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CONTENTS

Volume 331 Issue 6024

SPECIAL SECTION

Cancer Crusade at 40 REVIEWS 1553 Exploring the Genomes of Cancer Cells: Progress and Promise

INTRODUCTION 1539 Celebrating an Anniversary NEWS 1540 Cancer Research and the $90 Billion Metaphor

M. R. Stratton

1559

U.S. Cancer Trends

1565

Combining Targeted Drugs to Stop Resistant Tumors 1545 Can Treatment Costs Be Tamed? 1542

1548

A Push to Fight Cancer in the Developing World

Brothers in Arms Against Cancer

EDITORIAL 1491 The Challenge of Cancer

Bruce Alberts >> Cancer Crusade at 40 section p. 1539

NEWS OF THE WEEK 1499 A roundup of the week’s top stories NEWS & ANALYSIS 1502 1504

Cancer Immunoediting: Integrating Immunity’s Roles in Cancer Suppression and Promotion R. D. Schreiber et al.

Making Her Life an Open Book to Promote Expanded Care

1551

A Perspective on Cancer Cell Metastasis

C. L. Chaffer and R. A. Weinberg

DEVASTATION IN JAPAN

Nuclear Power’s Global Fallout Radiation Risks Outlined by Bombs, Weapons Work, and Accidents

>> See also Editorial p. 1491; Report p. 1612; and Science Translational Medicine including Research Article by L. Sequist et al., Science Signaling, Science Careers, Video, and Science Podcast at www.sciencemag.org/special/cancer2011/

NEWS FOCUS 1510 Peak Oil Production May Already Be Here 1512 Texas Site Conﬁrms Pre-Clovis Settlement of the Americas

page 1505

BOOKS ET AL. 1519 Principles of Social Evolution

A. F. G. Bourke, reviewed by S. West

1520

>> Report p. 1599

1513

Going Viral: Exploring the Role of Viruses in Our Bodies

LETTERS 1515 Dealing with Data: Fostering Fidelity

POLICY FORUM 1521 Marine Biodiversity and Gene Patents S. Arnaud-Haond et al.

Dealing with Data: Preserve Old Collections

PERSPECTIVES 1523 A New Focus on RNA in the Lens

Dealing with Data: Upgrading Infrastructure

1524

A. E. T. Finlayson

M. K. Duncan >> Research Article p. 1571

M. Greve and J.-C. Svenning

Candidate Radiation Drugs Inch Forward

Current Designs Address Safety Problems in Fukushima Reactors 1507 Fukushima Cleanup Will Be Drawn Out and Costly 1509 Japan’s Research Facilities Down But Not Out 1506

Dealing with Data: Training New Scientists

A. J. Severin

Antarctica’s Deep Frozen “Lakes”

S. Tulaczyk and S. Hossainzadeh >> Report p. 1592

W. Los and J. Wood

1525

On Dental Occlusion and Saber Teeth

J. Fröbisch >> Report p. 1603

Advanced Translational Research

B. Bolon et al. 1517

The Restless Plant

D. Koller, reviewed by S. E. Wyatt

CORRECTIONS AND CLARIFICATIONS

1528

Electronic Bonding Revealed by Electron Diffraction P. A. Midgley >> Report p. 1583

CONTENTS continued >>

COVER False-color scanning electron micrograph of cells from pancreatic cancer (cell in center is ~10 µm across), one of the cancer types whose incidence and mortality rates have not changed signiﬁcantly since the 1970s. The United States National Cancer Act, signed in 1971, aimed to eliminate cancer deaths through a massive increase in research funding. A special section beginning on p. 1539 examines the state of this Cancer Crusade 40 years later.

DEPARTMENTS 1487 1493 1496 1537 1631 1632

This Week in Science Editors’ Choice Science Staff AAAS News & Notes New Products Science Careers

Image: Anne Weston, Cancer Research UK, Visuals Unlimited, Inc.

www.sciencemag.org

SCIENCE

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Published by AAAS

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CONTENTS

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Selective Insulin Sensitizers

1592

J. Y. Kim-Muller and D. Accili >> Report p. 1621 1532

R. E. Bell et al. A large fraction of the ice at Dome A, Antarctica, did not form by the usual process of snowfall compaction. >> Perspective p. 1524

An Earlier Acheulian Arrival in South Asia R. Dennell >> Report p. 1596

1533

A Helix for the Final Cut C. Raiborg and H. Stenmark >> Report p. 1616

1596

C. Hemingway et al.

RESEARCH ARTICLE

1599

Mutations in the RNA Granule Component TDRD7 Cause Cataract and Glaucoma

S. A. Lachke et al. A Tudor domain protein mediates posttranscriptional control of gene expression and is required for eye-lens development. >> Perspective p. 1523

1603

Baryons at the Edge of the X-ray–Brightest Galaxy Cluster

From a Single-Band Metal to a High-Temperature Superconductor via Two Thermal Phase Transitions

1606

R.-H. He et al. Three techniques are used to probe the pseudogap state of cuprate high-temperature superconductors. 1583

1587

The Bonding Electron Density in Aluminum

P. N. H. Nakashima et al. A combination of microscopy and ﬁrst-principle calculations is used to study the bonding charge density in aluminum. >> Perspective p. 1528

Revealing Extraordinary Intrinsic Tensile Plasticity in Gradient Nano-Grained Copper

1609

CREDIT (TOP): RICHARD MAAS AND SALIL LACHKE

T. Liu et al. Differences in surface charge and water mobility allow slightly different inorganic macroions to self-assemble separately.

Social and Ecological Synergy: Local Rulemaking, Forest Livelihoods, and Biodiversity Conservation

pages 1528 & 1583

Impacts of Salmon on Riparian Plant Diversity

M. D. Hocking and J. D. Reynolds A survey of Canadian forests reveals the effects of nutrient subsidies from salmon on plant community structure. 1612

Self-Recognition Among Different Polyprotic Macroions During Assembly Processes in Dilute Solution

Dental Occlusion in a 260-Million-Year-Old Therapsid with Saber Canines from the Permian of Brazil

L. Persha et al. Participation in tropical forest governance by local people results in positive outcomes for conservation and subsistence.

T. H. Fang et al. Nanometer-sized grain copper conﬁned by a graded substrate leads to a material with both high strength and high ductility.

1590

pages 1523 & 1571

J. C. Cisneros et al. Tiarajudens extends the date of dental occlusion and suggests why the members of this Permian group were such diverse and successful herbivores. >> Perspective p. 1525

A. Simionescu et al. The Suzaku satellite provides a census of the gas, metals, and dark matter out to the outskirts of the Perseus Cluster. 1579

The Buttermilk Creek Complex and the Origins of Clovis at the Debra L. Friedkin Site, Texas

M. R. Waters et al. A large artifact assemblage dating to 15,000 years ago lies beneath a Clovis assemblage in central Texas. >> News story p. 1512

REPORTS 1576

Early Pleistocene Presence of Acheulian Hominins in South India

S. Pappu et al. Dates from a site in southeast India imply an early migration of Homo thrwough Eurasia about 1.1 to 1.5 million years ago. >> Perspective p. 1532

SCIENCE PRIZE ESSAY 1535 Building Botanical Literacy

1571

Widespread Persistent Thickening of the East Antarctic Ice Sheet by Freezing from the Base

CD40 Agonists Alter Tumor Stroma and Show Efﬁcacy Against Pancreatic Carcinoma in Mice and Humans

G. L. Beatty et al. CD40 immunotherapy shows efﬁcacy in treating pancreatic cancer in mice and humans by eliciting antitumor immunity. >> Cancer Crusade at 40 section p. 1539 1616

Cortical Constriction During Abscission Involves Helices of ESCRT-III–Dependent Filaments J. Guizetti et al. The process by which animal cells are physically separated after cell division is dissected in molecular detail. >> Perspective p. 1533

www.sciencemag.org

SCIENCE

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Published by AAAS

1621

FGF19 as a Postprandial, Insulin-Independent Activator of Hepatic Protein and Glycogen Synthesis

S. Kir et al. Fibroblast growth factor 19 regulates liver metabolism through a mechanism distinct from that of insulin. >> Perspective p. 1529 1624

Clr4/Suv39 and RNA Quality Control Factors Cooperate to Trigger RNAi and Suppress Antisense RNA K. Zhang et al. A histone methyltransferase and an RNA export protein team up to clobber aberrant RNAs in ﬁssion yeast.

CONTENTS continued >>

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CONTENTS

SCIENCEONLINE SCIENCESIGNALING

SCIENCEXPRESS

www.sciencexpress.org

Eosinophils Sustain Adipose Alternatively Activated Macrophages Associated with Glucose Homeostasis D. Wu et al.

Regulation of adipose tissue macrophages by eosinophils reveals an unexpected role for eosinophils in metabolic homeostasis. 10.1126/science.1201475

Conserved Eukaryotic Fusogens Can Fuse Viral Envelopes to Cells

O. Avinoam et al. A Caenorhabditis elegans cell-surface fusion protein can promote viral fusion with mammalian cells. 10.1126/science.1202333

AMP-Activated Protein Kinase Regulates Neuronal Polarization by Interfering with PI 3-Kinase Localization S. Amato et al. A bioenergy-sensing pathway determines axon initiation and growth in neurons. 10.1126/science.1201678

www.sciencesignaling.org The Signal Transduction Knowledge Environment 22 March issue: http://scim.ag/22Mar2011

RESEARCH ARTICLE: In Vivo Systems Analysis Identiﬁes Spatial and Temporal Aspects of the Modulation of TNF-α–Induced Apoptosis and Proliferation by MAPKs

K. S. Lau et al. A systems-level analysis of signaling in the mouse intestine identiﬁes critical mediators of inﬂammation and regionally speciﬁc responses.

PERSPECTIVE: CD69—An Unexpected Regulator of TH17 Cell-Driven Inﬂammatory Responses P. Martín and F. Sánchez-Madrid CD69 deﬁciency promotes differentiation of inﬂammatory TH 17 cells and exacerbates autoimmune diseases in mice.

MEETING REPORT: The Yin and Yang of Signaling in Tregs and TH17 Cells

F. Pan et al. This meeting in Shanghai focused on the relationship between regulatory and inﬂammatory T cells.

Global Trends in Wind Speed and Wave Height

I. R. Young et al. Wind speeds over the world’s oceans have increased over the past two decades, as have wave heights. 10.1126/science.1197219

Polarized Gamma-Ray Emission from the Galactic Black Hole Cygnus X-1

P. Laurent et al. This gamma-ray emission originates from a jet of relativistic particles that is formed in close proximity to the black hole. 10.1126/science.1200848

SCIENCENOW

www.sciencenow.org Highlights From Our Daily News Coverage

Long-Neglected Experiment Gives New Clues to Origin of Life

A 53-year-old ﬂask containing brown sludge may reveal how the ﬁrst amino acids were created. http://scim.ag/old-experiment

FUNDING SOURCES

Check out the new opportunities for funding signaling research and training.

SCIENCECAREERS

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

CTSciNet: Conducting Cancer Clinical Trials

K. Hede Training courses teach young cancer researchers the skills needed to design and carry out clinical trials. http://scim.ag/cancertrialstraining

CTSciNet Q&A: Finding and Exploiting Cancer’s Weaknesses

K. Travis Clinician-investigator David Solit studies the genetic basis of cancer tumors and looks for novel therapies that target speciﬁc mutations. http://scim.ag/solitqanda

Navy Sonar May Mimic Killer Whale Sounds

Cause of Lethal Disease in China Unmasked

COMMENTARY: A National Cancer Clinical Trials System for Targeted Therapies

A new bunyavirus poses some riddles. http://scim.ag/lethal-disease

F. Fang et al.

PODCAST

T. A. Chan and A. Colmone Breast cancer methylomes contribute to metastatic potential, modulate the metastasis transcriptome, and predict disease outcome.

RESEARCH ARTICLE: Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors L. V. Sequist et al. Lung cancers undergo dynamic genetic and histological changes upon developing resistance to EGFR inhibitors. >> Cancer Crusade at 40 section p. 1539

SCIENCEPODCAST

www.sciencemag.org/multimedia/podcast Free Weekly Show On the 25 March Science Podcast: targeted cancer therapy, progress with cancer genomes, mechanisms of metastasis, immunity’s role in cancer, and more.

SCIENCEINSIDER

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

VIDEOFEATURE

video.sciencemag.org

Sequencing Cancer Genomes: Targeted Cancer Therapies

Applying DNA sequencing to cancer genomes is providing insights that have allowed researchers to turn some cancers into chronic diseases rather than deadly ones. Still, the ultimate goal is to kill the cancer. >> Cancer Crusade at 40 section p. 1539 and www.sciencemag.org/special/cancer2011/

SCIENCETRANSLATIONAL MEDICINE

www.sciencetranslationalmedicine.org Integrating Medicine and Science 23 March issue: http://scim.ag/stm032311

This ﬁnding could explain cetacean strandings. http://scim.ag/whale-sonar

RESEARCH ARTICLE: Breast Cancer Methylomes Establish an Epigenomic Foundation for Metastasis

J. Mendelsohn Clinical cancer research requires a well-supported and efﬁcient national clinical trials network that engages in innovative trials of modern cancer therapies.

COMMENTARY: Accrual to Cancer Clinical Trials in the Era of Molecular Medicine

R. L. Schilsky In the testing of targeted cancer therapies, new issues can complicate the decision by patients or providers to participate in clinical trials.

www.sciencemag.org

SCIENCE

VOL 331

Published by AAAS

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 STELLA HURTLEY to the resolution of the cuprate high-temperature superconductivity puzzle. Whether the “transition” at T* is a real phase transition characterized by broken symmetry or a crossover, is one of the crucial unanswered questions. He et al. (p. 1579) used three techniques on the same sample to reveal an abrupt transition at the same temperature, consistent with the existence of a true phase transition at T*.

Large ice sheets, like the one that covers Antarctica, grow when, over long periods of time, falling snow accumulates faster than it disappears. However, this type of accumulation is not the only way that ice sheets can thicken. Bell et al. (p. 1592, published online 3 March; see the Perspective by Tulaczyk and Hossainzadeh) found that as much as half of the thickness at some locations of an ice sheet can result from freezing at the bottom. This process alters the structure of the ice column and the topography of the ice sheet surface and has implications for how we understand ice sheet movement and the paleoclimatological information contained within.

CREDITS (TOP TO BOTTOM): ROBIN E. BELL/LAMONT DOHERTY EARTH OBSERVATORY OF COLUMBIA UNIVERSITY; CISNEROS ET AL.

An Eye on RNA Granules During vertebrate organ development, gene expression is precisely regulated by controlling gene transcription, messenger RNA (mRNA) translation, or the stability of mRNAs. Lachke et al. (p. 1571; see the Perspective by Duncan) have determined that the Tudor Domain protein TDRD7, which forms RNA granules in developing lens tissue, is required for proper eye development and maintenance. TDRD7 deﬁciency in human patients and model organisms affects the expression of speciﬁc mRNAs and causes ocular defects including cataracts and glaucoma. Thus, regulation of posttranscriptional mRNA levels, like the regulation of gene transcription, is critical to mammalian organogenesis.

Pancreatic Cancer Immunotherapy Pancreatic ductal adenocarcinoma (PDA) is a particularly deadly form of cancer for which few therapies have shown efﬁcacy. The tumor microenvironment in PDA is largely immunosuppressive, blocking antitumor immunity. Beatty et al. (p. 1612) treated a small cohort of PDA patients with gemcitabine chemotherapy plus a monoclonal antibody that activates CD40, a protein known to promote T cell immunity. Because this combination showed efﬁcacy in a small number of patients, the same treatment was analyzed in a mouse model of PDA. A subset of CD40 antibody-treated mice also showed tu-

mor regressions. However, the antitumor effects depended not on T cells, but on macrophages. Macrophages that had inﬁltrated the tumors after antibody treatment were also tumoricidal in vitro. Thus, activation of macrophages by CD40 may promote antitumor immunity in PDA.

Cosmic Squeeze Baryonic matter is made up of protons and neutrons, but represents just a small fraction of the matter in the universe—most of the rest being dark matter. Using high-quality data from the Suzaku x-ray telescope, Simionescu et al. (p. 1576) measured the fraction of baryonic mass to total mass in the Perseus Cluster of galaxies. Within the inner region of the cluster, the baryonic fraction is consistent with that measured globally for the universe. At large radii, however, the baryonic fraction exceeds the global value, suggesting that the cluster has undergone a major merging event that caused a compression of the gas, where most of the cluster’s baryons reside, relative to the dark matter in the cluster outskirts.

Mind the Pseudogap Unlike conventional superconductors, the cuprate family of superconductors has an exotic phase above their transition temperature, Tc, where the material is neither superconducting nor in the normal state. This so-called pseudogap state commences at a higher-temperature T* and its nature is conjectured to hold the key

www.sciencemag.org SCIENCE VOL 331 Published by AAAS

A metal can be made stronger by reducing the size of its component crystalline grains. However, the material loses its ability to stretch and change shape, and will eventually fracture and fail completely. Because this sort of deformation precedes material failure, the loss of this plasticity has limited the application of nanocrystalline metals in the real world. Fang et al. (p. 1587, published online 17 February) conﬁned nanocrystalline copper within a substrate of coarsergrained copper, using a transition region where the grain size slowly decreased, and they were able to create specimens that retained plasticity but also showed high yield strength.

My, What New Teeth You Have Mammals evolved within a group of tetrapods, the therapsids, which arose during the lower Permian period about 270 million years ago. After the end of the Permian, a group of therapsid anomodonts (mammal-like reptiles) differentiated into an extremely diverse group of herbivores—from small burrowing animals to large browsers. Cisneros et al. (p. 1603; see the Perspective by Fröbisch) describe a new basal anomodont, Tiarajudens, from Brazil that provides some clues about how this group managed to become such successful herbivores. Dental occlusion is a key component of successful herbivory that facilitates efﬁcient processing of tough cellulosic plant materials through thorough grinding between hard teeth. Tiarajudens pushes the date of dental occlusion in this group back to 260 million years ago.

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Growing from Below

Strengthening Nanocrystalline Copper

Stone Tool Manufacturers Acheulian stone tools, including oval- and pearshaped handaxes, were ﬁrst manufactured in Africa about 1.6 million years ago. Comparable tools have been seen across Eurasia, but in

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This Week in Science Continued from page 1487

many cases their ages have been uncertain. Although the most conﬁdently dated sites are thought to be less than 1 million years old, earlier migrations of Homo from Africa have been suggested. Pappu et al. (p. 1596; see the Perspective by Dennell) have now obtained consistent cosmogenic ages from Acheulian tools in Southeast India of at least 1.1 million and up to 1.5 million years ago. Thus, an early Eurasian migration of Homo did indeed possess Acheulian technology.

Forest Community Commitment

Along the Riverbank Salmon are distributed around the Paciﬁc Rim from California to Korea, and the carcasses of salmon returning to their native streams to spawn provide substantial nutrient input to the surrounding riverside vegetation. Hocking and Reynolds (p. 1609) show that these subsidies cause detectable shifts in plant communities along streams. In a large-scale study of 50 watersheds in British Columbian rainforests, the impact of subsidies led to a simpliﬁcation of plant communities and a shift toward nutrient-demanding plant species. Thus, interactions across ecosystem boundaries can change ecological community structure and function, which will impact ecosystem-based management of salmon and their habitats.

Abscission in Glorious Technicolor Abscission represents the very ﬁnal stage of animal cell division, when daughter cells are physically separated from one another. Guizetti et al. (p. 1616, published online 10 February; see the Perspective by Raiborg and Stenmark) present three-dimensional electron tomographic reconstructions of intermediate stages of abscission in human cells, which reveal spirals of 17-nanometer-diameter ﬁlaments at a cortical constriction site within the intercellular bridge. Live-cell and structured illumination microscopy suggest that a protein complex involved in a variety of membrane-trafﬁcking processes and in cytokinesis, ESCRT-III, builds the abscission structure and mediates cortical ingression of the intercellular bridge.

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Tropical forests typically contain rich biodiversity with high conservation value, also providing important resources for human inhabitants and users. Persha et al. (p. 1606) used a social-ecological data set of 84 human-dominated landscapes in East Africa and South Asia to analyze the factors that shape the joint beneﬁts of forest biodiversity conservation and forest-based subsistence livelihoods. Jointly positive outcomes across these two potentially competing forest beneﬁts were far more likely when forest users participated in the rulemaking aspects of forest governance.

FGF19 and Liver Metabolism

CREDIT: IAN MCALLISTER/PACIFIC WILD

Insulin has been the main hormone described to regulate metabolism in the liver. Kir et al. (p. 1621; see the Perspective by Kim-Muller and Accili) now show that ﬁbroblast growth factor 19 (FGF19) appears to be another important regulator, promoting synthesis of glycogen and proteins. FGF19 is made in the small intestine in response to food intake, which stimulates receptors at the liver to regulate metabolism through a mechanism distinct from that of insulin. Unlike insulin, FGF19 did not promote lipogenesis in the liver. In a mouse model of diabetes, FGF19 promoted glycogen synthesis, and could thus provide a target for therapeutic control of insulin resistance.

RNA Quality Control Eukaryotic genomes are extensively transcribed into RNA by RNA polymerase II. Quality-control and surveillance systems must ensure that all this RNA is processed correctly. A ﬁssion yeast histone methyltransferase involved in heterochromatin assembly, Clr4, helps mediate exosome-dependent degradation of potentially dangerous antisense transcripts. Zhang et al. (p. 1624) now show that Clr4 interacts with and methylates the RNA export protein Mlo3. Mlo3 promotes Clr4’s ability to suppress aberrant antisense RNAs through its interaction with the RNA interference machinery and with a cofactor of the exosome nuclear surveillance system. Mlo3 and Clr4 may thus be part of a molecular sensor that differentiates aberrant RNAs from the real McCoy. www.sciencemag.org SCIENCE VOL 331

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EDITORIAL

The Challenge of Cancer

CREDIT: (RIGHT) MEDICALRF/PHOTO RESEARCHERS, INC.

THIS SPECIAL ISSUE OF SCIENCE FOCUSES ON CANCER, A DISEASE THAT TAKES A TERRIBLE HUMAN

toll. For scientists, preventing and curing this disease present an enormous challenge. We all know someone who suffers from cancer, and there is always pressure on researchers to focus on the immediate development of better treatments. But it is also essential to invest in innovative, longer-term efforts designed to generate more powerful approaches. The Reviews and analyses in this issue reveal why the task of treating the disease is so frustratingly complex (see p. 1539). By the time most cancers are detected, a tumor has grown to contain more than a billion cells. Through a process resembling mutation and natural selection stretching over many years, these cells have become altered in ways that allow them to escape from the large number of failsafe mechanisms that normally protect the human body. For example, the rogue cells have evaded the elaborate system of growth controls that keep most of the 80 trillion cells that form a human in a quiescent state, disrupted the cell suicide mechanisms that would otherwise eliminate any aberrant cell, and evolved a resistance to immune system surveillance. To make matters much worse, the cells in a cancer are constantly changing, having acquired a greatly enhanced ability to mutate and to alter the genes they express through other mechanisms. As a result, the population of cells in a tumor is quite heterogeneous, making it very unlikely that any single therapy can target them all. This presumably explains the repeated failure of powerful new treatments, such as those described on p. 1542, to permanently cure the disease: A small fraction of tumor cells resistant to the treatment continues to proliferate, eventually multiplying to a dangerous level. Attaining true cures for most cancers would therefore be expected to require that patients be treated with two or more drugs simultaneously.* The logic is simple: Whereas a tumor is likely to harbor some cells that are resistant to any one drug, it is unlikely that there will be cells resistant to two drugs that target different cellular pathways or multiple points within a single pathway. But the cells in different individual tumors will be different. How can we hope to ﬁnd a set of drugs that will effectively kill the cells in a particular patient’s tumor without damaging the vast number of normal cells needed to keep us alive? Powerful new approaches seem possible, given the great advances made in our ability to study and manipulate cells. In a traditional scientiﬁc meeting, many experts in cancer come together to present their research and exchange ideas. More likely to generate the needed innovation is a newer model of small workshops, in which ﬁve or so experts on cancer spend several days with 25 outstanding scientists and engineers who have expertise outside the cancer ﬁeld that is suspected to be relevant to new approaches. The effort begins with the experts framing the important unsolved problems for the nonexperts and answering their questions. Most of the remaining time is spent in a set of small breakout groups, each of which focuses on exploring a speciﬁc new approach, such as that emphasized in a previous editorial.** As a second example, what types of nanotechnologies might eventually be developed to allow a freshly excised tumor to be dispersed into many small groups of cells that maintain their normal physiological state, so that robotics can be used to rapidly administer many different combinations of drugs, growth factors, and antibodies, with automation used to monitor results? Science’s sister journal Science Translational Medicine will be cosponsoring a series of workshops of precisely this type in 2011, including workshops on Alzheimer’s disease and a speciﬁc aspect of cancer. If successful, we hope that many more such efforts can be undertaken in future years. – Bruce Alberts

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Bruce Alberts is Editorin-Chief of Science.

10.1126/science.1205711

*A. D. Levinson, Science 328, 137 (2010). **B. Alberts, Science 325, 1319 (2009).

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EDITORS’CHOICE sequencing costs go down and network analyses become more sophisticated, it is likely that these strategies will be increasingly used in public health efforts. — BJ N. Engl. J. Med. 364, 730 (2011). BIOMEDICINE

Lupus Casts a Complicated NET

BIOMEDICINE

Brainstem Tumor Development Brain cancer is the most common solid tumor in children. For children with medulloblastoma, survival rates have steadily improved as a result of optimized therapies. In contrast, children with an aggressive brainstem tumor called DIPG (for diffuse intrinsic pontine glioma) are far less fortunate, with death occurring usually within a year. Because biopsy specimens of human DIPG are rare and because there are no relevant animal models, little is known about the cellular and molecular origins of these tumors. A study by Monje et al. provides insight both into the likely cell of origin of DIPG and into a signaling pathway that may help promote tumor growth. The culprit cell appears to be a previously uncharacterized neural precursor cell in the normal human brainstem. The density of these cells peaks during the time of childhood, when DIPGs most commonly arise. In a cell culture model, human DIPG cells (above, overlaid on an MRI scan from a DIPG patient) showed activation of the Hedgehog (Hh) signaling pathway, which is critical to normal brain development and which is aberrantly activated in other human cancers, including medulloblastoma. Thus, DIPG probably arises through dysregulation of postnatal neurodevelopment, and the Hh signaling pathway may be a possible therapeutic target for this tumor. — PAK Proc. Natl. Acad. Sci. U.S.A. 108, 10.1073/pnas.1101657108 (2011).

EPIDEMIOLOGY

Tracking a TB Network Improvements in two very different methods of investigation led to better understanding of the dynamics of a tuberculosis (TB) outbreak in British Columbia, Canada. A 10-fold increase in TB cases was reported in a Canadian community between 2006 and 2008. Initial genotyping analysis by Gardy et al. suggested that the outbreak was clonal; however, whole-genome sequencing of M. tuberculosis isolates produced a different picture: The cases were the result of two outbreaks. Examination of historical isolates

indicated that the two lineages were present before the recent outbreak, which suggested that it was a social or environmental effect, not a genetic mutation, which triggered the increase in cases. Social network analysis was then used to build a picture of risk behavior, interactions, and social meeting places. When this was combined with the whole-genome data, the investigators were able to identify sources of the outbreak and a likely contribution of increased crack cocaine use. As

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SCIENCE

VOL 331

Published by AAAS

Systemic lupus erthymatosis is a debilitating autoimmune disease that is characterized by the accumulation of inﬂammatory immune complexes (antibodies bound to a person’s own nuclear structures) in tissues. Lupus patients exhibit alterations in cytokine production and in various immune cell numbers and functions, but how these changes contribute to disease pathogenesis is not well understood. Lande et al. and Garcia-Romo et al. analyze serum samples and cells isolated from lupus patients and report that immune complexes, type I interferons, and neutrophils are entwined in a vicious cycle that drives disease pathogenesis. This trio of immune mediators is elevated in lupus serum, and together they induce a type of cell death in neutrophils called NETosis, where neutrophils spew out their nuclear contents (NETs). NETs drive further production of interferons, which in turn induces more NETosis. NETs are also an antigenic source for the generation of more immune complexes. With only one targeted therapy available for lupus, new therapeutic targets such as these are badly needed. — KLM

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CREDIT (LEFT): MONJE ET AL., PROC. NATL. ACAD. SCI. U.S.A. 108, 10.1073/PNAS.1101657108 (2011). (DIPG IMAGE) MORGAN FRERET AND PHIL BEACHY; (RIGHT) NASA (FULL CREDIT INFORMATION AVAILABLE AT HTTP://APOD.NASA.GOV/)APOD/AP060824.HTML

EDITED BY KRISTEN MUELLER AND JAKE YESTON

Sci. Transl. Med. 3, 73ra19; 73ra20 (2011). ASTRONOMY

Bullets in the Dark Dark matter, amounting to 85% of the matter of the universe, interacts with ordinary matter through gravity and possibly through the weak nuclear force. Massey et al. propose a statistical method that could help constrain the extent to which dark matter and ordinary matter interact via the weak force. When two clusters of galaxies collide, as happened in the Bullet cluster, the gas composing most of the ordinary matter in the clusters is slowed down by drag forces, temporarily separating from the dark matter, which passes unimpeded through the collision. The degree of separation reﬂects the degree

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of interaction of dark matter, but constraints are limited by the rarity of cluster collisions. Numerical simulations of cosmic structure formation tell us that clusters are built gradually from smaller pieces of infalling matter. The new method treats these pieces as bullets, whose ordinary matter is expected to drag behind the dark matter. Although individual separations between ordinary and dark matter may not be signiﬁcant, averaging through a large number of clusters could provide constraints that are tighter than those provided by the collision in the Bullet cluster. — MJC Mon. Not. R. Astron. Soc. 10.1111/ j.1365-2966.2011.18246.x (2011). CHEMISTRY

Negative Get-Togethers When transition-metal salts dissolve in water, it’s not unusual for a number of the counterions to remain coordinated. In contrast, alkali metal ions such as sodium or potassium weren’t traditionally thought to behave this way; dissolution of their salts conjures images of a sea of water molecules keeping anions and cations thoroughly apart. Recently, however, precise spectroscopic studies have been uncovering a more complicated scenario for these simple salts, in which cation and anion continue to inﬂuence one another in solution. In one such study, Bian et al. now present evidence that concentrated solutions of alkali thiocyanates (SCN–) exhibit anion clustering. The authors used two-dimensional infrared spectroscopy to measure vibrational energy transfer between isotopically light and heavy SCN– solutes as a probe of their proximity, and they found that in 10 M KSCN, over 90% of the anions gathered in clusters. Rotational anisotropy measurements implicated cluster sizes of approximately 18 anions. Diluting the solution reduced the apparent proportion of anions that gathered in clusters, as did shifting to smaller cations (lithium or sodium); cesium—the largest stable alkali— correspondingly induced the highest clustering proportion. — JSY Proc. Natl. Acad. Sci. U.S.A. 108, 10.1073/ pnas.1019565108 (2011). GEOCHEMISTRY

CREDIT: MICHAEL PERFIT

Under the Ridge Oceanic crust forms along the spine of midocean ridges—massive mountain chains along the seaﬂoor—which churn out lava and push fresh crustal material outward in both directions. Discerning how the hot material delivered from the subsurface to the ridge eventually turns into www.sciencemag.org

crust depends on quantifying the cooling rate of fresh lavas. In search of new geochemical clues to constrain heat transfer processes, Schmitt et al. sampled lavas from a segment of the Juan de Fuca ridge off the coast of western North America. From the rock samples collected (such as the dacite shown below), they separated tiny crystallized zircon grains, which serve as an excellent thermometer for tracking the thermal history of the melts. Dates obtained using a variety of radiometric dating techniques translate to rapid cooling rates: over an order of magnitude faster than crust formed along other segments of the same ridge. These rates are too fast to support models by which the crust cools via conduction, instead implicating cooling by permeation of convective hydrothermal ﬂuids through the new crust. — NW Earth Planet. Sci. Lett. 302, 349 (2011). CELL SIGNALING

Cell Signaling Goes Live Elucidation of cellular signaling pathways holds the promise of revealing new therapeutic targets. Such an approach, however, requires a deeper understanding of how signaling pathways interact in regulatory networks and within their physiological niche in vivo. Lau et al. studied effects of the cytokine TNF-α, which contributes to chronic inﬂammation in diseases such inﬂammatory bowel disease. TNF-α administration to mice caused apoptosis of epithelial cells in the duodenum, whereas further along the small intestine in the ileum, TNF-α induced proliferation. By characterizing the phosphorylation signals from proteins known to act as critical nodes in cell signaling pathways, the authors constructed mathematical models of the variation in responses to TNF-α. These models predicted that the protein kinase ERK may regulate the distinct regional responses. Pharmacological inhibition of ERK signaling conﬁrmed the predictions and showed that modulation of ERK signaling affected the signaling network beyond the ERK pathway itself. The results show an effective strategy to characterize complex in vivo responses through modeling and then test predictions experimentally, a method that may enhance the development of new therapeutic strategies. — LBR

SCIENCE

Sci. Signal. 4, ra16 (2011).

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

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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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NEWS OF THE WEEK AROUND THE WORLD IXO

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CREDITS (TOP TO BOTTOM): NASA; THE TRISTAN CONSERVATION DEPARTMENT, WWW.TRISTANDC.COM

Scientists Welcome Proposed Changes to Libel Law

Scientiﬁc discourse may enjoy greater legal protection in the United Kingdom if the provisions in the draft Defamation Bill become law. The proposals include conferring more protection on statements dealing with matters of “public interest” and requiring that a statement must cause “substantial harm” before it becomes defamatory. The new Defamation Bill comes after several scientists and science writers have been ensnared in libel suits for discussing or writing about controversial matters (Science, 11 June 2010, p. 1348). Compared with other countries, the United Kingdom is generally considered more permissive to such lawsuits. Science minister David Willetts called the proposed legislation “good news for science,” but some advocates of libel reform, including Tracey Brown, managing director of Sense About Science, have so far reacted cautiously. More change is needed, said Brown in a statement, before scientists and journalists can “focus on the question ‘is it true?’ rather than ‘will they sue?’ ” http://scim.ag/libel-reform

Nightingale Island, Tristan da Cunha 2

Oil Spill Menaces Penguins A shipping disaster in one of the loneliest places in the world is threatening an already endangered penguin species. On 16 March, the Oliva, a Maltese cargo vessel carrying soybeans from Brazil to the Far East, ran ashore and broke in two at Nightingale Island, a speck midway between South Africa and Argentina. Nightingale is home to more than 200,000 penguins, including about 40% of the world’s population of Northern Rockhop-

pers (Eudyptes moseleyi), a threatened species. The ship’s crew was saved, but its 1500 tons of fuel oil have begun leaking out and have encircled the 3-square-kilometer island, which is administered by the United Kingdom. A local Web site has posted images of oil-covered penguins coming ashore (pictured); as Science went to press, the Southern African Foundation for the Conservation of Coastal Birds was considering whether to send a cleanup team. Another danger looms, says the U.K.’s Royal Society for the Protection of Birds (RSPB): Rats escaping from the ship could colonize Nightingale and create ecological havoc. “How a modern and fully-laden cargo vessel can sail straight into an island beggars belief,” RSPB research biologist Richard Cuthbert said in a statement on 21 March. Paris 3

ESA Flies Solo After NASA Yanks Support

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Not Your Average Ant Farm A model farm devoted to raising insects as food is set to open next week at the National University of Laos in Vientiane. The demonstration site is part of a larger research project sponsored by the U.N. Food and Agriculture Organization (FAO) designed to develop sustainable and proﬁtable techniques for raising “mini-livestock” such as crickets, mealworms, palm weevils, and weaver ants. Laos has several advantages as a place to establish insect farming, says Paul Vantomme, senior forestry ofﬁcer at FAO in Rome and an expert on entomophagy, the practice of eating insects. Wild-caught insects are a regular part of the local cuisine, he notes, and ﬁsh and chicken farms provide another potential market for the farm’s products. Several studies have demonstrated that insects can be a highly nutritious source of protein—with a low carbon footprint. Washington, D.C. 5

European space scientists are scrambling to rethink—and redesign—massive potential missions after it was conﬁrmed that NASA, whose budget is in disarray, won’t contribute signiﬁcant funding to any of the efforts. The European Space Agency (ESA) was supposed to decide in June whether to spend about $1 billion on one of three so-called L-class missions: the International X-ray Observatory (IXO), the Europa-Jupiter mission known as EJSM-Laplace, or a spacebased gravitational-wave detector called LISA. But each mission, which wouldn’t launch until the next decade, has been developed with NASA as a would-be partner.

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The beleaguered U.S. space agency has now told ESA it has higher priorities for its limited space science budget. The decision “means in principle that none of the three missions is feasible for ESA,” notes Xavier Barcons of the Cantabria Institute of Physics in Spain, who has helped develop plans for IXO. But ESA will press ahead on its own, delaying its choice until 2012. European scientists working on the three missions are now reviewing what can be cut from their projects. http://scim.ag/ﬂy-solo

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Guatemalans Demand Justice Seven Guatemalan plaintiffs ﬁled a class action lawsuit against a raft of U.S. government ofﬁcials last week, on behalf of hundreds of their compatriots who were part of unethical medical studies run by U.S.-funded scientists in Guatemala in the 1940s. Many of the study subjects were deliberately infected with syphilis. The class also includes thousands of others who, as children and partners of the subjects, were impacted. Six months ago, after a historian at Wellesley College discovered the decadesold research, the U.S. government apologized and pledged an inquiry. The plaintiffs’ >>

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Three Q’s Evan O’Dorney checked out his ﬁrst math book from the library when he was just 2 years old. Last week, the now 17-yearold homeschooler from Danville, California, won ﬁrst place and $100,000 Evan O’Dorney in the Intel Science Talent Search with an elegant formula that solves a mathematical stumper: When do two different methods for approximating square roots of integers, one based on inﬁnite fractions and the other on iterating functions, reach the same answer? Another nine precocious winners, whose discoveries included novel liver cancer treatments and insights into star formation, shared $530,000. O’Dorney, an avid pianist and composer, will study math at Harvard University this fall. Q: Why don’t other teenagers like math as much as you do? It seems like sometimes the teachers in the public schools make it dry and boring. When I went to the library, I found books that really exposed the fun and beauty of mathematics, but teachers often just want to teach the rote memorization and skills that [students] need for the test. Q: What’s your working style? For 2 weeks, when I was really getting the main ideas, I worked all day. … It’s just the excitement of the math. I never need [caffeine]. … I almost have the opposite problem. If I get into math, I have to remind myself that I’m hungry and I need to eat.

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After 6½ years in space, almost 8 billion kilometers traveled, six orbit-altering ﬂybys of planets, and 15 minutes of blasting its main engine, the MESSENGER spacecraft ﬁnally slipped into orbit around Mercury last week. The smallest, innermost planet is the last of the classical, naked-eye planets to get an orbiter. Tagged with the most contorted of acronyms (standing for MErcury Surface, Space ENvironment, GEochemistry, and Ranging), MESSENGER will probe everything from the mineral elements the sun continually blasts off the planet’s surface to its relatively huge metallic core, which occupies nearly half of its volume. Also of interest are the subsurface ice deposits thought to linger near the poles of the sun’s nearest neighbor. MESSENGER’s seven scientiﬁc instruments, battened down for the rocket burn (after taking images during three earlier Mercury ﬂybys), will be turned on and checked out for the start of science observations on 4 April.

Q: Did you ever have trouble explaining your project? There was one news reporter who … wanted to know what my project was about, so I ﬁrst stated the title [“Continued Fraction Convergents and Linear Fractional Transformations”]. … He was awestruck by it. … I tried to summarize, and after I had gotten half a sentence into it, he said, “I have no idea what that means.” He just ran off before I could ﬁnish.

Strong Medicine Three scientists whose work on stem cells ignited the ﬁeld of medicine will share this year’s Albany Medical Center Prize in Medicine and Biomedical Research and its accompanying $500,000. The award recognizes James Thomson, who holds appointments at the University of Wisconsin, Madison, the Morgridge Institute for Research in Madison, and the University of California, Santa Barbara, for pioneering the isolation of human embryonic stem cells and for his work on induced pluripotent stem cells, which are genetically reprogrammed from adult cells. Induced pluripotency, which researchers see as a powerful tool for understanding diseases and developing therapeutic treatments, was discovered by fellow prize winner Shinya Yamanaka of Kyoto University in Japan and the Gladstone Institute of Cardiovascular Disease in San Francisco, California.

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Elaine Fuchs of the Rockefeller University in New York City was also honored, for her investigations of skin stem cells and how they develop into skin and hair. That work has led to a better understanding of skin diseases and cancers.

