most of the tropics, is thought to have been a major source of hydrological variability during the late Quaternary (1–4). For example, ice sheet expansion forced the mean latitudinal position of the ITCZ southward along with other atmospheric circulation systems in the Northern Hemisphere during the Last Glacial Maximum (3), and abrupt North Atlantic cooling during deglacial melting and ice-

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1 Natural Sciences, Paul Smith’s College, Paul Smiths, NY 12970, USA. 2Climate Change Institute, University of Maine, Orono, ME 04469, USA. 3Centre for Hydrological and Ecosystem Science, Department of Geography, Loughborough University, Loughborough, LE11 3TU, UK. 4Institut des Sciences de l’Evolution de Montpellier, UMR 5554 Université Montpellier 2, Bâtiment 22, CC061, Place Eugène Bataillon 34095 Montpellier CEDEX 5, France. 5Department of Archaeology, History, Cultural Studies and Religion, University of Bergen, 5020 Bergen, Norway. 6Geophysical Institute, University of Bergen, Allégaten 70, 5007 Bergen, Norway. 7Bjerknes Centre for Climate Research, Allégaten 55, 5007 Bergen, Norway. 8European Commission, Joint Research Centre, Institute for Environment and Sustainability, Climate Change Unit, Ispra, Italy.

*To whom correspondence should be addressed. E-mail: [email protected]

rafting episodes such as Heinrich stadial 1 (HS-1), along with associated reductions of marine meridional overturning circulation (MOC), is also thought to have had a similar effect on rain belts associated with the ITCZ (1, 3, 4). Some model simulations of Northern Hemisphere climatic changes associated with HS-1 indicate a southward drift of up to 10 latitudinal degrees (2). Most of northern Africa became unusually dry around 16 to 17 thousand calendar years ago (ka) during the HS-1 ice-rafting peak of Heinrich event 1 (H1), including the Sahara and Sahel (5), Ethiopia (6), and the Red Sea region (7), as did most of southern Asia (8–11) (Figs. 1 and 2). Affecting most of the northern Old World tropics, this arid episode brought some of the most severe drought conditions of the past 50,000 years or more to many of the terrestrial sites that cover such long time periods in detail (Fig. 2 and SOM Text). Under such circumstances, a more southerly positioned ITCZ would presumably deliver less rain to the northern tropics while causing little change near the equator and wetter conditions in the southern tropics. However, a relative scarcity of high-resolution paleoclimate records from much of the inner and southern tropics has left

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18. 19. 20. 21. 22. 23.

A. K. Baker et al., Atmos. Environ. 42, 170 (2008). C. Warneke et al., J. Geophys. Res. 112, D10S47 (2007). F. Counillon, L. Bertino, Ocean Dyn. 59, 83 (2009). E. P. Chassignet et al., Oceanography 22, 64 (2009). W. H. White et al., Science 194, 187 (1976). We thank the flight crew of the NOAA WP-3D, as well as NOAA’s National Environmental Satellite, Data, and Information Service, for the oil spill maps in Fig. 1. Supported by the NASA Radiation Sciences Program (D.W.F., A.E.P., J.P.S., J.R.S., and L.A.W.), NSF grant 1048697 (C.P., M.L.H., and A.S.) and a U.S. Coast Guard Pollution Removal Funding Authorization to NOAA for flights over the oil spill made by the NOAA WP-3D.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1295/DC1 Materials and Methods Figs. S1 to S3 References 11 November 2010; accepted 3 February 2011 10.1126/science.1200320

this commonly cited hypothesis sparsely tested, particularly in Africa. This, in turn, has also limited understanding of the effects of major events such as H1 on global climates. We present a collection of new and recently published records from Africa that register severe aridity in the equatorial and southern tropics about 16 to 17 ka, thereby showing that the H1 megadrought extended far beyond the northern tropics and was therefore one of the most intense and far-reaching dry periods in the history of anatomically modern humans. Together, these records also show that southward drift of the ITCZ cannot have been the only cause of low-latitude drought during H1, and instead suggest that a substantial weakening of tropical rainfall systems also occurred. If the ITCZ did shift several degrees southward over Africa and Asia during H1, it should still have delivered rains to equatorial regions once or twice annually unless the latitudinal shift was unrealistically large, on the order of 20° or more. However, extreme equatorial drying centered on 16 to 17 ka also occurred in northern Tanzania [(12) and this study], Ghana (13), and the NigerSanaga and Congo watersheds (14, 15) (Fig. 1), as well as in Borneo on the opposite side of the Indian Ocean (16), much as it did in the more northerly reaches of the tropics from the Mediterranean Basin to the western Pacific (Figs. 1 to 3 and SOM Text). A dramatic event associated with these equatorial changes was the desiccation of Lake Victoria, East Africa (Fig. 1), which today is the world’s largest tropical lake. With rainfall over the watershed possibly reduced to less than a quarter of its present amount (7), the lake dried out twice between 15 and 18 ka, although the timing of the two low stands has previously been unclear (SOM Text). We present here radiocarbon dates and diatom records from two cores, which show that the first of these low stands occurred about 16 to 17 ka (Fig. 3D) (17). The disappearance of Lake Victoria would have had severe ecological impacts on regional ecosystems and cultures from

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reason, the SOA observed in polluted air cannot be unambiguously attributed to formation from SVOCs and IVOCs. The DWH oil spill provided a unique look at this chemistry because the emissions of VOCs, IVOCs, and SVOCs were spatially separated and the importance of SOA formation from IVOCs could be clearly demonstrated. These results form a well-constrained case to improve our quantitative understanding of IVOC chemistry, which will help to describe the importance of IVOCs for SOA formation in other polluted regions of the atmosphere.

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References and Notes 1. T. J. Crone, M. Tolstoy, Science 330, 634 (2010); 10.1126/science.1195840. 2. See supporting material on Science Online. 3. M. Kanakidou et al., Atmos. Chem. Phys. 5, 1053 (2005). 4. T. B. Ryerson et al., Geophys. Res. Lett. 10.1029/ 2011GL046726 (2011).

5. Q. Zhang et al., Geophys. Res. Lett. 34, L13801 (2007). 6. J. de Gouw, J. L. Jimenez, Environ. Sci. Technol. 43, 7614 (2009). 7. A. L. Robinson et al., Science 315, 1259 (2007). 8. J. A. de Gouw et al., J. Geophys. Res. 110, D16305 (2005). 9. R. Volkamer et al., Geophys. Res. Lett. 33, L17811 (2006). 10. H. Matsui et al., J. Geophys. Res. 114, D04201 (2009). 11. D. Johnson et al., Atmos. Chem. Phys. 6, 403 (2006). 12. N. M. Donahue, A. L. Robinson, S. N. Pandis, Atmos. Environ. 43, 94 (2009). 13. NOAA-NESDIS, Satellite Derived Surface Oil Analysis Products—Deepwater Horizon (www.ssd.noaa.gov/PS/ MPS/deepwater.html). 14. Spill Related Properties of MC 252 Crude Oil Sample ENT-052210-178, SL Ross Environmental Research Ltd. for British Petroleum (July 2010); see www.restorethegulf. gov/sites/default/files/documents/pdf/OilBudgetCalc_ Full_HQ-Print_111110.pdf, Appendix 8. 15. D. V. Spracklen, S. R. Arnold, J. Sciare, K. S. Carslaw, C. Pio, Geophys. Res. Lett. 35, L12811 (2008). 16. N. L. Ng et al., Atmos. Chem. Phys. 7, 3909 (2007). 17. C. E. Jordan et al., Atmos. Environ. 42, 8015 (2008).

Catastrophic Drought in the Afro-Asian Monsoon Region During Heinrich Event 1 J. Curt Stager,1,2* David B. Ryves,3 Brian M. Chase,4,5 Francesco S. R. Pausata6,7,8 Between 15,000 and 18,000 years ago, large amounts of ice and meltwater entered the North Atlantic during Heinrich stadial 1. This caused substantial regional cooling, but major climatic impacts also occurred in the tropics. Here, we demonstrate that the height of this stadial, about 16,000 to 17,000 years ago (Heinrich event 1), coincided with one of the most extreme and widespread megadroughts of the past 50,000 years or more in the Afro-Asian monsoon region, with potentially serious consequences for Paleolithic cultures. Late Quaternary tropical drying commonly is attributed to southward drift of the intertropical convergence zone, but the broad geographic range of the Heinrich event 1 megadrought suggests that severe, systemic weakening of Afro-Asian rainfall systems also occurred, probably in response to sea surface cooling. eridional repositioning of the intertropical convergence zone (ITCZ), the primary source of rainfall in most of the tropics, is thought to have been a major source of hydrological variability during the late Quaternary (1–4). For example, ice sheet expansion forced the mean latitudinal position of the ITCZ southward along with other atmospheric circulation systems in the Northern Hemisphere during the Last Glacial Maximum (3), and abrupt North Atlantic cooling during deglacial melting and ice-

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1 Natural Sciences, Paul Smith’s College, Paul Smiths, NY 12970, USA. 2Climate Change Institute, University of Maine, Orono, ME 04469, USA. 3Centre for Hydrological and Ecosystem Science, Department of Geography, Loughborough University, Loughborough, LE11 3TU, UK. 4Institut des Sciences de l’Evolution de Montpellier, UMR 5554 Université Montpellier 2, Bâtiment 22, CC061, Place Eugène Bataillon 34095 Montpellier CEDEX 5, France. 5Department of Archaeology, History, Cultural Studies and Religion, University of Bergen, 5020 Bergen, Norway. 6Geophysical Institute, University of Bergen, Allégaten 70, 5007 Bergen, Norway. 7Bjerknes Centre for Climate Research, Allégaten 55, 5007 Bergen, Norway. 8European Commission, Joint Research Centre, Institute for Environment and Sustainability, Climate Change Unit, Ispra, Italy.

*To whom correspondence should be addressed. E-mail: [email protected]

rafting episodes such as Heinrich stadial 1 (HS-1), along with associated reductions of marine meridional overturning circulation (MOC), is also thought to have had a similar effect on rain belts associated with the ITCZ (1, 3, 4). Some model simulations of Northern Hemisphere climatic changes associated with HS-1 indicate a southward drift of up to 10 latitudinal degrees (2). Most of northern Africa became unusually dry around 16 to 17 thousand calendar years ago (ka) during the HS-1 ice-rafting peak of Heinrich event 1 (H1), including the Sahara and Sahel (5), Ethiopia (6), and the Red Sea region (7), as did most of southern Asia (8–11) (Figs. 1 and 2). Affecting most of the northern Old World tropics, this arid episode brought some of the most severe drought conditions of the past 50,000 years or more to many of the terrestrial sites that cover such long time periods in detail (Fig. 2 and SOM Text). Under such circumstances, a more southerly positioned ITCZ would presumably deliver less rain to the northern tropics while causing little change near the equator and wetter conditions in the southern tropics. However, a relative scarcity of high-resolution paleoclimate records from much of the inner and southern tropics has left

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18. 19. 20. 21. 22. 23.

A. K. Baker et al., Atmos. Environ. 42, 170 (2008). C. Warneke et al., J. Geophys. Res. 112, D10S47 (2007). F. Counillon, L. Bertino, Ocean Dyn. 59, 83 (2009). E. P. Chassignet et al., Oceanography 22, 64 (2009). W. H. White et al., Science 194, 187 (1976). We thank the flight crew of the NOAA WP-3D, as well as NOAA’s National Environmental Satellite, Data, and Information Service, for the oil spill maps in Fig. 1. Supported by the NASA Radiation Sciences Program (D.W.F., A.E.P., J.P.S., J.R.S., and L.A.W.), NSF grant 1048697 (C.P., M.L.H., and A.S.) and a U.S. Coast Guard Pollution Removal Funding Authorization to NOAA for flights over the oil spill made by the NOAA WP-3D.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1295/DC1 Materials and Methods Figs. S1 to S3 References 11 November 2010; accepted 3 February 2011 10.1126/science.1200320

this commonly cited hypothesis sparsely tested, particularly in Africa. This, in turn, has also limited understanding of the effects of major events such as H1 on global climates. We present a collection of new and recently published records from Africa that register severe aridity in the equatorial and southern tropics about 16 to 17 ka, thereby showing that the H1 megadrought extended far beyond the northern tropics and was therefore one of the most intense and far-reaching dry periods in the history of anatomically modern humans. Together, these records also show that southward drift of the ITCZ cannot have been the only cause of low-latitude drought during H1, and instead suggest that a substantial weakening of tropical rainfall systems also occurred. If the ITCZ did shift several degrees southward over Africa and Asia during H1, it should still have delivered rains to equatorial regions once or twice annually unless the latitudinal shift was unrealistically large, on the order of 20° or more. However, extreme equatorial drying centered on 16 to 17 ka also occurred in northern Tanzania [(12) and this study], Ghana (13), and the NigerSanaga and Congo watersheds (14, 15) (Fig. 1), as well as in Borneo on the opposite side of the Indian Ocean (16), much as it did in the more northerly reaches of the tropics from the Mediterranean Basin to the western Pacific (Figs. 1 to 3 and SOM Text). A dramatic event associated with these equatorial changes was the desiccation of Lake Victoria, East Africa (Fig. 1), which today is the world’s largest tropical lake. With rainfall over the watershed possibly reduced to less than a quarter of its present amount (7), the lake dried out twice between 15 and 18 ka, although the timing of the two low stands has previously been unclear (SOM Text). We present here radiocarbon dates and diatom records from two cores, which show that the first of these low stands occurred about 16 to 17 ka (Fig. 3D) (17). The disappearance of Lake Victoria would have had severe ecological impacts on regional ecosystems and cultures from

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reason, the SOA observed in polluted air cannot be unambiguously attributed to formation from SVOCs and IVOCs. The DWH oil spill provided a unique look at this chemistry because the emissions of VOCs, IVOCs, and SVOCs were spatially separated and the importance of SOA formation from IVOCs could be clearly demonstrated. These results form a well-constrained case to improve our quantitative understanding of IVOC chemistry, which will help to describe the importance of IVOCs for SOA formation in other polluted regions of the atmosphere.

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eastern equatorial Africa to the Mediterranean coast. It is the largest water source for the Nile River during seasonal low-flood stages, and Lake Tana, Ethiopia, is the primary source of the Nile’s seasonal high floods; both lakes dried out completely at that time (6). In addition, an analysis of diatom assemblages in a core from Lake Tanganyika, Tanzania (17), supports geochemical evidence (18) that a major low stand occurred about 16 to 17 ka there, as well (Figs. 2D and 3F). We therefore link the synchronous regressions at Lakes Victoria and Tanganyika to the H1 ice-rafting peak that occurred about 16 to 17 ka during the longer Heinrich stadial period in the North Atlantic (3, 19), while recognizing that the ages assigned to these events are subject to the limitations of radiocarbon dating, variable carbon reservoir effects, and bioturbation. Together, these equatorial records demonstrate that a simple southward shift of the ITCZ cannot have been the only climatic mechanism to affect tropical rainfall substantially during H1. The occurrence of major droughts to the south of equatorial Africa during H1 even more clearly requires a mechanism other than southward drift of the ITCZ over the continent, which would be expected to make those regions wetter as the north became drier (Figs. 1 to 3). These sites included Lake Malawi (20), the Zambezi and Limpopo watersheds (21, 22), and other locations in southeastern Africa (Fig. 1 and SOM Text). In contrast, parts of southwestern Africa became wetter during H1 (23), but hydrology there can also be influenced by rainfall systems other than the ITCZ, such as winter storms carried on

the austral mid-latitude westerlies. The complexity of the interactions between subtropical and Southern Ocean dynamics is highlighted in a stable isotope record from the Western Cape, where changes in sea surface temperatures (SST) as a result of variability in MOC and/or the Agulhas Current caused progressively wetter conditions in that region across H1 (24) (Fig. 1). Further north, in the Kalahari, Burrough et al. (25) favored an easterly ITCZ rainfall source for the enlarged paleolake Makgadikgadi, proposed largely on the basis of sandy deposits on western shorelines, but droughts to the north and east, along with the possibility of distant runoff sources in addition to deflation and downwind sediment deposition during dry seasons, suggest an alternative interpretation as well. Wetter conditions in the Kalahari at that time might also be consistent with a northward extension of winter rains, which could have brought increased precipitation to Namibia during H1 (26). Wetter conditions could also reflect enhanced runoff from high stratiform clouds and fogs in the Angola highlands related to cooling along the Benguela coast (27), rather than a southward shift of the ITCZ alone. Hydrological conditions in the New World tropics are difficult to interpret in this context. Extreme aridity is registered in cores from the Cariaco Basin about 16 to 17 ka (1), and regional increases in precipitation occurred farther south in the tropical Andes and parts of Amazonia during H1 (1, 3). This pattern appears to be consistent with a southward shift of the mean position of the ITCZ, although it is not found universally (SOM Text). Most important in the context of this study, how-

ever, the development of wet conditions in numerous neotropical sites suggests that the proposed general weakening of rainfall systems over Africa did not occur in South America and that it apparently represented regional, rather than uniformly global, changes in tropical atmospheric circulation. General circulation models (GCMs) often have more difficulty in simulating precipitation than temperature, and GCM reconstructions of tropical rainfall are less well supported by historical instrumental weather data than those that focus on the northern temperate zone. Furthermore, to our knowledge, no modeling studies of deglacial climates have as yet been constrained by a detailed array of paleohydrological records spanning most of the African continent. Our findings are therefore useful for evaluating model reconstructions of past climates in Africa and of the global effects of H1, and we summarize several GCM simulations here to illustrate the difficulty of reconciling current GCM output with paleoclimatic reconstructions. For example, although Mulitza et al. (5) correctly simulated Sahel aridity in response to weakened MOC that was typical of the HS-1 interval, the model shows wetting over much of central Africa that is inconsistent with the data available. Kageyama et al. (2) correctly inferred Indian aridity but did not fully extend it to equatorial and southern Africa, whereas simulations by Thomas et al. (4), which identified wetting in the Angola-Kalahari region, did not completely capture the extreme aridity that occurred in much of the rest of the continent. Given the mismatches between recent GCM simulations and paleoclimate records of H1 in

Fig. 1. Site map of records showing hydrological conditions during the 16- to 17-ka interval (details in table S3). Red dots, reduced precipitationevaporation. Blue dots, increased precipitationevaporation. Vertically divided red/blue dots indicate signals of uncertain climatic importance. Horizontally divided dots indicate a trend of progressively moister climates across HS-1. Only the specific study sites and some of the major watersheds are indicated; the full geographic area affected by the H1 megadrought is not completely colored in. For example, records from marine sites 5 and 6 reflect climatic conditions in much of northwestern Africa. Purple shading in the Kalahari region indicates wetter conditions of uncertain origin, timing, and/or geographic extent.

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the Afro-Asian region, we suggest several possible causal mechanisms here. The occurrence of droughts throughout tropical Africa indicates that they most likely involved a reduction of convection and/or moisture content in the ITCZ, with or without a concurrent shift in its position. Surface warming in Lake Tanganyika during the driest

interval of a 60,000-year sediment record (18) (Fig. 2D), for example, might indicate reduced evaporative cooling and upwelling linked to a weakening of atmospheric circulation over East Africa during H1; a severe reduction of summer monsoon wind activity was also registered in the Arabian Sea then (8). It has been hypothesized

Fig. 2. Examples of terrestrial paleohydrological records in which the H1 signal was among the most intense of the past 50,000 years or more. (A) Dongge Cave, China, speleothem d18O (9). (B) Hulu Cave, China, speleothem d18O, composite time series (10). (C) Sofular Cave, Turkey, speleothem d18O (11). (D) Lake Tanganyika, East Africa, dD (18). Although these records do not mean that all intervening sites necessarily experienced uniquely intense drought during H1, they do establish that the pattern was widely distributed, spanning southern Asia and extending south of the equator in East Africa. Colored column represents the 13- to 19-ka time period illustrated in Fig. 3. Fig. 3. Paleoclimatic records of the 13- to 19-ka interval from Africa and Borneo, ordered from north to south (latitude on right for each site). (A) Lake Tana, Ethiopia, relative level, no units [as in (6)]. (B) Lake Bosumtwi magnetic mineral concentration (13). (C) Borneo speleothem, d18O series (16). (D) Lake Victoria relative lake level (this study),. (E) Congo basin soil pH (15). (F) Lake Tanganyika percentage of periphytic diatoms (inverted; this study). (G) Lake Malawi Aulacoseira nyassensis with lower percentage indicating less windy and/or drier conditions [as in (20)]. (H) Stalagmite T7 from Cold Air Cave, d13C series (31). Brown bar, approximate H1 interval. Dotted lines bracket the approximate HS-1 interval. All time series are arranged with drying trends oriented downward.

elsewhere that the southern limb of the tropical Hadley circulation system weakened during the longer HS-1 interval (3), which would also be consistent with the paleoclimate records indicating drought in equatorial and southern Africa. Cooler SSTs in the SE Atlantic and Indian oceans also represent plausible mechanisms for the inferred reductions of tropical rainfall because lower SSTs would tend to reduce the evaporative moisture content of the ITCZ. Cooling along the West African coast likely contributed to summer monsoon failure there (5, 27), and low SSTs in the western Indian Ocean (28) may likewise have contributed to aridity over eastern Africa. Conditions elsewhere along the margins of the Indian Ocean basin during H1 probably made SST cooling particularly widespread there as well. Stronger upwelling in the Southern Ocean may have cooled the southern margins of the Indian Ocean (29) and deflected cold, eastward-flowing water masses equatorward. Additionally, at that time much of today’s warm Pacific through-flow was blocked by land masses in the Indonesian region due to a sea level low stand, thereby reducing Pacific heat inputs into the area, and reduced SSTs in much of the northern Indian Ocean (8, 21) might have resulted from cooling by strong, southtrending winter monsoon winds over land masses that were concurrently chilled by conditions upwind in the Mediterranean and North Atlantic (3). More than half of all humanity is strongly influenced by Afro-Asian rainfall systems today, and anatomically modern humans evolved under their influence, yet the mechanisms behind precipitation variability in these regions remain relatively poorly understood and difficult to model. Furthermore, the unusual intensity and exceptionally broad geographic distribution of the H1 megadrought have not yet been widely recognized. The records presented here show that it was one of the most intense and extensive tropical dry periods of the past 50,000 years or more (Figs. 1 and 2), spanning roughly 60 latitudinal degrees, virtually all of southern Asia, and most of the African continent, and that it must have involved a systemic, as yet unexplained weakening of regional rainfall systems in addition to southward displacement of the ITCZ. Whatever its exact cause, such a catastrophic drought would have had powerful effects on Paleolithic cultures. For example, the desiccations of Lakes Tana and Victoria reorganized the distribution of wet and arid-environment resources in the region, Middle Eastern drying would have hindered overland migrations into or out of Africa, and aridity around this time period likely contributed to major reductions in human populations in southern Asia (30).

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References and Notes 1. L. C. Peterson, G. H. Haug, K. A. Hughen, U. Röhl, Science 290, 1947 (2000). 2. M. Kageyama et al., Clim. Past 5, 551 (2009). 3. G. H. Denton et al., Science 328, 1652 (2010). 4. D. S. G. Thomas, R. Bailey, P. A. Shaw, J. A. Durcan, J. S. Singarayer, Quat. Sci. Rev. 28, 526 (2009).

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REPORTS 18. J. E. Tierney et al., Science 322, 252 (2008). 19. E. Bard, F. Rostek, J.-L. Turon, S. Gendreau, Science 289, 1321 (2000). 20. F. Gasse, P. Barker, T. C. Johnson, in The East African Great Lakes: Limnology, Palaeoclimatology and Biodiversity, E. O. Odada, D. O. Olago, Eds. (Kluwer Academic Publishers, Dordrecht, 2002), pp. 393-414. 21. L. Dupont, T. Caley, B. Malaize, J. Girardeau, Geophys. Res. Abstr. 12, EGU2010-3892 (2010). 22. Y. Wang, T. Larsen, N. Andersen, T. Blanz, R. Schneider, Geophys. Res. Abstr. 12, EGU2010-11360 (2010). 23. B. M. Chase, M. E. Meadows, Earth Sci. Rev. 84, 103 (2007). 24. B. M. Chase et al., Geology 39, 19 (2010). 25. S. L. Burrough, D. S. G. Thomas, J. S. Singarayer, Earth Sci. Rev. 96, 313 (2009). 26. J.-B. W. Stuut et al., Mar. Geol. 180, 221 (2002). 27. L. M. Dupont, H. Behling, J. H. Kim, Clim. Past 4, 107 (2008). 28. E. Bard, F. Rostek, C. Sonzogni, Nature 385, 707 (1997). 29. R. F. Anderson et al., Science 323, 1443 (2009).

Complex Multicolor Tilings and Critical Phenomena in Tetraphilic Liquid Crystals Xiangbing Zeng,1 Robert Kieffer,2 Benjamin Glettner,2 Constance Nürnberger,2 Feng Liu,1 Karsten Pelz,3 Marko Prehm,3 Ute Baumeister,3 Harald Hahn,4 Heinrich Lang,4 Gillian A. Gehring,5 Christa H. M. Weber,5 Jamie K. Hobbs,5 Carsten Tschierske,2* Goran Ungar1,6* T-shaped molecules with a rod-like aromatic core and a flexible side chain form liquid crystal honeycombs with aromatic cell walls and a cell interior filled with the side chains. Here, we show how the addition of a second chain, incompatible with the first (X-shaped molecules), can form honeycombs with highly complex tiling patterns, with cells of up to five different compositions (“colors”) and polygonal shapes. The complexity is caused by the inability of the side chains to separate cleanly because of geometric frustration. Furthermore, a thermoreversible transition was observed between a multicolor (phase-separated) and a single-color (mixed) honeycomb phase. This is analogous to the Curie transition in simple and frustrated ferro- and antiferromagnets; here spin flips are replaced by 180° reorientations of the molecules. olid-state materials, particularly those performing useful functions, often have framework or cellular structures on the nanoscale (1). For example, crystalline zeolites (2) used in ion exchange or catalysis have frameworks whose shapes are determined by the covalent bond lengths and angles that tolerate little change. Each solid-state framework structure is specific to the individual compound. In contrast, in soft

S

1 Department of Materials Science and Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK. 2Institute of Chemistry, Organic Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 2, D-06120 Halle, Germany. 3Institute of Chemistry, Physical Chemistry, MartinLuther-University Halle-Wittenberg, Van-Danckelmann-Platz 4, D-06120 Halle, Germany. 4Institute of Inorganic Chemistry, Technische Universität Chemnitz, Strasse der Nationen 62, 09111 Chemnitz, Germany. 5Department of Physics and Astronomy, University of Sheffield, Hicks Building, Sheffield S3 7RH, UK. 6World Class University Program of Chemical Convergence for Energy & Environment, School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea.

*To whom correspondence should be addressed. E-mail: [email protected] (G.U.); [email protected] (C.T.)

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matter such as lyotropic liquid crystals (for example, surfactant-water systems) (3) or diblock copolymers (4), as well as in many biological systems, the shapes of the frameworks are determined primarily by the curvature of the interface between the two incompatible liquids (5). Hence they form a limited range of closed- or open-cell structures, typically micellar cubic or hexagonal (3). Liquid crystal (LC)–forming T-shaped block molecules bearing three types of mutually incompatible groups (triphilic) can be said to fall between those two extreme categories. Their selfassembly is governed by principles more general than those of crystals, yet the number of known framework LC structure types exceeds the number found in all lyotropics. Here we describe how the addition of a fourth incompatible group substantially broadens the range of cellular LC morphologies. Among the many LC phases of the T-shaped compounds are a series of honeycombs (Fig. 1, A to C) (6, 7). The aromatic rod-like cores (II in Fig. 1D) form cell walls connected at the “seams” by terminal hydrogen-bonding groups (I), typi-

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30. S. Kumar et al., BMC Evol. Biol. 8, 230 (2008). 31. K. Holmgren et al., Quat. Sci. Rev. 22, 2311 (2003). 32. This study was supported by National Science Foundation grant EAR-0822922 (P2C2). F.S.R.P. has been supported by the Norwegian Research Council through the Decadal to Century-Scale Variability in East Asia Climate (DecCen) and Arctic Records of Climate Change (ARCTREC) projects. Sediment core samples were provided by D. Livingstone, T. Johnson, and C. Scholz.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1198322/DC1 Materials and Methods SOM Text Figs. S1 and S2 Tables S1 to S3 References 27 September 2010; accepted 2 February 2011 Published online 24 February 2011; 10.1126/science.1198322

cally glycerol (7). The honeycomb cells of polygonal cross-section and infinite length are filled by the fluid lateral chains (III). Depending on the ratio of the cubic root of the volume of III to the length of II, cells ranging from triangular to hexagonal were obtained (Fig. 1, A to C) (6, 7). Projected on an Euclidian plane (8), most observed honeycombs are Archimedean tilings (all vertices are equal) or their duals (tile centers are replaced by vertices) (6). The recent example of kagome LC tiling (9), supported by simulation (9, 10), hints at the potential for substantial expansion of the range of honeycomb structures using X-shaped molecules bearing two different side chains (Fig. 1E). We prepared tetraphilic X-shaped LC compounds (Fig. 2) consisting of a terphenyl- (compounds A) or a larger oligo(phenyleneethinylene)-based (compounds B) rigid rod core (II in Fig. 1) with a glycerol group at each end (I), with two different types of flexible hydrophobic chains attached to either side of the rod-like core (III and IV) [for analytical details, see the supporting online material (SOM) text, section S1). III is a highly branched carbosilane-based hydrocarbon chain (Si chain), and IV is a semiperfluorinated chain (F chain). The chain volume to molecular core length ratios were chosen such (7) that triangular or square cells were expected in compounds B and hexagonal cells were expected in A. Complete segregation of the side chains of X-shaped molecules into different columns, resulting in two-color tilings (Fig. 1, F and G), should be possible for triangular and square cells, where “color” refers to the composition of the honeycomb cell interior. However, such complete segregation is frustrated for hexagonal cells, where some mixing of Si and F chains cannot be avoided. This is illustrated in Fig. 1H: among the three molecules sharing a node, at least one F chain will be mixed with a Si chain. Two uniform colorings of the partially phase-separated hexagonal structure are possible, one three-color [ p3m1 symmetry (Fig. 1I)] and the other twocolor [ p6mm (Fig. 1J)].

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5. S. Mulitza et al., Paleoceanography 23, PA4206 (2008). 6. H. F. Lamb et al., Quat. Sci. Rev. 26, 287 (2007). 7. W. S. Broecker, D. Peteet, I. Hajdas, J. Lin, E. Clark, Quat. Res. 50, 12 (1998). 8. H. Rashid, B. P. Flower, R. Z. Poore, T. M. Quinn, Quat. Sci. Rev. 26, 2586 (2007). 9. D. Yuan et al., Science 304, 575 (2004). 10. Y. J. Wang et al., Science 294, 2345 (2001). 11. D. Fleitmann et al., Geophys. Res. Lett. 36, L19707 (2009). 12. D. Verschuren et al.; CHALLACEA project members, Nature 462, 637 (2009). 13. J. A. Peck et al., Palaeogeogr. Palaeoclimatol. Palaeoecol. 215, 37 (2004). 14. S. Weldeab, D. W. Lea, R. R. Schneider, N. Andersen, Science 316, 1303 (2007). 15. J. W. H. Weijers, E. Schefuss, S. Schouten, J. S. Sinninghe Damsté, Science 315, 1701 (2007). 16. J. W. Partin, K. M. Cobb, J. F. Adkins, B. Clark, D. P. Fernandez, Nature 449, 452 (2007). 17. Materials and methods are available as supporting material on Science Online.

REPORTS 18. J. E. Tierney et al., Science 322, 252 (2008). 19. E. Bard, F. Rostek, J.-L. Turon, S. Gendreau, Science 289, 1321 (2000). 20. F. Gasse, P. Barker, T. C. Johnson, in The East African Great Lakes: Limnology, Palaeoclimatology and Biodiversity, E. O. Odada, D. O. Olago, Eds. (Kluwer Academic Publishers, Dordrecht, 2002), pp. 393-414. 21. L. Dupont, T. Caley, B. Malaize, J. Girardeau, Geophys. Res. Abstr. 12, EGU2010-3892 (2010). 22. Y. Wang, T. Larsen, N. Andersen, T. Blanz, R. Schneider, Geophys. Res. Abstr. 12, EGU2010-11360 (2010). 23. B. M. Chase, M. E. Meadows, Earth Sci. Rev. 84, 103 (2007). 24. B. M. Chase et al., Geology 39, 19 (2010). 25. S. L. Burrough, D. S. G. Thomas, J. S. Singarayer, Earth Sci. Rev. 96, 313 (2009). 26. J.-B. W. Stuut et al., Mar. Geol. 180, 221 (2002). 27. L. M. Dupont, H. Behling, J. H. Kim, Clim. Past 4, 107 (2008). 28. E. Bard, F. Rostek, C. Sonzogni, Nature 385, 707 (1997). 29. R. F. Anderson et al., Science 323, 1443 (2009).

Complex Multicolor Tilings and Critical Phenomena in Tetraphilic Liquid Crystals Xiangbing Zeng,1 Robert Kieffer,2 Benjamin Glettner,2 Constance Nürnberger,2 Feng Liu,1 Karsten Pelz,3 Marko Prehm,3 Ute Baumeister,3 Harald Hahn,4 Heinrich Lang,4 Gillian A. Gehring,5 Christa H. M. Weber,5 Jamie K. Hobbs,5 Carsten Tschierske,2* Goran Ungar1,6* T-shaped molecules with a rod-like aromatic core and a flexible side chain form liquid crystal honeycombs with aromatic cell walls and a cell interior filled with the side chains. Here, we show how the addition of a second chain, incompatible with the first (X-shaped molecules), can form honeycombs with highly complex tiling patterns, with cells of up to five different compositions (“colors”) and polygonal shapes. The complexity is caused by the inability of the side chains to separate cleanly because of geometric frustration. Furthermore, a thermoreversible transition was observed between a multicolor (phase-separated) and a single-color (mixed) honeycomb phase. This is analogous to the Curie transition in simple and frustrated ferro- and antiferromagnets; here spin flips are replaced by 180° reorientations of the molecules. olid-state materials, particularly those performing useful functions, often have framework or cellular structures on the nanoscale (1). For example, crystalline zeolites (2) used in ion exchange or catalysis have frameworks whose shapes are determined by the covalent bond lengths and angles that tolerate little change. Each solid-state framework structure is specific to the individual compound. In contrast, in soft

S

1 Department of Materials Science and Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK. 2Institute of Chemistry, Organic Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 2, D-06120 Halle, Germany. 3Institute of Chemistry, Physical Chemistry, MartinLuther-University Halle-Wittenberg, Van-Danckelmann-Platz 4, D-06120 Halle, Germany. 4Institute of Inorganic Chemistry, Technische Universität Chemnitz, Strasse der Nationen 62, 09111 Chemnitz, Germany. 5Department of Physics and Astronomy, University of Sheffield, Hicks Building, Sheffield S3 7RH, UK. 6World Class University Program of Chemical Convergence for Energy & Environment, School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea.

*To whom correspondence should be addressed. E-mail: [email protected] (G.U.); [email protected] (C.T.)

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matter such as lyotropic liquid crystals (for example, surfactant-water systems) (3) or diblock copolymers (4), as well as in many biological systems, the shapes of the frameworks are determined primarily by the curvature of the interface between the two incompatible liquids (5). Hence they form a limited range of closed- or open-cell structures, typically micellar cubic or hexagonal (3). Liquid crystal (LC)–forming T-shaped block molecules bearing three types of mutually incompatible groups (triphilic) can be said to fall between those two extreme categories. Their selfassembly is governed by principles more general than those of crystals, yet the number of known framework LC structure types exceeds the number found in all lyotropics. Here we describe how the addition of a fourth incompatible group substantially broadens the range of cellular LC morphologies. Among the many LC phases of the T-shaped compounds are a series of honeycombs (Fig. 1, A to C) (6, 7). The aromatic rod-like cores (II in Fig. 1D) form cell walls connected at the “seams” by terminal hydrogen-bonding groups (I), typi-

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30. S. Kumar et al., BMC Evol. Biol. 8, 230 (2008). 31. K. Holmgren et al., Quat. Sci. Rev. 22, 2311 (2003). 32. This study was supported by National Science Foundation grant EAR-0822922 (P2C2). F.S.R.P. has been supported by the Norwegian Research Council through the Decadal to Century-Scale Variability in East Asia Climate (DecCen) and Arctic Records of Climate Change (ARCTREC) projects. Sediment core samples were provided by D. Livingstone, T. Johnson, and C. Scholz.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1198322/DC1 Materials and Methods SOM Text Figs. S1 and S2 Tables S1 to S3 References 27 September 2010; accepted 2 February 2011 Published online 24 February 2011; 10.1126/science.1198322

cally glycerol (7). The honeycomb cells of polygonal cross-section and infinite length are filled by the fluid lateral chains (III). Depending on the ratio of the cubic root of the volume of III to the length of II, cells ranging from triangular to hexagonal were obtained (Fig. 1, A to C) (6, 7). Projected on an Euclidian plane (8), most observed honeycombs are Archimedean tilings (all vertices are equal) or their duals (tile centers are replaced by vertices) (6). The recent example of kagome LC tiling (9), supported by simulation (9, 10), hints at the potential for substantial expansion of the range of honeycomb structures using X-shaped molecules bearing two different side chains (Fig. 1E). We prepared tetraphilic X-shaped LC compounds (Fig. 2) consisting of a terphenyl- (compounds A) or a larger oligo(phenyleneethinylene)-based (compounds B) rigid rod core (II in Fig. 1) with a glycerol group at each end (I), with two different types of flexible hydrophobic chains attached to either side of the rod-like core (III and IV) [for analytical details, see the supporting online material (SOM) text, section S1). III is a highly branched carbosilane-based hydrocarbon chain (Si chain), and IV is a semiperfluorinated chain (F chain). The chain volume to molecular core length ratios were chosen such (7) that triangular or square cells were expected in compounds B and hexagonal cells were expected in A. Complete segregation of the side chains of X-shaped molecules into different columns, resulting in two-color tilings (Fig. 1, F and G), should be possible for triangular and square cells, where “color” refers to the composition of the honeycomb cell interior. However, such complete segregation is frustrated for hexagonal cells, where some mixing of Si and F chains cannot be avoided. This is illustrated in Fig. 1H: among the three molecules sharing a node, at least one F chain will be mixed with a Si chain. Two uniform colorings of the partially phase-separated hexagonal structure are possible, one three-color [ p3m1 symmetry (Fig. 1I)] and the other twocolor [ p6mm (Fig. 1J)].

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5. S. Mulitza et al., Paleoceanography 23, PA4206 (2008). 6. H. F. Lamb et al., Quat. Sci. Rev. 26, 287 (2007). 7. W. S. Broecker, D. Peteet, I. Hajdas, J. Lin, E. Clark, Quat. Res. 50, 12 (1998). 8. H. Rashid, B. P. Flower, R. Z. Poore, T. M. Quinn, Quat. Sci. Rev. 26, 2586 (2007). 9. D. Yuan et al., Science 304, 575 (2004). 10. Y. J. Wang et al., Science 294, 2345 (2001). 11. D. Fleitmann et al., Geophys. Res. Lett. 36, L19707 (2009). 12. D. Verschuren et al.; CHALLACEA project members, Nature 462, 637 (2009). 13. J. A. Peck et al., Palaeogeogr. Palaeoclimatol. Palaeoecol. 215, 37 (2004). 14. S. Weldeab, D. W. Lea, R. R. Schneider, N. Andersen, Science 316, 1303 (2007). 15. J. W. H. Weijers, E. Schefuss, S. Schouten, J. S. Sinninghe Damsté, Science 315, 1701 (2007). 16. J. W. Partin, K. M. Cobb, J. F. Adkins, B. Clark, D. P. Fernandez, Nature 449, 452 (2007). 17. Materials and methods are available as supporting material on Science Online.

Fig. 1. (A to C) Singlecolor triangular, square, and hexagonal honeycombs in triphilic T-shaped compounds (D) with increasing relative sidechain volume. The three incompatible groups are I, hydrogen-bonding group; II, rod-like rigid aromatic core; and III, flexible lateral chain. (E) Tetraphilic molecules (IV, flexible lateral chain incompatible with III) and their expected two-color (F, G, and J) or three-color (I) honeycombs. For hexagonal honeycombs, the orientation of the side chains is shown as arrows pointing in the direction of the Si chain (H). Doubleheaded arrows in ( J) represent molecules undergoing 180° flips and spending half the time in each orientation. RSi /RF ratios in the light green cells in (I) and ( J) are 1:1 and 3:1, respectively. Fig. 2. Compounds used in this work, volumes of their lateral chains (VR = total volume of III + IV), and core lengths l (II + 2I).

Table 1. Transition temperatures T (in °C) and transition enthalpies [DH/kJ mol−1]*, lattice parameters ahex/nm, and critical exponents b. Compound

Tm

A1

58 [105]

A2

<20

A3 B1 B2

52 [2.5]‡ 99|| [20.6] 83|| [10.9]

Tc 80† [0.15§] 81.1‡ 61† [<0.05] 56.4‡ – 68.0‡ [0.0] 84.7‡ [0.0]

Ti

ahexlow-T

ahexhigh-T

b

103 [3.4]

7.03 (60°C)

4.05 (100°C)

0.17

93 [4.7]

6.98 (30°C)

4.02 (80°C)

0.18

76 [3.0] 197 [5.8] 189 [5.7]

7.00 (70°C) 4.67 (40°C) 4.67 (65°C)

4.66 (80°C) 4.63 (96°C)

0.38 0.36

*Measured on solution-precipitated, dried, and annealed sample; Tm = melting point; Tc = Colhexlow-T to Colhexhigh-T transition; Ti = †Determined by differential scanning calorimetry heating scans, 2 K min−1. ‡Critical Colhex to isotropic liquid transition. temperature Tc determined by fitting the I(10) curve, as shown in Fig. 4A. §A very small endotherm indicates a very weak first||Observed order transition, probably associated with the core-shell structure of the Colhexhigh-T phase of A1 (SOM text S2.2.1). only in the first heating run; no crystallization was observed on cooling and no melting on subsequent heating.