Northern Lights Seven medical researchers have won Canada’s Gairdner Foundation Awards, valued at CAD $100,000 each. Jules Hoffmann of France’s National Center for Scientiﬁc Research (CNRS) in Strasbourg and Shizuo Akira, director of the Immunology Frontier Research Center in Osaka, Japan, were recognized for identifying proteins involved in pathogen recognition. Developmental biologists Howard Cedar and Aharon Razin of the Hebrew University of Jerusalem and geneticist Adrian Bird of the University of Edinburgh were honored for their discoveries in the ﬁeld of DNA methylation and related gene expression. Robert Black of Johns Hopkins University in Baltimore, Maryland, won a special Global Health award for his research on reducing childhood deaths from diarrheal diseases, while geneticist Michael Hayden of the University of British Columbia, Vancouver, was lauded for leadership in “medical genetics, entrepreneurship and humanitarianism.” Since the awards were created in 1959, 76 Gairdner winners have also garnered Nobel prizes.

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NEWSMAKERS

MESSENGER Arrives Safely at Mercury

CREDITS (TOP TO BOTTOM): NASA/JHU APPLIED PHYSICS LABORATORY/CARNEGIE INSTITUTION OF WASHINGTON; LAURENCE GENON/INTEL

attorneys, who ﬁled suit in the U.S. District Court for the District of Columbia, say the research violated the Nuremberg Code and the U.S. Constitution’s Eighth Amendment, which prohibits cruel and unusual punishment. The plaintiffs will ask a jury to award damages for pain and suffering, medical expenses, and other hardships. The named plaintiffs include former Guatemalan soldiers inoculated with syphilis and their children. Attorneys argue that the successors to the U.S. ofﬁcials who oversaw the experiments are legally accountable, including the heads of the Department of Health and Human Services and the Centers for Disease Control and Prevention.

NEWS

17% Percentage of the Massachusetts Institute of Technology’s science and engineering faculty who are women. That’s up from 7% in 1995.

5000 Number of homes that

could run on an underwater turbine array just authorized by the Scottish government. Planned for a narrow strait between the islands of Islay and Jura, the turbines will also power several distilleries. FINDINGS

Cause of Lethal Disease In China Unmasked

CREDITS: THINKSTOCK

BEIJING—Each spring since 2006, a new

disease in China has been killing up to 30% of its victims. Scientists aren’t entirely sure how it spreads or kills, but researchers now at least know the face of this enemy. In an article published online last week in The New England Journal of Medicine (NEJM), two teams described a new virus that appears to cause severe fever with thrombocytopenia syndrome (SFTS). SFTS came to light when people in Anhui Province in central China began dying of an illness characterized by high fever, gastrointestinal distress, and a depressed platelet count. The disease has since spread to six provinces. In December 2009, Xue-jie Yu, an expert on tick-borne diseases at the University of Texas Medical Branch at Galveston, isolated from a patient’s blood a new bunyavirus, part of a family that includes hantavirus and Rift Valley fever virus. Then last spring and summer, researchers at the Chinese Center for Disease Control and Prevention (CDC) here detected SFTS bunyavirus RNA, speciﬁc antiviral antibodies, or both in 171 out of 241 people hospitalized for SFTS. Chinese CDC virologist Li Dexin and colleagues isolated

Random Sample

Proof Positive Space scientists at the University of Leicester are working to protect the people of Britain from a terrifying scourge: counterfeit whisky. Detecting fake whisky actually isn’t that hard. “The trick with highvalue whiskies is to measure there’s something wrong without opening the bottle” and ruining its value, says George Fraser, whose day job is working on instruments that go on space probes. So he and colleagues are working on a device, based on another one that they developed to detect counterfeit pharmaceuticals, that measures whisky the same way an astronomer measures the spectrum of a star. “It’s basically holding the object up to the light in a technological fashion,” Fraser says. A spectrometer records which wavelengths of light pass through the bottle of whisky, and researchers can then compare them with the spectrum transmitted by, say, a 15-year-old Glenlivet. (The technique can also ﬂag watered-down spirits.) And, because it’s tough for counterfeiters to print a label exactly the same color as the real thing, the scientists are also measuring the wavelengths reﬂected off the label. The group hopes to have the instrument on the market in 18 months. In the meantime, Fraser doesn’t mind leaving the bottles closed. He’s a native of Scotland’s Spey Valley, the home of some of the world’s ﬁnest whisky, but says, “I don’t much like it, actually.”

11 strains of the bunyavirus from these samples. With their NEJM article, the Texas and Beijing teams, former rivals, share credit for the discovery. Scientists will probe whether ticks are the vector and other questions when SFTS presumably strikes this spring. http://scim.ag/SFTS-virus

Allergic to Peanuts? For Some Kids, Eating Them Helps A new study is adding to a small but growing pile of evidence that kids with food allergies can beneﬁt when exposed, under a doctor’s supervision, to the very food they react poorly to. Last week, a team of British researchers reported that 22 children with peanut allergies generally did well when given higher and higher doses of peanut ﬂour, mixed into chocolate bars, over several months. After 30 weeks, the children got about 32 roasted peanuts to eat. Fourteen tolerated that dose; on average, the peanut serving size the children could handle grew 1000-fold. The work was led by pediatric allergist Andrew Clark at Addenbrooke’s Hospital in Cambridge, U.K., and published online 18 March in Clinical & Experimental Allergy. It’s not the last word, and doctors warn that parents shouldn’t try this on their own; the study was small and had no control group. But it builds on similar evidence for

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BY THE NUMBERS $753 million Cost to ferry U.S. astronauts to the International Space Station aboard Soyuz spacecraft under a new contract with Russia for 2014 to June 2016. That’s $63 million per astronaut.

egg and milk allergies. There’s also hope that eating peanuts can help prevent allergies to them in the ﬁrst place. A different U.K. study, expected to end in 2014, is trying to thwart peanut allergies in hundreds of kids who are at high risk.

Sperm From a Test Tube Generating sperm is a complicated biological feat that researchers have been trying to recreate in the lab for a century. In a paper published online in Nature this week, Takehiko Ogawa and colleagues at Yokohama City University in Japan describe a simple method that accomplishes just that. They hope the method will allow infertility researchers to study sperm development more effectively. The researchers took testes from 2- or 3-day-old mice and grew the organs for about a month in a petri dish with media that contained a compound often used for stem cell cultures. The testes looked relatively normal as they developed and eventually started producing mature sperm. When the researchers extracted the sperm and artiﬁcially inseminated female mice, healthy pups were born. Using the same culture method, the researchers were even able to produce sperm from young testes that had been frozen for a month. They are now trying to recreate the process with tissue samples from larger animal, including humans. http://scim.ag/test-tube

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DEVASTATION IN JAPAN

Nuclear Power’s Global Fallout

UNITED KINGDOM The government has asked its chief nuclear inspector to compile a report on the implications of the Fukushima accident for Britain’s current and future nuclear plants. Energy and climate change secretary Chris Huhne said: “We should not rush to judgment. It is important that we have the full facts at our disposal.” Current policy is for future reactors to be built with private funds.

The crisis at the Fukushima Daiichi nuclear power plant, like the accidents at Three Mile Island and Chernobyl, is prompting countries around the world to reassess the safety of their plants and their nuclear aspirations. The map on this page provides a snapshot of the number of nuclear reactors in operation (dark blue) and under construction (green), locations of power plants in relation to seismic hazard zones, and reactions to events in Japan in some countries. The following pages examine what we have learned about radiation risks from previous exposures (p. 1504), improvements in safety since the boiling water designs at Fukushima (p. 1506), what to do with the wrecked reactors (p. 1507), and damage to research facilities from the earthquake (p. 1509). –NEWS STAFF

CANADA 18 0 U.S. 104 1

MEXICO 2 0

MAP LEGEND

UNITED STATES

Reactor sites*

The United States is the world’s largest producer of nuclear power, but no new reactor has been built there for 3 decades. The Nuclear Regulatory Commission has ordered a safety review of the 104 existing U.S. plants, some of which are in seismically active areas. The Obama Administration has proposed expanding nuclear capacity largely by stimulating new construction with loan guarantees; opposition to that plan is likely to strengthen.

COUNTRY Number of operating reactors Number of reactors under construction *Some power plants have multiple reactors

Seismic zones Low

Moderate

High

BRAZIL 2 1

Very High

BOILING WATER REACTORS

Finland Germany India Japan Mexico Spain Sweden Switzerland Taiwan United States

BRAZIL Mines and Energy Minister Edison Lobão said Brazil’s federal government will review security at the country’s twin nuclear reactors at Angra and halt construction of a third, due to go on line in 2015, until the review is completed. The Fukushima crisis has renewed calls to create an independent regulatory agency for nuclear power in Brazil.

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ARGENTINA Events in Japan have reopened debate over Argentina’s aging reactors. Its oldest plant dates to 1974, and another has been under construction since 1981 but is not yet complete. Critics call it a “Model T.”

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ARGENTINA 2 1

MAP SOURCE: GLOBAL SEISMIC HAZARD ASSESSMENT PROGRAM; REACTOR TOTALS: EUROPEAN NUCLEAR SOCIETY; BOILING WATER REACTOR TABLE: IAEA

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The French government has promised a safety audit of the country’s 58 nuclear reactors. But the government has made clear that nuclear energy will remain the cornerstone of France’s 40-year-old policy of energy independence and has rejected calls for a referendum on atomic energy. France generates over 75% of its electricity with nuclear power, more than any other country in the world.

NEWS GERMANY

BELGIUM

SWITZERLAND

ITALY

Chancellor Angela Merkel suspended for 3 months a newly enacted law that postponed Germany’s planned phase out of nuclear power. Several older reactors, which got a reprieve under the law, are now being shut down, and some are unlikely to come back on line. During the 3-month moratorium, the government will reassess the safety of all 17 reactors and is expected to propose amendments to the new law in accordance with the ﬁndings.

The Belgian government decided in 2003 to phase out the country’s seven nuclear reactors, which produce half of Belgium’s electricity. But the ﬁrst phase, shutting the three oldest reactors by 2015, was recently pushed back 10 years. Environmental groups hope the Fukushima disaster will swing the pendulum back in their direction.

Switzerland generates 40% of its electricity with its ﬁve nuclear reactors. Last week, the government suspended feasibility studies for three potential reactors, and politicians across the political spectrum have spoken in favor of a nuclear power phaseout, though most have been vague about a timeline.

A 1987 referendum, inﬂuenced by the Chernobyl disaster, led to Italy shutting down its four nuclear power plants between 1987 and 1990. Silvio Berlusconi’s government has supported plans to build at least four new reactors, starting in 2013. A referendum this spring could block those plans.

NETHERLANDS 1 BELGIUM 0 7 0 FRANCE 58 1

SWEDEN 10 0

FINLAND 4 1 CZECH. REP. 6 0

U.K. 19 0

SWITZERLAND 5 0 SPAIN 8 0 SLOVENIA 1 0

ARMENIA 1 0

UKRAINE 15 2

RUSSIA 32 11

ROMANIA 2 0 SLOVAKIA 4 2

CHINA 13 27

HUNGARY 4 0 BULGARIA 2 2

INDIA 20 5

IRAN 0 1

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GERMANY 17 0

RUSSIA Prime Minister Vladimir Putin ordered safety checks at Russia’s nuclear power plants and a review of the country’s nuclear plans. Russia has 32 operating reactors and another 11 under construction.

JAPAN 54 2 REP. KOREA 21 TAIWAN 5 6 2

PAKISTAN 2 1

SOUTH AFRICA 2 0

CHINA

INDIA Prime Minister Manmohan Singh ordered a safety review of India’s 20 operating nuclear reactors. In an interview with Science, Srikumar Banerjee, chair of India’s Atomic Energy Commission, said no shutdowns are planned. India has ﬁve plants under construction and aims to produce 25% of its electricity from nuclear power by 2050.

Premier Wen Jiabao announced a temporary halt to assessment and approval of nuclear power projects that are in the planning stage. The government will conduct a comprehensive review of all nuclear facilities, draw up nuclear safety regulations, and adjust its 15-year nuclear power–development plan. China has the world’s most ambitious nuclear power program, with 13 reactors in commercial operation, 27 under construction, and as many as 50 more in the pipeline. The temporary halt applies only to projects yet to be approved; China’s growing reliance on nuclear power is expected to continue.

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SOUTH KOREA South Korean President Lee Myung-bak ordered safety reviews of the country’s nuclear reactors as well as procedures for handling emergencies. The country’s 21st nuclear reactor started commercial power production at the end of February, another ﬁve reactors are under construction, and further expansion is planned. Knowledge Economy Minister Choi Joong-kyung said now is not the time to review the country’s nuclear power policy.

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CREDITS (TOP TO BOTTOM): WIKIMEDIA COMMONS; DOE PHOTO; WIKIMEDIA COMMONS

The ongoing leaks from Japan’s crippled Fukushima Daiichi nuclear power plant have raised concern that some workers and even the public could be exposed to dangerous levels of radiation. So far, ofﬁcials have said that levels outside the plant are low. But how do they know how much radiation is harmful? Risk calculations are based heavily on a 63-year study of 94,000 people who survived the two atomic bombs dropped on Japan in August 1945. It is one of the largest, longest population studies ever done; for radiation safety, it is the gold standard. Its breadth and precision are “magniﬁcent,” says John Boice, scientiﬁc director of the International Epidemiology Institute in Rockville, Maryland, and former chief of radiation epidemiology at the U.S. National Cancer Institute (NCI) in Bethesda, Maryland. Up to 200,000 people in Nagasaki and Hiroshima died soon after the bomb blasts— some from radiation sickness—but more survived. To understand the delayed effects of radiation, the U.S. National Academy of Sciences launched a joint study with Japan of bomb survivors, using a 1950 census to track them down. Initial case reports of cataracts, leukemia, and birth defects eventually became a long-term study to follow cancer and other illnesses in about 94,000 survivors and 26,000 unexposed residents. It was run by a U.S. and Japanese– funded agency eventually named the Radiation Effects Research Foundation (RERF). Researchers “spent a huge amount of time reconstructing where people were ATB, at time of blast,” says epidemiologist Richard Monson of Harvard University. They asked subjects whether they were inside or outdoors, near a window, upstairs or downstairs, and which direction they faced. They even constructed a mock Japanese village in the Nevada desert, hoisted a uranium reactor up in a tower, and

the study who received signiﬁcant exposures developed solid cancers, only 850 cases, or 11%, have been attributed to radiation. (The cancer risk was about 50% higher for those who received at least 1 sievert of radiation; the measured the neutrons it spewed to study the risk drops with dose to 2% below 0.1 sievert. movement of radiation through the air and into Lifetime risks are lower; for an exposure of buildings. To ﬁne-tune gamma-radiation esti- 0.250 sieverts, the allowed limit for workers mates, researchers tested ceramic roof tiles on at the Daiichi plant, the increased risk of ever Japanese houses for a high-energy electron developing cancer is about 2.5%, Boice says.) signature, says Tore Straume of NASA Ames Data from hundreds of medical studResearch Center in Mountain View, Califor- ies have been used to bolster the A-bomb– nia. The dosimetry underwent several revi- survivor results. In the early 20th century, sions over the decades. before the risks were recognized, radiation Other RERF researchers monitored the was used to diagnose or treat everything from health of survivors. Using death records and mastitis to tonsillitis—and some patients cancer registries, they soon documented developed cancer, Boice notes. Studies leukemias, particularly in the young, tally- of workers—such as women who applied ing 219 deaths by 2002 in people receiving radium paint to clock dials and later devela signiﬁcant exposure—a 45% rise above oped bone cancer—also proved useful. the number expected. It appeared to peak Studies of about 21,000 workers exposed to in 1950. By the 1970s, researchers were radiation starting in 1948 at the former Soviet tracking an elevated rate of solid cancers; Union’s Mayak nuclear weapons plant, and “it looks like it persists for a lifetime,” says of 30,000 villagers nearby along the Techa NCI epidemiologist Kiyohiko Mabuchi. River, are proving “important,” Preston says: Still, “people’s perceptions of cancer The cohorts received a wide range of radiacaused are probably different from reality,” tion doses, and many workers inhaled plutosays biostatistician Dale Preston of Hiro- nium, a long-lived radioisotope absent in the soft International, who worked at RERF for Japanese A-bomb survivors. 23 years. Leukemias were relatively rare, and These studies and others of nuclear workalthough by 1998 about 7851 survivors in ers “in general have supported estimates from the A-bomb survivors,” says NCI’s Ethel Gilbert. Controversy remains, MAJOR RADIATION EVENTS however, about whether the bomb survivors’ brief, one1945 Hiroshima-Nagasaki atomic bombs time exposure would be Population: 94,600 survivor cohort as harmful if spread over Health effects: 45% increase in leukemia many years. “It’s the one deaths, 11% increase in solid cancers major unanswered question,” Boice says. 1950s Nevada nuclear tests, U.S. Studies of nuclear acciPopulation: 160 million U.S. citizens dents have been less useHealth effects: Possible increase in ful for estimating dose thyroid cancer responses, although they confirm that it’s hard to 1948–72 Mayak-Techa River, USSR see health effects from Population: 21,000 workers, low-level exposures. The 30,000 villagers 1979 accident at Three Health effects: Consistent with Mile Island in PennsylA-bomb results vania exposed the nearby population to a “triv1979 Three Mile Island, U.S. ial” amount of radiation, Population: 2 million Boice says; health effects Health effects: not detected were not detected. The 1986 Chernobyl acci1986 Chernobyl, Ukraine, USSR dent, on the other hand, Population: 5 million in immediate area spewed iodine-131 and Health effects: 6000 thyroid cancers, cesium-137 for 10 days in mainly from contaminated milk a plume that reached 5 million people. Researchers

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Radiation Risks Outlined by Bombs, Weapons Work, and Accidents

DEVASTATION IN JAPAN | NEWS&ANALYSIS expect that 4000 excess cancer deaths will eventually result. But precise dose information is lacking even for the “liquidators,” the 600,000 workers who helped clean up, says Mabuchi, making it difﬁcult to link exposure to disease. The only clear health effect among the public from Chernobyl so far has been more than 6000 cases of thyroid cancer (15 of them fatal), mainly in people who as children

and adolescents drank milk from cows that fed on grass tainted with iodine-131. This should “not be a problem in Japan” because contaminated milk and vegetables are being removed from the food supply, says radiologist Fred Mettler of the University of New Mexico, Albuquerque, a consultant to the United Nations on the Chernobyl disaster. Studies of the A-bomb survivors (40% of the original group are still living) continue at

RERF. A few of its 45 researchers are helping Japanese ofﬁcials monitor the population near the Daiichi plant, says RERF vice chair and research chief Roy Shore. They’re also discussing a possible study of the hundreds of workers involved in keeping the plant under control. If the Fukushima crisis becomes another Chernobyl, Shore says, “we’d certainly want to compile all the data we could.” –JOCELYN KAISER

CREDIT: TOKYO ELECTRIC POWER CO. (TEPCO) VIA KYODO NEWS/AP PHOTO

Candidate Radiation Drugs Inch Forward

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Beyond potassium iodide, whose utility is limited to warding off thyroid cancer, the pharmaceutical industry has no proven drugs to ward off the devastating health effects of a large radiation exposure. Over the past 5 years, however, a few promising candidates have begun to undergo animal, and even human, testing. Still, only a few companies and academic groups are addressing this unmet need. The obvious lack of a commercial market may explain why. But some also blame the U.S. government’s relative indifference toward the development of radiological countermeasures compared with biodefense. “Radiation has been a stepchild,” says Ramesh Kumar, a microbiologist and CEO of Onconova Therapeutics Inc., headquartered in Newtown, Pennsylvania, which is developing a so-called radioprotectant. Major radiation exposures sicken, and typically kill, victims by inﬂicting damage on the bone marrow and the gastrointestinal (GI) tract. It affects both targets by generating free radicals—a reactive oxygen species—which damage the DNA of cells, causing them to self-destruct. Researchers are trying different approaches to counter Unprotected? With few medical options if exposed to signiﬁcant radiation, workers at the damaged Fukushima nuclear plant wear protective gear. those processes. Cleveland BioLabs Inc. in Buffalo, New York, is testing CBLB502, essentially a modiﬁed fragment of the Salmonella ﬂagellum. Injected radiation sickness. It inhibits certain enzymes called kinases, “encouraging into the body, the molecule tricks cells into believing that they are under cells that have suffered DNA damage to repair themselves,” Kumar says. attack from a Salmonella infection, making them produce nuclear fac- The company, which has received more than $10 million in U.S. defense tor κB, a protein that switches on genes behind processes that block cells grants, has shown that mice injected with the drug and exposed to a norfrom self-destructing (Science, 11 April 2008, p. 226). It promoted sur- mally lethal radiation dose had an 88% survival rate 30 days after the vival in mice and monkeys when administered before radiation exposure exposure. The company has also demonstrated the compound’s radioas well as up to 48 hours after exposure. The drug also triggers the pro- protective properties on human cells and lung tissue. duction of molecules that mop up free radicals, limiting DNA damage, A team led by Richard Kolesnick of the Memorial Sloan-Kettering says Andrei Gudkov, a molecular geneticist and chief scientiﬁc ofﬁcer at Cancer Center in New York City has shown that radiation promotes the Cleveland BioLabs. The company’s work has been supported with more overproduction of a lipid called ceramide in GI blood vessel cells, trigthan $50 million in grants from the departments of Defense and Health gering their death and the breakdown of the vessels. Based on this, the and Human Services. researchers have developed an anticeramide antibody called 2A2 that After providing evidence of the drug’s efﬁcacy in primate trials and they say could confer protection to the GI tract even at acute doses of its safety in human trials, the company is hoping CBLB502 will fall radiation. They’ve shown that the antibody works in mice; more studies under the Food and Drug Administration’s (FDA’s) so-called animal are under way. rule, which permits the approval of drugs and vaccines that cannot be These developments are promising, but death from radiation “is a comshown to work in humans through traditional clinical trials, because plicated problem that involves failure of multiple organs,” cautions Mark that would involve sickening human subjects deliberately. Under the Whitnall, the program adviser for radiation countermeasures at the Armed rule, drug developers must convince FDA that a candidate drug with a Forces Radiobiology Research Institute in Bethesda, Maryland. Reducing demonstrated efﬁcacy in animals has the same mechanism of action in the problem to “bone marrow syndrome and GI syndrome is a simpliﬁcahealthy humans. Gudkov says that his company has completed part of tion; what causes actual mortality is more complicated,” he says. this requirement by injecting CBLB502 into some 150 human subjects The panic caused by the nuclear accident in Fukushima could acceland showing that it leads to increased levels of granulocyte colony- erate work on developing radioprotectants, says Onconova’s Kumar. He stimulating factor and interleukin-6, two biomarkers associated with has already been summoned for meetings in Washington, D.C., with the the drug’s radioprotective mechanism. Department of Homeland Security as well as congressional staffers. Ex-RAD, developed by Onconova, is intended as a prophylactic against –YUDHIJIT BHATTACHARJEE

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Despite the severity of the accident at the Fukushima I plant, nuclear reactor designers don’t expect the same type of backlash against the nuclear industry as occurred a generation ago after Three Mile Island and Chernobyl. Their confidence rests on the fact that the reactors being built or planned today are quite different—and they say much safer—than those that are still smoldering in Japan. Andrew Sherry, director of the Dalton Nuclear Institute at the University of Manchester, U.K., likens the differences to those between a car built during the 1960s and a car built today. Crumple zones, safety cages, three-point seat belts, and multiple airbags allow passengers today to walk away from a headon crash almost unharmed, he says; not so in a 1960s car. The Fukushima I reactors are very old reactor designs, direct copies of “the hairy edge of the ﬁrst commercial plants in the U.S.” in the 1960s, says nuclear engineer Tony Roulstone of the University of Cambridge in the U.K. The new machines, using so-called Generation III+ designs, “have the beneﬁt of 50 years of design evolution and operational practice,” Sherry says. Modern reactors have multiple layers of defense, use natural forces such as gravity and convection to move cooling water rather than rely on pumps, and employ automatic valves that kick in extra measures if necessary. Their manufacturers claim the reactors can be left for days and not overheat. “You can walk away from [such a reactor]. It’s designed to cope with decay heat,” Sherry says. The principles for designing a safe reactor haven’t changed, however. “The essence of the safety case for any nuclear reactor,” says nuclear engineer Barry Marsden of the University of Manchester in the U.K., “is ﬁrst to shut [the ﬁssion reaction] down and, second, to cool the fuel.” As at Three Mile Island, the three operating reactors at Fukushima (three were ofﬂine for refueling or repairs) executed the ﬁrst of these tasks automatically following the earthquake. In boiling water reactors (BWRs) of the type used there, neutron-absorbing control rods are pushed upward from below the core to between the fuel rods, killing the fission chain reaction. This still requires power,

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however. In some modern reactors, the rods are held above the core with electromagnets so that a power outage will release them and gravity will do the rest. But stopping the chain reaction doesn’t neutralize heat production in the core. The radioactive decay of the fuel and fission products keeps generating as much as 7%

Hands-off cooling. GE-Hitachi’s Economic Simpliﬁed BWR features depressurization valves (at the top of the pressure vessel), upper pools to top up the core cooling water by gravity, and lower pools to condense steam from the core.

of the full thermal capacity of the reactor, and cooling water must be actively pumped through the core to prevent overheating. “With a complete loss of power, decay heat did the rest” at Fukushima, Roulstone says. The reactor designers of the 1960s considered a limited number of accident scenarios and devised systems to deal with each one. “Three Mile Island and Chernobyl changed the way people looked at safety,” Roulstone says. People realized that accidents can be complicated, have multiple causes, and that operators could do more harm than good. The lesson from those disasters: Expect the unexpected and prepare for the worst. “You have to accept there will be events that will overwhelm your systems,” says nuclear engineer Michael Golay of the Massachusetts Institute of Technology in Cambridge. The design philosophy for reactors now is “defense in depth.” That approach requires

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duplicate systems and different technologies that can’t, or shouldn’t, all fail together. In the case of power supply, that might mean having more than one connection to the grid, an oversupply of diesel generators in different places, and batteries as a backup, along with possibly a ﬂywheel system. Loss of power, the main ongoing problem in Fukushima, is one of the most studied scenarios in the industry, Roulstone says, because in some designs, no power leads to an inability to keep the coolant moving and shifting heat out of the reactor. Consequently, all Generation III+ designs emphasize moving coolant with gravity and convection, driving pumps with steam, activating valves with DC battery power or no power at all, and keeping humans out of the loop. One Gen III+ contender dispenses with pumps altogether to circulate the coolant. An updated BWR design known as the Economic Simpliﬁed BWR, for which GE-Hitachi is currently seeking regulatory approval, required extensive modeling to ensure that convection alone could move enough heat out of the reactor vessel. Other safety measures include automatic pressure-release valves, a large “suppression tank” to which steam can escape when pressure gets high, and water reservoirs above the reactor to top up the coolant—via gravity—if its level drops too low. Westinghouse’s AP1000 strives to make its systems as simple as possible by cutting down on piping, cables, and valves to reduce the possibility of problems. China is building the ﬁrst four of this Gen III+ design, which uses convection to shift heat out of the reactor vessel into a massive tank of cooling water nearby. Decay heat is managed naturally without the need even for diesel generators. “It buys a grace period, giving operators some time to manage the situation,” Sherry says. Golay hopes that Fukushima will spur designers to think harder about the problem of storing spent fuel. Modern reactor designs don’t keep fuel in an elevated pool above the reactor like BWRs do but move it into a separate earthquake-proof building. No country has faced up to the spent-fuel problem and created centralized storage away from reactors or a long-term underground repository. “Maybe now attitudes will change,” he says. Roulstone says the days may be numbered for old BWRs like those at Fukushima. But he thinks the imperatives of peak oil and climate change will keep nuclear in the mix of future energy sources: “I think people will face up to [the problems] and bite the bullet.” –DANIEL CLERY

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Current Designs Address Safety Problems in Fukushima Reactors

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NEWS&ANALYSIS

DEVASTATION IN JAPAN | NEWS&ANALYSIS

Once the crisis at the Fukushima Daiichi nuclear power plant eases, authorities will begin plotting the endgame: how to dismantle the contaminated complex and dispose of its nuclear fuel. They are unlikely to have to resort to the desperate measures taken at the Chernobyl nuclear plant, where a reactor exploded and burned 25 years ago. “I don’t see that as necessary unless the situation deteriorates unexpectedly” and the primary containment vessels of one or more reactors are extensively breached, says Tony Irwin, a nuclear technology expert who lectures at Australian National University and the University of Sydney. Instead, the Fukushima operation is more likely to resemble the protracted cleanup at the Three Mile Island Nuclear Generating Station in Pennsylvania, where one reactor experienced a partial meltdown in 1979. The extent of the damage at Fukushima was uncertain as Science went to press. Explosions triggered by hydrogen gas had rocked three of the six reactor halls. Fireﬁghters snuffed out the blazes, and the steel vessels encasing the uranium oxide fuel assemblies appeared to be intact. A big question is whether spent fuel rods stored at the plant remain submerged in their cooling basins; some experts worry that the earthquake may have cracked one pond. If the zirconium-clad rods lose their cooling bath, radioactivity from the fuel and ﬁssion products would melt the cladding and be released. On 21 March, smoke apparently rising from a fuel pond prompted authorities to evacuate workers. The spiraling events at Fukushima conjure memories of Chernobyl—the calamity against which all nuclear mishaps are measured. On 26 April 1986 in Ukraine, then a Soviet republic, the power plant’s number 4 reactor exploded during an unauthorized test. Graphite control rods burned for 10 days, spewing a radioactive plume that dumped fallout across a swath of Europe. Twenty-four plant workers and six ﬁreﬁghters died from off-the-chart radiation doses. Thousands of Soviet soldiers were sent to tame the seething inferno. Some “liquidators” made timed dashes onto the reactor hall roof to shovel chunks of smoking graphite back into the core. Helicopters dumped 5000 tons of clay, sand, and other materials onto the burning core, while coal miners dug a tunnel under it

reactor suffered a partial meltdown after valves in its cooling system malfunctioned. The laborious cleanup, in which about 150 tons of contaminated materials were shipped to Idaho National Laboratory for storage, took 14 years and cost nearly $1 billion. The cleanup challenge at Fukushima is threefold: the possible partial meltdown of fuel rods inside the reactors, the spent-fuel cooling ponds, and contaminated structures and soil in and around the plant. The technical complexity will be somewhere between that of Three Mile Island and Chernobyl, says Murray Jennex, a mechanical engineer at San Diego State University in California and former U.S. Navy nuclear power ofﬁcer. “The reactor vessel cleanup will be more like Three Mile Island; the spent-fuel pool cleanup will be more like Chernobyl,” he says. If the containment vessels are found to be intact, any melted fuel rods would have pooled like lava inside. The best solution for the spent fuel rods, says Irwin, formerly with the Australian Nuclear Science and Technology Organisation in Lucas Heights, is to transfer them to dry storage casks. Residual radiation at the plant will dictate the pace of this operation. “The dose to the workers will be the limiting factor,” he says. Fukushima’s reactor complex has suffered far more structural damage than that seen at Three Mile Island, so disposal of contaminated materials and remediation of the landscape will be much more arduous. Earlier this week, Japanese authorities reported that iodine-131, a short-lived radioisotope, and cesium-137, a radioisotope with a halflife of 30 years, had been detected in spinach, leeks, and milk on farms near the Fukushima plant. Cesium-137 is a lingering headache at Chernobyl, but there the contamination was far heavier. No one expects that it will take an army of liquidators to dismantle Fukushima—if, in fact, the worst is over.

Eerie resemblance? Concerns over contamination from spent fuel rods at Fukushima (left) kindle memories of Chernobyl, where work has just commenced (right) on a massive structure to safely dismantle a ruined reactor.

to pump in liquid nitrogen. After the ﬂames were doused, Soviet engineers hastily erected a concrete structure, called the sarcophagus, over the destroyed reactor. Work started last autumn on the foundations of the New Safe Confinement, a $1.4 billion steel and concrete structure that will be taller than the Statue of Liberty and will slide into place over the sarcophagus. Expected to be completed as early as 2014, it will enable engineers to gradually dismantle the sarcophagus using remote-controlled cranes and dispose of the still-hot debris. “Bad as it is” at Fukushima, the situation there “is dramatically less catastrophic than Chernobyl,” says Matthew Bunn, a nuclear energy expert at Harvard University. Ongoing efforts to cool the stricken reactors appear to have kept the primary containment vessels intact. To moderate the neutron ﬂux that drives ﬁssion of the uranium fuel, Fukushima’s reactors used water rather than graphite—a ﬂammable material—so the risk of the core catching ﬁre is much lower than it was at Chernobyl. And most important, Fukushima’s reactors are still encased in their containment vessels; Chernobyl’s primitive design lacked that crucial safety feature. As of 22 March, experts say there is no need for last-ditch measures at Fukushima such as dumping sand on the reactors or entombing the complex in concrete. “We are not thinking of this as a realistic option,” Hidehiko Nishiyama, deputy director general of Japan’s Nuclear and Industrial Safety Agency, said last week. He says engineers intend to restore Fukushima’s cooling systems and then study the options. At this point, a more apt comparison is Three Mile Island, where the plant’s number 2

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Fukushima Cleanup Will Be Drawn Out and Costly

–RICHARD STONE

With reporting by Dennis Normile.

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Japan’s Research Facilities Down But Not Out pled Fukushima nuclear plant, Japan’s scientiﬁc community has been quietly taking stock of how extensively the magnitude-9 earthquake and subsequent tsunami on 11 March damaged facilities and experiments. The news is mixed. Two of Japan’s major physics facilities were knocked out indefinitely, and many laboratories along the eastern seaboard have lost instruments and research materials or are shuttered due to rolling blackouts. In a glimmer of good news, the university hardest hit by the quake suffered no fatalities at its campuses. Tohoku University, one of the country’s top research schools, is in Sendai, just 130 kilometers west of the epicenter. Although the quake and tsunami hammered Sendai, Tohoku’s ﬁve campuses are at the western, inland end of town and were not inundated. “We didn’t have a single death or serious injury” on campus among students or staff members, says Yukihisa Kitamura, a Tohoku vice president. Classes had ended for the academic year, which runs from 1 April to 31 March, so few students were around. The university has set up a Web site for students to report whether they are safe. As of 18 March, 10,500 were confirmed to be okay. Some 3800 are unaccounted for. Although no university buildings collapsed, an initial safety check last week ﬂagged several as off-limits pending further study, Kitamura says. Inside safe buildings, faculty and staff members have been sifting through wrecked labs. “We can’t yet put a [monetary] figure on the damage,” Kitamura says. At the medical school, meanwhile, “all the clinicians are volunteering to help out at other area hospitals,” says Noriyuki Kasai, a veterinarian who heads the school’s Institute for Animal Experimentation. His facility and its 20,000 mice and rats escaped mostly unscathed. That’s not the case for two premier physics centers. The Japan Proton Accelerator Research Complex (J-PARC) in Tokai, on the coast 200 kilometers south of Sendai, was spared tsunami damage because it sits on high ground; the building was placed there to preserve shore dunes and vegetation. The 2-year-old, $1.5 billion lab features a 50-gigaelectronvolt synchrotron supporting work on a range of physics, materials science, and biomedical studies. Extensive liquefaction, which turns silty soils into quicksand, damaged roads to and within the complex, but J-PARC buildings,

Out of commission. Major facilities facing months of repairs after the 11 March disaster include the Chikyu ocean drilling vessel (top) and accelerators at J-PARC in Tokai (middle) and KEK in Tsukuba (bottom).

which sit on deep piles, are intact. That’s also the case at the High Energy Accelerator Research Organization (KEK) in Tsukuba, a science city 260 kilometers southwest of Sendai. KEK is Japan’s workhorse for particle and neutrino physics. The accelerators and instruments at both KEK and J-PARC were damaged, says particle physicist Yohei Morita, KEK’s press ofﬁcer. With the electricity supply limited, it will take several weeks to carry out a damage assessment. Although neither facility suffered casualties, experiments are out of the question. “I think we can get these facilities back on line,” Morita says, “but don’t know the schedule yet.” Other experimentalists are also experiencing enforced idleness. To cope with potential blackouts, the RIKEN Omics Science Center

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in Yokohama has delayed starting genomicsequencing runs, which can take 10 days to complete. And with commuter train operations curtailed, many employees are having a hard time getting to work. “We’ve stopped all research activity,” says the center’s director, Yoshihide Hayashizaki. Another major research asset now out of commission is the Chikyu, a deep-sea drilling vessel that was docked at Hachinohe, 250 kilometers north of Sendai, on 11 March. Chikyu scientists were hosting a class of schoolchildren on a ﬁeld trip that day. “The tsunami washed into the harbor; there was no way to escape,” says Asahiko Taira, a vice president of the Japan Agency for Marine-Earth Science and Technology (JAMSTEC). Children and scientists were evacuated safely. But the vessel was less fortunate. During the turbulence, the Chikyu scraped bottom, snapping off one of six thrusters that hold the ship in position while drilling. As a result, an Integrated Ocean Drilling Program expedition to study the deep coal-bed biosphere off Shimokita, Japan, has been canceled. Taira expects that it will take 2 to 3 months to repair Chikyu and get it back out to sea. In the meantime, JAMSTEC has urgent business to attend to. Immediately after the earthquake the agency sent another vessel, Kairei, to deploy seabed seismometers to monitor aftershocks and better understand the fault zone. “There is a lot to be learned,” Taira says. Debris swept out to sea by the tsunami, he says, is impeding work. Many other survey plans have been put on hold. To avoid exposing researchers to high radiation levels, the University of Tokyo’s Earthquake Research Institute has postponed dispatching a team to investigate the tsunami’s extent and its impact on infrastructure until after the Fukushima crisis is resolved. Other scientists are reluctant to travel to the stricken area at this time because they fear their presence would exacerbate trafﬁc congestion and gasoline shortages that are bedeviling efforts to supply relief goods to shelters housing hundreds of thousands of displaced people. “Some faculty members think it is unethical to do scientiﬁc research [in the disaster area] now,” says Shuji Yoshida, a geologist at Chiba University. Already a humanitarian disaster, the Tohoku earthquake is taking a toll on science as well.

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CREDITS (TOP TO BOTTOM): PHOTO TAKEN AT YOKOSUKA NEW PORT, KANAGAWA, JAPAN, BY GLEAM/WIKIMEDIA COMMONS; PATRICK DEP/WIKIMEDIA COMMONS; HITOKAZU EZAKI/WIKIMEDIA COMMONS

TOKYO—With attention riveted on the crip-

–DENNIS NORMILE

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NEWSFOCUS The hard way. Depletion of con-

Outside of OPEC’s vast resources, oil production has leveled off, and it’s looking like it may never rise again FIVE YEARS AGO, MANY OIL EXPERTS SAW trouble looming. In 10 years or so, they said, oil producers outside the Organization of the Petroleum Exporting Countries (OPEC) would likely be unable to pump oil any faster (Science, 18 November 2005, p. 1106). Non-OPEC oil production would peak, no matter the effort applied. All the hightechnology exploration and drilling, all the frontier-pushing bravado of the oil industry would no longer stave off the inevitable as OPEC gains an even stronger hand among the world’s oil producers. Five years on, it appears those experts may have been unduly optimistic—non-OPEC oil production may have been peaking as they spoke. Despite a near tripling of world oil prices, non-OPEC production, which accounts for 60% of world output, hasn’t increased signiﬁcantly since 2004. And many of those same experts, as well as some major oil companies, don’t see it increasing again— ever. In their view, it’s stuck on a ﬂat-topped peak or plateau at present levels of production for another decade or so before starting to decline. “Stable [non-OPEC] production is

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the best we can hope for,” says energy economist Robert Kaufmann of Boston University. “I have trouble seeing it increase more. It’s a wake-up call.” Optimists remain. Some experts still see production from new frontiers, such as Kazakhstan, the deep waters off Brazil, and the oil sands of Canada, pushing production above the current plateau in the next few years. But time’s running out to prove that newly discovered ﬁelds and new technology can more than compensate for ﬂagging production from the rapidly aging ﬁelds beyond OPEC. Running to stay in place There’s no debate about the reality of the 6-year-and-counting plateau of non-OPEC production. Output stagnated at about 40 million barrels a day beginning in 2004 after rising from an earlier plateau in the early 1990s, one induced by a low price for oil. But prices have been anything but low lately. They have gone from about $35 a barrel early in the past decade to double and nearly triple that. Normally, higher prices would encourage more production, but not this time. Since 2004,

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Peak Oil Production May Already Be Here

“there’s been a tremendous increase in price, yet this is all we get for it, stable production,” Kaufmann says. “It’s quite stark.” The problem up to this point, all agree, has been increasing difﬁculties extracting conventional oil. That’s the easiest oil to get at, oil that freely ﬂows out of a well of its own accord or with a minimum of encouragement, such as pumping it out or pushing it out with water. Production of conventional oil from any one well or field typically increases, peaks, and then goes into decline. Larger producing regions behave the same way. Production from the United States, once the world’s largest oil producer, peaked in 1970 as rising output from newly discovered ﬁelds failed to compensate for declines in old ﬁelds. Mexico’s production peaked in 2004 as its huge, aging Cantarell ﬁeld went into steep decline. North Sea production peaked in 1999, just 28 years after starting up. The same pattern now seems to be emerging across much of the world. “We believe— and pretty much everybody else believes— that non-OPEC [conventional] production has plateaued,” says oil analyst Michael Rodgers, a partner with PFC Energy in Kuala Lumpur. “Arguing that you’re going to get continued and sustained growth of conventional oil is a very hard case to make.” PFC Energy has just done a complete reassessment of the prospects for non-OPEC conventional production, he says. As in most oil outlooks, a country-by-country or even ﬁeld-by-ﬁeld survey of what producers are planning for the next 5 to 10 years was combined with an educated guess of how much oil remains to be discovered in each region. That forecast of added production is balanced against how fast production from existing ﬁelds is declining. In the past decade, analysts have realized that rather than the 2% to 3% per year decline once assumed, production from existing ﬁelds is declining 4% to 5% per year. Some believe the depletion is even faster. The balance between added and declining production, in the PFC Energy assessment, is a plateau, though the plateau may undulate from year to year. “You

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ventional oil fields outside of OPEC is driving the mining of oil sands in Alberta, Canada.

NEWSFOCUS

Tough oil to the rescue? But what about unconventional oil, the hardto-get-at oil that’s only extractable using the latest in high technology? There’s the oil beneath kilometers of seawater far offshore of the U.S. Gulf Coast, Brazil, and West Africa. It wasn’t reachable until development of the necessary deep-water drilling and production technology. There is also the oil—more like tar—that is so viscous that steam must be piped underground to thin it before pumping it out. In Alberta, Canada, huge shovels just dig up the “oil sands” so it can be trucked to oil-extraction plants. And American drillers have lately taken to drilling into rock formations that would normally only dribble oil and fracturing the rock with high-pressure ﬂuids in order to wrest worthwhile amounts from the rock. That’s how drillers have been “fracking” stingy natural gas formations (Science, 25 June 2010, p. 1624). Such unconventional oil is out there in abundance, everyone agrees, and more will be produced than in the past. However, some major oil companies as well as other analysts don’t see unconventional oil boosting nonOPEC production much in the next 20 years. In their most recent annual energy outlooks to 2030, both ExxonMobil and BP—two of the world’s largest independent oil companies— forecast that non-OPEC production will more or less hold its own, no better. “It’s quite an accomplishment to keep non-OPEC supply ﬂat level,” says analyst Kyle Countryman, who as a member of ExxonMobil’s energy and economics group in Dallas, Texas, helped put the outlook together. Adds his colleague, group manager Robert Gardner: “We’re not optimistic we’ll see a signiﬁcant increase in unconventional liquids.” The problem with unconventional oil is that, by deﬁnition, it is hard to extract. “It’s a matter of timing,” Gardner says. “It depends on the pace of technology development.” And even after the essential technology is developed, unconventional oil will still be difﬁcult—as well as expensive—to extract, limiting the rate at which it can be produced. All in all, “technology matters, economics matters, but geology really does matter,” says oil analyst David Greene of the U.S. Department of Energy’s Oak Ridge National Laboratory in Tennessee. “Progress in technology is not fast enough to keep up with depletion” of oil reservoirs. Oil analyst Richard Nehring of Nehring Associates in Colorado Springs, Colorado, is more optimistic about prospects

on oil’s frontiers and how fast some kinds of unconventional oil can be brought online, but he still ﬁnds that “non-OPEC will be stable or at the very best slowly increasing” over the next couple of decades.

Liquids supply

Millions of oil-equivalent barrels per day

100

~36

80

~35 ~29

Biofuels Optimism not dead ~27 60 “We’re a little more bullish Other petroleum OPEC crude about non-OPEC than some others,” says Peter Jack40 son of Cambridge Energy Canadian oil sands Research Associates (CERA) in London. Along with the U.S. Energy Information 20 Non-OPEC crude and condensate Agency (EIA), CERA sees real promise in underdevel0 oped oil provinces such as 1980 1990 2000 2010 2020 2030 offshore Brazil and Kazakhstan. Likewise, if prices stay Running flat out. An ExxonMobil outlook has non-OPEC oil high, unconventional oil will (orange plus blue) plateauing. Natural gas–derived liquids (green) contribute substantially, both and biofuels (yellow) will help, but OPEC (purple) must pitch in. ﬁnd, especially the Canadian oil sands. Beyond the next few years, “we’re demand might be met by several sources. In seeing a gradual increase in non-OPEC decreasing order of reliability, production supply,” Jackson says. of another sort of petroleum liquid, natural Such optimism has not always served fore- gas liquids (NGLs), is expected to increase. casters well. In 2005, Jackson and his CERA NGLs are the lighter-weight hydrocarbons colleague Robert Esser of the New York ofﬁce that condense from natural gas when it predicted that “global oil production capacity cools. The expected increase in global natis actually set to increase dramatically” up to ural gas production—at least half of which 2010. It didn’t; both OPEC and non-OPEC oil would come from OPEC—would lead to production remained steady. Likewise, in its increased production of NGLs. 2005 outlook, EIA projected a jump in nonOPEC would, it is fervently hoped outside OPEC production by 2010 if prices were high, of the cartel, be willing and able to boost its which they mostly were. But 2010 production output of conventional oil. ExxonMobil has was about 40 million barrels per day, right OPEC production rising from about 29 milwhere it was in 2005. lion barrels per day today to about 36 million So what if the pessimists turn out to be barrels per day in 2030. That would increase realists and non-OPEC producers can’t OPEC’s share of oil production even further, answer the call for more oil? Demand will but Kaufmann, among others, expects that increase in this decade, mainly from devel- OPEC will see an opportunity to make more oping countries like China and India as pop- money from its oil by curbing production ulations grow and incomes rise. That rising and driving prices up. That would tend to encourage production of liquid biofuels, but whether output could be ramped up quickly enough to bring relief remains unclear. The clearest outcome, according to Greene, is likely to be continued or even greater volatility in the price of oil with all the economic downsides that would entail. Perhaps the most sobering outcome of a non-OPEC plateau might be reminding everyone that even planet-scale resources have their limits. And that when you are consuming them at close to 1000 gallons a second, the limits can catch you unaware. The next 5 years, assuming oil prices A remaining hope. Some analysts see untapped oil ﬁelds in frontier areas, such as offshore Brazil remain on the high side, should show who (above), pushing up non-OPEC production. the realists are. –RICHARD A. KERR

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bring on a [new] 100,000-barrel-a-day ﬁeld,” Rodgers says, “and somewhere else you’ve lost a 100,000-barrel-a-day ﬁeld.”

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sites. And the dates of the overlying horizons correlated with those of their diagnostic tools. “The OSL dates are almost perfect top to bottom,” says archaeologist Dennis Jenkins of the Museum of Natural and Cultural History in Eugene, Oregon, who was not involved in the study. Very ancient stone tools help conﬁrm what many have long suspected: Clovis hunters, The team did four other analyses, such as with their distinctive spear points, were not the ﬁrst to people the Americas examining whether artifacts were sorted by size, to be sure that artifacts from upper levNear the headwaters of a small creek, a The new paper claims to change all that. els had not fallen down into the pre-Clovis group of hunter-gatherers made their camp In 2006, Waters and his team began excavat- layer. “All [the analyses] pointed to an intact and began to craft stone tools. The river- ing at the Debra L. Friedkin site in central sequence,” says Sandweiss, another outsider banks rang with their blows, as they struck Texas, not far from a known Clovis site. The familiar with the ﬁndings. ﬂakes off chert nodules to create tools for site lies near a year-round water supply and Use-wear studies on tools from the lowest cutting hard materials such as bone; they close to an abundant source of high-quality layer reveal that the pre-Clovis people used also knapped small blades for processing chert for toolmaking. Repeated incremental the tools to work bone, wood, or ivory and to hides. They left thousands of sharp stone deposits of clay from ﬂooding over the mil- cut or process hides. And their blades, bladeﬂakes and chips discarded on the ground. lennia offered good preservation. lets, and bifaces bear some important resemAt one time or another, similar scenes As the team dug down into the clay, they blances to the later Clovis toolkit, pointing have played out almost the whole world over. found a sequence of cultural horizons, each to continuity between the two groups. “The But the remarkable thing about this one, as with diagnostic stone tools in correct strati- thing I find really neat,” Waters says, “is detailed on page 1599 of this issue, is that that we found 12 [preit happened near Buttermilk Creek, Texas— Clovis] bifaces, and about 15,500 years ago. That’s long before they were making them the Clovis hunters, once thought to be the by bifacial reduction, very ﬁrst people in America, had appeared. similar to the technique Lead author Michael Waters of Texas A&M used by Clovis people.” University in College Station says this Longtime Clovissite “tells us for once and for all that we f irst advocate Gary can abandon this Clovis-ﬁrst model.” The Haynes of the Univerancient tools also offer a ﬁrst glimpse into sity of Nevada, Reno, is how the distinctive ﬂuted Clovis points may impressed by the paper, have developed over millennia. saying that it includes Although some previous claims of prekey stratigraphic analClovis artifacts have been controversial, yses missing from other archaeologists say the new research is studies of other poshighly convincing. “The many distinct and sible pre-Clovis sites superbly documented lines of evidence … in North America. And offer pretty unequivocal conﬁrmation that “there’s no question people were in interior North America south that the artifacts are of the ice sheets before the Clovis radiation,” really stone tools,” he says David Anderson of the University of says, a problem raised Tennessee, Knoxville, founder of the online Time team. Excavators unearthed pre-Clovis stone tools, including (inset, at other pre-Clovis Paleoindian Database of the Americas. sites. “These are really top to bottom) a biface, ﬂake tool, and ﬂake core. Archaeologists have been locked in an things that could be acrimonious debate over the early peopling graphic order, ranging from Late Prehistoric technologically and logically ancestral to of the Americas for nearly 30 years. Advo- artifacts in the uppermost horizon to Folsom, Clovis.” But Haynes isn’t sure the tools date cates of the Clovis-ﬁrst theory argued that Clovis, and ﬁnally pre-Clovis in the three to pre-Clovis times and would like radiothe big-game–hunting Clovis people were the lowest levels. But they found no charcoal or carbon dates to be certain. ﬁrst to arrive from Asia, about 13,200 years other organic material, which is often poorly Despite such doubts, Anderson expects ago. Other archaeologists reported nearly two preserved in this region, and so had no way to that the new ﬁndings will move studies of the dozen pre-Clovis sites in North America. In radiocarbon-date the layers. ﬁrst Americans into a new phase. “As more each case, however, the evidence was incomThe team opted to use optically stimu- attention focuses on pre-Clovis lifeways, plete or ﬂawed. Even so, says archaeologist lated luminescence (OSL), a technique we can begin to get a better handle on many Dan Sandweiss of the University of Maine, that measures the amount of light energy major questions: how did the colonization Orono, the pre-Clovis theory “has been get- trapped in quartz and feldspar grains in proceed, [and] how widely did these folks ting more and more traction.” But advocates the clay and dates the last time they were range over the landscape,” he concludes. have had trouble pointing to a particular exposed to sunlight. The lowest tool-laden –HEATHER PRINGLE North American site that offered strong proof horizon dated to 15,500 years ago—more Heather Pringle is a contributing editor at Archaeology in one place. than 2 millennia before the first Clovis magazine.

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Texas Site Conﬁrms Pre-Clovis Settlement of the Americas

M I C R O B I O LO G Y

adults. These volunteers were sequestered for 10 days and fed either a high-fat or a low-fat diet, and the researchers analyzed their stool samples for bacteria and viruses. Overall, the median number of different kinds of phages was 44 per sample, Bushman reported at the meeting. But within 24 hours of a person starting the new eating regimen, the community of phages and bacteria began changing. Because the phages live in the bacteria, one would expect the number and kinds of bacteria and phages to change in parallel, but that was not always the case, Bushman noted. There was also a lot of variation between individuals, with the number of viral types differing by as much as 40-fold in the samples. But in those

Going Viral: Exploring the Role Of Viruses in Our Bodies

CREDITS: ADAPTED FROM OMAR JABADO AND W. IAN LIPKIN; (PHOTO) CDC

‘Virome’ surveys reveal our vast number and variety of viruses

more diverse than the bacterial communities within the same individuals. But each person’s viral community remained stable over the course of the year. WU microbiologist Kristine Wylie and her colleagues have begun to look at how the virome may inﬂuence health, in particular what role it might play in unexplained fevers in infants. For children under 900,000 3, fevers are the most common 800,000 Growth of cause of emergency room visViral Database its, but almost 90% of the time 700,000 Retroviridae there’s no clear cause for the Orthomyxoviridae 600,000 high temperatures. Flaviviridae Hepadnaviridae Wylie, Weinstock, WU’s 500,000 Picornaviridae Gregory Storch, and their colParamyxoviridae 400,000 leagues sequenced the DNA Herpesviridae obtained from nasal swabs or Other (59 Families) 300,000 blood plasma of 151 individuals, 200,000 about half of whom had unexplained fevers. The team esti100,000 mated there were 10,000 viral 0 sequences in the plasma samples 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 Year of the children with fever and only about 1000 in healthy chil- Expanding universe. The tally of known viruses is exploding, dren. Some of the viruses found and this graph doesn’t even include the incredible number that were common human pathogens, prey on bacteria. such as herpes and cold viruses. But there also seem to be unusual viruses, people eating the same foods, the repertoire including an astrovirus, in the mix, Wylie of viruses tended to converge. reported. “There were new isolates that you These studies drive home that the human might not have thought would be associated virome needs to be studied more closely, with febrile illness,” says Frederic Bushman, Suttle says. But there are many challenges. a virologist at the University of Pennsylvania. For one, most of the viral sequences that Much more work is needed to establish have turned up so far don’t have matches that any of these viruses explain the fevers, in any known databases, so the viruses but proving that could mean fewer anti- can’t be characterized. And all the virome biotics for infants, Wylie points out. To be survey techniques have their drawbacks. on the safe side, physicians tend to prescribe Bushman ﬁltered out all the human and bacantibiotics for unexplained fevers, but such teria cells and cleared out all nonviral DNA drugs are ineffective against viruses. before sequencing any remaining DNA in his While Wylie has focused on the viruses samples. “What [that approach] probably that infect human cells, Bushman has homed does is throw out most of the virus that was in on the bacteriophages, viruses that attack there,” Suttle says. Wylie and her colleagues, bacteria. For every bacterium in our body, on the other hand, sequenced all of the DNA there’s probably 100 phages, with an esti- in the stool samples and used computer promated 10 billion of these viruses packed into grams to sort sequences into human, bacteeach gram of human stool. As part of a study rial, and viral bins. But with that strategy, “you of the interplay of diet, human gut microbes, usually wind up with a big bin of unknown and Crohn’s disease, an inﬂammation of the [sequences],” Suttle adds. Bottom line, says digestive system, Bushman and his colleagues WU’s Herbert “Skip” Virgin, “Bacteria are have looked at the viromes of six healthy easier to count.” –ELIZABETH PENNISI Nucleotide sequences

VANCOUVER, CANADA—In the past decade, scientists have come to appreciate the vast bacterial world inside the human body. They have learned that it plays a role in regulating the energy we take in from food, primes the immune system, and performs a variety of other functions that help maintain our health. Now, researchers are gaining similar respect for the viruses we carry around. For a start, the variety and sheer number of viruses that inhabit us put our bacterial companions to shame. Many of the viruses prey on the bacteria in our bodies, altering their numbers and diversity and shuffling genes—including genes for antibiotic resistance—from one bacterium to another. “Ultimately, those viruses are incredibly important in driving what’s going on” in the human microbiota, says Curtis Suttle, a virologist at the University of British Columbia (UBC), Vancouver, in Canada. “To understand the bacteria associated with humans, you can’t do that without looking at the viruses as well,” he says. Studies presented here at the International Human Microbiome Congress earlier this month have begun to do just that. One provocative, albeit preliminary, finding has already emerged: Infants with unexplained fevers harbor many more viruses than healthy infants. For years, virologists have documented speciﬁc viral infections, including HIV and SARS, by detecting identiﬁable viral DNA within blood or other tissues. But to do a comprehensive survey of the viruses in the body—the so-called virome—“is a true frontier,” says B. Brett Finlay, a microbiologist at UBC Vancouver. Last July, Jeffrey Gordon, a microbiologist at Washington University (WU) School of Medicine in St. Louis, and his colleagues described one such effort in a group of seemingly healthy people. They isolated and characterized the viromes of adult identical twins and their mothers, sampling stools for viral genetic material three times over the course of a year. The overall conclusion: Healthy people “are full of viruses,” says WU’s George Weinstock. By one measure, the number of distinct viruses in the stool samples ranged from 52 to 2773. The viromes varied signiﬁcantly from one individual to the next; they were even

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NEWSFOCUS

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COMMENTARY Bonding in aluminum

SPORE Prize Essay

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LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES

LETTERS edited by Jennifer Sills

Dealing with Data: Fostering Fidelity

ALEXANDER EDWARD THOMAS FINLAYSON

Department of Global Health, King’s College London, London, UK. E-mail: alexanderﬁ[email protected]

References

1. A. E. Finlayson et al., J. Telemed. Telecare 16, 181 (2010). 2. A. Leather et al., Lancet 368, 1119 (2006).

CREDIT: Y. FITZPATRICK, USING WWW.WORDLE.NET/SCIENCE

Dealing with Data: Preserve Old Collections THE SPECIAL SECTION ON DEALING WITH DATA (11 February, p. 692) highlighted the challenges of storing, accessing, sharing, and reusing electronic data. We would like to point out that in addition to electronic data, data in the form of biological (and other) collections housed in herbaria and museums for decades or even centuries are in dire need of preservation. These collections continue to be of importance to biologists, who often find new applications for the data as new methods become available (1). For example, in recent years DNA has successfully been extracted from specimens that were collected long before anybody could have envisaged such a use for their collections (2). In another example, geographical information from

specimen labels is used for inferring speciesenvironment relationships, now an important basis for climate impact predictions for species globally (3). Finally, as the collections themselves are stored, much of the data extracted from them remains veriﬁable. In the face of funding cuts that threaten many such institutions (1), it is essential these collections continue to be maintained.

MICHELLE GREVE* AND JENS-CHRISTIAN SVENNING

Department of Biological Sciences, Aarhus University, 8000, Denmark. *To whom correspondence should be addressed. E-mail: [email protected]

References

1. G. H. Pyke, P. R. Ehrlich, Biol. Rev. 85, 247 (2010). 2. S. Lehtonen, M. J. M. Christenhusz, Biologia 65, 204 (2010). 3. J. Elith, J. Leathwick, Divers. Distrib. 13, 265 (2007).

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IN HER PERSPECTIVE “ADVANCING GLOBAL HEALTH RESEARCH THROUGH DIGITAL TECHNOLOGY and sharing data” (special section on Dealing with Data, 11 February, p. 714), T. Lang neglects to mention a critical barrier to health information sharing. Before the global healthcare workforce will fully trust a uniﬁed IT architecture for research, we must develop a robust system to verify global professional ﬁdelity. The healthcare community must be assured that data is reliable, conﬁdential, and safe from abuse by those who have access to it. Unfortunately, the Hippocratic Oath cannot guarantee protection of patients in developed countries. Internet disclaimers and online ratings are too ambiguous, and local institutions that could provide professional veriﬁcation are nonexistent in many areas most in need of local health research. Medical establishments and governments must work together to provide doctors, nurses, and laboratory workers with training and well-deﬁned standards that are acceptable to the international community [e.g., (1, 2)]. Only then will trustworthy collaboration between professionals in geographically and culturally disparate countries be possible. Viral, open-source Internet activity alone is insufﬁcient to solve the problems of global health. Online solutions must be augmented in the real world and we must be willing to confront the massive practical and political challenge of deﬁning who is qualiﬁed to contribute and access this information by creating a global online passport for healthcare.

Dealing with Data: Upgrading Infrastructure THE SPECIAL SECTION ON DEALING WITH DATA (11 February, p. 692) showed that data currently reside in a clumsy network of poorly linked databases of unknown quality. It is a cottage industry trying to participate in an international environment. We need to replace this poorly organized network with an international framework for a collaborative data infrastructure (1). The new system should provide secure persistent storage, data identiﬁers, authenticity and workﬂow support for data mining, and other applications to access data for multidisciplinary interests. It should be not a grand uniﬁed service but a number of separate ones; data should be controlled by the generators and owners and operated by a number of public and/or private services who adhere to common standards. The drive for this service will become reality when researchers make it a priority for economic and efﬁciency reasons. Along with adopting the new data structure, we must develop the appropriate legal and ﬁnan-

25 MARCH 2011

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LETTERS

INTRODUCING

AAAS MemberCentral

cial models to foster trust in the service. We must also discuss the plan internationally and agree on next steps to coordinate the change.

WOUTER LOS1* AND JOHN WOOD2

Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, NL-1098XH, Amsterdam, Netherlands. 2 Association of Commonwealth Universities, London WC1H 9HF, UK. 1

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

Reference

Dealing with Data: Training New Scientists The exclusive new website for the AAAS member community.

Summer Internships Students with Disabilities To meet the challenge of the competitive economy in the new millennium, AAAS started Entry Point!, a program that offers students with disabilities competitive internship opportunities in science, engineering, mathematics, computer science, and some ﬁelds of business. Join us. Together we can make a difference.

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THE DATA DELUGE IS ALREADY HERE (SPECIAL section on Dealing with Data, 11 February, p. 692). As a community, we should encourage young scientists to prepare for careers, such as bioinformatics, that will help address data issues. We should also find ways to move beyond specialization in one ﬁeld and teach interdisciplinary ﬂexibility. Scientists are needed who can communicate with the molecular biologists, pathologists, and geneticists as well as the bio informaticians, programmers, and IT specialists. Unfortunately, the best way to train this next generation of scientists is unclear. Current interdisciplinary PhD programs do not ensure that coursework is balanced across disciplines. One alternative would be to encourage wet-lab trained PhD students to apply for post-doc positions in bioinformatics. This would provide the foundation to understand and communicate with both the bench scientist and bioinformatician, but it also requires that we as a community be willing to take a chance and hire benchtrained PhDs with some bioinformatics afﬁnity over the bioinformatics-only trained PhDs who are currently in short supply.

ANDREW J. SEVERIN

Department of Agronomy, Iowa State University, Ames, IA 50011, USA. E-mail: [email protected]

Advancing Translational Research THE NATIONAL INSTITUTES OF HEALTH (NIH) proposal to launch a National Center for Advancing Translational Sciences (NCATS) by dismembering the National Center for Research Resources (NCRR) (“Collins sparks furor with proposed NIH reshufﬂing,”

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1. High Level Expert Group on Scientiﬁc Data, “Riding the wave: How Europe can gain from the rising tide of scientiﬁc data” (European Commission, October 2010); http://cordis.europa.eu/fp7/ict/e-infrastructure/docs/ hlg-sdi-report.pdf.

LETTERS

BRAD BOLON,1* BRUCE ALTROCK,2 STEPHEN W. BARTHOLD,3 NICOLE BAUMGARTH,3 DAVID BESSELSEN,4 GREGORY BOIVIN,5 KELLI L. BOYD,6 CORY BRAYTON,7 ROBERT D. CARDIFF,3 SUZANA COUTO,8 KATHRYN A. EATON,9 ODED FOREMAN,10 STEPHEN M. GRIFFEY,3 KRISTA LA PERLE,11 MICHAEL D. LAIRMORE,11 CHEN LIU,12 DAVID K. MEYERHOLZ,13 ALEXANDER YU. NIKITIN,14 TRENTON R. SCHOEB,15 DENISE SCHWAHN,16 RANI S. SELLERS,17 JOHN P. SUNDBERG,18 RAVI TOLWANI,19 VICTOR E. VALLI,20 M. CHRISTINE ZINK7

GEMpath Inc., 2867 Humboldt Circle, Longmont, CO 80503, USA. 2Center for Genomic Pathology, Davis, CA 95616, USA. 3 Center for Comparative Medicine, University of California— Davis, Davis, CA 95616, USA. 4University Animal Care, University of Arizona, Tucson, AZ 85721, USA. 5Laboratory Animal Resources, Wright State University, Dayton, OH 45435, USA. 6Department of Pathology and Comparative Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. 7Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 8Genentech Inc., South San Francisco, CA 1

94080, USA. 9Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA. 10The Jackson Laboratory, Sacramento, CA 95838, USA. 11Department fo Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA. 12Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA. 13Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. 14 Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA. 15Department of Genetics and Comparative Pathology Laboratory, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 16Research Animal Resources Center, University of Wisconsin-Madison, Madison, WI 53726, USA. 17Albert Einstein College of Medicine, Bronx, NY 10461, USA. 18The Jackson Laboratory, 610 Main Street, Bar Harbor, ME 04609, USA. 19Comparative Bioscience Center, The Rockefeller University, New York, NY 10065, USA. 20 VDx Pathology, Davis, CA 95616, USA. *To whom correspondence should be addressed. E-mail: [email protected]

References

1. National Center for Research Resources Program Overview (www.ncrr.nih.gov/about_us/program_overview/index.asp). 2. Feedback NIH, Proposed National Center for Advancing Translational Sciences (http://feedback.nih.gov/index. php/category/ncats/). 3. Feedback NIH, “Archived NCATS comments, 12/9/10– 1/13/11” (http://feedback.nih.gov/index.php/ncats/ ncats-comments/#comments). 4. Feedback NIH, “Comments to NCRR task force straw model” (http://feedback.nih.gov/index.php/ncats/ straw-model/#comments). 5. J. Mervis, “Collins’s plan to reshufﬂe NIH draws more ﬂak,” ScienceInsider (28 January 2011). 6. M. Wadman, “Collins defends decision to dismantle NIH center,” The Great Beyond, 21 January 2011; http://blogs. nature.com/news/thegreatbeyond/2011/01/ collins_defends_decision_to_di.html. 7. M. Wadman, “NIH revamp rushes ahead,” Nature News, 1 March 2011; www.nature.com/news/2011/110301/ full/471015a.html. 8. Feedback NIH, “Comments to NCRR Task Force Recommendations” (http://feedback.nih.gov/index.php/ncats/ task-force-recs/#comments).

CORRECTIONS AND CLARIFICATIONS Essay: “Penguins and polar bears integrates science and literacy” by J. Fries-Gaither and K. Lightle (28 January, p. 413). The portrait photographer’s name is Margaux Baldridge. Reports: “Intravascular danger signals guide neutrophils to sites of sterile inﬂammation” by B. McDonald et al. (15 October 2010, p. 362). In the penultimate paragraph, the following sentence was imprecise: “In contrast to recent reports (17), we show that ATP does not function as a chemoattractant….” The study by Chen et al. (reference 17) did not show that ATP is itself a chemoattractant, but rather that ATP signals are essential for chemotaxis by amplifying chemotactic signals and directing cell orientation toward the chemoattractant.

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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J. Kaiser, News and Analysis, 28 January, p. 386) is more likely to hinder than bolster translational research. Developing new treatments requires understanding of disease mechanisms. Such studies depend on superior facilities, resources, and training, which for years have been effectively supported by the NCRR Division of Comparative Medicine (DCM) (1). The original NCATS proposal sought to divide core NCRR functions, including those in DCM, between NCATS and a hodgepodge Interim Infrastructure Unit (2). The updated proposal preserves major DCM roles within an Infrastructure Entity (2). This revision clearly concedes that reducing disruption to existing NCRR resources is a surer way for NIH to sustain its historical vigor in mechanistic research while also boosting its translational science attainments than an impulsive shift to chemical screening and preclinical testing by an untried NCATS. Furthermore, the utility of the NCATS compared with the proven value of NCRR has not been adequately considered by the scientiﬁc community. To date, the NCATS concept and original “Straw Model” have received hundreds of comments, many of which are critical (3–7); deliberations over the revised NCATS proposal have barely begun (8). Hurried implementation of NCATS over such widespread objections will immediately call into question the credibility of the new Center. As veteran comparative biologists, we believe that the best way to rapidly advance NIH translational science efforts will be to build rather than break the successful, integrated program within the NCRR. Any other choice should be recognized for what it is: good politics, but bad policy.

BOOKS ET AL. Szathmáry did not offer a uniﬁed conceptual framework but instead provided a mixed bag of explanations for the different transitions. Building on Hamilton’s theory and the more recent work in this area, especially that of David Queller and Koos Boomsma, Bourke has ﬁxed that problem. In an important step forward, he has made the major-transitions approach both more approachable and more obviously fundamental. Principles of Social Evolution provides an accessible, comprehensive, and highly readable overview, which will be invaluable in undergraduate teaching (although I would have liked to have found more ﬁgures). In addition, Bourke clariﬁes the future research that is required, making the book equally suitable for frontline researchers from postgraduate to professorial levels. In dividing the major transitions into three successive steps,

EVOLUTION

A Better View of Eras of Life Stuart West

CREDIT: CAROLINA BIOLOGICAL/VISUALS UNLIMITED/CORBIS

M

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any biology classdriven by either a direct benPrinciples of Social Evolution rooms and lecefit to the individual conby Andrew F. G. Bourke ture theaters are cerned or an indirect benOxford University Press, adorned with posters that eﬁt from helping relatives. Oxford, 2011. 279 pp. $117, £65. depict the history of life on The beauty of this approach ISBN 9780199231157. Paper, $52.95, Earth as a succession of difis that it allows enlighten£29.95. ISBN 9780199231164. Oxford ferent taxonomic groups. ing comparisons across Series in Ecology and Evolution. These posters tell the stanthe transitions. Particularly dard story of the age of inverlucid, and worth the price of tebrates giving rise to the age of ﬁshes, which the book alone, is Bourke’s exposition of how gave rise to the age of reptiles, which gave the factors shaping the evolution of complex rise to the age of mammals, which culminates multicellular organisms and eusocial sociwith us at the pinnacle. In Principles of Social eties are remarkably similar. Both instances Evolution, Andrew Bourke makes an excel- involve social groups that develop from a lent case for replacing this history, which is single propagule (a sinbiased toward relatively humanlike orga- gle cell that then repronisms, with a hierarchical approach based on duces clonally or a single how organisms come together to form higher- monogamously mated level collectives, tracing the steps from genes female), the same type to complex societies. He has written a superb of social group formabook, one that should change how we teach tion (offspring stay with and think about life on our planet. parents), and a collection Bourke, an evolutionary biologist at of overlapping and nonthe University of East Anglia, takes a two- overlapping ecological pronged approach. First, he builds on previ- factors (such as defense ous work by other researchers (1–3) to empha- against predators). size how evolution has involved a number As well as offering a of major transitions, through which a group good explanation for the of individuals that could previously repli- major transitions, Bourke cate independently come together to coop- clariﬁes why such transieratively form a new, more complex form of tions have not developed individual that can only replicate as a whole. in other cases. For examGenes joined to form genomes, prokaryotic ple, slime molds and From many, one. In the colonial green algae Volvox aureus (here cells incorporated protobacteria as mitochon- allodapine bees can have releasing a daughter colony), individual cells work together in a coordidria to become eukaryotic cells, single-celled life cycles with, respec- nated fashion and exhibit cellular differentation. organisms joined together to become multi- tively, some multicellucellular organisms, multicellular organisms larity and sociality. But it isn’t surprising that he demonstrates how we understand the ﬁrst became eusocial societies, and multicellular they haven’t gone all the way to the next level, two (the formation and maintenance of social species joined with either unicellular species because they have a method of group forma- groups) fairly well and the third (the transforor other multicellular species to become inter- tion different from groups that have. In these mation to the next level of individual) relaspeciﬁc mutualisms. Over several chapters, slime molds and bees, same-generation indi- tively poorly. The book does exhibit a bias the author compares and contrasts these tran- viduals come together to form social groups, toward the social insects. Although Bourke sitions, dissecting them into three principal and so relatedness isn’t as high as when off- suggests this may reﬂect his background, the steps through which social groups are initially spring stay with parents. This stresses that tilt is probably more a reﬂection of the fact formed, maintained, and then transformed while there is a continuum of sociality, there that theirs is the transition that has attracted into the new level of individuality. is something special (and predictable) about the most attention. Hopefully, his book will Second, Bourke uses Hamilton’s inclu- what carries groups all the way through a resolve that problem. sive ﬁtness theory to provide an overarch- major transition. References ing conceptual framework to explain all of Bourke’s book will no doubt be compared 1. L. W. Buss, The Evolution of Individuality (Princeton Univ. these major transitions. This elucidates how with John Maynard Smith and Eörs SzathPress, Princeton, NJ, 1988). the cooperation that drives these transitions is máry’s The Major Transitions in Evolution 2. E. G. Leigh, Trends Ecol. Evol. 6, 257 (1991). 3. J. Maynard Smith, E. Szathmáry, The Major Transitions in (1). That earlier book did an excellent job of Evolution (Freeman Spektrum, Oxford, 1995). setting out the major-transitions view as an The reviewer is at the Department of Zoology, University approach to understanding life’s evolutionof Oxford, South Parks Road, Oxford OX1 3PS, UK. E-mail: 10.1126/science.1203273 [email protected] ary history. However, Maynard Smith and

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BOOKS ET AL. PLANT SCIENCE

A Ballet of Plant Movement Sarah E. Wyatt

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I

n the Northern Hemisphere, winter is ending and the world of plants seems dead and lifeless. However spring is on the way, and the ever-present plants will again resurface. Their return will herald a re-greening of Earth, a ballet of germination and sprouting. But for most people, plants seem as if inanimate objects. Trees stand majestically, grass carpets a lawn, and ﬂowers provide a backdrop of color, yet all appear to stand lifeless except for swaying in the Revealing movements. Flowers of water hyacinths (Eichhornia crassipes) undergo diurnal cycles of wind. Intellectually, you know they are alive. opening and closing by means of reversing the curvature of the perianth leaves at their base. You learned that in school. You also learned they are sessile—they don’t move, at least naea muscipula) snap shut when presented driven responses in speciﬁc cells (pulvini) not in the animal-centric sense of the word. with prey. Whereas many have seen these that are ﬁlled or drained of water as needed As a child, few of us stopped long enough demonstrations, few have witnessed them in for movement. For the slower growth moveto appreciate the row of lawn chairs lined nature. They are treated as novelties, excep- ments, the tropisms, the motors are growing up facing our neighbor’s house. Every eve- tions to the rules of plant life. But they aren’t cells within speciﬁc regions of the plant. Koller presents a thorough survey of the ning, adults from our neighborhood would the exception; they just provide a showcase sit there, facing the “house” as the sun set, that too often begins and ends discussion. positioning of plant organs (stems, leaves, chatting about the day’s events and waiting The Restless Plant presents a “guided tour flowers, and roots) in response to gravity for the moonﬂowers (Ipomoea alba) planted of plant movements.” Koller starts with the and light; of how plants interpret interacting there to open. Childhood rumblings of paint classic, rapid leaf movements of the sensi- and competing signals; of prey-driven movetive plant and flytrap but then provides a ments; and of the liberation and invasion of drying and life in a small town abounded. It is this misconception that the late Dov broader understanding of plant movement seeds. He details the signaling pathways, at Koller puts to rest in The Restless Plant. that includes growth responses, expansion least what is known for each. Although many of plant organs, and move- of the puzzle pieces have been identified, Koller (who was a botanist ments of individual cells and some of these signaling mechanisms remain at the Hebrew University of The Restless Plant organelles. The world of plants mysterious. For example, when considering Jerusalem) provides an inby Dov Koller. Elizabeth Van becomes a fascinating dance gravitropism, a quote by Robert Fulghum depth and compelling arguVolkenburgh, Ed. with many movements: con- still rings true: “Remember the seed in the ment for what many plant Harvard University Press, tractile roots pulling a bulb Styrofoam cup: the roots go down and the biologists know: plants move. Cambridge, MA, 2011. into the soil; the folding of plant goes up and nobody really knows how They just do so on their own 224 pp. $39.95, £29.95, €36. leaves and ﬂowers at nightfall; or why” (2). time scale and in their own ISBN 9780674048638. leaves and ﬂowers tracking the At times the text may contain a few too way. With the advent of timeSun; roots searching for water many technical terms for the casual reader. lapse photography, even these slower plant movements have become the and nutrients; the explosion of seeds into the However, the glossary will help with the terstars of video clips and movies, for example, world at large; and growth responses to light, minology, and the effort is worth the time. Koller sets the stage for an amazing interpreRoger Hangarter’s Plants-in-Motion Web site gravity, water, temperature, and touch. From the germination of its seed, a plant tation of the world around us. The Restless (1) and David Attenborough’s 1995 BBC documentary series The Private Life of Plants needs input from the environment to give it Plant is not just a litany of examples of plant (now partially available in YouTube clips). direction: the roots go down, the shoots go movement, although that alone is worth the The image of a restless plant ﬁdgeting, ﬁnd- up. This direction is required for the sheer read, but also an exploration of the mechasurvival of an organism that is literally rooted nisms and physiology that support those ing its way in the world, is apropos. The classic examples, those pulled out for into the soil and dependent on sunlight for movements, the intricacies and beauty of school children, have rapid movements: the food and energy. Driven by both internal, their responses. You will never look at plants sensitive (or, in Chinese, shy) plant (Mimosa autonomous signals and external, environ- the same way again. pudica) collapses its leaves upon touch; the mental stimuli, plant movements are necesReferences traps of the carnivorous Venus ﬂytrap (Dio- sary for life. “Motors” provide these move1. http://plantsinmotion.bio.indiana.edu/index.html. ments, and, although the use of the term for 2. R. Fulghum, All I Really Need to Know I Learned in Kinsome of the responses is not without controdergarten (Villard, New York, 1990). The reviewer is at the Department of Environmental and versy, the analogy is sound. For more rapid, Plant Biology, Ohio University, Athens, OH 45701, USA. E-mail: [email protected] reversible movements, motors involve turgor10.1126/science.1203705

POLICYFORUM GLOBAL GENETIC RESOURCES

Marine Biodiversity and Gene Patents

Ten countries account for 90% of patent claims associated with marine genes, including some from international waters.