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Honeycombs with multicolor hexagonal tiling are indeed found in compounds A when examined by small-angle x-ray scattering (SAXS): A1 to A3 exhibit a hexagonal columnar phase (Colhex) with unit cell parameter ahex = 7.00 T 0.03 nm. In addition, compounds A1 and A2 exhibit another Colhex phase at higher temperatures. The two Colhex phases are separated by a thermoreversible transition at temperature Tc (Table 1 and Fig. 3, A to C). The fact that only diffuse x-ray scattering is observed in the range of interatomic distances (d < 0.6 nm, fig. S4), as well as the typical optical textures (fig. S1), confirm the LC character of both phases. The unit cell parameters of Colhexlow-T and Colhexhigh-T are related as ahexlow-T (7.0 nm) ≈ √3 ahexhigh-T (4.0 nm) ≈ 3 × l, where l = 2.3 nm is the molecular length. This suggests that the Colhexlow-T phase is either three-color or two-color (Fig. 1, I and J), whereas the Colhexhigh-T phase is singlecolor (Fig. 1C). We have evaluated the energies and entropies of the three candidate side-chain distributions in the hexagonal phase. All nine pairs of chains in the unit cell (assuming a = 3l) are arranged randomly in the single-color phase; this is reduced to three for the two-color phase, and to zero for the three-color phase. Consequently the entropies of the three phases are 9ln2, 3ln2, and 0, respectively. At the same time, the energy differences between these phases are estimated assuming only pairwise interactions between side chains within one hexagon. The energies are, respectively, S, F, and M between two Si, two F, and an Si and an F chain. We find that, relative to the single-color phase (1c), the energies of the two-color (2c) and three-color (3c) phases are DE2c=1c ¼ −7x and DE3c=1c ¼ −9x respectively, where x ¼ 34 ½2M − S − F. These energy and entropy differences predict the phase sequence with increasing temperature to be three-color → two-color → single-color, with the (mean field) transition temperatures related as T2c/1c = 1.75 T3c/2c (for details, see SOM text S3). Thus, the mean field theory predicts that the three-color phase could only appear far below the temperatures of the current experiments. The reconstructed electron density (ED) maps of compound A2 are shown in Fig. 3, F and G, for the two- and single-color phases. The maps confirm that the crystallographic unit cell of the two-color phase contains one RSi-rich and two RF-rich (for an explanation of RSi and RF, see Fig. 2) honeycomb cells, whereas for the singlecolor phase, Si and F chains are mixed in all cells. For the two-color phase, comparison of observed x-ray and neutron diffraction intensities from A1 with those calculated from models also supports the conclusion that the odd one out of the three cylinders in the crystallographic cell is rich in RSi rather than RF; the two RF-rich cells contain an approximately 3:1 RF:RSi molar mixture on average. The above is further supported by atomic force microscopy (AFM). In phase images of the single-color form, the bright spots arranged on a

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On the basis of the mapping to the spin-½ Ising model, it can be shown that for hexagonal systems, I(10) is directly linked to sublattice mag-

netization M, via I(10) º M2 (for details, see SOM text S4.1). Thus, the continuous change of I(10) in Fig. 4A is analogous to the critical

Fig. 3. Two-color [(F) and (I)] to single-color [(G) and (H)] tiling transition in hexagonal (A to G) and triangular (H and I) LC honeycombs. (A) SAXS patterns of powder and [(B) and (C)] surface-aligned compound A2 versus temperature. (D) and (E) AFM phase images of A1 recorded at 85° and 55°C (Fourier-filtered). (F) and (G) ED maps of the low-T and high-T phase of A2. (H) ED map of the high-T and (I) low-T phase of B2. The maps refer to the color scale on the right. See SOM text S2.3 for details. Fig. 4. (A) Intensity of the {10} Bragg diffraction versus T for A2 and B2; lines are fits to A(Tc – T )2b (A is a constant). (B) T dependence above Tc of quasielastic scattering from A2 powder around q(10) of the low-T phase. (C to F) GISAXS patterns of thin film of A1 on Si substrate at Tc – 8.8, Tc – 0.3, Tc + 0.9, and Tc + 6.0 K. (G and H) Hexagonal and triangular honeycombs and their mapping onto spin-1/2 Ising models on a kagome lattice. (I) Equivalence of the six orientations of the RSi side chains with up or down spins.

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triangular lattice (Fig. 3D) are attributed to the centers of the mixed columns. On cooling, the pattern becomes consistent with that of the twocolor phase (Fig. 3E). Cells rich in the harder RF chains are brighter than the softer RSi cells (11), hence six bright cells surround each dark one. The intensity of the Bragg {10} reflection, I(10), for both A1 and A2 decreases continuously to zero as Tc is approached from below (for A2, see Fig. 4A). Close to Tc, I(10) obeys a power law relationship with (Tc – T ) (12). For A1 and A2, we find I(10) º (Tc – T )0.34 T 0.01 (Fig. 4A and Table 1). Another feature of SAXS from A1 and A2 is the diffuse scattering centered at q(10), the position of the low-temperature {10} Bragg peak. This scatter occurs both below (superimposed on the Bragg peak) and above Tc (Fig. 4, B to F). It has a Lorentzian profile (Fig. 4B) and is due to local fluctuations. Thus, for instance, above Tc it arises from local clusters of two-color tiling that increase in size as Tc is approached. At Tc, the height of the diffuse peak reaches a maximum and its width a minimum. The above behavior is reminiscent of the classical spontaneous magnetization of a ferromagnet. The direction of the side chains for each X-shaped molecule, and their interactions in the hexagonal phase, can be mapped onto a spin-½ Ising system on a kagome lattice (Fig. 4G), where the nearest-neighbor interactions are antiferromagnetic. In such a model there is an infinite number of ways of arranging spins to achieve the same minimum energy, and there are no critical order-disorder transitions (13, 14). In that respect, the situation is similar to that of crystalline ice with its shuttling hydrogens rather than flipping spins (15) and of various forms of spin-ice materials (16, 17). However, for our systems, all pairwise interactions inside a hexagon, both ferromagnetic and antiferromagnetic, should be included. Energetically, this favors the arrangement of spins around a hexagon in the kagome lattice to be + – + – + – (Fig. 4G, right); in the LC phase this is equivalent to a hexagonal cylinder preferring all its side chains to be of the same type. We have not found any published results for an Ising model on a kagome lattice with pairwise interactions defined as above (see also SOM text S3). However, an Ising model on a kagome lattice, with antiferromagnetic nearestneighbor and ferromagnetic next-nearest-neighbor interactions, has been shown to exhibit critical transitions (18). The hexagons filled purely with Si chains in the Colhexlow-T phase cannot be adjacent to each other (Fig. 1J), just as krypton atoms adsorbed on the basal plane of graphite, because of their size, cannot occupy adjacent carbon hexagons. Such a system undergoes an order-disorder transition related to our two-color–single-color transition and has the symmetry of the two-dimensional (2D) three-state Potts model (19). A similar frustrated system on a larger length scale is a compressed raft of colloidal spheres on water, where some spheres have to “escape to the third dimension” (20).

behavior of M, which scales as (Tc – T )b close to the Curie point (12). The critical exponent b = 0.17 for compounds A1 and A2 should be compared with b = 0.11 expected for the 2D three-state Potts model. Compounds B1 and B2, with the longer core, display triangular honeycombs, as deduced from their ahex ≈ l relation (Fig. 1, A and F); here ahex = 4.6 nm and l = 4.3 nm. Whether the triangular honeycomb is single-color (Fig. 1A, p6mm) or two-color (Fig. 1F, p3m1) cannot be established unequivocally from diffraction alone, because Laue symmetry is the same for both. However, B1 and B2 display a clear secondorder phase transition, as shown by I(10) versus T plots (for B2 see Fig. 4A and fig. S7). In B1 and B2, I(10) does not vanish at Tc but retains a small residual value Ir , whereas I(11) barely changes, as expected from a transition between models in

Fig. 1, A and F. Based on structure factor phases obtained from best-fit triangular models, ED maps of the high- and low-T phases of B2 are shown in Fig. 3, H and I (for details, see SOM text S2.3.2). The two-color triangular phase is not frustrated. It can also be mapped onto a kagome net, but as a ferromagnet (Fig. 4H). Here I(10) is linked to magnetization through I(10) – Ir º M 2 (SOM text S4.2), which is again related to (Tc – T ) through a power law. Tc and b for B1 and B2 are listed in Table 1. The critical exponent b = 0.37, determined here for the ferromagnetic triangular phases, is close to that of the 3D Ising model (0.33); and that for the antiferromagnetic hexagonal phase, 0.17, clearly belongs to a 2D system (19). Even though both systems are 2D in symmetry, they are physically 3D because they consist of columns infinite along z. Because the distance be-

Fig. 5. Complex tiling patterns in LC honeycombs of compound B3. (A and B) GISAXS patterns at (A) 150°C (p2mm phase) and (B) 175°C (c2mm phase), with overlaid reciprocal lattice nets (the different orientations are color-coded mirror images with dashed lines). (C and F) ED maps of the p2mm and c2mm phases, with schematic molecules overlaid (gray rods denote aromatic cores, green dots the glycerol groups, and red and blue dots the Si and F chains); black rectangles are unit cells; and the color scale as in Fig. 3. (D and G) Tiling patterns corresponding to the above ED maps. (E and H) The three and five types of tiles which, through tessellation, form the p2mm and c2mm honeycombs. www.sciencemag.org

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tween molecules along z is much shorter than that in the lateral plane, ordering along z is expected to be decoupled from that in the xy plane. Qualitatively, however, one would expect that in-plane coupling would be stronger in the triangular phase, where the angle between molecular backbones is 60° rather than 120°, resulting in a shorter distance between side chains. The critical behavior of these soft-matter systems is of considerable interest, because it reiterates the applicability of statistical mechanics of model spin systems to supramolecular selfassembly. The observed behavior implies a highly dynamic LC system, with molecular flips replacing spin flips and side-chain interaction replacing magnetic interaction. The dynamics of this motion are fast, and the structures are very close to equilibrium, as indicated by the very small hysteresis of the phase transition (fig. S2). In this respect, the LC honeycombs differ from the related cellular 2D structures in three-arm star polymers, where slow dynamics seem to inhibit the attainment of true equilibrium (21). Analogies with spin systems have also been used to describe phase transitions in amphiphilic self-assembling systems (22). Because of the large difference in area between an equilateral triangle and a square (a factor of 2.3), compounds with side chains larger than those in B2, such as B3, experience additional geometrical frustration of triangular cells being too small and square ones being too large. A compromise single-color honeycomb in T-shaped LCs was reported to contain both triangles and squares in a ratio of 2: 1 (6). However, the structure in X-shaped compound B3 must simultaneously relieve the frustrations of its side chains being both oversized for triangular cells and mutually incompatible. B3 was found to display two new LC honeycombs of unusual complexity, separated by a first-order transition at 167°C. Thin-film grazing incidence SAXS (GISAXS) patterns are shown in Fig. 5, A and B, and powder patterns showing the same reflections are in fig. S10. The plane groups of the low- and high-T phases are p2mm and c2mm, respectively, with rectangular unit cells as large as 11.20 × 4.46 nm and 21.30 × 11.08 nm. ED maps are displayed in Fig. 5, C and F (for details, see SOM text S2.3.3). The overlaid molecular arrangement in (Fig. 5, C and D) shows the partitioning of the p2mm honeycomb into three types of cells: a triangular, a rhombic and a square (Fig. 5, D and E). These have different compositions, and a good fit with the map and with the overall 1:1 RSi /RF molar ratio is obtained by assuming that the triangles, rhombi, and squares contain pure Si chains, pure F chains, and a 1:3 mixture of the two, respectively. In contrast, to interpret the ED map of the high-T c2mm phase (Fig. 5F), five different tile types are required, with different shapes and colors (Fig. 5, G and H). Two are triangles, two are rhombi, and one is a square. After the variation in cell wall lengths is minimized (SOM text S2.3.3), the mean side length of all tiles in Fig. 5, D and G, is 4.24 nm,

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which is in good agreement with l (Fig. 2), the standard deviation being 0.10 nm. The unit cell of the low-T phase of B3 contains four honeycomb cells, two triangular and two quadrangular, whereas that of the high-T phase has 18 cells, of which 10, or 56%, are quadrangular. The slightly increased quadrangular fraction above the transition is consistent with thermal expansion of the flexible side chains, hence with an increased area/wall ratio. Moreover, apportioning the area of the tiles to Si and F chains according to the tilings in Fig. 5 gives the ratio of effective volumes VSi chain /VF chain of 0.69 and 0.76 for the low- and high-T phases, which is consistent with thermal expansion of RSi being higher than that of RF. The complexity of the self-assembled softmatter superstructures of B3 is unprecedented. Compounds similar to B3 were found to form as yet unidentified 2D phases with even larger unit cells. In geometrically frustrated 3D structures of metal alloys (23) and dendrimers (24), increasing unit cell size eventually leads to a breakdown of periodicity and the formation of quasicrystals (25). By analogy, we anticipate that polyphilic LCs may ultimately produce quasiperiodic honeycombs. The ellipses drawn in Fig. 5F hint at the possibility of dodecagonal order developing in a related LC system. Overall, design based on segregation and geometric frus-

tration paves the way to self-assembly of small molecules into elaborate multicompartment softmatter structures for complex 2D nanopatterning. References and Notes 1. A. F. Wells, Three-Dimensional Nets and Polyhedra (Wiley, New York, 1977). 2. C. Baerlocher, L. B. McCusker, Database of Zeolite Structures, www.iza-structure.org/databases/. 3. J. N. Israelachvili, D. J. Mitchell, B. W. Ninham, J. Chem. Soc. Faraday Trans. 2. Mol. Chem. Phys, 72, 1525 (1976). 4. I. W. Hamley, The Physics of Block-Copolymers (Oxford Univ. Press, Oxford, 1998). 5. S. Hyde et al., The Language of Shape: The Role of Curvature in Condensed Matter—Physics, Chemistry, and Biology (Elsevier, Amsterdam, 1997). 6. B. Chen, X. Zeng, U. Baumeister, G. Ungar, C. Tschierske, Science 307, 96 (2005). 7. C. Tschierske, Chem. Soc. Rev. 36, 1930 (2007). 8. B. Grünbaum, G. C. Shephard, Tilings and Patterns (Freeman, New York, 1987). 9. B. Glettner et al., Angew. Chem. Int. Ed. 47, 9063 (2008). 10. M. A. Bates, M. Walker, Phys. Chem. Chem. Phys. 11, 1893 (2009). 11. R. García, R. Pérez, Surf. Sci. Rep. 47, 197 (2002). 12. J. M. Yeomans, Statistical Mechanics of Phase Transitions (Oxford Univ. Press, Oxford, 1992). 13. I. Syôzi, Prog. Theor. Phys. 6, 306 (1951). 14. K. Kanô, S. Naya, Prog. Theor. Phys. 10, 158 (1953). 15. L. C. Pauling, The Nature of the Chemical Bond (Cornell Univ. Press, Ithaca, NY, 1945). 16. S. T. Bramwell, M. J. P. Gingras, Science 294, 1495 (2001). 17. D. J. P. Morris et al., Science 326, 411 (2009). 18. M. Wolf, K. D. Schotte, J. Phys. Math. Gen. 21, 2195 (1988). 19. M. Kardar, A. N. Berker, Phys. Rev. Lett. 48, 1552 (1982).

A Planar Rhombic Charge-Separated Tetrasilacyclobutadiene Katsunori Suzuki,1 Tsukasa Matsuo,1* Daisuke Hashizume,2 Hiroyuki Fueno,3 Kazuyoshi Tanaka,3 Kohei Tamao1* The cyclobutadiene (CBD) molecule C4H4 deviates from a high-symmetry square geometry to compensate for its antiaromatic electronic structure. Here, we report a CBD silicon analog, Si4(EMind)4 (1), stabilized by the bulky 1,1,7,7-tetraethyl-3,3,5,5-tetramethyl-s-hydrindacen-4-yl (EMind) groups, obtained as air- and moisture-sensitive orange crystals by the reduction of (EMind)SiBr3 with three equivalents of lithium naphthalenide. X-ray crystallography reveals a planar and rhombic structure of the Si4 four-membered ring, with alternating pyramidal and planar configurations at the silicon atoms. The large 29Si chemical shift differences (Dd > 350 parts per million) in the solid-state nuclear magnetic resonance spectra suggest a contribution of an alternately charge-separated structure. The rhombic-shaped charge-separated singlet state of compound 1 thus stabilizes its cyclic 4p-electron antiaromaticity in a manner that contrasts sharply with the bond-length alternation, characterizing the rectangular distortion of carbon-based CBD. ust as benzene’s hexagonal arrangement of alternating double bonds confers unusual stability, or aromaticity, the tetragonal arrangement of cyclobutadiene (CBD) confers the opposite

J

1 Functional Elemento-Organic Chemistry Unit, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 2 Advanced Technology Support Division, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 3Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan.

*To whom correspondence should be addressed. E-mail: [email protected] (T.M.); [email protected] (K.T.)

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property. Delocalization of four p-electrons over the four-membered ring results in a destabilization of the total energy. In 1965, Petit et al. reported the generation and low-temperature trapping of a parent CBD (C4H4) by the oxidative treatment of (h4-C4H4)Fe(CO)3 with CeIV ions (1). Since then, CBD has continued to fascinate both experimentalists and theoreticians in their quest to understand the various stabilizing factors of antiaromatic species (2–4). Theoretical and experimental studies of the parent CBD (2–11) demonstrated that a squareshaped triplet of the highest symmetry with two

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20. Y. Han et al., Nature 456, 898 (2008). 21. Y. Matsushita, A. Takano, K. Hayashida, T. Asari, A. Noro, Polymer 50, 2191 (2009). 22. M. Girardi, W. Figueiredo, Phys. A 319, 421 (2003). 23. D. P. Shoemaker, C. B. Shoemaker, Acta Crystallogr. 42, 3 (1986). 24. X. B. Zeng et al., Nature 428, 157 (2004). 25. M. Senechal, Quasicrystals and Geometry (Cambridge Univ. Press, Cambridge, 1995). 26. This work was supported as part of the European Science Foundation Eurocores Program SONS 2 (Self-Organised NanoStructures 2), project SCALES, by the Engineering and Physical Sciences Research Council and Deutsche Forschungsgemeinschaft (DFG) and the European Commission Sixth Framework Programme (grant NERASCT-2003-989409); by the government of Saxonia-Anhalt in the framework of the Cluster of Excellence “Nanostructured Materials”; the DFG-funded Research Unit FG 1145; and the World Class University program through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology (grant R31-10013). For their help in setting up synchrotron experiments, we thank N. Terrill, C. Pizzey, S. Collins, and A. Bombardi at beamlines I22 and I16, Diamond Light Source, and D. Mannix, S. Brown, and P. Thompson at beamline BM28 at the European Synchrotron Research Facility. We are grateful to A. Perkins and B. Deme at beamline D16, Institut Laue-Langevin, for their help with neutron-scattering experiments.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1302/DC1 SOM Text S1 to S5 Figs. S1 to S25 Tables S1 to S4 1 June 2010; accepted 3 February 2011 10.1126/science.1193052

degenerate nonbonding orbitals (Fig. 1A) is not the ground state but rather a less stable structure that is subjected to second-order Jahn-Teller (J-T) distortion for stabilization. There are two major linearly independent modes of distortion: One produces a rectangular-shaped singlet, keeping two unsaturated bonds with the frontier molecular orbitals (MOs) delocalized on all four atoms (covalent perturbation) (Fig. 1B); the other produces a planar rhombic structure, causing an alternating charge separation on four atoms, with the out-of-phase frontier MOs localized on each set of diagonal atoms (polar perturbation) (Fig. 1C) (12). Experimentally, some CBD derivatives bearing rectangular structures with two isolated C=C double bonds (because of the covalent J-T distortion) have been prepared and structurally characterized by taking advantage of kinetic (steric) and/or thermodynamic (electronic) stabilization effects (2–4, 13–19). We now report that a silicon analog of CBD is stabilized by the polar J-T distortion and has the planar rhombic structure. A variety of unsaturated silicon molecules, including silaaromatics (20), trisilaallene (21), tetrasilabutadiene (22), disilyne (23), and disilicon(0) (24), have previously been isolated by the introduction of bulky protecting groups on the basis of the kinetic stabilization concept introduced by West and Brook in 1981 (25, 26). Quite recently, a tricyclic aromatic valence isomer of hexasilabenzene was reported (27). The parent tetrasilacyclobutadiene (SiH)4 has been the subject of theoretical studies (28). A

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which is in good agreement with l (Fig. 2), the standard deviation being 0.10 nm. The unit cell of the low-T phase of B3 contains four honeycomb cells, two triangular and two quadrangular, whereas that of the high-T phase has 18 cells, of which 10, or 56%, are quadrangular. The slightly increased quadrangular fraction above the transition is consistent with thermal expansion of the flexible side chains, hence with an increased area/wall ratio. Moreover, apportioning the area of the tiles to Si and F chains according to the tilings in Fig. 5 gives the ratio of effective volumes VSi chain /VF chain of 0.69 and 0.76 for the low- and high-T phases, which is consistent with thermal expansion of RSi being higher than that of RF. The complexity of the self-assembled softmatter superstructures of B3 is unprecedented. Compounds similar to B3 were found to form as yet unidentified 2D phases with even larger unit cells. In geometrically frustrated 3D structures of metal alloys (23) and dendrimers (24), increasing unit cell size eventually leads to a breakdown of periodicity and the formation of quasicrystals (25). By analogy, we anticipate that polyphilic LCs may ultimately produce quasiperiodic honeycombs. The ellipses drawn in Fig. 5F hint at the possibility of dodecagonal order developing in a related LC system. Overall, design based on segregation and geometric frus-

tration paves the way to self-assembly of small molecules into elaborate multicompartment softmatter structures for complex 2D nanopatterning. References and Notes 1. A. F. Wells, Three-Dimensional Nets and Polyhedra (Wiley, New York, 1977). 2. C. Baerlocher, L. B. McCusker, Database of Zeolite Structures, www.iza-structure.org/databases/. 3. J. N. Israelachvili, D. J. Mitchell, B. W. Ninham, J. Chem. Soc. Faraday Trans. 2. Mol. Chem. Phys, 72, 1525 (1976). 4. I. W. Hamley, The Physics of Block-Copolymers (Oxford Univ. Press, Oxford, 1998). 5. S. Hyde et al., The Language of Shape: The Role of Curvature in Condensed Matter—Physics, Chemistry, and Biology (Elsevier, Amsterdam, 1997). 6. B. Chen, X. Zeng, U. Baumeister, G. Ungar, C. Tschierske, Science 307, 96 (2005). 7. C. Tschierske, Chem. Soc. Rev. 36, 1930 (2007). 8. B. Grünbaum, G. C. Shephard, Tilings and Patterns (Freeman, New York, 1987). 9. B. Glettner et al., Angew. Chem. Int. Ed. 47, 9063 (2008). 10. M. A. Bates, M. Walker, Phys. Chem. Chem. Phys. 11, 1893 (2009). 11. R. García, R. Pérez, Surf. Sci. Rep. 47, 197 (2002). 12. J. M. Yeomans, Statistical Mechanics of Phase Transitions (Oxford Univ. Press, Oxford, 1992). 13. I. Syôzi, Prog. Theor. Phys. 6, 306 (1951). 14. K. Kanô, S. Naya, Prog. Theor. Phys. 10, 158 (1953). 15. L. C. Pauling, The Nature of the Chemical Bond (Cornell Univ. Press, Ithaca, NY, 1945). 16. S. T. Bramwell, M. J. P. Gingras, Science 294, 1495 (2001). 17. D. J. P. Morris et al., Science 326, 411 (2009). 18. M. Wolf, K. D. Schotte, J. Phys. Math. Gen. 21, 2195 (1988). 19. M. Kardar, A. N. Berker, Phys. Rev. Lett. 48, 1552 (1982).

A Planar Rhombic Charge-Separated Tetrasilacyclobutadiene Katsunori Suzuki,1 Tsukasa Matsuo,1* Daisuke Hashizume,2 Hiroyuki Fueno,3 Kazuyoshi Tanaka,3 Kohei Tamao1* The cyclobutadiene (CBD) molecule C4H4 deviates from a high-symmetry square geometry to compensate for its antiaromatic electronic structure. Here, we report a CBD silicon analog, Si4(EMind)4 (1), stabilized by the bulky 1,1,7,7-tetraethyl-3,3,5,5-tetramethyl-s-hydrindacen-4-yl (EMind) groups, obtained as air- and moisture-sensitive orange crystals by the reduction of (EMind)SiBr3 with three equivalents of lithium naphthalenide. X-ray crystallography reveals a planar and rhombic structure of the Si4 four-membered ring, with alternating pyramidal and planar configurations at the silicon atoms. The large 29Si chemical shift differences (Dd > 350 parts per million) in the solid-state nuclear magnetic resonance spectra suggest a contribution of an alternately charge-separated structure. The rhombic-shaped charge-separated singlet state of compound 1 thus stabilizes its cyclic 4p-electron antiaromaticity in a manner that contrasts sharply with the bond-length alternation, characterizing the rectangular distortion of carbon-based CBD. ust as benzene’s hexagonal arrangement of alternating double bonds confers unusual stability, or aromaticity, the tetragonal arrangement of cyclobutadiene (CBD) confers the opposite

J

1 Functional Elemento-Organic Chemistry Unit, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 2 Advanced Technology Support Division, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. 3Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan.

*To whom correspondence should be addressed. E-mail: [email protected] (T.M.); [email protected] (K.T.)

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property. Delocalization of four p-electrons over the four-membered ring results in a destabilization of the total energy. In 1965, Petit et al. reported the generation and low-temperature trapping of a parent CBD (C4H4) by the oxidative treatment of (h4-C4H4)Fe(CO)3 with CeIV ions (1). Since then, CBD has continued to fascinate both experimentalists and theoreticians in their quest to understand the various stabilizing factors of antiaromatic species (2–4). Theoretical and experimental studies of the parent CBD (2–11) demonstrated that a squareshaped triplet of the highest symmetry with two

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20. Y. Han et al., Nature 456, 898 (2008). 21. Y. Matsushita, A. Takano, K. Hayashida, T. Asari, A. Noro, Polymer 50, 2191 (2009). 22. M. Girardi, W. Figueiredo, Phys. A 319, 421 (2003). 23. D. P. Shoemaker, C. B. Shoemaker, Acta Crystallogr. 42, 3 (1986). 24. X. B. Zeng et al., Nature 428, 157 (2004). 25. M. Senechal, Quasicrystals and Geometry (Cambridge Univ. Press, Cambridge, 1995). 26. This work was supported as part of the European Science Foundation Eurocores Program SONS 2 (Self-Organised NanoStructures 2), project SCALES, by the Engineering and Physical Sciences Research Council and Deutsche Forschungsgemeinschaft (DFG) and the European Commission Sixth Framework Programme (grant NERASCT-2003-989409); by the government of Saxonia-Anhalt in the framework of the Cluster of Excellence “Nanostructured Materials”; the DFG-funded Research Unit FG 1145; and the World Class University program through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology (grant R31-10013). For their help in setting up synchrotron experiments, we thank N. Terrill, C. Pizzey, S. Collins, and A. Bombardi at beamlines I22 and I16, Diamond Light Source, and D. Mannix, S. Brown, and P. Thompson at beamline BM28 at the European Synchrotron Research Facility. We are grateful to A. Perkins and B. Deme at beamline D16, Institut Laue-Langevin, for their help with neutron-scattering experiments.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1302/DC1 SOM Text S1 to S5 Figs. S1 to S25 Tables S1 to S4 1 June 2010; accepted 3 February 2011 10.1126/science.1193052

degenerate nonbonding orbitals (Fig. 1A) is not the ground state but rather a less stable structure that is subjected to second-order Jahn-Teller (J-T) distortion for stabilization. There are two major linearly independent modes of distortion: One produces a rectangular-shaped singlet, keeping two unsaturated bonds with the frontier molecular orbitals (MOs) delocalized on all four atoms (covalent perturbation) (Fig. 1B); the other produces a planar rhombic structure, causing an alternating charge separation on four atoms, with the out-of-phase frontier MOs localized on each set of diagonal atoms (polar perturbation) (Fig. 1C) (12). Experimentally, some CBD derivatives bearing rectangular structures with two isolated C=C double bonds (because of the covalent J-T distortion) have been prepared and structurally characterized by taking advantage of kinetic (steric) and/or thermodynamic (electronic) stabilization effects (2–4, 13–19). We now report that a silicon analog of CBD is stabilized by the polar J-T distortion and has the planar rhombic structure. A variety of unsaturated silicon molecules, including silaaromatics (20), trisilaallene (21), tetrasilabutadiene (22), disilyne (23), and disilicon(0) (24), have previously been isolated by the introduction of bulky protecting groups on the basis of the kinetic stabilization concept introduced by West and Brook in 1981 (25, 26). Quite recently, a tricyclic aromatic valence isomer of hexasilabenzene was reported (27). The parent tetrasilacyclobutadiene (SiH)4 has been the subject of theoretical studies (28). A

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Fig. 1. Schematic representations of molecular orbital diagrams of CBD. (A) Square-shaped triplet. (B) Rectangular-shaped singlet from covalent second-order Jahn-Teller (J-T) distortion. (C) Rhombic-shaped singlet from polar second-order J-T distortion.

Fig. 3. (Top) Synthesis of 1 and representation of its charge-separated canonical form 1’. (A) The molecular structure of 1 (50% probability ellipsoids). Hydrogen atoms are not shown. Selected bond lengths (angstroms) and angles (degrees): Si1-Si2 = 2.2877(8), Si1-Si4 = 2.2924(8), Si2-Si3 = 2.2671(8), Si3-Si4 = 2.2846(8), Si2-Si1-Si4 = 103.00(3), Si1-Si2-Si3 = 76.76(3), Si2-Si3-Si4 = 103.90(3), and Si1-Si4-Si3 = 76.32(3). (B) Electrostatic potential maps of (SiH)4 (C2h symmetry). Blue and red regions represent positive and negative potentials, respectively. www.sciencemag.org

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puckered D2d structure and its valence isomer, tetrasilatetrahedrane, were calculated to be local energy minima; a planar rectangular D2h structure with two Si=Si double bonds and a planar rhombic C2h structure (29) emerged as saddle point structures (Fig. 2). Experimentally, among the valence isomers the tetrasilatetrahedranes have been isolated by the introduction of the isotropically bulky tBu3Si (where tBu is tertiary butyl) or dendritically bulky [(Me3Si)2CH]2MeSi (where Me is methyl) groups on the silicon atoms (30, 31). A square planar tetrasilacyclobutadiene has also been reported to be stabilized as a transition metal complex (32, 33). We anticipated that bulky 1,1,3,3,5,5,7,7-octa-R-substituted s-hydrindacen-4-yl (Rind)–protecting groups might have the ability to stabilize a free and planar tetrasilacyclobutadiene, in view of our previous observations of highly coplanar p-conjugated core frameworks comprising Si=Si and Si=P double bonds (34, 35). We now report the isolation and characterization of the silicon CBD analog Si4(EMind)4 (1), stabilized by the appropriately designed bulky 1,1,7,7-tetraethyl-3,3,5,5-tetramethyl-s-hydrindacen4-yl (EMind) group. The Si4 ring in compound 1 has a planar rhombic structure, with alternating pyramidal and planar configurations at the four silicon atoms. Clearly, the fused-ring bulky EMind groups on the silicon atoms can stabilize the planar rhombic four-membered ring as the ground state, which is in contrast to the less stable saddle point C2h structure theoretically predicted for the parent (SiH)4. The reduction of the EMind-substituted tribromosilane, (EMind)SiBr3, with three equivalents of lithium naphthalenide (LiNaph) in dry tetrahydrofuran (THF), led to the formation of a dark brown solution from which the tetrasilacyclobutadiene, Si4(EMind)4, 1 was isolated as airand moisture-sensitive orange crystals in 9% yield [maximum wavelength absorption (lmax) = 448 nm, molar extinction coefficient (dm3 mol –1 cm–1) (e) = 29000 in hexane] (Fig. 3, top) (36). Compound 1 is not thermally very stable; decomposition occurred in solution and even in the solid state at room temperature. The half-life was estimated

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Fig. 2. Theoretically predicted isomers of (SiH)4.

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to be about 3 days in C6D6 at 298 K according to monitoring by 1H nuclear magnetic resonance (NMR). Nevertheless, single crystals of 1 suitable for x-ray crystallographic analyses were obtained through recrystallization from a saturated solution in hexane/toluene (1:1 ratio) at 243 K. The molecular structure of 1 (Fig. 3A, bottom), based on x-ray diffraction data collected at 100 K (36), features a planar central Si4 ring: The sum of the internal bond angles is 360.0°. The four Si-Si bond lengths differ by less than 0.03 Å and are intermediate between typical Si-Si single (2.34 Å) and double (2.14 Å) bonds. The four silicon atoms have different configurations. Whereas the Si1 and Si3 atoms are planar with sums of surrounding bond angles of 360.0° (sp2-hybridization), the Si2 and Si4 atoms are pyramidal with sums of surrounding bond angles of 338.8° and 335.1° (sp3-like hybridization). The positions of the ipsocarbon atoms (C25 and C73) of the EMind groups on the Si2 and Si4 atoms deviate up and down from the plane of the Si4 ring by 32.87(7)° and 35.80(7)°, respectively. The diagonal distances are 2.8280(8) Å for Si1···Si3 and 3.5843(7) Å for Si2···Si4, both of which are shorter than the sum of the van der Waals radii of the silicon atom (4.20 Å). Space-filling models show that the Si4 ring is covered by the four EMind groups, thus producing an EMind-meshed molecular gear (fig. S4). The 1H NMR spectrum of 1 in C6D6 solution indicates a symmetrical D4 structure at ambient temperature: Only one set of resonances due to the EMind groups with C2 symmetry is observed. The spectrum suggests that a length-width interconversion of the rhombic forms occurs on the NMR time scale, thus maintaining the gear arrangement of the four EMind groups. This dynamic process persists down to 213 K, as observed in 1H NMR spectra in C7D8. This geometryswitching process is suppressed in the crystalline state, as observed in the x-ray molecular structure. Accordingly, in the solid state 29Si crosspolarization magic angle spinning (CP-MAS) NMR spectrum of 1, two sets of two resonances are found in the higher field region [chemical shifts (d) = –52 and –50 parts per million (ppm)] and in the lower field region (d = 300 and 308 ppm) (37). The former are assignable to the pyramidal silicon atoms (Si2 and Si4) and the latter to the planar silicon atoms (Si1 and Si3). The large chemical shift differences (Dd > 350 ppm) suggest the contribution of a canonical form 1’ with charge-separation on the Si centers associated with the configurational difference. To elucidate the electronic nature of the CBD silicon analog, density functional theory (DFT) computations were performed for 1 at the B3LYP/631G(d,p) level by using the Gaussian 03 suite of programs. In addition to the most stable structure 1, a planar rectangular singlet 1(re) (C1 symmetry) having two Si=Si double bonds and a square planar triplet 1(sq) (C1 symmetry) are also found at 6.41 kcal mol–1 and 2.09 kcal mol–1 higher energy levels, respectively (figs. S11 to S13). The optimized closed-shell structure well reproduces

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the x-ray molecular structure: The most stable isomer is a singlet with an Si4 rhombic framework, as determined by the internal bond angles of 77.2°, 78.8°, 101.4°, and 102.5° and the Si-Si bond lengths of 2.296 to 2.355 Å (average of 2.321 Å). The bond orders based on the Wiberg bond index (WBI) of the four Si-Si bonds of 1 are almost equal (1.070 to 1.179), in sharp contrast to those of 1(re) (0.884, 0.885, 1.795, and 1.797), which exhibits bond length alternation (figs. S15 and S16). The WBI of the diagonal Si1-Si3 (0.372) and Si2-Si4 (0.269) in 1 are larger than those in 1(re) (0.055 and 0.062), which is suggestive of a certain degree of bonding interaction. The complete active space self-consistent field (CASSCF) (4,4) calculation for 1 gave occupation numbers of 1.849 and 0.153 in the highest occupied natural orbital (HONO) and the lowest unoccupied NO (LUNO), respectively, indicating a low diradical character (38) on the Si2 and Si4 atoms (fig. S20). The calculated 29Si chemical shifts (d = –50.3, –33.0, 371.1, and 373.2 ppm) by the gauge-independent atomic orbital (GIAO) method at the B3LYP/6-311+G(d,p)//B3LYP/631G(d,p) level show good agreement with the

experimental values. The natural population analysis (NPA) charge distribution of 1 exhibits a considerable charge difference on silicon atoms with slightly positive Si2(+0.144) and Si4(+0.167) centers and more highly positive Si1(+0.647) and Si3(+0.637) centers; the former and the latter are, respectively, more negative and positive than those of 1(re) (+0.339 ~ +0.398), which is consistent with the contribution of the chargeseparated canonical form 1’. The charge-separation is much more evident in the parent (SiH)4 C2h structure with positive +0.370 for Si1 and Si3 and negative –0.077 for Si2 and Si4 (fig. S8), and is visualized by the electrostatic potential maps shown in Fig. 3B, bottom. The NICS(1) value (39) of 1 at the geometrical center of the Si4 ring is estimated to be –0.9 ppm, suggesting the lack of a diatropic or paratropic ring current by the delocalization of p-electrons in the four-membered skeleton. In other words, 1 is neither aromatic nor antiaromatic, but nonaromatic, which is consistent with the charge-separated structure (fig. S19). Figure 4 shows the four pertinent p-like MOs of 1, which correspond to the four orbitals schematically represented in Fig. 1C. The HOMO

Fig. 4. Four pertinent molecular orbitals and their energy levels for 1.

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and LUMO of 1 essentially involve mutually outof-phase orbitals in the plane with the large coefficients localized on the diagonal silicon atoms. The LUMO involves a p*-like interaction between the p-orbitals on the planar Si1 and Si3 atoms, whereas in the HOMO the out-of-phase orbitals on the pyramidalized Si2 and Si4 atoms lean in one direction to provide a degree of inphase interaction between the back lobes, resulting in overall stabilization. The absorption peak at 448 nm in the ultraviolet-visible (UV-vis) spectrum of 1 is assignable to a mixture of two allowed p-p* transitions (HOMO→LUMO+1 and HOMO-1→LUMO) on the basis of timedependent DFT (TD-DFT) calculations (fig. S18). The HOMO→LUMO transition is calculated to give a very weak absorption peak at 1306 nm because of a forbidden transition. Although Si=Si double bonds ordinarily display an essentially nonpolar character between the constituent atoms, as is the case with C=C double bonds, the Si=Si double bonds involved in the silicon CBD analog 1 are polarized as a result of a polar J-T distortion to counteract antiaromaticity. The polar J-T distortion is induced by alternate electronegativity differences at the silicon centers, which is realized in practice by the pyramidalization of the Si2 and Si4 atoms: The HOMO lobes there exhibit a higher s-character and thus make these atoms more electronegative than the LUMO lobes on the Si1 and Si3 atoms, which are of nearly p-character. Thus in contrast to carbon-based CBD, which is stabilized through bond-length alternation, the tetrasilacyclobutadiene is stabilized as a planar rhombic charge-separated structure because of the weaker Si=Si p-bond, the lower capacity for s-p hybridization at silicon (40, 41), and the rather flexible silicon s-bond framework relative to carbon.

References and Notes 1. L. Watts, J. D. Fitzpatrick, R. Pettit, J. Am. Chem. Soc. 87, 3253 (1965). 2. T. Bally, S. Masamune, Tetrahedron 36, 343 (1980). 3. G. Maier, Angew. Chem. Int. Ed. Engl. 27, 309 (1988). 4. T. Bally, Angew. Chem. Int. Ed. 45, 6616 (2006). 5. C. Y. Lin, A. Krantz, J. Chem. Soc. Chem. Commun. 1972, 1111 (1972). 6. D. J. Cram, M. E. Tanner, R. Thomas, Angew. Chem. Int. Ed. Engl. 30, 1024 (1991). 7. A. A. Deniz, K. S. Peters, G. J. Snyder, Science 286, 1119 (1999). 8. A. Fattahi, L. Lis, Z. Tian, S. R. Kass, Angew. Chem. Int. Ed. 45, 4984 (2006). 9. A. M. Orendt et al., J. Am. Chem. Soc. 110, 2648 (1988). 10. B. R. Arnold, J. G. Radziszewski, A. Campion, S. S. Perry, J. Michl, J. Am. Chem. Soc. 113, 692 (1991). 11. A. Balková, R. J. Bartlett, J. Chem. Phys. 101, 8972 (1994). 12. Push-pull perturbed CBDs provide the localized MOs (38). 13. H. Kimling, A. Krebs, Angew. Chem. Int. Ed. Engl. 11, 932 (1972). 14. H. Irngartinger, H. Rodewald, Angew. Chem. Int. Ed. Engl. 13, 740 (1974). 15. L. T. J. Delbaere, M. N. G. James, N. Nakamura, S. Masamune, J. Am. Chem. Soc. 97, 1973 (1975). 16. G. Maier, S. Pfriem, U. Schäfer, R. Matusch, Angew. Chem. Int. Ed. Engl. 17, 520 (1978). 17. E. J. Petersson et al., J. Am. Chem. Soc. 119, 11122 (1997). 18. J. I. Wu, F. A. Evangelista, P. R. Schleyer, Org. Lett. 12, 768 (2010). 19. Y.-M. Legrand, A. van der Lee, M. Barboiu, Science 329, 299 (2010). 20. K. Wakita, N. Tokitoh, R. Okazaki, S. Nagase, Angew. Chem. Int. Ed. 39, 634 (2000). 21. S. Ishida, T. Iwamoto, C. Kabuto, M. Kira, Nature 421, 725 (2003). 22. M. Weidenbruch, S. Willms, W. Saak, G. Henkel, Angew. Chem. Int. Ed. Engl. 36, 2503 (1997). 23. A. Sekiguchi, R. Kinjo, M. Ichinohe, Science 305, 1755 (2004). 24. Y. Wang et al., Science 321, 1069 (2008). 25. A. G. Brook, F. Abdesaken, B. Gutekunst, G. Gutekunst, R. K. Kallury, J. Chem. Soc. Chem. Commun. 1981, 191 (1981). 26. R. West, M. J. Fink, J. Michl, Science 214, 1343 (1981). 27. K. Abersfelder, A. J. P. White, H. S. Rzepa, D. Scheschkewitz, Science 327, 564 (2010). 28. B. F. Yates, D. A. Clabo Jr., H. F. Schaefer III, Chem. Phys. Lett. 143, 421 (1988).

Tomography of Reaction-Diffusion Microemulsions Reveals Three-Dimensional Turing Patterns Tamás Bánsági Jr., Vladimir K. Vanag, Irving R. Epstein* Spatially periodic, temporally stationary patterns that emerge from instability of a homogeneous steady state were proposed by Alan Turing in 1952 as a mechanism for morphogenesis in living systems and have attracted increasing attention in biology, chemistry, and physics. Patterns found to date have been confined to one or two spatial dimensions. We used tomography to study the Belousov-Zhabotinsky reaction in a microemulsion in which the polar reactants are confined to aqueous nanodroplets much smaller than the scale of the stationary patterns. We demonstrate the existence of Turing patterns that can exist only in three dimensions, including curved surfaces, hexagonally packed cylinders, spots, and labyrinthine and lamellar patterns. n his seminal paper, Alan Turing (1) aimed to provide a mechanism for self-regulated pattern formation in biology by showing that sets of reaction-diffusion equations with appropriate kinetics and diffusion coefficients could

I

spontaneously evolve to spatially periodic structures. The main requirements for Turing-pattern formation in a reaction-diffusion medium are longrange reaction inhibition (fast diffusion of an inhibitor) and short-range activation (slow diffusion

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29. This C2h structure was also found in B3LYP/6-31G(d,p) level calculations (36). 30. N. Wiberg, C. M. M. Finger, K. Polborn, Angew. Chem. Int. Ed. Engl. 32, 1054 (1993). 31. M. Ichinohe, M. Toyoshima, R. Kinjo, A. Sekiguchi, J. Am. Chem. Soc. 125, 13328 (2003). 32. K. Takanashi, V. Ya. Lee, T. Matsuno, M. Ichinohe, A. Sekiguchi, J. Am. Chem. Soc. 127, 5768 (2005). 33. V. Ya. Lee, K. Takanashi, T. Matsuno, M. Ichinohe, A. Sekiguchi, J. Am. Chem. Soc. 126, 4758 (2004). 34. A. Fukazawa, Y. Li, S. Yamaguchi, H. Tsuji, K. Tamao, J. Am. Chem. Soc. 129, 14164 (2007). 35. B. Li et al., J. Am. Chem. Soc. 131, 13222 (2009). 36. Materials and methods are available as supporting material on Science Online. 37. A downfield 29Si NMR chemical shift of 226.7 ppm was observed for (Mes)3Si+ (where Mes is 2,4,6-trimethylphenyl) in the solid state (42). 38. V. Bonačić-Koutecký, J. Koutecký, J. Michl, Angew. Chem. Int. Ed. Engl. 26, 170 (1987). 39. Z. Chen, C. S. Wannere, C. Corminboeuf, R. Puchta, P. R. Schleyer, Chem. Rev. 105, 3842 (2005). 40. W. Kutzelnigg, Angew. Chem. Int. Ed. Engl. 23, 272 (1984). 41. R. C. Fischer, P. P. Power, Chem. Rev. 110, 3877 (2010). 42. K.-C. Kim et al., Science 297, 825 (2002). 43. Supported by a Grant-in-Aid for Specially Promoted Research (19002008) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Numerical calculations were partly performed at the Supercomputer Laboratory, Institute for Chemical Research, Kyoto University. We thank Y. Hongo and T. Nakamura (RIKEN) for their kind help with the mass spectrometry and solid-state NMR spectroscopy. We also thank J. Michl, R. Noyori, A. Sekiguchi, and R. West for their valuable discussions. Metric data for the solid-state structure of 1 are available from the Cambridge Crystallographic Data Centre under reference number CCDC-786049.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1306/DC1 Materials and Methods Figs. S1 to S20 Tables S1 and S2 References 2 November 2010; accepted 24 January 2011 10.1126/science.1199906

of an activator) (1). The first experimental demonstration of a Turing pattern (2, 3) occurred in the chlorite–iodide–malonic acid (CIMA) reaction (4), in which complexation of the activator species (iodine) with the indicator (starch) generated the necessary separation between the effective diffusion constants of activator and inhibitor. Recent advances in molecular biology have made it possible to establish the Turing mechanism in several biological systems (5–7), but many important aspects of the reaction-diffusion processes in such systems remain to be clarified (8, 9). One key and still largely unexplored aspect affecting Turing-pattern formation is dimensionality; that is, how the number of spatial dimensions influences the pattern behavior. Actual patterns can never be truly planar [two-dimensional (2D)] at a molecular level, as is often assumed. This

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Department of Chemistry, MS 015, Brandeis University, Waltham, MA 02454, USA. *To whom correspondence should be addressed. E-mail: [email protected]

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and LUMO of 1 essentially involve mutually outof-phase orbitals in the plane with the large coefficients localized on the diagonal silicon atoms. The LUMO involves a p*-like interaction between the p-orbitals on the planar Si1 and Si3 atoms, whereas in the HOMO the out-of-phase orbitals on the pyramidalized Si2 and Si4 atoms lean in one direction to provide a degree of inphase interaction between the back lobes, resulting in overall stabilization. The absorption peak at 448 nm in the ultraviolet-visible (UV-vis) spectrum of 1 is assignable to a mixture of two allowed p-p* transitions (HOMO→LUMO+1 and HOMO-1→LUMO) on the basis of timedependent DFT (TD-DFT) calculations (fig. S18). The HOMO→LUMO transition is calculated to give a very weak absorption peak at 1306 nm because of a forbidden transition. Although Si=Si double bonds ordinarily display an essentially nonpolar character between the constituent atoms, as is the case with C=C double bonds, the Si=Si double bonds involved in the silicon CBD analog 1 are polarized as a result of a polar J-T distortion to counteract antiaromaticity. The polar J-T distortion is induced by alternate electronegativity differences at the silicon centers, which is realized in practice by the pyramidalization of the Si2 and Si4 atoms: The HOMO lobes there exhibit a higher s-character and thus make these atoms more electronegative than the LUMO lobes on the Si1 and Si3 atoms, which are of nearly p-character. Thus in contrast to carbon-based CBD, which is stabilized through bond-length alternation, the tetrasilacyclobutadiene is stabilized as a planar rhombic charge-separated structure because of the weaker Si=Si p-bond, the lower capacity for s-p hybridization at silicon (40, 41), and the rather flexible silicon s-bond framework relative to carbon.

References and Notes 1. L. Watts, J. D. Fitzpatrick, R. Pettit, J. Am. Chem. Soc. 87, 3253 (1965). 2. T. Bally, S. Masamune, Tetrahedron 36, 343 (1980). 3. G. Maier, Angew. Chem. Int. Ed. Engl. 27, 309 (1988). 4. T. Bally, Angew. Chem. Int. Ed. 45, 6616 (2006). 5. C. Y. Lin, A. Krantz, J. Chem. Soc. Chem. Commun. 1972, 1111 (1972). 6. D. J. Cram, M. E. Tanner, R. Thomas, Angew. Chem. Int. Ed. Engl. 30, 1024 (1991). 7. A. A. Deniz, K. S. Peters, G. J. Snyder, Science 286, 1119 (1999). 8. A. Fattahi, L. Lis, Z. Tian, S. R. Kass, Angew. Chem. Int. Ed. 45, 4984 (2006). 9. A. M. Orendt et al., J. Am. Chem. Soc. 110, 2648 (1988). 10. B. R. Arnold, J. G. Radziszewski, A. Campion, S. S. Perry, J. Michl, J. Am. Chem. Soc. 113, 692 (1991). 11. A. Balková, R. J. Bartlett, J. Chem. Phys. 101, 8972 (1994). 12. Push-pull perturbed CBDs provide the localized MOs (38). 13. H. Kimling, A. Krebs, Angew. Chem. Int. Ed. Engl. 11, 932 (1972). 14. H. Irngartinger, H. Rodewald, Angew. Chem. Int. Ed. Engl. 13, 740 (1974). 15. L. T. J. Delbaere, M. N. G. James, N. Nakamura, S. Masamune, J. Am. Chem. Soc. 97, 1973 (1975). 16. G. Maier, S. Pfriem, U. Schäfer, R. Matusch, Angew. Chem. Int. Ed. Engl. 17, 520 (1978). 17. E. J. Petersson et al., J. Am. Chem. Soc. 119, 11122 (1997). 18. J. I. Wu, F. A. Evangelista, P. R. Schleyer, Org. Lett. 12, 768 (2010). 19. Y.-M. Legrand, A. van der Lee, M. Barboiu, Science 329, 299 (2010). 20. K. Wakita, N. Tokitoh, R. Okazaki, S. Nagase, Angew. Chem. Int. Ed. 39, 634 (2000). 21. S. Ishida, T. Iwamoto, C. Kabuto, M. Kira, Nature 421, 725 (2003). 22. M. Weidenbruch, S. Willms, W. Saak, G. Henkel, Angew. Chem. Int. Ed. Engl. 36, 2503 (1997). 23. A. Sekiguchi, R. Kinjo, M. Ichinohe, Science 305, 1755 (2004). 24. Y. Wang et al., Science 321, 1069 (2008). 25. A. G. Brook, F. Abdesaken, B. Gutekunst, G. Gutekunst, R. K. Kallury, J. Chem. Soc. Chem. Commun. 1981, 191 (1981). 26. R. West, M. J. Fink, J. Michl, Science 214, 1343 (1981). 27. K. Abersfelder, A. J. P. White, H. S. Rzepa, D. Scheschkewitz, Science 327, 564 (2010). 28. B. F. Yates, D. A. Clabo Jr., H. F. Schaefer III, Chem. Phys. Lett. 143, 421 (1988).

Tomography of Reaction-Diffusion Microemulsions Reveals Three-Dimensional Turing Patterns Tamás Bánsági Jr., Vladimir K. Vanag, Irving R. Epstein* Spatially periodic, temporally stationary patterns that emerge from instability of a homogeneous steady state were proposed by Alan Turing in 1952 as a mechanism for morphogenesis in living systems and have attracted increasing attention in biology, chemistry, and physics. Patterns found to date have been confined to one or two spatial dimensions. We used tomography to study the Belousov-Zhabotinsky reaction in a microemulsion in which the polar reactants are confined to aqueous nanodroplets much smaller than the scale of the stationary patterns. We demonstrate the existence of Turing patterns that can exist only in three dimensions, including curved surfaces, hexagonally packed cylinders, spots, and labyrinthine and lamellar patterns. n his seminal paper, Alan Turing (1) aimed to provide a mechanism for self-regulated pattern formation in biology by showing that sets of reaction-diffusion equations with appropriate kinetics and diffusion coefficients could

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spontaneously evolve to spatially periodic structures. The main requirements for Turing-pattern formation in a reaction-diffusion medium are longrange reaction inhibition (fast diffusion of an inhibitor) and short-range activation (slow diffusion

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29. This C2h structure was also found in B3LYP/6-31G(d,p) level calculations (36). 30. N. Wiberg, C. M. M. Finger, K. Polborn, Angew. Chem. Int. Ed. Engl. 32, 1054 (1993). 31. M. Ichinohe, M. Toyoshima, R. Kinjo, A. Sekiguchi, J. Am. Chem. Soc. 125, 13328 (2003). 32. K. Takanashi, V. Ya. Lee, T. Matsuno, M. Ichinohe, A. Sekiguchi, J. Am. Chem. Soc. 127, 5768 (2005). 33. V. Ya. Lee, K. Takanashi, T. Matsuno, M. Ichinohe, A. Sekiguchi, J. Am. Chem. Soc. 126, 4758 (2004). 34. A. Fukazawa, Y. Li, S. Yamaguchi, H. Tsuji, K. Tamao, J. Am. Chem. Soc. 129, 14164 (2007). 35. B. Li et al., J. Am. Chem. Soc. 131, 13222 (2009). 36. Materials and methods are available as supporting material on Science Online. 37. A downfield 29Si NMR chemical shift of 226.7 ppm was observed for (Mes)3Si+ (where Mes is 2,4,6-trimethylphenyl) in the solid state (42). 38. V. Bonačić-Koutecký, J. Koutecký, J. Michl, Angew. Chem. Int. Ed. Engl. 26, 170 (1987). 39. Z. Chen, C. S. Wannere, C. Corminboeuf, R. Puchta, P. R. Schleyer, Chem. Rev. 105, 3842 (2005). 40. W. Kutzelnigg, Angew. Chem. Int. Ed. Engl. 23, 272 (1984). 41. R. C. Fischer, P. P. Power, Chem. Rev. 110, 3877 (2010). 42. K.-C. Kim et al., Science 297, 825 (2002). 43. Supported by a Grant-in-Aid for Specially Promoted Research (19002008) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Numerical calculations were partly performed at the Supercomputer Laboratory, Institute for Chemical Research, Kyoto University. We thank Y. Hongo and T. Nakamura (RIKEN) for their kind help with the mass spectrometry and solid-state NMR spectroscopy. We also thank J. Michl, R. Noyori, A. Sekiguchi, and R. West for their valuable discussions. Metric data for the solid-state structure of 1 are available from the Cambridge Crystallographic Data Centre under reference number CCDC-786049.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1306/DC1 Materials and Methods Figs. S1 to S20 Tables S1 and S2 References 2 November 2010; accepted 24 January 2011 10.1126/science.1199906

of an activator) (1). The first experimental demonstration of a Turing pattern (2, 3) occurred in the chlorite–iodide–malonic acid (CIMA) reaction (4), in which complexation of the activator species (iodine) with the indicator (starch) generated the necessary separation between the effective diffusion constants of activator and inhibitor. Recent advances in molecular biology have made it possible to establish the Turing mechanism in several biological systems (5–7), but many important aspects of the reaction-diffusion processes in such systems remain to be clarified (8, 9). One key and still largely unexplored aspect affecting Turing-pattern formation is dimensionality; that is, how the number of spatial dimensions influences the pattern behavior. Actual patterns can never be truly planar [two-dimensional (2D)] at a molecular level, as is often assumed. This

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Department of Chemistry, MS 015, Brandeis University, Waltham, MA 02454, USA. *To whom correspondence should be addressed. E-mail: [email protected]

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longer than the characteristic time for mass exchange via fission-fusion of water droplets. We used BZ reactant concentrations and microemulsion parameters [the water-surfactant molar ratio w = [H2O]/[AOT] (where [H2O] and [AOT] are the concentrations of H2O and AOT, respectively) and ϕd, the volume fraction of droplets] that give rise to Turing patterns in 2D. To improve visualization, we replaced the octane used as the oil phase in earlier experiments with cyclooctane, which requires a small readjustment of the concentrations to remain in the Turing domain. Cyclooctane has nearly the same index

of refraction as quartz, which minimizes refraction at the curved interfaces between the capillary and its surroundings. For the 2D experiments, we used a protocol described earlier (22), in which we sandwiched the BZ-AOT emulsion between two optical windows separated by an 80-mm-thick polytetrafluoroethylene (Teflon, DuPont, Wilmington, Delaware) gasket. Patterns were observed via a microscope equipped with a charge-coupled–device camera. Stationary Turing structures emerged after initial transient bulk oscillations and/or waves and persisted for 1 hour or more. At longer times, we observed changes (for example, from labyrinthine

Fig. 1. Snapshots of stationary 2D spots (A) and stripes (B) in a thin layer and 2D images of the corresponding 3D structures (A→B, C; B→E to G) in a capillary. Initial concentrations: [malonic acid] = 0.25 M; [H2SO4] = 0.20 M (A, B, D to G), 0.15 M (C); [NaBrO3] = 0.16 M (A and B), 0.20 M (D to G), 0.21 M (C); [ferroin] = 10 mM. w = 13.9; ϕd = 0.348. Inner diameter of capillaries: (B and C) 0.6 mm; (E to G) 0.3 mm. Bright regions correspond to high concentrations of the oxidized form of the catalyst. Dark bands at the capillary walls are due to a slight mismatch in refractive index. Scale bar, 1 mm. Fig. 2. Tomographically reconstructed concentration fields for 3D Turing patterns. Spots (A), hexagonal close-packing (B), horizontal cross sections (C) taken in the vertical direction through data array in (B), labyrinthine pattern (D), tube (E), halfpipe (F), lamellar pattern (G), and concentric hemispherical lamellae (H). Experimental parameters for (A), (B), and (D) to (F) correspond to (B), (C), and (E) to (G), respectively, in Fig. 1, for (G) and (H): [malonic acid]0 = 0.30 M; [H2SO4]0 = 0.18 M, [NaBrO3]0 = 0.23 M, [ferroin] = 5 mM. The inner diameter of a capillary is 0.6 mm for (A) to (D), (G), and (H) and 0.3 mm for (E) and (F). A short segment at the bottom of the front half of pattern (E) has been removed to reveal the inner structure.

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3D-to-2D simplification is adequate only when the characteristic Turing wavelength (lT) of the pattern is sufficiently (at least two times) greater than the thickness of the medium; for example, in the case of skin patterns (9, 10). If the linear size of the system in all three dimensions exceeds lT, then 3D Turing patterns may differ from the wellstudied 2D patterns. The issue of possible differences between 2D and 3D patterns was raised for the first time in connection with some of the earliest experiments on 2D Turing patterns (2, 3). A number of theoretical works predict a variety of possible shapes for 3D patterns (11–13)—including bodycentered cubic (bcc) arrays of spots, hexagonally close-packed (hcp) cylinders), and lamellae— that are not simply extensions of 2D patterns into 3D. However, experiments in continuously fed unstirred reactors (CFURs) on the CIMA system or others (14) that are able to generate Turing patterns have been effectively confined to 2D patterns in a thin layer or to 3D patterns in a thick layer with large concentration gradients (i.e., an inhomogeneous system) (15). Moreover, the CFUR configuration does not lend itself to the analysis of 3D patterns (16) by tomographic methods, which have successfully been applied to the study of 3D scroll waves (17–19), a straightforward extension of 2D spiral waves into 3D media. Visualization of 3D patterns is a challenging task; simple visual observation or microscopy yields a 2D projection, which will obscure the structure in all but the simplest patterns. To generate 3D Turing patterns in a homogeneous medium while simultaneously overcoming the technical problems that hinder their observation, we explore the Belousov-Zhabotinsky (BZ) reaction incorporated into a water-in-oil aerosol OT (AOT) microemulsion, the so-called BZ-AOT system (20, 21). This medium has already been shown to give rise to 2D Turing patterns that are stable for 1 to 2 hours without external feeding (22). The ~10-nm–diameter aqueous droplets of the microemulsion are surrounded by a monolayer of surfactant, sodium bis(2-ethylhexyl)sulfosuccinate and are dispersed in a continuous oil phase. The polar BZ reagents (malonic acid, sodium bromate, sulfuric acid, and ferroin) reside in the water droplets. When the reaction begins, apolar intermediates (notably the inhibitor species, Br2) are produced in the droplets and diffuse through the oil phase. The diffusion of Br2 occurs at rates up to two orders of magnitude greater than that of the polar species, including the BZ activator HBrO2, which diffuse together with entire water droplets. Note that the microheterogeneous BZ-AOT system can be considered as homogeneous on the chemically relevant length scale, because the characteristic wavelength of the patterns observed (lT = 0.1 to 0.3 mm) is several orders of magnitude greater than the size and spacing of the of water droplets (~10 nm). The characteristic times of the key chemical processes in the BZ reaction, typically on the order of seconds, are also much

or lamellar to spot patterns) and, ultimately, the disappearance of structured patterns. We conducted the 3D experiments in a cylindrical quartz capillary with an inner diameter of 0.3 to 0.6 mm (i.e., several lT). The capillary was filled with the BZ-AOT mixture, sealed at the bottom, and vertically submerged in a rectangular cuvette containing cyclooctane, the oil used in the BZ-AOT medium. Visualization of the 3D Turing structures was then implemented by optical tomography performed on a series of absorption images taken within a full rotation of the capillary. The samples were typically rotated at 0.25 revolutions per second with a frame capture rate of 20 frames per second. Concentration fields (oxidized form of the catalyst) were obtained by inverse Radon transformation of the filtered image data (23). The 2D and 3D stationary structures were moderately stable to vibration and shear stress caused by starting the rotation motor or removing the sample from the holder. More details are available in the supporting online material. We obtained 2D and 3D patterns in experiments using microemulsions with the same composition. In 2D, stationary Turing structures generally consist of imperfect hexagonal arrays of spots or labyrinthine arrangements of stripes (Fig. 1, A and D). In 3D, we observed a variety of new patterns (Fig. 1, B, C, and E to G). When the BZAOT system was in the central portion of the 2D spot region in the space of initial concentrations (Fig. 1A), we also observed spots in 3D (Fig. 1B). A 3D reconstruction of the patterns seen in Fig. 1B is presented in Fig. 2A. As we changed the initial concentrations to move closer to the boundary between the 2D spot and stripe regions, we observed long, hexagonally packed cylinders in 3D; i.e., an hcp pattern (pattern in

Fig. 1C and its 3D reconstruction in Fig. 2, B and C). In the parameter regime of 2D labyrinthine stripes (as in Fig. 1D), the 3D configuration yielded labyrinthine patterns (Fig. 1E and Fig. 2D), closed tubular surfaces (Fig. 1F and Fig. 2E), and half-pipe–shaped structures (Fig. 1G and Fig. 2F). Note that such patterns as hcp (Fig. 2B) and stationary surfaces (Fig. 2, E to H) can exist only in 3D. Curved surfaces appear to be favored by narrow capillaries, where the influence of the lateral boundary is strong. The labyrinthine structure in Fig. 2D is interconnected, a feature clearly visible in a capillary with larger diameter. The patterns in Fig. 2, D to F, were obtained at the same average initial concentrations, but at slightly different local initial conditions, and these distinct patterns can coexist in the same capillary but in different regions along its length. Differences in initial conditions resulted from the waves initially appearing in the capillary before the Turing patterns arose. These waves generated an inhomogeneous concentration distribution from which the stationary Turing patterns gradually emerged. Although these patterns are stationary for a time that corresponds to dozens of cycles of the BZ-AOT system under oscillatory conditions, our patterns were necessarily transient because the system was closed. In an effort to better understand our experimental results and to test whether these patterns could indeed be stable, we performed 3D simulations using a two-variable (x, z) Oregonator-like model of the BZ-AOT system that is very good at reproducing many of the 2D structures observed in the BZ-AOT system (24). In Fig. 3, A and B, we present 3D spots and hexagonal close-packing obtained at

Fig. 3. Stationary structures in numerical simulations. Spots (A), hexagonal close-packing (B), labyrinthine (C), tube (D), half-pipe (E), and lamellar (F) emerging from asymmetric [(A), (B), and (E)], symmetric [(D) and (F)], and random (C) initial conditions in a cylindrical domain. Numerical results are obtained from the model: dx/dt = (1/e)[fz(q – x)/(q + x) + x(1 – mz)/(e1 + 1 – mz) – x2] + ∇2x; dz/dt = x(1 – mz)/(e1 + 1 – mz) – z + dz∇2z, where x and z denote the activator, HBrO2 and the oxidized form of the catalyst, respectively; dz is the ratio of diffusion coefficients Dz/Dx; and t is the dimensionless time. Parameters (dimensionless units): q = 0.0002; m = 0.0007; e1 = 0.02; e = 2.2; f = (A) 1.1, (B) 0.93, [(C) to (F)] 0.88; and dz = 10. Size of domains (dimensionless): diameter = 20 [(A) to (C) and (F)]; 14 [(D) and (E)]; height = 40.

model parameters that give spots in 2D. As in the experiments, hexagonal close-packing (Fig. 3B) emerged when the system was near the boundary in parameter space between the 2D spotand stripe regions; patterns corresponding to the experimentally observed patterns in Fig. 2, D to F, are shown in Fig. 3, C to E. In agreement with the experiments, these patterns were obtained with model parameters (the same for all patterns) at which stripes emerged in 2D. The simulations differed only in the initial distribution of x: random for labyrinthine (Fig. 3C), radially symmetric Gaussian in the horizontal plane for tubes (Fig. 3D), Gaussian symmetric with respect to a vertical plane through the cylinder axis for lamellae (Fig. 3F), and asymmetric (offcenter radially symmetric Gaussian) for the halfpipe (Fig. 3E). Our results demonstrate the potential importance of dimensionality and initial conditions for pattern formation in nature. Pattern selection is circumscribed by the number of available spatial dimensions, and the characteristics of a stationary pattern in a given region of the system are determined by the local conditions present at the early stages of pattern formation. The possibilities for spatial multistability are considerably greater in three dimensions than in two, and pattern selection via gradients established during early stages of development seems an eminently plausible scenario in living systems. Theorists predict many other 3D Turing patterns, such as bcc, face-centered cubic, or perforated lamellae (25). Experimental detection of such patterns— which depend, among other factors, on whether the Turing instability is sub- or supercritical— may be expected. Biological Turing patterns in 3D have not yet been reported, even though there has been considerable effort to find Turing-driven morphological phenomena. With the growing interest in Turing’s theory as a model for morphogenesis (1) and emerging examples of 2D Turing patterns in living systems—for example, disposition of feather buds in chicks (6) and hair follicles in mice (7)—the near future seems likely to unveil evidence of 3D self-regulated patterns in living systems. Candidates include skeletal pattern formation in developing chick limbs (26) and the head regeneration process in Hydra (27), a species that inspired Turing in developing his groundbreaking theory of morphogenesis. In addition, 3D Turing patterns [especially those in the subcritical regime, where 3D localized patterns can exist (28)] may be useful vehicles for information storage (29).

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References and Notes 1. A. M. Turing, Philos. Trans. R. Soc. London. Ser. B 237, 37 (1952). 2. V. Castets, E. Dulos, J. Boissonade, P. De Kepper, Phys. Rev. Lett. 64, 2953 (1990). 3. Q. Ouyang, H. L. Swinney, Nature 352, 610 (1991). 4. P. De Kepper, I. R. Epstein, K. Kustin, M. Orbán, J. Phys. Chem. 86, 170 (1982). 5. T. X. Jiang, H. S. Jung, R. B. Widelitz, C. M. Chuong, Development 126, 4997 (1999).

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REPORTS 17. A. T. Winfree, S. Caudle, G. Chen, P. McGuire, Z. Szilagyi, Chaos 6, 617 (1996). 18. U. Storb, C. R. Neto, M. Bär, S. C. Müller, Phys. Chem. Chem. Phys. 5, 2344 (2003). 19. T. Bánsági Jr., O. Steinbock, Chaos 18, 026102 (2008). 20. V. K. Vanag, D. V. Boulanov, J. Phys. Chem. 98, 1449 (1994). 21. V. K. Vanag, I. R. Epstein, Science 294, 835 (2001). 22. V. K. Vanag, I. R. Epstein, Phys. Rev. Lett. 87, 228301 (2001). 23. A. S. Kak, M. Slaney, Principles of Computerized Tomographic Imaging (IEEE Press, New York, 1988). 24. A. Kaminaga, V. K. Vanag, I. R. Epstein, J. Chem. Phys. 122, 174706 (2005). 25. H. Shoji, K. Yamada, D. Ueyama, T. Ohta, Phys. Rev. E 75, 046212 (2007). 26. S. A. Newman, H. L. Frisch, Science 205, 662 (1979). 27. R. Augustin et al., Dev. Biol. 296, 62 (2006). 28. M. Leda, V. K. Vanag, I. R. Epstein, Phys. Rev. E 80, 066204 (2009).

Creating Favorable Geometries for Directing Organic Photoreactions in Alkanethiolate Monolayers Moonhee Kim,1,2 J. Nathan Hohman,1,2 Yang Cao,1 Kendall N. Houk,1* Hong Ma,3 Alex K.-Y. Jen,3* Paul S. Weiss1,2,4* The products of photoreactions of conjugated organic molecules may be allowed by selection rules but not observed in solution reactions because of unfavorable reaction geometries. We have used defect sites in self-assembled alkanethiolate monolayers on gold surfaces to direct geometrically unfavorable photochemical reactions between individual organic molecules. High conductivity and stochastic switching of anthracene-terminated phenylethynylthiolates within alkanethiolate monolayers, as well as in situ photochemical transformations, have been observed and distinguished with the scanning tunneling microscope (STM). Ultraviolet light absorbed during imaging increases the apparent heights of excited molecules in STM images, a direct manifestation of probing electronically excited states. egioselectivity of organic reactions is critically important for organic synthesis, and a wide variety of strategies are used to restrict the geometry of approaching reactants in solution to favor the preferred products (for example, bulky ligands on metal complex catalysts to direct incoming reactants). Geometric control can also be exerted in solution by encapsulating reactants in host-guest complexes. In the context of surface reactions, the formation of adsorbed monolayers can restrict the relative motion of molecules and may afford a route to regioselective chemistry. For example, n-alkanethiolate self-assembled monolayers (SAMs) on gold surfaces have been used to prepare well-defined matrices for studying molecules or nanoparticles in locally con-

R

1 California NanoSystems Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA. 2Department of Chemistry and Department of Physics, 104 Davey Laboratory, The Pennsylvania State University, University Park, PA 16802, USA. 3Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA. 4Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA.

*To whom correspondence should be addressed. E-mail: [email protected] (K.N.H.); [email protected] (A.K.-Y.J.); [email protected] (P.S.W.)

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trolled environments (1–3). The stability, simple fabrication, and fundamental understanding of alkanethiolate SAMs provide an excellent system to control the placement, orientation, and reactions of molecules. Here, we detail our strategy to control molecular placement and environment, enabling selection of a photocycloaddition reaction that is geometrically unfavorable in solution yet allowed by selection rules, the [4+4] between

29. P. Coullet, C. Riera, C. Tresser, Chaos 14, 193 (2004). 30. We thank M. Hauser for helpful discussions, J. Carballido-Landeira for preliminary experiments, and F. Mello for assistance in constructing the tomography apparatus. This work was funded by the NSF under grants CHE-0615507, CHE-0526866, and NSF Materials Research Science and Engineering Center grant DMR-0820492 and a U.S.-Hungarian Cooperative Grant. I.R.E. thanks the Radcliffe Institute for a fellowship.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200815/DC1 Materials and Methods Figs. S1 and S2 23 November 2010; accepted 26 January 2011 Published online 10 February 2011; 10.1126/science.1200815

adjacent 9-(4-mercapto-phenylethynyl)anthracene (MPEA, Fig. 1) molecules on a Au surface. A thiol form of 9-phenylethynylanthracene (PEA, Fig. 2) is readily inserted singly, in pairs, or in groups into the defects of preformed alkanethiolate SAMs (4–6). The rigid spacer, phenylethynyl backbone was buried with Au-S surface attachment, leaving anthracene exposed at the surface of the alkanethiolate monolayer. Precise control over the molecular assembly enables us to trap molecules in an environment where they are constrained to undergo a regioselective [4+4] cycloaddition under ultraviolet (UV) illumination, and imaging with a scanning tunneling microscope (STM) enables us to screen for molecules in welldefined environments. The use of the STM to track reactions between upright, photoreactive molecules inserted in SAMs in situ is complicated by the dynamics of the individual molecules. Inserted molecules display frequent changes in apparent height, commonly referred to as stochastic switching (7, 8), whereas imaging under illumination can cause electronic excitation or reactions in the molecules (9–12). The aromaticity and photoreactivity of anthracene make it an important candidate material for organic electronics, optoelectronics, and surface photochemistry (13–16). Detailed studies of 2-anthracenethiolate (Fig. 1, shown as the thiol) SAMs on gold emphasize the importance of struc-

Fig. 1. Structures of 2-anthracenethiol and 10-thiodecyl 2-anthryl ether are compared to structures of 9-(4-mercaptophenylethynyl)anthracene (MPEA, 1), 9-phenylethynylanthracene disulfide (2), and oligo(phenylene-ethynylene)thiol (OPE, 3).

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6. H. S. Jung et al., Dev. Biol. 196, 11 (1998). 7. S. Sick, S. Reinker, J. Timmer, T. Schlake, Science 314, 1447 (2006); 10.1126/science.1130088. 8. P. K. Maini, R. E. Baker, C.-M. Chuong, Science 314, 1397 (2006). 9. S. Kondo, T. Miura, Science 329, 1616 (2010). 10. R. T. Liu, S. S. Liaw, P. K. Maini, Phys. Rev. E 74, 011914 (2006). 11. T. K. Callahan, Physica D 188, 65 (2004). 12. A. De Wit, P. Borckmans, G. Dewel, Proc. Natl. Acad. Sci. U.S.A. 94, 12765 (1997). 13. T. Leppänen, M. Karttunen, K. Kaski, Int. J. Mod. Phys. B 17, 5541 (2003). 14. J. Horváth, I. Szalai, P. De Kepper, Science 324, 772 (2009). 15. E. Dulos, P. Davies, B. Rudovics, P. De Kepper, Physica D 98, 53 (1996). 16. P. K. Moore, W. Horsthemke, Chaos 19, 043116 (2009).

REPORTS 17. A. T. Winfree, S. Caudle, G. Chen, P. McGuire, Z. Szilagyi, Chaos 6, 617 (1996). 18. U. Storb, C. R. Neto, M. Bär, S. C. Müller, Phys. Chem. Chem. Phys. 5, 2344 (2003). 19. T. Bánsági Jr., O. Steinbock, Chaos 18, 026102 (2008). 20. V. K. Vanag, D. V. Boulanov, J. Phys. Chem. 98, 1449 (1994). 21. V. K. Vanag, I. R. Epstein, Science 294, 835 (2001). 22. V. K. Vanag, I. R. Epstein, Phys. Rev. Lett. 87, 228301 (2001). 23. A. S. Kak, M. Slaney, Principles of Computerized Tomographic Imaging (IEEE Press, New York, 1988). 24. A. Kaminaga, V. K. Vanag, I. R. Epstein, J. Chem. Phys. 122, 174706 (2005). 25. H. Shoji, K. Yamada, D. Ueyama, T. Ohta, Phys. Rev. E 75, 046212 (2007). 26. S. A. Newman, H. L. Frisch, Science 205, 662 (1979). 27. R. Augustin et al., Dev. Biol. 296, 62 (2006). 28. M. Leda, V. K. Vanag, I. R. Epstein, Phys. Rev. E 80, 066204 (2009).

Creating Favorable Geometries for Directing Organic Photoreactions in Alkanethiolate Monolayers Moonhee Kim,1,2 J. Nathan Hohman,1,2 Yang Cao,1 Kendall N. Houk,1* Hong Ma,3 Alex K.-Y. Jen,3* Paul S. Weiss1,2,4* The products of photoreactions of conjugated organic molecules may be allowed by selection rules but not observed in solution reactions because of unfavorable reaction geometries. We have used defect sites in self-assembled alkanethiolate monolayers on gold surfaces to direct geometrically unfavorable photochemical reactions between individual organic molecules. High conductivity and stochastic switching of anthracene-terminated phenylethynylthiolates within alkanethiolate monolayers, as well as in situ photochemical transformations, have been observed and distinguished with the scanning tunneling microscope (STM). Ultraviolet light absorbed during imaging increases the apparent heights of excited molecules in STM images, a direct manifestation of probing electronically excited states. egioselectivity of organic reactions is critically important for organic synthesis, and a wide variety of strategies are used to restrict the geometry of approaching reactants in solution to favor the preferred products (for example, bulky ligands on metal complex catalysts to direct incoming reactants). Geometric control can also be exerted in solution by encapsulating reactants in host-guest complexes. In the context of surface reactions, the formation of adsorbed monolayers can restrict the relative motion of molecules and may afford a route to regioselective chemistry. For example, n-alkanethiolate self-assembled monolayers (SAMs) on gold surfaces have been used to prepare well-defined matrices for studying molecules or nanoparticles in locally con-

R

1 California NanoSystems Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA. 2Department of Chemistry and Department of Physics, 104 Davey Laboratory, The Pennsylvania State University, University Park, PA 16802, USA. 3Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA. 4Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA.

*To whom correspondence should be addressed. E-mail: [email protected] (K.N.H.); [email protected] (A.K.-Y.J.); [email protected] (P.S.W.)

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trolled environments (1–3). The stability, simple fabrication, and fundamental understanding of alkanethiolate SAMs provide an excellent system to control the placement, orientation, and reactions of molecules. Here, we detail our strategy to control molecular placement and environment, enabling selection of a photocycloaddition reaction that is geometrically unfavorable in solution yet allowed by selection rules, the [4+4] between

29. P. Coullet, C. Riera, C. Tresser, Chaos 14, 193 (2004). 30. We thank M. Hauser for helpful discussions, J. Carballido-Landeira for preliminary experiments, and F. Mello for assistance in constructing the tomography apparatus. This work was funded by the NSF under grants CHE-0615507, CHE-0526866, and NSF Materials Research Science and Engineering Center grant DMR-0820492 and a U.S.-Hungarian Cooperative Grant. I.R.E. thanks the Radcliffe Institute for a fellowship.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200815/DC1 Materials and Methods Figs. S1 and S2 23 November 2010; accepted 26 January 2011 Published online 10 February 2011; 10.1126/science.1200815

adjacent 9-(4-mercapto-phenylethynyl)anthracene (MPEA, Fig. 1) molecules on a Au surface. A thiol form of 9-phenylethynylanthracene (PEA, Fig. 2) is readily inserted singly, in pairs, or in groups into the defects of preformed alkanethiolate SAMs (4–6). The rigid spacer, phenylethynyl backbone was buried with Au-S surface attachment, leaving anthracene exposed at the surface of the alkanethiolate monolayer. Precise control over the molecular assembly enables us to trap molecules in an environment where they are constrained to undergo a regioselective [4+4] cycloaddition under ultraviolet (UV) illumination, and imaging with a scanning tunneling microscope (STM) enables us to screen for molecules in welldefined environments. The use of the STM to track reactions between upright, photoreactive molecules inserted in SAMs in situ is complicated by the dynamics of the individual molecules. Inserted molecules display frequent changes in apparent height, commonly referred to as stochastic switching (7, 8), whereas imaging under illumination can cause electronic excitation or reactions in the molecules (9–12). The aromaticity and photoreactivity of anthracene make it an important candidate material for organic electronics, optoelectronics, and surface photochemistry (13–16). Detailed studies of 2-anthracenethiolate (Fig. 1, shown as the thiol) SAMs on gold emphasize the importance of struc-

Fig. 1. Structures of 2-anthracenethiol and 10-thiodecyl 2-anthryl ether are compared to structures of 9-(4-mercaptophenylethynyl)anthracene (MPEA, 1), 9-phenylethynylanthracene disulfide (2), and oligo(phenylene-ethynylene)thiol (OPE, 3).

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6. H. S. Jung et al., Dev. Biol. 196, 11 (1998). 7. S. Sick, S. Reinker, J. Timmer, T. Schlake, Science 314, 1447 (2006); 10.1126/science.1130088. 8. P. K. Maini, R. E. Baker, C.-M. Chuong, Science 314, 1397 (2006). 9. S. Kondo, T. Miura, Science 329, 1616 (2010). 10. R. T. Liu, S. S. Liaw, P. K. Maini, Phys. Rev. E 74, 011914 (2006). 11. T. K. Callahan, Physica D 188, 65 (2004). 12. A. De Wit, P. Borckmans, G. Dewel, Proc. Natl. Acad. Sci. U.S.A. 94, 12765 (1997). 13. T. Leppänen, M. Karttunen, K. Kaski, Int. J. Mod. Phys. B 17, 5541 (2003). 14. J. Horváth, I. Szalai, P. De Kepper, Science 324, 772 (2009). 15. E. Dulos, P. Davies, B. Rudovics, P. De Kepper, Physica D 98, 53 (1996). 16. P. K. Moore, W. Horsthemke, Chaos 19, 043116 (2009).

ture, phase, and stability (17). Exposure to UV light induces distinct structural changes by dimerization within the monolayer (18). However, the close proximity of the anthracene moiety to the Au surface and the perpendicular orientation of the anthracene moiety to the substrate limit SAM ordering and stability and trap molecules in unfavorable arrangements for photodimerization. Photolysis on thioalkyl 2-anthryl ether (Fig. 1, shown as the thiol) monolayers has shown reversible photodimerization of adjacent anthracene moieties, as confirmed by ensemble techniques, including infrared spectroscopy, contact angle, and x-ray photoelectron spectroscopy (19). Well-ordered, n-dodecanethiolate (C12) monolayers were assembled on Au{111} substrates by vapor annealing, where substrates were held above 100 ml of 1 mM ethanolic n-dodecanethiol solution at 78°C for 24 hours. These conditions lead to the formation of high-quality, tightly packed SAMs with large, ordered domains (20). The SAM matrix was placed in a solution containing ~1 mM MPEA (1) or the disulfide form of PEA (2, Fig. 1), allowing insertion and exchange at defects. Insertion of the disulfide molecules assured adjacent placement of molecules (21–23). We note that the same strategy can be used with asymmetric disulfides to place two different molecules in proximity by insertion. Images were collected under ambient conditions with our custom-built STM. Samples were imaged continuously at –1 V sample bias voltage and 1 pA tunneling current. The tun-

neling junction was illuminated with low-power 365 nm UV light (~1 mW cm–2), and inserted molecules were monitored in situ for reactions between and excitations of MPEA thiolates (see UV-visible absorption spectra of 1 in fig. S1). In solution, anthracene and its 9-substituted derivatives undergo UV-activated dimerization at the 9 and 10 positions via [4+4] cycloaddition (14, 24, 25). The reaction is reversible by irradiation at 254 nm or by mild heating (14). However, irradiation of PEA in solution triggers a [4+2] Diels-Alder addition reaction between the ethynyl moiety and the central ring of the other anthracene system (Fig. 2) (26). The regiochemistry of this reaction is governed by the more favorable centrosymmetric-oriented complex. Electronic excitation is not limited only to the anthracene chromophore (25, 27). Connecting a phenylethynyl backbone to anthracene substantially increases electron delocalization through the ethynyl group (fig. S3). Thus, photodimerization between one anthracene and one ethynyl moiety are more favorable in solution. To investigate photoreactions between adjacent PEA molecules on surfaces, we designed MPEA (1); a rigid spacer, phenylethynyl backbone introduces separation between the anthracene moiety and the gold. 9-(4-mercapto-phenylethynyl) anthracene was inserted into preformed C12 SAMs (Fig. 3). The calculated length of MPEA is slightly greater than the thickness of C12 molecular layer (28). Because of the steric hindrance between the

Fig. 2. Schematic view of the photoreaction of 9-phenylethynylanthracene (PEA) in solution (26).

bulky anthracene and surrounding methyl groups, upright orientation of MPEA with the anthracene parallel to the substrate is the most stable configuration (29). If two upright MPEA molecules are adjacent on the surface, UV illumination can drive the dimerization of anthracene moieties via [4+4] photocycloaddition rather than the [4+2] reaction favored in solution, as shown schematically in Fig. 3. For the reaction to occur, MPEA molecules must be parallel (30) and sufficiently close for strong p-p stacking interactions (28). This conformation and the limited mobility forced by a compact matrix increase the likelihood of the [4+4] photocycloaddition between two adjacent anthracenes. Long-wave UV irradiation at 365 nm does not cause rapid photooxidation in the host SAMs, although exposure to short-wave UV light (254 nm) rapidly damages the alkanethiolate matrix before the reverse reaction can progress appreciably (31). Inserted MPEA molecules in STM images appear to protrude from their surrounding host matrices as a result of both their higher conductivity and their geometric height differences. Insertion occurs at defects, predominantly in SAM domain boundaries, around substrate vacancy islands, and along substrate step edges (4, 6). The number of inserted molecules is limited by using tightly packed C12 matrices, short insertion times (~5 min), and low insertion solution concentration (~1 mM). Isolated MPEA molecules displayed stochastic switching activity during imaging. Similar molecules are well known to undergo transitions between the more-conductive state (on) and the less-conductive state (off ) in sequential STM imaging. The on-state molecules appear as protrusions, and the off-state molecules appear as depressions relative to the surrounding matrix (or at the same apparent height as the matrix) in STM images. Images were screened for switching activity (5, 7). The observed number of transitions from the on state to the off state and from the off state to the on state were 76 and 65, respectively, from 148 selected images with 1000 Å–by–1000 Å scan areas containing an average of 10 isolated MPEA molecules per imaged area. The limited amount of stochastic switching observed is attributed to the tight SAM matrices used (6, 7, 32). Two typical sequences of conductance switching are highlighted in Fig. 4. One pair of molecules on the left is observed initially (Fig. 4A, yellow box). A second pair of switches is on in

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Fig. 3. Schematic view of the photoreaction of 9-(4-mercaptophenylethynyl)anthracene on a Au{111} surface.