Sophie Arnaud-Haond,1* Jesús M. Arrieta,2 Carlos M. Duarte23

Marine biodiversity contains most phylogenetic and genomic diversity on Earth (e.g., 34 of 36 animal phyla hitherto described versus 17 on land). Progress in marine and molecular technologies has facilitated “bioprospecting,” with the number of patent claims associated with genes of marine organisms growing at 12% per year (2). Although patenting does not always result in effective exploitation, the existence or lack of patent claims and their relative distribution offer a reaIfremer (French Research Institute for Exploration of the Sea) Centre de Brest; Department DEEP-LEP; 29280 Plouzané, France. 2Department of Global Change Resarch, Mediterranean Institute for Advanced Studies, jointly governed by Consejo Superior de Investigaciones Cientíﬁcas and Universidad de las Islas Baleares: IMEDEA (CSIC-UIB), 07190 Esporles, Mallorca, Spain. 3Oceans Institute, University of Western Australia, Crawley 6009, Australia. 1

*Author for correspondence. E-mail: sophie.arnaud@ ifremer.fr

Patent claims for a gene of marine origin with source Country

Marine organism patent claims

USA

199

Germany

149

Japan

128

France

34

United Kingdom

33

Denmark

24

Belgium

17

Netherland

13

Switzerland

11

Norway

9

100 10 1 0.1

1

10

100 200

Number of patent claims

Patent claims associated with genes of marine origin. Cumulative distribution of patent claims showing the proportion of countries (y axis) at the origin of x or more patent claims. See SOM.

in marine bioprospecting (11, 12), broadening the gap in oceanographic and biotechnological capacities among countries. A similar distribution for patent claims associated with human genes or key plant crops (table S1 and ﬁg. S1) suggests differential access to molecular technologies as the main advantage. This inequality calls for policies targeting capacity-building in countries that lag behind and stresses the need for an internationally recognized framework governing MGRs. The Legal Gap

With 95% of claims filed after 2000, the growth of marine gene patents is a recent phenomenon, but it has matured well beyond “proof of concept.” The global market for marine biotechnology was estimated at U.S. $2.4 billion in 2004, with estimated average growth of 5.9% per year from 1999 to 2007 (13). Marine molecules include cancer- and HIV-ﬁghting agents representing $1 billion and $125 million, respectively, annual markets in 2005 (5). Most MGRs are derived from organisms sampled in territorial waters. However, source organisms can be shared by several different EEZs and/or may disperse across international waters during their life cycles. Exploration of extreme ecosystems, such as hydrothermal vents and polar oceans, mostly located in ABNJs, has disclosed a broad spectrum of molecules of biotechnological interest (2, 6). Examples include thermostable enzymes for molecular biology or the food industry (2, 5) and amylase isolated from a black smoker hydrothermal vent used for biofuel

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Ownership of Patent Claims

sonable ranking of countries’ accessing of resources. We screened records in the patent division of GenBank (7) to extract international claims valid in all countries subscribing to the World Trade Organization (WTO) agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) and deposited in the World International Property Organization (WIPO). Among 677 international claims of marine gene patents deposited between 1991 and 2009, 8648 sequences from 520 species were found. Gene patent claims from marine organisms make up only 2% of the WIPO gene patents (table S1), whereas claims associated with human genes dominated (35%), closely followed by the most frequently raised cultivars (wheat, rice, maize, and barley). Claims associated with marine genes originate from only 31 of the 194 countries in the world. Ten countries own 90% of the patents deposited with marine genes, with 70% belonging to the top three (see the table). These 10 nations represent only about 20% of the world’s coastline, but they benefit from access to advanced technologies required to explore the vast genetic reservoir of the oceans. The power law describing the distribution of “ownership” of MGRs across countries (see the ﬁgure) (table S1), ﬁts the Pareto principle describing the “rich get richer” distribution of wealth in society (8–10). Beneﬁts derived from application of MGR patents may fuel further investment

Percentage of countries

T

he October 2010 Nagoya conference of the United Nations (UN) Convention on Biological Diversity (CBD) saw establishment of the protocol for improved access to genetic resources and fair and equitable sharing of beneﬁts arising from their utilization (1). This allows effective implementation of provisions in Article 15 of CBD regulating access to genetic resources through mutual agreements between countries of origin of resources and those acquiring them. Yet the principle of sovereign rights of states underlying the CBD does not apply to Marine Genetic Resources (MGRs) in Areas Beyond National Jurisdiction (ABNJs), international waters encompassing 65% of the ocean; thus, no consensus could be reached to include them in this new protocol. Although recent CBD efforts tackled terrestrial genetic resources and those distributed in Exclusive Economic Zones (EEZs), where states holds rights over marine resources, the increasing industrial use of MGRs, particularly those extracted from ABNJs, occurs in a legal void because of the lack of an internationally accepted framework to ensure ethical and equitable access, and sharing of beneﬁts (2–6). We describe imbalances in ownership of patent claims on MGRs and propose steps toward addressing gaps in governance.

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POLICYFORUM

Prospects

The governance framework for MGRs in ABNJs will require a multifaceted approach to improve coordination of the protection of biodiversity and of intellectual property (IP) regimes to promote equity. Of the genes associated with WIPO patents, 17% are of unknown taxonomic origin, and almost none of the patent claims examined disclosed the geographic origin of material. Although states compromised in promoting establishment of sharing agreements under CBD, this is not a legally binding agreement and so does not imply that companies will necessarily comply. A sustainable CBD regime of access and benefit-sharing of genetic resources, terrestrial or marine, would proﬁt from complementary efforts from the WTO to require geographic and taxonomic origin of resources associated with a patent under

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TRIPS, allowing identiﬁcation of the country (or countries) or geographical areas of origin of the genes involved. This would support application of Article 15 of CBD and the associated Nagoya Protocol for sharing between countries at the origin of resources and those exploiting them. This may also help ease constraints on bioprospecting for academic and environmental research that stem from national laws (e.g., Brazil) (17) protecting against “biopiracy” of resources. Addressing inequality in exploitation of MGRs requires an international consensus over MGR status. The UN Universal Declaration on the Human Genome and Human Rights recognized that the human genome is a common heritage of humankind. Yet this has not prevented the patenting of human genes or given special status for sharing of beneﬁts from those patents (18). But within the UNCLOS, there is speciﬁc meaning and signiﬁcance of common heritage applied to mineral resources of the seabed. If this meaning could be applied to MGRs, they, too, could be managed by the International Seabed Authority. Alternatively, a new authority could be established that would beneﬁt from the joint expertise of the CBD regarding conservation measures and access and beneﬁtsharing protocols and from the International Seabed Authority large-scale management of resources in vast areas beyond national jurisdictions, as well as the promotion of knowledge transfer. MGRs should be regulated by an internationally negotiated regime that includes payment of fees to a common fund when exploiting resources, or any option promoting access and beneﬁt-sharing. Such a regime may encompass other genetic resources, as inequality in the appropriation of biological resources through gene patents is not unique to MGRs but applies also to human genes and cultivars as well. Recent court cases in North America and the UK (19) involving human genes have challenged the legal basis for patenting genes that exist in nature. Also, patenting has been claimed to discourage rather than promote scientiﬁc progress (16, 20). Solutions are emerging, involving patent pools (21–23) to facilitate access to new technologies and more equitable use and beneﬁts from IP rights. The idea of licensing all patents in a pool collectively, and sharing risks and royalties, such as was recently applied in HIV research (23), may save time and money for both claimers and users, and facilitate access to the information. A patent pool for genetic resources managed by a UN authority could ensure fair reward of research efforts and equitable shar-

ing of beneﬁts derived from resources that we believe should be considered a common good. In turn, coordination of instruments required to address governance of MGRs may inspire solutions for IP and access and beneﬁt-sharing around other gene patenting, including human ones. We must pursue a framework in which exploration of marine life, and the biotechnological potentials it contains, serve to improve the lives of all humans rather than generate wealth for a few. References and Notes

1. CBD, Nagoya Protocol, COP 10 Decision X/1, Access to genetic resources and the fair and equitable sharing of beneﬁts arising from their utilization, CBD, Nagoya, Japan, 2010; www.cbd.int/decision/cop/?id=12267. 2. J. M. Arrieta, S. Arnaud-Haond, C. M. Duarte, Proc. Natl. Acad. Sci. U.S.A. 107, 18318 (2010). 3. S. Arico, C. Salpin, Bioprospecting of Genetic Resources in the Deep Seabed: Scientiﬁc, Legal and Policy Aspects (United Nations University–Institute of Advanced Studies, Yokohama, Japan, 2005). 4. Reports on the work of the United Nations Open-Ended Informal Consultative Process on Oceans and the Law of the Sea at its eight and ninth meetings (UN, New York, 2007 and 2008); www.un.org/Depts/los/consultative_process/consultative_process.htm. 5. D. Leary, M. Vierros, G. Hamon, S. Arico, C. Monagle, Mar. Policy 33, 183 (2009). 6. K. E. Zewers, Loyola Univ. Chicago Intl Law Rev. 5, 151 (2007). 7. Materials and methods are available as supporting material on Science Online. 8. A. Dra˘gulescu, V. M. Yakovenko, Physica A 299, 213 (2001). 9. M. E. J. Newman, Contemp. Phys. 46, 323 (2005). 10. M.-B. Hu, R. Jiang, Q. S. Wu, Y. H. Wu, Eur. Phys. J. B 53, 273 (2006). 11. G. M. Grossman, E. Helpman, J. Econ. Perspect. 8, 23 (1994). 12. P. M. Romer, J. Polit. Econ. 98, (S5), S71 (1990). 13. Douglas Westwood Ltd., Marine Industries Global Market Analysis (Marine Foresight Series No. 1, Marine Institute, Galway, Ireland, 2005). 14. R. J. Blaustein, Bioscience 60, 408 (2010). 15. T. F. Molinski, D. S. Dalisay, S. L. Lievens, J. P. Saludes, Nat. Rev. Drug Discov. 8, 69 (2009). 16. C. Garrison, KEStudies 1, 1 (2007); www.kestudies.org/ ojs/index.php/kes/article/view/21/38. 17. S. I. Martinez, S. Biber-Klemm, Curr. Opin. Environ. Sustain. 2, 1 (2010). 18. P. N. Ossorio, J. Law Med. Ethics 35, 425 (2007). 19. M. Francisco, Nat. Biotechnol. 28, 300 (2010). 20. T. Editors, Biotechniques 48, 347 (2010). 21. J. Clark et al., Patent Pools: A Solution to the Problem of Access in Biotechnology Patents? (U.S. Patent and Trademark Ofﬁce, Washington, DC, 2000); www.uspto.gov/ web/ofﬁces/pac/dapp/opla/patentpool.pdf. 22. T. Caulﬁeld, E. Einsiedel, J. F. Merz, D. Nicol, Nat. Biotechnol. 24, 1352 (2006). 23. J. Bermudez, E. ’t Hoen, Open AIDS J. 4, 37 (2010). 24. This is a contribution to the Malaspina 2010 project, funded by the CONSOLIDER-Ingenio 2010 program of the Spanish Ministry of Science and Technology, the DIVERSITAS program, and the European Union Framework Programme 7 Hermione project. We thank S. Teixeira, E. Jarmache, P. Boudry, B. Guilloux, K. Gjerde, J. Rochette, and Institut du développement durable et des relations internationals (IDDRI).

Supporting Online Material

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production with an annual market value of about $150 million (5). The scope for bioprospecting of MGRs surpasses national jurisdictions, a situation that does not apply to terrestrial resources. Frameworks in place for regulating the use of genetic resources (e.g., the UN Food and Agriculture Organization and CBD) were agreed on in the context of sovereign rights of states and therefore apply only to land areas and EEZs. The International Seabed Authority, a body within the UN Convention on the Law of the Sea (UNCLOS), manages activities linked to mineral resources in the seabed and subsoil in ABNJs. It is not clear why parties’ agreement on mineral resource governance under UNCLOS was not extended to a similar framework for governance of genetic resources. Thus, lacking regulation, MGRs in ABJNs are accessed on a “ﬁrst-come, ﬁrstserved” basis (2, 5, 14). The applications that MGRs offer to beneﬁt humankind (2, 5, 15, 16) emphasize the need for an equitable solution. The issues of conservation and fair exploitation of oceans are increasingly present on the UN agenda to progress toward a governance framework for biodiversity in ABNJs (4). Although nations recognize the urgency of facilitating protection, the process is stalled by the lack of agreement regarding the status and governance of MGRs (4). One group of nations, including developing countries, proposes that MGRs in international waters should be considered common heritage of humankind, whereas another wishes to maintain the status quo of “freedom” of exploitation. These positions emerged in Nagoya where some states’ proposal to include ABNJs was not retained in the ﬁnal protocol.

PERSPECTIVES DEVELOPMENT

A New Focus on RNA in the Lens

A protein forms granules with RNA to control gene expression and mammalian eye lens development.

Melinda K. Duncan

CREDIT: M. K. DUNCAN AND A. ALDOSSARY

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igh-resolution vision proteins that are not translated Lens epithelium requires the focus of light into proteins until the onset of onto cells that translate this ﬁber cell differentiation (13), information into a form that can be which is also when the onset of understood by the brain. Land verTdrd7 expression occurs. Furtebrates, including humans, accomther, if βB2 crystallin mRNA plish the task of light refraction (which produces 14% of the through the use of a curved transtotal soluble protein of young parent window to the environment, human lens ﬁbers) is ectopithe cornea, whereas further focus cally expressed in embryLens fibers is accomplished by a transparent onic lens ﬁber cells, it is not but cellular tissue internal to the translated efficiently ( 14). All clear. (Left) The vertebrate eye lens is a highly transparent tissue (cow lens eye, the lens (1). The lens must have is shown). (Right) It is composed of two cell types, the lens epithelium and lens Thus, another possible role a very high refractive index to be ﬁbers (mouse embryonic lens shown). Fiber cells express high amounts of struc- for TDRD7-containing RNA functional in the aqueous environ- tural proteins with restricted gene expression, including the water channel aqua- granules would be to regument of the eye while also retain- porin 0 (stained in red). DNA is stained in blue. late the translation of ﬁber cell ing its transparency for decades (2). structural proteins. Lachke et Most known human mutations leading to the that result in cataract formation. TDRD7 al. found that reduced TDRD7 expression congenital cloudiness of the lens, known as is a member of a large family of Tudor in a lens epithelial cell line led to statisticataract, occur in genes that encode struc- domain–containing proteins that interact cally signiﬁcant changes in expression level tural proteins of the lens. Mutations in genes with methylated arginine residues on other for 6% of the genes expressed in those cells, encoding transcription factors (such as cMaf, proteins. Many members of the TDRD fam- including some crystallins. Further, lenses Hsf4, and Pitx3) that regulate the expression ily, including TDRD7, are found in RNA from mutant mice lacking TDRD7 expresof these structural genes account for another granules—cytoplasmic complexes of pro- sion also had large numbers of differentially subset of mutations causing human congeni- tein and RNA that regulate gene expres- expressed genes, including several regulatory tal cataracts (3). On page 1571 of this issue, sion by inﬂuencing RNA degradation, RNA genes with known relationships to cataract, Lachke et al. (4) propose that RNA-contain- stability, and the subcellular localization of although further work is needed to elucidate ing granules could regulate both the sub- RNA (9). TDRD7 transcripts and protein the precise mechanism behind this differencellular localization and processing of lens are found in a variety of cells, especially tial mRNA expression. mRNAs that are important for lens transpar- of the male germ line, but TDRD7 expresAlthough RNA granules are found in all ency. This refutes the long-standing dogma sion is very high in lens ﬁber cells and may eukaryotes, until now they were best underthat lens gene expression is largely controlled control the production of extremely high stood to function in the germ cells of mulat the level of transcription (5). amounts of lens structural proteins. ticellular eukaryotes. In particular, TDRD1, The structure of the vertebrate lens Lachke et al. show that TDRD7 is found TDRD4, and TDRD6 are all essential for requires a very high concentration (about 350 in a granular distribution in lens ﬁber cells the assembly of RNA granules in male germ mg/ml) of specialized cytoplasmic proteins, and colocalizes with concentrations of RNA cells and thus spermatogenesis. TDRD1 the crystallins; a specialized intermediate- found in the cytoplasm of these cells. These also is important for repression of retﬁlament cytoskeleton to stabilize lens struc- granules appear molecularly distinct from rotransposons whose activation could damture (6); and cellular membranes with high other known RNA granules, although they age the germ line (10). Lachke et al. show concentrations of both the lens-speciﬁc water sometimes appear to be docked to these other that mutant male mice lacking TDRD7 also channel, aquaporin 0, and lens-preferred granule types, including processing bodies, have defects in spermatogenesis, revealing connexins, which allow the lens to transport which regulate translational repression and a commonality between the RNA granules nutrients and waste products despite its lack mRNA decay (10), as well as Staufen-con- found in somatic and germ cells. However, of blood vessels (7) (see the ﬁgure). Many of taining ribonuclear protein particles, which further work is necessary to show whether the genes encoding these proteins are highly are implicated in RNA transport in neurons TDRD7 has similar or distinct functions in expressed in the lens (between 1 and 4% of (11). Simple RNA diffusion within lens ﬁbers the lens and spermatogenesis. total mRNA), leading to a highly biased tran- would presumably be inefﬁcient because of Overall, Lachke et al. demonstrate that scriptome (8). the relatively long distance between the the vertebrate lens contains several classes Lachke et al. have identiﬁed two inde- lens ﬁber cell nucleus and the ﬁber cell tips of RNA granules and show that TDRD7-conpendently occurring mutations in the human as well as the high concentration of pro- taining granules are crucial for normal lens Tudor domain–containing 7 (TDRD7) gene teins in the ﬁber cell cytoplasm (12). Thus, function. Further, they show that TDRD7and one mutation in the mouse gene Tdrd7 a requirement for a lens-preferred RNA containing granules are a novel class of ribotransport mechanism may not be surprising nuclear protein particle, although they appear in retrospect. Notably, cells of the lens epi- to interact with other known classes, sugDepartment of Biological Sciences, University of Delaware, thelium express mRNAs for lens structural gesting some functional overlap. These ﬁndNewark, DE 19716, USA. E-mail: [email protected]

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PERSPECTIVES These future investigations may provide a new paradigm for the posttranscriptional regulation of gene expression in somatic cells. References and Notes

1. M. F. Land, Contemp. Phys. 29, 435 (1988). 2. B. K. Pierscionek, R. C. Augusteyn, Lens Eye Toxic. Res. 8, 229 (1991). 3. J. F. Hejtmancik, Semin. Cell Dev. Biol. 19, 134 (2008). 4. S. A. Lachke et al., Science 331, 1571 (2011). 5. M. K. Duncan, A. Cvekl, M. Kantorow, J. Piatigorsky, in Development of the Ocular Lens, F. J. Lovicu, M. L. Robinson, Eds. (Cambridge Univ. Press, Cambridge, 2004), pp. 119–150. 6. S. Song et al., J. Clin. Invest. 119, 1837 (2009). 7. P. Donaldson, J. Kistler, R. T. Mathias, News Physiol. Sci. 16, 118 (2001).

8. G. Wistow et al., Mol. Vis. 8, 171 (2002). 9. P. Anderson, N. Kedersha, Nat. Rev. Mol. Cell Biol. 10, 430 (2009). 10. A. L. Arkov, A. Ramos, Trends Cell Biol. 20, 482 (2010). 11. T. Miki et al., Cell Struct. Funct. 30, 51 (2005). 12. P. P. Fagerholm, B. T. Philipson, B. Lindström, Exp. Eye Res. 33, 615 (1981). 13. X. Wang, C. M. Garcia, Y. -B. Shui, D. C. Beebe, Invest. Ophthalmol. Vis. Sci. 45, 3608 (2004). 14. J. R. Taube et al., Transgenic Res. 11, 397 (2002). 15. K. J. Lampi et al., J. Biol. Chem. 272, 2268 (1997). 16. Supported by National Eye Institute grants EY12221 and EY15279 and National Center for Research Resources grants RR027273-01 and RR016472-10. I thank A. Aldossary for providing the image of the mouse lens. 10.1126/science.1204205

GEOCHEMISTRY

Antarctica’s Deep Frozen “Lakes”

Airborne radar surveys performed over East Antarctica reveal massive and widespread bodies of accreted basal ice.

Slawek Tulaczyk and Safﬁa Hossainzadeh

T

he Antarctic ice sheet is made of meteoric ice formed by compression and recrystallization of snow. On page 1592 of this issue, Bell et al. (1) report new radar data that shows that this ostensibly obvious statement is not fully correct. They demonstrate that over a large fraction of East Antarctica the deepest section of the ice sheet contains thick basal accreted ice. This ice did not originate as surface snow but developed when subglacial meltwater was frozen onto the underside of the ice sheet to form frozen analogs of Antarctic subglacial lakes. These accreted ice masses expose the existence of an internal hydrological system, which accomplishes appreciable redistribution of mass and heat within the Antarctic ice sheet. Because basal accreted ice does not develop from surface snow, it does not contain direct records of past climates. Hence, its unexpectedly high abundance in the interior of Antarctica will affect the ongoing search for the oldest ice on Earth. Frozen-on basal ice layers provide, however, a new, exciting archive of spatial and temporal dynamics in subglacial hydrology and microbial life habitats. Accreted basal ice may be softer than regular glacial ice; its widespread existence may make the Antarctic ice sheet more susceptible to changes in velocity and mass balance than current models recognize. Accreted basal ice layers are known from past Antarctic studies, but the newly discovered ones are considerably thicker and more Department of Earth and Planetary Sciences, University of California, Santa Cruz, CA 95064, USA. E-mail: tulaczyk@ pmc.ucsc.edu

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widespread. Boreholes in West Antarctica encountered accreted ice that is just 5 to 15 m thick (2, 3). In contrast, Bell et al. imaged accreted ice that is hundreds of meters thick. Basal conditions favor freezing over approximately half of the area of the Antarctic ice sheet (4) (see the ﬁgure). If the average thickness of Antarctic accreted ice is in the range of 10 m to 100 m, its volume is around 100,000 to 1,000,000 km3, i.e., much larger than the estimated volume of Antarctic subglacial lakes, 10,000 km3 (5). It may even exceed the volume of all glaciers on Earth outside of the two polar ice sheets, 180,000

km3 (6). If the amount of frozen-on subglacial water estimated above would drain to the ocean, the global sea level would be higher by tens of centimeters to a few meters. Discovery of large accreted ice masses is unexpected because glaciological models assume that the direction of subglacial water ﬂow follows the ice surface slope. Under this assumption, models predict that subglacial water escapes toward the ocean (4). Regions of basal freezing are predicted to be dry, with basal temperatures below the freezing point of water (4). The growth of thick accreted ice requires an internally draining hydro5 4 3 2 1 0 –1 –2 –3 –4 –5

Mapping Antarctica. Estimated Antarctic basal melting and freezing rates in mm yr–1. Melting is positive and shown in warm colors; freezing is negative and in cold colors. Values higher/lower than ±5 mm yr–1 have been truncated. Calculations were performed using methodology analogous to the one described in (4) and publicly available data sets for ice sheet modeling (http://websrv.cs.umt.edu/isis/index.php/Present_Day_Antarctica).

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ings open up a new ﬁeld of investigation that will need to elucidate both the physiological and biochemical functions that RNA granules control in the lens. Such functions could include the selective translation of mRNAs for structural genes to facilitate the production of 10% or more of the vertebrate lens proteome from mRNA produced by a single-copy gene (15), the transport of mRNAs through the dense lens ﬁber cell cytoplasm to a site of local translation, and ﬁne control of gene expression necessary to set up the refractive index gradient, which corrects for spherical aberration in biological lenses (2).

PERSPECTIVES jecture that Antarctic subglacial aquatic environments hold a large pool of bacterial carbon in diverse microbial environs (5, 9). Whereas Lake Vostok appears to contain relatively fresh water, solute exclusion during freezeon of accreted ice may generate more saline water bodies and drive geochemical heterogeneity of subglacial microbial habitats (10, 11). Basal ice represents a key mechanical boundary layer controlling the rate of ice sheet motion over geologic substrata. Most ice deformation happens just above the base of a polar ice sheet, either as a result of laminar shear or in association with basal ice sliding. Much research is currently focused on improving ice sheet models to reﬁne predictions of future sea level changes (6). These models typically use only rheological parameters of meteoric ice. However, basal accreted ice may be considerably weaker than meteoric ice due to its higher content of water, sediments, and ionic impurities (12). Softer ice deforms faster and will yield greater changes in ice velocity than stiffer ice in response to the same stress change. Hence, the widespread presence of thick, accreted basal ice layers may make the Antarctic ice sheet more susceptible to rapid changes in geometry than previously thought. The discovery of thick, widespread accreted ice layers changes in fundamental ways our understanding of the Antarctic ice sheet. Further mapping and modeling of these

ice bodies is necessary to aid the ongoing search for the oldest ice on Earth. New models of subglacial water generation, ﬂow, and freezing will have to be developed to account for this large internal mass and heat redistribution within the ice sheet. Future coring and sampling of Antarctica’s deep, frozen “lakes” will unlock a new archive of spatial and temporal changes in deep Antarctic microbial habitats and water drainage systems. Ice sheet models will explore the potential impact of soft basal ice on the sensitivity of ice sheet mass balance to climate changes. References

1. R. E. Bell et al., Science 331, 1592 (2011); 10.1126/science.1200109. 2. A. J. Gow, S. Epstein, W. Sheehy, J. Glaciol. 23, 185 (1979). 3. P. Christoffersen, S. Tulaczyk, A. Behar, J. Geophys. Res. 115, F03034 (2010); 10.1029/2009JF001430. 4. F. Pattyn, Earth Planet. Sci. Lett. 295, 451 (2010). 5. J. C. Priscu et al., in Polar Limnology, W. Vincent, J. Laybourn-Parry, Eds. (Oxford Univ. Press, New York, 2008), pp. 119–135. 6. I. Allison, R. B. Alley, H. A. Fricker, R. C. Thomas, R. C. Warner, Antarct. Sci. 21, 413 (2009). 7. J. Severinghaus, E. W. Wolff, E. J. Brook, Eos Trans. AGU 91, 357 (2010). 8. B. C. Christner et al., Limnol. Oceanogr. 51, 2485 (2006). 9. B. Lanoil et al., Environ. Microbiol. 11, 609 (2009). 10. J. A. Mikucki et al., Science 324, 397 (2009). 11. M. Skidmore, M. Tranter, S. Tulaczyk, B. Lanoil, Hydrol. Process. 24, 517 (2010). 12. D. Cohen, J. Glaciol. 46, 611 (2000).

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logical system operating over thousands of years or longer (1). This system transfers subglacial water from areas of basal melting to areas of basal freezing. The resulting internal mass and heat redistribution is currently not accounted for by ice sheet models. The lowermost part of the Antarctic ice sheet is of particular importance to ice core scientists searching for new drill sites with climate records beyond the recently achieved ~800,000 years ago (7). Simulations predict that meteoric ice that originated as surface snow ~1 million years ago is now in the lowermost tens of meters of the ice sheet (4). Prior to the work of Bell et al., their study area, the Gamburtsev Mountains, represented one of the prime targets for ﬁnding the oldest ice on Earth. However, the search for ancient climate records in ice will be complicated wherever the accreted basal ice is tens of meters thick. The Antarctic accreted ice layers are an attractive target for scientiﬁc exploration—as illustrated by the scientiﬁc insights into biology, hydrology, and geochemistry of subglacial Lake Vostok gained from the analyses of accreted lake ice found in the Vostok ice core (8). Even though subglacial Lake Vostok has yet to be penetrated and explored, its potential as a microbial habitat is supported by the fact that accreted lake ice samples have bacterial concentrations up to seven times as high as the overlying meteoric ice (8). This evidence provides also the key underpinning for the con-

10.1126/science.1202888

PALEONTOLOGY

On Dental Occlusion and Saber Teeth

An early mammal relative from Brazil offers insight into the early evolution of herbivory.

Jörg Fröbisch

S

tudies of fossil vertebrates belonging to the group Synapsida are central to understanding mammalian origins. Synapsida includes mammals and is one of the two major clades of amniotes (all fully terrestrial vertebrates). The other is Reptilia, which includes modern turtles, snakes, lizards, crocodiles, and birds. The therapsids, one major group of nonmammalian synapsids (historically but erroneously known as “mammal-like reptiles”) have been particularly important to understanding the acquiMuseum für Naturkunde–Leibniz-Institut für Evolutionsund Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Invalidenstrasse 43, 10115 Berlin, Germany. E-mail: [email protected]

sition of mammalian characteristics. One of the key features within the evolutionary history of synapsids is the morphological differentation of their dentition (teeth) over time (1). On page 1603 of this issue, Cisneros et al. (2) describe a new therapsid fossil from South America, Tiarajudens eccentricus, which displays a unique dentition, including broad chewing teeth on the palate and a pair of extremely long saber canines. The discovery provides novel insights into early tooth differentiation in synapsids and into the evolution of herbivory (plant eating) and its accompanying complex social interactions. Nonmammalian synapsids are important to our understanding of the evolution of ter-

restrial ecosystems. Numerous clades of nowextinct synapsids dominated life on land during most of the late Paleozoic and early Mesozoic eras. Although several synapsid clades (Anomodontia, Therocephalia, and Cynodontia) survived the most severe extinction event in Earth’s history at the end of the Permian, only nonmammalian cynodonts flourished until the Cretaceous. This group gave rise to mammals near the Triassic-Jurassic boundary (3). In the Late Paleozoic, a drastic faunal shift occurred in the terrestrial realm. A Late Carboniferous and Early Permian fauna dominated by “pelycosaurs,” a basal group of synapsids, gave way to therapsid-dominated faunas in the Middle and Late Permian. This

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Neogene

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Placentalia

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Non-mammalian cynodonts

Therocephalia

Gorgonopsia

Anomodontia

Dinocephalia

Biarmosuchia

Edaphosauridae

Ophiacodontidae

Varanopidae

Caseidae*

Eothyrididae

Reptilia

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Marsupialia

Monotremata

Paleogene

Sphenacodontidae

Mesozoic

Jurassic Permian Carboniferous

Paleozoic

Triassic

Millions of years (age)

Cretaceous

Cenozoic

shift was accompanied by a major change in ecosystem structure. Early 23.0 terrestrial vertebrate ecosystems primarily consisted of insectivorous and carnivorous forms with few herbi65.5 vores. In contrast, Late Permian ecosystems display the earliest evidence Late for a modern pattern of trophic inter99.6 actions, with large numbers of herbivores supporting a relatively small Early number of top predators (4). Over time, therapsids show an 145.5 increasing development of mamLate 161.2 malian characters. These include the Middle evolution of the mammalian middle 175.6 ear, a bony secondary palate, and a Early mammalian “phalangeal formula” 199.6 (the number of phalangeal bones Late in each digit). They also develop 228.0 a more upright posture in the fore Middle 245.0 and hind limbs and begin to explore Early 251.0 new habitats (5–7). Various clades Late 260.4 Middle of synapsids, such as anomodonts 270.6 during most of the Late Permian Early and Early Triassic, independently 299.0 evolved a herbivorous mode of life Late and often represented the major pri318.1 mary consumers among vertebrates Early Synapsida of their times (8). Tiarajudens eccentricus belongs Dental occlusion and saber teeth in synapsids. Green and red boxes indicate temporal ranges of major subgroups to Anomodontia, the most abunwithin Synapsida, and connecting lines describe their evolutionary relationships. Carnivorous forms in red; herbivodant and speciose clade among rous forms in green. Subgroups in red and green include both herbivorous and carnivorous forms. Herbivory is genernonmammalian synapsids. Anomo- ally associated with dental occlusion, except in caseids (asterisk). Saber teeth depict the presence of enlarged canines donts were the major herbivores of (including saber teeth, tusks, and simply enlarged canines). Tiarajudens eccentricus (from Brazil) is the only herbivore the Late Permian and Early Triassic. with saber teeth in the Paleozoic era. They achieved a cosmopolitan distribution, with specimens known from every Anomocephaloidea stands in contrast to the beaked dicynodonts, Tiarajudens shows that present-day continent, reﬂecting their great venyukovioids, a well-known clade of basal anomodonts displayed by far the most dispataxonomic diversity (9, 10). This is mirrored anomodonts that lived on the northern super- rate feeding strategies of any group of Paleozoic herbivores. by an enormous morphological disparity; continent of Laurasia. The evolution of saber teeth is comparaThe early evolution of dental occlusion anomodonts included small fossorial (molelike burrowing) forms, large browsing and (the contact between upper and lower teeth) bly rare among tetrapods and exceedingly “grazing” forms, and semiaquatic (amphib- and saber canines in Tiarajudens is of special uncommon in herbivorous forms (see the ious) and arboreal (tree-climbing) body interest. The evolution of herbivory is often ﬁgure). The presence and extent of the saber plans (11–13). This diversity is unparalleled accompanied by dental occlusion (14) (see teeth in the Brazilian anomodont is extraordiby any other clade of Permian-Triassic ter- the ﬁgure). In fact, the evolution of high-ﬁber nary, in particular when considering its paralrestrial tetrapods. In addition, anomodonts herbivory and dental occlusion in basal ano- lel evolution to the continuously growing tusk were among the first synapsids to display modonts has previously been documented in displayed by a group of derived anomodonts, an increasing development of mammalian the slightly younger and specialized climber the dicynodonts (16). In fact, the tusks of the characters. Indeed, “mammalian characters” Suminia (15). However, this close relative of similarly herbivorous dicynodonts evolved could instead be termed “anomodontian Tiarajudens lacks any enlarged canines and even before the saber teeth of the Brazilcharacters.” shows dental occlusion between its marginal ian form, and their behavioral implications The discovery of Tiarajudens, in Middle dentition in the upper and lower jaws. There- have been discussed in detail for the sexually Permian deposits in Brazil, sheds new light fore, the degree of heterodonty (tooth differ- dimorphic Diictodon (17). on the early diversiﬁcation of therapsids and entiation) in Tiarajudens is remarkable. In These ﬁndings raise a question: When is particularly anomodonts. Tiarajudens is of particular, the transversely expanded palatal a saber tooth a saber tooth, and when is it a particular importance because of its unique, teeth on the pterygoid and ectopterygoid are tusk or simply an enlarged canine? The existspecialized dentition. Also, it seems to belong unique among synapsids. Together with other ing literature is quite imprecise, but saber to a new aberrant clade of basal anomodonts, recently discovered basal anomodonts, such teeth tend to be laterally compressed, whereas called Anomocephaloidea, that lived on as Suminia (from Russia) and Biseridens tusks tend to be rather round in cross section the southern supercontinent of Gondwana. (from China), as well as the highly successful and continuously growing, such as in modern

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PERSPECTIVES history of synapsids. Nonetheless, future research applying integrative and quantitative approaches to the study of herbivory will be needed to further investigate its signiﬁcance for the evolution of terrestrial vertebrate ecosystems. References

1. B. Peyer, Comparative Odontology (University of Chicago Press, Chicago, 1968). 2. J. C. Cisneros, F. Abdala, B. S. Rubidge, P. C. DentzienDias, A. de Oliveira Bueno, Science 331, 1603 (2011). 3. T. S. Kemp, The Origin and Evolution of Mammals (Oxford Univ. Press, Oxford, 2005). 4. E. C. Olson, Ecology 47, 291 (1966). 5. J. A. Hopson, J. Vertebr. Paleontol. 15, 615 (1995). 6. Z.-X. Luo, Nature 450, 1011 (2007). 7. C. A. Sidor, J. A. Hopson, Paleobiology 24, 254 (1998).

8. H.-D. Sues, R. R. Reisz, Trends Ecol. Evol. 13, 141 (1998). 9. J. Fröbisch, PLoS ONE 3, e3733 (2008). 10. J. Fröbisch, Earth Sci. Rev. 95, 119 (2009). 10.1371/ journal.pone.0003733. 11. C. B. Cox, in Studies in Vertebrate Evolution, K. A. Joysey, T. S. Kemp, Eds. (Oliver and Boyd, Edinburgh, 1972), pp. 173–189. 12. J. Fröbisch, R. R. Reisz, Proc. R. Soc. B-Biol. Sci. 276, 3611 (2009). 13. S. Ray, A. Chinsamy, S. Bandyopadhyay, Palaeontology 48, 1169 (2005). 14. R. R. Reisz, J. Exp. Zool. B Mol. Dev. Evol. 306B, 261 (2006). 15. N. Rybczynski, R. R. Reisz, Nature 411, 684 (2001). 16. J. Fröbisch, R. R. Reisz, J. Vertebr. Paleontol. 28, 770 (2008). 17. C. Sullivan et al., Proc. Biol. Sci. 270, 173 (2003). 10.1126/science.1204206

MATERIALS SCIENCE

Electronic Bonding Revealed by Electron Diffraction

A new electron diffraction method reveals a tetrahedral bond network in aluminum that can account for the directional nature of its mechanical properties.

Paul A. Midgley

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hen atoms come together to form a crystal, a redistribution of electron charge creates bonds that govern almost all of the crystal’s physical and chemical properties. Ab initio calculations can provide theoretical determination of the bonding charge density, but experimental veriﬁcation can be fraught with difficulty because the change in the total charge density, as measured via diffraction experiments, is very small. For example, in aluminum (Al), most of the electrons are highly delocalized and Department of Materials Science and Metallurgy, University of Cambridge, Pembroke Street, Cambridge CB2 3QZ, UK. E-mail: [email protected]

A

form a free electron gas, but some of the electron density forms highly directional bonds. On page 1583 of this issue, Nakashima et al. (1) demonstrate the use of ultrasensitive convergent-beam electron diffraction (CBED) to map the bonding charge density of Al to an unrivaled accuracy. These results lead to a greater understanding of this metal’s mechanical properties. For many decades, efforts have been made to determine bonding charge density with both x-ray and electron diffraction. Crystallographic studies measure the pattern of different intensities of the scattered reﬂections. These patterns are modeled by determining structure factors, which sum up the individual

B e

scattering contributions of each atom that is positioned in the unit cell, the repeating block within the crystal. For x-rays, the structure factors are associated with the electron charge density; for electrons, they are associated with the electrostatic potential, which can yield the charge density via Poisson’s equation. The charged nature of the electron ensures that the interaction between the electron and the crystal is far stronger than the equivalent x-ray interaction (by a factor of 100 to 1000), and that charge-density effects are more likely to be observed. The stronger interaction means that electrons scatter many times before exiting the crystal. Unlike most x-ray diffraction data, electron diffraction patterns

C

_

Specimen

o t

002

0 0-2 0 0 0 1 -1 -1 2 -2-2 1 -1 1 2 -2 0 2 -2 2

Making a difference with diffraction. (A) Schematic representation of convergent-beam electron diffraction (CBED) showing the formation of patterncontaining disks of different diffraction beams (the beam order is given by the three-digit Miller indices). (B) An experimental CBED pattern from aluminum

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determined by Nakashima et al. (C) The corresponding bonding charge density they determined using a differential form of the CBED pattern, along with an illustration of the octahedral (o) and tetrahedral (t) sites in the lattice (the latter is the bond position that matches the pattern of excess charge density).

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elephants, wild boars, and walruses. Finally, the distinction of saber teeth and tusks from ordinary large canines appears to be vague and primarily based on length. Tiarajudens seems to further blur this distinction, since anomodonts evolved both approaches (saber teeth and tusks) to enlarge their canines, even though they might have had similar functions, such as deterring predators and intraspeciﬁc display or combat (2, 17). The discovery of extraordinary fossils such as Tiarajudens eccentricus provides new insights into the dental diversification and early evolution of herbivory in tetropods and the complex evolutionary

PERSPECTIVES electrons that have lost only a small amount of energy (on the order of tens of millielectron volts) associated with scattering from lattice vibrations (phonons). Such “thermal diffuse scattering” is seen over the whole diffraction pattern, predominantly as a slowly varying background. The differential method removes this background and allows elastic simulations to be matched to a remarkably accurate level. Moreover, by using this differential method, Nakashima and Muddle had previously shown that in many cases, energy ﬁltering is not needed at all (9). In the present work, Nakashima et al. recorded more than 100 unﬁltered CBED patterns comprising ~106 data points to determine 14 structure factors. The result of this enormous overdetermination is high accuracy and conﬁdence in the structure factor values obtained and in the uniqueness of the solution. For the lowest-order structure factor in Al, this accuracy approaches 1 part in 1000 (better than equivalent x-ray data by a factor of ~10). Using these highly accurate structure factors, they found the excess electron charge (the “bond”) to be in the tetrahedral “hole” between the Al atoms (see the ﬁgure, panel C). Much of the previous work, both theory and experiment, had indicated that the bond would be (at least in part) octahedral in nature. Nakashima et al. show how this tetrahedral

bond can be related to the mechanical properties of Al by equating the “shape” of the bond to the anisotropy of the elastic constants. In studies of bonding charge, electron experiments have two advantages over x-rays: sensitivity and spatial resolution. Nakashima et al. have illustrated beautifully that highly accurate structure factors can be obtained from volumes of material much smaller than is possible even with synchrotron x-rays. This powerful combination offers the possibility of mapping bonding characteristics across heterogeneous samples (e.g., composites or quantum wells) that may not be achievable with any other method. References

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can be interpreted fully only by a complex dynamical theory that takes multiple scattering events into account. When determining an unknown structure, the effects of dynamical scattering can be minimized by using, for example, only the high-order reflections at high scattering angles (2), very thin or weakly scattering crystals (3), or special geometries, as seen with precession electron diffraction (4). However, once a structure is known, dynamical effects can yield diffraction data that are very sensitive to changes in the electrostatic scattering potential, and the lowestorder reﬂections in the diffraction pattern are the ones most sensitive to the bonding charge density. The wavelength of the diffracting electron depends on the energy imparted by an accelerating voltage, and at so-called “critical voltages,” distinct diffraction features (intensity minima in diffracted disks) persist at every crystal thickness (5). By matching these data to simulations, or through analytical methods, the ratios of structure factor magnitudes can be determined. However, for many materials, such critical voltages lie beyond the usable range of most microscopes. With the advent of greater computer power, best-ﬁt CBED pattern matching that can use full dynamical theory (rather than making assumptions about scattering) has become the preferred way to determine structure factors. The basic geometry of a CBED pattern (see the ﬁgure, panel A) focuses electrons to a spot on the sample perhaps only a few nanometers wide. The CBED pattern is composed of reﬂections in the form of disks, the detail within each being a “map” of diffracted intensity as a function of beam orientation (see the ﬁgure, panel B). Dynamical simulations of the electrons in a CBED pattern that undergo elastic scattering (which preserves their energy) are relatively straightforward, but inelastically scattered electrons that have lost energy when scattered are also present and are much harder to incorporate accurately into simulations. Energy ﬁlters can be used to remove inelastic scattering from experimental patterns, and experiments in the 1990s yielded some success in determining the bonding charge densities of semiconductors (6), oxides (7), and metals (8). Nakashima et al. introduced a new approach in which a difference CBED pattern (derived with respect to orientation) is constructed and a dynamical simulation ﬁtted to that difference pattern. Commercial energy ﬁlters can remove the majority of inelastic scattering (arising primarily from plasmon excitations), but they cannot filter out the

1. P. N. H. Nakashima, A. E. Smith, J. Etheridge, B. C. Muddle, Science 331, 1583 (2011). 2. R. Vincent, D. Bird, J. W. Steeds, Philos. Mag. A 50, 745 (1984). 3. R. Henderson, P. N. Unwin, Nature 257, 28 (1975). 4. R. Vincent, P. A. Midgley, Ultramicroscopy 53, 271 (1994). 5. D. Watanabe, R. Uyeda, A. Fukuhara, Acta Crystallogr. A24, 580 (1968). 6. P. A. Midgley, M. Saunders, Contemp. Phys. 37, 441 (1996). 7. J. M. Zuo, M. Kim, M. O’Keeffe, J. C. H. Spence, Nature 401, 49 (1999). 8. M. Saunders, A. G. Fox, P. A. Midgley, Acta Crystallogr. A55, 471 (1999). 9. P. N. H. Nakashima, B. C. Muddle, Phys. Rev. B 81, 115135 (2010). 10.1126/science.1203614

CELL BIOLOGY

Selective Insulin Sensitizers Ja Young Kim-Muller and Domenico Accili Can the liver’s insensitivity to insulin in diabetes be overcome with another hormone?