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the subsequent image (Fig. 4B, yellow box). The off-state molecules appear at nearly the same apparent height as surrounding C12 domains. The measured apparent height change between the on and the off conductance states was ~2 Å at these tunneling conditions. Inserted groups of molecules can appear with larger apparent height depending on the number of molecules, which is attributed to intermolecular p-p interactions (33). Then the second pair changed shape in the next image (Fig. 4C, yellow box). This pair was more active than the other in this area. Some molecules do not appear to switch over the experimental time scale (Fig. 4, arrows). Distortion in molecular shape by a sudden contrast change was also detected (Fig. 4B, red box), attributed to molecular switching in the presence of the STM tip. Later, this molecule returned to the on state (Fig. 4C, red box). Stochastic conductance switching occurs spontaneously at moderate sample bias, T1.0 V, and occurs on time scales faster than typical STM image acquisition (8, 34). We anticipated that, as in our earlier work on functionalized oligo(phenylene-ethynylene)thiols (OPEs, 3, Fig. 1), switching is the result of changes in the hybridization of the Au-thiolate bond as the molecules tilt (6, 7, 21). All data recorded are consistent with this observation (35). Inserted MPEA molecules were irradiated with long-wave UV light (~365 nm) for ~4 hours of 1 mW cm–2. In situ time-lapse STM images were collected during irradiation. Heating induced by direct illumination introduces noise and thermal drift, so relatively large scan areas (1500 Å by 1500 Å) were monitored to track the changes in many molecules simultaneously and to ease the required compensation for thermal drift. Figure 5 shows photoreactivity of isolated MPEA molecules at longer illumination time. Measurements were restricted to one atomically flat substrate terrace to maintain a consistent background height. Several sets of STM image frames containing ~20 to 30 MPEA molecules were used to measure apparent height changes by photodimerization of anthracene moieties. Ultraviolet irradiation stimulates molecules to switch to the on state (Fig. 5 arrows) and leads to substantial increases in conductivity; MPEA molecules appear more protruding under illumination relative to the surrounding matrix. The apparent height difference between the isolated MPEA molecules and the surrounding C12 matrix doubles to ~4 Å, compared to the onstate apparent height without illumination. Because topographic STM images represent a convolution of the physical and electronic structures of the molecules (7, 36) and conformation is restricted by the matrix, this change is attributed to a steady-state fraction of molecules in an electronically excited state (fig. S3). Continuous illumination makes molecules appear protruding, despite the short time scale of individual excitation events; the calculated absorption cross section of the anthracene moiety with a molar extinction coefficient of 9700 M−1 cm−1 at 356.25 nm

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is 3.7 × 10−17 cm2 (37). As the illumination time increases, more molecules switch on, although the rate of appearance does not strongly correlate with increased illumination time. Light-induced electronic excitation can be detected with singlemolecule resolution without appreciable quenching by gold, and it may be distinguished from switching events characteristic of such molecules. Irradiation also induces permanent apparent height decreases in groups of molecules (colored boxes in Fig. 5, B and C). We attribute this change to the photodimerization of adjacent anthracene moieties, shown schematically in Fig. 3. This reaction breaks the extended p conjugation network, resulting in a significant decrease in conductivity. By constraining the degrees of freedom in orientation in photodimerized molecules, conductance switching is inhibited. This photodimerization can be reversed by exposure to 254-nm light (or mild heat), but such illumination appreciably damages the surrounding C12 SAM. [The photo-

oxidation of anthracene can be discounted, because it usually occurs in substituted anthracenes at both the 9 and 10 positions (38, 39).] We observe three distinct phenomena on anthracene-terminated phenylethynyl thiolates molecules on Au{111}: stochastic switching, increased conductance for molecules in excited states, and drops in conductance attributed to photochemical reactions. The change in molecular configuration and conformation modulate the conductivity by changing the p conjugation and the electron distributions in the highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels. STM imaging under illumination probes photoinduced conductance. Long illumination (~3 hours) drives molecules to photodimerize between two anthracene moieties in adjacent MPEA molecules, resulting in apparent depressions in STM images. Molecular orientation, packing, and the proximity of other molecules all influence the photoreaction efficiency.

Fig. 4. (A to C) Sequential STM images of inserted MPEA molecules in a C12 SAM matrix. Yellow and red boxes show the apparent height changes of molecules. (D to I) Cropped sequential STM images of molecular switching. The arrows at the bottom of images (A) to (C) highlight molecules that show no substantial conductance changes during imaging. Imaging conditions were sample bias voltage (Vsample) = –1.0 V and tunneling current (Itunnel) = 1.0 pA.

Fig. 5. (A to C) STM images of MPEA molecules inserted into a C12 SAM matrix and then exposed to UV light (~365 nm). Images were collected during irradiation after 24 min (A), 124 min (B), and 220 min (C). Imaging conditions were Vsample = –1.0 V and Itunnel = 1.0 pA. The UV illumination increases the apparent height of photoreactive molecules, and more molecules appear as protrusions at longer irradiation (arrows). Several pairs of molecules show substantial decreases in apparent height, attributed to photodimerization [boxes in (B) and (C)].

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Exercising control over molecular reactivity by confinement has great potential for both understanding and measuring complex chemical reactions (40, 41). Regioselective photoreactions between individual molecules by molecular design and self-assembly with in situ monitoring are powerful demonstrations of chemical control via the local environment (7, 42, 43). The extraordinary resolution of the STM enables monitoring molecular motions and reactions. Controlling the chemical environment and monitoring such selective reactions between individual molecules will be important elements in directing chemistry with this approach. References and Notes 1. R. K. Smith et al., J. Am. Chem. Soc. 128, 9266 (2006). 2. D. I. Gittins, D. Bethell, D. J. Schiffrin, R. J. Nichols, Nature 408, 67 (2000). 3. A. Vaish et al., ACS Chem. Neurosci. 1, 495 (2010). 4. M. T. Cygan et al., J. Am. Chem. Soc. 120, 2721 (1998). 5. A. M. Moore et al., Nano Lett. 5, 2292 (2005). 6. P. S. Weiss, Acc. Chem. Res. 41, 1772 (2008). 7. Z. J. Donhauser et al., Science 292, 2303 (2001). 8. A. M. Moore et al., J. Am. Chem. Soc. 129, 10352 (2007). 9. S. J. van der Molen et al., Nano Lett. 9, 76 (2009). 10. W. R. Browne et al., J. Phys. Chem. C 112, 1183 (2008). 11. S. W. Wu, N. Ogawa, W. Ho, Science 312, 1362 (2006). 12. A. S. Kumar et al., Nano Lett. 8, 1644 (2008).

13. J. E. Anthony, Chem. Rev. 106, 5028 (2006). 14. H. Bouas-Laurent, A. Castellan, J.-P. Desvergne, R. Lapouyade, Chem. Soc. Rev. 29, 43 (2000). 15. D. K. James, J. M. Tour, Chem. Mater. 16, 4423 (2004). 16. J. Reichert et al., Phys. Rev. Lett. 88, 176804 (2002). 17. D. Käfer, G. Witte, P. Cyganik, A. Terfort, C. Wöll, J. Am. Chem. Soc. 128, 1723 (2006). 18. M. H. Zareie, J. Barber, A. M. McDonagh, J. Phys. Chem. B 110, 15951 (2006). 19. M. A. Fox, M. D. Wooten, Langmuir 13, 7099 (1997). 20. Z. J. Donhauser, D. W. Price II, J. M. Tour, P. S. Weiss, J. Am. Chem. Soc. 125, 11462 (2003). 21. A. M. Moore et al., J. Am. Chem. Soc. 128, 1959 (2006). 22. E. Delamarche, B. Michel, H. A. Biebuyck, C. Gerber, Adv. Mater. 8, 719 (1996). 23. T. Ishida et al., Langmuir 13, 3261 (1997). 24. H. D. Becker, Chem. Rev. 93, 145 (1993). 25. T. Wolff, N. Müller, G. von Bünau, J. Photochem. 22, 61 (1983). 26. H. D. Becker, K. Andersson, J. Photochem. 26, 75 (1984). 27. D. R. Maulding, B. G. Roberts, J. Org. Chem. 34, 1734 (1969). 28. M. H. Zareie, H. Ma, B. W. Reed, A. K. Y. Jen, M. Sarikaya, Nano Lett. 3, 139 (2003). 29. R. F. Dou et al., Langmuir 22, 3049 (2006). 30. C. Gonzalez, E. C. Lim, Chem. Phys. Lett. 322, 382 (2000). 31. N. J. Brewer, S. Janusz, K. Critchley, S. D. Evans, G. J. Leggett, J. Phys. Chem. B 109, 11247 (2005). 32. P. A. Lewis et al., J. Am. Chem. Soc. 126, 12214 (2004). 33. J. U. Nielsen, M. J. Esplandiu, D. M. Kolb, Langmuir 17, 3454 (2001).

A Circadian Rhythm Orchestrated by Histone Deacetylase 3 Controls Hepatic Lipid Metabolism Dan Feng,1* Tao Liu,2* Zheng Sun,1 Anne Bugge,1 Shannon E. Mullican,1 Theresa Alenghat,1 X. Shirley Liu,2 Mitchell A. Lazar1† Disruption of the circadian clock exacerbates metabolic diseases, including obesity and diabetes. We show that histone deacetylase 3 (HDAC3) recruitment to the genome displays a circadian rhythm in mouse liver. Histone acetylation is inversely related to HDAC3 binding, and this rhythm is lost when HDAC3 is absent. Although amounts of HDAC3 are constant, its genomic recruitment in liver corresponds to the expression pattern of the circadian nuclear receptor Rev-erba. Rev-erba colocalizes with HDAC3 near genes regulating lipid metabolism, and deletion of HDAC3 or Rev-erba in mouse liver causes hepatic steatosis. Thus, genomic recruitment of HDAC3 by Rev-erba directs a circadian rhythm of histone acetylation and gene expression required for normal hepatic lipid homeostasis. n mammals, metabolic processes in peripheral organs display robust circadian rhythms, coordinated with the daily cycles of light and nutrient availability (1, 2). Circadian misalignment causes metabolic dysfunction, and people engaged in night-shift work suffer from higher incidences of obesity, diabetes, and metabolic syndrome (3–5). The molecular basis of this is unknown, but genetic disruption of circadian clock components in mice leads to altered glucose and lipid metabolism (6–10). Gene expression profiles in multiple metabolic organs have revealed a circadian control of the

I

transcriptome, which might be mediated by regulation of histone acetylation (11–13) that alters the structure of the epigenome. Regulation of histone acetylation is complex, involving multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) (14). Histone deacetylase 3 (HDAC3) functions in the regulation of circadian rhythm and glucose metabolism (15). We report diurnal recruitment of HDAC3 to the mouse liver genome detected by chromatin immunoprecipitation with an HDAC3-specific antibody (fig. S1) and massively parallel DNA sequencing (ChIP-seq).

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34. V. Iancu, S. W. Hla, Proc. Natl. Acad. Sci. U.S.A. 103, 13718 (2006). 35. Other proposed mechanisms for switching have included relative ring rotation. The calculated barrier to rotation about the ethynyl bond in MPEA is only ~1 kcal mol–1 (fig. S2), similar to the value calculated for phenyl ring rotation in OPEs, so the observed stability of the conductance status is inconsistent with ring rotation being responsible for conductance switching. 36. P. S. Weiss, D. M. Eigler, Phys. Rev. Lett. 71, 3139 (1993). 37. I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules (Academic Press, New York, 1971). 38. M. A. Fox, S. Olive, Science 205, 582 (1979). 39. M. A. Meador, H. Hart, J. Org. Chem. 54, 2336 (1989). 40. S. P. Sullivan, A. Schnieders, S. K. Mbugua, T. P. Beebe Jr., Langmuir 21, 1322 (2005). 41. M. F. Perutz, A. J. Wilkinson, M. Paoli, G. G. Dodson, Annu. Rev. Biophys. Biomol. Struct. 27, 1 (1998). 42. C. Chen, C. A. Bobisch, W. Ho, Science 325, 981 (2009). 43. T. Ye et al., ACS Nano 4, 3697 (2010). 44. We thank S. Claridge for insightful discussions and the U.S. Department of Energy (no. DE-FG02-07ER15877), the NSF, the Air Force Office of Scientific Research, and the Kavli Foundation for financial support.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1312/DC1 Materials and Methods SOM Text Figs. S1 to S3 References 23 November 2010; accepted 2 February 2011 10.1126/science.1200830

At ZT10 [where ZT is Zeitgeber time (light on at ZT0 and off at ZT12)], in the light period when mice are inactive, HDAC3 bound to over 14,000 sites in adult mouse liver (the HDAC3 ZT10 cistrome) (fig. S2A); a majority of these binding sites were distant from transcription start sites (TSS) or present in introns (fig. S2, B and C). However, at ZT22, in the dark period when mice are active and feeding, the HDAC3 signal was dramatically reduced at ZT10 sites, with only 120 specific peaks (Fig. 1A and figs. S2, A and D, and S3). HDAC3 recruitment oscillated in a 24hour cycle (Fig. 1B), and this rhythm was retained in constant darkness (Fig. 1C), suggesting that it was controlled by the circadian clock. The liver clock is entrained by food intake (16), and, indeed, the pattern of HDAC3 enrichment was reversed when food was provided only during the light period (Fig. 1D), further supporting the conclusion that the rhythm of HDAC3 genomic recruitment was controlled by the circadian clock. Despite its known role in histone deacetylation and transcriptional repression, HDAC3

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1 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, and the Institute for Diabetes, Obesity, and Metabolism (IDOM), University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA 02115, USA.

*These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]

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Exercising control over molecular reactivity by confinement has great potential for both understanding and measuring complex chemical reactions (40, 41). Regioselective photoreactions between individual molecules by molecular design and self-assembly with in situ monitoring are powerful demonstrations of chemical control via the local environment (7, 42, 43). The extraordinary resolution of the STM enables monitoring molecular motions and reactions. Controlling the chemical environment and monitoring such selective reactions between individual molecules will be important elements in directing chemistry with this approach. References and Notes 1. R. K. Smith et al., J. Am. Chem. Soc. 128, 9266 (2006). 2. D. I. Gittins, D. Bethell, D. J. Schiffrin, R. J. Nichols, Nature 408, 67 (2000). 3. A. Vaish et al., ACS Chem. Neurosci. 1, 495 (2010). 4. M. T. Cygan et al., J. Am. Chem. Soc. 120, 2721 (1998). 5. A. M. Moore et al., Nano Lett. 5, 2292 (2005). 6. P. S. Weiss, Acc. Chem. Res. 41, 1772 (2008). 7. Z. J. Donhauser et al., Science 292, 2303 (2001). 8. A. M. Moore et al., J. Am. Chem. Soc. 129, 10352 (2007). 9. S. J. van der Molen et al., Nano Lett. 9, 76 (2009). 10. W. R. Browne et al., J. Phys. Chem. C 112, 1183 (2008). 11. S. W. Wu, N. Ogawa, W. Ho, Science 312, 1362 (2006). 12. A. S. Kumar et al., Nano Lett. 8, 1644 (2008).

13. J. E. Anthony, Chem. Rev. 106, 5028 (2006). 14. H. Bouas-Laurent, A. Castellan, J.-P. Desvergne, R. Lapouyade, Chem. Soc. Rev. 29, 43 (2000). 15. D. K. James, J. M. Tour, Chem. Mater. 16, 4423 (2004). 16. J. Reichert et al., Phys. Rev. Lett. 88, 176804 (2002). 17. D. Käfer, G. Witte, P. Cyganik, A. Terfort, C. Wöll, J. Am. Chem. Soc. 128, 1723 (2006). 18. M. H. Zareie, J. Barber, A. M. McDonagh, J. Phys. Chem. B 110, 15951 (2006). 19. M. A. Fox, M. D. Wooten, Langmuir 13, 7099 (1997). 20. Z. J. Donhauser, D. W. Price II, J. M. Tour, P. S. Weiss, J. Am. Chem. Soc. 125, 11462 (2003). 21. A. M. Moore et al., J. Am. Chem. Soc. 128, 1959 (2006). 22. E. Delamarche, B. Michel, H. A. Biebuyck, C. Gerber, Adv. Mater. 8, 719 (1996). 23. T. Ishida et al., Langmuir 13, 3261 (1997). 24. H. D. Becker, Chem. Rev. 93, 145 (1993). 25. T. Wolff, N. Müller, G. von Bünau, J. Photochem. 22, 61 (1983). 26. H. D. Becker, K. Andersson, J. Photochem. 26, 75 (1984). 27. D. R. Maulding, B. G. Roberts, J. Org. Chem. 34, 1734 (1969). 28. M. H. Zareie, H. Ma, B. W. Reed, A. K. Y. Jen, M. Sarikaya, Nano Lett. 3, 139 (2003). 29. R. F. Dou et al., Langmuir 22, 3049 (2006). 30. C. Gonzalez, E. C. Lim, Chem. Phys. Lett. 322, 382 (2000). 31. N. J. Brewer, S. Janusz, K. Critchley, S. D. Evans, G. J. Leggett, J. Phys. Chem. B 109, 11247 (2005). 32. P. A. Lewis et al., J. Am. Chem. Soc. 126, 12214 (2004). 33. J. U. Nielsen, M. J. Esplandiu, D. M. Kolb, Langmuir 17, 3454 (2001).

A Circadian Rhythm Orchestrated by Histone Deacetylase 3 Controls Hepatic Lipid Metabolism Dan Feng,1* Tao Liu,2* Zheng Sun,1 Anne Bugge,1 Shannon E. Mullican,1 Theresa Alenghat,1 X. Shirley Liu,2 Mitchell A. Lazar1† Disruption of the circadian clock exacerbates metabolic diseases, including obesity and diabetes. We show that histone deacetylase 3 (HDAC3) recruitment to the genome displays a circadian rhythm in mouse liver. Histone acetylation is inversely related to HDAC3 binding, and this rhythm is lost when HDAC3 is absent. Although amounts of HDAC3 are constant, its genomic recruitment in liver corresponds to the expression pattern of the circadian nuclear receptor Rev-erba. Rev-erba colocalizes with HDAC3 near genes regulating lipid metabolism, and deletion of HDAC3 or Rev-erba in mouse liver causes hepatic steatosis. Thus, genomic recruitment of HDAC3 by Rev-erba directs a circadian rhythm of histone acetylation and gene expression required for normal hepatic lipid homeostasis. n mammals, metabolic processes in peripheral organs display robust circadian rhythms, coordinated with the daily cycles of light and nutrient availability (1, 2). Circadian misalignment causes metabolic dysfunction, and people engaged in night-shift work suffer from higher incidences of obesity, diabetes, and metabolic syndrome (3–5). The molecular basis of this is unknown, but genetic disruption of circadian clock components in mice leads to altered glucose and lipid metabolism (6–10). Gene expression profiles in multiple metabolic organs have revealed a circadian control of the

I

transcriptome, which might be mediated by regulation of histone acetylation (11–13) that alters the structure of the epigenome. Regulation of histone acetylation is complex, involving multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) (14). Histone deacetylase 3 (HDAC3) functions in the regulation of circadian rhythm and glucose metabolism (15). We report diurnal recruitment of HDAC3 to the mouse liver genome detected by chromatin immunoprecipitation with an HDAC3-specific antibody (fig. S1) and massively parallel DNA sequencing (ChIP-seq).

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34. V. Iancu, S. W. Hla, Proc. Natl. Acad. Sci. U.S.A. 103, 13718 (2006). 35. Other proposed mechanisms for switching have included relative ring rotation. The calculated barrier to rotation about the ethynyl bond in MPEA is only ~1 kcal mol–1 (fig. S2), similar to the value calculated for phenyl ring rotation in OPEs, so the observed stability of the conductance status is inconsistent with ring rotation being responsible for conductance switching. 36. P. S. Weiss, D. M. Eigler, Phys. Rev. Lett. 71, 3139 (1993). 37. I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules (Academic Press, New York, 1971). 38. M. A. Fox, S. Olive, Science 205, 582 (1979). 39. M. A. Meador, H. Hart, J. Org. Chem. 54, 2336 (1989). 40. S. P. Sullivan, A. Schnieders, S. K. Mbugua, T. P. Beebe Jr., Langmuir 21, 1322 (2005). 41. M. F. Perutz, A. J. Wilkinson, M. Paoli, G. G. Dodson, Annu. Rev. Biophys. Biomol. Struct. 27, 1 (1998). 42. C. Chen, C. A. Bobisch, W. Ho, Science 325, 981 (2009). 43. T. Ye et al., ACS Nano 4, 3697 (2010). 44. We thank S. Claridge for insightful discussions and the U.S. Department of Energy (no. DE-FG02-07ER15877), the NSF, the Air Force Office of Scientific Research, and the Kavli Foundation for financial support.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1312/DC1 Materials and Methods SOM Text Figs. S1 to S3 References 23 November 2010; accepted 2 February 2011 10.1126/science.1200830

At ZT10 [where ZT is Zeitgeber time (light on at ZT0 and off at ZT12)], in the light period when mice are inactive, HDAC3 bound to over 14,000 sites in adult mouse liver (the HDAC3 ZT10 cistrome) (fig. S2A); a majority of these binding sites were distant from transcription start sites (TSS) or present in introns (fig. S2, B and C). However, at ZT22, in the dark period when mice are active and feeding, the HDAC3 signal was dramatically reduced at ZT10 sites, with only 120 specific peaks (Fig. 1A and figs. S2, A and D, and S3). HDAC3 recruitment oscillated in a 24hour cycle (Fig. 1B), and this rhythm was retained in constant darkness (Fig. 1C), suggesting that it was controlled by the circadian clock. The liver clock is entrained by food intake (16), and, indeed, the pattern of HDAC3 enrichment was reversed when food was provided only during the light period (Fig. 1D), further supporting the conclusion that the rhythm of HDAC3 genomic recruitment was controlled by the circadian clock. Despite its known role in histone deacetylation and transcriptional repression, HDAC3

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1 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, and the Institute for Diabetes, Obesity, and Metabolism (IDOM), University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA 02115, USA.

*These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]

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B

HDAC3 ZT10 ZT22

2.0

HDAC3 Enrichment (% of Input)

Fig. 1. Circadian rhythm of genomic HDAC3 recruitment in mouse liver. (A) Heat map of HDAC3 binding signal at ZT10 (left) and ZT22 (right) from –1 kb to +1 kb surrounding the center of all the HDAC3 ZT10 binding sites, ordered by strength of HDAC3 binding at ZT10. ChIP-seq with antibody against HDAC3 (anti-HDAC3) was performed, and data were analyzed as described in (31). Each line represents a single HDAC3 binding site, and the color scale indicates the HDAC3 signal (reads encompassing each locus per million total reads). A read is a unique sequence obtained in ChIP-seq and then aligned to the mouse genome. ZT, Zeitgeber time (light on at ZT0, off at ZT12). (B) HDAC3 recruitment at two selected genomic sites over a 24-hour cycle by ChIP–polymerase chain reaction (PCR). Immunoprecipitated DNA was normalized to input. Values are mean T SEM (n = 4 or 5). (C) HDAC3 diurnal genomic recruitment is maintained in constant darkness. Six HDAC3 binding sites were assessed by ChIP-PCR (n = 4 or 5), and the Bmal1 promoter served as a positive control (23); regions close to the TSS of the Arbp and Ins genes served as negative controls. CT, circadian time. (D) The rhythm of HDAC3 recruitment is reversed by daytime feeding (n = 4 or 5). RF, food was provided only from ZT3 to ZT11 every day for 2 weeks.

Arbp HDAC3 Site 1 HDAC3 Site 2

1.6 1.2 0.8 0.4 0

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6

12

30

CT10

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CT22 1.0 0.5

CT10 RF

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Signal Per Million Reads

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Pol II ZT10

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HDAC3 Enrichment (% of Input)

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0.5 0.4 0.3 0.2 0.1 0

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Bound by HDAC3 at ZT10 (%)

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Constant Darkness

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Fig. 2. Orchestration of genome-wide rhythms of histone acetylation, Pol II recruitment, and gene expression by HDAC3. (A) Heat map of the H3K9 acetylation (H3K9Ac) signal in WT liver at ZT22 (left), ZT10 (middle), and ZT10 in liver depleted of HDAC3 (KO) (right) from –1 kb to +1 kb surrounding the center of all the HDAC3 ZT10 binding sites, ordered by k means clustering of H3K9Ac signal. Each line represents a single HDAC3 binding site, and the color scale indicates the H3K9Ac signal per million total reads. HDAC3-depleted liver was removed from HDAC3fl/fl mice 1 week after injection of AAV-Cre as in (31). (B) Average H3K9Ac signal from –1 kb to +1 kb surrounding the center of all the HDAC3 ZT10 binding sites. The y axis represents the H3K9Ac signal per million total reads. bp, base pairs. (C) Heat map of Pol II signal at ZT10 (left) and ZT22 (right) from –1 kb to +1 kb surrounding the TSS of genes with HDAC3 recruitment within 10 kb of the TSS, ordered by strength of Pol II binding at ZT22. Each line represents a single HDAC3bound gene, and the color scale indicates the Pol II signal per million total reads. Green marks denote 130 genes under the Gene Ontology term “Lipid Biosynthetic Process” listed in table S1. (D) Genes up-regulated in liver depleted of HDAC3 are significantly enriched for HDAC3 binding at ZT10. Expression arrays of WT and HDAC3 KO liver were performed and analyzed as described in (31), and the percentage of HDAC3-bound genes in each category was calculated. *P ~ 10−156 based on hypergeometic distribution as in (31).

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recruitment has been reported to be associated with high histone acetylation, RNA polymerase II (Pol II) recruitment, and gene expression in human primary T cells (17). In mouse liver, HDAC3 recruitment at ZT10 was also enriched around active genes (fig. S4A), and many of these display circadian expression patterns (18) (fig. S4B). Thus, HDAC3 may have an important role in transient regulation of these active genes by the circadian clock. Consistent with this hypothesis, we observed decreases in acetylation of histone H3 lysine 9 (H3K9) at ZT10 compared with that at ZT22, the inverse of HDAC3

A

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recruitment to these sites (Fig. 2, A and B). Deletion of hepatic HDAC3 by tail vein injection of adeno-associated virus expressing cre-recombinase (AAV-Cre) into adult C57Bl/6 mice homozygous for a floxed HDAC3 allele (HDAC3fl/fl) (fig. S1A) led to H3K9 acetylation at ZT10 comparable to that of wild-type mice at ZT22 (Fig. 2, A and B). Accompanying the decreased H3K9 acetylation at ZT10 was a decrease in binding of RNA polymerase II (Pol II) at the TSS of genes with HDAC3 binding within 10 kb, indicating that they were actively repressed (Fig. 2C). Indeed, the majority of genes whose transcripts were increased

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Fig. 3. Recruitment of HDAC3 to the genome by Rev-erba. (A) Immunoblot of HDAC3 and Rev-erba over a 24-hour cycle in mouse liver. Heat shock protein 90 (Hsp90) levels are shown as loading control. (B) The HDAC3 cistrome at ZT10 largely overlaps with the Rev-erba cistrome at ZT10. ChIP-seq with anti-Rev-erba was performed and analyzed as described in (31). (C) Heat map of Rev-erba at ZT10 and ZT22 (left) and of NCoR at ZT10 and ZT22 (right), both at HDAC3 ZT10 sites ordered as in Fig. 1A. Each line represents a single HDAC3 binding site, and the color scale indicates the signal per million total reads. (D and E) HDAC3 (D) and NCoR (E) recruitment to six binding sites (as in Fig. 1C) were interrogated by ChIP-PCR in liver from mice lacking Rev-erba. The Bmal1 promoter was used as a positive control (23); regions close to the TSS of the Arbp and Ins genes served as negative controls. Values are mean T SEM (n = 3). www.sciencemag.org

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1 week after HDAC3 deletion in liver displayed HDAC3 binding within 10 kb of their TSS in WT mice at ZT10 (Fig. 2D). Thus, genome-wide diurnal recruitment of HDAC3 directs a rhythm of epigenomic modification, Pol II recruitment, and gene expression. Although HDAC3 recruitment to the genome is diurnal, the abundance of HDAC3 was constant throughout the light/dark cycle (Fig. 3A). HDAC3 enzyme activity requires interaction with nuclear receptor (NR) corepressors (19), and de novo motif analysis of the HDAC3 binding sites revealed the classical motif recognized by a number of NRs (fig. S5). The NR Rev-erba is a transcriptional repressor that is expressed in a circadian manner (20), and the abundance of Rev-erba protein oscillated in phase with HDAC3 recruitment (Fig. 3A). We used a Rev-erba–specific antibody (fig. S6A) to determine the Rev-erba binding sites (fig. S6, B and C). At ZT10, the Rev-erba binding sites overlapped with the majority of HDAC3 binding sites (Fig. 3B). Furthermore, Rev-erba bound to the majority of HDAC3 ZT10 sites at ZT10 but not ZT22 (Fig. 3C). The extent of overlap of HDAC3 with Rev-erba was surprising given that other NRs can interact with corepressors and HDAC3 (21). However, HDAC3 recruitment was diminished at many sites in Reverba knockout (KO) mice (Fig. 3D), consistent with a critical role for Rev-erba, although residual HDAC3 binding suggests that other factors also contribute to its recruitment. Rev-erba recruits HDAC3 via the nuclear receptor corepressor (NCoR) (22, 23). NCoR was recruited to HDAC3 sites with a diurnal rhythm (Fig. 3C), which was attenuated in the Rev-erba KO mice (Fig. 3E). Moreover, HDAC3 bound together with NCoR as well as Rev-erba at the majority of ZT10 sites (fig. S7). We addressed the biological role of the circadian genomic recruitment of HDAC3 in mouse liver. The set of genes bound by Rev-erba and HDAC3, and up-regulated in livers depleted of HDAC3, was enriched for genes encoding proteins that function in lipid metabolic processes (Fig. 4A). Indeed, in liver of chow-fed mice in which HDAC3 was deleted for 2 weeks, Oil Red O staining for neutral lipid was dramatically increased (Fig. 4B), and liver triglyceride content was increased nearly 10-fold (Fig. 4C), with serum transaminase activity increasing only modestly (fig. S8A). This was consistent with a fatty liver phenotype of mice depleted of hepatic HDAC3 in utero (24) (fig. S9). The majority of genes up-regulated in liver depleted of Rev-erba (25) were bound by HDAC3 as well as Rev-erba at ZT10 (fig. S10). Indeed, chow-fed C57Bl/6 mice genetically lacking Reverba (26) had normal serum transaminase activity (fig. S8B), but Oil Red O staining of liver was increased (Fig. 4D) and hepatic triglyceride content was nearly double that of wild-type mice (Fig. 4E). The relatively modest hepatic steatosis in the Rev-erba–deleted mice likely reflects a role for HDAC3 in mediating effects of

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other NRs—including Rev-erbb, whose circadian expression pattern is similar to that of Rev-erba (27)—but could also reflect a compensatory effect of Rev-erba KO in other tissues. Nevertheless the finding that depletion of either Rev-erba or HDAC3 led to a fatty liver phenotype supports the conclusion that circadian Rev-erba recruitment of HDAC3 to lipid metabolic genes plays a critical physiological role. Rev-erba and HDAC3 colocalized at >100 lipid biosynthetic genes at ZT10, including Fasn

B

Genes bound by HDAC3 and Rev-erbα and up-regulated in HDAC3 KO

Biological Processes

p-value

Lipid metabolic process

1.72E-24

Metabolic process

8.22E-21

Cellular amino acid and derivative metabolic process

8.33E-19

Primary metabolic process

8.30E-17

Coenzyme metabolic process

1.84E-14

Carbohydrate metabolic process

1.78E-11

HDAC3fl/fl /GFP

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HDAC3fl/fl /Cre

70 60 50 40 30 20 10 0

Hepatic TG (mg/g)

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and Acaca (table S1), and Pol II recruitment increased from ZT10 to ZT22 at the TSS of many of these genes (Fig. 2C), suggesting that they were directly repressed. Assessment of palmitate synthesis after injection of deuterated water revealed increased de novo lipogenesis in mice lacking hepatic HDAC3 (Fig. 4F) and in Rev-erba KO mice (fig. S11), thus revealing a molecular mechanism underlying the observation that hepatic lipogenesis in mice follows a diurnal rhythm (28) that is antiphase to Rev-

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Fig. 4. Regulation of hepatic lipid homeostasis by HDAC3. (A) Gene Ontology analysis of the HDAC3and Rev-erba–bound genes that were up-regulated in liver depleted of HDAC3 was performed as described in (31). (B) Oil Red O staining of liver from 12-week-old HDAC3fl/fl mice 2 weeks after tail vein injection of AAV–green fluorescent protein (GFP) or AAV-Cre. (C) Hepatic triglyceride (TG) levels in mice treated as in (B). (D) Oil Red O staining of livers from 9-week-old WT and mice lacking Rev-erba (Rev-erba KO). (E) Hepatic TG levels in livers from 9-week-old WT and Rev-erba KO. Values are mean T SEM (n = 4). *P < 0.05 by Student’s t test. (F) Hepatic de novo lipogenesis (DNL) in 12-week-old HDAC3fl/fl mice 1 week after infection with AAV-Cre or with AAV-GFP. Hepatic DNL is measured as newly synthesized 2Hlabeled palmitate. Values are mean T SEM (n = 7 or 8). *P < 0.05 by Student’s t test. (G) Model depicting the mechanistic links between the daily cycles of Rev-erba expression (orange oval), HDAC3 genomic recruitment, epigenomic status, and hepatic lipogenesis.

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erba and HDAC3 recruitment to the mouse genome. These findings demonstrate the existence of circadian changes in histone acetylation whose dysregulation has the potential to cause major perturbations in normal metabolic function. Each day, low concentrations of Rev-erba lead to reduced HDAC3 association with the liver genome while the organism is active and feeding, altering the epigenome to permit lipid synthesis and accumulation until abundance of Rev-erba increases HDAC3 recruitment to liver metabolic genes and halts the lipid build-up (Fig. 4G). When either Rev-erba or HDAC3 is depleted, this cycle does not occur, and fatty liver ensues. Misalignment of fasting/feeding and sleep/wake cycles with endogenous circadian cycles could disrupt the rhythm of HDAC3 association with target genes and contribute to the fatty liver observed in rotating shift workers as well as people with genetic variants of molecular clock genes (29, 30).

SCIENCE

1. C. B. Green, J. S. Takahashi, J. Bass, Cell 134, 728 (2008). 2. E. Maury, K. M. Ramsey, J. Bass, Circ. Res. 106, 447 (2010). 3. F. A. Scheer, M. F. Hilton, C. S. Mantzoros, S. A. Shea, Proc. Natl. Acad. Sci. U.S.A. 106, 4453 (2009). 4. A. Pietroiusti et al., Occup. Environ. Med. 67, 54 (2010). 5. D. De Bacquer et al., Int. J. Epidemiol. 38, 848 (2009). 6. B. Marcheva et al., Nature 466, 627 (2010). 7. F. W. Turek et al., Science 308, 1043 (2005); 10.1126/science.1108750. 8. K. A. Lamia, K. F. Storch, C. J. Weitz, Proc. Natl. Acad. Sci. U.S.A. 105, 15172 (2008). 9. K. Eckel-Mahan, P. Sassone-Corsi, Nat. Struct. Mol. Biol. 16, 462 (2009). 10. R. D. Rudic et al., PLoS Biol. 2, e377 (2004). 11. S. Panda et al., Cell 109, 307 (2002). 12. J. P. Etchegaray, C. Lee, P. A. Wade, S. M. Reppert, Nature 421, 177 (2003). 13. M. Doi, J. Hirayama, P. Sassone-Corsi, Cell 125, 497 (2006). 14. B. D. Strahl, C. D. Allis, Nature 403, 41 (2000). 15. T. Alenghat et al., Nature 456, 997 (2008). 16. F. Damiola et al., Genes Dev. 14, 2950 (2000). 17. Z. Wang et al., Cell 138, 1019 (2009). 18. M. E. Hughes et al., PLoS Genet. 5, e1000442 (2009). 19. M. G. Guenther, O. Barak, M. A. Lazar, Mol. Cell. Biol. 21, 6091 (2001). 20. N. Preitner et al., Cell 110, 251 (2002). 21. M. Goodson, B. A. Jonas, M. A. Privalsky, Nucl. Recept. Signal. 3, e003 (2005). 22. I. Zamir et al., Mol. Cell. Biol. 16, 5458 (1996). 23. L. Yin, M. A. Lazar, Mol. Endocrinol. 19, 1452 (2005). 24. S. K. Knutson et al., EMBO J. 27, 1017 (2008). 25. B. Kornmann, O. Schaad, H. Bujard, J. S. Takahashi, U. Schibler, PLoS Biol. 5, e34 (2007). 26. P. Chomez et al., Development 127, 1489 (2000). 27. A. C. Liu et al., PLoS Genet. 4, e1000023 (2008). 28. D. A. Hems, E. A. Rath, T. R. Verrinder, Biochem. J. 150, 167 (1975). 29. E. M. Brunt, Nat. Rev. Gastroenterol. Hepatol. 7, 195 (2010). 30. S. Sookoian, G. Castaño, C. Gemma, T. F. Gianotti, C. J. Pirola, World J. Gastroenterol. 13, 4242 (2007). 31. Materials and methods are available as supporting material on Science Online.

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REPORTS ( J. Johnston) of the Penn Diabetes Endocrinology Research Center (NIH DK19525) for deep sequencing and virus preparation; the Morphology Core (J. Katz and G. Swain) (NIH DK49210) for tissue preparation and staining; and the Penn Bioinformatics Core (J. Tobias) and Microarray Core (D. Baldwin) for gene expression analysis. Supported by the Cox Institute, NIH DK45586, DK43806, and RC1DK08623 (to M.A.L.) and NIH HG4069 (to X.S.L.).

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response Alice Lebreton,1,2,3 Goran Lakisic,4 Viviana Job,5 Lauriane Fritsch,6 To Nam Tham,1,2,3 Ana Camejo,7 Pierre-Jean Matteï,5 Béatrice Regnault,8 Marie-Anne Nahori,1,2,3 Didier Cabanes,7 Alexis Gautreau,4 Slimane Ait-Si-Ali,6 Andréa Dessen,5 Pascale Cossart,1,2,3* Hélène Bierne1,2,3* Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)–stimulated genes (ISGs). IFN-l expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/– mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-l−mediated immune response to control bacterial colonization of the host.

L

isteria monocytogenes is a food-borne pathogen that can cause serious illness in pregnant women and immunocompromised individuals (1). This intracellular bacterium uses an arsenal of effectors to exploit cellular functions in various ways (2). Host cells respond to this invasion by turning on appropriate defense transcriptional programs (3). Listeria and other pathogens can manipulate chromatin to reprogram host transcription (4, 5). However, very few bacterial molecules have been shown to enter eukaryotic cell nuclei, and knowledge about microbial factors that may act directly on the chromatinregulatory machinery is limited (6). To identify factors involved in bacterial pathogenicity, we screened the L. monocytogenes strain EGDe genome for genes encoding secreted proteins absent in nonpathogenic Listeria species. lmo0438/lntA (listeria nuclear targeted protein A) was one such gene (fig. S1A). lntAwas expressed 1 Institut Pasteur, Unité des Interactions Bactéries Cellules, Paris F-75015, France. 2Inserm, U604, Paris F-75015, France. 3INRA, USC2020, Paris F-75015, France. 4CNRS UPR3082, Laboratoire d’Enzymologie et de Biochimie Structurales, Gif-sur-Yvette F-91198, France. 5Institut de Biologie Structurale, Bacterial Pathogenesis Group, UMR 5075 (CNRS/CEA/UJF), Grenoble F-38027, France. 6CNRS UMR7216, Université Paris DiderotParis 7, Paris F-75013, France. 7Institute for Molecular and Cellular Biology, Porto 4150, Portugal. 8Institut Pasteur, Génopole, Paris F-75015, France.

*To whom correspondence should be addressed. E-mail: [email protected] (H.B.); [email protected] (P.C.)

at very low levels by the EGDe strain grown in brain-heart infusion (BHI) medium (fig. S1B) (7). Two major regulators of virulence genes, PrfA and sB, were required for basal lntA transcription (Fig. 1A). lntA expression was significantly higher in bacteria harvested from spleens of infected mice, 48 hours after intravenous inoculation, compared with that of bacteria grown in BHI (Fig. 1B). In addition, deletion of lntA led to a decrease in bacterial colonization of spleens and livers, as well as blood bacteraemia (Fig. 1B). lntA thus contributes to L. monocytogenes virulence. It encodes a 205–amino acid basic protein with a N-terminal signal peptide but no sequence similarity with any known polypeptide. The 2.3 Å resolution structure of LntA reveals a compact a-helical fold (fig. S2) [Protein Data Bank (PDB) ID no. 2xl4]. Consistent with low lntA transcription levels in vitro, LntA was undetectable in either total extracts or supernatants of wild-type (WT) bacteria grown in BHI (Fig. 1C). To address the role of LntA during L. monocytogenes cellular infection, we generated strains that constitutively expressed lntA under the control of a heterologous promoter, either on the chromosome (lntAc+ ) or on a plasmid in fusion with the V5 tag (lntAV5+ ) (tables S1 and S2). Both strains produced and secreted LntA (Fig. 1C and fig. S1C) and showed no noticeable difference in entry or multiplication in cultured cells compared

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Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1315/DC1 Materials and Methods Figs. S1 to S11 Tables S1 to S3 References 22 September 2010; accepted 6 January 2011 10.1126/science.1198125

with the WT or lntA-deficient strains (DlntA, lntA–, or lntAc–) (table S3A and fig. S3). Secreted LntA accumulated in the nucleus of fibroblasts after 22 hours of infection with lntAV5+ bacteria (Fig. 1D and fig. S4A). We thus assessed whether LntA interacted with nuclear proteins in a largescale yeast two-hybrid screen of a human cDNA library. One of the strongest LntA interactors was BAHD1, a silencing factor that orchestrates heterochromatin assembly at specific genes such as that for insulin-like growth factor 2 IGF2 (8). A fusion of LntA with glutathione S-transferase (GST) pulled down V5-tagged BAHD1 from nuclear extracts, which confirmed the capacity of LntA to specifically interact with BAHD1 (Fig. 1E). When produced ectopically in human fibroblasts, LntA colocalized with heterochromatin nuclear foci that were induced by overexpression of BAHD1 tagged with yellow fluorescent protein (BAHD1YFP) (8), both in fixed (LntA-V5) (Fig. 1F) and in living cells (LntA tagged with cyan fluorescent protein, LntA-CFP) (fig. S4B). Because BAHD1 is involved in gene silencing, LntA might control host gene expression. To assess this hypothesis, we performed a transcriptome analysis of colon carcinoma epithelial LoVo cells infected for 24 hours with either lntAV5+ or lntA– bacteria (GEO database, GSE26414). The lntAV5+ bacteria specifically up-regulated the expression of a subset of genes, out of which 39 displayed a more than twofold induction (table S4). Of these genes, 83% belonged to the interferon-inducible genes regulon: 28 are known interferon-stimulated genes (ISGs), including three genes (IL29, IL28A, and IL28B) that encode type III interferons (IFN-l1, -l2, and -l3), and four are predicted ISGs. LntA may thus play a role in the IFN-III–mediated immune response. This pathway controls various viral infections, especially in epithelial tissues (9–13). We confirmed that WT L. monocytogenes triggered the expression of IFN-l2 in intestinal LoVo and placental JEG-3 epithelial cells (Fig. 2A and fig. S5A), while type I IFN-b1 was induced a little and type II IFN-g was undetectable. However, the induction of downstream ISGs was modest (Fig. 2A), except for CCL5, which, like IFN-l genes, is controlled both by nuclear factor kB (NF-kB) and interferon regulatory factors (IRFs) (14). These data suggested that interferon signaling was down-regulated in infected cells. We wondered whether the host factor BAHD1 could act as a repressor of ISGs, as it does for IGF2 (fig. S5B) (8). Knockdown (depletion) of BAHD1 had no or minor effect on ISG expression

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32. ChIP-seq and microarray data have been deposited in the Gene Expression Omnibus (GSE25937 and GSE26345) database. We thank B. Vennström for providing Rev-erba KO mice; M. Brown, D. Steger, and members of the Lazar lab for helpful discussions; D. Zhuo for technical assistance; the Penn IDOM Metabolism Resource (J. Millar) for help with de novo lipogenesis experiments; the Functional Genomics Core ( J. Schug and K. Kaestner) and the Viral Vector Core

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REPORTS ( J. Johnston) of the Penn Diabetes Endocrinology Research Center (NIH DK19525) for deep sequencing and virus preparation; the Morphology Core (J. Katz and G. Swain) (NIH DK49210) for tissue preparation and staining; and the Penn Bioinformatics Core (J. Tobias) and Microarray Core (D. Baldwin) for gene expression analysis. Supported by the Cox Institute, NIH DK45586, DK43806, and RC1DK08623 (to M.A.L.) and NIH HG4069 (to X.S.L.).

A Bacterial Protein Targets the BAHD1 Chromatin Complex to Stimulate Type III Interferon Response Alice Lebreton,1,2,3 Goran Lakisic,4 Viviana Job,5 Lauriane Fritsch,6 To Nam Tham,1,2,3 Ana Camejo,7 Pierre-Jean Matteï,5 Béatrice Regnault,8 Marie-Anne Nahori,1,2,3 Didier Cabanes,7 Alexis Gautreau,4 Slimane Ait-Si-Ali,6 Andréa Dessen,5 Pascale Cossart,1,2,3* Hélène Bierne1,2,3* Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)–stimulated genes (ISGs). IFN-l expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1+/– mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-l−mediated immune response to control bacterial colonization of the host.

L

isteria monocytogenes is a food-borne pathogen that can cause serious illness in pregnant women and immunocompromised individuals (1). This intracellular bacterium uses an arsenal of effectors to exploit cellular functions in various ways (2). Host cells respond to this invasion by turning on appropriate defense transcriptional programs (3). Listeria and other pathogens can manipulate chromatin to reprogram host transcription (4, 5). However, very few bacterial molecules have been shown to enter eukaryotic cell nuclei, and knowledge about microbial factors that may act directly on the chromatinregulatory machinery is limited (6). To identify factors involved in bacterial pathogenicity, we screened the L. monocytogenes strain EGDe genome for genes encoding secreted proteins absent in nonpathogenic Listeria species. lmo0438/lntA (listeria nuclear targeted protein A) was one such gene (fig. S1A). lntAwas expressed 1 Institut Pasteur, Unité des Interactions Bactéries Cellules, Paris F-75015, France. 2Inserm, U604, Paris F-75015, France. 3INRA, USC2020, Paris F-75015, France. 4CNRS UPR3082, Laboratoire d’Enzymologie et de Biochimie Structurales, Gif-sur-Yvette F-91198, France. 5Institut de Biologie Structurale, Bacterial Pathogenesis Group, UMR 5075 (CNRS/CEA/UJF), Grenoble F-38027, France. 6CNRS UMR7216, Université Paris DiderotParis 7, Paris F-75013, France. 7Institute for Molecular and Cellular Biology, Porto 4150, Portugal. 8Institut Pasteur, Génopole, Paris F-75015, France.

*To whom correspondence should be addressed. E-mail: [email protected] (H.B.); [email protected] (P.C.)

at very low levels by the EGDe strain grown in brain-heart infusion (BHI) medium (fig. S1B) (7). Two major regulators of virulence genes, PrfA and sB, were required for basal lntA transcription (Fig. 1A). lntA expression was significantly higher in bacteria harvested from spleens of infected mice, 48 hours after intravenous inoculation, compared with that of bacteria grown in BHI (Fig. 1B). In addition, deletion of lntA led to a decrease in bacterial colonization of spleens and livers, as well as blood bacteraemia (Fig. 1B). lntA thus contributes to L. monocytogenes virulence. It encodes a 205–amino acid basic protein with a N-terminal signal peptide but no sequence similarity with any known polypeptide. The 2.3 Å resolution structure of LntA reveals a compact a-helical fold (fig. S2) [Protein Data Bank (PDB) ID no. 2xl4]. Consistent with low lntA transcription levels in vitro, LntA was undetectable in either total extracts or supernatants of wild-type (WT) bacteria grown in BHI (Fig. 1C). To address the role of LntA during L. monocytogenes cellular infection, we generated strains that constitutively expressed lntA under the control of a heterologous promoter, either on the chromosome (lntAc+ ) or on a plasmid in fusion with the V5 tag (lntAV5+ ) (tables S1 and S2). Both strains produced and secreted LntA (Fig. 1C and fig. S1C) and showed no noticeable difference in entry or multiplication in cultured cells compared

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Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1315/DC1 Materials and Methods Figs. S1 to S11 Tables S1 to S3 References 22 September 2010; accepted 6 January 2011 10.1126/science.1198125

with the WT or lntA-deficient strains (DlntA, lntA–, or lntAc–) (table S3A and fig. S3). Secreted LntA accumulated in the nucleus of fibroblasts after 22 hours of infection with lntAV5+ bacteria (Fig. 1D and fig. S4A). We thus assessed whether LntA interacted with nuclear proteins in a largescale yeast two-hybrid screen of a human cDNA library. One of the strongest LntA interactors was BAHD1, a silencing factor that orchestrates heterochromatin assembly at specific genes such as that for insulin-like growth factor 2 IGF2 (8). A fusion of LntA with glutathione S-transferase (GST) pulled down V5-tagged BAHD1 from nuclear extracts, which confirmed the capacity of LntA to specifically interact with BAHD1 (Fig. 1E). When produced ectopically in human fibroblasts, LntA colocalized with heterochromatin nuclear foci that were induced by overexpression of BAHD1 tagged with yellow fluorescent protein (BAHD1YFP) (8), both in fixed (LntA-V5) (Fig. 1F) and in living cells (LntA tagged with cyan fluorescent protein, LntA-CFP) (fig. S4B). Because BAHD1 is involved in gene silencing, LntA might control host gene expression. To assess this hypothesis, we performed a transcriptome analysis of colon carcinoma epithelial LoVo cells infected for 24 hours with either lntAV5+ or lntA– bacteria (GEO database, GSE26414). The lntAV5+ bacteria specifically up-regulated the expression of a subset of genes, out of which 39 displayed a more than twofold induction (table S4). Of these genes, 83% belonged to the interferon-inducible genes regulon: 28 are known interferon-stimulated genes (ISGs), including three genes (IL29, IL28A, and IL28B) that encode type III interferons (IFN-l1, -l2, and -l3), and four are predicted ISGs. LntA may thus play a role in the IFN-III–mediated immune response. This pathway controls various viral infections, especially in epithelial tissues (9–13). We confirmed that WT L. monocytogenes triggered the expression of IFN-l2 in intestinal LoVo and placental JEG-3 epithelial cells (Fig. 2A and fig. S5A), while type I IFN-b1 was induced a little and type II IFN-g was undetectable. However, the induction of downstream ISGs was modest (Fig. 2A), except for CCL5, which, like IFN-l genes, is controlled both by nuclear factor kB (NF-kB) and interferon regulatory factors (IRFs) (14). These data suggested that interferon signaling was down-regulated in infected cells. We wondered whether the host factor BAHD1 could act as a repressor of ISGs, as it does for IGF2 (fig. S5B) (8). Knockdown (depletion) of BAHD1 had no or minor effect on ISG expression

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32. ChIP-seq and microarray data have been deposited in the Gene Expression Omnibus (GSE25937 and GSE26345) database. We thank B. Vennström for providing Rev-erba KO mice; M. Brown, D. Steger, and members of the Lazar lab for helpful discussions; D. Zhuo for technical assistance; the Penn IDOM Metabolism Resource (J. Millar) for help with de novo lipogenesis experiments; the Functional Genomics Core ( J. Schug and K. Kaestner) and the Viral Vector Core

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REPORTS ture regulators. Except for HP1 proteins, found as BAHD1 partners (8) and repressing ISGs (15), there is no reported link between BAHD1 and STAT signaling. To address whether BAHD1 was associated with other proteins involved in ISG regulation, we purified the BAHD1-associated complex from the chromatin fraction of human embryonic kidney (HEK293) cells expressing His6−protein C−tagged BAHD1 (HPT-BAHD1)

by tandem affinity chromatography (Fig. 2C and fig. S6). Mass spectrometry analysis of the complex revealed several polypeptides involved in chromatin and transcriptional regulation, including KAP1, HP1g, and histone deacetylases HDAC1 and 2, as confirmed by immunoblots (Fig. 2C). HDAC1 and 2 directly bind STAT (16), as does the scaffolding protein KAP1, which represses both basal and IFN-I–mediated STAT-driven tran-

Fig. 1. The secreted virulence factor LntA targets the nuclear protein BAHD1. (A) lntA is regulated by PrfA and sB. Real-time reverse transcription quantitative polymerase chain reaction (RT-QPCR) analysis of lntA levels in WT, ∆lntA, ∆prfA, or ∆sigB strains. (B) lntA is up-regulated and contributes to virulence 48 hours post infection in an intravenous mouse model. (Left) RTQPCR analysis of lntA and control lmo2845 levels in WT Listeria extracted from spleens. (Right) Bacteria were numerated in organ or mL blood from mice infected with WT or ∆lntA strains. (A and B) **P < 0.01; ***P < 0.001 (two-tailed t tests). (C) LntA is a secreted protein. Bacterial total extracts (TE) and supernatants (Sn) of WT or lntAV5+ strains were analyzed by immunoblot, with ActA and InlC used as controls. WT bacteria do not produce LntA in BHI. P, precursor; S, secreted. (D) LntA localizes to the nucleus of C3SV40 fibroblasts. V5 immunolabeling and 4′,6′-diamidino-2-phenylindole (DAPI) staining in cells infected for 22 hours with lntA– or lntAV5+ bacteria. (E) Purified LntA binds BAHD1. GST or GST-LntA were incubated with nuclear extracts from CFP-V5– or BAHD1-V5–expressing HEK293 cells. Immunoblots of inputs and eluted fractions were probed with antibodies against V5 (a-V5) or GST. (F) LntA localizes to BAHD1-induced heterochromatin foci. BAHD1-YFP and either LntA-V5 or CFP-V5 were cotransfected into C3SV40 cells and detected by immunofluorescence. (D and F) Scale bars, 5 mm.

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Fig. 2. The BAHD1 complex represses ISGs in Listeria-infected epithelial cells. (A) RT-QPCR analysis of IFN and ISG expression in response to Listeria infection in LoVo cells infected for 49 16 hours with WT L. monocytogenes, compared with noninfected cells. (Left) Type I (IFN-b1), 17 II (IFN-g), and III (IFN-l2) interferon genes. (Right) Various ISGs. †, below detection limits. 14 (B) Quantification of ISGs mRNA in LoVo cells treated for 72 hours with control small interfering RNA (siRNA), siRNA against BAHD1 or KAP1 and infected for 16 hours (WT) or not (NI). (C) Tandem affinity purification of the BAHD1-associated complex. Solubilized chromatin extracts from HEK293 cells expressing the HPT-BAHD1 fusion or control cells were first purified on anti–protein C affinity matrix, followed by polishing on nickel-Sepharose. Eluted fractions from the first (E1) and second (E2) affinity columns were analyzed by colloidal Coomassie staining (left) or immunoblot (right). I, input; FT, flow-through from first column. Histone H4 was a control for nonspecific binding of chromatin components. 38 -

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in noninfected LoVo cells (Fig. 2B). However, infection of these BAHD1-depleted cells with L. monocytogenes induced the expression of several ISGs, which highlighted that BAHD1 could act as a negative regulator of this pathway after bacteria-triggered signaling (Fig. 2B and fig. S5B). ISG expression is governed by IRF-STAT (signal transducer and activator of transcription) transcriptional activators and by chromatin struc-

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Fig. 3. LntA impairs BAHD1-mediated repression of ISGs. (A) LntA in4 1 duces ISGs. mRNA levels 5 3 were estimated by RT0.5 QPCR on total RNA from 2 LoVo cells infected for 0 0 1 1 1 16 hours with lntA– or 4 3 3 2 M M 4 BAHD1 T T F I I T T D I I G F F I I I C lntAV5+, compared with IF IF noninfected cells. (B) LntA impairs BAHD1 recruitment and increases acetyl-H3 levels at ISGs. ChIP analysis was performed on LoVo cells infected as in (A), with antibodies against BAHD1 or H3K9Ac. Promoter DNA levels were assessed by QPCR and normalized to cognate levels in histone H3-ChIP. (C) Constitutive expression of lntA in Listeria decreases bacterial burden during murine systemic listeriosis and triggers overproduction of IFN-III. Mice were infected intravenously with either ∆lntA or lntAc+ strains. (Left) Colony-forming units (CFUs) per organ were numerated at 72 hours postinfection. (Right) Mouse IFN-l3 concentration was quantified by enzyme-linked immunosorbent assay (ELISA) in clarified total extracts of infected spleens and livers. (D) BAHD1+/– mice are less sensitive to systemic listeriosis. BAHD1+/+ or isogenic BAHD1+/– mice were infected intravenously with L. monocytogenes EGDe strain. CFUs per organ were numerated at 72 hours postinfection. (C and D) *P < 0.05; **P < 0.01; ***P < 0.001.

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scription (17, 18). We investigated whether KAP1 also repressed ISGs during infection with Listeria. KAP1 knockdown induced ISG expression in noninfected cells, and bacterial infection greatly enhanced this induction (Fig. 2B and fig. S5C). The BAHD1-KAP1 corepressor complex thus inhibits ISGs downstream of IFN-III stimulation during L. monocytogenes infection (fig. S7). Because WT L. monocytogenes does not express lntA in vitro, we further explored the role played by LntA in the IFN-III signaling pathway using lntA constitutive strains. In agreement with the transcriptome data (table S4), ISG expression was higher in LoVo cells infected with lntAV5+ (Fig. 3A) or lntAc+ (fig. S5D) bacteria, compared with noninfected cells or lntA–-infected cells. This effect was observed only in epithelial cell lines (fig. S5E). The expression of CCL5 (Fig. 3A) and IFN-l2 (fig. S5F), which are ISGs themselves, was also increased upon infection with lntA constitutive strains. Thus, LntA can activate ISGs specifically in Listeria-infected epithelial cells, phenocopying BAHD1 depletion (Fig. 2B and fig. S5B). As LntA interacted with BAHD1, we addressed whether it inhibited BAHD1-mediated silencing. Chromatin immunoprecipitation (ChIP) revealed that the recruitment of BAHD1 at the promoter of representative ISGs (IFIT3 and IFITM1) was impaired in lntAV5+-infected cells, compared with lntA–-infected cells (Fig. 3B). This correlated with an enrichment of acetylated histone H3 at Lys9 (H3K9Ac) at these genes, consistent with increased transcriptional activity. Thus, by displacing the BAHD1-HDAC complex from ISGs,

LntA derepresses these genes in infected cells (for a model, see fig. S7). Neither lntA expression (fig. S3 and table S3) nor cellular stimulation with recombinant IFN-l2 (fig. S8) altered bacterial infection in tissuecultured LoVo cells. We thus assessed the consequences of LntA-BAHD1 interactions on the outcome of infection in vivo. To this end, (i) we infected BALB/c mice with lntA constitutive or lntA-deficient bacteria, and (ii) we generated C57BL/6 BAHD1+/– mice (figs. S9 and S10) and infected them with WT bacteria. We observed a strong decrease in bacterial burden in spleens and livers of BALB/c mice infected with lntAc+ or lntAV5+ relative to ∆lntA or WT bacteria, whereas the IFN-l3 concentration increased in infected organs (Fig. 3C and fig. S11). Thus, constitutive expression of lntA promotes the IFN-III response and decreases bacterial colonization in vivo. Moreover, in BAHD1+/– mice infected with WT L. monocytogenes, the bacterial burden in organs was reduced compared with that of BAHD1+/+ mice (Fig. 3D). Thus, increasing lntA expression in Listeria had effects similar to impairing BAHD1 expression in the host, i.e., decreasing infection. Furthermore, although controlled secretion of LntA by WT bacteria is beneficial to the pathogen, either its constitutive secretion or its absence is detrimental. We propose that a tight control of lntA expression during infection allows Listeria to finetune localized immune responses and to escape antibacterial response (19). Given the tropism of Listeria (20) and IFN-III (9–13) for epithe-

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1. O. Dussurget, Int. Rev. Cell. Mol. Biol. 270, 1 (2008). 2. P. Cossart, A. Toledo-Arana, Microbes Infect. 10, 1041 (2008). 3. S. C. Corr, L. A. O'Neill, Cell. Microbiol. 11, 703 (2009). 4. M. A. Hamon, P. Cossart, Cell Host Microbe 4, 100 (2008). 5. K. Paschos, M. J. Allday, Trends Microbiol. 18, 439 (2010). 6. A. P. Bhavsar, J. A. Guttman, B. B. Finlay, Nature 449, 827 (2007). 7. Materials and methods are available as supporting material on Science Online. 8. H. Bierne et al., Proc. Natl. Acad. Sci. U.S.A. 106, 13826 (2009). 9. M. Li, X. Liu, Y. Zhou, S. B. Su, J. Leukoc. Biol. 86, 23 (2009). 10. R. P. Donnelly, S. V. Kotenko, J. Interferon Cytokine Res. 30, 555 (2010). 11. M. Mordstein, T. Michiels, P. Staeheli, J. Interferon Cytokine Res. 30, 579 (2010). 12. G. Gallagher et al., J. Interferon Cytokine Res. 30, 603 (2010). 13. J. E. Pulverer et al., J. Virol. 84, 8626 (2010). 14. A. Casola et al., J. Virol. 75, 6428 (2001). 15. M. Lavigne et al., PLoS Genet. 5, e1000769 (2009). 16. I. Nusinzon, C. M. Horvath, Proc. Natl. Acad. Sci. U.S.A. 100, 14742 (2003). 17. S. Kamitani et al., Biochem. Biophys. Res. Commun. 370, 366 (2008). 18. R. Tsuruma et al., Oncogene 27, 3054 (2008). 19. Supporting discussion is available on Science Online. 20. M. Lecuit, Clin. Microbiol. Infect. 11, 430 (2005). 21. We are very grateful to O. Dussurget for help in virulence assays. We thank E. Gouin for antibodies against LntA; J.-Y. Coppée for microarrays facilities at the IP Genopole; S. Jacquot and M. C. Birling at the Targeted Mutagenesis and Transgenesis department of the Mouse Clinical Institute (MCI/ICS), where the BAHD1+/– mouse line was generated. Work in the Cossart laboratory received financial support from the Pasteur Institute, French National Institute for Agricultural Research (INRA), INSERM, The French National Research Agency (ANR)–European Research Area (ERA)–NETPathoGenomics (grant SPATELIS), French Ligue Nationale Contre le Cancer (LNCC RS10/75-76 Bierne), and European Research Council (Advanced Grant 233348). P.-J.M. received a Ph.D. fellowship from Région Rhône Alpes. Work in the Cabanes laboratory was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC-SAU/MII/65406/2006; Ph.D. fellowship to A.C. SFRH/BD/29314/2006) and ERANETPathoGenomics (grant SPATELIS). P.C. is an international research scholar of the Howard Hughes Medical Institute. The LntA molecular structure data are deposited at the Worldwide Protein Data Bank (http://www.wwpdb.org), ID no. 2xl4, structure factor file no. r2xl4sf.

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Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200120/DC1 Materials and Methods SOM Text Figs. S1 to S11 Tables S1 to S7 References 8 November 2010; accepted 12 January 2010 Published online 20 January 2010; 10.1126/science.1200120

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Mechanistic Basis of Resistance to PCBs in Atlantic Tomcod from the Hudson River Isaac Wirgin,1* Nirmal K. Roy,1 Matthew Loftus,1 R. Christopher Chambers,2 Diana G. Franks,3 Mark E. Hahn3 The mechanistic basis of resistance of vertebrate populations to contaminants, including Atlantic tomcod from the Hudson River (HR) to polychlorinated biphenyls (PCBs), is unknown. HR tomcod exhibited variants in the aryl hydrocarbon receptor 2 (AHR2) that were nearly absent elsewhere. In ligand-binding assays, AHR2-1 protein (common in the HR) was impaired as compared to widespread AHR2-2 in binding TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and in driving expression in reporter gene assays in AHR-deficient cells treated with TCDD or PCB126. We identified a six-base deletion in AHR2 as the basis of resistance and suggest that the HR population has undergone rapid evolution, probably due to contaminant exposure. This mechanistic basis of resistance in a vertebrate population provides evidence of evolutionary change due to selective pressure at a single locus. rom 1947 to 1976, two General Electric facilities 315 km upstream of the mouth of the Hudson River (HR) released 590,000 kg of polychlorinated biphenyls (PCBs) into the river. Atlantic tomcod Microgadus tomcod is an abundant, bottom-dwelling, and resident finfish of the HR (1). Levels of PCBs and polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) [including the

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1 Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA. 2Howard Marine Sciences Laboratory, Northeast Fisheries Science Center, National Oceanic and Atmospheric Administration Fisheries Service, 74 Magruder Road, Highlands, NJ 07732, USA. 3Biology Department, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

most toxic congener, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD)] in tomcod livers from the HR and Hackensack River in the western HR estuary are among the highest known in nature (2). Most toxicities from halogenated aromatic hydrocarbons (HAHs) such as PCBs and PCDD/Fs and induction of xenobiotic metabolizing enzymes such as cytochrome P4501A (CYP1A) are mediated by the aryl hydrocarbon receptor (AHR) (3). The pathway is activated when ligands bind cytoplasmic AHR at its ligand-binding domain (LBD), and the AHR-ligand complex translocates to the nucleus, where it binds dioxin response elements in the promoter of genes such as CYP1A and activates their transcription. There are two AHRs in fishes (4), of which AHR2 is the more functionally active (5). CYP1A expres-

sion is 100-fold less sensitive to induction by PCBs and TCDD in all tissues (6, 7) and life stages of tomcod (1) from throughout the HR (8) as compared to tomcod from cleaner locales. Decreased inducibility is heritable to at least the F2 generation, is co-dominant (fig. S1), and co-occurs with a 100-fold reduction in the sensitivity of HR tomcod to early–life stage toxicities (1). We characterized full-length AHR2 cDNAs by sequencing overlapping AHR2 amplicons in 10 tomcod from each of three locales: the HR and two reference locales, one distant from [the Miramichi River (MR) in New Brunswick, Canada] and one proximal to [Shinnecock Bay (SB) in New York] the HR. Four of five polymorphisms that were observed showed fixed differences between tomcod from the HR and the other locales. These were two synonymous base substitutions; one nonsynonymous base substitution [nucleotide (nt) 3274 in exon 11: A (Asn) in the HR; T (Tyr) in SB and the MR]; and a six-nt deletion (nts 1314 to 1319 in exon 10; TTCCTC) in HR fish that resulted in a two–amino acid (Phe-Leu) deletion located 43 amino acids downstream of the amino terminal of the AHR2 LBD. We screened for the AHR2 deletion and the nt 3274 base substitution in tomcod from larger collections (n = 31 to 62 specimens per site) from seven Atlantic Coast estuaries ranging from the HR to the St. Lawrence (Fig. 1, A and B). The AHR2-1 allele [with the 6–base pair (bp) deletion and nt 3274 substitution] was detected only in tomcod from the four southernmost populations [frequencies were as follows: 99% in the HR; 92% in the Hackensack River, New Jersey (western HR estuary); 6% in the Niantic River, Connecticut; and 5% in SB]. Populations more distant from

Fig. 1. (A) Frequencies of variant AHR2-1 and AHR2-2 alleles in tomcod from Atlantic coast estuaries. n indicates the number of specimens analyzed per estuary. (B) Restriction fragment length polymorphism analysis of variant tomcod AHR2 alleles. Lanes 1 to 7 contained the AHR2-1 allele (with the 6-bp deletion), and lanes 8 to 14 contained the AHR2-2 allele (without the 6-bp deletion). M, molecular weight marker.

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the HR were monomorphic for AHR2-2; in the HR estuary, AHR2-2 was seen only in heterozygotes. In the Niantic River and SB, near the HR, AHR2-1 was observed only in heterozygotes. The presence of AHR2-1 at low frequencies in tomcod from these two cleaner nearby estuaries within 150 km of the mouth of the HR suggests that AHR2-1 was present as a standing variant at low frequency in the HR before anthropogenic disturbances. The presence of AHR2-2 at low frequencies in the HR suggests that its frequency was altered recently through selection. To evaluate the functional significance of the variant AHR2 alleles, we screened a subset of specimens from these estuaries for sequence variation at the selectively neutral mitochondrial DNA control region (mtDNA CR). The HR population exhibited the most haplotypes (n = 15) and had the greatest haplotypic diversity (0.853)

(table S1), which is consistent with recent Pleistocene glaciation. Thus, any broad-scale mortality episode that may have occurred in the HR because of contaminants did not result in a genetic bottleneck. Haplotype frequencies between the main stem of the HR and the Hackensack River were similar, consistent with an absence of barriers to gene flow between the two locales in the HR estuary. In contrast, significant haplotype frequency differences (P < 0.01) were observed among all U.S. collections (except between the HR and Squamscott River), consistent with the reproductive isolation of these populations and the year-round residency of tomcod in their natal estuaries (Fig. 2 and table S1). The extent of genetic differentiation in haplotype frequencies between the HR and its most proximal neighbors did not approach that seen at the AHR2 locus. Furthermore, a direct association was observed

Fig.2.UPGMA (unweighted pair group method with arithmetic mean) population dendrogram based on genetic distances from mtDNA control region sequence data for tomcod from seven Atlantic Coast estuaries. Bootstrap support is on the dendrogram nodes.

between mtDNA and geographic distances (Mantel’s test, R = 0.583; P = 0.013), which is consistent with patterns of mtDNA diversity among populations reflecting stochastic mutation and genetic drift. Furthermore, we sequenced 901 bp of AHR2 intron 8, exon 9, and intron 9 immediately upstream of the deletion polymorphism in exon 10 in a subset of specimens from five of the seven collections (n = 34 HR specimens and 29 from other rivers) and failed to detect any additional polymorphisms. The full-length in vitro–synthesized AHR2-1 and AHR2-2 proteins (Fig. 3A, inset) were evaluated for their abilities to bind [3H]-radiolabeled TCDD in ligand-binding assays. The AHR2 proteins differed in their abilities to bind [3H]TCDD (Fig. 3A). AHR2-1 was approximately one-third as effective in binding [3H]TCDD as was AHR2-2 (Fig. 3B). Saturation binding analysis demonstrated that the AHR2-2 protein bound [3H]TCDD with an apparent equilibrium dissociation constant (Kd) of 1.27 nM. In contrast, the specific binding to the AHR2-1 protein was lower at all concentrations of [3H]TCDD, and a Kd could not be calculated because a plateau was not achieved under the conditions of the assay (Fig. 3C). The Kd value for AHR2-1 is likely to be near or above the highest concentration of free [3H]TCDD present in our assay (~7 nM), suggesting that the affinity of AHR2-1 for TCDD is at least five times lower than that of AHR2-2. Differences in ligand affinity of this magnitude can result in much larger differences in sensitivity to toxicants in vivo (9)

Fig. 3. Comparison of variant AHR2 proteins in binding [3H]TCDD. (A) In vitro–expressed AHR2-1 and AHR2-2 proteins were incubated with [3H]TCDD, and specific binding was assessed by velocity sedimentation on sucrose gradients. Nonspecific binding was measured as the binding of [3H]TCDD to unprogrammed lysate containing vector with no insert. Synthesis was evaluated by autoradiography of 35S-labeled AHR2 proteins and Western blotting using antibody to tomcod AHR2 (inset). (B) Specific binding of tomcod AHR2-1 and AHR2-2 proteins to [3H]TCDD (means T SD of three replicates). (C) Saturation binding analysis. AHR2-1 and AHR2-2 proteins were incubated with [3H]TCDD (0.1 to 12 nM), and specific binding was assessed by velocity sedimentation on sucrose gradients. dpm, disintegrations per minute. The Kd for binding of [3H]TCDD to AHR2-2 is 1.27 nM. The Kd for AHR2-1 could not be calculated.

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We compared the variant AHR2 proteins in reporter gene assays in AHR-deficient murine c12 hepatoma cells. Cells were transfected with pSB-AHR2 (AHR2-2) or pHR-AHR2 (AHR2-1), tomcod ARNT1, and the firefly luciferase reporter gene construct pGudluc 6.1, and treated with graded doses of TCDD or PCB126. The complete dose-response curves for the two AHR2 proteins were compared for each chemical by counting the number of doses at which the mean response for AHR2-1 was smaller than for AHR22. The difference was significant (binomial test) for each of the two chemicals: for TCDD, seven of eight (7/8) doses (P = 0.035), and for PCB126, 8/8 doses (P = 0.004) (Fig. 4A). For both treatments, cells transfected with pSB-AHR2 were significantly more responsive than those transfected with pHR-AHR2. To compare the effects of the 6-bp deletion and of the nt 3274 single-nucleotide polymorphism (SNP) on TCDD-induced gene expression, pSB-AHR2 was mutated to have an A at nt 3274 in pSB-A-AHR2, and pHR-AHR2 was mutated to have a T at nt 3274 in pHR-T-AHR2. Both pSBAHR2 and pSB-A-AHR2 were effective in driving reporter gene expression in TCDD-treated cells (Fig. 4B). In contrast, TCDD failed to induce significant luciferase expression in either pHRAHR2– or pHR-T-AHR2–transfected c12 cells. This indicates that the 6-bp AHR2 deletion, rather than the nt 3274 SNP, was the basis of resistance to inducible gene expression. The mechanistic basis of variation in sensitivities to polycyclic aromatic hydrocarbons (PAHs) and TCDD between inbred mouse strains (10) and among avian species (9, 11) resulted from one or two amino acid substitutions at critical residues within the AHR LBD that reduce the binding affinities to ligand. We also observed substantially reduced binding affinity for TCDD in resistant tomcod, but this did not result from variation within the LBD. It is possible that the two–amino acid deletion altered the conformation of AHR2 and thus the accessibility of ligand to the LBD. Consistent with this possibility, the region of AHR2 in which this deletion is located

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and nt 3274 substitution in conferring resistance in reporter gene assays. Cells were transfected with pSB-AHR2 (without deletion; T at nt 3274), pHR-AHR2 (with deletion; A at nt 3274), pSB-A-AHR2 (without deletion; A at nt 3274), or pHR-T-AHR2 (with deletion; T at nt 3274) and treated with TCDD (1 ppb) (means T SD of four replicates). DMSO, dimethyl sulfoxide.

is the site of interaction with the immunophilinrelated chaperone AIP/Ara9/XAP2, which stabilizes AHR and enhances its ability to bind ligand and activate transcription (12, 13). Thus, altered AHR protein stability may also contribute to the resistant phenotype. Resistance to TCDD, PCBs, and PAHs has also been observed in populations of Atlantic killifish, Fundulus heteroclitus, from three Atlantic Coast estuaries contaminated with aromatic hydrocarbons (14, 15). Sensitivity to PCB126induced embryonic toxicity and CYP1A expression in F1 and F2 killifish embryos whose parents were collected at different locales varied >10,000 fold and was adaptive to levels of PCBs at their residence sites (16). The resistance response was heritable between the F1 and F2 generation in most but not all killifish populations (16). Resistance in killifish was unrelated to altered expression of components of the AHR pathway (17). AHR1 (18) and AHR2 (19) showed evidence of selection, but variants were functionally indistinguishable in vitro. Multiple mechanisms of resistance to HAHs may exist among fish populations, including mechanisms that are both AHRdependent and AHR-independent. Although our results indicate a primary role for the AHR2 deletion in the resistance of HR tomcod to HAHs, we cannot exclude the possibility that AHRindependent mechanisms may also contribute to the resistant phenotype. Our results indicate that recent evolution has occurred in the HR population as a result of selective pressures. The bioaccumulation of high levels of persistent HAHs, minimal gene flow with populations in nearby estuaries (20), panmixia within the HR, and strong selective pressures lead us to conclude that HR tomcod have experienced rapid evolutionary change in the 50 to 100 years since the release of these contaminants. Our results indicate that resistance can be due to one structural change in the coding region of a single gene and that evolutionary change in anthropogenically challenged natural populations can be rapid. AHR2 is probably a sensitive target for selection because of its regulatory role in the

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metabolism of contaminants and in the activation of most HAH-induced toxicities. It would have been informative to date the onset of resistance in the HR population, but the only archived samples collected before the introduction of contaminants were preserved in formalin, which makes analyses of their DNA problematic. Rapid evolutionary change at AHR2 most likely resulted from selective pressure against sensitive phenotypes of early life stages of tomcod: acute embryo mortality or increased prevalence of malformations that were incompatible with survival. Resistance is believed to be accompanied by evolutionary costs that are manifest either as heightened sensitivity to other stressors or impaired performance in common life history traits (21). We have yet to detect evolutionary costs in resistant tomcod, but given the role of AHR in binding endogenous ligand(s) and its pleiotrophic effects in AHR knockout models, it is likely that costs do exist. References and Notes 1. I. I. Wirgin, R. C. Chambers, in Hudson River Fishes and their Environment, J. R. Waldman, K. E. Limburg, D. Strayer, Eds. (American Fisheries Society Symposium 51, American Fisheries Society, Bethesda, MD, 2006), pp. 331–364. 2. M. P. Fernandez, M. G. Ikonomou, S. C. Courtenay, I. I. Wirgin, Environ. Sci. Technol. 38, 976 (2004). 3. Q. Ma, Curr. Drug Metab. 2, 149 (2001). 4. M. E. Hahn, S. I. Karchner, M. A. Shapiro, S. A. Perera, Proc. Natl. Acad. Sci. U.S.A. 94, 13743 (1997). 5. A. L. Prasch et al., Toxicol. Sci. 76, 138 (2003). 6. Z. Yuan, S. Courtenay, I. Wirgin, Aquat. Toxicol. 76, 306 (2006). 7. I. I. Wirgin et al., Mar. Environ. Res. 34, 103 (1992). 8. Z. Yuan, S. C. Courtenay, R. C. Chambers, I. Wirgin, Environ. Health Perspect. 114, 77 (2006). 9. S. I. Karchner, D. G. Franks, S. W. Kennedy, M. E. Hahn, Proc. Natl. Acad. Sci. U.S.A. 103, 6252 (2006). 10. A. Poland, D. Palen, E. Glover, Mol. Pharmacol. 46, 915 (1994). 11. J. A. Head, M. E. Hahn, S. W. Kennedy, Environ. Sci. Technol. 42, 7535 (2008). 12. J. J. LaPres, E. Glover, E. E. Dunham, M. K. Bunger, C. A. Bradfield, J. Biol. Chem. 275, 6153 (2000). 13. B. K. Meyer, G. H. Perdew, Biochemistry 38, 8907 (1999). 14. P. A. Van Veld, D. E. Nacci, in The Toxicology of FIshes, R. T. Di Giulio, D. E. Hinton, Eds. (CRC Press, Boca Raton, FL, 2008), pp. 597–641.

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Fig. 4. (A) Functional evaluation of AHR2 proteins in reporter gene assays. Expression was quantified in AHR-deficient cells transfected with pSB-AHR2 or pHR-AHR2expressing AHR2 proteins and treated with graded doses of TCDD or PCB126. Values are the ratio of firefly to Renilla luciferase units (means T SD of four replicates). ppb, part per billion. (B) Evaluation of the AHR2 6-bp deletion

REPORTS 20. K. A. Able, M. P. Fahay, The First Year of Life of Estuarine Fishes in the Middle Atlantic Bight (Rutgers Univ. Press, Rutgers, NJ, 1998). 21. N. M. Van Straalen, A. A. Hoffmann, in Demography of Ecotoxicology (Wiley, New York, 2000), pp. 147–161. 22. We acknowledge support of R01ES015447, P42ES007381, ES00260, the Hudson River Foundation, and the Northeast Fisheries Science Center. We thank M. Mattson, K. Sullivan, D. Danila, S. Courtenay, C. Burnett, L. Upchurch, P. Roy, S. Karchner, and A. Nadas for assistance. Accession nos. FJ215751 to FJ215756.

Aging in the Natural World: Comparative Data Reveal Similar Mortality Patterns Across Primates Anne M. Bronikowski,1 Jeanne Altmann,2,3 Diane K. Brockman,4 Marina Cords,5 Linda M. Fedigan,6 Anne Pusey,7 Tara Stoinski,8 William F. Morris,9 Karen B. Strier,10 Susan C. Alberts3,9* Human senescence patterns—late onset of mortality increase, slow mortality acceleration, and exceptional longevity—are often described as unique in the animal world. Using an individual-based data set from longitudinal studies of wild populations of seven primate species, we show that contrary to assumptions of human uniqueness, human senescence falls within the primate continuum of aging; the tendency for males to have shorter life spans and higher age-specific mortality than females throughout much of adulthood is a common feature in many, but not all, primates; and the aging profiles of primate species do not reflect phylogenetic position. These findings suggest that mortality patterns in primates are shaped by local selective forces rather than phylogenetic history. umans are thought to age more slowly than other mammalian taxa [(1), but see (2)] on the basis of their low early-adult mortality, slow mortality acceleration, and long life span. However, it is not known if these human features are unique or are shared with other primates (3, 4). The rapid increase in human life expectancy in the 20th century (5) has increased the proportion of individuals in older age classes (6), raising ques-

H

tions about the flexibility of human aging patterns and the limits of the human life span [e.g., (7–9)]. These questions necessitate a deeper understanding of natural aging patterns in other primates, which represent our closest living relatives (10). Nonhuman primates, like humans, are cognitively and socially complex and behaviorally flexible. However, their long lives and the challenges of continuous, long-term observation make longitudi-

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1197296/DC1 Materials and Methods Fig. S1 Tables S1 to S3 References and Notes

2 September 2010; accepted 26 January 2011 Published online 17 February 2011; 10.1126/science.1197296

nal demographic data on nonhuman primates uncommon, especially for wild populations [(11); see also (12)]. We compiled rare data sets from seven species that span the Primate Order [one Indriid (a Madagascan prosimian), two New World monkeys, two Old World monkeys, and two great apes] and carried out a comparative demographic analysis of mortality. Our analyses used data from 226 observation-years of births and deaths on more than 2800 individually recognized male and female primates (13, 14). We produced species-specific mortality tables for each sex and computed actuarial estimates of age-specific survival and mortality for each of 1 Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, IA 50011, USA. 2Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 88001, USA. 3Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya. 4Department of Anthropology, University of North Carolina, Charlotte, NC 28223, USA. 5Department of Ecology, Evolution and Environmental Biology, Columbia University, New York, NY 10027, USA. 6 Department of Anthropology, University of Calgary, Calgary, T2N 1N4 Canada. 7Department of Evolutionary Anthropology, Duke University, Durham, NC 27708, USA. 8 The Dian Fossey Gorilla Fund International and Zoo Atlanta, Atlanta, GA 30315, USA. 9Department of Biology, Duke University, Durham, NC 27708, USA. 10Department of Anthropology, University of Wisconsin-Madison, Madison, WI 53706, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1. Age-specific mortality at age x, ux, for each study species, illustrating high infant mortality, low juvenile mortality, and mortality increasing with age over the adult life span. No sexspecific first-year mortality estimates are available for sifaka because individuals were not sexed and individually identified until their first birthday. For blue monkey males and both sexes of capuchins and muriquis, mortality estimates extend only through age 6, 20, and 32 years, respectively; in each case, this is much less than the suspected full life span, making it difficult to estimate the shape of the mortality curve at the end of life.

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15. I. Wirgin, J. R. Waldman, Mutat. Res. 55, 1897 (2004). 16. D. E. Nacci, D. Champlin, S. Jayaraman, Estuaries Coasts 33, 853 (2010). 17. W. H. Powell, R. Bright, S. M. Bello, M. E. Hahn, Toxicol. Sci. 57, 229 (2000). 18. M. E. Hahn, S. I. Karchner, D. G. Franks, R. R. Merson, Pharmacogenetics 14, 131 (2004). 19. M. E. Hahn et al., Mechanism of PCB and Dioxin Resistance in Fish in the Hudson River Estuary. Role Of Receptor Polymorphisms (Hudson River Foundation, New York, 2005).

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REPORTS 20. K. A. Able, M. P. Fahay, The First Year of Life of Estuarine Fishes in the Middle Atlantic Bight (Rutgers Univ. Press, Rutgers, NJ, 1998). 21. N. M. Van Straalen, A. A. Hoffmann, in Demography of Ecotoxicology (Wiley, New York, 2000), pp. 147–161. 22. We acknowledge support of R01ES015447, P42ES007381, ES00260, the Hudson River Foundation, and the Northeast Fisheries Science Center. We thank M. Mattson, K. Sullivan, D. Danila, S. Courtenay, C. Burnett, L. Upchurch, P. Roy, S. Karchner, and A. Nadas for assistance. Accession nos. FJ215751 to FJ215756.

Aging in the Natural World: Comparative Data Reveal Similar Mortality Patterns Across Primates Anne M. Bronikowski,1 Jeanne Altmann,2,3 Diane K. Brockman,4 Marina Cords,5 Linda M. Fedigan,6 Anne Pusey,7 Tara Stoinski,8 William F. Morris,9 Karen B. Strier,10 Susan C. Alberts3,9* Human senescence patterns—late onset of mortality increase, slow mortality acceleration, and exceptional longevity—are often described as unique in the animal world. Using an individual-based data set from longitudinal studies of wild populations of seven primate species, we show that contrary to assumptions of human uniqueness, human senescence falls within the primate continuum of aging; the tendency for males to have shorter life spans and higher age-specific mortality than females throughout much of adulthood is a common feature in many, but not all, primates; and the aging profiles of primate species do not reflect phylogenetic position. These findings suggest that mortality patterns in primates are shaped by local selective forces rather than phylogenetic history. umans are thought to age more slowly than other mammalian taxa [(1), but see (2)] on the basis of their low early-adult mortality, slow mortality acceleration, and long life span. However, it is not known if these human features are unique or are shared with other primates (3, 4). The rapid increase in human life expectancy in the 20th century (5) has increased the proportion of individuals in older age classes (6), raising ques-

H

tions about the flexibility of human aging patterns and the limits of the human life span [e.g., (7–9)]. These questions necessitate a deeper understanding of natural aging patterns in other primates, which represent our closest living relatives (10). Nonhuman primates, like humans, are cognitively and socially complex and behaviorally flexible. However, their long lives and the challenges of continuous, long-term observation make longitudi-

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1197296/DC1 Materials and Methods Fig. S1 Tables S1 to S3 References and Notes

2 September 2010; accepted 26 January 2011 Published online 17 February 2011; 10.1126/science.1197296

nal demographic data on nonhuman primates uncommon, especially for wild populations [(11); see also (12)]. We compiled rare data sets from seven species that span the Primate Order [one Indriid (a Madagascan prosimian), two New World monkeys, two Old World monkeys, and two great apes] and carried out a comparative demographic analysis of mortality. Our analyses used data from 226 observation-years of births and deaths on more than 2800 individually recognized male and female primates (13, 14). We produced species-specific mortality tables for each sex and computed actuarial estimates of age-specific survival and mortality for each of 1 Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, IA 50011, USA. 2Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 88001, USA. 3Institute of Primate Research, National Museums of Kenya, Nairobi, Kenya. 4Department of Anthropology, University of North Carolina, Charlotte, NC 28223, USA. 5Department of Ecology, Evolution and Environmental Biology, Columbia University, New York, NY 10027, USA. 6 Department of Anthropology, University of Calgary, Calgary, T2N 1N4 Canada. 7Department of Evolutionary Anthropology, Duke University, Durham, NC 27708, USA. 8 The Dian Fossey Gorilla Fund International and Zoo Atlanta, Atlanta, GA 30315, USA. 9Department of Biology, Duke University, Durham, NC 27708, USA. 10Department of Anthropology, University of Wisconsin-Madison, Madison, WI 53706, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1. Age-specific mortality at age x, ux, for each study species, illustrating high infant mortality, low juvenile mortality, and mortality increasing with age over the adult life span. No sexspecific first-year mortality estimates are available for sifaka because individuals were not sexed and individually identified until their first birthday. For blue monkey males and both sexes of capuchins and muriquis, mortality estimates extend only through age 6, 20, and 32 years, respectively; in each case, this is much less than the suspected full life span, making it difficult to estimate the shape of the mortality curve at the end of life.