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ype 2 diabetes mellitus and its complications are, with cardiovascular diseases, leading threats to public health in the 21st century. In the United States, type 2 diabetes care accounts for a third of federal health insurance (Medicare) expenditures— nearly half of it to treat associated macrovascular problems (1). The cornerstone of type 2 diabetes is insulin resistance—a decreased sensitivity of tissues and organs, such as the liver, to the metabolic effects of the hormone insulin (the other major cause is failure of the pancreas to produce insulin). Yet, except for thiazolidinediones—whose checkered safety history, troublesome side effects, and regulatory setbacks stiﬂed widespread adopDepartment of Medicine and Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. E-mail: [email protected]

tion by clinicians—treatment options for insulin resistance have generally remained unchanged since the 1940s. Although insulin signaling pathways in cells have been largely deciphered (2), the key mediators of insulin signaling are poor drug targets because they either lack a suitable ligand-binding domain, or are shared with other cellular pathways that regulate cell growth and proliferation. This realization spawned research into “alternative pathways” that control insulin resistance. On page 1621 of this issue, Kir et al. (3) find that human fibroblast growth factor 19 (FGF19) can boost certain effects of insulin on the mammalian liver, raising interest and questions about possible therapies involving this molecule. After food intake, blood glucose concentration increases, triggering the secretion of insulin. Glucose is taken up by liver cells

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PERSPECTIVES

Pancreas

Intestine

Liver

ing of the effects of insulin on VLDL secretion. Although in fasting conditions insulin appears to drive VLDL secretion from the liver, especially in obese individuals with insulin resistance, it also inhibits this process in the postprandial state, independent of its ability to shunt free fatty acids away from liver into adipocytes (6). And in isolated liver cells, insulin can suppress processing, maturation, and degradation of the main VLDL protein, apolipoprotein B (6). Hence, the net effect of an insulin sensitizer on VLDL secretion by the liver cannot be predicted from its ability to promote lipogenesis. Although Kir et al. did not analyze hepatic glucose production, FGF19 is likely to be effective in treating its abnormal elevation in the diabetic liver (7). FGF19 may help restore the liver’s ability to prevent excessive glucose release by modulating the relative contributions of glycogen breakdown and de novo glucose production to this process. The clinical future of FGFs is uncertain. Peptide-based diabetes therapeutics, once considered impractical and viewed as an obstacle to initiating insulin therapy (because of the complexity of measuring the correct dose as well as patient aversion to needles), have gained renewed attention from the experience with injectable ago-

nists that mimic the effect of an incretin peptide hormone called glucagon-like peptide–1 (Glp-1), which stimulates insulin release. But FGF19 production is normal in diabetics, raising doubts about the beneﬁts of boosting its actions. A similar concern, however, could have been raised over incretin-based treatments, but hasn’t prevented their rapid adoption. Moreover, FGF19 production falls in response to administration of bile acid absorption inhibitors ( 8), a moderately effective and seemingly safe, if somewhat impractical, antidiabetic and lipid-lowering treatment. These data suggest that the therapeutic beneﬁts of FGF19 may be secondary to its ability to restrict the expanded bile acid pool in diabetics. Notably, Kir et al. draw attention to the crucible of insulin signaling and nutrientactivated nuclear receptors (those that control the transcription of genes involved in glucose and fat metabolism) that likely holds the key to unraveling the paradox of the diabetic liver, in which resistance to the actions of insulin on glucose production seemingly coexists with preserved sensitivity to the lipogenic effects of insulin. The looming threat of young type 2 diabetics coming of age with their cardiovascular comorbidities lends urgency to the search for remedies. 1. 2. 3. 4. 5. 6.

References

J. J. Caro et al., Diabetes Care 25, 476 (2002). D. Accili, Diabetes 53, 1633 (2004). S. Kir et al., Science 331, 1621 (2011). N. Itoh, Cell Tissue Res. 342, 1 (2010). R. A. Haeusler, D. Accili, Cell Metab. 8, 7 (2008). M. Adiels, S. O. Olofsson, M. R. Taskinen, J. Borén, Arterioscler. Thromb. Vasc. Biol. 28, 1225 (2008). 7. D. -J. Shin, T. F. Osborne, J. Biol. Chem. 284, 11110 (2009). 8. G. Brufau et al., Hepatology 52, 1455 (2010).

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Integrated pathways in the liver. In response to food intake and an increase in blood glucose concentration, the hormones FGF19 and insulin control hepatic protein synthesis and glycogen production. Insulin also controls liver lipogenesis. In the diabetic liver, the effects of insulin are blocked. Treatment with FGF19 may selectively rescue glycogen and protein production without affecting lipid synthesis.

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(hepatocytes), where insulin then stimulates its conversion into glycogen (as an energy reserve). Insulin also stimulates protein and lipid production in the liver (see the ﬁgure). Made in the small intestine in response to food intake, FGF19 acts on the liver to inhibit the synthesis of bile acids, thereby regulating cholesterol and triglyceride levels in the plasma. It also promotes energy expenditure, by acting on the central nervous system and by stimulating fat oxidation (4). Kir et al. reveal that like insulin, FGF19 also regulates hepatic glycogen and protein buildup in the mouse liver, through seemingly independent and possibly synergistic signaling pathways with insulin. Therapeutically, this could be beneficial to the insulininsensitive diabetic liver. However, there are two key aspects of hepatic insulin resistance that must be considered. The diabetic liver overproduces glucose and atherogenic lipoproteins [very-low-density lipoproteins (VLDLs) and small dense LDLs, resulting in lower concentrations of high-density lipoproteins (HDLs)], which cause microvascular and macrovascular complications, respectively. Understanding the linkage between these two conditions would likely provide insight into potential treatments for type 2 diabetes. A leading view is that increased hepatic glucose production stems from impaired insulin action, whereas increased lipoprotein secretion stems from the preserved ability of insulin to promote lipogenesis (5). If so, redressing this balance to boost some actions of insulin, while curtailing others, would require identiﬁcation of selective agonists of insulin signaling (similar to selective agonists developed for G protein–coupled receptors or nuclear receptors in other diseases). FGF19 could be viewed as one such agonist, in view of the demonstration by Kir et al. that it stimulates hepatic glycogen and protein synthesis without promoting lipogenesis. But this conclusion remains conjectural to the extent that it attributes the increase in VLDL secretion in diabetes to increased de novo lipogenesis, which is usually a minor contributor to the process of lipoprotein assembly and secretion (6). In fact, there remain yawning gaps in our understand-

10.1126/science.1204504

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PERSPECTIVES ARCHAEOLOGY

An Earlier Acheulian Arrival in South Asia

Early stone tools found in India alter our view of how a key technology spread from Africa.

Robin Dennell

Millions of years ago Millions of years ago

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outh Asia has rarely Spreading technology. (Top) AccordEurope Levant India China featured in recent ing to the consensus view, Acheulian bifacial technology originated in East discussions of paleo0.5 Africa around 1.6 Ma and dispersed by anthropology. On page the movement of hominins (or ideas) at 1596 of this issue, howleast twice into the Levant, at ~1.4 Ma ever, Pappu et al. (1) report Bose GBY and ~0.8 Ma. It did not disperse into a breakthrough, showEurope and South Asia until 0.5 to 0.6 1.0 ing that an assemblage Ma. In this scenario, the Acheulian-like of Acheulian stone tools bifaces from Bose, China (dated to ~0.8 found in India can be dated Ma) are likely to have been an indigto at least 1 million years ‘Ubeidiya enous development. (Bottom) New 1.5 ago (Ma) and are perhaps evidence from Attirampakkam, India, implies that Acheulian bifacial techas ancient as ~1.5 Ma— Africa nology dispersed as far east as India far older than previously shortly after it first appeared in the shown. Acheulian tools are Levant by the movement of hominins the product of a distinctive (or ideas) from either East Africa or the set of tool-making techLevant. This raises the possibility that Europe Levant India China niques that originated in the Acheulian-like bifaces from China Africa and then spread to indicate subsequent dispersal from 0.5 Europe and Asia. The exact India. The European Acheulian now chronology of this spread, appears substantially younger than that ? however, has been a longin Southwest and South Asia. Bose GBY standing puzzle. In India, 1.0 researchers recently dated magnetic ﬁeld, which occurred at a few sites with Acheu0.78 Ma. There was no evidence lian tools to more than 0.6 of two short periods of normal Ma (2, 3), but these dates polarity, known as the Jaramillo ‘Ubeidiya Attirampakkam 1.5 are problematic (4). At the (0.97 to 1.07 Ma) and Olduvai same time, most of the few (1.77 to 1.95 Ma) subchrons. This Africa dates available for Indian means that the deposits and their Acheulian sites were artifacts likely date from 1.07 to obtained years ago through 1.77 Ma. Pappu et al. obtained a method that measures a more precise age estimate by isotopes of thorium and uranium (230Th/234U) ever, as indicated by the discovery of more applying a burial dating technique that mea(5), and there are good reasons to suspect than 3500 stone artifacts, including more sures aluminum and beryllium isotopes that it seriously underestimated their true than 70 Acheulian hand axes and cleav- (26Al/10Be) to six artifacts from layers 6 and ages. Pappu’s team has performed a notable ers, and several hundred retouched ﬂakes 8; a pooled average of the results suggested service in demonstrating unequivocally that and waste ﬂakes. The artifacts were found a probable age of 1.51 ± 0.07 Ma. the South Asian Acheulian extends back into in layers 6 to 8 of trench T8, as deep as 7 How does this new evidence affect our the Early Pleistocene, more than 0.78 Ma. m below the surface. The artifacts are fresh understanding of the South Asian AcheuFirst, the evidence. The site, Attirampak- and appear to be in primary context; there lian? Previously, the general consensus was kam, is in the Kortallaiyar Basin in South is no evidence that they intruded into the that the Indian Acheulian was less than 0.6 India (where, incidentally, researchers dis- clay from overlying deposits. The standards to 0.5 Ma (5) and was thus much younger covered the ﬁrst paleolithic artifacts from of excavation and recording appear to have than that in the Levant (eastern MediterraIndia in 1863). Pappu et al. excavated three been impeccable. The researchers took 49 nean). There, the earliest dates of 1.4 Ma, trenches in a series of waterlain red clays at paleomagnetic measurements from a pro- from ‘Ubeidiya in Israel, probably indicate least 9 m thick. Previous studies had mapped ﬁle of the clays that was 8 m in depth and a dispersal of hominins from Africa (6). A the clays as Cretaceous in age (~145.5 to comprised layers 6 and 8, and all showed a second inﬂux of African immigrants is indi65.6 Ma). They clearly were younger, how- reversed polarity (relative to Earth’s current cated by the discovery of African types of magnetic ﬁeld). This means that the clays cleavers and hand axes at Gesher Benot predate the Brunhes chron, the name given Ya’aqov (GBY), in Israel, dated to 0.78 Ma Department of Archaeology, Shefﬁeld University, Shefﬁeld to the period after the last reversal of Earth’s (7). This evidence implied that the AcheuS1 4ET, UK. E-mail: r.dennell@shefﬁeld.ac.uk

PERSPECTIVES gies from the Levant occurred in two phases, by two paleospecies of hominin: initially by Homo erectus eastward into South Asia shortly after the Acheulian ﬁrst appeared in East Africa, and much later by H. heidelbergensis northward and westward into Europe. This is consistent with other dispersal events from Africa, in that southern Asia was colonized ﬁrst and Europe later. Two obvious examples are the initial expansion of hominins from Africa, in which Europe appears to have been colonized after Asia, and much later, the expansion of Homo sapiens, which colonized Europe ~35,000 years later, several scores of millennia later than South Asia and Australia. The dates from Attirampakkam have three other consequences. The ﬁrst concerns the Acheulian-like bifaces from the Bose Basin, South China, which are dated to ~0.8 Ma (12). Previously, it was difﬁcult to envisage how these tools resulted from the diffusion of ideas or the dispersal of hominins, because the South Asian Acheulian was younger than the Bose bifaces. Although these Chinese bifaces may be an indigenous development (5), the case for diffusion from the west is now strengthened by the dating of Attirampakkam. The second is that the duration of the Acheulian record of India is now more than doubled. This, in turn, means that

the intensity of settlement in South Asia by Acheulian groups now appears to have been much lower, creating gaps in the archaeological record. The extent to which this happened will only become apparent when more Acheulian sites in India are dated (or redated) by state-of-the-art techniques. Finally, this new evidence from Attirampakkam makes it all the more important that we ﬁnd out what type of hominin ﬁrst brought Acheulian artifacts to South Asia. References and Notes

1. S. Pappu et al., Science 331, 1596 (2011). 2. C. Gaillard, S. Mishra, M. Singh, S. Deo, R. Abbas, Quat. Int. 223–224, 234 (2010). 3. K. Paddayya et al., Curr. Sci. 83, 641 (2002). 4. P. R. Chauhan, Quat. Int. 223–224, 248 (2010). 5. R. Dennell, The Palaeolithic Settlement of Asia (Cambridge Univ. Press, Cambridge, 2009). 6. O. Bar-Yosef, N. Goren-Inbar, The Lithic Assemblages of ‘Ubeidiya: A Lower Palaeolithic Site in the Jordan Valley (Hebrew University, Jerusalem, 1993). 7. N. Goren-Inbar et al., Science 289, 944 (2000). 8. W. Roebroeks, J. Hum. Evol. 41, 437 (2001). 9. G. P. Rightmire, in Human Roots: Africa and Asia in the Middle Pleistocene, L. Barham, K. Robson-Brown, Eds. (Western, Bristol, UK, 2001), pp. 123–133. 10. S. Antón, C. C. Swisher, III, Annu. Rev. Anthropol. 33, 271 (2004). 11. G. P. Rightmire, D. Lordkipanidze, A. Vekua, J. Hum. Evol. 50, 115 (2006). 12. H. Yamei et al., Science 287, 1622 (2000).

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lian dispersed eastward toward South Asia only several hundred millennia after it ﬁrst appeared in the Levant. It also implied that the spread of Acheulian bifacial technologies into South Asia was broadly contemporaneous with its ﬁrst appearance in Europe, where the earliest sites date from ~0.5 to 0.6 Ma (8). Some have attributed this expansion of the Acheulian into South Asia and Europe to Homo heidelbergensis. This Middle Pleistocene type of hominin is known mostly from Europe, where it was ﬁrst deﬁned, but is also recognized by some (but not all) researchers at African sites such as Bodo, Ethiopia, and Kabwe, Zambia, and even at some sites in China (9). The new evidence from Attirampakkam invalidates much of this scenario, because the South Asian Acheulian now considerably predates the ﬁrst appearance of H. heidelbergensis. More likely, the Acheulian in South Asia was derived from an earlier population in either East Africa or Southwest Asia. Although we do not know what type of hominin inhabited the Levant or India at 1.5 Ma, it was likely H. erectus sensu lato (i.e., in a general sense), as that species was present in East Africa (10) at that time, and also at Dmanisi, Georgia, at ~1.75 Ma (11). The new evidence from Attirampakkam implies that the expansion of Acheulian bifacial technolo-

10.1126/science.1203806

CELL BIOLOGY

The constriction of a protein helix and the action of a microtubule-severing enzyme trigger the ﬁnal stage of separation during cell division.

A Helix for the Final Cut Camilla Raiborg1,2 and Harald Stenmark1,2

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he ability of cells to divide is essential for the proliferation and development of organisms ranging from bacteria to humans. Despite spectacular progress in knowledge about the molecular dynamics of cell division, one key issue still puzzles scientists: How is the connection between two dividing cells ﬁnally cut (1)? On page 1616 of this issue, Guizetti et al. (2) identify a contractile helix made up of a 17-nm-thick protein ﬁlament that accomplishes abscission between two human cells separating during cell division, in concert with an enzyme that breaks up the bundles of cytoskeletal microtubules that hold the pair together. Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0310 Oslo, Norway. 2Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310 Oslo, Norway. E-mail: [email protected] 1

The separation between two daughter cells that arise from a single dividing cell begins with the formation of a cleavage furrow that uses the reshaping force of the ﬁlamentous cytoskeletal protein actin and actinbased motors to ingress (3). When the cleavage furrow has ingressed to the point at which the two daughter cells are almost separated, the cells remain connected by a thin membrane bridge ﬁlled with microtubules (see the ﬁgure). A microtubule is a 30-nm-thick cytoskeletal element that helps move and position chromosomes, membrane vesicles, and macromolecular complexes during cell division. Therefore, to ﬁnally separate the two daughter cells, both the membrane bridge and its resident microtubules must be severed. Several alternative models have been proposed for such severing, including mechanical rupture or addition of membrane vesicles from within the bridge (1), but ﬁrm evidence for

any particular mechanism in mammalian cell-cell abscission has been lacking. Recent studies have allowed a fresh look at the mechanisms of membrane abscission during cell separation by identifying components of the so-called endosomal sorting complex required for transport (ESCRT) machinery as crucial for this process (4, 5). The ESCRT machinery participates in sorting membrane proteins into intraluminal vesicles of endosomes, the intracellular compartments that receive material internalized from the plasma membrane (6). The machinery consists of four protein complexes called ESCRT0, -I, -II and -III (7). Studies with genetically engineered ESCRT components and model membranes have revealed that ESCRT-0 is involved in cargo sequestration, ESCRT-I and -II in membrane invagination, and ESCRT-III in intraluminal vesicle abscission (8). Under in vitro conditions, and when expressed at

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PERSPECTIVES

ESCRT-I ESCRT-II

Vesicle

Breaking apart. (Top) Daughter cells that arise from cell division are separated by a thin membrane bridge that contains microtubule bundles. A helical ﬁlament, probably consisting of ESCRT III polymers, spans the bridge. Spastin is recruited to the narrowest part of the microtubule bundles (the constriction zone) to sever them. This causes the helix to constrict, which leads to membrane abscission at the same site. (Bottom) The budding of intraluminal vesicles into multivesicular endosomes is a membrane abscission process topologically equivalent to cell division. It is thought that a helical ﬁlament of ESCRT III is also involved.

high levels in cells, certain ESCRT-III complexes oligomerize into ﬁlaments that assume spiral-shaped conformations (9–11). This has spurred the hypothesis that an ESCRT-III spiral is contractile and might form a spring that cuts the vesicle neck from the limiting endosomal membrane, thereby releasing the vesicle into the lumen. Given the topological similarities between intraluminal vesicle abscission and cell-cell abscission, it has been hypothesized that the same machinery might sever the membrane tube connecting daughter cells as well, but evidence for this has been missing so far. Using state-of-the-art light and electron microscopy, Guizetti et al. identiﬁed ESCRTdependent ﬁbers that likely form the core of an abscission machinery. The authors used ﬂuorescently tagged tubulin (the monomeric unit of a microtubule) to study cell division in live human cells and found that bundles of microtubules in the membrane bridge compress to a diameter of about 1 µm before they disassemble on one side of the midbody, an enigmatic bulky structure in the middle of the intercellular bridge. This microtubule disassembly coincided with cell-cell abscission, providing an important clue that localized

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severing of the microtubule bundles is crucial for abscission. Guizetti et al. found that an ATP-consuming enzyme called Spastin, previously shown to bind ESCRT-III and to sever microtubules in the intercellular bridge (12, 13), is highly enriched in the area of microtubule bundle severing. At ultrahigh resolution in three dimensions (using electron tomography), Guizetti et al. further observed that at the site of microtubule disassembly, the intercellular bridge narrows to form a thin stalk, called the constriction zone. Strikingly, a 17-nm-thick ﬁlament formed a membrane-proximal helix spanning the intercellular bridge. This helix may represent ESCRT-III polymers because of its structural characteristics and localization and because its formation requires ESCRT-III. The model that emerges from these observations is that the spiral-shaped ESCRT-III ﬁlament that assembles in the intercellular bridge has a dual function: to recruit Spastin at the appropriate site to sever microtubules, and to constrict, thereby causing the intercellular bridge to narrow sufﬁciently so that abscission occurs. But how is the 17-nm ﬁlament recruited to the intercellular bridge in the ﬁrst place? A

References

1. F. A. Barr, U. Gruneberg, Cell 131, 847 (2007). 2. J. Guizetti et al., Science 331, 1616 (2011); 10.1126/ science.1201847. 3. M. Glotzer, Science 307, 1735 (2005). 4. J. G. Carlton, J. Martin-Serrano, Science 316, 1908 (2007). 5. E. Morita et al., EMBO J. 26, 4215 (2007). 6. D. J. Katzmann, M. Babst, S. D. Emr, Cell 106, 145 (2001). 7. C. Raiborg, H. Stenmark, Nature 458, 445 (2009). 8. T. Wollert, J. H. Hurley, Nature 464, 864 (2010). 9. S. Lata et al., Science 321, 1354 (2008). 10. P. I. Hanson, R. Roth, Y. Lin, J. E. Heuser, J. Cell Biol. 180, 389 (2008). 11. S. Ghazi-Tabatabai et al., Structure 16, 1345 (2008). 12. A. Roll-Mecak, R. D. Vale, Nature 451, 363 (2008). 13. D. Yang et al., Nat. Struct. Mol. Biol. 15, 1278 (2008). 14. M. Fabbro et al., Dev. Cell 9, 477 (2005). 15. A. P. Sagona, H. Stenmark, FEBS Lett. 584, 2652 (2010).

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Cell cytoplasm

10.1126/science.1204208

CREDIT: Y. HAMMOND/SCIENCE

ESCRT-0

centrosomal protein, CEP55, which translocates to the midbody during the late phase of cell division, functions as a scaffold for components of the abscission machinery (14). CEP55 interacts with Alix, an ESCRT-III– binding protein required for cell division (4, 5). Using super-resolution light microscopy, Guizetti et al. found that CEP55 and Alix are conﬁned to the midbody, whereas ESCRT-III is found on the cortical side of the midbody as well as in the constriction zone. CEP55 is recruited to the midbody before Alix and ESCRT-III; thus, ESCRT-III polymers may nucleate from midbodies, anchored by Alix, which in turn is recruited by CEP55. The discovery of a helical ﬁlament represents great progress in the ﬁeld of cell division and may be the ﬁrst demonstration of a natural ESCRT-III polymer. However, deﬁnitive proof that the 17-nm ﬁlament is composed of ESCRT-III proteins will require analysis by immunoelectron microscopy with appropriate antibodies. A key question is how the 17-nm filament is attached to the plasma membrane of the intercellular bridge, which is required to constrict the bridge from inside. And how would helix constriction lead to membrane abscission? At some point the 17-nm ﬁlament itself will have to be severed, and there must be mechanisms controlling this. It will also be important to examine the function of the ESCRT machinery in cell division in vivo, because current analyses have been restricted to cell culture models. The relatively mild cell division phenotypes obtained in such models may well reﬂect incomplete depletion of ESCRT complexes under experimental conditions (4, 5), but the possibility that cells use additional systems for separating from one another cannot be ruled out. Faulty cellcell abscission is associated with cancer (15), providing plenty of motivation to continue exploring the cutting mechanisms.

ESSAY SPORE* SERIES WINNER

Building Botanical Literacy

Online mentors inspire interest in science while engaging students in thinking about plant biology.

Claire Hemingway,1 William Dahl,1 Chris Hauﬂer,2 Carol Stuessy3

CREDITS: (TOP) ANDREW BARNES; (BOTTOM) CLAIRE HEMINGWAY

tinuing U.S. crisis in science literacy, although some underlying causes, such as little exposure to plants in school and preferences for animals, are unique to botany (3). Textbook coverage is biased toward animals, and teachers use animal examples with which they are more familiar. There is also a general human tendency toward “plant blindness”—overlooking their presence in the environment (4). Plants, in their favor, have distinct advantages for generating excitement about science discovery. Inexpensive, easy to keep, and noncontroversial subjects for experimentation, plants are adaptable classroom organisms. At the 2003 Botany Education Forum, Bruce Alberts, then president of the National Academy of Sciences, challenged the Botanical Society of America (BSA) to enhance science classroom experiences. Stakeholder meetings of plant scientists, middle school and high school teachers, and teacher leaders with the National Research Council identified curriculum standards teachers could introduce with plant investigations. Scientiﬁc Inquiry through Plants, a pilot program enabling teachers to extend inquiry learning beyond the classroom through interactive and integrated technology tools, was launched in 2005 (5).

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B

etsy Justus, a doctoral student at Ohio University, has a fan club. It is a team of students from Cheyenne Central High School investigating spore germination in their classroom and posting research information to their team Web page. Betsy has been mentoring her team through online guidance and encouragement, posing questions throughout the inquiry process, and providing insights on what scientists know and how they think. Similarly, Eric Jones, a Florida State Multimedia sharing among students, scientists, and teachers. A University doctoral candi- student-generated photomicrograph of a C-Fern gametophyte docudate, hooked his St. Sebas- ments the team ﬁndings. tian middle school team into an intriguing conversation about their inves- will emerge from breakthroughs in plant bioltigations on pollinator visits to ﬂowers. His ogy (1). Meeting these challenges requires reward was in experiencing his students’ preparing future scientists and science-literate motivation to learn, expressed in statements citizens. To rebuild botanical knowledge that such as, ‘We are sad that our experiments are has been declining across academic, private, over,” and ‘We have developed a whole new and government sectors, strengthening educainterest in ﬂowers!” tion about plants across grade levels is recomBetsy and Eric are among the more than mended (2). This decline is part of the con500 scientists from 14 professional plantrelated organizations volunteering as online mentors and personalizing an inquiry experience for student teams. Plant biology research projects and student dialogue with online mentors about the student-generated research are part of the PlantingScience (www.plantingscience.org) online learning community (see the ﬁrst ﬁgure). PlantingScience makes science experts accessible to secondary school classrooms with the goal of improving understanding of science while fostering an awareness of plants. Plants are essential to our everyday lives, and society faces major food, fuel, and environmental challenges, some of whose solutions Botanical Society of America, St. Louis, MO 63166, USA. Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA. 3Department of Teaching, Learning, and Culture, Texas A&M University, College Station, TX 77843, USA. 1

2

*SPORE, Science Prize for Online Resources in Education; www.sciencemag.org/site/special/spore/. Author for correspondence. E-mail: [email protected]

Workshops for teachers. Teachers in the 2010 Summer Institute discuss celery morphology before beginning team investigations.

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ESSAY

From left to right: Rob Brandt, Claire Hemingway, and William Dahl. Not pictured are Chris Hauﬂer and Carol Stuessy. C. Hemingway, W. Dahl, and C. Stuessy are co-principal investigators on the project. C. Hauﬂer is BSA at-large director for education. R. Brandt is BSA and project information technology manager.

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for using online and classroom discourse and science notebooks as they design an implementation plan for their own students. The “digital generation” is often connected 24 hours, 7 days a week, and digital learning is now on the national education agenda (9). PlantingScience students voluntarily post ﬁndings and communicate with mentors on weekends and evenings. The Web site received 1,628,164 visitors between August 2005 and November 2010. Interest in the model has spread internationally, with a Dutch translation of the Web site managed independently, and Dutch–U.S. collaboration under way. Science and society will benefit from piquing children’s interest in plants at an early age and nurturing their thinking about how science works. The personal connection with an online mentor also holds promise for inspiring individual students. There is power in the collective commitment and expertise of scientist-school partnerships to efﬁciently raise engaging collaborative science learning to a national scale. References and Notes

1. National Research Council, A New Biology for the 21st Century (National Academies Press, Washington, DC, 2009). 2. K. Havens, A. T. Kramer, B. Zorn-Arnold, Assessing Botanical Capacity to Address Grand Challenges in the United States (Botanic Gardens Conservation International, Surrey, UK, 2010); www.bgci.org/usa/bcap. 3. G. Uno, Am. J. Bot. 96, 1753 (2009). 4. J. Wandersee, E. Schlusser, Am. Biol. Teach. 61, 84 (1999). 5. C. Hauﬂer, M. Sundberg, Am. J. Bot. 96, 1751 (2009). 6. American Bryological and Lichenological Society, American Fern Society, American Institute for Biological Sciences, American Phytopathological Society, American Society of Agronomy, American Society of Plant Taxonomists, Canadian Society of Botany Crop Science Society of America, Ecological Society of America, Society for Economic Botany, Society for the Study of Evolution, Soil Science Society of America, Wisconsin Fast Plants, and 4-H Youth Science, Engineering and Technology Programs (4-H SET). 7. S. Michaels, A. W. Shouse, H. A. Schweingruber, Ready, Set, Science! Putting Research to Work in K–8 Science Classrooms (National Academies Press, Washington, DC, 2008). 8. S. R. Singer, M. L. Hilton, H. A. Schweingruber, America’s Lab Report: Investigations in High School Science Classrooms (National Academies Press, Washington, DC, 2006). 9. President’s Council of Advisors on Science and Technology, Prepare and Inspire: K–12 Science, Technology, Engineering, and Mathematics (STEM) Education (Executive Ofﬁce of the President, Washington, DC, 2010); www.whitehouse.gov/sites/default/ﬁles/microsites/ostp/ pcast-stem-ed-ﬁnal.pdf. 10. PlantingScience is possible because of the exceptional efforts of teachers and volunteer mentors, contributions of advisers and collaborators, and partner societies (full list on is on the Web site). The BSA, the Monsanto Fund, and the NSF (DRL-0733280) have funded this ongoing effort.

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About the authors

options for proﬁtable lines of investigation. Mentor materials—for example, the Power of Sunlight tip sheet—include common misconceptions about photosynthesis, and suggestions for helping students tweak experimental set-ups and make sense of their data. The key tool for reﬂection and discourse is the student team Web page where students and mentors post messages asynchronously. Talking online with a scientist is exciting and motivating to students. Teachers commonly relate that their students develop a new level of conﬁdence and responsibility toward their experiments. Preliminary results of before and after tests showed significant improvement in student attitudes toward enjoying studying plants and plant biology. Many students report that tending to and observing their plants is exciting and rewarding. Teachers often encourage students to use the searchable research gallery to evaluate questions investigated by other teams; they report that seeing other students tackle similar topics opens them to the scientists’ world of peer scrutiny. A search of Wonder of Seeds projects in the archive reveals that students from middle school through college have asked a wide range of questions about germination and growth— such as what is the effect of pH of soil, does the presence of earthworms inﬂuence growth, and how do seeds respond to gravity while germinating. Flexibility within a framework allows teachers to tailor investigations for their students. We aim to shift curricula from repetitive lab exercises with predicable outcomes into the real world of science where ambiguity, messy data, and creativity reside (8). Modules range from widely accessible, open inquiries to more structured inquiries. Some incorporate the established classroom models of Wisconsin Fast Plants, C-Fern, and Arabidopsis. In drafting modules, scientist-teacher teams work with the curriculum coordinator. Classroom fieldtesting follows, with some mentors also assessing protocols. Scientists from the module development teams then lead a 5-day inquiry immersion that opens the 9-day institute for teachers who wish to deepen their understanding of plant biology, inquiry learning, and online community platform use (see the second figure). Sixteen teachers are selected to experience the plant inquiries as learners. Plant scientists provide content background and teachers can become familiar with the interactive tools. The second week provides focused time for teachers to share strategies

10.1126/science.1196979

CREDIT: WILLIAM DAHL

The American Society of Plant Biologists signed on in 2006. Today, a big part of what makes PlantingScience special is our many partners (6). Over recent years, 108 Master Plant Science Team mentors, primarily graduate students, have made year-long mentoring commitments. Online mentoring allows scientists, from graduate students to professor emeriti, to contribute to school science without leaving their ofﬁces and to remain in constant contact with their teams. Partnerships permeate PlantingScience. Societies unite volunteer efforts in a national network to address education needs: Online mentors and classroom teachers collaboratively support student teams; these teams develop curricula and Web site materials and provide professional development, and the Texas A&M University and Biological Sciences Curriculum Study colleagues investigate how online mentorship affects learning. Over 9000 students, 2500 research teams, and teachers in 34 states have thus far experienced inquiry science as a community endeavor with plant biologists. About 60% of the classes are in high schools, and 38% are in middle schools. College classes and 4-H clubs also participate. Educators seek to engage their students in collaboration, communication, and innovation—21st-century skills for success—and inquiry experiences that mirror the practices of scientists. Participating in the science enterprise and communication are vital to science learning proﬁciency (7). Resources for reﬂection and argumentation are critical for students to construct their understanding. Teacher materials include guiding questions and

EDITED BY EDWARD W. LEMPINEN

AAAS ANNUAL MEETING

Researchers Shape New Strategies For a Time of Budget Turmoil Just 2 years ago, American science seemed to be on the cusp of a new era, with Washington strongly backing research to drive economic growth and address a range of challenges. But as scientists and policy experts gathered recently for the AAAS Annual Meeting, they were confronting a new landscape where budget crises and shifting politics could jeopar- A continuing commitment. John P. Holdren, the White House science dize the nation’s power of and technology adviser, told a standing-room-only crowd that President Obama considers R&D funding crucial for U.S. economic strength. innovation. It was a recurring theme throughout the 5-day meeting: Whether In an address marking the end of her the subject was new energy sources, the term as AAAS president, Alice S. Huang university of the future, or particle physics, described how her own practice of science discussions frequently turned to the potential diplomacy—in the lab and in work with impact of federal and state budget cuts—and Asian nations—has conto the need for new strategies and alliances vinced her of its power to that will allow science to move ahead. engage others and even Addressing a standing-room-only crowd address challenges such of more than 1500 people, White House scias poverty and women’s ence and technology adviser John P. Holdren economic development. said that President Barack Obama remains But for U.S. science ﬁrmly committed to scientiﬁc research. And, diplomacy to be effeche added, Obama’s concerns reach beyond tive, said the Caltech research spending to supporting the larger sci- Alice S. Huang virologist, “we need to ence enterprise, from education to the comavoid arrogance and mercialization of discoveries. Western-centric views and behave as true “It’s not automatic that the United States partners in advancing international science will be No. 1 in science, technology, and as well as the welfare of all citizens.” innovation,” Holdren told reporters before In an increasingly global science culture, his address. “This is something that has to experts said at the meeting, cuts in U.S. scibe cultivated. It has to be invested in. The ence would likely be felt worldwide: fewer president has been very clear that he wants cooperative research projects; diminished us to out-innovate, out-educate, and out- ability to compete against rising science build the competition.” and technology powers; and eroding U.S. The 177th AAAS Annual Meeting con- influence in areas such as education and vened in Washington, DC, from 17 to 21 Feb- research integrity. ruary, drawing several thousand researchers, Compromise remains possible, but the policy experts, and educators, plus hundreds prospects are not bright—indeed, some anaof U.S. and foreign journalists. Under the lysts see the risk of a government shutdown theme “Science Without Borders,” the meet- later this spring. ing explored the global frontiers of research Patrick Clemins, director of the AAAS and contemporary policy issues. R&D Budget and Policy Program, out-

lined critical elements of the conflict during a budget symposium: Obama in fiscal year 2012 would hold overall R&D spending to an increase of 0.5% over 2010 levels; nondefense R&D, however, would rise 6.5%. Republicans, in their plan for 2011, propose cuts of 4.4% in overall R&D, and 5.1% for nondefense R&D from current spending levels. The continuing economic crisis is already creating unprecedented f inancial pressure at state universities and colleges, said Howard Gobstein, executive vice president for research, innovation, and STEM education at the Association of Public and Landgrant Universities. In 2009, 85% of higher education institutions reported state funding cuts averaging 11%. Last year, 60% reported reductions averaging 7%. This year, Gobstein said, cuts exceeding 20% have been imposed at some institutions. Gobstein is confident that universities can adapt and preserve their commitments to teaching, research, and civic engagement. Still, he worries that education at public research universities “will become less and less accessible to most of our citizens as tuition soars to partially replace cuts in government support.” With change happening so broadly and so swiftly, many at the Annual Meeting emphasized the importance of adapting, of ﬁnding new ways to have an impact. In an event organized by AAAS and 10 other science organizations, 35 climate scientists came to Washington, DC, and spent a day meeting with members of Congress, including some in the new Republican majority in the House who have supported drastic reductions in funding for conservation and research on energy. The scientists took an unconventional approach: Rather than debate the existence of climate change, they focused on related local issues such as childhood asthma and agricultural pests. Sometimes the reception was chilly, but other meetings with climate skeptics ended with agreements to keep an open line of communication. In the view of Rob Young, a professor specializing in coastal geology at Western Carolina University, that was good news. “Success,” he said, “from my point of view, will really just be an e-mail asking about coastline erosion—anything to bridge the gap.”

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AAASNEWS&NOTES

–Becky Ham, Amy Maxmen, and Edward W. Lempinen

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SPECIALSECTION

INTRODUCTION

CREDIT: ANNE WESTON/CANCER RESEARCH UK/VISUALS UNLIMITED INC.

In this issue of Science, we commemorate the 40th anniversary of the U.S. National Cancer Act, which provided a massive stimulus for cancer research. At the start of this “Cancer Crusade,” researchers were already tackling some tough questions, as reﬂected in papers published by Science in 1971. Among them: How do abnormalities in chromosome number arise in tumor cells? Can tissue-speciﬁc markers be used to determine the epithelial versus mesenchymal origin of a solid tumor? Can the immune system be manipulated so that it recognizes tumor cells as foreign invaders that must be eliminated from the body? Do viruses play a role in human cancer? Skeptics might argue that 40 years later, cancer researchers continue to grapple with the same questions. Perhaps there’s some truth in this. But our hope is that the selection of articles in this special section of Science will explain why many of these questions have proved so challenging and, more importantly, how contemporary cancer research is providing a clearer view of the biology that will lead to answers. Stratton (p. 1553) discusses international efforts to sequence the complete genomes of a wide range of human tumor types and the impact that this sequence information is anticipated to have on our understanding of cancer biology as well as our ability to detect, diagnose, and treat the disease. A working model for cancer cell metastasis is presented by Chaffer and Weinberg (p. 1559), who highlight the important role of cancer stem cells and a developmental process called the epithelial-mesenchymal transition. Schreiber et al. (p. 1565) describe “cancer immunoediting,” a conceptual framework that integrates the immune system’s dual roles in inhibiting and promoting cancer growth. News reports examine other signiﬁcant challenges for the ﬁeld: Jocelyn Kaiser (p. 1542) describes how even the best new drugs eventually seem to fail and how they might be made more effective. David Malakoff (p. 1545) outlines a key social issue: the fast-rising cost of care. Martin Enserink (p. 1548) reports on efforts to close the huge disparity between cancer treatment in rich and developing countries. And Mitch Leslie (p. 1551) describes how researchers are taking a new look at the role of p53 and the related proteins p63 and p73 in tumors. A video report by Robert Frederick appears online at www.sciencemag. org/special/cancer2011/, along with links to additional reading material. Science Careers features an article on cancer clinical trials training (http://scim. ag/cancertrialstraining) and a Q&A with Memorial Sloan-Kettering clinicianinvestigator David Solit (http://scim.ag/solitqanda). It is worth noting that at least one of the questions that concerned cancer researchers writing in Science back in 1971 has been deﬁnitively answered. We now know that viruses do in fact play a causal role in certain human cancers, and, thanks to decades of tumor virology research, vaccines against these viruses have been developed into successful cancer-preventive agents. That’s something to celebrate.