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15. I. Wirgin, J. R. Waldman, Mutat. Res. 55, 1897 (2004). 16. D. E. Nacci, D. Champlin, S. Jayaraman, Estuaries Coasts 33, 853 (2010). 17. W. H. Powell, R. Bright, S. M. Bello, M. E. Hahn, Toxicol. Sci. 57, 229 (2000). 18. M. E. Hahn, S. I. Karchner, D. G. Franks, R. R. Merson, Pharmacogenetics 14, 131 (2004). 19. M. E. Hahn et al., Mechanism of PCB and Dioxin Resistance in Fish in the Hudson River Estuary. Role Of Receptor Polymorphisms (Hudson River Foundation, New York, 2005).

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the primate populations (15). Analysis of mortality rates revealed the expected pattern for mammals: high infant mortality, followed by a period of low mortality during the juvenile stage, and an extended period of increasing age-specific mortality during mid to late life (Fig. 1). We focused on mid- to late-life demography and modeled initial mortality rate at the start of adulthood for each species, defined in Table 1, through the last age interval for which we had census data. For humans, we used published male and female age-atdeath data, from age 15 through 100 years, from the U.S. Department of Health and Human Services life tables (16) and repeated the analyses with a second, independent life table for humans (17), which confirmed our findings. Understanding flexibility and constraints in the expression and evolution of aging requires a careful analysis of key aging metrics (1, 18, 19). We used a maximum-likelihood framework for estimating two metrics that, together, describe the pattern of senescence for a population: the initial adult mortality rate (IMR, the risk of death at onset of adulthood) and the rate of aging (RoA, the

rate of increase in the age-specific mortalities with advancing adult age). These aging metrics are often best estimated by fitting the Gompertz model of increasing failure time. We thus tested among competing models for accelerating risk of death with advancing age on the basis of the Gompertz family of models in program WinModest (20) model fitting as described in (21). Our tests included a standard two-parameter Gompertz model and the Gompertz-Makeham and Logistic models. In all but 2 of the 13 species and sex comparisons we examined, the standard twoparameter Gompertz model yielded the best fit to the nonhuman primate data. In the other two cases (sifaka females and capuchin males), the Gompertz-Makeham model was recommended, but because of particular features of those two data sets (see table S1), we proceeded with the standard Gompertz model for males and females of all species. Our model was of the form ux = IMR × e(RoA)x, where ux is the age-specific mortality, i.e., instantaneous mortality probability, at age x (results in Tables 2 and 3 for females and males, respectively).

We found significantly positive values for RoA in all study species, indicating that mortality rate increased with advancing age [Tables 2 and 3 and fig. S1; see also (22, 23)]. Notably, humans fell along a continuum with the other primate species for both IMR and RoA (Fig. 2). Furthermore, in neither females nor males did we find evidence of a negative correlation between IMR and RoA, which would be indicative of a trade-off between these two parameters (Fig. 2). Instead, our data suggest that they can evolve independently. Humans had low values for both parameters, which explains their exceptional longevity. For females, we identified four distinct groups of IMR across the eight species (Fig. 2A). All species comparisons were computed on the basis of c2 tests of pairwise comparisons of the loglikelihood ratio of models with unique versus identical Gompertz parameters (table S2). We identified three significant groups for RoA (Fig. 2A). The coefficient of variation among species for female IMR was 111%, much greater than that for RoA, which was 30%; females of these primate species exhibited a wide range of IMR values,

Table 1. Summary of study populations. Details about and references for study sites are in (15). Common name

Species

Family

Country

Avg. annual rainfall (mm)*

Start Sample size year † M F

Life-style

Sifaka Propithecus verreauxi Indriidae Madagascar 578 Arboreal 1984 Northern Brachyteles hypoxanthus Atelidae Brazil 1180 Arboreal 1983 Muriqui Capuchin Cebus capucinus Cebidae Costa Rica 1736 Arboreal 1983 Yellow Papio cynocephalus Cercopithecidae Kenya 347 Semi-terrestrial 1971 baboon Blue monkey Cercopithecus mitis Cercopithecidae Kenya 1962 Arboreal 1979 Chimpanzee Pan troglodytes Hominidae Tanzania 1330 Semi-terrestrial 1963 Gorilla Gorilla beringei Hominidae Rwanda 1358 Terrestrial 1967

Adult age‡ M

F

Mean age Predominant of first dispersing dispersal sex (years)

291 192

219 212

5–6 6–7

6–7 8–9

M F

4–5 6–7

98 489

58 437

6–7 7–8

6–7 5–6

M¶ M

4–5 7–8

128 122 128

194 8–9 7–8 144 14–15 14–15 120 15–16 9–10

M F M# F

7–8 12–13 15–16 7–8

*Average annual rainfall for each study, representative of the study years. Rainfall data for gorillas were collected by the Rwandan Government Meteorological Office at a location several kilometers from the field site and at a lower elevation. Rainfall data for other studies were collected at the study site. †Year study was established. Latest census date for all populations in these analyses was December 2008. ‡Mean age class at which adulthood is attained for each sex. Male onset of adult stage was defined as mean age of likely first reproduction (using physical criteria such as copulation with ejaculation, behavioral criteria such as the onset of mate guarding behavior, or genetically confirmed paternity). Female onset of adult stage is defined as the mean age of first live birth. ¶Twelve percent of female capuchins disperse. The average age interval of dispersing capuchin females is 6 to 7 years. #Both sexes disperse in gorillas.

Table 2. Gompertz estimates of female mortality parameters and life-span summary statistics. Adult age interval is the age interval containing the mean age of first live birth; IMR (= Gompertz a) is the Gompertz estimate of instantaneous mortality rate at the first adult age interval [with its 95% confidence

Species Sifaka Muriqui Capuchin Baboon Blue monkey Chimpanzee Gorilla Human‡

Adult age interval (years) 6–7 8–9 6–7 5–6 7–8 14–15 9–10

IMR (/year) 0.0278 0.00170 0.0415 0.0285 0.00723 0.00774 0.00028 0.00009

95% CI [0.019, [0.00042, [0.0150, [0.020, [0.00367, [0.0038, [0.00004, [0.00008,

0.0410] 0.00685] 0.114] 0.040] 0.0143] 0.0156] 0.00214] 0.00009]

RoA 0.0991 0.129 0.165 0.123 0.160 0.0992 0.211 0.0961

interval (CI)]; RoA (= Gompertz b) is the adult rate of aging estimated with Gompertz acceleration (with its 95% CI); MRDT is the mortality rate doubling time during adulthood; Oldest age reached is the age class of the oldest observed individual; Median age is the 50% survival age with its range. MRDT (no. of years)

95% CI [0.072, [0.0722, [0.055, [0.0926, [0.123, [0.070, [0.148, [0.0956,

0.136] 0.230] 0.494] 0.165] 0.209] 0.140] 0.300] 0.0967]

7.0 5.4 4.2 5.6 4.3 7.0 3.3 7.2

Oldest age reached (years) Estimated

Known*

31–32 40–41 26–27 27–28 33–34 53–54 43–44 100+

23–24 26–27 19–20† 27–28 26–27 38–39 38–39 100+

Median age 10 25 11 8 18 16 33 83.5

*Oldest individual with known date of birth. †Truncated at 18–19 for mortality analysis because of relatively smaller sample sizes of deaths and transitions in later age classes. from (16), modeled beginning at age interval 15–16 years through 99–100 years.

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[Range] [9, [18, [10, [7, [17, [10, [31,

12] 33] 13] 9] 22] 25] 35]

‡Data

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whereas RoA was less variable (equality of variance test: F7,7 = 6.44, P = 0.02). Moreover, all combinations of high and low IMR with high and low RoA were found in the females of the seven nonhuman species. For example, female chimpanzees were characterized by both low IMR and low RoA, whereas female sifaka exhibited high IMR but relatively low RoA. In contrast, female gorillas had low IMR and high RoA, while female capuchins exhibited both high IMR and high RoA. The RoA for human females was statistically indistinguishable from that of the four other slowly aging female primates (Fig. 2A and table S2). Human females had one of the two lowest IMRs (statistically indistinguishable from gorilla; Fig. 2A and table S2), but this trait is arguably more reflective of environmental plasticity than is RoA (24). This similarity between humans and nonhuman primates indicates that aging in humans is not evolutionarily divergent from that in other

primate species [see also (1)]. This similarity is particularly noteworthy given that our humannonhuman comparison was a conservative one, in that it used data from modern human populations rather than hunter-foragers or historical populations [which might resemble wild nonhuman primates more than modern humans do (23, 25)]. Among males, the coefficient of variation for IMR was 107%, much greater than the coefficient of variation in RoA, which was 40% (equality of variance F6,6 = 26.0, P = 0.001). Males and females showed similar variation in IMR, but males showed greater variation than females in RoA. Males exhibited fewer combinations of IMR and RoA than females: Baboon, sifaka, and capuchin males were characterized by high IMR and high RoA, whereas gorilla, muriqui, and chimpanzee males had intermediate IMR and intermediate RoA. Like females, males exhibited four significant groupings of IMR and three signif-

Table 3. Gompertz estimates of male mortality and life-span summary statistics. Adult age interval is the mean age class of likely first reproduction. IMR (= Gompertz a) is the instantaneous mortality at adulthood (with its 95%CI); RoA (= Gompertz b) is the rate of aging estimated with Species †Sifaka Muriqui Capuchin†‡ Baboon†‡ Blue monkey Chimpanzee Gorilla Human||

Adult age interval (years) 5–6 6–7 6–7 7–8 8–9 14–15 15–16

IMR (/year) 0.0201 0.00187 0.010 0.0371 No est. 0.00787 0.00594 0.00024

95% CI [0.0140 0.0290] [0.00044, 0.00784] [0.0027, 0.036] [0.0266, 0.0517] [0.00346, 0.0179] [0.00139, 0.0254] [0.00023, 0.00025]

Gompertz acceleration (with its 95% CI); MRDT is the mortality rate doubling time during adulthood; Oldest age reached is the age class of the oldest observed individual; Median age is the 50% survival age with its range.

RoA 0.186 0.148 0.294 0.213 No est. 0.137 0.182 0.086

icant groupings of RoA (Fig. 2B and table S2). RoA in human males, unlike in human females, was significantly lower than the next closest value, that of chimpanzees, and the IMR for human males was relatively even lower (Fig. 2B). Males of monogamous animal species tend to age at rates similar to those of females, whereas males of polygynous species exhibit increased aging rates relative to females (26, 27). All of the nonhuman primate species studied here are polygynous (or more accurately polygynandrous, as multiple mating is exhibited by females as well as males). Further, six of the seven experience relatively intense male-male competition for access to mates [see (28) for genus-level data on Cebus, Cercopithecus, Gorilla, Papio, and Pan; (29) for data on Propithecus]. The exception is the muriqui, a sexually monomorphic species in which malemale competition for access to females appears to be absent (30). In the species with relatively

95% CI [0.155, [0.0820, [0.159, [0.177,

0.222] 0.266] 0.542] 0.256]

[0.0980, 0.190] [0.106, 0.313] [0.0854, 0.0863]

MRDT (no. of years) 3.73 4.70 2.36 3.26 5.07 3.81 8.07

Oldest age reached (years) Estimated

Known*

26–27 33–34 24–25¶ 24–25 19–20 43–44 38–39 100+

19–20 26–27 12–13 22–23 19–20 40–41 34–35 100+

Median age 12 24 4 10 No est. 11 23 79.5

[Range] [12, [22, [3, [9,

13] 27] 5] 11]

[9, 14] [22, 29]

*Oldest individual with known date of birth. †Distribution of deaths imputed from onset of adulthood with age structure. See methods in the Supporting Online Material (SOM). ‡Age structure corrected for population growth. See methods in the SOM. ¶Truncated at 18–19 for mortality analysis because of relatively smaller samples sizes of deaths and transitions in later age classes. ||Data from (16), modeled beginning at age interval 15–16 years through 99–100 years.

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Fig. 2. IMR versus RoA for (A) females and (B) males. Phylogenetic relationships among species are shown in (C). Letters over bars denote statistically significant groupings. [Female IMR: human, gorilla (A) ≤ gorilla, muriqui (B) < blue monkey, chimpanzee (C) < sifaka, baboon, capuchin (D); female RoA: human, chimpanzee, sifaka, baboon, muriqui (A) ≤ muriqui, blue monkey, capuchin (B) ≤ blue monkey, capuchin, gorilla (C); male IMR: human (A) < muriqui, gorilla, chimpanzee, capuchin (B) ≤ capuchin, sifaka (C) < baboon (D); male RoA: human (A) < chimpanzee, muriqui, gorilla, sifaka (B) ≤ muriqui, gorilla, sifaka, baboon, capuchin (C).] See table S2 for tests of pairwise comparisons of IMR and RoA.

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intense male-male competition for mates, males and females showed significant differences in either IMR or RoA, and male life span was shorter than female life span (baboons, sifaka, gorillas, chimpanzees, and capuchins; we lacked mortality data for male blue monkeys; see Fig. 1 and table S3). In contrast, male and female muriquis were indistinguishable in their IMRs, RoAs, and life spans (table S3). This male-female similarity in muriqui aging patterns, combined with the observation of multiple mating by both sexes in all of our study species, suggests that the male-male competitive environment, not just multiple mating by males, may be a key factor driving faster aging in males in polygynandrous species [see also (26)]. If demographic patterns of aging were evolutionarily constrained, we would expect closely related species of primates to exhibit similar aging patterns. Instead, the species rankings of IMR and RoA in males and females showed no relationship to phylogeny (Fig. 2C and fig. S1). This implies that the study species have not been constrained phylogenetically to high or low aging rates, and have the flexibility to respond to evolutionary forces at the species level or potentially even the local population level. Furthermore, within-species comparisons of baboons (31), chimpanzees (23, 32), and humans (23, 25) all support the view that both IMR and RoA can vary substantially among populations within a species. Notably, in all three species, populations existing in more demanding habitats, without benefit of modern medical intervention (e.g., hunter-forager humans and wild as opposed to captive primates), exhibit higher IMR and, for both chimpanzees and humans, higher RoA. That is, aging appears to be both evolutionarily labile and phenotypically plastic. The slowing of aging-related disease under dietary restriction (33) is further evidence of the flexibility of aging rates in primates. We examined our data for the existence of mortality plateaus (34), a subject of much recent interest in the aging literature, but none of the age-specific mortality relationships in our nonhuman primate analyses demonstrated the type of leveling off that has been shown in human and fly data sets [e.g., (35)]. Whether additional long-term data from natural primate populations will demonstrate a generalized mortality deceleration in old age remains an open question that should motivate future comparative analyses of aging in other natural populations. References and Notes 1. C. E. Finch, M. C. Pike, M. Witten, Science 249, 902 (1990). 2. R. E. Ricklefs, Proc. Natl. Acad. Sci. U.S.A. 107, 10314 (2010). 3. C. Finch, Longevity, Senescence and the Genome (Univ. of Chicago Press, Chicago, 1990). 4. G. A. Sacher, in Handbook of the Biology of Aging, C. E. Finch, L. Hayflick, Eds. (Van Nostrand Reinhold, New York, 1977), pp. 582–638. 5. W. C. Sanderson, S. Scherbov, Science 329, 1287 (2010). 6. W. Lutz, W. Sanderson, S. Scherbov, Nature 451, 716 (2008).

1328

7. N. S. Gavrilova et al., Hum. Biol. 70, 799 (1998). 8. J. Evert, E. Lawler, H. Bogan, T. Perls, J. Gerontol. A Biol. Sci. Med. Sci. 58, 232 (2003). 9. R. G. J. Westendorp, T. B. L. Kirkwood, Nature 396, 743 (1998). 10. T. B. L. Kirkwood, Nature 451, 644 (2008). 11. A. K. Brunet-Rossinni, S. N. Austad, in Handbook of the Biology of Aging, E. J. Masoro, S. N. Austad, Eds. (Elsevier, Amsterdam, 2006), pp. 243–266. 12. T. Clutton-Brock, B. C. Sheldon, Science 327, 1207 (2010). 13. W. F. Morris et al., Am. Nat. 177, E14 (2011). 14. K. B. Strier et al., Methods Ecol. Evol. 1, 199 (2010). 15. Supporting material is provided on Science Online. 16. E. Arias, United States life tables, 2004. National Vital Statistics Reports (National Center for Health Statistics, Hyattsville, MD, 2007), vol. 56. 17. R. N. Anderson, United States life tables, 1997. National Vital Statistics Reports (National Center for Health Statistics, Hyattsville, MD, 1999), vol. 47. 18. K. W. Wachter, Popul. Dev. Rev. 29 (suppl.), 270 (2003). 19. K. W. Wachter, C. E. Finch, Eds., Between Zeus and the Salmon: The Biodemography of Longevity (National Academy Press, Washington, DC, 1997). 20. S. D. Pletcher, J. Evol. Biol. 12, 430 (1999). 21. A. M. Bronikowski, D. E. L. Promislow, Trends Ecol. Evol. 20, 271 (2005). 22. T. B. Gage, Annu. Rev. Anthropol. 27, 197 (1998). 23. K. Hawkes, K. R. Smith, S. L. Robson, Am. J. Hum. Biol. 21, 578 (2009). 24. D. E. L. Promislow, M. Tatar, A. A. Khazaeli, J. W. Curtsinger, Genetics 143, 839 (1996). 25. M. Gurven, H. Kaplan, Popul. Dev. Rev. 33, 321 (2007). 26. T. H. Clutton-Brock, K. Isvaran, Proc. Biol. Sci. 274, 3097 (2007). 27. J. Allman, A. Rosin, R. Kumar, A. Hasenstaub, Proc. Natl. Acad. Sci. U.S.A. 95, 6866 (1998).

28. J. C. Mitani, J. Gros-Louis, A. F. Richard, Am. Nat. 147, 966 (1996). 29. R. R. Lawler, A. F. Richard, M. A. Riley, J. Hum. Evol. 48, 259 (2005). 30. K. B. Strier, Behaviour 130, 151 (1994). 31. A. M. Bronikowski et al., Proc. Natl. Acad. Sci. U.S.A. 99, 9591 (2002). 32. K. Hill et al., J. Hum. Evol. 40, 437 (2001). 33. R. J. Colman et al., Science 325, 201 (2009). 34. J. W. Vaupel et al., Science 280, 855 (1998). 35. R. Rau, E. Soroko, D. Jasilionis, J. W. Vaupel, Popul. Dev. Rev. 34, 747 (2008). 36. The National Evolutionary Synthesis Center (NESCent) and the National Center for Ecological Analysis and Synthesis (NCEAS) jointly supported the Primate Life Histories Working Group. H. Lapp and X. Liu at NESCent provided expert assistance in designing and implementing the Primate Life Histories Database (PLHD). J. Moorad commented on the manuscript. The governments of Brazil, Costa Rica, Kenya, Madagascar, Rwanda, and Tanzania provided permission for our field studies, and all research complied with guidelines in the host countries. For study-specific acknowledgments and Institutional Animal Care and Use Committee compliance, see http://demo.plhdb.org. The nonhuman primate data used in these analyses are available in the Dryad database (http://dx.doi.org/10.5061/dryad.8682).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1325/DC1 Methods Figs. S1 to S8 Tables S1 to S10 References 13 December 2010; accepted 1 February 2011 10.1126/science.1201571

Positive Supercoiling of Mitotic DNA Drives Decatenation by Topoisomerase II in Eukaryotes J. Baxter,1,4* N. Sen,1† V. López Martínez,2† M. E. Monturus De Carandini,2† J. B. Schvartzman,2 J. F. X. Diffley,3 L. Aragón1* DNA topoisomerase II completely removes DNA intertwining, or catenation, between sister chromatids before they are segregated during cell division. How this occurs throughout the genome is poorly understood. We demonstrate that in yeast, centromeric plasmids undergo a dramatic change in their topology as the cells pass through mitosis. This change is characterized by positive supercoiling of the DNA and requires mitotic spindles and the condensin factor Smc2. When mitotic positive supercoiling occurs on decatenated DNA, it is rapidly relaxed by topoisomerase II. However, when positive supercoiling takes place in catenated plasmid, topoisomerase II activity is directed toward decatenation of the molecules before relaxation. Thus, a topological change on DNA drives topoisomerase II to decatenate molecules during mitosis, potentially driving the full decatenation of the genome. n eukaryotes, most topological links between the DNA strands are removed during DNA replication by topoisomerases I and II (fig. S1A) (1). However, many links are converted into double-stranded DNA intertwines or catenanes during the completion of replication (2, 3). These can only be resolved by topoisomerase II (fig. S1) (4). Passage through mitosis is required for complete decatenation, because topoisomerase II activity is essential during mitosis as late as anaphase (fig.

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S1B) (4–6). Because mitotic spindles are required to complete decatenation, it is assumed to occur only after chromosome segregation during anaphase (4). However, sister chromatids appear to be fully decatenated before their physical separation by spindles (7, 8). Potential alternate mechanisms, in which decatenation is promoted by supercoiling, have been proposed in prokaryotes (9–11). These mechanisms prompted us to study whether mitotic changes to DNA topology help drive decatenation in eukaryotes.

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intense male-male competition for mates, males and females showed significant differences in either IMR or RoA, and male life span was shorter than female life span (baboons, sifaka, gorillas, chimpanzees, and capuchins; we lacked mortality data for male blue monkeys; see Fig. 1 and table S3). In contrast, male and female muriquis were indistinguishable in their IMRs, RoAs, and life spans (table S3). This male-female similarity in muriqui aging patterns, combined with the observation of multiple mating by both sexes in all of our study species, suggests that the male-male competitive environment, not just multiple mating by males, may be a key factor driving faster aging in males in polygynandrous species [see also (26)]. If demographic patterns of aging were evolutionarily constrained, we would expect closely related species of primates to exhibit similar aging patterns. Instead, the species rankings of IMR and RoA in males and females showed no relationship to phylogeny (Fig. 2C and fig. S1). This implies that the study species have not been constrained phylogenetically to high or low aging rates, and have the flexibility to respond to evolutionary forces at the species level or potentially even the local population level. Furthermore, within-species comparisons of baboons (31), chimpanzees (23, 32), and humans (23, 25) all support the view that both IMR and RoA can vary substantially among populations within a species. Notably, in all three species, populations existing in more demanding habitats, without benefit of modern medical intervention (e.g., hunter-forager humans and wild as opposed to captive primates), exhibit higher IMR and, for both chimpanzees and humans, higher RoA. That is, aging appears to be both evolutionarily labile and phenotypically plastic. The slowing of aging-related disease under dietary restriction (33) is further evidence of the flexibility of aging rates in primates. We examined our data for the existence of mortality plateaus (34), a subject of much recent interest in the aging literature, but none of the age-specific mortality relationships in our nonhuman primate analyses demonstrated the type of leveling off that has been shown in human and fly data sets [e.g., (35)]. Whether additional long-term data from natural primate populations will demonstrate a generalized mortality deceleration in old age remains an open question that should motivate future comparative analyses of aging in other natural populations. References and Notes 1. C. E. Finch, M. C. Pike, M. Witten, Science 249, 902 (1990). 2. R. E. Ricklefs, Proc. Natl. Acad. Sci. U.S.A. 107, 10314 (2010). 3. C. Finch, Longevity, Senescence and the Genome (Univ. of Chicago Press, Chicago, 1990). 4. G. A. Sacher, in Handbook of the Biology of Aging, C. E. Finch, L. Hayflick, Eds. (Van Nostrand Reinhold, New York, 1977), pp. 582–638. 5. W. C. Sanderson, S. Scherbov, Science 329, 1287 (2010). 6. W. Lutz, W. Sanderson, S. Scherbov, Nature 451, 716 (2008).

1328

7. N. S. Gavrilova et al., Hum. Biol. 70, 799 (1998). 8. J. Evert, E. Lawler, H. Bogan, T. Perls, J. Gerontol. A Biol. Sci. Med. Sci. 58, 232 (2003). 9. R. G. J. Westendorp, T. B. L. Kirkwood, Nature 396, 743 (1998). 10. T. B. L. Kirkwood, Nature 451, 644 (2008). 11. A. K. Brunet-Rossinni, S. N. Austad, in Handbook of the Biology of Aging, E. J. Masoro, S. N. Austad, Eds. (Elsevier, Amsterdam, 2006), pp. 243–266. 12. T. Clutton-Brock, B. C. Sheldon, Science 327, 1207 (2010). 13. W. F. Morris et al., Am. Nat. 177, E14 (2011). 14. K. B. Strier et al., Methods Ecol. Evol. 1, 199 (2010). 15. Supporting material is provided on Science Online. 16. E. Arias, United States life tables, 2004. National Vital Statistics Reports (National Center for Health Statistics, Hyattsville, MD, 2007), vol. 56. 17. R. N. Anderson, United States life tables, 1997. National Vital Statistics Reports (National Center for Health Statistics, Hyattsville, MD, 1999), vol. 47. 18. K. W. Wachter, Popul. Dev. Rev. 29 (suppl.), 270 (2003). 19. K. W. Wachter, C. E. Finch, Eds., Between Zeus and the Salmon: The Biodemography of Longevity (National Academy Press, Washington, DC, 1997). 20. S. D. Pletcher, J. Evol. Biol. 12, 430 (1999). 21. A. M. Bronikowski, D. E. L. Promislow, Trends Ecol. Evol. 20, 271 (2005). 22. T. B. Gage, Annu. Rev. Anthropol. 27, 197 (1998). 23. K. Hawkes, K. R. Smith, S. L. Robson, Am. J. Hum. Biol. 21, 578 (2009). 24. D. E. L. Promislow, M. Tatar, A. A. Khazaeli, J. W. Curtsinger, Genetics 143, 839 (1996). 25. M. Gurven, H. Kaplan, Popul. Dev. Rev. 33, 321 (2007). 26. T. H. Clutton-Brock, K. Isvaran, Proc. Biol. Sci. 274, 3097 (2007). 27. J. Allman, A. Rosin, R. Kumar, A. Hasenstaub, Proc. Natl. Acad. Sci. U.S.A. 95, 6866 (1998).

28. J. C. Mitani, J. Gros-Louis, A. F. Richard, Am. Nat. 147, 966 (1996). 29. R. R. Lawler, A. F. Richard, M. A. Riley, J. Hum. Evol. 48, 259 (2005). 30. K. B. Strier, Behaviour 130, 151 (1994). 31. A. M. Bronikowski et al., Proc. Natl. Acad. Sci. U.S.A. 99, 9591 (2002). 32. K. Hill et al., J. Hum. Evol. 40, 437 (2001). 33. R. J. Colman et al., Science 325, 201 (2009). 34. J. W. Vaupel et al., Science 280, 855 (1998). 35. R. Rau, E. Soroko, D. Jasilionis, J. W. Vaupel, Popul. Dev. Rev. 34, 747 (2008). 36. The National Evolutionary Synthesis Center (NESCent) and the National Center for Ecological Analysis and Synthesis (NCEAS) jointly supported the Primate Life Histories Working Group. H. Lapp and X. Liu at NESCent provided expert assistance in designing and implementing the Primate Life Histories Database (PLHD). J. Moorad commented on the manuscript. The governments of Brazil, Costa Rica, Kenya, Madagascar, Rwanda, and Tanzania provided permission for our field studies, and all research complied with guidelines in the host countries. For study-specific acknowledgments and Institutional Animal Care and Use Committee compliance, see http://demo.plhdb.org. The nonhuman primate data used in these analyses are available in the Dryad database (http://dx.doi.org/10.5061/dryad.8682).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1325/DC1 Methods Figs. S1 to S8 Tables S1 to S10 References 13 December 2010; accepted 1 February 2011 10.1126/science.1201571

Positive Supercoiling of Mitotic DNA Drives Decatenation by Topoisomerase II in Eukaryotes J. Baxter,1,4* N. Sen,1† V. López Martínez,2† M. E. Monturus De Carandini,2† J. B. Schvartzman,2 J. F. X. Diffley,3 L. Aragón1* DNA topoisomerase II completely removes DNA intertwining, or catenation, between sister chromatids before they are segregated during cell division. How this occurs throughout the genome is poorly understood. We demonstrate that in yeast, centromeric plasmids undergo a dramatic change in their topology as the cells pass through mitosis. This change is characterized by positive supercoiling of the DNA and requires mitotic spindles and the condensin factor Smc2. When mitotic positive supercoiling occurs on decatenated DNA, it is rapidly relaxed by topoisomerase II. However, when positive supercoiling takes place in catenated plasmid, topoisomerase II activity is directed toward decatenation of the molecules before relaxation. Thus, a topological change on DNA drives topoisomerase II to decatenate molecules during mitosis, potentially driving the full decatenation of the genome. n eukaryotes, most topological links between the DNA strands are removed during DNA replication by topoisomerases I and II (fig. S1A) (1). However, many links are converted into double-stranded DNA intertwines or catenanes during the completion of replication (2, 3). These can only be resolved by topoisomerase II (fig. S1) (4). Passage through mitosis is required for complete decatenation, because topoisomerase II activity is essential during mitosis as late as anaphase (fig.

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S1B) (4–6). Because mitotic spindles are required to complete decatenation, it is assumed to occur only after chromosome segregation during anaphase (4). However, sister chromatids appear to be fully decatenated before their physical separation by spindles (7, 8). Potential alternate mechanisms, in which decatenation is promoted by supercoiling, have been proposed in prokaryotes (9–11). These mechanisms prompted us to study whether mitotic changes to DNA topology help drive decatenation in eukaryotes.

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wt

pRS316

20

0

80

60

40

80

20

0

100

pRS316

time after release (mins)

120

120

time after release (mins)

top2-td

pRS316

time after release (mins)

120

top2-td

pRS316

100

wt

100

A

pRS316

60 40

CatC

20 OCm CatC*

0 o

25 C exp 1C 2C

1C 2C 100 80 60 40 20 0

CCCm

0 20 40 60 80 100 120

native conditions

0 20 40 60 80 100 120

% of cells in population

time after release 37°C

B

anaphase nucleus budded single nucleus separated nuclei

top2-td cdc20-td pRS316

+ NOCODAZOLE

top2-td cdc20-td pRS316

120

100

80

60

40

20

120

+ NOCODAZOLE

100

80

60

40

0

120

20

time after release (mins)

pRS316

100

time after release (mins)

80 CatC

60 40

OCm

20 0

CatC*

1C 2C

o

25 C exp 1C 2C

+ NOCODAZOLE

CCCm

native conditions

0 20 40 60 80 100 120

100 % of cells 80 60 in population 40 20 0

0 20 40 60 80 100 120

Fig. 1. Mitosis and spindle formation induce a topological change in catenated plasmid DNA in vivo. (A) WT pRS316 and top2-td pRS316 cells were synchronized in G1 and top2-td-depleted of Top2. The cells were synchronously released into the cell cycle, and regular samples were taken for analysis. (Histograms at left) Fluorescence-activated cell sorting analysis of DNA content and the histogram of kinetics of nuclear division and budding index. (Right panel) Autoradiogram of a blot containing cellular DNA electrophoretically resolved in agarose and probed for pRS316 sequences. Electrophoretic mobilities of CCCm (covalently closed circular monomers), OCm (open circle monomers), and CatC (covalently closed catenated dimers) are indicated. The region where the topologicallymodifiedCatC-CatC* resolve is also indicated. (B) Double degron cdc20td top2-td pRS316 strains were processed as in (A), but were arrested before anaphase entry by either depletion of Cdc20 or treatment with nocodazole, in addition to Cdc20 depletion.

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*To whom correspondence should be addressed. E-mail: [email protected] (L.A.); [email protected] (J.B.) †These authors contributed equally to this work.

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1 Cell Cycle Group, Medical Research Council (MRC) Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. 2Department of Cell Proliferation and Development, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. 3 Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD, UK. 4Genome Damage and Stability Centre, Science Park Road, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, UK.

vitro topoisomerase Ib treatment confirmed that the catenated plasmids were topoisomers of the CatC dimers that formed after replication (fig. S6A). We refer to the premitotic form as C-type catenanes (CatC) and to the mitotic form as C*type catenanes (CatC*). The transition from CatC to CatC* coincided with chromosome segregation. In cells arrested in metaphase by depletion of the anaphase-promoting– complex activator Cdc20 (top2-td cdc20-td), we observed the CatC-to-CatC* transition, which indicates that it occurred before anaphase onset (Fig. 1B). We detected CatC* dimers until cells exited mitosis (fig. S3), demonstrating that the CatC* catenanes occur when both mitotic cyclindependent kinases are active and mitotic spindles are present. However, CatC* dimer formation did not take place in cells arrested in metaphase with the microtubule-depolymerizing drug nocodazole

shortly after DNA replication in the presence of wild-type (WT) levels of topoisomerase II (Fig. 1A). Therefore, we used the top2-td degron strain (6) to ensure an enriched population of catenated DNA that could be examined through the later stages of the cell cycle. Passage through DNA replication in the absence of topoisomerase II converted the supercoiled monomer pRS316 plasmid (CCCm) into catenated plasmid dimer (CatC). Both CCCm and CatC were negatively supercoiled, consistent with normal chromatinization (6). Assaying the electrophoretic mobility of these plasmids throughout the rest of the cell cycle showed that their mobility changed significantly between 40 and 80 min as the cells started entering anaphase (Fig. 1A). Chloroquine intercalation confirmed that the catenated plasmids were still composed of intact plasmids (fig. S2), and in

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We assayed in vivo DNA topology with circular plasmids, which retain topological information after DNA extraction (12). We used yeast plasmids that contain yeast-origin and centromeric sequences whose segregation behavior generally mimics that of endogenous chromosomes. However, these small plasmids are mostly decatenated

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in vivo topoisomerases are extremely efficient in relaxing DNA topological perturbations. But depletion of topoisomerase II, which is used to generate catenated DNA, will also reduce the supercoiling relaxation activity of the cell. This could possibly stabilize induced positive supercoiling, promoting its detection in top2 depleted cells. To investigate this, we repeated the experiment but depleted Top2 specifically during the period when mitotic spindles were reformed. We then observed that a substantial proportion of the negatively supercoiled monomer plasmids observed in nocodazole became highly positively supercoiled when spindles formed (Fig. 2C, bottom panel). Therefore, a potent positive supercoiling activity is induced on both decatenatedmonomer (Fig. 2C) and catenated-dimer plasmids (Fig. 2, A and B, and fig. S6) during mitosis. However, the induced positive supercoiling is normally rapidly relaxed by topoisomerase II (Fig. 2C). Mitotic spindles are required to complete topoisomerase II–mediated decatenation of chromosomes during mitosis (4, 5). Here, we demonstrate that in mitosis, spindles induce a potent positive supercoiling activity on plasmid DNA. In prokaryotes, intrachromosomal supercoiling promotes decatenation (9–11). Therefore, the mitotic positive supercoiling activity could be required to promote decatenation by topoisomerase II. We treated DNA enriched for either the nega-

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demonstrating that CatC dimers responded to chloroquine intercalation as if negatively supercoiled, whereas CatC* dimers responded as if positively supercoiled. Thus, the CatC-to-CatC* transition induced by mitotic spindles is due to a change from negative supercoiling (in CatC) to positive supercoiling (in CatC*). Because the plasmids enter mitosis in a highly catenated state (due to topoisomerase II depletion), it is possible that positive supercoiling only occurs in this experimental situation. Therefore, we analyzed the supercoiling of plasmids that were decatenated by topoisomerase II immediately after DNA replication in both the absence and presence of mitotic spindles. Cells were allowed to replicate their DNA in the presence of Top2 activity before being arrested in metaphase by nocodazole treatment. Cdc20 was depleted to prevent entry into anaphase, and nocodazole was washed off to allow mitotic spindles to form. DNA was isolated from cells in nocodazole [without spindles] and from cells after the wash-off of nocodazole [with spindles], and the supercoiling of monomer plasmids was compared. Spindle formation shifted the supercoiling distribution to a more positive state (Fig. 2C, top panel). However, the extent of positive supercoiling appeared minor compared with the increase in positive supercoiling previously observed in catenated dimers generated in the absence of Top2 (Fig. 2B and fig. S6B). Normally,

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(Fig. 1B). Therefore, mitotic spindles are required for the transition from CatC to CatC*. The noncentromeric plasmid pRS426 did not undergo the distinct transition to CatC* in the absence of Top2 (fig. S4), suggesting that spindles must be engaged with kinetochores in cis for the transition to occur. The CatC-to-CatC* transition was not due to residual Top2 decatenation activity in mitosis, because in vitro nicking of the two types of catenated dimers generated very similar populations of relaxed catenanes (fig. S5), indicative of similar catenation states. To examine whether the CatC-to-CatC* transition correlated with any changes in supercoiling, we treated DNA samples enriched for either of the catenated dimers (CatC enriched by nocodazole arrest and CatC* enriched by cdc20 arrest) with eukaryotic topoisomerase I (topo IB), which relaxes both negative and positive supercoils, or Escherichia coli topoisomerase I (topo IA), which relaxes only negative supercoils. Topo IB relaxed catenated dimers from both CatC and CatC* samples (fig. S6A). However, topo IA relaxed CatC but not CatC* catenanes (Fig. 2A and fig. S6A), indicating that the intertwined plasmids in CatC* dimers are positively supercoiled, whereas those in CatC are negatively supercoiled. We used both titration analysis (Fig. 2B) and high-resolution analysis of partially nicked catenanes (fig. S6B) to confirm this finding by

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without spindles Fig. 2. Plasmids become positively supercoiled in mitosis when spindles are present. (A) Catenated DNA enriched for CatC* C Nocodazole plasmids was treated with either E. coli topoisomerase I (topo IA) arrested or eukaryotic topoisomerase I (topo IB). The products were visualized by two-dimensional (2D) agarose gel electrophoresis with native conditions in the first dimension and 20 mg/ml chloro- WT expression quine in the second dimension. Different plasmid forms, including of Top2 partially nicked catenanes (CatB), are indicated. (B) top2-td pRS314 cells were processed as in Fig. 1 but with the purified DNA resolved in gels containing different concentrations of chloroquine, as indicated. (C) Both cdc20-td pRS316 or cdc20-td top2-td pRS316 cells were synchronously arrested in mitosis using nocodazole to prevent formation of mitotic spindles. Cdc20 (top) or Cdc20 and Top2 proteins (bottom) were then degraded before washing off of nocodazole to allow mitotic Top2 depleted spindles to form. Purified DNA was analyzed in 2D chloro- during mitosis quine gels (12) to reveal the supercoiling distribution of the monomer plasmids. A cartoon representation of how the plasmid distribution relates to supercoiling status is shown (right).

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erated significant levels of CatC dimers (Fig. 4A). However, no transition from CatC to CatC* could be observed (Fig. 4A), despite the normal formation of mitotic spindles in this background (fig. S7). Furthermore, Smc2 function was required for the positive supercoiling of decatenated monomer plasmids. Formation of mitotic spindles in the top2-td strain with WT Smc2 function generated high levels of positive supercoiling in the monomer plasmids (Fig. 4B, left panel). However, positively supercoiled plasmids could not be observed when Smc2 function was disrupted (Fig. 4B, middle panel). The absence of positively supercoiled monomers was not due to a lack of functional spindles, because both the smc2-8 top2-td and top2-td cells had similar numbers of cells undergoing anaphase and nuclear segregation after formation of mitotic spindles (Fig. 4B, right panels). Thus, we conclude that Smc2 function is required for the mitotic positive-supercoiling activity.

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Fig. 3. CatC* catenated plasmids are preferentially decatenated by topoisomerase II in vitro. Catenated DNA enriched for CatC plasmids (nocodazole-arrested) or catenated DNA enriched for CatC* plasmids (cdc20arrested) was treated in vitro with recombinant topoisomerase IIa, and the products were analyzed on agarose gels. Autoradiograms of blots where the DNA was treated with 5 to 40 units [(A) and (C)] or 1 to 10 units of enzyme [(B) and (D)] are shown. Products were resolved in either native agarose [(A) and (B)] or agarose containing 0.5 mg/ml EtBr [(C) and (D)] to confirm that plasmids remained covalently closed during treatment. 5 × 10 −9 grams of purified (E. coli) supercoiled andrelaxed(topoI–treated) pRS316 was also resolved on each gel.

monomers at low topoisomerase concentrations, and we observed relaxation of the resultant supercoiled monomers only after complete decatenation (Fig. 3B). Therefore, although positive supercoiling of decatenated monomer plasmids is rapidly followed by topoisomerase II relaxation, induction of positive supercoiling on catenated plasmids drives topoisomerase II to resolve any intermolecular catenated links before positivesupercoil relaxation. We surmised that kinetochore attachment may indirectly induce topological change through activation of centromere-associated complexes that can modulate DNA topology. One such candidate complex is the condensin SMC2/4 complex (13, 14). We asked whether SMC2 activity was required for the positive supercoiling that characterizes CatC* by assessing whether the transition from CatC to CatC* occurs in the condensin smc2-8 mutant background (15). DNA replication in the absence of SMC2 and TOP2 function gen-

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tively supercoiled CatC dimers or the positively supercoiled CatC* dimers with different amounts of recombinant topoisomerase II. Topoisomerase II treatment of CatC dimers (nocodazole-arrested) initially produced catenanes with progressively slower electrophoretic mobilities. Resolved monomers were only observed at relatively high enzyme concentrations (>20 units) (Fig. 3, A and C). The kinetics that we observed are consistent with topoisomerase II concurrently relaxing negative supercoils and decatenating the interplasmid links in CatC dimers, so that when the final catenanes are resolved, only a small number of supercoils remain on the monomer plasmids. In contrast, the positively supercoiled CatC* plasmids (cdc20-arrested) were fully decatenated at the lowest topoisomerase II concentration tested (Fig. 3A). Decatenation of the CatC* dimers required one order of magnitude fewer units of enzyme (Fig. 3, B and D). The CatC* catenanes were resolved directly into highly supercoiled

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Fig. 4. Condensin SMC2 is required to positively supercoil plasmids during mitosis. (A) smc2-8 top2-td pRS316 cells were processed as in Fig. 1 to assess the electrophoretic mobility of the plasmid after disruption of both SMC2 and TOP2 functions. (B) top2-td and smc2-8 top2-td cells were processed as in Fig. 2C to specifically disrupt TOP2 and SMC2 during the period when mitotic Extrapolating from our data and previously proposed hypothetical models (10, 16) of how SMC-mediated compaction may facilitate chromosome decatenation, we propose a model for how complete decatenation of the genome by topoisomerase II is accomplished during mitosis (outlined in fig. S8). Although Top2 can actively remove catenations between sister chromatids postreplication, residual catenanes are present at the time of mitosis (hence, the observed requirement for Top2 at this stage of the cell cycle). We propose that extensive positive supercoiling induced by mitotic spindles and condensin generates a topology on catenated chromosomes where intermolecular crossovers are isolated from intramolecular crossovers with a geometry that maximizes topoisomerase II decatenation activity. Once the sister chromatids are fully decatenated, topoisomerase II rapidly relaxes the positive supercoiling generated, returning the intrachromosomal topology of the sister chromatids to a state comparable to that observed before

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spindles were formed in the cells after washing off of nocodazole and anaphase entry. (Left) Autoradiograms of plasmid supercoiling in nocodazole and 20 min after washing off are shown. Highly positively supercoiled monomers are indicated by arrows. (Right) Histogram indicating nuclear morphology of cells before and after nocodazole wash-off is shown.

the transition, except that now, crucially, all catenated links have been removed. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13.

J. C. Wang, Nat. Rev. Mol. Cell Biol. 3, 430 (2002). O. Sundin, A. Varshavsky, Cell 21, 103 (1980). O. Sundin, A. Varshavsky, Cell 25, 659 (1981). C. Holm, T. Goto, J. C. Wang, D. Botstein, Cell 41, 553 (1985). T. Uemura et al., Cell 50, 917 (1987). J. Baxter, J. F. Diffley, Mol. Cell 30, 790 (2008). R. A. Oliveira, R. S. Hamilton, A. Pauli, I. Davis, K. Nasmyth, Nat. Cell Biol. 12, 185 (2010). F. Uhlmann, D. Wernic, M. A. Poupart, E. V. Koonin, K. Nasmyth, Cell 103, 375 (2000). E. L. Zechiedrich, A. B. Khodursky, N. R. Cozzarelli, Genes Dev. 11, 2580 (1997). C. D. Hardy, N. J. Crisona, M. D. Stone, N. R. Cozzarelli, Philos. Trans. R. Soc. London Ser. B Biol. Sci. 359, 39 (2004). M. L. Martínez-Robles et al., Nucleic Acids Res. 37, 5126 (2009). Materials and methods are available as supporting material on Science Online. K. Kimura, M. Hirano, R. Kobayashi, T. Hirano, Science 282, 487 (1998).

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14. J. St-Pierre et al., Mol. Cell 34, 416 (2009). 15. L. Freeman, L. Aragon-Alcaide, A. Strunnikov, J. Cell Biol. 149, 811 (2000). 16. T. Hirano, Annu. Rev. Biochem. 69, 115 (2000). 17. We thank members of the Aragon laboratory, M. Merkenschlager, A. Carr, and J. Murray for discussions and critical reading of the manuscript. We also thank K. Nasmyth and S. Henikoff for helpful discussions, as well as anonymous referees for useful suggestions. This work was funded by European Research Council starting grant 202337 (L.A. and J.B.), the Royal Society (J.B.), the Spanish government grant BFU2008-00408/BMC (J.B.S.), Cancer Research UK (J.F.X.D.), and the MRC UK (L.A.). Extended figure legends are available in the supporting online material.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1328/DC1 Materials and Methods SOM Text Figs. S1 to S8 Table S1 References 10 December 2010; accepted 28 January 2011 10.1126/science.1201538

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Function of Rhodopsin in Temperature Discrimination in Drosophila Wei L. Shen,1 Young Kwon,1 Abidemi A. Adegbola,2 Junjie Luo,1 Andrew Chess,2,3,4 Craig Montell1* Many animals, including the fruit fly, are sensitive to small differences in ambient temperature. The ability of Drosophila larvae to choose their ideal temperature (18°C) over other comfortable temperatures (19° to 24°C) depends on a thermosensory signaling pathway that includes a heterotrimeric guanine nucleotide–binding protein (G protein), a phospholipase C, and the transient receptor potential TRPA1 channel. We report that mutation of the gene (ninaE) encoding a classical G protein–coupled receptor (GPCR), Drosophila rhodopsin, eliminates thermotactic discrimination in the comfortable temperature range. This role for rhodopsin in thermotaxis toward 18°C was light-independent. Introduction of mouse melanopsin restored normal thermotactic behavior in ninaE mutant larvae. We propose that rhodopsins represent a class of evolutionarily conserved GPCRs that are required for initiating thermosensory signaling cascades. emperature sensation in animals is mediated largely by direct activation of transient receptor potential (TRP) ion channels (1–3). An exception is a TRP channel in Drosophila larvae that functions indirectly in the selection of their optimal temperature (18°C) over other comfortable temperatures (19° to 24°C) and does so through a signaling cascade that includes a heterotrimeric guanine nucleotide–binding protein (G protein) Gq, phospholipase C (PLC), and the TRPA1 channel (4). A thermosensory signaling cascade is

T

1 Departments of Biological Chemistry and Neuroscience, Center for Sensory Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2Center For Human Genetic Research and Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. 3Broad Institute, Cambridge, MA, 02142, USA. 4Department of Developmental and Regenerative Biology and Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1. Requirement for ninaE for larval thermotaxis. (A) Temperature preferences by using the binary choice assay. Larvae were given a choice between 18°C (top) and other temperatures (14° to 32°C) (bottom). Preferences for 18°C or the alternative temperature result in positive or negative PIs, respectively. (B) Assays of preference for 18° versus 24°C with multiple ninaE alleles. (C) Assays of preference for 18° versus 24°C with indicated genotypes. Error bars represent SEMs. Unless indicated otherwise, differences were relative to wild-type [*P < 0.05; (A) Tukey’s analysis of variance (ANOVA); (B and C) Dunnett’s ANOVA]. See tables S2 to S4 for statistics.

also required in Caenorhabditis elegans, which includes guanylate cyclases and a guanosine 3′,5′monophosphate (cGMP)–gated channel (5–7). Thermosensory signaling cascades may contribute to amplification of small temperature differences and to adaptation to temperatures that are less than optimal, but still permissive for survival (4, 8). G protein–coupled receptors (GPCRs) are candidates to initiate thermosensory cascades because they couple to pathways that include Gq, PLC, and TRP channels, as well as to cascades that engage guanylate cyclases and cGMP-gated channels. However, there are up to 200 hundred GPCRs encoded in flies (9) and over one thousand in worms (10), and there is no precedent for a GPCR that functions in thermosensation. We wondered whether the canonical GPCR (rhodopsin) might be required for thermosensation, even though it is thought to function exclusively in light sensation. The basis for this proposal is that the same Gq (Ga49B) and PLC [No Receptor Potential A (NORPA)] that function

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in light sensation and link rhodopsin to activation of TRP channels are required for larvae to move preferentially toward the 18°C region when the alternative zone is held at another temperature in the 19° to 24°C range (4). If this behavior requires rhodopsin, it would be a light-independent function, because thermotaxis takes place effectively in the dark (4). To test temperature selection, we placed larvae on a plate between two temperature zones, one of which was kept at 18°C and the other at an alternative temperature (11) (fig. S1A). After 10 min, we counted the larvae in each zone and calculated the preference index (PI) (fig. S1A). A lack of temperature bias results in a PI of 0, whereas a complete preference for 18°C or the alternative temperature results in a PI of 1.0 or –1.0, respectively. Wild-type larvae select 18°C over any other temperature, including other temperatures in their comfortable range (20° to 24°C) (Fig. 1A). To address whether the major opsin (Rh1) encoded by the ninaE gene was required for thermotaxis in their comfortable temperature range, we tested flies with a deletion that removed the ninaE coding region (ninaEI17). The ability to distinguish 18° from 24°C was impaired in ninaE I17 larvae (Fig. 1, A and B) and in animals containing the ninaEI17 mutation in trans with another deletion (Df) that removed ninaE on the homologous chromosome (Fig. 1A). This phenotype was indistinguishable from the thermotaxis deficits resulting from mutations disrupting PLC (norpAP24) or the TRPA1 channel (trpA11) (4). Flies with any of five of six additional ninaE alleles showed deficits in discrimination between 18° and 24°C (Fig. 1B and fig. S1B), but not between 18°C and cooler or very warm temperatures (fig. S2, A and B). Larvae with one missense allele, ninaEP332, strongly preferred 18°C over 24°C (Fig. 1B), although the bias for 18°C was eliminated

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when the alternative temperature was either 20° or 22°C (fig. S2C). To confirm that the thermotaxis defect was due to mutation of ninaE, we tested for rescue of the phenotype with a wild-type transgene, using

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the GAL4-UAS system (12). This approach employs the yeast GAL4 transcription factor that binds to the upstream activation sequence (UAS) to promote transcription. Only ninaE17 larvae containing both the ninaE-GAL4 and UAS-ninaE

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transgenes effectively chose 18°C over 24°C (Fig. 1C). Another GPCR (serotonin receptor; UAS-5-HT2), which is most similar to mammalian Gq-coupled serotonin receptors (13, 14), does not rescue the ninaEI17 deficit (Fig. 1C). Similar to the norpAP24 and trpA11 phenotypes, loss of ninaE impaired discrimination between 18°C and other temperatures in the comfortable range, 20° or 22°C, but not selection of 18°C over cooler (14° or 16°C) or warmer temperatures (26° to 32°C) (Fig. 1A). In Drosophila, the vitamin A–derived chromophore stably binds to the opsin and is required for Rh1 to exit the endoplasmic reticulum (15). Wildtype larvae grown on food depleted of vitamin A, or mutant larvae (santa maria1) missing a scavenger receptor required for chromophore generation (16), showed impaired temperature discrimination in the 18°C to 24°C range (Fig. 2, A and B, and fig. S2D). The defect in santa maria1 was reversed by adding all trans-retinal to the food (R+) (Fig. 2B). To address whether Rh1 might function in the same cells as other components involved in 18° to 24°C thermotaxis, we expressed UAS-RNAi transgenes under the transcriptional control of the ninaE-GAL4 or the trpA1-GAL4. Expression of Ga49B, norpA, or trpA1 RNA interference (RNAi) transgenes using the ninaE-GAL4 reduced the biases toward 18°C over 22° or 23°C (Fig. 3A). Similarly, the preference for 18°C was diminished in larvae expressing the ninaE RNAi under control of the trpA1-GAL4 (Fig. 3B and fig. S3). Expression of UAS-ninaE+ under control of the trpA1-GAL4 restored 18° versus 24°C temperature discrimination in ninaEI17 larvae (Fig. 1C). Because rhodopsin is a light sensor, we tested whether thermotactic behavior is altered by light.

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Fig. 2. Effects of chromophore and light on temperature selection. (A) Wild-type larvae were reared on retinal-deficient food (vit A depleted) or on the same food supplemented with all-trans-retinal (R+) and allowed to select 18° or 24°C. (B) Wild-type larvae were reared on instant fly food. santa maria1 larvae were reared on instant food or on instant food supplemented with all-trans-retinal (R+). (C) Assays of selection of 18° versus 24°C under ambient light (~0.035 mW/cm2) or in the dark. (D) Wild-type larvae and mutants blind to moderate light (norpAP24;;trpA1-GAL4/UAS-norpA) were placed on plates maintained uniformly at room temperature (RT; ~20°C) or on plates with two zones (18° and 23°C). The zones were kept in the dark or exposed to blue light (6.75 mW/cm2). Error bars represent SEMs. Unless indicated otherwise, asterisk indicates significant differences relative to wild type [P < 0.05; (A, C, D), t test; (B), Tukey’s ANOVA]. See tables S5 to S8 for statistics.

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Fig. 3. Requirements for signaling components in the same cells. (A) Thermotactic behavior in larvae with UASRNAi transgenes expressed under control of the ninaE-GAL4. (B) Temperature selection after expression of UAS-ninaE RNAi under control of the ninaE-GAL4 or the trpA1-GAL4. (C) RT-PCR analysis after manual dissection of GFP-positive neurons from the body wall or the anterior region of the larvae (trpA1-GAL4 and UAS-mCD8-GFP), or after dissection of GFP-negative cells, which were close to v (vbd) or d (dbd). The reporter marked two morphologically similar neurons (dbd and vbd) in each body segment and two neurons near the anterior tip (A and B). The PCR primers spanned introns to distinguish products generated from RNA (white triangles) and genomic DNA (hollow triangles). Error bars represent SEMs. Unless indicated otherwise, asterisk indicates significant differences from wild-type [P < 0.05; (A), Tukey’s ANOVA; (B), Dunnett’s ANOVA]. See tables S9 and S10 for statistics.

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REPORTS

Wild-type larvae chose 18° over 24°C equally well in the light or dark (Fig. 2C). Moreover, ninaEI17 displayed similar thermotactic impairments in the presence or absence of light (Fig. 2C). Thus, selection of 18° over 24°C was light-independent. We also characterized larvae that were unresponsive to light. Wild-type early third instar larvae avoid white or blue, but not orange, light (Fig. 2D and fig. S4) (17). For larvae given a choice between 18° and 23°C, the aversion to light overcame the preference for 18°C (Fig. 2D and fig. S4B). Bolwig’s organs, which consist of larval photoreceptor cells that function in the avoidance of moderate light intensities, do not express the trpA1-GAL4 (fig. S5). norpAP24 animals are not negatively phototactic, and expression of UAS-norpA, under the control the trpA1-GAL4 does not restore negative phototaxis (Fig. 2D and fig. S4B). These larvae discriminated temperatures in the 18° to 23°C range, and this behavior was not affected by light. The ninaE gene appeared to be expressed at an exceptionally low level, because we were unable to detect a signal in larvae with Rh1 antibodies or using the ninaE-GAL4 to drive UAS-GFP. Low amounts of Rh1 might prevent efficient light activation of Rh1 in thermosensory neurons, which might impair thermotactic discrimination. To provide additional evidence that ninaE was coexpressed with trpA1, we dissected neurons from the body wall and the anterior region that expressed the trpA1-reporter (trpA1-GAL4 and UAS-mCD8-GFP; mCD8 is the mouse CD8 receptor), and we performed reverse transcription polymerase chain reaction (RT-PCR). We detected ninaE RT-PCR products in 5 out of 15 green fluorescent protein (GFP)–positive neurons (3 out of 8 from the body wall; 2 out of 7, anterior region),

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atures tested (18° to 24°C) (fig. S6C). We obtained similar results with the ninaEI17, norpAP24, and trpA11 larvae (fig. S6C). Thus, turning at 24°C was dependent on prior exposure to 18°C. Several results argue strongly against a developmental defect underlying the thermotaxis impairment in the comfortable range. First, although ninaEP332 larvae were impaired in selecting 18°C over 20° or 22°C, they were able to choose 18°C over 24°C (fig. S2C). Second, multiple ninaE missense mutations, including ninaEP332 and ninaEP318, have no apparent effects on morphogenesis and are not associated with retinal degeneration (20), which suggests that these alleles do not affect development of the thermosensory neurons. Third, we found indistinguishable numbers and morphological appearances of GFPpositive cells in wild-type and ninaEI17 larvae that expressed UAS-mCD8-GFP under control of the trpA1-GAL4 (table S1 and fig. S7A). We took advantage of the slightly higher PI exhibited by ninaEP332 (18° versus 24°C) to test whether other genes required for thermotaxis functioned subsequent to ninaE. Introduction of the Ga49B1, norpAP24 or trpA11 mutations into the ninaEP332 background prevented 18°C selection over 24°C (fig. S7B). Another mutation that causes a higher-than-normal PI disrupts the rhodopsin phosphatase (rdgC306) (4). The combination of ninaEI17 or Ga49B1 with rdgC 306 eliminated the bias for 18° over 24°C (fig. S7B). These analyses indicate that Gq, PLC, and TRPA1 function in a pathway downstream of Rh1. Drosophila encodes additional opsins (Rh2-6) (15). To determine whether other opsins could substitute for Rh1, we expressed Rh2-6 under control of the ninaE promoter in ninaEI17 flies and assayed 18° versus 24°C selection. With the

but not in any dissected GFP negative neurons (0 of 11; P < 0.05, Fisher’s exact test) (Fig. 3C). Selection of 17.5° to 18°C over cooler temperatures occurs through avoidance that results from increased turning at slightly lower temperatures (18, 19). To test whether the preference for 18° over slightly higher temperatures occurred through a similar mechanism, we tracked larvae. Wild-type larvae appeared to progress only a short distance into the 24°C area before they paused, stretched their heads (movie S1), and initiated their first turns (fig. S6A). However, ninaEI17, norpAP24, and trpA11 mutant larvae did not appear to turn until they traversed far into the 24°C zone (fig. S6A). To quantify turning behavior, we developed a simple assay. We demarcated the 24°C zone with 20 lines (Fig. 4A), released the larvae on the 18°C side near the 24°C interface, and tabulated the last line crossed before the larvae made their first turn. We only counted larvae that moved perpendicular to the lines (≤5° deviation). Wild-type larvae turned near line 3 (Fig. 4B). However, the mutant larvae traveled to near line 14 in the 24°C area before turning (Fig. 4B). The much greater distances traveled by the mutants before turning did not appear to be due to increased movement speeds, because all the larvae moved at similar rates (fig. S6B). In a reciprocal experiment, we placed larvae on the 24°C side and monitored animals that crossed perpendicular to the lines demarcating the 18°C zone. Wild-type larvae did not turn until line 10, and there were only small variations between wild-type and mutant animals (Fig. 4B). We tested whether the higher rate of larval turning at 24°C was dependent on prior exposure to a lower temperature. Wild-type larvae placed on a plate uniformly held at a single temperature showed similar turning frequencies at all temper-

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Fig. 4. Temperature-dependent turning and thermotactic behavior of larvae expressing other opsins in place of Rh1. (A) Set-up for quantitative analysis of turning behavior. To assay turning behavior from 18° to 24°C, the 24°C side was demarcated with 20 lines. We released larvae within the 18°C zone and tracked larvae that crossed the midline (line 0) and moved perpendicular to the lines (T5°). We tabulated the last line crossed before the larvae made the first turn. To assay turning from 24° to 18°C, we released larvae on the 24°C www.sciencemag.org

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side. (B) Last line crossed before larvae made the first turn at 18° to 24°C and 24° to 18°C. (C) Rescue of ninaEI17 thermotactic defect by expression of other fly opsins or mouse Opn4 under control of the ninaE promoter. (D) Thermal preferences with Rh4 and Rh6 in place of Rh1 (18°C versus the indicated temperature). Error bars indicate SEMs. Asterisks indicate significant differences from wild-type (P < 0.05; Dunnett’s ANOVA test). See tables S11 to S13 for detailed statistics. VOL 331

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REPORTS to consider the question as to whether the archetypal role for rhodopsin was in light sensation or in thermosensation. References and Notes 1. M. J. Caterina, Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R64 (2007). 2. M. Bandell, L. J. Macpherson, A. Patapoutian, Curr. Opin. Neurobiol. 17, 490 (2007). 3. K. Venkatachalam, C. Montell, Annu. Rev. Biochem. 76, 387 (2007). 4. Y. Kwon, H. S. Shim, X. Wang, C. Montell, Nat. Neurosci. 11, 871 (2008). 5. C. M. Coburn, C. I. Bargmann, Neuron 17, 695 (1996). 6. H. Komatsu, I. Mori, J. S. Rhee, N. Akaike, Y. Ohshima, Neuron 17, 707 (1996). 7. H. Inada et al., Genetics 172, 2239 (2006). 8. D. Ramot, B. L. MacInnis, M. B. Goodman, Nat. Neurosci. 11, 908 (2008). 9. T. Brody, A. Cravchik, J. Cell Biol. 150, F83 (2000). 10. C. I. Bargmann, Science 282, 2028 (1998). 11. Materials and methods are available as supporting material on Science Online. 12. A. H. Brand, N. Perrimon, Development 118, 401 (1993). 13. J. F. Colas, J. M. Launay, O. Kellermann, P. Rosay, L. Maroteaux, Proc. Natl. Acad. Sci. U.S.A. 92, 5441 (1995). 14. M. Filip, M. Bader, Pharmacol. Rep. 61, 761 (2009).

A Polarized Epithelium Organized by b- and a-Catenin Predates Cadherin and Metazoan Origins Daniel J. Dickinson,1 W. James Nelson,1,2,3* William I. Weis1,3,4* A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex that contains a classical cadherin, the Wnt-signaling protein b-catenin and the actin-binding protein a-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an a-catenin ortholog that binds a b-catenin–related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion, and proper multicellular morphogenesis. Thus, the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins. simple epithelium is the most basic tissue type in metazoans (multicellular animals). It is the first overt sign of cellular differentiation during embryogenesis and is important for the morphogenesis of many tissues and homeostasis in the adult (1). A simple epithelium comprises a cell monolayer surrounding a luminal space. The cells have a polarized organization of plasma membrane proteins, or-

A 1

Program in Cancer Biology, Stanford University, Stanford, CA 94305, USA. 2Department of Biology, Stanford University, Stanford, CA 94305, USA. 3Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA. 4Department of Structural Biology, Stanford University, Stanford, CA 94305, USA. *To whom correspondence should be addressed. E-mail: [email protected] (W.J.N.); [email protected] (W.I.W.)

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ganelles, and cytoskeletal networks that together regulate the directional absorption and secretion of proteins and other solutes (1). The structural integrity and functional polarity of epithelial tissues in higher animals require cell-cell adhesion mediated by classical cadherins (2). Adhesion provides a spatial cue that initiates cell polarization via recruitment of cadherinassociated cytosolic proteins (3), including the Wnt-signaling protein b-catenin (4) and the actinbinding protein a-catenin (5). Classical cadherins, which have extracellular cadherin repeats (6) and a conserved cytoplasmic domain that can bind b-catenin (7), are found in all multicellular animals, including sponges, but not in choanoflagellates (8–10), which suggests that classical cadherins are restricted to metazoans. However, the evolutionary history of the catenins is unknown, and

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15. T. Wang, C. Montell, Pflugers Arch. 454, 821 (2007). 16. T. Wang, Y. Jiao, C. Montell, J. Cell Biol. 177, 305 (2007). 17. P. H. Strange, J. Exp. Biol. 38, 237 (1961). 18. Y. Kwon, W. L. Shen, H. S. Shim, C. Montell, J. Neurosci. 30, 10465 (2010). 19. L. Luo et al., J. Neurosci. 30, 4261 (2010). 20. J. P. Kumar, D. F. Ready, Development 121, 4359 (1995). 21. I. Provencio, G. Jiang, W. J. De Grip, W. P. Hayes, M. D. Rollag, Proc. Natl. Acad. Sci. U.S.A. 95, 340 (1998). 22. R. C. Hardie et al., Neuron 36, 689 (2002). 23. We thank Y. Liu for advice with the statistical analyses, the Bloomington Stock Center, FlyBase, and the Harvard TRiP. A.A.A. received support from a NARSAD Young Investigator Award. This study was supported by a grant to C.M. from the National Institute of General Medical Sciences, NIH (GM085335).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1333/DC1 Materials and Methods Figs. S1 to S8 Tables S1 to S17 References Movie S1 12 October 2010; accepted 10 January 2011 10.1126/science.1198904

thus how the cadherin-catenin complex evolved to mediate epithelial polarity in metazoans is unclear. The non-metazoan social amoeba Dictyostelium discoideum undergoes multicellular morphogenesis in response to starvation: Single cells aggregate and undergo culmination to form a fruiting body, which comprises a rigid stalk that supports a collection of spores (Fig. 1A) (11). The mechanical rigidity of the stalk is due to the stalk tube, which contains cellulose and the extracellular matrix proteins EcmA/B (Fig. 1B) (12, 13). Harwood and colleagues described a ring of cells surrounding the stalk tube at the tip of the culminant and speculated that these cells might contribute to stalk formation during culmination (14, 15). However, the subcellular organization and function of tip cells have not been characterized. We confirmed the earlier observation (14) that the tip consists of an organized monolayer of cells surrounding the stalk (Fig. 1, A and B, and movie S1). Additionally, we found that these cells have a distinctive polarized organization: Centrosomes and Golgi localized to a stalk side of nuclei (Fig. 1C), and the transmembrane protein cellulose synthase [encoded by the dcsA gene (12)] localized to the plasma membrane domain adjacent to the stalk tube (Fig. 1D). Thus, D. discoideum tip cells have a subcellular organization that is characteristic of a simple polarized epithelium (fig. S1), and we refer to these cells as the tip epithelium. In metazoans, b-catenin and a-catenin are essential for the formation of polarized simple epithelia (16, 17). A b-catenin–related protein called Aardvark has been identified in D. discoideum (fig. S2) (9, 14). We identified a member of the a-catenin family in this organism, which we

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exception of Rh3, other opsins could replace Rh1 (Fig. 4C). However, the transgenic flies showed significant differences from wild type when given a choice between 18° and 20° to 22°C (Fig. 4D). Another GPCR coupled to Gq [5-hydroxytryptamine (5-HT2)] did not function in place of Rh1 (Fig. 1C). The mammalian opsin that is most similar to Drosophila Rh1 is melanopsin (OPN4) (21). Expression of Opn4 under control of the ninaE promoter did not reverse the phototransduction defect in adult ninaEI17 (fig. S8). However, Opn4 enabled the ninaEI17 larvae to distinguish between 18°C and 24°C (Fig. 4C). The observations that Rh1 is required for thermosensory discrimination and that OPN4 could substitute for Rh1 suggest that Rh1 and related opsins might be intrinsic thermosensors. However, the intrinsic rate of thermal activation, which is ~1/min in fly photoreceptor cells (22), is far too low to account for the requirement for Rh1 for thermosensation. We suggest that an accessory factor might interact with Rh1 and accelerates its intrinsic thermal activity. Finally, because rhodopsin has dual roles, it is interesting

REPORTS to consider the question as to whether the archetypal role for rhodopsin was in light sensation or in thermosensation. References and Notes 1. M. J. Caterina, Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R64 (2007). 2. M. Bandell, L. J. Macpherson, A. Patapoutian, Curr. Opin. Neurobiol. 17, 490 (2007). 3. K. Venkatachalam, C. Montell, Annu. Rev. Biochem. 76, 387 (2007). 4. Y. Kwon, H. S. Shim, X. Wang, C. Montell, Nat. Neurosci. 11, 871 (2008). 5. C. M. Coburn, C. I. Bargmann, Neuron 17, 695 (1996). 6. H. Komatsu, I. Mori, J. S. Rhee, N. Akaike, Y. Ohshima, Neuron 17, 707 (1996). 7. H. Inada et al., Genetics 172, 2239 (2006). 8. D. Ramot, B. L. MacInnis, M. B. Goodman, Nat. Neurosci. 11, 908 (2008). 9. T. Brody, A. Cravchik, J. Cell Biol. 150, F83 (2000). 10. C. I. Bargmann, Science 282, 2028 (1998). 11. Materials and methods are available as supporting material on Science Online. 12. A. H. Brand, N. Perrimon, Development 118, 401 (1993). 13. J. F. Colas, J. M. Launay, O. Kellermann, P. Rosay, L. Maroteaux, Proc. Natl. Acad. Sci. U.S.A. 92, 5441 (1995). 14. M. Filip, M. Bader, Pharmacol. Rep. 61, 761 (2009).

A Polarized Epithelium Organized by b- and a-Catenin Predates Cadherin and Metazoan Origins Daniel J. Dickinson,1 W. James Nelson,1,2,3* William I. Weis1,3,4* A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex that contains a classical cadherin, the Wnt-signaling protein b-catenin and the actin-binding protein a-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an a-catenin ortholog that binds a b-catenin–related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion, and proper multicellular morphogenesis. Thus, the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins. simple epithelium is the most basic tissue type in metazoans (multicellular animals). It is the first overt sign of cellular differentiation during embryogenesis and is important for the morphogenesis of many tissues and homeostasis in the adult (1). A simple epithelium comprises a cell monolayer surrounding a luminal space. The cells have a polarized organization of plasma membrane proteins, or-

A 1

Program in Cancer Biology, Stanford University, Stanford, CA 94305, USA. 2Department of Biology, Stanford University, Stanford, CA 94305, USA. 3Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA. 4Department of Structural Biology, Stanford University, Stanford, CA 94305, USA. *To whom correspondence should be addressed. E-mail: [email protected] (W.J.N.); [email protected] (W.I.W.)

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ganelles, and cytoskeletal networks that together regulate the directional absorption and secretion of proteins and other solutes (1). The structural integrity and functional polarity of epithelial tissues in higher animals require cell-cell adhesion mediated by classical cadherins (2). Adhesion provides a spatial cue that initiates cell polarization via recruitment of cadherinassociated cytosolic proteins (3), including the Wnt-signaling protein b-catenin (4) and the actinbinding protein a-catenin (5). Classical cadherins, which have extracellular cadherin repeats (6) and a conserved cytoplasmic domain that can bind b-catenin (7), are found in all multicellular animals, including sponges, but not in choanoflagellates (8–10), which suggests that classical cadherins are restricted to metazoans. However, the evolutionary history of the catenins is unknown, and

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15. T. Wang, C. Montell, Pflugers Arch. 454, 821 (2007). 16. T. Wang, Y. Jiao, C. Montell, J. Cell Biol. 177, 305 (2007). 17. P. H. Strange, J. Exp. Biol. 38, 237 (1961). 18. Y. Kwon, W. L. Shen, H. S. Shim, C. Montell, J. Neurosci. 30, 10465 (2010). 19. L. Luo et al., J. Neurosci. 30, 4261 (2010). 20. J. P. Kumar, D. F. Ready, Development 121, 4359 (1995). 21. I. Provencio, G. Jiang, W. J. De Grip, W. P. Hayes, M. D. Rollag, Proc. Natl. Acad. Sci. U.S.A. 95, 340 (1998). 22. R. C. Hardie et al., Neuron 36, 689 (2002). 23. We thank Y. Liu for advice with the statistical analyses, the Bloomington Stock Center, FlyBase, and the Harvard TRiP. A.A.A. received support from a NARSAD Young Investigator Award. This study was supported by a grant to C.M. from the National Institute of General Medical Sciences, NIH (GM085335).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6022/1333/DC1 Materials and Methods Figs. S1 to S8 Tables S1 to S17 References Movie S1 12 October 2010; accepted 10 January 2011 10.1126/science.1198904

thus how the cadherin-catenin complex evolved to mediate epithelial polarity in metazoans is unclear. The non-metazoan social amoeba Dictyostelium discoideum undergoes multicellular morphogenesis in response to starvation: Single cells aggregate and undergo culmination to form a fruiting body, which comprises a rigid stalk that supports a collection of spores (Fig. 1A) (11). The mechanical rigidity of the stalk is due to the stalk tube, which contains cellulose and the extracellular matrix proteins EcmA/B (Fig. 1B) (12, 13). Harwood and colleagues described a ring of cells surrounding the stalk tube at the tip of the culminant and speculated that these cells might contribute to stalk formation during culmination (14, 15). However, the subcellular organization and function of tip cells have not been characterized. We confirmed the earlier observation (14) that the tip consists of an organized monolayer of cells surrounding the stalk (Fig. 1, A and B, and movie S1). Additionally, we found that these cells have a distinctive polarized organization: Centrosomes and Golgi localized to a stalk side of nuclei (Fig. 1C), and the transmembrane protein cellulose synthase [encoded by the dcsA gene (12)] localized to the plasma membrane domain adjacent to the stalk tube (Fig. 1D). Thus, D. discoideum tip cells have a subcellular organization that is characteristic of a simple polarized epithelium (fig. S1), and we refer to these cells as the tip epithelium. In metazoans, b-catenin and a-catenin are essential for the formation of polarized simple epithelia (16, 17). A b-catenin–related protein called Aardvark has been identified in D. discoideum (fig. S2) (9, 14). We identified a member of the a-catenin family in this organism, which we

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Downloaded from www.sciencemag.org on March 10, 2011

exception of Rh3, other opsins could replace Rh1 (Fig. 4C). However, the transgenic flies showed significant differences from wild type when given a choice between 18° and 20° to 22°C (Fig. 4D). Another GPCR coupled to Gq [5-hydroxytryptamine (5-HT2)] did not function in place of Rh1 (Fig. 1C). The mammalian opsin that is most similar to Drosophila Rh1 is melanopsin (OPN4) (21). Expression of Opn4 under control of the ninaE promoter did not reverse the phototransduction defect in adult ninaEI17 (fig. S8). However, Opn4 enabled the ninaEI17 larvae to distinguish between 18°C and 24°C (Fig. 4C). The observations that Rh1 is required for thermosensory discrimination and that OPN4 could substitute for Rh1 suggest that Rh1 and related opsins might be intrinsic thermosensors. However, the intrinsic rate of thermal activation, which is ~1/min in fly photoreceptor cells (22), is far too low to account for the requirement for Rh1 for thermosensation. We suggest that an accessory factor might interact with Rh1 and accelerates its intrinsic thermal activity. Finally, because rhodopsin has dual roles, it is interesting

named Dda-catenin on the basis of structural and functional characteristics (9). Dda-catenin is approximately 35% homologous to human a-catenins and their paralog vinculin (Fig. 2A and figs. S3 to S5). Dda-catenin was expressed at low levels in single D. discoideum cells but was up-regulated during multicellular develop-

ment (Fig. 2B). Endogenous Dda-catenin localized to cell-cell contacts in the slug and fruiting body (Fig. S6 and fig. S7A) and especially in columnar cells of the tip epithelium (Fig. 2C). We examined whether Dda-catenin is similar to metazoan a-catenin or vinculin, or both (9). Like metazoan a-catenin, Dda-catenin bound and

Fig. 1. (A) D. discoideum developmental process. M, mound; Sl, slug; C, culminant; FB, fruiting body. (B) Confocal section of the tip of a wild-type culminant. Brackets indicate the tip epithelium; arrowheads indicate the stalk tube; S indicates the stalk. (C) Maximum-intensity projections showing Golgi (left), centrosomes (right), and nuclei (4´,6´-diamidino-2-phenylindole stain) in the entire tip (top) or tip epithelium (bottom). (D) Confocal section of the tip (top) and tip epithelium (bottom) in a wild-type culminant expressing cellulose synthase (mRFP-dcsA). In tip epithelial cells, mRFP-dcsA localizes to the tip epithelial cell membrane adjacent to the stalk (arrowheads). mRFP-dcsA is also expressed in the stalk cells. Scale bars, [(B) to (D)] 10 mm in lower-magnification views and [(C) and (D)] 2 mm in higher-magnification views. In views of the tip epithelium, the top of the images faces the stalk.

Fig. 2. (A) Primary structures of Dda-catenin and human a-catenin and vinculin. Regions of homology are shaded gray. NTD, N-terminal domain; M, M-domain; ABD, actinbinding domain; P, prolinerich region. (B) Western blot for Dda-catenin at the indicated developmental time points. (C) Confocal sections of the tip epithelium in a wildtype culminant and an Aardvark knockout (14). Asterisks indicate nonspecific signal on the exterior of the culminant (fig. S8). (D) High-speed pelleting assay demonstrating binding of 5 mM full-length (FL) or the isolated tail domain of Ddacatenin to 5 mM F-actin. (E) Negative-stain electron micrographs of actin filaments in the absence or presence of 5 mM Dda-catenin. Scale bar, 500 nm. (F) Bead-bound fractions from a glutathione S-transferase (GST) pull-down www.sciencemag.org

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bundled actin filaments (Fig. 2, D and E). Ddacatenin bound to the D. discoideum b-catenin– related protein Aardvark (Fig. 2F) and mouse b-catenin (fig. S9), and its localization to cellcell contacts in vivo was Aardvark-dependent (Fig. 2C and fig. S7). Unlike mammalian aEcatenin, but like the C. elegans a-catenin ortholog HMP-1 (18), purified Dda-catenin was monomeric in solution (fig. S10), and it did not inhibit the actin-nucleating activity of the Arp2/3 complex (Fig. 2G). In contrast to its overall similarity to metazoan a-catenin, Dda-catenin lacked key properties of metazoan vinculin (figs. S11 and S12) (9). Because Dda-catenin represents the most basally branching members of the a-catenin/vinculin family (fig. S4), these data indicate that the ancestral member of this protein family was probably a-catenin-like. To test whether Dda-catenin and its binding partner Aardvark are involved in the polarized organization of the tip epithelium, we depleted Ddacatenin using RNA interference (fig. S13). When Dda-catenin was depleted below a level that could be detected by means of immunofluorescence, multicellular development arrested at the onset of culmination (Fig. 3A). Tip cells were disorganized, and the stalk and tip epithelium were absent (Fig. 3, A and B). Moreover, the distributions of Golgi and centrosomes were not polarized (Fig. 3C and fig. S14), and cellulose synthase was mislocalized intracellularly (Fig. 3D). Culminants with partial Dda-catenin knockdown exhibited a milder phenotype: A distinct stalk and tip epithelium formed, but the epithelium appeared disorganized and was more than one cell layer thick (Fig. 3B), and organelles (Fig. 3C and fig. S14, arrowheads) and cellulose synthase (Fig. 3D) were not correctly polarized. Prestalk cell differentiation was unaffected in Dda-catenin

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assay demonstrating binding of Dda-catenin (10 mM) to GST-Aardvark (~0.3 mM). 5 mM GST is a negative control. (G) Pyrene actin polymerization assays were performed in the presence of N-WASp VCA domain and the indicated additional proteins. aE-catenin or Dda-catenin concentrations were 5 mM. VOL 331

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Fig. 3. (A) Early culminants formed by wildtype and Dda-catenin knockdown cells. (B) Confocal sections of the tip in culminants of the indicated cells. Severe and mild Dda-catenin knockdown phenotypes are distinguished by the absence or presence, respectively, of a nascent stalk. Asterisk indicates nonspecific signal on the exterior of the culminant (fig. S8). (C) Maximum intensity projections showing centrosomes and nuclei. Arrowheads indicate centrosomes that are mislocalized relative to wild type (Fig. 1C). (Bottom) Higher-magnification views of the boxed regions. (D) Confocal sections of the (top) tip and (bottom) tip epithelium in culminants of the indicated cells expressing mRFP-dcsA (cellulose synthase). Arrowheads indicate residual localization of mRFP-dcsA in mild Dda-catenin knockdowns and Aardvark knockouts. Scale bars, (A) 25 mm, [(B) to (D)] 10 mm in lower-magnification views, or [(C) and (D)] 2 mm in higher-magnification views.

Fig. 4. (A) Confocal sections of the tip epithelium in culminants of the indicated cells. Arrows indicate deposition of small amounts of extracellular cellulose and EcmA/B in a nascent stalk tube. Arrowheads indicate intracellular accumulation of EcmA/B. (B) Confocal section of the tip epithelium in a culminant of cellulose synthase (dcsA) knockout cells (12). (C) Confocal sections of tip epithelia in culminants of the indicated cells. Arrowheads indicate Sec15 localization. Asterisks indicate nonspecific signal on the exterior of the culminant (fig. S8). Scale bars, 2 mm.

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REPORTS

REPORTS organized stalk tube, we tested whether the secretory pathway was polarized in wild-type and mutant strains. Sec15, a component of the Exocyst complex involved in polarized exocytosis in diverse systems (20), localized adjacent to the stalk tube (Fig. 4C)—reminiscent of Exocyst localization in polarized mammalian epithelial cells (21)—and this distribution was strongly disrupted in Dda-catenin knockdowns and Aardvark knockouts (Fig. 4C). The molecular mechanisms underlying the polarized organization of the Exocyst in D. discoideum are unknown, but the catenins have been reported to associate in a complex with Exocyst components in mammalian cells (22). Taken together with earlier results (14), our work shows that the non-metazoan D. discoideum has a bona fide polarized epithelium consisting of a single layer of structurally and functionally polarized cells that secrete proteins into a luminal space (fig. S1). Epithelial polarity in both metazoans and D. discoideum requires homologs of a-catenin and b-catenin, indicating a close evolutionary relationship between D. discoideum and metazoan epithelia. Because D. discoideum lacks cadherins, Wnt-signaling components, and polarity proteins of the PAR, Crumbs, and Scribble complexes (9), the conserved catenin complex appears to be an ancient functional module that mediates epithelial polarity in the absence of the more complicated machinery found in metazoans (1). The fact that the catenin complex is essential for epithelial polarity in both D. discoideum and metazoans indicates that this complex probably functioned in cell polarity before the divergence of social amoebae and metazoans. It is possible that the catenins evolved initially to mediate cell polarity in a unicellular organism and then were used to organize cell polarity in a multicellular context in both social amoebae and metazoans. Alternatively, the last common ancestor of social amoebae and metazoans may have formed a polarized epithelial tissue organized by the catenin complex, but epithelial polarity was lost in some intervening lineages (9). In either case, our results identify unexpected similarities in tissue organization between two groups of distantly related organisms that were thought to have independently evolved multicellularity (23), and thus reveal molecular factors and organizational principles that may have contributed to the early evolution and diversification of animals.

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References and Notes 1. D. M. Bryant, K. E. Mostov, Nat. Rev. Mol. Cell Biol. 9, 887 (2008). 2. W. J. Nelson, Nature 422, 766 (2003). 3. W. J. Nelson, Biochem. Soc. Trans. 36, 149 (2008). 4. H. Clevers, Cell 127, 469 (2006). 5. D. L. Rimm, E. R. Koslov, P. Kebriaei, C. D. Cianci, J. S. Morrow, Proc. Natl. Acad. Sci. U.S.A. 92, 8813 (1995). 6. P. Hulpiau, F. van Roy, Int. J. Biochem. Cell Biol. 41, 349 (2009). 7. A. H. Huber, W. I. Weis, Cell 105, 391 (2001). 8. M. Srivastava et al., Nature 466, 720 (2010). 9. Materials and methods and supporting text are available as supporting online material on Science Online. 10. M. Abedin, N. King, Science 319, 946 (2008). 11. H. Urushihara, Dev. Growth Differ. 50, (Suppl 1), S277 (2008). 12. R. L. Blanton, D. Fuller, N. Iranfar, M. J. Grimson, W. F. Loomis, Proc. Natl. Acad. Sci. U.S.A. 97, 2391 (2000). 13. S. J. McRobbie, R. Tilly, K. Blight, A. Ceccarelli, J. G. Williams, Dev. Biol. 125, 59 (1988). 14. M. J. Grimson et al., Nature 408, 727 (2000). 15. H. P. Williams, A. J. Harwood, Curr. Opin. Microbiol. 6, 621 (2003). 16. M. Watabe, A. Nagafuchi, S. Tsukita, M. Takeichi, J. Cell Biol. 127, 247 (1994). 17. M. Torres et al., Proc. Natl. Acad. Sci. U.S.A. 94, 901 (1997). 18. A. V. Kwiatkowski et al., Proc. Natl. Acad. Sci. U.S.A. 107, 14591 (2010). 19. J. C. Coates et al., Mech. Dev. 116, 117 (2002). 20. B. He, W. Guo, Curr. Opin. Cell Biol. 21, 537 (2009). 21. K. K. Grindstaff et al., Cell 93, 731 (1998). 22. C. Yeaman, K. K. Grindstaff, W. J. Nelson, J. Cell Sci. 117, 559 (2004). 23. N. King, Dev. Cell 7, 313 (2004). 24. We thank numerous colleagues for reagents (9); T. Soldati for sharing Sec15 antibodies before publication; C. Carswell-Crumpton, N. Ghori, J. Perrino, and T. Weiss for technical assistance; and D. Ehrhardt, N. King, D. N. Robinson, T. Soldati, J. A. Spudich, M. Tsujioka, H. Warrick, and members of the Nelson and Weis laboratories for discussions. This work was supported by a Stanford Graduate Fellowship and an NSF Graduate Research Fellowship (D.J.D.), NIH GM035527 (W.J.N.), and NIH GM56169 (W.I.W.). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, supported by the U.S. Department of Energy and the National Institute of General Medical Sciences.