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Celebrating an Anniversary Cancer Crusade at 40 CONTENTS News 1540

Cancer Research and the $90 Billion Metaphor U.S. Cancer Trends

1542

Combining Targeted Drugs to Stop Resistant Tumors

1545

Can Treatment Costs Be Tamed

1548

A Push to Fight Cancer in the Developing World

1551

Brothers in Arms Against Cancer

Reviews 1553

Exploring the Genomes of Cancer Cells: Progress and Promise M. R. Stratton

1559

A Perspective on Cancer Cell Metastasis C. L. Chaffer and R. A. Weinberg

1565

Cancer Immunoediting: Integrating Immunity’s Roles in Cancer Suppression and Promotion R. D. Schreiber et al.

See also Editorial p. 1491; Report p. 1612; and Science Translational Medicine including Research Article by L. Sequist et al., Science Signaling, Science Careers, Video, and Science Podcast at www.sciencemag.org/special/cancer2011/.

– PAULA KIBERSTIS AND ELIOT MARSHALL

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CANCER CCrusade ANCatE40R U.S. Cancer Trends

Cancer Research and The $90 Billion Metaphor

Lung & Bronchus

reports and debates attached itself to the U.S. program that began when President Richard Nixon signed the National Cancer Act in December 1971. The law made big promises and gave the U.S. National Cancer Institute (NCI) a token measure of independence. It also encouraged the idea that cancer could be targeted, like a trip to the moon, and cured. The law was important for research, RAND medical historian and policy analyst Richard Rettig has written: It stopped a decline in NCI’s budget. This reversal began in Congress. Urged on by health activists such as Mary Lasker, leading Democrats in 1970 adopted curing cancer as their cause. Aware that it might become a national issue, Nixon embraced it, too. The resulting legislation raised NCI’s budget almost overnight by 23% to $233 million; NCI’s funding has continued to climb since then to more than $5 billion per year— although in recent years inﬂation has grown faster. Since the 1971 act, NCI has spent about $90 billion on science, treatment, and prevention of cancer. The war metaphor remains part of this legacy—and a target for skeptics. In 1986, former NCI biostatistician John Bailar stirred controversy with a bleak analysis in The New England Journal of Medicine, noting that cancer incidence and mortality rates hadn’t changed fundamentally in 15 years. He suggested that the nation was “losing the war against cancer.” Sweeping declarations continued. In 2003, then–NCI Director Andrew von Eschenbach set a goal of ending suffering and death from cancer “by 2015.” Such broad claims invite balloon-pricking. But if one sets aside the rhetoric, says Allen Lichter, executive director of the American Society of Clinical Oncology, it’s evident that the cancer campaign has changed therapy and saved lives (see indicators for the seven deadliest cancers, right). It is true that for certain cancers—of the pancreas, brain, and liver, for example—the picture has not improved. But the overall U.S. cancer mortality rate began to decline in the 1990s. And clinical care “looks nothing like it did 40 years ago,” says Lichter, who began training as an oncologist in the 1970s. He speaks of a revolution that took lessons from the 1960s advances against childhood leukemia to develop “adjuvant therapy”: the “crazy idea” that chemotherapy should be given to patients in remission to treat “presumed microscopic disease” that has spread. His list of beneﬁts continues with breast-conserving and microscopic surgery, imaging for diagnosis and disease management, molecular analysis of tumors and targeted drug therapy, longer survival times, and much better palliative care. Looking for progress in cancer can feel “like watching the hands of a clock,” Lichter admits. “But things are deﬁnitely moving in the right direction.”

–ELIOT MARSHALL

Incidence per

80 100,000 70 60 50 40 Deaths per 100,000 30 20 10 0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

2010 Estimated Deaths

157,300

5-Year Mortality Trend

-1.6%

Colon & Rectum The U.S. National Cancer Institute (NCI) spotlighted declining colorectal cancer trends in its 2010 Annual Report to the Nation on the Status of Cancer. Improved diet and screening with colonoscopy, among other early-detection techniques, are helping to control the second-deadliest cancer. NCI’s modeling predicts that overall mortality could drop 50% by 2020. Incidence per 100,000

70 60 50 40 30 20 10

Deaths per 100,000

0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

2010 Estimated Deaths

51,370

5-Year Mortality Trend

-3.0%

SOURCE FOR CANCER STATISTICS: NCI SURVEILLANCE RESEARCH PROGRAM; PHOTOS (LEFT TO RIGHT): LINDA BARTLETT/NCI; BILL BRANSON/NCI; NCI

40 Years of the War on Cancer

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1971

1973

1978

President Richard Nixon signs the National Cancer Act promoting the National Cancer Institute.

NCI launches Surveillance Epidemiology and End Results program to collect U.S. cancer data.

Clinical testing begins of interferon-α, the ﬁrst biological cancer therapy.

FDA approves tamoxifen to prevent breast cancer recurrence.

1979

1980

Researchers discover p53, the mutated gene most often seen in tumors.

Robert Gallo and others isolate human T-cell lymphotropic virus-1, a cause of cancer.

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THERE NEVER WAS AN OFFICIAL “WAR ON CANCER.” THAT PHRASE FROM NEWS

The good news: Lung cancer incidence among men began to decline in the early 1980s; the death rate, in the early 1990s. This shows the power of prevention—through a campaign to curb tobacco use. The bad news: The death rate is far higher for African-American men than for white men, and deaths among women (of all races) continued to climb until recent years.

SPECIALSECTION

2010 Estimated Deaths

39,840

5-Year Mortality Trend

120 90 60 30

-2.2%

Pancreas

36,800

12 10

4 2 0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

240

5-Year Mortality Trend

200

120 80 40

-3.3%

Death rates for the four main types of leukemia have slowly declined, thanks largely to treatments that combine chemotherapy drugs. The survival rate is now about 80% for childhood acute lymphoblastic leukemia.

12

21,840

10 8

Deaths per 100,000

6 4 2

-1.3%

Mortality and incidence for liver and bile duct cancers have risen steadily, linked to infections with hepatitis B and C, which are top risk factors, along with alcohol abuse. Because tumors usually cannot be removed with surgery, post-diagnosis survival is brief.

18,910

Incidence per 100,000

14

Liver

2010 Estimated Deaths

Deaths per 100,000

0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

16

2010 Estimated Deaths

Incidence per 100,000

160

Leukemia

5-Year Mortality Trend

Deaths per 100,000

8 6

5-Year Mortality +0.6% Trend

The incidence of prostate cancer, the second most common cancer in men, spiked in the early 1990s after regulators approved the prostate-speciﬁc antigen (PSA) screening test. Most men treated after a PSA test had nonlethal tumors.

32,050

Incidence per 100,000

14

Prostate

2010 Estimated Deaths

Deaths per 100,000

0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

In part because it is difﬁcult to detect early, pancreatic cancer remains the fourth-deadliest cancer, and incidence and mortality have hardly budged. The average patient diagnosed with advanced disease will live only 6 months. 2010 Estimated Deaths

Incidence per 100,000

150

Efforts to detect and treat invasive breast cancer partly explain why incidence rose dramatically in the 1980s, reaching a peak for all races in 1999. Death rates from breast cancer have been declining steadily since 1989–90, although 5-year survival continues to be far higher for whites than African Americans.

Downloaded from www.sciencemag.org on March 24, 2011

Breast (female)

5-Year Mortality +2.2% Trend

0

‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

8 7 6

Incidence per 100,000

5 4 3 2 Deaths per 100,000 1 0 ‘75 ‘77 ‘79 ‘81 ‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01 ‘03 ‘05 ‘07

SOURCE FOR CANCER STATISTICS: NCI SURVEILLANCE RESEARCH PROGRAM; PHOTOS (LEFT TO RIGHT): ISTOCKPHOTO; THINKSTOCK; NCI; PAUL SAKUMA/AP

1981

1983

1985

1986

1989

First cancerprevention vaccine introduced— against human hepatitis B virus.

Researchers create severe combined immunodeﬁcient mice, a model for cancer research.

Randomized trial shows that lumpectomy plus radiation are as effective as mastectomy for breast cancer.

Biostatistician John Bailar writes in The New England Journal of Medicine, “We are losing the war against cancer.”

Nobel Prize for discovering the ﬁrst proto-oncogene (Src) awarded to Harold Varmus and Michael Bishop.

www.sciencemag.org SCIENCE VOL 331 25 MARCH 2011 Published by AAAS

> 1541

Combining Targeted Drugs To Stop Resistant Tumors Even the most successful targeted therapies lose potency with time. Researchers hope to ﬁgure out how tumors escape; they aim to turn months of survival into years

patients’ lives, they never completely eliminate the cancer. For reasons still not well understood, the initial success is only “a foot in the door,” says cancer geneticist Michael Stratton of the Wellcome Trust Sanger Institute in Cambridge, U.K., whose team has found several genetic weak spots in tumors. Researchers are still working out exactly what to do next. On the surface, the answer is straightforward: Identify the ways that tumors resist the drug, then ﬁnd or develop second-generation drugs that block these escape routes. With the right drugs on hand, researchers envision designing a cocktail— perhaps two, three, or more drugs—that, if given when a patient is ﬁrst diagnosed, could stop tumors from ever evading the blockade. This approach has worked for patients infected with HIV, who usually take three antiviral drugs. Sawyers and many other researchers say there’s no reason it shouldn’t work for cancer. But getting a therapy to work and getting it to endure are two different things. Even if a combination therapy stops tumor growth, it may not buy patients more than a temporary reprieve, researchers admit. To stretch the beneﬁt over years, it might be necessary to devise one complex cocktail after another, each tailored to a patient’s evolving tumors.

cer researcher at Memorial Sloan-Kettering drugs in the past decade comes from a simple Cancer Center (MSKCC) in New York City. idea: Find a weak point in a tumor’s molec- Sawyers is a pioneer of the targeted approach ular machinery and throw a well-aimed and co-developer of the drug Gleevec that wrench into it. The strategy has led to some has been spectacularly effective against dramatic successes, stopping the growth of chronic myelogenous leukemia (CML). certain cancers in their tracks while doing But then came the letdown: After about little or no harm to healthy tissue. But the 7 months, most melanoma patients on the pursuit of what’s known as “targeted ther- PLX4032 pill saw their tumors begin to apy” has taken many of those involved on grow again; many died. Last fall, trying to a roller-coaster ride. The new treatments, learn what enabled that puzzling regrowth, after a brilliant debut, tend to lose potency several research teams showed that resistant as tumors develop resistance. After a pause tumor cells had found ways to switch back lasting weeks or months, the cancer may on the cell pathway that the PLX4032 drug begin to grow again. had jammed. They gave some of the relapsed Understanding why this occurs and devis- patients another new drug that blocks the ing therapies that will overcome resistance pathway at another point, hoping their are the main focus of a growing community tumors would shrink under the dual assault. of cancer researchers. A recent example of The results are still coming in. their quest involves a drug designed to stop In the past few years, researchers have metastatic melanoma, a highly aggressive reported dramatic responses to a handdisease for which there is no effective treat- ful of new drugs that are given to patients Tossing in the wrench ment today. with a speciﬁc mutation in their tumors. But Molecular targeting builds on what researchIn late 2007, Roche and a biotech com- the emerging pattern is that although these ers have learned from 30 years of work on pany called Plexxikon began testing a new drugs can shrink solid tumors and extend the genetic changes behind cancer. Uncontargeted drug called PLX4032 for patients with advanced POSSIBLE COCKTAILS OF TARGETED CANCER DRUGS melanoma. When researchers presented the first scans Median time Possible from treated patients, audiDrug Cancer Target to resistance cocktails ences were stunned: In some Gleevec chronic myelogenous BCR-ABL fusion 5 years Dasatinib or nilotinib cases, the tumors had almost leukemia protein (17% of patients) + T315I inhibitor disappeared. Eighty percent of patients got better, a remarkIressa, non-small cell lung EGFR receptor 12 months Tarceva + T790M able response that seemed to Tarceva cancer with inhibitor + (MET inhibitor EGFR mutation or PI3K inhibitor) validate the concept of targeted therapy. There was “jubilaPLX4032 melanoma with V600E BRAF protein 7 months PLX4032 + MEK inhibitor tion about how well it works,” BRAF mutation says Charles Sawyers, a can-

ONE OF THE BEST HOPES FOR ANTICANCER

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1991

1992

1993

National Breast Cancer Coalition launched, in the AIDS activist style.

FDA approves synthetic yew bark derivative, Taxol (paclitaxel), for breast cancer.

Congress orders study of environmental causes of breast cancer on Long Island; the 10-year study will yield no signiﬁcant ﬁndings.

Science names p53 “Molecule of the Year.”

1994

1996

1998

BRCA1 gene, identiﬁed as a risk for breast and ovarian cancer, is cloned; BRCA2 cloned the next year.

American Cancer Society and others report the “ﬁrst sustained decline” in overall U.S. cancer deaths, a drop of 2.6% from 1991 to 1995.

FDA approves Herceptin (trastuzumab), a monoclonal antibody, for metastatic breast tumors that overproduce HER2.

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

Downloaded from www.sciencemag.org on March 24, 2011

NEWS

CREDITS (TOP TO BOTTOM): COPYRIGHT ®NOVARTIS AG; COURTESY ASTRAZENECA; WIKIMEDIA COMMONS; (TABLE SOURCE) CHARLES SAWYERS/MSKCC AND JEFFREY ENGELMAN/MGH; GEORGE MCGREGOR/NCI; SCIENCE

CANCER CCrusade ANCatE40R

trolled cell growth is often driven by an A aberrant protein in the cell membrane that transmits a spurious signal to the nucleus, instructing it to divide. Antibodies or small molecules can be used to block these overactive cell receptors, or a mutated protein farther down the signaling chain, causing tumors to shrink dramatically. (By contrast, stanB dard chemotherapy targets all dividing cells in the body, which makes it much more toxic.) But because tumors are genetically diverse, resistance seems inevitable. Once a targeted drug wipes out the bulk of a tumor, cells harboring “resistance genes,” or alternative growth instructions, have a chance to C grow. Even a tiny population of resistant cells can expand and take over. “Every single targeted therapy will select for resistance” in this way, says Carlo Maley, who studies the evolution of cancer at the University of California, San Francisco. Gleevec is the classic example. In 95% of patients with CML, the cancer is driven by a gene called BCR-ABL that is formed when two chromosomes swap pieces, making a fused segment known as the Philadelphia chromosome. Gleevec, made by Novartis, is a small molecule that blocks the BCR-ABL fusion protein. The drug was approved for CML by regulators 10 years ago this May, and many patients on it live for at least a decade. But about 17% of patients develop resistance within 5 years. Gleevec’s developers anticipated this, they say. Sawyers and others have shown that in most cases resistance results from mutated versions of the BCR-ABL protein that are not affected by the drug and continue to tell cells to grow. Companies developed other specific drugs, dasatinib and nilotinib, that block most forms of the mutated enzyme and are given to patients who relapse. Trials published last year show that the drugs work so well as an alternative to Gleevec as initial therapy for CML

Sawyers says. Gleevec has been used with good effect to treat another cancer, gastrointestinal stromal tumor (GIST), a P P p85 rare disease usually caused by mutaP13K p85 tions in genes called PDFGRA or p110 P13K KIT, says Michael Heinrich of OHSU. p110 Akt But resistance mutations can appear in the KIT protein. Although a drug Two ways out. (A) The lung EGFR called sunitinib targets some of them, cancer drug Tarceva (blue T790M ERBB3 there’s no current drug that can “patch spheres) blocks the EGFR up all the holes,” Heinrich says. And receptor from transmitting a because GIST patients live a relatively signal. (B) The T790M mutaP P p85 tion prevents the drug from long time on Gleevec—5 years versus P13K binding. (C) Tumor cells can 15 months on chemotherapy—it’s hard p110 also overexpress the MET to interest companies in testing a cockreceptor, which takes over Akt tail for GIST, he says. when EGFR is blocked. Resistance is a more urgent problem for lung cancer patients treated MET EGFR ERBB3 MET ERBB3 with Iressa and Tarceva (geﬁtinib and erlotinib), the ﬁrst big success for targeted therapy after Gleevec. These P P p85 P p85 nearly identical drugs block a cell P13K P13K receptor called EGFR that transmits P P p110 p110 growth signals. The drugs didn’t help most patients in trials for non-small Akt Akt cell lung cancer, but researchers realpatients that they may delay resistance for ized that they work extremely well on the years, Sawyers says. roughly 10% of patients who have an EGFR However, no cocktail for CML has yet mutation in their tumors (they tend to be been tested as an initial therapy in a clinical women, never-smokers, or Asian). Some of trial. One reason, Sawyers says, is that the these patients’ tumors almost vanish when cocktail mix is not quite complete. There is they receive an EGFR inhibitor. However, one important mutant version of the BCR- the average patient develops resistance after ABL protein, T315I, that no existing drugs about a year. target. (A promising candidate is in clinical As with Gleevec for leukemia and GIST, trials, though.) Furthermore, patients aren’t the trouble is often that tumor cells appear that interested in enrolling in a combina- in which the EGFR receptor has a speciﬁc tion trial because Gleevec alone works very new mutation (T790M) that prevents the well for most patients, says Gleevec co- drugs from binding well. Companies are still developer Brian Druker of Oregon Health moving toward clinical trials of drugs that and Science University (OHSU) in Portland. block EGFR proteins with this mutation, And as a researcher, Druker says he ﬁnds it which about half of all patients develop, says hard to ask people to take an experimental Jeffrey Engelman of Massachusetts General drug as well as Gleevec when only a small Hospital (MGH) in Boston. Lung cancer fraction will likely do any better than they cells have another way of evading the drug, would on Gleevec alone. “Would I treat 90 moreover: They can make more of a differpeople for the beneﬁt of 10?” Druker asks. ent cell receptor, called MET, that can take “People are so comfortable with one drug,” over for EGFR and maintain the growth sigEGFR

ERBB3

1998

2001

2003

2004

Nobelist James Watson tells The New York Times that blocking the growth of tumor blood vessels (antiangiogenesis) can “cure cancer in 2 years.”

FDA approves Gleevec (imatinib), a targeted drug, for chronic myelogenous leukemia; Time calls it a “magic bullet.”

NCI Director Andrew von Eschenbach vows to “eliminate suffering and death from cancer by 2015.”

FDA approves Avastin, an antiangiogenesis drug, for colon cancer, with chemotherapy.

2005 Childhood cancer landmark: nearly 80% of those treated for acute lymphoblastic leukemia are free of cancer “events” for 5 years or more.

www.sciencemag.org SCIENCE VOL 331 25 MARCH 2011 Published by AAAS

NIH launches The Cancer Genome Atlas to catalog genomic changes in tumors.

Downloaded from www.sciencemag.org on March 24, 2011

CREDITS (TOP TO BOTTOM): ADAPTED FROM ENGELMAN & JÄNNE, CLINICAL CANCER RESEARCH 14 (15 MAY 2008); NCI; GLOGAU PHOTOGRAPHY/NCI; NCI

SPECIALSECTION

> 1543

CANCER CCrusade ANCatE40R

Engelman

Flaherty

Retargeting. Gleevec co-developer Charles Sawyers, lung cancer researcher Jeffrey Engelman, and mela-

noma trial co-leader Keith Flaherty are working on ways to overcome acquired resistance to targeted drugs.

Ingram Cancer Center in Nashville. Pao is optimistic about another early clinical trial, however, that is treating patients who became resistant to Tarceva or Iressa with a new, more potent EGFR inhibitor and an approved EGFR-blocking antibody called cetuximab; the combination shrank T790M-carrying tumors in mice. Initial results are expected this summer. New escape routes How melanoma tumors become resistant to the initially powerful PLX4032 drug is more complex. Plexxikon began developing the drug after Stratton’s team reported 9 years ago that tumors in half of advanced melanoma patients have the same mutation in a protein called BRAF. This protein is part of a key growth signaling pathway. Because

1544

to use three different escape routes. This was disappointing, but some also saw good news: Two of these forms of resistance function in the same way—restoring the growth pathway by activating a downstream protein called MEK. This suggests, says David Solit of MSKCC, that combining a BRAF inhibitor and a drug that blocks MEK could block both escape routes. These studies “still don’t account for the lion’s share” of resistance cases, Flaherty says; there is more work to be done. But they have inspired GlaxoSmithKline, which makes a BRAF inhibitor similar to PLX4032, to launch a trial combining its drug with a MEK inhibitor. This “will provide proof of concept” of the cocktail idea, Sawyers says, within a couple of years. Another cocktail combining PLX4032

2006

2007-2008

2009

2010

FDA approves Gardasil vaccine to prevent HPV infection, which can lead to cervical cancer.

Breast cancer incidence declines, attributed to better screening and reduced use of hormone replacement therapy.

James Watson writes that it’s time to turn from cancer genetics to “understanding the chemical reactions within cancer cells,” or cell metabolism.

National Lung Cancer Screening Trial ﬁnds that helical CT screening can reduce cancer deaths among smokers.

with an immunotherapy drug developed at MSKCC is also under study (Science, 22 October 2010, p. 440). Cocktails with caveats Testing combinations of drugs is not new in cancer research. But these cocktails would be different because they would be “rationally designed” to block tumor escape routes, researchers say. Although researchers are eager to begin, they will need to overcome some barriers. One is commercial competition. Many drugs that target the newly found resistance pathways are still in development. Companies are loath to test two unapproved drugs simultaneously, especially if one comes from a business rival. They worry that side effects from one product will “bloody” a drug that is safe on its own, Flaherty says. “People in my line of work are all about trying to make this happen because the biology is so obvious,” he says, but drug development is another matter. Flaherty and others are encouraged, however, by recent examples of companies teaming up: In 2009, Merck and AstraZeneca agreed in a groundbreaking decision to test a Merck MEK inhibitor and a drug blocking another key pathway, PI3K/ Akt. Since then, a couple more companies have signed such agreements. The motivation is not just to overcome resistance but also to explore an exciting possibility: It has become “increasingly clear” from cell studies that pairing two drugs aimed at different pathways can result in synergistic effects, says D. Gary Gilliland, senior vice president and franchise head for oncology at Merck in North Wales, Pennsylvania. He also praises new draft U.S. Food and Drug Administration guidelines that allow for ﬂexibility for testing combinations. Researchers testing combinations must also face the fact that tumors are constantly evolving, Engelman notes. His group published a study on non-small cell lung cancer this week in Science Translational Medicine (STM) that illustrates this complexity.

2011 FDA approves Provenge, an immune treatment for metastatic prostate cancer. It extends life about 4 months and costs $93,000.

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

PLX4032, a targeted cancer drug, extends life in patients with advanced melanoma.

Downloaded from www.sciencemag.org on March 24, 2011

Sawyers

PLX4032 blocks mutated BRAF and inhibits this pathway only in tumor cells, it can be given at high doses (Science, 18 December 2009, p. 1619). But nobody was surprised, says trial co-leader Keith Flaherty of MGH, when patients on PLX4032 whose tumors melted away eventually relapsed. But researchers were surprised to ﬁnd that biopsied tumors from patients who developed resistance didn’t have mutations in the BRAF protein. Instead, several teams reported recently that tumor cells appear

CREDITS (TOP TO BOTTOM): COURTESY MEMORIAL SLOAN-KETTERING CANCER CENTER; COURTESY MASSACHUSETTS GENERAL HOSPITAL (2); JUPITER IMAGES/THINKSTOCK

naling pathway. These escape routes and a couple of others are problematic but “a manageable list,” says Jeffrey Settleman, who left MGH last year for Genentech in South San Francisco, California. Hoping to close off two escape routes at once, a few companies are running trials combining an EGFR inhibitor and a MET inhibitor. Early results are mixed, Engelman says. One problem is that the cocktails didn’t target the T790M mutation, says William Pao of Vanderbilt-

SPECIALSECTION “pulse” of a cocktail for several days. As his STM study suggests, clinicians would need to constantly biopsy patients and tailor the cocktail to the mutations in their tumor. This could stretch responses out to years, Engelman says. “A lot of this is going to have to be done by trial and error,” Pao agrees. Sawyers and others point out, however, that such combination therapies developed in the 1960s and ’70s eventually vanquished most cases of childhood leukemia, Hodgkin lymphoma, and testicular cancer. Major cancer centers are already routinely genotyping biopsies from patients throughout their treatment using procedures that are less invasive than surgery, such as collecting a few tumor cells with a thick needle. Engelman says this is essential: “We can’t be afraid to rebiopsy to see what’s going on.” Cancer researchers acknowledge that coming up with cocktails to corner cancer will be much more difﬁcult than it was with HIV. Cancer geneticist Bert Vogelstein

of Johns Hopkins University in Baltimore, Maryland, points out that cancer is different from AIDS: Among other things, tumors vary more from patient to patient than HIV does; the genetic heterogeneity of cancer cells within a single patient is “orders of magnitude greater” than HIV genotypes in patients, so tumors have a “much larger reservoir of resistance mechanisms” that go beyond those already uncovered. “Unfortunately, we’ve got billions of cancer cells ready to become resistant, and it takes 15 years to develop each new drug,” Vogelstein says. Moreover, to wipe out a tumor completely, he and others say, a cocktail might also have to include a drug that targets stem cell–like cells in a tumor that continuously give rise to tumor cells. Turning cancer into a manageable disease, however, “would be wonderful,” Vogelstein says, if it can be done. Advocates of targeted therapy think they will get there. “Melanoma was completely untreatable 18 months ago,” says Neal Rosen of MSKCC. “Give us a chance.” –JOCELYN KAISER

Downloaded from www.sciencemag.org on March 24, 2011

His team analyzed 37 biopsy samples from patients with the EGFR mutation who were given Iressa or Tarceva and later became resistant. Although some resistance mutations were known, others were new, and for 30% of the samples, his team could not identify the mechanism. Some tumors even morphed into a different type of lung cancer that requires an entirely different treatment. In addition, biopsies from three patients collected during the course of treatment showed that tumors changed: Some that developed resistance mutations later lost them. “It’s very, very complex. It’s not ﬁtting into the simple boxes that we’ve made until now,” Engelman says. Cocktail therapy will face another issue that has not been well explored so far: the risk that combining two drugs—particularly ones that target different pathways used by normal cells—can lead to unacceptable side effects. Engelman thinks that for this reason, patients will be able to tolerate a cocktail for only a short time. He envisions putting them on a single drug, then intermittently giving them a

come with sticker-shock prices of $50,000 or more per patient. New and more costly, however, haven’t necessarily meant better. Although targeted treatments, which attack a molecuMore patients and the rising costs of new cancer treatments spark debate over how lar weak spot in the tumor’s support sysmuch is too much—and who should decide tem, have helped improve survival rates for many cancers, some extend life for just a THE AFTERNOON JEFF GUSTAFSON LOST HIS according to a recent estimate. By 2020, it few weeks or months (see p. 1542). And the life to stomach cancer, a rainbow appeared could grow by as much as 66%, to $207 bil- prices can be sobering: more than $1.2 milabove his Arlington, Virginia, hospice. lion. Multiple forces are driving the spiral: lion to extend a lung cancer patient’s life for Even if the shimmering arc had led to a a growing and aging population, more peo- 1 year in one scenario involving a costly but pot of gold, however, it’s not clear whether ple living longer with cancer, and new “per- common drug. That example is unusual, but the treasure would have covered the cost sonalized,” or “targeted,” therapies that can such numbers have sparked a growing—and of treating the 48-year-old sometimes feisty—debate PROJECTED COSTS OF TOTAL U.S. CANCER CARE, 2010–20 builder, who was known for his over how best to calculate the wit and compassion. The bills beneﬁts of new cancer treat220 for just 4 months of dogged, ments, whether their use will 5% increase in annual costs, first and last year of care full-tilt treatment totaled more lower or raise per-patient 200 than $350,000—including expenses, and who should 2% increase in annual costs one drug that cost more than decide whether using them is 180 $12,000 per dose. worth the cost. “The question Population growth only Gustafson’s story isn’t that is, ‘Are we spending too much 160 Current trends in incidence unusual to experts who track for too little?’ ” says oncoloand survival, projected 140 the costs of treating cancer in gist Antonio Tito Fojo of the the United States—and are U.S. National Cancer Institute 120 increasingly worried about (NCI) in Bethesda, Maryland. where they are headed. Over 100 the past 3 decades, total U.S. Demographic drivers 2010 2020 spending on cancer care has Tallying the current and more than quadrupled, reach- Four scenarios. Experts predict total U.S. spending on cancer care could rise by future costs of treating caning $125 billion last year, or as much as 66% by 2020, depending on shifts in disease incidence and survival, cer isn’t easy. “Cancer” now 5% of the nation’s medical bill, and treatment costs. includes more than 100 difNEWS

U.S. $ (billions)

CREDIT (GRAPH SOURCE): A. MARIOTTO, ET AL., JOURNAL OF THE NATIONAL CANCER INSTITUTE 103, 2 (19 JANUARY 2011)

Can Treatment Costs Be Tamed?

www.sciencemag.org SCIENCE VOL 331 25 MARCH 2011 Published by AAAS

1545

SPECIALSECTION “pulse” of a cocktail for several days. As his STM study suggests, clinicians would need to constantly biopsy patients and tailor the cocktail to the mutations in their tumor. This could stretch responses out to years, Engelman says. “A lot of this is going to have to be done by trial and error,” Pao agrees. Sawyers and others point out, however, that such combination therapies developed in the 1960s and ’70s eventually vanquished most cases of childhood leukemia, Hodgkin lymphoma, and testicular cancer. Major cancer centers are already routinely genotyping biopsies from patients throughout their treatment using procedures that are less invasive than surgery, such as collecting a few tumor cells with a thick needle. Engelman says this is essential: “We can’t be afraid to rebiopsy to see what’s going on.” Cancer researchers acknowledge that coming up with cocktails to corner cancer will be much more difﬁcult than it was with HIV. Cancer geneticist Bert Vogelstein

of Johns Hopkins University in Baltimore, Maryland, points out that cancer is different from AIDS: Among other things, tumors vary more from patient to patient than HIV does; the genetic heterogeneity of cancer cells within a single patient is “orders of magnitude greater” than HIV genotypes in patients, so tumors have a “much larger reservoir of resistance mechanisms” that go beyond those already uncovered. “Unfortunately, we’ve got billions of cancer cells ready to become resistant, and it takes 15 years to develop each new drug,” Vogelstein says. Moreover, to wipe out a tumor completely, he and others say, a cocktail might also have to include a drug that targets stem cell–like cells in a tumor that continuously give rise to tumor cells. Turning cancer into a manageable disease, however, “would be wonderful,” Vogelstein says, if it can be done. Advocates of targeted therapy think they will get there. “Melanoma was completely untreatable 18 months ago,” says Neal Rosen of MSKCC. “Give us a chance.” –JOCELYN KAISER

Downloaded from www.sciencemag.org on March 24, 2011

His team analyzed 37 biopsy samples from patients with the EGFR mutation who were given Iressa or Tarceva and later became resistant. Although some resistance mutations were known, others were new, and for 30% of the samples, his team could not identify the mechanism. Some tumors even morphed into a different type of lung cancer that requires an entirely different treatment. In addition, biopsies from three patients collected during the course of treatment showed that tumors changed: Some that developed resistance mutations later lost them. “It’s very, very complex. It’s not ﬁtting into the simple boxes that we’ve made until now,” Engelman says. Cocktail therapy will face another issue that has not been well explored so far: the risk that combining two drugs—particularly ones that target different pathways used by normal cells—can lead to unacceptable side effects. Engelman thinks that for this reason, patients will be able to tolerate a cocktail for only a short time. He envisions putting them on a single drug, then intermittently giving them a

come with sticker-shock prices of $50,000 or more per patient. New and more costly, however, haven’t necessarily meant better. Although targeted treatments, which attack a molecuMore patients and the rising costs of new cancer treatments spark debate over how lar weak spot in the tumor’s support sysmuch is too much—and who should decide tem, have helped improve survival rates for many cancers, some extend life for just a THE AFTERNOON JEFF GUSTAFSON LOST HIS according to a recent estimate. By 2020, it few weeks or months (see p. 1542). And the life to stomach cancer, a rainbow appeared could grow by as much as 66%, to $207 bil- prices can be sobering: more than $1.2 milabove his Arlington, Virginia, hospice. lion. Multiple forces are driving the spiral: lion to extend a lung cancer patient’s life for Even if the shimmering arc had led to a a growing and aging population, more peo- 1 year in one scenario involving a costly but pot of gold, however, it’s not clear whether ple living longer with cancer, and new “per- common drug. That example is unusual, but the treasure would have covered the cost sonalized,” or “targeted,” therapies that can such numbers have sparked a growing—and of treating the 48-year-old sometimes feisty—debate PROJECTED COSTS OF TOTAL U.S. CANCER CARE, 2010–20 builder, who was known for his over how best to calculate the wit and compassion. The bills beneﬁts of new cancer treat220 for just 4 months of dogged, ments, whether their use will 5% increase in annual costs, first and last year of care full-tilt treatment totaled more lower or raise per-patient 200 than $350,000—including expenses, and who should 2% increase in annual costs one drug that cost more than decide whether using them is 180 $12,000 per dose. worth the cost. “The question Population growth only Gustafson’s story isn’t that is, ‘Are we spending too much 160 Current trends in incidence unusual to experts who track for too little?’ ” says oncoloand survival, projected 140 the costs of treating cancer in gist Antonio Tito Fojo of the the United States—and are U.S. National Cancer Institute 120 increasingly worried about (NCI) in Bethesda, Maryland. where they are headed. Over 100 the past 3 decades, total U.S. Demographic drivers 2010 2020 spending on cancer care has Tallying the current and more than quadrupled, reach- Four scenarios. Experts predict total U.S. spending on cancer care could rise by future costs of treating caning $125 billion last year, or as much as 66% by 2020, depending on shifts in disease incidence and survival, cer isn’t easy. “Cancer” now 5% of the nation’s medical bill, and treatment costs. includes more than 100 difNEWS

U.S. $ (billions)

CREDIT (GRAPH SOURCE): A. MARIOTTO, ET AL., JOURNAL OF THE NATIONAL CANCER INSTITUTE 103, 2 (19 JANUARY 2011)

Can Treatment Costs Be Tamed?

www.sciencemag.org SCIENCE VOL 331 25 MARCH 2011 Published by AAAS

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Worth the cost? The impending d e m o g r a p h i c a l ly driven increases are “inevitable,” the authors note. Harder to pin down, however, is the future impact of technological changes that, overall, have been rapidly pushing up costs. In particular, chemotherapy prices have been escalating faster than other medical costs, largely because of One life. After standard treatments failed to stop Jeff Gustafson’s stomach can- the introduction of cer, his insurer approved the experimental use of one expensive drug, but it targeted drugs that come with price tags failed to extend his life. higher than those of last year of life, but with relatively less spent many older compounds. Although studies in between. For instance, men under 65 with suggest that cancer drugs typically account stomach cancer—such as Gustafson—spent for less than 15% of a patient’s total treatan average of $94,000 per year on “initial” ment costs, experts say that is changing. care in 2010 and $161,000 in the last year of For instance, an increasingly used drug life, but just $4200 per year for continuing called bevacizumab—sold under the name care. In part, that’s because stomach can- Avastin—can cost from $30,000 to $62,000 cer can kill quickly, and for those who don’t per patient per course of treatment, dependdie early, end-stage costs can be very high. ing on the targeted cancer, according to But the numbers also reﬂect the fact that estimates compiled by NCI’s Fojo and colyounger patients typically get more aggres- leagues. In contrast, some drugs used at the sive—and more costly—care than older beginning of the war on cancer in 1971 cost ones, perhaps because society perceives a just a few hundred dollars per patient. greater potential beneﬁt to extending the Part of the problem, Fojo and his collife of a younger person. Patients under 65 leagues have argued in a pair of provocative now make up about 40% of all cancer cases, papers, is that the new, costlier drugs often the JNCI team estimated, and typically fail to improve survival appreciably. They receive care that costs about 35% more than don’t work for all patients, and when they that of older patients. do work, the effect is often limited. To highlight such problems, in both a 2009 JNCI U.S. CANCER PREVALENCE paper and a 2010 paper published in Clinical Cancer Research, Fojo’s team focused on a Number of people living with cancer “breakthrough” treatment that was touted at a major annual cancer research meeting Younger than 65 2010 in 2008. A study had shown that non-small 65 and older cell lung cancer patients treated with cetuximab (sold as Erbitux) lived, on average, 1.2 months longer than those receiving standard treatments. One scenario, which called 2020 for using cetuximab for 18 weeks at a cost of $40,000, translated to a cost of $496,000 for one extra “quality-adjusted life year” 0 2 4 6 8 10 12 (QALY). In contrast, they noted, kidney Millions dialysis, a lifesaving technology Medicare Surviving. One of the major drivers of increased made universally available in 1972, costs costs is that more people are living longer with $129,090 per QALY. Another scenario, using bevacizumab cancer.