Supporting Online Material

Downloaded from www.sciencemag.org on March 10, 2011

knockdowns, indicating that the lack of a stalk was not due to a failure of the developmental program to correctly specify cell types (fig. S15). Similar results were obtained with an Aardvark knockout strain (14) (Fig. 3, B to D, and fig. S14), indicating that both Dda-catenin and Aardvark are required to organize and polarize the tip epithelium during culmination. Harwood and colleagues reported that Aardvark was necessary for formation of actin-associated cell-cell junctions in tip cells that appeared similar to adherens junctions at the ultrastructural level (14, 15, 19). However, we found that Aardvark knockouts formed junctions similar to wild-type, as did Dda-catenin knockdowns (fig. S16). Because these junctions do not require Dda-catenin or Aardvark, and D. discoideum does not have classical cadherins, we conclude that these junctions are unlikely to be molecularly equivalent to metazoan adherens junctions (9) and are not involved in the developmental phenotypes described above. To better understand the developmental mechanism underlying impaired stalk formation in Dda-catenin knockdowns and Aardvark knockouts, we examined whether the stalk tube components cellulose and EcmA/B were correctly distributed. Accumulation of cellulose and EcmA/B in the stalk tube was absent in severe Dda-catenin knockdowns and was strongly reduced in mild Dda-catenin knockdowns and Aardvark knockouts (Fig. 4A and fig. S17, A and B) (19). Cellulose synthase (compare Figs. 3D and 1D) and EcmA/B (Fig. 4A and figs. S17, A and B, arrowheads) were mislocalized intracellularly in tip epithelial cells but were unchanged in stalk cells, indicating that tip epithelial cells are the primary source of secreted cellulose and EcmA/B in the stalk tube. Confirming this interpretation, we observed rare cases in which half of the tip epithelium was better organized than the other half, and in those culminants cellulose and EcmA/B accumulated in the stalk tube adjacent to the better-organized tip epithelial cells (fig. S18). In cellulose synthase knockouts, which do not form a stalk tube (12), the tip epithelium was morphologically normal, and EcmA/B were secreted (Fig. 4B and fig. S17C), demonstrating that tip epithelial polarity is genetically upstream of stalk tube formation. Because tip epithelial cells appear to secrete cellulose and EcmA/B directionally to form an

www.sciencemag.org/cgi/content/full/331/6022/1336/DC1 Materials and Methods SOM Text Figs. S1 to S19 Table S1 Movies S1 and S2 27 October 2010; accepted 21 January 2011 10.1126/science.1199633

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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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Stem Cells ELISpot Kits Flow Cytometry

R&D Systems 41.8%

Cell Selection

17.3% 7.9% 3.7% 2.5% 2.1% 1.6% 1.4%

Approximately 42% of Referenced Immunoassays are Developed and Manufactured by R&D Systems. A survey of 860 manuscripts from 44 journals was conducted to compare the number of citations specifying the use of R&D Systems ELISAs to the number citing ELISAs from other commercial sources. A total of 433 ELISA citations referencing immunoassays from 66 different vendors were identified in the survey.

 α1-Acid Glycoprotein  Angiopoietin-like 3  Cathepsin V  Clusterin  Dkk-1  EGF R/ErbB1  EG-VEGF/PK1  Fetuin A  FGF-21  Galectin-3  Gas 6  GDF-15  IL-17A/F Heterodimer  IL-19  Lipocalin-2/NGAL  MBL  Proprotein Convertase 9/PCSK9  Periostin/OSF-2  Progranulin  ST2/IL-1 R4  Thrombomodulin/CD141  Tie-1  TIM-1/KIM-1

For more information visit our website at www.RnDSystems.com/go/ELISA For research use only. Not for use in diagnostic procedures. R&D Systems, Inc. www.RnDSystems.com R&D Systems Europe, Ltd. www.RnDSystems.co.uk R&D Systems China Co., Ltd. www.RnDSystemsChina.com.cn

Vice Provost for Research

s r e e r a C e c n Sciedvertising A For full advertising details, go to ScienceCareers.org and click For Employers, or call one of our representatives. Tracy Holmes Worldwide Associate Director Science Careers Phone: +44 (0) 1223 326525

UNITED STATES & CANADA E-mail: [email protected] Fax: 202-289-6742

Oakland University (OU) is seeking an innovative and energetic academic leader to serve as Vice Provost for Research. Oakland University is recognized by the Carnegie Foundation as one of the nation's 82 doctoral/research universities. The university offers 134 bachelor's degree programs and 124 graduate degree and certificate programs. Dedicated to delivering a distinctive undergraduate experience to more than 19,000 students, complemented by the strength of its graduate offerings and research accomplishments, Oakland University is organized into the College of Arts and Sciences and the Schools of Business Administration, Education and Human Services, Engineering and Computer Science, Health Sciences, and Nursing, as well as the recently established Oakland University William Beaumont School of Medicine. See our web page at http://www.oakland.edu. As the university’s chief advocate for research, the selected individual will lead the development of a clear vision for research at OU, inclusive of all disciplines, including incentive programs to facilitate activities to enhance the research infrastructure of the institution and to raise the level of external funding. The Vice Provost for Research reports to the Provost and oversees the Office of Grants, Contracts and Sponsored Research which manages pre- and post-award processes as well as regulatory compliance activities to ensure the ethical and responsible conduct of research. The successful candidate will also be responsible for the further development and management of policies related to intellectual property and technology transfer. This individual will be expected to establish and cultivate relationships both within and outside the university in order to expand collaborations with industry and government, as well as educational and research institutions. This leader will be expected to foster inter- and multi-disciplinary research initiatives and to facilitate entrepreneurial opportunities. Required Qualifications: The successful candidate must have an earned doctorate and academic accomplishments consistent with a tenured appointment at the university and evidence a passion for research. Additional expectations include: a strong record in the application for and management of externally funded grants and/or contracts; demonstrated leadership abilities; administrative/supervisory experience; ability to communicate and collaborate effectively with diverse internal and external constituencies; experience with research compliance issues; and, broad knowledge of research funding sources. Applications/Nominations: Interested individuals should submit their curriculum vitae, a letter of application that describes how their qualifications and interests meet those of the position, and a list of five references with complete contact information (address, phone, email). References will not be contacted until late in the process. All material must be submitted electronically as a single pdf file to [email protected]. Nominations should go to the same email address and should include the name and contact information for the nominee as well as a nomination statement. Review of applications will begin on March 1, 2011 and will continue until the position is filled. The appointment is anticipated to begin on or about August 15, 2011.

Tina Burks Midwest/West Coast/ South Central/Canada Phone: 202-326-6577

Locale: Rochester, Michigan is located twenty-five miles north of downtown Detroit in Oakland County. With a population of 75,000 residents and the characteristics of a small town, Rochester provides an excellent family environment. The Detroit metropolitan area has a population of over 3,000,000 residents and offers a wide diversity of cultural, sports, and entertainment activities.

Elizabeth Early East Coast & Industry Phone: 202-326-6578

Oakland University is an Equal Opportunity Employer and encourages applications from women and minorities.

Marci Gallun Sales Administrator Phone: 202-326-6582 Online Job Posting Questions Phone: 202-326-6577

EUROPE & REST OF WORLD E-mail: [email protected] Fax: +44 (0) 1223 326532 Alex Palmer Phone: +44 (0) 1223 326527 Susanne Kharraz Phone: +44 (0) 1223 326529 Dan Pennington Phone: +44 (0) 1223 326517 Lisa Patterson Phone: +44 (0) 1223 326528

JAPAN ASCA Corporation Jie Chin Phone: +81-3-6802-4616 Fax: +81-3-6802-4615 E-mail: [email protected]

CHINA & TAIWAN Ruolei Wu Phone: +86-1367-1015-294 E-mail: [email protected] All ads submitted for publication must comply with applicable U.S. and non-U.S. laws. Science reserves the right to refuse any advertisement at its sole discretion for any reason, including without limitation for offensive language or inappropriate content, and all advertising is subject to publisher approval. Science encourages our readers to alert us to any ads that they feel may be discriminatory or offensive.

POSTDOCTORAL FELLOWSHIP OPPORTUNITIES AT DUKE UNIVERSITY The Duke Cancer Institute (DCI) at Duke University Medical Center invites applications for postdoctoral fellowships for an NCI Cancer Education and Career Development Program entitled Integrating Population and Basic Science in Cancer Research. This three-year postdoctoral training program has a strong interdisciplinary focus in population science with a goal of training basic/experimental scientists who are interested in cancer research that spans laboratory and population sciences. Duke University will recruit two candidates for this program during the next few months. Applications are now being accepted and reviewed, with the decision to be made by June 1, 2011, with an anticipated start date of August 15, 2011. The deadline for all candidate materials is April 15, 2011. Candidates must have a PhD or equivalent in basic cancer biology, pharmacology, genetics or related disciplines and be engaged in cancer research. Applicants must be citizens of the United States, or have been lawfully admitted to the United States for permanent residence. Further information may be found on the DCI website (cancer.duke.edu/CECD). Candidates should send a letter describing their research interests (three-page maximum) and their Curriculum Vitae by e-mail to Ms. Sydnee Crankshaw ([email protected]). Duke University and Health System is an Equal Opportunity/Afﬁrmative Action Employer.

Institut de Pharmacologie Moléculaire et Cellulaire, Sophia-Antipolis, France Our research group has been recently awarded an ERC advanced grant. Through this prestigious program, we are seeking candidates for two post doctoral (1 to 4 years), one PhD (3 years), and one technician (1 to 4 years) positions/fellowship. Field: Remodeling of cellular membranes and sensors of membrane curvature. Approaches: biochemistry, cell biology and/ or molecular dynamics. www.ipmc.cnrs.fr/?page=antonny. Contact: [email protected]

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Oakland University

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Founded in 1911, The University of Hong Kong is committed to the highest international standards of excellence in teaching and research, and has been at the international forefront of academic scholarship for many years. Ranked 21st among the top 200 universities in the world by the UK’s Times Higher Education, the University has a comprehensive range of study programmes and research disciplines spread across 10 faculties and about 100 sub-divisions of studies and learning. There are over 23,400 undergraduate and postgraduate students coming from 50 countries, and more than 1,200 members of academic and academicrelated staff, many of whom are internationally renowned.

Post-doctoral Fellowships and Research Assistant Professorships Applications are invited for a number of positions as Post-doctoral Fellow (PDF) (Ref: 20110119) and Research Assistant Professor (RAP) (Ref: 20110120), at the University of Hong Kong, on or before February 29, 2012. Appointments will be made for a period of 2 to 3 years. PDF and RAP posts are created speciﬁcally to bring new impetus and vigour to the University’s research enterprise. Positions are available from time to time to meet the strategic research needs identiﬁed by the University. Positions are available in the following Departments: • Real Estate and Construction • School of English • School of Humanities (Philosophy) and Department of Psychology • Centre for the Humanities and Medicine • Faculty of Dentistry • Faculty of Education • Electrical and Electronic Engineering • Mechanical Engineering • Biochemistry • Centre for Cancer Research • Eye Institute

• Research Centre of Heart, Brain, Hormone and Healthy Aging • Research Centre of Infection and Immunology • Medicine • Public Health Research Centre • Centre for Reproduction, Development and Growth • School of Biological Sciences • Chemistry • Psychology • The State Key Laboratory of Synthetic Chemistry

Post-doctoral Fellows PDFs are expected to devote full-time to research. Applicants should be doctoral degree holders having undertaken original research that has contributed to the body of knowledge. A highly competitive salary commensurate with qualiﬁcations and experience will be offered. Annual leave and medical beneﬁts will also be available. Research Assistant Professors The main focus of an RAP’s duty is research. RAPs can however be assigned some teaching duties, up to 50% of the normal teaching load. Applicants should be research active and have a proven publication record. A highly competitive salary commensurate with qualiﬁcations and experience will be offered, with a contract-end gratuity and University contribution to a retirement beneﬁts scheme (totalling up to 15% of basic salary). Annual leave, and medical/dental beneﬁts will also be offered. Procedures Prospective applicants are invited to visit the following webpage to view the full list of the research areas and their home Faculties/Departments/Schools/Centres for which PDF/RAP positions are currently available. Before preparing an application they should contact the Head of the appropriate academic unit to ascertain that their research expertise matches the research area for which a vacant PDF/RAP post is available. Applicants must submit a completed University application form, which should clearly state which position they are applying for; and in which academic discipline. They should also provide further information such as details of their research experience, publications, research proposals, etc. Further particulars and application forms (152/708) can be obtained at http://www.hku.hk/apptunit/; or from the Appointments Unit (Senior), Human Resource Section, Registry, The University of Hong Kong, Hong Kong (fax: (852) 2540 6735 or 2559 2058; e-mail: [email protected]). Closes April 15, 2011. Candidates who are not contacted within 3 months of the closing date may consider their applications unsuccessful. The University is an equal opportunity employer and is committed to a No-Smoking Policy

GRADUATE PROGRAM

International PhD program Frontiers in Genetics proposes an international PhD program supported by the Swiss National Science Foundation. The program is based at the University of Geneva and includes participating members from the Universities of Lausanne and Zurich, the Swiss Federal Institute of Technology in Lausanne and the Friedrich Miescher Institute (Basel). The program starts in October 2011 and provides a strong background in molecular genetics and genomics for the study of modern biological problems. We are looking for outstanding candidates with a degree in biological sciences and a commitment to a career in research. The selected students will receive stipends for four years, subject to completion of all program requirements. Applicants should send the registration form, a letter describing their interests, background and research experience, official transcripts of their university courses and grades, copy of diplomas, and 3 letters of recommendation, to: NCCR Frontiers in Genetics International PhD Program Sciences III - University of Geneva 30, quai Ernest-Ansermet CH - 1211 Geneva 4, Switzerland Participating members: Stylianos E. Antonarakis, Konrad Basler, Bart Deplancke, Emmanouil T. Dermitzakis, Denis Duboule, Susan M. Gasser, Marcos González-Gaitán, Monica Gotta, Thanos Halazonetis, Nouria Hernandez, Winship Herr, Pedro L. Herrera, François Karch, Ulrich K. Laemmli, Joachim Lingner, Robbie Loewith, Serge Nef, Ivan Rodriguez, Botond Roska, Ariel Ruiz i Altaba, Ueli Schibler, David Shore, Françoise Stutz, Bernard Thorens, Didier Trono, Walter Wahli

Registration form: www.frontiers-in-genetics.org

Application deadline: May 1, 2011

Tenure-Track/Tenure-Eligible Investigator Hepatitis Virus Vaccine Research

The incumbent will be expected to conduct a vigorous research program that includes molecular virology, studies of protective immunity, production and characterization of candidate vaccines, and testing of vaccines and antibodies in animal models including nonhuman primates. The incumbent will benefit from unique opportunities to interact with other members of LID and DIR who perform basic and translational research on hepatitis and other viruses. LID has a diverse portfolio of virus vaccine research and an extensive collection of hepatitis specimens from humans and nonhuman primates. Access to nonhuman primates, stateof-the-art core research support, and the NIH Clinical Center—a premier research hospital on the main NIH campus—is available.

Additional information about LID is available online at www.niaid.nih.gov/LabsAnd Resources/labs/aboutlabs/lid. DIR provides support for salary, technical personnel, postdoctoral fellows, equipment, and research supplies. Salary is dependent on experience and qualifications.

Applicants must have an M.D., Ph.D., M.D./Ph.D., or equivalent degree in a relevant field with extensive postdoctoral experience, as well as a strong publication record demonstrating potential for creative research. Interested candidates may contact Jeffrey Cohen, LID Chief, at 301-496-5265 or [email protected] for additional information about this position.

To apply, submit your curriculum vitae, bibliography, and a

NIAID

The Laboratory of Infectious Diseases (LID), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases is seeking an outstanding individual to study the molecular biology and pathogenesis of hepatitis C virus and to develop and test candidate vaccines, immunotherapy, and immunoprophylaxis strategies. Studies of other human hepatitis viruses are also encouraged.

detailed statement of how your expertise can contribute to the success of the hepatitis virus program to Ms. Bao-Hahn Ngo at [email protected]. In addition, three letters of reference must be sent directly from the referee to Dr. Bernard Moss, Chair, NIAID Search Committee, c/o Ms. Bao-Hahn Ngo at [email protected] or 10 Center Drive, MSC 1356, Building 10, Room 4A22, Bethesda, MD 20892-1356. E-mail is preferred. Applications will be reviewed starting on April 25, 2011 and will be accepted until the position is filled. Further information regarding DIR laboratories is available at www.niaid.nih.gov/about/organization/dir, and information on working at NIAID is available at www.niaid.nih.gov/careers/dat. A full package of benefits (including retirement and health, life, and long-term care insurance) is available. Women and minority candidates are especially encouraged to apply. U.S. citizenship is not required.

National Institute of Allergy and Infectious Diseases

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health

National Institute of Allergy and Infectious Diseases Proud to be Equal Opportunity Employers

The Department of Molecular and Cellular Biology (MCB) in the College of Science is seeking a new department head. MCB has an interactive and congenial work environment with vigorous basic and translational research programs with an emphasis on the use of model organisms. The new MCB Department Head will be expected to lead efforts to bridge basic discovery science with biomedical and translational research. A successful candidate will be expected to have strong leadership skills, an active research program, and to take an active role in furthering the department’s undergraduate and graduate education missions. We are particularly interested in individuals whose research programs complement existing strengths on campus in cancer biology, epigenetics, regeneration, or aging. The University of Arizona is located in sunny Tucson and is a major Research I University consistently ranked in the top 20 based on research funding. To apply, submit an online faculty application for job number 46702 at www.uacareertrack.com. Please also be prepared to attach a curriculum vitae, a 1-2 page statement of research interests and the names and contact information of four references. Review of applications will begin Arpil 10, 2011, and continue until the position is ﬁlled. The University of Arizona is an EEO/AA Employer/M/W/D/V and is seeking individuals who are able to work with diverse students and colleagues with experience in a variety of teaching methods and curricular perspectives.

COMPUTER BIOLOGIST AND HEAD OF SCIENTIFIC INFORMATION DIVISION The Institut Pasteur, a leading center for biomedical research focusing on infectious diseases, neurosciences, developmental biology, immunology and structural biology, is seeking to strengthen its computer biology capacity within a new Direction for Information Systems. An important step in this process will be the recruitment of a Head of the Computer Biology Division, who will take overall responsibility for the management and development of several informatics platforms specializing in genomic analysis, data banks, imaging informatics, and software development, and will also lead an independent research group in bioinformatics, integrative biology, systems biology, modelling or imaging. The position is open to leading computer biologists who wish to pursue their own research programme while developing the informatics resources used by scientists throughout the Paris campus of the institute. The Application should comprise the following (in order) in a single PDF file: 1. A brief introductory letter explaining why the candidate wishes to join the Institut Pasteur. 2. A curriculum vitae and a full publication list. 3. A description of past and present research activities and computer biology and management experience (up to 8 pages with 1.5 spacing). 4. The proposed research project (up to 5 pages with 1.5 spacing). 5. The names of 3 computer biologists from whom letters of recommendation can be sought. The completed application should be sent by e-mail to applyDIS@ pasteur.fr by April 30, 2011.

Assistant and Associate Professor

Research Fellows

The Department of Cellular Biology and Anatomy at the LSU Health Sciences Center School of Medicine in Shreveport, LA is currently recruiting faculty members with a Ph.D. or equivalent degree for tenure-track positions at the level of Assistant and Associate Professor in the area of Cardiovascular Sciences or related ﬁeld. These positions are being ﬁlled as part of a major expansion of the research program in the department with a new chairperson. The positions will be supported by state-of-the-art laboratory space, competitive salaries and signiﬁcant start-up packages. The requirements for Assistant Professor include a minimum of two years of post-doctoral research experience and for the Associate Professor a minimum of three years of experience at the level of Assistant Professor. The successful candidates are expected to have a distinguished record of scholarly activity including teaching experience in Cellular Biology and Anatomy, or related basic science areas. For the Associate Professor level, extramural funding (NIH R01 or equivalent) is expected.

The Regulatory Systems Biology and the Nuclear Receptor Transcription laboratories are seeking Postdoctoral Scientists. The Cancer Research UK, Cambridge Research Institute, is an exciting initiative focused on translational cancer research based on the University of Cambridge’s Addenbrooke’s Biomedical Campus and supported by superb core facilities.

This is an exciting opportunity for appropriate candidates to play a signiﬁcant role in an invigorated department. Numerous outstanding research core facilities are available staffed by dedicated research associates. Exciting opportunities to interact with investigators of similar interests are available.

Position 1 - The Regulatory Systems Biology lab (Duncan Odom) employs experimental high-throughput tools to explore the evolution of transcriptional regulation and gene expression among mammals. Prior experience in evolutionary/ developmental biology or comparative genomics would be desirable.

Please submit application with full curriculum vitae and names of three references via emails to: Dr. William G. Mayhan ([email protected]), Professor and Chair, Department of Cellular Biology and Anatomy, LSU Health Sciences Center, School of Medicine in Shreveport, Shreveport, LA 71130. Please include a letter that summarizes your future research plans. Review of applications will begin immediately and will continue until all positions are ﬁlled. Louisiana State University, Medical School in Shreveport is an Equal Opportunity Educator and Employer.

Health Sciences Center SCHOOL OF MEDICINE IN SHREVEPORT

Cambridge Research Institute £26,650 - £34,150 pa inc

Applicants must hold a relevant PhD, be intellectually and technically flexible, with excellent molecular biology skills and preferably an understanding of transcriptional mechanisms. We welcome accomplished applicants seeking to switch fields.

Registered charity no 1089464

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DEPARTMENT HEAD MOLECULAR AND CELLULAR BIOLOGY

Position 2 -The Nuclear ReceptorTranscription lab (Jason Carroll) uses genomic, molecular and proteomic tools to define and characterise the underlying features of Estrogen Receptor (ER)-mediated transcription in breast cancer. Prior tissue culture and/or genomic/proteomics expertise would be desirable. For more details or to apply, please visit jobs.cancerresearchuk.org quoting the reference number (10853). Closing date: 22nd March 2011.

Laboratory Head Positions at Janelia Farm

Appointments may be made at either of two levels:

At Janelia Farm, we pursue challenging basic biomedical problems for which future progress requires technological innovation. We focus on two research areas: the identification of general principles that govern how information is processed by neuronal circuits and the development of imaging technologies and computational methods for image analysis. We have decided to broaden our foci at the Fellow level—we also seek very promising, early career stage scientists with interests beyond these two major foci. We expect that Janelia would be attractive to people with scientific programs that could benefit from collaborators or technologies already at Janelia. We value people with new perspectives who will contribute to our intellectual community.

Fellows

Examples might include:

We invite applications from biochemists, biologists, chemists, computer scientists, engineers, geneticists, mathematicians, neurobiologists, physicists, and statisticians at an early career stage who are passionate in their pursuit of important problems in basic scientiﬁc and technical research.

Fellows are independent scientists with labs of up to two additional members. Appointments are for five years. Group Leaders Group leaders are independent scientists, similar to HHMI investigators, with labs of up to six additional members. The initial appointment is for six years. Thereafter, group leaders will be reviewed for reappointment every five years.

There are two application deadlines per year and the next are: July 15, 2011, and December 15, 2011

For more information and to submit an application: www.hhmi.org/labhead/sci

A cell biologist looking to apply super-resolution optical microscopy. A computer scientist interested in machine vision. A physicist interested in instrument development. A biochemist interested in single-molecule imaging. Janelia Farm is now home to a growing, multidisciplinary community of more than 40 research groups and four project teams. Our scientists are supported by outstanding shared resource facilities within a unique campus less than an hour from Washington, D.C. All research is internally funded, without extramural grants. Janelia Farm offers a highly interactive working environment with on-site childcare, a fitness center, and dining facilities. Individual research groups are limited in size. We value research collaboration between groups as a mechanism to enable long-range innovative science and encourage the self-assembly of interdisciplinary teams of scientists. In addition, we support external collaborative science through a scientific visitor program.

The Howard Hughes Medical Institute is an equal opportunity employer. Women and members of racial and ethnic groups traditionally underrepresented in the biomedical sciences are encouraged to apply.

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HOWARD HUGHES MEDIC AL INSTITUTE

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Chief Operating Officer Position

FACULTY POSITION IN THE DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY The Department of Physiology and Pharmacology at Des Moines University seeks to ﬁll a tenure track faculty position. Highly desirable applicants will have preparation and expertise in pharmacology with an interest in teaching in the medical, podiatric, and health sciences curricula. Additionally, it is expected that the individual develop an innovative and extramurally funded research program that compliments the department’s current research emphasis in cardiovascular or cell signaling research. Applicants must have an earned Ph.D. or equivalent and relevant postdoctoral experience. For full consideration, candidates are invited to submit a letter of application stating their interest along with their curriculum vitae, a concise statement of teaching and research interests, educational philosophy and contact information for three references using the online applicant tracking system at www.dmu.edu/employment. Review of applications will begin March 1, 2011 and continue until a successful candidate is identiﬁed and hired. Candidates with questions speciﬁc to this position may contact the Search Committee Chair, Dr. Julia Moffitt at 515-271-1512 or julia.mofﬁ[email protected]. For complete job description, Faculty beneﬁt summary and/or information on Des Moines University, please visit www.dmu.edu/employment. DMU is an EOE Employer.

W E L C O M I N G

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Vaccine and Gene Therapy Institute (VGTI) is conducting some of the most important scientific research and clinical testing underway in the world today. The VGTI Florida research team is focused on the development of new vaccines and novel therapies to boost the immune response among the elderly, treat and ultimately eliminate AIDS, enhance immune responses to cancer, tuberculosis, and other infectious diseases, including those caused by dangerous, emerging viruses. We are part of the budding research technology area on the beautiful coastline in Florida. Our new 100,000 sq. ft. research building will be open soon. It’s non-fiction and it’s happening right here, right now at VGTI Florida. The COO provides leadership and strategic development of the key operational and administrative components of the Institute. VGTI consists of a multidisciplinary team of scientists focusing on developing vaccines and therapeutics for infectious diseases. The vision of VGTI Florida is to create synergies with existing institutes in Florida and the region. The COO will be responsible for all internal systems, and operations. The COO will lead through a period of rapid growth and there is need to evolve operational procedures to fit VGTI’s new size and throughput. This is a pivotal role and VGTI seeks a creative and accomplished leader with a passionate concern for research. The successful COO candidate will be extraordinarily skillful in evolving and guiding complex organizational systems and will assume stewardship of operations and driving continuous improvement. In this role you will be working with the VGTI-FL President and other members of the VGTI-FL leadership to oversee strategic planning, lead and manage the internal operations of VGTI. The COO will establish highly efficient and effective internal organizational processes and infrastructure that will allow VGTI to continue to operate at a high level and fulfill its mission of translating research into health.

W O R L D - C L A S S

Winthrop-University Hospital is leading the way in innovative treatments by using the most sophisticated medical technology available. Join Team Winthrop & Surround Yourself with the Best!

DIRECTOR

BONE AND MINERAL METABOLISM – LONG ISLAND, NY –

The Winthrop-University Hospital Research Institute is actively recruiting an MD or PhD scientist to join the current group of clinical and basic researchers studying bone and mineral metabolism.The scientist will direct the laboratory effort of the bone and mineral research program.The successful candidate will be capable of developing an innovative program of research focused on the causes and complications of osteoporosis,and other disorders of bone and mineral metabolism. It is expected that senior candidates will have an established track record of and existing funding and junior candidates will have a high degree of potential for becoming successful independent investigators. Appointments from the Assistant to Professor level will be made commensurate with the level of academic achievements.As the Nassau County clinical campus of Stony Brook School of Medicine, Winthrop provides a vibrant, interactive environment offering numerous opportunities for multidisciplinary collaborative investigations. Construction of a new research facility and a generous recruitment package including support for a team of researchers in clinical, basic, translational and outcomes research, create an exceptionally exciting and unique opportunity. Winthrop is located in a highly desirable geographic location within easy access to New York City and the beaches of Long Island. Please send letter of interest & curriculum vitae,to: John F.Aloia, MD, Chief Academic Officer. Email: [email protected], or call for more information: 516-663-2442.

Institute of Immunology and Translation Medicine

(IITM) Institute of Immunology and Translation Medicine (IITM) invites applications for Research Director, Professor and Associate Professors for the following research laboratories: 1) Tumor immunology 2) Stem cell and immune cell therapy 2) System Biology 3) Inflammation Research 4) Allergy Research 5) Molecular Immunology IITM is a new Institute affiliated with Jilin University and The First Hospital of Jilin University. IITM is established under the leadership of Executive Scientific Board: Yong-Jun Liu (Chair): Professor and Chairman, Department of Immunology; Director, Center for Cancer Immunology Research. University of Texas, M. D. Anderson Cancer Center. Xue Tao Cao (Co-Chair): Professor and Director, Institute of Immunology; Director, National Key Laboratory of Medical Immunology; Second Military Medical University. The mission of IITM is to understand the pathophysiology of human diseases by conducting basic and translational immunology research using cell and molecular biology, system biology, genomic and proteiomic approaches. The goal of IITM is to develop new methods of diagnosis and more effective treatment for human diseases including infectious diseases, autoimmune and inflammatory diseases and cancers. IITM is house in a 9,000 SM new building equipped with the state of the art cutting edge core facilities, including flowcytometry, imaging, antibody production, microarry and system biology, Mass Spectrometry and immunohistochemstry.

WUH is committed to affirmative action, equal opportunity, and the diversity of its workforce. EOE-AA-m/f/d/v

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Salary Negotiable. Please electronically send your CV, a summary of research plan and name of three references to e-mail: [email protected]

The H.C. Ørsted Postdoc Program at the Technical University of Denmark (DTU) DTU invites highly talented young researchers to apply for one of the stipends under the H.C. Ørsted Postdoc program. The H.C. Ørsted Postdoc program is named after the founder of the university, H.C. Ørsted, who discovered Electromagnetism. We seek candidates who have already obtained outstanding results during their PhD studies and who have demonstrated excellence and potential in their field of study. In order to be considered, applications must include a confirmation letter signed by the relevant DTU department head, stating that the department and the candidate have agreed upon the research plan. Applications must be based on the details of the full text announcement. Application deadline: 12 May 2011

DTU is a leading technical university rooted in Denmark but international in scope and standard. Our total staff of 4,500 is dedicated to create value and to promote welfare for the benefit of society through science and technology; and our 6,000 students are being

trained to address the technological challenges of the future. While safeguarding academic freedom and scientific independence we collaborate with business, industry, government, public agencies as well as other universities around the world.

Further details: dtu.dk/vacancy

A future worth working for

To support our growth in Neurology Research and Development, we are hiring Senior Investigators and Research Associates in the following areas: Behavioral Neuroscience

Breaking new ground in therapeutic areas. As well as careers. ABOUT BIOGEN IDEC

Blood Brain Barrier Transport In Vitro Assay Development

Biogen Idec uses cutting-edge science to discover, develop, manufacture and market biological products for the treatment of serious diseases with a focus on neurological disorders. Founded in 1978, we are the world’s oldest independent biotechnology company. Patients worldwide benefit from our leading multiple sclerosis therapies, and we generate more than $4 billion in annual revenues.

Myelin Repair

THE OPPORTUNITIES

Synaptic Plasticity

As part of our Framework for Growth, we are renewing our commitment to scientific innovation in the pursuit of new therapies for Progressive multiple sclerosis and other important neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, ALS and more. With this renewed commitment, we are expanding our Neurology Discovery and Translational Medicine teams, which use cutting-edge research and imaging technologies to investigate novel disease pathways, identify new drug candidates, and establish novel translational strategies to improve the likelihood of drug development success in neurodegenerative diseases. We invite you to be part of our culture of innovation, collaboration and discovery. It’s a future — and a company — worth working for. To review our current openings and apply online, please visit www.biogenidec.jobs/science. EOE

Neurophysiology Protein Misfolding

Translational Neuroimaging

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H.C. ØRSTED POSTDOC

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Faculty Position in Human Genetics and Genome Science in Toronto The Genetics and Genome Biology Program in The Hospital for Sick Children (SickKids) Research Institute is seeking an outstanding scientist to ﬁll a new faculty position in human genetics and genome science. Individuals with speciﬁc expertise in human genetics/genomics, informatics, statistical genetics, or genetic epidemiology are particularly encouraged to apply. Candidates must have a PhD and/or MD, or equivalent degree; have completed signiﬁcant postdoctoral training; and have an outstanding record of research productivity. The successful candidate will receive a competitive salary/startup package and a faculty appointment in an appropriate academic department of the University of Toronto. The candidate will have access to state of the art core facilities and is expected to establish a strong extramurally-funded research program as well as participate in undergraduate, graduate and postdoctoral training. The Hospital for Sick Children is Canada’s largest paediatric health care and research centre and belongs to the Toronto Academic Health Sciences Network, one of the world’s largest biomedical research and teaching communities. The Genetics and Genome Biology Program’s 24 faculty are internationally recognized for their achievements in a broad range of basic, translational and clinical research in human genetics and genomics. Junior investigators are preferred but more senior applicants will also be considered. All qualiﬁed candidates are encouraged to apply; however Canadian citizens and permanent residents of Canada will be given priority. SickKids hires on the basis of merit and we are committed to the principle of equity in employment. We welcome diversity and encourage applications from all qualiﬁed women and men, including persons with disabilities, members of visible minorities, sexual minorities and Aboriginal persons. Interested individuals should e-mail their application, preferably in PDF format, to: [email protected] by April 7, 2011. The application should include a curriculum vitae, a description of past research, a description of proposed research, and copies of three representative publications. Candidates should also arrange to have three signed letters of recommendation sent by e-mail to: [email protected].

Physiology Position in the Colleges of Veterinary Medicine and Medicine The Colleges of Medicine and Veterinary Medicine at the University of Illinois at Urbana-Champaign invite applications for a tenure-track position at the Assistant or Associate Professor level. We seek candidates with an established or strong potential for developing an independent, innovative research program in an area broadly encompassing cardiovascular physiology. Candidates will have the opportunity to form collaborative, interdisciplinary relationships with a research faculty strong in bioengineering, biomedical imaging, stem cell research, aging, neuroscience, nutritional science and translational science (http://biomedical.illinois.edu/) as well as interface with the local medical community including the Carle Heart and Vascular Institute (http: //www.carle.org/Services/heart/index.aspx). The successful applicant will have demonstrated teaching experience and be expected to provide cardiovascular physiology instruction to professional students at both Colleges. The position will be based within the Department of Comparative Biosciences, College of Veterinary Medicine (http://vetmed.illinois.edu/cb/) with a joint appointment in the College of Medicine (http://www.med.illinois.edu/). The position is a full-time, 9-month tenure track appointment, available August 2011. Candidates must possess a Ph.D., D.V.M, or M.D, or equivalent. Candidates with postdoctoral or advanced research training are preferred Salary and rank will be commensurate with qualiﬁcations. Qualiﬁed applicants should electronically submit a cover letter, a statement of research and teaching experience, CV, and contact information for three references to Illinois Human Resources (https://jobs.illinois.edu), Search Coordinator. For inquiries, contact Dr. David Bunick, search chair, at [email protected] or 217/333-2318. In order to ensure full consideration, applications must be received by April 15, 2011. Illinois is an Afﬁrmative Action/Equal Opportunity Employer and welcomes individuals with diverse backgrounds, experiences, and ideas who embrace and value diversity and inclusivity. (www.inclusiveillinois. illinois.edu).

We seek an established Scientific Coordinator for the P2ALS consortium of researchers working toward a cure for amyotrophic lateral sclerosis (ALS). P2ALS is the largest single consortium worldwide focused on preclinical research on this devastating disease. It comprises more than 15 laboratories across the US working together to discover new genes, disease mechanisms and therapeutic targets. The main ﬁelds of research are motor neuron and glial biology, human genetics, human stem cell biology and drug screening.

p2 als

The role of the Scientiﬁc Coordinator will be to maximize collaboration across the consortium to accelerate translation of new data toward the clinic. Working together with the P2ALS directors and advisors, he/she will conceive and impulse new goal-oriented multicenter projects, establish collaborations with the pharmaceutical and biotech industry, enhance drug screening and target validation platforms and interact with multiple institutions to facilitate sharing of intellectual property. The Scientiﬁc Coordinator will also organize group meetings and other interactions to enhance progress, and will oversee scientiﬁc reporting by the network as a whole. The successful candidate will have demonstrated experience in running of complex milestone-driven projects and a deep knowledge of the therapeutic development process. Expertise in neurodegenerative diseases and ALS will be an advantage. The position is available immediately and will be based in New York but will require frequent travel to institutions and meetings across the country. Salary commensurate with high level experience. Contact: Prof. C.E. Henderson Columbia University Motor Neuron Center 630 West 168th Street, P&S 5-420 New York, NY 10032 [email protected] Tel: 212-342-4086

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Our World-Class Research Institute Is Looking for Scientific Leaders Since its inception, The Methodist Hospital Research Institute has challenged the notion of “by-the-book” medical research. Led by Mauro Ferrari, Ph.D., President and CEO, the Research Institute is a 440,000-square-foot research enterprise for The Methodist Hospital System in Houston, TX, and is affiliated with the Weill Cornell Medical College in New York City. Methodist is transforming medicine with emerging techniques, and a staff that is developing real treatments and cures every day. Our laboratories are equipped with advanced technology and facilities that include a cyclotron, pre-clinical and clinical imaging, flow cytometry and microscopy, small and large animal vivariums; and a GMP facility for nanoparticles, contrast agents, vaccines, and therapeutic molecules. Our facility is a vertically integrated state-of-the-art laboratory for translational and clinical research where translational researchers and physician scientists bring ideas to clinical applications.

Health  Sustainability  Policy  Technology

LIVING THE

PROMISE

We are now searching for research professionals to serve in a variety of capacities.

Program leaders in the fields of: • Neurodegenerative Diseases and Repair of the Nervous System (Methodist Neurological Institute) • Cardiovascular Science (Methodist DeBakey Heart & Vascular Center) • Cancer Biology (Methodist Cancer Center)

Senior scientists in the fields of: • Diabetes and Metabolic Disorders (Methodist Center for Diabetes, Obesity and Lipids) • Transplant Immunology (Methodist Transplant Center) Candidates should be nationally and internationally recognized leaders with an outstanding track record of scientific discovery, funded research, programmatic leadership and academic mentorship. We will provide you with a position in the epicenter of medical research. You’ll discover an excellent research environment, state-of-the-art equipment, and the chance to follow your research from discovery to clinical application in a single facility. Applicants should submit a Statement of Scientific Interest, a Curriculum Vitae, and the names of three references to: Tong Sun, Director of Central Research Administration, The Methodist Hospital Research Institute, 6670 Bertner St., M.S. R2-216, Houston, TX 77030, or email [email protected] (please specify applying field in the subject line of email). Our success as an organization is due to the diversity of our team. We are an equal opportunity employer.

www.tmhri.org

www.MethodistHealth.com

Houston, TX

The Methodist Hospital System is the ofﬁcial health care provider of the Houston Texans, Houston Astros, Houston Dynamo, Rice Athletics, Houston Ballet, Houston Grand Opera and Houston Symphony.

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ENDOWED CHAIR in Bioinformatics University of Arkansas The University of Arkansas has funded an Endowed Chair of Bioinformatics in the Department of Biological Sciences. We seek to recruit an established researcher to be appointed at the level of ASSISTANT or ASSOCIATE PROFESSOR. The Department requests applications from or nominations of persons emphasizing modern areas of bioinformatics in genomics, proteomics, transcriptomics, or metabolomics, which would strengthen research programs at the University. Focus areas of interest include, but are not limited to, conservation biology, phylogeny, agriculture, or pathway analysis. Applicants must have a Ph.D. and postdoctoral experience. The successful candidate will establish an extramurally supported research program, supervise graduate and undergraduate research, and teach at the graduate and undergraduate levels. Additional details on this position are available (website: http://biology.uark.edu/search). Review of completed applications will begin March 24, 2011, and will continue until the position is filled. Applications must include curriculum vitae, a statement of current and future research plans and teaching interests, and three letters of recommendation. Nominations or applications (electronic or paper) should be addressed to: Dr. Douglas Rhoads (e-mail: [email protected]), Search Committee Chair, Department of Biological Sciences, 601 SCEN, 1 University of Arkansas, Fayetteville, AR 72701. The University of Arkansas is an Equal Opportunity/Affirmative Action Employer. Applicants must have proof of legal authority to work in the United States at the time of hire.

MICROBIOLOGIST XEUS in Boston, Massachusetts seeks a Microbiologist. Position requires an exceptional understanding of microbial fermentation, along with a solid working knowledge of common microorganisms utilized in the biofuels area. Applicant will team with Bio-Processing Engineers in the scaling and commercialization of our cellulosic biofuels technology. The candidate preferably has a Ph.D. in Microbiology or the equivalent. Send curriculum vitae to e-mail: [email protected].

ASSISTANT PROFESSOR of Biology (Environmental Biologist) The Department of Biology and Geology at the University of South Carolina Aiken (website: http:// www.usca.edu/biogeo) seeks applications for a fulltime, tenure-track Assistant Professor of Biology. Ph.D. required; postdoctoral experience preferred. Candidates with expertise in the human health aspects of environmental remediation preferred. Start date: January 2012. Primary teaching responsibilities include undergraduate courses in introductory biology, physiology, and a specialty area within our new Environmental Remediation and Restoration Program. The successful candidate will also develop an active research program that includes mentoring of undergraduate research projects. The Department has a history of funded research and places high value on undergraduate research. Application must be made online at website: https://uscjobs. sc.edu/applicants/Central?quickFind066208 and include a cover letter, curriculum vitae, teaching philosophy, research goals, and contacts for three references. Review of applications will begin on April 15, 2011 and continue until position is filled. USCA seeks to attract and retain a diverse faculty consistent with its student body and the surrounding community. Women and minorities are encouraged to apply. USCA is an Affirmative Action/Equal Opportunity Employer.

The University of Oklahoma Health Sciences Center is seeking a RESEARCH ASSOCIATE to perform clinical cytogenetics/molecular diagnostic testing. This position will conduct research in the area of clinical cytogenetic studies and will assist the laboratory director in managing the daily functions, results, and reports of the laboratory. Applicant must hold a Master_s degree in Cytogenetics or Plant Physiology and 60 months experience in clinical cytogenetics or Ph.D. and 36 months exp. Do not apply in person. Send curriculum vitae to: Dr. Shibo Li at OUHSC Department of Pediatrics, 941 SL Young Boulevard Room 224, OKC, OK 73104. Equal Opportunity Employer.

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TENURE-TRACK POSITIONS in Eye and Vision Research The Medical College of Wisconsin Eye Institute invites applications for tenure-track positions at all academic ranks. The area of eye and vision research is open, but scientists involved in retina-related research and those using in vivo imaging methods are especially encouraged to apply. For applicants at the ASSOCIATE or FULL PROFESSOR level, priority will be given to those with NEI funding. Applications (PDF format) and inquiries should be electronically sent to the Search Committee (e-mail: [email protected]). Include a cover letter, statement of research interests, and curriculum vitae including a complete list of publications. Applicants at the level of ASSISTANT PROFESSOR should also have three letters of reference electronically sent to the Search Committee. The Medical College of Wisconsin is an Affirmative Action/ Equal Opportunity Employer, and is located in a suburban setting about 7 miles west of downtown Milwaukee with easy highway access from all surrounding communities. TENURE-TRACK POSITION, IMMUNOLOGY— The Department of Biomedical Sciences at Oregon State University invites applications for a tenure-track ASSISTANT PROFESSOR position in Immunology. We seek an immunologist with outstanding potential for developing an extramurally funded research program in chronic diseases. For further details of the position and to apply, visit website: http://oregonstate.edu/ jobs; posting# 0006939. Full consideration: application materials must be received by April 10, 2011.

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