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ferent diseases that affect more than 13 million people in the United States each year, and there is no single source of uniform statistics. To come up with their recent forecasts, a team led by NCI’s Angela Mariotto turned to a massive trove of data kept by the federal government’s Medicare health insurance program, which now covers some 40 million Americans over age 65. Sieving the data, they were able to extract a rough snapshot of the costs associated with treating nearly 1.8 million Medicare patients diagnosed with cancer between 1975 and 2005. Overall, they examined incidence, survival, and cost trends for 13 cancers in men and 16 cancers in women, and estimated costs for patients younger than 65. The result, published last January in the Journal of the National Cancer Institute (JNCI), reveals some of the complex demographic and technological forces that are pushing costs down, even as the overall total rises. Earlier studies, for instance, have suggested that average per-patient costs for many cancers have declined slightly in recent years, largely because outpatient treatment has often replaced more expensive in-hospital stays. The JNCI study, however, shows that those savings are being swamped, in part, by the growing number of older people, who are more likely to get cancer. Medicare predicts its rolls will nearly double, to 70 million people, by 2020. And the JNCI study forecasts that some 16% of these older Americans— about 11.4 million people—will have cancer, up from 8 million today. Another 6.6 million younger people will also be living with cancer. Even if all other trends—such as the cost of individual treatment—don’t change, they estimate that those demographic changes alone will push national cancer care costs up 27% by 2020. Ironically, another factor driving up costs is that people are now surviving cancers that might have killed them quickly decades ago. Some of the largest projected cost increases, for instance, are linked to providing “continuing care” for people living with breast and prostate cancer, a group expected to grow by as much as 41%, to nearly 8 million in 2020. It will cost some $18 billion to provide continuing care for these patients, the team estimates, some 30% to 40% more than current costs. The study notes, however, that spending tends to follow a u-shaped curve over the period of cancer treatment, with the highest costs coming just after diagnosis and in the

CREDITS (TOP TO BOTTOM): COURTESY MOLLY SIM; (GRAPH SOURCE): A. MARIOTTO, ET AL., JOURNAL OF THE NATIONAL CANCER INSTITUTE 103, 2 (19 JANUARY 2011)

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at a cost of $30,000, translated to a QALY say. They view this as a “radically egalitar- a number of CED experiments, and the new cost of $1.2 million. And such drugs are “not ian” approach that forces insurers to choose health care reform law authorizes extensive alone among treatments offering marginal between drugs they “can afford for all, as new efforts to compare the effectiveness of beneﬁt at very high cost,” they wrote: More opposed to those they will fund for no one.” different medical treatments, including cancer than 90% of the cancer drugs approved by They also argue that preventing doctors from drugs. In 2014, it also requires private insurthe U.S. Food and Drug Administration using drugs off-label would hobble the proven ers to cover the routine costs of enrolling can(FDA) in the previous 4 years had cost more practice of freeing doctors to ﬁnd promising cer patients in clinical trials, removing a major than $20,000 for 12-week treatments. “We new uses for existing drugs. And it would barrier to their participation. must stop deluding ourselves into thinking stand “in stark contrast with clinical practice.” that prescribing … expensive chemothera- Studies, for instance, suggest that up to 75% Agonizing decisions pies and tests are an aberration, a temporary of anticancer drugs are already used off-label. Although such studies didn’t help Gustafson, deviation from an otherwise reasonable cost And price controls would, they argue, ulti- his treatment did reﬂect both the challenge trajectory,” they concluded. mately cause investors to reduce funding for and opportunity inherent in efforts to tamp Along with their critique, Fojo and his col- research into new drugs because they couldn’t down the costs of cancer care. His oncololeagues offer solutions. They argue that Medi- be sure of recouping their costs. There is one gists, for instance, talked with him and his care and other U.S. insurers, for instance, thing the two sides do agree on: Better, more wife, Molly Sim, about the costs they were shouldn’t pay for drugs that would cost more organized studies could help steer the right likely to encounter. That’s something that a than $129,090 per QALY. That would still drugs to the right patients. Better genetic tests, recent statement from the American Society make the United States somewhat more gen- for instance, could identify patients unlikely of Clinical Oncologists says needs to happen erous than other nations, including the United to beneﬁt from a particular drug. They could more often, given how many families exhaust Kingdom, Australia, and Canada, whose gov- also help researchers design smaller, less their savings ﬁghting cancer. “We were forernment-run health services routinely reject costly trials that better identify smaller groups tunate, we had really good insurance,” says the use of new cancer drugs because they of patients likely to respond; current trials Sim, who ended up paying less than $6000 can’t meet certain QALY out of pocket. THE PRICE OF TREATMENT costs. Fojo’s group also Gustafson’s doctors says that drug compaalso gave him genetic Initial Continuing Last year nies shouldn’t fund tritests to determine 350,000 als designed to detect whether he should be survival improvements given one expensive but 300,000 of fewer than 2 months promising drug; unforunless the drug will cost tunately, the tests indi250,000 less than $20,000 and cated that he wouldn’t 200,000 should even consider benefit. But when he reducing charges if a failed to respond to 150,000 drug doesn’t work in a standard treatments, particular patient. Docthey proposed a last100,000 tors should use costly ditch strategy: Sim suc50,000 drugs only in the subset cessfully appealed to of patients proven to gain her insurer to pay for 0 benefit, they say, and off-label use of AvasFemale-Breast Female-Brain Male-Stomach Male-Lung Female- Maleavoid using new drugs tin at a cost of nearly (under (65 and (under (65 and (under (65 and (under (65 and Pancreas Pancreas 65) over) 65) over) 65) over) 65) over) (under (65 and “off-label,” i.e., to treat $40,000. Gustafson 65) over) cancers other than those died in November 2009, for which the drug was Varying costs. The average annual cost of treatment can vary, depending on the patient’s age and before he could comapproved by FDA. Such cancer type. In general, costs are higher just after initial diagnosis and in the last year of life. plete the treatment. steps, Fojo says, “would It’s a story that focus our attention on using drugs that deliver often “wash out” these drugs because the ben- Fojo—who urges caution in using expensive proven benefits” until researchers can find eﬁts seem statistically negligible. drugs—says he knows all too well. “In the better ways to efﬁciently develop and use tarAnother idea gaining ground is “cover- abstract, you say using these marginal drugs geted therapies. age with evidence development” (CED), is crazy,” he said recently, shortly after comSome disagree sharply. Such ideas are in which insurers link payments for certain ing off his rounds treating cancer patients. based on “ﬂawed premises,” Joshua Cohen drugs to efforts to collect data on compara- “But when you have the patient in front of and William Looney of Tufts University tive effectiveness, with the aim of discontinu- you, and nothing else is working, well, I School of Medicine in Boston argue in a ing coverage for drugs that don’t work. Cohen might try the crazy thing, too. We used to do November 2010 response published in Nature and Looney even suggest that CED could be it with cheap drugs, but now we are doing it Biotechnology. Using QALY calculations combined with “risk-sharing arrangements,” with really expensive drugs. The problem is to rule out therapy, for instance, is a biased, in which insurers and manufacturers agree they really don’t work any better.” “blunt instrument” that ends up denying to link a drug’s price to its performance. U.S. –DAVID MALAKOFF drugs to patients who need them, the authors Medicare managers have already launched David Malakoff is a writer in Alexandria, Virginia. Annualized mean net costs in 2010 dollars

CREDIT (GRAPH SOURCE): A. MARIOTTO, ET AL., JOURNAL OF THE NATIONAL CANCER INSTITUTE 103, 2 (19 JANUARY 2011)

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A Push to Fight Cancer in the Developing World

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on infectious diseases. Now, some argue, it’s time to start closing an equally unconscionable gap between rich and poor nations in cancer prevention, diagnosis, and treatment. The numbers speak volumes. A child suffering from leukemia in Western Europe has an 85% chance of survival; in the 25 poorest countries in the world, it’s just over 10%. For a man with testicular cancer, the numbers are about 95% and just over 40%. Estimates suggest that less than 5% of the world’s cancer resources are spent in the developing world. In many countries, even painkillers are hard or impossible to come by—“a violation of human rights,” Shulman says. In a bid to change that, oncologists at topﬂight centers in the United States and Europe are now taking time out NEWS to help improve cancer care in low- and middle-income countries. In an article last month, World Health Organization DirectorGeneral Margaret Chan said that cancer needs to be “acknowledged as a vital part of the global health agenda.” Cancer will also feature on the agenda at the United Nations Cancer and other chronic diseases have received little attention from global health High-level Meeting on Non-Communicable advocates. That’s beginning to change Diseases in September in New York City. Shulman and Harvard School of Public BOSTON—In one of his PowerPoint presenIt’s a message that is beginning to be Health Dean Julio Frenk co-chair the new tations, Lawrence Shulman has a series of heard. Global health has gained in promi- Global Task Force on Expanded Access photographs that are hard to forget. One nence on political agendas in recent years, but to Cancer Care and Control in Developshows Tushime, an 11-year-old girl in attention has been overwhelmingly focused ing Countries (GTF.CCC), which aims to Rwanda suffering from rhabdomyosarcoma, move cancer up on the global THE CANCER SURVIVAL GAP a rare cancer of the muscles. A tumor resempriorities list. The group— bling a cauliflower is growing out of her which combines expertise in 0.8 right cheek. cancer, global health, eco0.7 The good news comes in Shulman’s nomics, ﬁnance, and policy— 0.6 next three slides. Over a 10-week treatment published a 10-page call to 0.5 course with drugs donated by a U.S. proaction last year in The Lancet 0.4 gram, the mass started to shrink until evenand is now working on a col0.3 tually it could be removed surgically. The lection of papers for the same 0.2 last picture shows Tushime, standing with journal that details what needs 0.1 her happy family and—despite a somewhat to be done. 0.0 lopsided face—looking healthy. The obstacles are major, and Low Lower middle Upper middle High Shulman is chief medical ofﬁcer at the some people question whether Country income Dana-Farber Cancer Institute here, which battling cancer is the wisest use Breast Cervix uteri Testicular Prostate Non-Hodgkin lymphoma Hodgkin lymphoma is afﬁliated with Harvard Medical School of scarce global-health money. Colorectal Thyroid Leukemia (0-14 years of age) (HMS), and to him the pictures carry a powSimilar doubts were once raised erful message: Given that treatments are Life and death. For many cancers, the case fatality rate (of which about infectious diseases, readily available, how can you not treat a the ratio of mortality to incidence is a proxy) is much higher in says HMS physician and GTF. child suffering from a very curable cancer? CCC member Paul Farmer: poor countries than in rich countries.

CREDITS (TOP TO BOTTOM): COURTESY OF PARTNERS IN HEALTH; (GRAPH SOURCE) GTF.CCC, MEXICAN HEALTH FOUNDATION; ESTIMATES BASED ON IARC GLOBOCAN DATA FOR 2002 AND 2008

for cervical cancer at a Partners In Health clinic in Rwanda.

SPECIALSECTION 15 years ago people questioned the logistical and ﬁnancial feasibility of treating HIV and multidrug-resistant tuberculosis (TB) in poor countries. Yet as Farmer points out, both are now being addressed on a large scale. Great strides have been made recently in a range of other tropical diseases, too. So why can’t it be done for cancer?

A more difﬁcult job Cancer takes a markedly different toll depending on the country. Lung cancer, a major killer in the West, is rarer in Africa, where fewer people smoke and life expectancy is shorter. Several virus-related cancers, on the other hand, are much more prevalent in poor countries. Cervical can-

cer, caused by the human papillomavirus (HPV), has become quite rare in rich countries thanks to screening with Pap smears. But it is common in the developing world, where 93% of the estimated 273,000 annual deaths from this disease occur. Taken together, however, the burden of cancer is still lower in the poorer coun-

CREDIT: KENT DAYTON/HSPH/©PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Making Her Life an Open Book to Promote Expanded Care she thought, so she wrote a frank book about her experience. The resulting TV interviews with her and Frenk made waves. She and Frenk took the campaign to the next level after he was appointed dean of HSPH in 2008 and she became director of the Harvard Global Equity Initiative, a research program founded by Nobel laureate Amartya Sen. She had the idea to start a global task force to expand cancer care in poor countries. Frenkk I sickness and in health. In now co-chairs it withh Felicia Knaul—with husn, Lawrence Shulman, band, Julio Frenk—wrote chief medical ofﬁcer of about her disease. cer the Dana-Farber Cancer Institute in Boston. Many of their well-placed friends signed on: former UNAIDS chief Peter Piot, director of the London School of Hygiene and Tropical Medicine; Columbia University economist and poverty warrior Jeffrey Sachs; and CNN chief medical correspondent Sanjay Gupta. “They have really brought this to the doorstep of many people at high levels,” says Carlos Rodriguez, a pediatric oncologist at Dana-Farber. The task force is now collecting evidence on how cancer care in developing countries can be improved, she says. To do so, she has helped recruit a series of papers for publication in The Lancet (see main text, p. 1548). Her own story, she realizes, is atypical for a woman in Mexico. Still, it reinforces her message, she says: “When you say at a meeting, ‘I have cancer,’ people listen in a way that happens with very few other diseases.” Her frankness is part of her strategy. Mexican couples thanking her for her book will sometimes mention “chapter 18,” she says, in which she discusses how the chemotherapy-induced menopause shut down her sex life. At her art-ﬁlled house 30 kilometers west of Boston, Knaul also discussed, matter-of-factly, why, in her case, reconstructive surgery was not successful. (“The implant dropped a couple of inches,” she says, because of a lack of tissue to hold it up.) Her own marriage didn’t suffer, she adds—on the contrary, the disease brought the two closer, and for the book, Frenk contributed fragments of four love letters written when she was ill. As Knaul wrote, “I had my boyfriend back.” –M.E.

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SUDBURY, MASSACHUSETTS—”You can ask me absolutely anything,” says Felicia Knaul—and she’s serious. Knaul, a health economist at Harvard Medical School in Boston, doesn’t mind telling in detail how breast cancer changed her marriage, her family, and her career. In her mission to shatter taboos around the disease and improve the lives of patients in developing countries, her professional and private lives have become one. Knaul is one half of a Mexican-Canadian power couple that aims to end the neglect of cancer as a disease of the poor—and will succeed, if anyone can, say colleagues. The other half is Julio Frenk, the former health minister of Mexico and a much admired reformer, who is now dean of the Harvard School of Public Health (HSPH). Knaul, born and raised in Toronto, says she has always had a keen interest in poverty and health. She lived in Bogotá for 2 years in the early ‘90s, working on a project for street children and helping the Colombian government reform its health system. After meeting Frenk, who comes from a family of doctors, she moved to Mexico; she now considers it home. She joined the Mexican Health Foundation in Mexico City, where she still leads a research group. When Frenk became minister in Vicente Fox’s Cabinet in 2000, she worked pro bono to help him push through a major reform that extended basic health coverage to the country’s poorest and took effect in 2003. That didn’t prepare her for her own terrifying brush with illness. In October 2007, a technician in Cuernavaca discovered a lump in Knaul’s left breast. It was malignant. After weeks of anguish, she underwent a mastectomy and started on chemotherapy. Knaul says she was lucky. She had access to the best doctors in Mexico, and—because her husband took a job at the Bill and Melinda Gates Foundation after he left the government in 2006—she even had insurance coverage in the United States. Knaul used it to seek additional treatment at the Seattle Cancer Care Alliance. She now rates her 5-year survival chance at between 85% and 90%. That’s much more than most Mexican women with breast cancer can expect. Coverage for breast cancer treatment was added to Frenk’s insurance plan for the poorest in 2007; in reality, some women still don’t get treatment, for instance, if they live far from a hospital. Early diagnosis is rare. Many women forgo mammograms even where they are available, Knaul says, and a culture of machismo often leads men to abandon their wives or girlfriends if they lose a breast. Knaul recalls a woman recently diagnosed with breast cancer at one public event saying: “A woman without breasts is ugly. I don’t want to be ugly.” Knaul decided to start a program—it is now a not-for-proﬁt group— called Cáncer de Mama: Tómatelo a Pecho (Breast Cancer: Take It to Heart) that aims to raise awareness of and improve access to prevention, early detection, and treatment. Sharing her own story might help other women,

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and its chief technologist is training staff members. “We’re totally committed to doing this,” Shulman says. Going horizontal Expanding cancer care will cost money. Take the new HPV vaccines. So far they have been introduced in wealthy countries, but cancer experts say they would prevent far more cases of cervical cancer in poor countries, where Pap smears are seldom done. Their cost, more than $300 per vaccinated teenage girl in the West, has been a barrier to their introduction in the developing world. But with the economy in a global dip, it’s hard to see where the money will come from. Cancer is competing for attention with the infectious diseases, which have seen a major increase in program support over the past decade but are still underfunded. Meanwhile, other diseases such as diabetes and mental illness are vying for attention as well. Mental health advocates are already disappointed that they won’t have a seat at the September U.N. meeting on noncommunicable diseases, where the task force is trying to make cancer well-represented. But it’s not one or the other, Farmer argues: “We have to get away from the whole notion of choosing between diseases.” Gene Bukhman, head of Harvard’s Program in Global Non-communicable Disease and Social Change, advocates building up health systems broadly so that they can deal with not just cancer but also diabetes, heart disease, and a variety of other chronic ailments that each make up a small percentage of the disease burden. But this approach— sometimes dubbed “horizontal” as opposed to “vertical” disease-speciﬁc programs—isn’t particularly popular. The Bill and Melinda Gates Foundation is spending its billions mostly vertically, focusing on specific diseases. Likewise, many advocacy groups want to extend their work—say, on breast or prostate cancer—to the world’s poor, but they’re less interested in helping ﬂedgling health systems. Farmer, too, wants to channel the enthusiasm generated by single diseases into help for a better health system: “The breast cancer advocate needs to see that without a proper health system we’re never going to get early detection or good treatment.” Emotional appeals can help, Bukhman says, and that is where Tushime’s pictures come in. “I don’t think we’ve said this enough. When you see someone dying needlessly of cancer, it is an obscene inequality”—no different from seeing someone die from TB or HIV.

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sarcoma, an HIV-related cancer of the skin. The price of drugs is not insurmountable, says HMS health economist and breast cancer advocate Felicia Knaul, who runs the task force’s secretariat (see sidebar, p. 1549). Of a list of 27 essential cancer drugs compiled by the task force, 24 are offpatent, and prices could be brought down further through negotiations with the pharmaceutical industry, Knaul says. A recent study showed that drugs needed to treat a case of Burkitt’s lymphoma—a cancer that primarily affects African children and is associated with the Epstein-Barr virus— cost less than $50, a bargain in terms of life years saved per dollar. The biggest challenges in combatAREAS OF OPPORTUNITY ing cancer—as for other diseases—are to build up expertise and infrastructure Here are some cancer types for which experts say major progress could be made in developing and extend care to the poorest people. countries: Some private groups are intervening directly. For the past 18 years, pediatCurable with early detection and treatment ric oncologists at St. Jude Children’s ● Breast cancer Research Hospital in Memphis, Ten● Cervical cancer nessee, which sets aside about 1% of its annual budget for global health, have Curable with inexpensive chemotherapy pioneered so-called twinning programs ● Non-Hodgkin lymphoma that provide assistance to hospitals in ● Hodgkin lymphoma ● Testicular cancer 20 countries to improve cancer care for ● Sarcoma in children children. Several other pediatric hospi● Acute lymphoblastic leukemia in children tals have followed suit. The impact is real: In El Salvador, where the twinPalliation with systemic treatment ning started, the 5-year survival rate for ● Advanced breast cancer children with acute lymphoblastic leu● Kaposi sarcoma kemia went from 10% to 60%, and is still climbing. Preventable Partners In Health (PIH), best ● Tobacco-related: lung cancer, head and known for its pioneering work ﬁghtneck cancer, bladder cancer ● Vaccine-preventable: cervical cancer ing AIDS and TB in Haiti, has long (HPV) and liver cancer (hepatitis B) treated cancer patients as well, says co-founder and director Farmer: “They often go from one clinic to the next, in short supply; oncologists are extremely seeking care.” But PIH is also working with scarce. There aren’t enough surgeons or governments to strengthen their health sysoperating rooms; 30 countries—half of tems—and expanding cancer care is a key them in Africa—don’t have a single radia- goal. The Rwandan government is very keen tion therapy machine. on it, Farmer says. Still, in The Lancet paper, Shulman and Currently, the PIH network still relies on colleagues at the task force identiﬁed a list Boston’s medical infrastructure for backup. of cancers for which a lot can be done even Brigham and Women’s Hospital’s patholin places with poor infrastructure (see table). ogy department examines specimens to These measures include battling tobacco identify tumors, for instance, an indispensuse—which increases the risk of various able part of diagnosis. (Shulman himself cancers as well as cardiovascular disease— has returned from Malawi with a suitcase vaccinating against HPV and hepatitis B, full of specimens. “Fortunately, nobody at improving early detection, treating eminently customs checked,” he says.) But the plan is curable tumors such as Tushime’s sarcoma to develop pathology expertise and infraand childhood leukemia, and improving life- structure locally. Brigham and Women’s has extending treatment for cancers like Kaposi donated equipment for Haiti and Rwanda,

CREDIT: THINKSTOCK; TABLE SOURCE: L. SHULMAN

tries, which is one reason why it has failed to get global health policymakers’ full attention. Unfortunately, the developing world is catching up. In many middle-income countries, life expectancy is increasing, and obesity and smoking are on the rise, all of which lead to more cases of cancer. Women are having their children later in life and breastfeeding for a shorter time, which increases their risk of breast cancer. And treating cancer is “much more difﬁcult” than treating malaria or TB, Shulman says. Diagnosis is complex, requiring competently staffed and well-equipped pathology labs. Physicians in many countries are

SPECIALSECTION 15 years ago people questioned the logistical and ﬁnancial feasibility of treating HIV and multidrug-resistant tuberculosis (TB) in poor countries. Yet as Farmer points out, both are now being addressed on a large scale. Great strides have been made recently in a range of other tropical diseases, too. So why can’t it be done for cancer?

A more difﬁcult job Cancer takes a markedly different toll depending on the country. Lung cancer, a major killer in the West, is rarer in Africa, where fewer people smoke and life expectancy is shorter. Several virus-related cancers, on the other hand, are much more prevalent in poor countries. Cervical can-

cer, caused by the human papillomavirus (HPV), has become quite rare in rich countries thanks to screening with Pap smears. But it is common in the developing world, where 93% of the estimated 273,000 annual deaths from this disease occur. Taken together, however, the burden of cancer is still lower in the poorer coun-

CREDIT: KENT DAYTON/HSPH/©PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Making Her Life an Open Book to Promote Expanded Care she thought, so she wrote a frank book about her experience. The resulting TV interviews with her and Frenk made waves. She and Frenk took the campaign to the next level after he was appointed dean of HSPH in 2008 and she became director of the Harvard Global Equity Initiative, a research program founded by Nobel laureate Amartya Sen. She had the idea to start a global task force to expand cancer care in poor countries. Frenkk I sickness and in health. In now co-chairs it withh Felicia Knaul—with husn, Lawrence Shulman, band, Julio Frenk—wrote chief medical ofﬁcer of about her disease. cer the Dana-Farber Cancer Institute in Boston. Many of their well-placed friends signed on: former UNAIDS chief Peter Piot, director of the London School of Hygiene and Tropical Medicine; Columbia University economist and poverty warrior Jeffrey Sachs; and CNN chief medical correspondent Sanjay Gupta. “They have really brought this to the doorstep of many people at high levels,” says Carlos Rodriguez, a pediatric oncologist at Dana-Farber. The task force is now collecting evidence on how cancer care in developing countries can be improved, she says. To do so, she has helped recruit a series of papers for publication in The Lancet (see main text, p. 1548). Her own story, she realizes, is atypical for a woman in Mexico. Still, it reinforces her message, she says: “When you say at a meeting, ‘I have cancer,’ people listen in a way that happens with very few other diseases.” Her frankness is part of her strategy. Mexican couples thanking her for her book will sometimes mention “chapter 18,” she says, in which she discusses how the chemotherapy-induced menopause shut down her sex life. At her art-ﬁlled house 30 kilometers west of Boston, Knaul also discussed, matter-of-factly, why, in her case, reconstructive surgery was not successful. (“The implant dropped a couple of inches,” she says, because of a lack of tissue to hold it up.) Her own marriage didn’t suffer, she adds—on the contrary, the disease brought the two closer, and for the book, Frenk contributed fragments of four love letters written when she was ill. As Knaul wrote, “I had my boyfriend back.” –M.E.

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SUDBURY, MASSACHUSETTS—”You can ask me absolutely anything,” says Felicia Knaul—and she’s serious. Knaul, a health economist at Harvard Medical School in Boston, doesn’t mind telling in detail how breast cancer changed her marriage, her family, and her career. In her mission to shatter taboos around the disease and improve the lives of patients in developing countries, her professional and private lives have become one. Knaul is one half of a Mexican-Canadian power couple that aims to end the neglect of cancer as a disease of the poor—and will succeed, if anyone can, say colleagues. The other half is Julio Frenk, the former health minister of Mexico and a much admired reformer, who is now dean of the Harvard School of Public Health (HSPH). Knaul, born and raised in Toronto, says she has always had a keen interest in poverty and health. She lived in Bogotá for 2 years in the early ‘90s, working on a project for street children and helping the Colombian government reform its health system. After meeting Frenk, who comes from a family of doctors, she moved to Mexico; she now considers it home. She joined the Mexican Health Foundation in Mexico City, where she still leads a research group. When Frenk became minister in Vicente Fox’s Cabinet in 2000, she worked pro bono to help him push through a major reform that extended basic health coverage to the country’s poorest and took effect in 2003. That didn’t prepare her for her own terrifying brush with illness. In October 2007, a technician in Cuernavaca discovered a lump in Knaul’s left breast. It was malignant. After weeks of anguish, she underwent a mastectomy and started on chemotherapy. Knaul says she was lucky. She had access to the best doctors in Mexico, and—because her husband took a job at the Bill and Melinda Gates Foundation after he left the government in 2006—she even had insurance coverage in the United States. Knaul used it to seek additional treatment at the Seattle Cancer Care Alliance. She now rates her 5-year survival chance at between 85% and 90%. That’s much more than most Mexican women with breast cancer can expect. Coverage for breast cancer treatment was added to Frenk’s insurance plan for the poorest in 2007; in reality, some women still don’t get treatment, for instance, if they live far from a hospital. Early diagnosis is rare. Many women forgo mammograms even where they are available, Knaul says, and a culture of machismo often leads men to abandon their wives or girlfriends if they lose a breast. Knaul recalls a woman recently diagnosed with breast cancer at one public event saying: “A woman without breasts is ugly. I don’t want to be ugly.” Knaul decided to start a program—it is now a not-for-proﬁt group— called Cáncer de Mama: Tómatelo a Pecho (Breast Cancer: Take It to Heart) that aims to raise awareness of and improve access to prevention, early detection, and treatment. Sharing her own story might help other women,

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Brothers in Arms Against Cancer Cancer researchers are trying to harness siblings of p53, the famous tumor-blocking protein

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He was a physician, inventor, and philosopher of some repute. Familiar with Cassandra Austen, the amateur painter and Jane’s sister? Probably not. Famous brothers and sisters often overshadow their siblings. The same thing happens in molecular families. Take p53, the tumor-suppressor protein that was named Science’s Molecule of the Year in 1993 and has been dubbed “guardian of the genome.” Few nonspecialists know that the celebrated p53 is closely related to two other proteins, p63 and p73. Yet these unheralded siblings are grabbing the attention of cancer biologists. New research suggests that p63 and p73 are ﬁerce cancer killers that deserve equal billing with p53. Instead of a single genome protector, “there’s a family of guardians,” says cancer biologist Elsa Flores of M. D. Anderson Cancer Center in Houston, Texas. Because efforts to exploit p53 in cancer therapies haven’t yet paid off, some researchers are now looking to p73 and p63 as alternative tumor treatments. Researchers have shrunk or prevented tumors in animals by targeting p73, and the first clinical trials—attempting to use p73 to combat a hard-to-treat type of breast cancer—have already started. “It would be very attractive to ﬁnd a way to activate these proteins” in people with tumors, says cancer biologist Alexander Zaika of Vanderbilt University Medical Center (VUMC) in Nashville. Strategies that capitalize on the tumor-ﬁghting capability of the p53 family “belong in the armamentarium against cancer,” adds molecular oncologist Waﬁk El-Deiry of the Penn State Hershey Cancer Institute in Pennsylvania. p53, the hard target When a cell suffers DNA damage that can lead to uncontrolled growth, p53 comes to the rescue. The p53 protein can trigger DNA repair, stop the dodgy cell from dividing, or, when the damage is grievous, prompt it to commit suicide. Because p53 is so potent, cells normally keep levels of the protein low. Spurring cancer cells to produce more protein, the argument goes, could prompt tumors to self-destruct. “p53

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it, for example, by overproducing enzymes that prompt the protein’s destruction. Attacking many kinds of tumors through p53 will require pharmacological feats— restoring the gene or reshaping the malformed protein—that are tougher than the standard tactic of blocking a molecule’s unwanted function. “Many of us appreciated it [p53] would be difﬁcult to target,” says cancer biologist Leif Ellisen of Harvard Medical School (HMS) in Boston. “Throwing a wrench into the system is much easier than ﬁxing the system.” Yet many researchers remain conﬁdent that they will eventually overcome these obstacles. “My personal bet is still on p53,” says cancer biologist Anna Mandinova, also of HMS. But in the meantime, researchers are looking at other options, namely, p63 and p73.

YOU’VE HEARD OF CHARLES DARWIN, BUT do you know of his elder brother, Erasmus?

is a great therapeutic target,” says cancer biologist Kevin Ryan of the Beatson Insti- Band of molecular brothers tute for Cancer Research in Glasgow, U.K. Scientists discovered the p53 protein in “Its involvement in tumor suppression is 1979 but didn’t recognize its importance for cancer until 10 years later. In 1997, biolowithout question.” Scientists are pursuing a number of gists identiﬁed p73 as a molecular relative, strategies to enlist p53 in the cancer ﬁght after discovering a DNA sequence closely (Science, 2 March 2007, p. 1211). They resembling the gene for p53. “That came have completed or are running several clini- as a bit of a surprise,” says cancer biologist cal trials for gene therapy approaches, which Gerry Melino of the University of Rome involve introducing extra copies of the p53 “Tor Vergata” in Italy. “Nobody expected gene into cancer cells. Researchers at six a protein so close to p53.” Yet researchers institutions in the United States and the reported another relative, p63, the next year. Although p53 was the ﬁrst family member United Kingdom have also begun safety trials on a compound, developed by the phar- discovered, p63 and p73 are the older siblings, maceutical company Roche, that hikes p53 evolutionarily speaking. Comparisons of levels in cells by impeding the protein’s nat- their genes suggest that p53 evolved from the ural recycling. ancestral version of p63 and p73 more than So far, however, no p53-based treat- 450 million years ago. These elders have a ments have been approved for clinical use range of responsibilities. Unlike p53, p63 and in the United States. The practical difﬁcul- p73 are essential during embryonic developties are formidable. For one thing, thanks to ment. Shaping limbs and giving the skin its a variety of mutations in p53’s gene, more layered structure are among p63’s tasks in than half of all tumors an embryo. Formation of Total PubMed Citations don’t carry a working brain regions such as the version of the protein. hippocampus and corSome harbor misshapen tex depends on p73, as mutants that are inert or does maturation of the that turn traitor and subimmune system. Both vert antitumor defenses. proteins are also necesp53 p63 p73 Even when cancer cells sary for female fertility. have a functional form of For cancer research2149 1541 ers, p63 and p73 have p53, they often neutralize 56,939

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Taking on tumors p63 and p73 battle cancer in several ways. Like their famous brother, the proteins cull cells that carry potentially cancer-causing DNA damage, activating their apoptotic, or cell suicide, pathways. Fortuitously, some

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cancer cells into mice and administered RETRA. The treatment cut the number 37AA Disengages p73 Shrinks tumors of tumors that sprouted in from inhibitor in mice the animals, the scientists NSC176327 Boosts p73 Kills cancer cells revealed in the Proceedproduction in culture ings of the National AcadAntisense Curtails production Curbs growth of emy of Sciences. gapmers of p73’s ∆N isoforms melanoma tumors To liberate p73 from a in mice different inhibitor that is Everolimus Increases p73 levels Trials in progress abundant in cancer cells, RETRA Separates p73 from Impedes tumor one known as iASPP, Ryan mutant p53 formation in mice and colleagues ironically On the attack. Researchers are testing antitumor compounds that rely turned to p53 for inspiration. They developed a on p73. snippet of p53, just 37 of chemotherapy already takes advantage of its nearly 400 amino acids, that in the test this ability, causing DNA damage that spurs tube separates p73 from iASPP. Dosing cancer-ridden mice with the snippet, known as p63 or p73 to kill tumor cells. Recent studies suggest that p63 also reins 37AA, shrank the rodent’s tumors, the team in metastasis, the migration of tumor cells to reported in The Journal of Clinical Investia new location in the body; that’s what usu- gation in 2007. RETRA’s effects were fairly weak, and ally kills cancer patients. In 2009 in Cell, a team led by Stefano Piccolo of the Univer- 37AA was fragile, so neither is likely to sity of Padua School of Medicine in Italy become a drug. But the importance of studrevealed that some mutant forms of p53 ies like these, says El-Deiry, is that they found in cancer cells prevent p63 from acti- show it’s possible to uncover compounds that vating two genes that curtail metastasis. And rouse p53’s siblings to attack cancer cells. last fall, in a study in Nature, Flores and col- Scientists are hunting for other molecules leagues showed that the TA isoforms of p63 that might spur p73 and p63 into action and curb metastasis through another mechanism: that could make effective drugs. El-Deiry’s boosting levels of microRNAs, RNA snip- group, for example, is in the middle of a projpets that turn down gene activity. The team ect to screen thousands of small molecules— discovered that p63’s TA isoforms increase they’ve assessed more than 70,000 so far—in production of Dicer, an enzyme that snips hopes of ﬁnding ones that switch on genes in and activates inert microRNAs. The isoforms the p53 pathway. Some of the candidates, he also raise levels of a specific microRNA, says, appear to work by activating p73. At least one group has started clinimiR-130b, that prevents cells from moving on. p63 may be the master regulator of cal trials. Medical oncologist Ingrid Mayer metastasis, says Flores, and activating it and cancer biologist Jennifer Pietenpol might increase levels of several metastasis- of VUMC are targeting a form of breast halting microRNAs by ﬂipping on Dicer. cancer—known as triple-negative because The big question is whether drug design- the tumor cells lack three key receptor proers can capture p63’s and p73’s cancer- teins—that deﬁes standard treatments such quelling talents. The research directed at as tamoxifen and Herceptin. The tumor cells this goal isn’t as intense or advanced as the harbor large quantities of the ∆N isoforms of work on p53, but scientists can claim some p63, which the researchers suspect prevent encouraging ﬁndings. p73 from killing the cells. Along with stanOne therapeutic strategy attempts to dard chemotherapy, patients will receive an reduce sibling rivalry in the p53 fam- existing drug, everolimus, that boosts p73 ily. The mutant p53s found in cancer cells levels by inhibiting a p73 blocker called often latch onto and neutralize p63 and p73. mTOR. The goal is to overcome p63’s interThree years ago, a research group from the ference and enable p73 to kill the tumor cells. Cleveland Clinic in Ohio screened more It’s too late for Erasmus Darwin to match than 46,000 compounds and pinpointed his brother’s fame. But these clinical trials one, named RETRA, that in test tubes and the surge of research on p63 and p73 breaks mutant p53’s embrace of p73. To suggest that the proteins are ﬁnally stepping test whether freeing p73 destroys tumors, out of the shadow of their famous sibling. the researchers then transplanted human –MITCH LESLIE Mechanism

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a big advantage over their sibling: Their genes are almost never mutated in or lost from cancer cells. So p63 and p73 could in theory take over for p53 in almost all cancers, and researchers wouldn’t have to fret about restoring a lost gene or reshaping a distorted protein. That strategy will work, of course, only if p73 and p63 are tumor suppressors like their brother. But that turned out to be surprisingly tough to conﬁrm. A standard experiment for gauging a molecule’s relevance to cancer—deleting its gene from mice— didn’t clarify the issue. Whereas mice missing p53 live to adulthood and are beset by tumors, mice lacking p63 or p73 die young from other causes, revealing little about their cancer susceptibility. Complicating the matter, although some studies found that levels of p73 or p63 fell in cancer cells, suggesting that the proteins are antitumor, other work indicated that their levels soared, implying that they foster abnormal growth. The solution to that apparent contradiction may lie in the discovery more than 10 years ago that cells don’t manufacture just one kind of each protein. They can fashion at least 12 variants, or isoforms, of the p73 protein, and at least eight isoforms of the p63 protein. Scientists divide these varieties into the longer, or TA, isoforms and the shorter, or ∆N, isoforms. In 2008, Melino, molecular geneticist Tak Mak of the University of Toronto in Canada, and colleagues engineered mice that lack all of the TA isoforms of p73 but retain the ∆N versions. The mice were prone to cancer, though they weren’t as vulnerable as rodents lacking p53. The TA isoform-deﬁcient mice “really convinced people that these genes are acting as tumor suppressors,” says Flores. Researchers now hypothesize that in cancer, TA isoforms are generally good guys, suppressing unchecked cell growth. The ∆N isoforms, for the most part, are bad guys. They can latch on to and disable p53 and the good TA isoforms, thus aiding cancer. For example, cancer biologist Alea Mills of Cold Spring Harbor Laboratory in New York and colleagues reported in February in Cell Stem Cell that a common ∆N isoform of p63 spurs the growth of skin tumors.

REVIEW

Exploring the Genomes of Cancer Cells: Progress and Promise Michael R. Stratton* The description and interpretation of genomic abnormalities in cancer cells have been at the heart of cancer research for more than a century. With exhaustive sequencing of cancer genomes across a wide range of human tumors well under way, we are now entering the end game of this mission. In the forthcoming decade, essentially complete catalogs of somatic mutations will be generated for tens of thousands of human cancers. Here, I provide an overview of what these efforts have revealed to date about the origin and behavioral features of cancer cells and how this genomic information is being exploited to improve diagnosis and therapy of the disease. uch of our current understanding of cancer is based on the central tenet that it is a genetic disease, arising as a clone of cells that expands in an unregulated fashion because of somatically acquired mutations (1). These somatic mutations include base substitutions, insertions and deletions (indels) of bases, rearrangements caused by breakage and abnormal rejoining of DNA, and changes in the copy number of DNA segments. They also often include epigenetic changes that are stably inherited over mitotic DNA replication, for example, alterations in methylation of cytosine residues (2). Whether a mature cancer clone emerges in an individual person is influenced by environmental and life-style factors, as well as by the set of genomic sequence variants present in the fertilized egg from which the individual develops and that are therefore found in all somatic cells. These socalled constitutional or “germline” mutations can influence cancer susceptibility in a number of ways, including directly altering growth of the cancer clone, altering the mutation rate in somatic cells, or modulating the metabolism of carcinogens. Somatic mutations are thought to occur in the genomes of all normal cells as they proceed through the rounds of cell division that take place during development in utero and during replenishment of body tissues in postnatal life. Additional somatic mutations continue to accumulate in cancer cells as they divide. The rate of acquisition and the types of somatic mutation that accrue can be increased by exogenous and endogenous exposures that cause DNA damage and are mitigated by DNA repair processes. Indeed, in the event that DNA repair fails, the somatic mutation rate may also increase. Somatic mutations are more or less randomly distributed throughout the genome. However, in the cell that undergoes clonal expansion to become

M

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. *E-mail: [email protected]

a cancer, a subset—termed “driver mutations”— have by chance fallen in a set of key genes, called “cancer genes,” and have thus subverted normal control of cell proliferation, differentiation, death, and other homeostatic interactions with the tissue microenvironment (3). Driver mutations confer growth advantage upon the neoplastic clone, allowing it to expand more than normal cells from the same tissue, invade into surrounding tissue, and, in many cases, metastasize. The number of driver mutations in a cancer cell reflects the number of mutated cancer genes and thus the deregulation of cell biological processes required to convert a normal cell into a symptomatic cancer clone. The remaining—and often the large majority of— mutations are “passengers,” which, by definition, do not confer growth advantage. The number of passenger mutations in a cancer genome primarily reflects the number of mitotic cell divisions between the fertilized egg and the cancer cell and the mutation rate at each of these cell divisions. Thus, the catalog of somatic mutations in the genome of a cancer cell represents genomic changes that usually accumulate over several decades. It includes the mutations responsible for conferring the various aspects of the neoplastic phenotype and bears the imprints of the mutational processes that caused the disease in the first place. Cataloging Mutations in Human Cancer Genomes Over the past half-century a series of technologies have been deployed to characterize systematically, at ever-increasing levels of resolution, the state of cancer genomes across the range of cancer types (Fig. 1). The earliest, and still one of the most influential in its impact on cancer science, was cytogenetic studies of chromosomes from cancer cells. These revealed abnormalities of chromosome copy number and the presence of somatically acquired rearrangements (chromosomal translocations). They showed that some cancer types had very disordered genomes whereas others displayed few genomic abnormalities. They also

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yielded evidence that certain positions in the genome were recurrently rearranged in particular cancer types, from which it was inferred that a cancer gene resided at the rearrangement breakpoints. After the widespread adoption of recombinant DNA technology in the 1980s, it became possible to isolate and sequence the genome in the vicinity of these recurrently rearranged regions, leading to the identification of many rearranged cancer genes, particularly in leukemias, lymphomas, and sarcomas (4). The next major suite of technologies primarily provided evidence of copy number change in cancer genomes, but at higher resolution than was generally possible by cytogenetics. These approaches confirmed the variation in extent of copy number change between individual cancer genomes and highlighted regions showing recurrent increases or reductions in copy number. Subsequent studies focusing on these recurrently abnormal regions provided a further harvest of new cancer genes (5, 6). These technologies had their limitations. Most obviously, they could not directly detect base substitutions or small indels. The emergence of the draft human genome sequence in 2000 empowered the study of cancer genomes in many ways. In particular, it provided a template for the design of polymerase chain reaction (PCR) primer pairs to amplify and sequence (by conventional sequencing technology) the coding exons of large numbers of protein-coding genes. This facilitated more extensive sequencing of cancer genomes, including whole gene families and subsequently most coding exons (7–28). These studies systematically sampled cancer genomes for somatic base substitutions and small indels, providing, for the first time, insights into their prevalence. However, exploration of noncoding areas of the genome and larger numbers of cases was still restricted by high cost and limited sequencing capacity. The recent arrival of second-generation DNA sequencing technologies (29) has further transformed investigation of cancer genomes. These technologies are being applied in a number of ways. Because most of the currently known driver mutations change the coding sequences of proteincoding genes and because protein-coding exons account for only ~1% of the human genome, sequencing is often being thriftily targeted at these (30–32). Use of technologies that extract subsets of DNA sequences from the whole genome (33), in combination with second-generation sequencing, has already allowed sequencing of the protein-coding exons of roughly 2000 individual cancers worldwide. This strategy will find base substitutions and indels in coding exons (and potentially copy number changes) but will miss these types of mutation in noncoding regions and require other analyses of the same genomes to report most rearrangements. To a similar end, after extraction of RNA, the transcriptomes of many

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hundreds of cancers have been sequenced (34–36). This approach will report substitutions in genes that have sufficiently high levels of mRNA and can report rearrangements that are transcribed. Again, however, abnormalities of noncoding regions will generally be missed, and protein-truncating mutations may be difficult to find if they activate nonsensemediated RNA decay. In the longer term, however, the major impact of these remarkable technology shifts will be to permit the sequencing of whole cancer genomes (37–43). This strategy, in which genomic DNA from a cancer (and, in parallel, DNA isolated from normal tissue of the same person) is randomly fragmented and hundreds of millions of fragments are sequenced, can reveal all classes of somatic change (base substitutions, indels, rearrangements, copy number changes, and even potentially epigenetic alterations) in all sectors of the genome (exons, introns, and intergenic regions). It has paved the way to the generation of almost complete catalogs of somatic mutation for individual cancers (39, 40), allowing us to set aside our preconceptions of where the important mutations that cause the disease might lie and, by acquisition of large numbers of mutations from individual cases, empowering deeper study of the mutational processes that have been operative. A few hundred

whole cancer genomes have already been generated by sequencing machines worldwide and are in the process of being analyzed. The Number of Mutations in Cancer Genomes As noted above, cytogenetic and copy number studies revealed that the number of genomic rearrangements and copy number changes can differ markedly between individual cancers. Until the recent advent of systematic sequencing studies, however, we had little insight into the numbers of somatic base substitutions and indels and the extent of their variation. We now know that there are usually between 1000 and 10,000 somatic substitutions in the genomes of most adult cancers, including breast, ovary, colorectal, pancreas, and glioma (10, 21). There are cancer types that generally carry relatively few mutations—for example, medulloblastomas, testicular germ cell tumors, acute leukemias, and carcinoids (10, 16)—whereas others, such as lung cancers and melanomas, have many more mutations (occasionally more than 100,000) (9, 10, 27, 39, 40). Even within a particular cancer type, individual tumors often display wide variation in the prevalence of base substitutions. Two major factors account for these differences in mutation prevalence: differences between

individual cancers in mutation rate at the cell divisions that have taken place between the fertilized egg and the cancer cell and differences in the number of mitoses in this lineage. The basis for the high prevalence of somatic substitutions observed in some cancers is likely to be overwhelming mutagenic exposure such as ultraviolet (UV) light (in melanoma) or tobacco carcinogens (in lung cancer); the presence of defective DNA repair mechanisms (e.g., in colorectal, stomach, and other cancers with defective DNA mismatch repair); and therapy with DNA-damaging agents (e.g., in gliomas treated with the alkylating agent temozolomide) (10, 11). However, there are individual cancer cases in which the large number of base substitution mutations remains unexplained (18). The reason that some cancer types have relatively few mutations is not completely clear. Some are tumors of children or young adults, and therefore it is conceivable that the neoplastic cell has been through relatively few DNA replications. Alternatively, it may be that most cancers, including those with the typical mutation prevalence, have experienced an elevated mutation rate compared to normal cells and that cancers with low mutation prevalence are the exceptions that have evolved without it. Because we currently know little about the prevalence of somatic base

Second-generation sequencing technologies

First sequence of all exons in a cancer

Human genome sequence

First observations that the material of inheritance was abnormal in cancer cells and consequent proposal that cancers are clones arising due to somatic changes

First complete cancer genome sequence

Description of the double helical structure of DNA

400 known cancer genes

First recurrent chromosomal rearrangement in cancer

First somatic driver mutation and first cancer gene identified

Identification of DNA as the material of inheritance

1900

1920

1940

Thousands of complete cancer genome sequences

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Cancer genome sequences as a routine diagnostic?

2000

Fig. 1. Time line showing key events in the investigation of the cancer genome.

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substitutions in normal cells, the importance of an elevated base substitution mutation rate in cancer development remains controversial (44, 45). However, for indels in cancer cases with DNA mismatch-repair deficiency and for rearrangements and copy number changes in cancers that have large numbers of these changes, an increased mutation rate is likely to have been operative. Systematic sequencing studies have also provided our first comprehensive insights into the proportions of driver and passenger mutations. Thus far, the large majority of base substitutions in most cancer genomes appear to be passengers (10, 21). However, these studies also suggest that there may be many more drivers than can be unambiguously identified by current approaches. If the latter interpretation is correct, a substantial number of cancer genes remain to be discovered, albeit many contributing infrequently to cancer development (10, 21). The Repertoire of Human Cancer Genes Studies of driver mutations in cancer genes have yielded many insights into the molecular and cellular events that convert a healthy cell into a cancer cell. In recent years, the proteins altered by driver mutations have become targets for successful anticancer drug development (46). Identification of new mutated cancer genes is, therefore, one of the most important deliverables that emanates from exploration of cancer genomes. The primary analytic approach to the identification of driver mutations and cancer genes has assumed that passenger mutations are randomly distributed throughout the genome, whereas drivers (by definition) are clustered in the subset of genes that are cancer genes. The strategy is thus to search in a large number of samples of a specific cancer type, for genes that have a higher prevalence of somatic mutations than would be expected by chance alone, followed by verification of biological activity in experimental systems. This basic strategy has been highly effective over decades and remains the mainstay of cancer gene discovery today. However, it has become clear that passenger mutations are not always randomly distributed in the genome; their clustering can mimic that of driver changes, and thus additional filtering strategies may sometimes be required to avoid errors in cancer gene identification (47). The search for cancer genes through systematic exploration of cancer genomes by cytogenetics, copy number analyses, and sequencing has been supplemented by targeted somatic mutational analyses of genes previously identified as cancer susceptibility genes, by mutational analyses of biologically plausible candidates, and by biological assays of transforming activity, notably DNA transfection through NIH3T3 cells. Collectively, these varied approaches have identified ~400 somatically mutated cancer genes that contribute to neoplastic change in one or more types of cancer. This number corresponds to roughly 2% of

the protein-coding genes in the human genome (5, 6) (www.sanger.ac.uk/genetics/CGP/Census/). Cancer genes are often classified according to whether they function in a dominant or recessive manner at the level of the cancer cell. Dominant cancer genes require only one of the two parental alleles present in a normal cell to be mutated, and the encoded protein is usually constitutively activated by the mutations. Recessive cancer genes (also known as tumor suppressor genes) require mutation of both parental alleles, and these usually result in inactivation of the encoded protein. More than 80% of the currently known cancer genes are dominantly acting; mostly these are genes that are rearranged in the myriad recurrent chromosomal translocations particularly found in leukemias, lymphomas, and sarcomas (www. sanger.ac.uk/genetics/CGP/Census/). The current predominance of dominantly acting cancer genes is in part due to ascertainment bias, and the real balance remains to be determined. Most of the known cancer genes were found through primary cytogenetic analyses, with the wave of ever higher resolution copy number studies bringing a further substantial yield. Recent systematic sequencing of cancer genomes has provided a new harvest of cancer genes identified directly through an elevated prevalence of base substitutions and small indels. These include several dominant cancer genes, such as BRAF, EGFR, ERBB2, PIK3CA, IDH1, IDH2, EZH2, FOXL2, PPP2R1A, and JAK2 (8, 12–15, 17, 34, 36, 48, 49) (some of which were also found by alternative approaches). Some are on biological pathways previously implicated in cancer development. Others— for example, IDH1, which encodes isocitrate dehydrogenase 1, a component of the Krebs cycle; or FOXL2, which encodes a tissue-specific transcription factor—would not have featured on many candidate gene lists. Several recessive cancer genes (and others for which the dominant or recessive status is unclear) have also emerged through systematic sequencing, including SETD2, KDM6A, KDM5C, PBRM1, BAP1, ARID1A, DNMT3A, GATA3, DAXX, ATRX, and MLL2 (7, 12, 16, 19, 20, 30–32, 35, 50). Many of the proteins encoded by this set of genes (and, in addition, EZH2 and IDH1 among the dominant cancer genes mentioned above) are involved in chromatin modification and remodeling. For example, SETD2, EZH2, and MLL2 are histone H3 methylases, whereas KDM6A and KDM5C are histone H3 demethylases. ARID1A, PBRM1, BAP1, ATRX, and DAXX are components of protein complexes that restructure chromatin, and DNMT3A is involved in maintenance of cytosine methylation in DNA. Although this sector of cell biology was previously known to be disrupted through mutation in some cancers, these discoveries have placed new emphasis on its role in a range of adult and childhood solid tumor types and highlight a potentially important link between somatic mutation and epigenetic changes

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that are present in many cancers. This area of biology promises to be a major focus of activity in the development of new cancer therapeutics. Some newly discovered cancer genes—for example, BRAF, JAK2, ARID1A, EZH2, BAP1, PBRM1, and DNMT3A—are mutated in a substantial proportion of cases of a particular cancer type. Others, such as SETD2 and KDM5C, are mutated in only a small fraction of cancers of any class. This appears to be an emerging feature of the landscape of somatically mutated cancer genes, of which a relatively limited set are commonly mutated and a substantial number mutated infrequently. From the standpoint of novel drug discovery, the latter presents obvious challenges. An important perspective on the evolution of the cancer clone is provided by the number of mutated cancer genes required to generate an individual human cancer. It is often speculated that five mutated cancer genes are necessary (51). However, higher estimates have been suggested, and for some hematopoietic neoplasms fewer may be required. The presence of two to four driver mutations has been demonstrated in many cases of various cancer types. In a few years, we will be able to estimate this core metric of cancer biology, and the extent to which it varies, more accurately. Once large numbers of cancer genomes have been completely sequenced with all classes of somatic mutation harvested and once most cancer genes have been identified, robust direct assessments of the number of mutated cancer genes in individual cancers will become achievable. The Cancer Genome and Drug Discovery The central role of mutated cancer genes in the genesis and maintenance of cancer clones renders them potential “Achilles’ heels” to be exploited for drug discovery. There are now several celebrated examples of anticancer drugs that act by inhibiting the aberrantly activated proteins encoded by mutated cancer genes (46). A paradigm of such strategies is the development of imatinib and subsequent generations of small-molecule inhibitors of the constitutively activated ABL kinase engendered by the chromosome 9:22 translocation in chronic myeloid leukemia (CML) (52). This advance has transformed the treatment of CML and, on the way, has helped to revolutionize cancer therapeutics. Small-molecule drugs against mutated versions of EGFR, ERBB2, KIT, PDGFRA, PML-RARA, MET, and ALK are either already in clinical use or being evaluated in clinical trials (46, 53). Similarly, a therapeutic antibody (trastuzumab) directed against HER2, the protein encoded by a gene amplified in about 20% of breast cancers, has had a major impact on treatment of these cancers (54). An illustrative example of the combined power of modern genomics, biology, and drug discovery is that of BRAF. Somatic mutations of BRAF were discovered in an early systematic sequenc-

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ing screen in 2002 (8). BRAF encodes a serinethreonine kinase and is mutated in 50 to 70% of malignant melanomas, 10 to 15% of colorectal cancers, 50% of papillary thyroid cancers, and at a lower frequency in other cancer types. A single mutation, V600E (substitution of valine 600 with glutamic acid), accounts for more than 90% of mutations and results in constitutive activation of the BRAF kinase. Being a kinase (with a deep ATP-binding pocket in which inhibitors can sit) and being activated by its mutations made mutated BRAF an attractive target for drug development. Inhibitors of V600E mutant BRAF have been tested in phase 1 trials and have produced encouraging responses in 80% of patients with metastatic malignant melanomas carrying the V600E mutation (55). Unfortunately, minor subclones resistant to BRAF inhibitors appear to be present in many V600Epositive malignant melanomas, and these grow out as recurrences. Nevertheless, investigation into the genomes of recurrences has already identified some of the mutations that confer resistance, proffering new avenues for therapeutic intervention (56, 57). Thus, in the decade since the discovery of BRAF as a mutated cancer gene, the field has seen small-molecule inhibitors identified and developed into orally available drugs, the drugs put through clinical trials and shown to have anticancer activity, and mechanisms of resistance to the drugs elucidated. Although we collectively aspire to even more rapid progress in the future, this is a remarkable achievement. In some cancers, direct targeting and inhibition of constitutively activated proteins encoded by mutated cancer genes may not be possible. For example, in clear cell renal cancer, mutated and activated kinases have not been found. Indeed, all the operative cancer genes appear to be recessive (7, 20, 31). Because the proteins encoded by these genes are already inactivated by their mutations, other strategies—for example, the development of drugs that exhibit synthetic lethality with particular mutated cancer genes (58)—will have to be adopted. Genomic Evidence of Mutagenic and Repair Processes The patterns of somatic mutation found in a cancer genome reflect the DNA damage and mutagenic processes that have been operative and the repair mechanisms that have mitigated their impact. Thus, the cancer genome can be likened to an archaeological record bearing the imprint of these processes. The mutational patterns (often called mutational spectra) incorporate many types of information, including the numbers of each class of mutation, the DNA sequences around

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each mutated base, and, in transcribed regions, whether the transcribed or the untranscribed strand is preferentially mutated. In the past, mutational spectra have been assembled with the use of mutations found in frequently mutated cancer genes, notably the tumor suppressor gene TP53. In such studies, each informative cancer case usually contributes a single mutation, and the spectrum is established by grouping together mutations from multiple cases of the same cancer type. This approach demonstrated that lung cancers exhibit many C:G>A:T

transversion mutations, a pattern similar to that induced in experimental systems by tobacco carcinogens; that hepatocellular cancers also show C:G>A:T mutations that are likely to be induced by aflatoxins, known etiological agents in liver cancer development; and that skin cancers predominantly show C:G>T:A mutations of the pattern known to be caused by UV light (59). However, a major limitation of these studies is that mutational spectra generated in this way are composites of all the mutational spectra present in a tumor class. Thus, although well powered to report a strong exposure that dominates a particular cancer type, they cannot untangle the diverse mutational processes and patterns that may be present in some cancer types. By contrast, partial or complete catalogs of mutations from individual cancer genomes, which usually number several thousand somatic mutations per case, can report with extraordinary resolution the mutational spectra of individual cancers, thus revealing the diverse mutational and repair processes operative within a class of cancer and even within individual cases. This type of analysis is in its infancy, but some examples illustrate its potential. Early systematic sequencing studies revealed the presence in some breast cancers of a mutational process characterized by C>T and C>G mutations that occur almost exclusively at cytosines that follow a thymine [i.e., at TpC dinucleotides (18)]. The nature of the mutagenic process underlying this pattern of mutations remains mysterious, but future epidemiological studies correlating its presence

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with exogenous exposures and studies replicating the pattern by examining the effects of chemicals or DNA repair defects in experimental systems may elucidate its origin. In the case of melanoma and lung cancers, the use of essentially complete catalogs of thousands of mutations from the genomes revealed the predominant mutational classes expected of the known exposures underlying these cancers (39, 40). Although UVexposure accounted for most mutations in melanoma, there was evidence for at least one additional mutational process, the spectrum of which suggested that it may have been due to reactive oxygen species (39). Similarly, by examining the DNA sequences around somatically mutated bases in a case of lung cancer, it was possible to tease out multiple distinct mutational processes that may reflect the complexity of the carcinogen mixture present in cigarette smoke (40). Traces of DNA repair processes are also embedded in mutational spectra. For example, in individual melanoma and lung cancer cases, evidence has been found for past activity of transcription-coupled repair, a subclass of nucleotide excision repair that is directed at the transcribed strand of each gene (38–40). These completely sequenced cancer genomes also revealed that nucleotide excision repair had been preferentially deployed to the untranscribed strand of genes, that repair correlated with the expression level of the target gene, and that 5′ ends of genes had been more effectively repaired than 3′ ends. Sequencing of cancer genomes has revealed unexpected features of mutational processes beyond those that cause base substitutions. For example, some cancers display many more genomic rearrangements than would have been predicted on the basis of cytogenetic studies (60). Indeed, different types of rearrangement architecture predominate in different cancer types. In some breast cancers, for example, there are frequent tandem duplications of DNA (60), whereas in pancreatic cancers this pattern is rare (61). The genetic defects, or possibly environmental exposures, that underlie these distinctive patterns of genomic rearrangement are unknown. Insights have also emerged with respect to the timing of mutations. In principle, some mutational processes may cause steady accumulation of mutations over decades, whereas others may be characterized by a sharp burst over a short period. A small proportion of cancer genomes exhibit a distinctive pattern characterized by extraordinary numbers of rearrangements localized to a small segment of the genome. In such regions, the genome appears to have been shattered and subsequently reassembled by the cell, albeit in a disordered manner. The structure of these dense aggregates suggests that the rear-

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Revealing the Tree of Clonal Evolution in Cancer Although each cancer derives from a single normal cell, the population of neoplastic cells constituting the final cancer often has a complex evolutionary history, which can be visualized in the following way. Multiple waves of clonal expansion are thought to be required, each brought about by an additional driver mutation, to generate the dominant subclone that manifests as the symptomatic cancer. Along the way, additional branching subclones with further drivers may have been spun off that failed to outcompete the dominant subclone. The dominant subclone itself may have spawned a further minor subclone with an additional driver mutation that in time would dominate. Some of these minor subclones may have been completely extinguished, but others may persist. Thus, the final population of cancer cells is composed of the dominant subclone accompanied by relics of its evolutionary past, outstripped rivals, and portents of its future. Somatic mutations acquired by cancer cells as they divide can serve as markers of clonal origin and thus allow retrospective reconstruction of the evolutionary tree of individual cancers. These analyses have revealed the complex subclonal structure of certain cancers and have demonstrated that minor subclones at initial presentation of the cancer are often the source of the major clone that recurs after treatment (63–66). Metastases have been analyzed by similar methods, and these studies indicate that they are usually subclones of the primary cancer. Comparison of somatic changes in metastases to those of the primary tumors from which they originated has revealed that many likely passed through a clonal bottleneck, continued to acquire somatic changes, and diverged from the primary cancer (37, 41, 61, 67). Comparing genomic changes in different metastases from the same patient has yielded provocative insights. For example, in some cases multiple distinct metastases apparently originated from the same minor subclone of the primary tumor, suggesting that this subclone possessed enhanced metastatic potential compared to the bulk of the primary tumor (37, 61). Unexpected relationships between metastases have also surfaced. For example, in some pancreatic cancers metastatic to the lung and to the abdomen, the lung metastases shared a set of somatic changes with each other, and the abdominal metastases shared a different set (in addition to the mutations both groups of metastases shared with the primary cancer). These results suggest that, rather than each metastasis being a direct offshoot of the primary cancer, a single seed of pancreatic cancer

reached the lung and then reseeded further in this organ to generate the multiple metastases observed, and similarly, a single metastasis seeded in the abdomen and then reseeded elsewhere in the peritoneal cavity (61). The Cancer Genome as a Personalized Diagnostic As noted above, the successful development of drugs against proteins encoded by somatically mutated cancer genes has helped to revolutionize cancer therapeutics in the past decade. In many cases, such drugs are only effective against cancers carrying the relevant mutated cancer gene. Testing for the presence of the mutated gene in biopsies as a prelude to administering the drug is therefore a

tection of recurrence. Similar approaches have been used for many years to monitor disease burden in several types of leukemia. In these diseases, they have been applicable because of the presence of common recurrent driver rearrangements (translocations) that allow design of PCR assays across the rearrangement junction; these assays serve as sensitive and specific tests that are straightforward to implement clinically. This mode of diagnostic has not generally been employed in solid tumors, in part because there are relatively few examples of common recurrent rearrangements. However, most solid tumors do carry multiple passenger rearrangements that are specific to each individual cancer. The possibil-

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rangements occurred more or less synchronously, possibly within a single catastrophic cell cycle, rather than in a serial manner over many cell divisions (62).

Fig. 2. Cancer genome analysis is expected to have a far-reaching impact on our understanding of cancer biology and will likely prompt new approaches to the detection, diagnosis, treatment, and possibly prevention of the disease. rapidly expanding area of cancer diagnostics that seems certain to be integrated into future clinical practice. There are additional ways in which knowledge of the cancer genome can potentially be used to improve patient care. Many cancers leak DNA into the circulation as cells die. Detection of somatic changes present in the cancer genome can, in principle, distinguish circulating DNA originating from the cancer from circulating DNA derived from normal cells. Such tests would potentially allow monitoring of tumor burden from measurements on blood samples and might have utility in a variety of circumstances, including evaluation of response to treatment and early de-

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ity of sequencing a cancer genome as a real-time diagnostic to find these rearrangements offers the potential of developing customized tests of circulating DNA to determine the tumor burden of most cancer patients. Early proof-of-principle studies indicate that this approach is technically feasible (68, 69), and its benefit for patients is being evaluated. The leakage of mutated DNA from cancers into blood or other body fluids also raises the possibility of early diagnosis by detection of circulating cancer-derived DNA before a cancer becomes symptomatic and the tumor burden high. This is a longer-term vision with additional attendant technical challenges. As with all screening

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approaches, its utility in clinical practice will ultimately depend on its sensitivity, false-positive rate, and impact on mortality. The Future The march toward exhaustive sequencing of cancer genomes across the range of tumor classes is now under way. This global enterprise is being conducted under the auspices of the International Cancer Genome Consortium (70) and currently includes large-scale sequencing initiatives in Australia, Canada, China, France, Germany, India, Italy, Japan, Mexico, Spain, the United Kingdom, and the United States. Sequencing of several hundred cases of each major cancer subtype is envisaged to provide sufficient statistical power to detect cancer genes that may be operative in only 5% of cases. Over the next 5 to 7 years, it is realistically anticipated that tens of thousands of cancer genomes will be sequenced. Initially, there will continue to be a diversity of approaches, with some studies sequencing the DNA of exons of protein-coding genes while others continue to analyze transcriptomes. It is likely, however, that these large-scale initiatives will ultimately converge on whole-genome sequencing, coupled to exploration of the transcriptome and epigenome from the same cases. This convergence will be encouraged by the falling cost of whole-genome sequencing; by the convenience of harvesting all classes of somatic mutation in one experiment; and by the insight that, albeit large, the human genome is finite and that we should exploit this advantageous attribute. The only way of being sure that nothing important has been missed is to examine it all. Collectively, the outcomes of these studies are expected to have an overarching influence on our understanding of cancer biology and prompt new approaches to therapy and potentially prevention (Fig. 2). They will reveal the full repertoire of mutated cancer genes that operate across the most common forms of human cancer, will provide us with a clear picture of the number and combinations of mutated cancer genes required to generate each individual cancer, and will shed light on the mutational and repair processes that have been operative in generating neoplastic clones in the first place. Through analysis of samples from early preinvasive lesions, from metastases, from recurrences after therapy, and from patients with known exposures or epidemiological risk factors, these studies should also provide insights into disease pathogenesis, progression, and mechanisms of drug resistance. These studies will also establish a new, comprehensive, and biologically rational classification of human cancer based on genomic abnormalities. As with any new classification, it will be necessary to evaluate, in a further wave of research, the ability of this genomic classification scheme to predict the key features of tumor behavior of most concern to us, notably progression and response

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to therapeutics. We already know that the presence of certain mutated genes determines response to some therapies, and mutational testing of specific genes has already been introduced into some clinical trials, particularly when the mutated gene is the target of the new therapy being evaluated. However, with an essentially complete set of cancer genes to be revealed in a few years, and the plausibility that some are likely to influence the clinical behavior of cancer, the ultimate goal should be to examine the prognostic and predictive effectiveness of all mutated cancer genes present in each cancer type, much as similar waves of research in the past have correlated cancer behavior with clinical parameters, pathology, or specific biomarkers. In principle, this assessment should be implemented systematically for both existing and new patient treatment protocols. What sort of test design could accomplish this? Each cancer type is driven by different, although often overlapping, sets of mutated cancer genes. Thus, customized tests for each cancer class might be an option. However, a single test that could be applied to all types of cancer and access all the relevant information in each type is an especially attractive prospect. The complete catalog of somatic mutations provided by the sequence of the cancer genome fits that description. Although currently expensive for routine implementation, it is unlikely to remain so for long, and the costs of performing a cancer genome sequence in 10 years will be insignificant compared to other aspects of conducting clinical trials. Thus, a full cancer genome sequence may well turn out to be a pragmatic test design for this next phase of research. One should not, however, underestimate the technical, scientific, and analytic challenges intrinsic to this proposal. Moreover, such a test is unlikely to replace all other intrinsic predictors of cancer behavior. Nevertheless, given the rich seam of information that we know is buried in each cancer genome, the extraordinary pace of technological advance in sequencing, and the practical advantage of using a single test in diverse clinical contexts (including many outside oncology), it seems reasonable to look forward to a time in the not-so-distant future when we will consider a cancer genome sequence as a routine adjunct in clinical trials and a test we will perform on many newly diagnosed cancers. References and Notes 1. M. R. Stratton, P. J. Campbell, P. A. Futreal, Nature 458, 719 (2009). 2. P. W. Laird, Hum. Mol. Genet. 14 (suppl. 1), R65 (2005). 3. D. Hanahan, R. A. Weinberg, Cell 100, 57 (2000). 4. F. Mitelman, B. Johansson, F. Mertens, Nat. Rev. Cancer 7, 233 (2007). 5. P. A. Futreal et al., Nat. Rev. Cancer 4, 177 (2004). 6. T. Santarius, J. Shipley, D. Brewer, M. R. Stratton, C. S. Cooper, Nat. Rev. Cancer 10, 59 (2010). 7. G. L. Dalgliesh et al., Nature 463, 360 (2010). 8. H. Davies et al., Nature 417, 949 (2002). 9. L. Ding et al., Nature 455, 1069 (2008). 10. C. Greenman et al., Nature 446, 153 (2007).

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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.

C. Hunter et al., Cancer Res. 66, 3987 (2006). S. Jones et al., Science 330, 228 (2010). R. L. Levine et al., Cancer Cell 7, 387 (2005). J. G. Paez et al., Science 304, 1497 (2004). D. W. Parsons et al., Science 321, 1807 (2008). D. W. Parsons et al., Science 331, 435 (2011). Y. Samuels et al., Science 304, 554 (2004). P. Stephens et al., Nat. Genet. 37, 590 (2005). J. Usary et al., Oncogene 23, 7669 (2004). G. van Haaften et al., Nat. Genet. 41, 521 (2009). L. D. Wood et al., Science 318, 1108 (2007). T. Sjöblom et al., Science 314, 268 (2006). R. McLendon et al., Cancer Genome Atlas Research Network, Nature 455, 1061 (2008). H. Davies et al., Cancer Res. 65, 7591 (2005). D. W. Parsons et al., Nature 436, 792 (2005). A. Bardelli et al., Science 300, 949 (2003). Z. Kan et al., Nature 466, 869 (2010). L. M. Scott et al., N. Engl. J. Med. 356, 459 (2007). J. Zhao, S. F. Grant, Curr. Pharm. Biotechnol. 12, 293 (2010). J. W. Harbour et al., Science 330, 1410 (2010). I. Varela et al., Nature 469, 539 (2011). Y. Jiao et al., Science 331, 1199 (2011). L. Mamanova et al., Nat. Methods 7, 111 (2010). R. D. Morin et al., Nat. Genet. 42, 181 (2010). K. C. Wiegand et al., N. Engl. J. Med. 363, 1532 (2010). S. P. Shah et al., N. Engl. J. Med. 360, 2719 (2009). L. Ding et al., Nature 464, 999 (2010). W. Lee et al., Nature 465, 473 (2010). E. D. Pleasance et al., Nature 463, 191 (2010). E. D. Pleasance et al., Nature 463, 184 (2010). S. P. Shah et al., Nature 461, 809 (2009). E. R. Mardis et al., N. Engl. J. Med. 361, 1058 (2009). T. J. Ley et al., Nature 456, 66 (2008). L. A. Loeb, Semin. Cancer Biol. 20, 279 (2010). W. Bodmer, J. H. Bielas, R. A. Beckman, Cancer Res. 68, 3558, discussion 3560 (2008). D. Stuart, W. R. Sellers, Curr. Opin. Cell Biol. 21, 304 (2009). G. R. Bignell et al., Nature 463, 893 (2010). P. Stephens et al., Nature 431, 525 (2004). H. Yan et al., N. Engl. J. Med. 360, 765 (2009). T. J. Ley et al., N. Engl. J. Med. 363, 2424 (2010). C. Hornsby, K. M. Page, I. P. Tomlinson, Lancet Oncol. 8, 1030 (2007). B. J. Druker, Blood 112, 4808 (2008). E. L. Kwak et al., N. Engl. J. Med. 363, 1693 (2010). F. J. Esteva, D. Yu, M. C. Hung, G. N. Hortobagyi, Nat. Rev. Clin. Oncol. 7, 98 (2010). K. T. Flaherty et al., N. Engl. J. Med. 363, 809 (2010). R. Nazarian et al., Nature 468, 973 (2010). C. M. Johannessen et al., Nature 468, 968 (2010). F. L. Rehman, C. J. Lord, A. Ashworth, Nat Rev. Clin. Oncol. 7, 718 (2010). G. P. Pfeifer, Mutat. Res. 450, 1 (2000). P. J. Stephens et al., Nature 462, 1005 (2009). P. J. Campbell et al., Nature 467, 1109 (2010). P. J. Stephens et al., Cell 144, 27 (2011). P. J. Campbell et al., Proc. Natl. Acad. Sci. U.S.A. 105, 13081 (2008). C. G. Mullighan et al., Science 322, 1377 (2008). F. Notta et al., Nature 469, 362 (2011). K. Anderson et al., Nature 469, 356 (2011). S. Yachida et al., Nature 467, 1114 (2010). D. J. McBride et al., Genes Chromosomes Cancer 49, 1062 (2010). R. J. Leary et al., Sci. Transl. Med. 2, 20ra14 (2010). International Cancer Genome Consortium, Nature 464, 993 (2010). M.R.S. thanks A. Futreal, P. Campbell, U. McDermott, N. Rahman, and many other colleagues for conversations over the years that have clarified ideas that have found their way into this Review. Supported by the Wellcome Trust under grant reference 077012/Z/05/Z.

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A Perspective on Cancer Cell Metastasis Christine L. Chaffer1,3* and Robert A. Weinberg1,2,3* Metastasis causes most cancer deaths, yet this process remains one of the most enigmatic aspects of the disease. Building on new mechanistic insights emerging from recent research, we offer our perspective on the metastatic process and reflect on possible paths of future exploration. We suggest that metastasis can be portrayed as a two-phase process: The first phase involves the physical translocation of a cancer cell to a distant organ, whereas the second encompasses the ability of the cancer cell to develop into a metastatic lesion at that distant site. Although much remains to be learned about the second phase, we feel that an understanding of the first phase is now within sight, due in part to a better understanding of how cancer cell behavior can be modified by a cell-biological program called the epithelial-to-mesenchymal transition.

etastasis is responsible for as much as 90% of cancer-associated mortality, yet it remains the most poorly understood component of cancer pathogenesis. During metastatic dissemination, a cancer cell from a primary tumor executes the following sequence of steps: It locally invades the surrounding tissue, enters the microvasculature of the lymph and blood systems (intravasation), survives and translocates largely through the bloodstream to microvessels of distant tissues, exits from the bloodstream (extravasation), survives in the microenvironment of distant tissues, and finally adapts to the foreign microenvironment of these tissues in ways that facilitate cell proliferation and the formation of a macroscopic secondary tumor (colonization) (1). Here we suggest that this complex metastatic cascade can be conceptually organized and simplified into two major phases: (i) physical translocation of a cancer cell from the primary tumor to the microenvironment of a distant tissue and then (ii) colonization (Fig. 1). We propose that an understanding of physical dissemination is in sight, whereas the second phase, colonization, involves complex interactions that may still require several years of research before they come into clear view. From a therapeutic standpoint, understanding the mechanisms of physical translocation is likely to be important for preventing metastasis in patients who are diagnosed with early cancer lesions, whereas understanding the mechanisms leading to successful colonization may lead to effective therapies for patients with already-established metastases.

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1 Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA. 2Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA. 3Ludwig MIT Center for Molecular Oncology, Cambridge, MA 02139, USA.

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

In the discussion that follows, we provide our perspective on a selection of issues relevant to contemporary cancer metastasis research. Physical Translocation from the Primary Tumor to the Site of Dissemination In order for individual or small groups of cancer cells to break away from the primary tumor and initiate the metastatic process, these cells must acquire the ability to migrate and invade. These traits enable cells to degrade and move through the extracellular matrix of the surrounding tissue toward blood and lymphatic vessels, which in turn provide highways for their passage to distant secondary sites. The first clinical indication of metastatic dissemination may come from the presence of cancer cells in the draining lymph nodes—those connected directly with the site of primary tumor formation through lymphatic vessels; more often than not, these draining lymph nodes seem to represent dead ends rather than temporary stopping points from which more distant metastases are launched (2). In fact, spread to the anatomically distant sites seems to occur almost entirely through the blood via the process of hematogenous dissemination (3, 4). Carcinomas, the tumors on which we focus in this review, arise in epithelial tissues. Normally, the cells forming the epithelial sheets in these tissues are tightly bound to neighboring cells and to underlying basement membranes by adherens junctions, tight junctions, desmosomes and hemi-desmosomes, effectively immobilizing them in these sheets. These tight physical constraints encumber not only normal epithelial cells, but also those within many benign carcinomas. However, as a tumor progresses, carcinoma cells liberate themselves from these associations and begin to strike out on their own, first by dissolving underlying basement membranes and then invading adjacent stromal compartments. This invasiveness seems to empower carcino-

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ma cells to both intravasate and subsequently extravasate (5). A central question, as yet unaddressed, is whether this acquisition of malignant traits occurs as an almost-inevitable consequence of primary tumor progression or as an accidental product thereof. A widely accepted but still unproven model of primary tumor formation posits that cancer cells acquire a sequence of genetic and epigenetic alterations, each of which confers one or another form of increased fitness (6). Each of these alterations can trigger a clonal expansion of the cells that have acquired it, leading to a succession of clonal expansions that resembles, at least formally, a scheme of Darwinian evolution. This working model raises the question of whether the development by cancer cells of aggressive traits, such as invasiveness and metastatic dissemination, reflects the fact that these traits are locally advantageous for carcinoma cells within the confines of a primary tumor. The alternative is more subtle: that multistep clonal evolution selects for cells that have greatly enhanced powers of survival and proliferation within primary tumors. Once formed, such cells—as an almost-accidental, unselected consequence of their phenotypic state—can now respond to contextual signals that induce them to express highly malignant traits. We do not resolve between these alternatives here, although observations described below may help to illuminate this question. Cancer stem cells and metastasis. One critical input into this discussion comes from recent observations that the neoplastic cells within individual tumors are not homogeneous. An important source of intratumoral heterogeneity has been revealed by the discovery that populations of cells within a tumor, like those in the corresponding normal tissues, are organized hierarchically (7, 8). Thus, the scheme of self-renewing stem cells (SCs), partially differentiated transitamplifying (i.e., progenitor) cells, and fully differentiated end-stage cells seems to be recapitulated in many carcinomas and other tumor types (9). The discovery of these cancer stem cells (CSCs) has forced a major rethinking of tumor biology, because a variety of cancer-associated traits that were at one time ascribed to tumor cell populations as a whole must now be associated with one or another of these subpopulations: the nonCSCs and CSCs within each tumor. (For clarification, we do not equate the biological properties of CSCs with normal tissue SCs; instead, we use the term to define a subpopulation of cancer cells with greatly enhanced tumor-initiating potential relative to other cancer cells within a tumor. CSCs should also display self-renewal potential and the ability to spawn non-CSC progeny.) These considerations become especially critical in the present discussion, because many of the biological traits of high-grade malignancy have now been traced specifically to the subpopulations

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would empower them to disseminate from primary tumors and seed metastases (15); hence, it is an attractive solution to understanding the mechanics of dissemination. Moreover, the heightened resistance to apoptosis that is integral to cells generated by an EMT is surely critical to the ability of carcinoma cells to survive the rigors of the voyage from primary tumors to sites of dissemination (18). In addition, the CSC-like state approached by carcinoma cells that have passed through an EMT may be critical in their sites of dissemination for launching new colonies of cancer cells. Merely because the EMT is an attractive solution does not make it a unique one, however, and it remains possible that other, still-undiscovered cell-biological programs operate in certain carcinoma cells as drivers of malignancy. Activation of an EMT program during tumorigenesis often requires signaling between cancer cells and neighboring stromal cells (19). Islands of cancer cells in advanced primary carcinomas are thought to recruit a variety of cell types into the surrounding stroma, such as fibroblasts, myofibroblasts, granulocytes, macrophages, mesenchymal stem cells, and lymphocytes; these recruited cells create a “reactive” stroma—in effect, an inflammatory microenvironment that appears to result in the release of EMT-inducing signals. The carcinoma cells respond to these contextual signals by activating expression of certain transcription factors (EMT-TFs) that proceed to orchestrate EMT programs within these cells. This scenario implies that activation of an EMT program flows from two major sources. First, the Physical translocation Colonization biology of the cancer cell-of-origin from primary tumor to distant organ before its transformation conspires E Survival at with the genetic and epigenetic A Acquisition secondary site changes sustained during primary of invasive C phenotype tumor formation to generate carcinoCTCs transit to ma cells that are responsive to EMTdistant organ inducing signals. Second, inductive signals released by the reactive stroma impinge on these responsive B D carcinoma cells, causing them to exLocal invasion CTCs extravasate press various EMT-TFs and thereby F cells invade into surrounding and invade into the activate previously latent EMT prostroma, then intravasate to enter Adaptation and parenchyma of grams. When portrayed in this way, hematogenous circulation proliferation to foreign tissue form metastases the EMT-associated traits are not direct products of genetic and epiDifferentiated Transitioning Cancer Inflammatory cell Stromal cell genetic evolution in primary tumors; cancer cell cancer cell stem cell instead, they represent adaptations to Fig. 1. The metastatic cascade. Metastasis can be envisioned as a process that occurs in two major phases: (i) physical contextual signals experienced once translocation of cancer cells from the primary tumor to a distant organ and (ii) colonization of the translocated cells primary tumors have formed. Though it is appealing in conwithin that organ. (A) To begin the metastatic cascade, cancer cells within the primary tumor acquire an invasive cept, the role of the EMT in enabling phenotype. (B) Cancer cells can then invade into the surrounding matrix and toward blood vessels, where they intravasate to enter the circulation, which serves as their primary means of passage to distant organs. (C) Cancer cells metastatic dissemination remains traveling through the circulation are CTCs. They display properties of anchorage-independent survival. (D) At the largely unproven, in part because of distant organ, CTCs exit the circulation and invade into the microenvironment of the foreign tissue. (E) At that foreign the technical difficulties of capturing site, cancer cells must be able to evade the innate immune response and also survive as a single cell (or as a small this transitory process in human cancluster of cells). (F) To develop into an active macrometastatic deposit, the cancer cell must be able to adapt to the cer patients. Moreover, the EMT may microenvironment and initiate proliferation. only operate in a tiny fraction of of CSCs within carcinomas (10–12). Thus, traits such as motility, invasiveness, and self-renewal, which are central to malignancy, may in fact be the ref lection of the actions of the elusive CSC subpopulations within larger populations of neoplastic cells. In many tumors, such cells may represent a tiny fraction of the total cellularity of individual tumors, yet these CSCs may be the critical drivers of their malignant progression. At a more practical level, CSCs have been defined by their most central trait: the ability to seed new tumors when experimentally implanted into appropriate animal hosts. Though the representation of CSCs, assessed in this way, may fluctuate wildly from one tumor to another (13, 14), it is clear that carcinoma cells can reside in two, or even three, alternative states of differentiation within a tumor. Such subpopulations, when separated by fluorescence-activated cell sorting according to distinct cell-surface antigen profiles, show drastic differences in tumor-initiating ability. Because these cell fractionations stratify tumor cell populations that have not previously been subjected to experimental manipulation, it is clear that residence in these alternative states preexisted in primary tumors before their excision from hosts. At one level, the critical role of CSCs in metastasis is obvious: Tumor initiation by experimentally implanted cells is theoretically analogous to tumor initiation by disseminated cancer cells; both processes depend on the ability of cancer cells to function as founder cells that spawn essen-

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tially unlimited numbers of descendants. Hence, the very traits that are used to define CSCs—selfrenewal and tumor-initiating ability—would seem to be inextricable elements of successful metastasis formation. Less intuitive is the observation that CSCs exhibit yet other traits that are relevant to metastasis: notably motility, invasiveness, and heightened resistance to apoptosis (10–12). This implies that there is a multifaceted cell-biological program packaged together to empower cancer cells within primary tumors to execute multiple steps of the invasion-metastasis cascade. The epithelial-to-mesenchymal transition and metastasis. Over the past three decades, developmental biologists have defined a cell-biological program—the epithelial-to-mesenchymal transition (EMT)—that plays critical roles in early embryonic morphogenesis (15). This transdifferentiation program, driven by EMT-inducing transcription factors (EMT-TFs), is deployed during a number of critical steps of morphogenesis, enabling cells of epithelial phenotype to generate mesenchymal derivatives. Importantly, in many embryonic contexts, the EMT is reversible; thus, cells that were recently induced to assume a mesenchymal phenotype may revert back to an epithelial state via mesenchymal-to-epithelial transitions. Recent studies have demonstrated that the EMT can induce non-CSCs to enter into a CSClike state (16, 17 ). As such, the EMT confers on epithelial cells precisely the set of traits that

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cancer cells that are in intimate contact with induced CSCs may be a feature of tumors able standing how cells are able to survive in the adjacent reactive stroma. This implies that ex- to recruit a reactive stroma capable of inducing circulation. Importantly, the presence of CTCs pression of an EMT program cannot possibly an EMT. This raises the question of whether the and of extravasated, disseminated tumor cells be discerned by examining the altered genome two types of CSCs play identical roles in tumor (DTCs), such as those in the bone marrow of patients with breast cancer, correlates with inprogression and differ only in their origins. of an unfractionated tumor sample. Plasticity between epithelial and mesenchy- creased metastatic burden, aggressive disease, A clear resolution of the role of EMTs in high-grade malignancies is complicated by one mal states will surely be important as we try to and a decreased time to relapse (22). In the longer term, a more thorough underfurther consideration: Although this program is understand the dynamics of how metastatic coloften depicted as a bi-stable switch that causes onies are initiated after disseminated carcinoma standing of CTC and DTC biology may gencells to flip from one state into the other, the cells extravasate into tissue parenchyma. Such erate highly useful diagnostic and prognostic biological reality is likely to be more subtle. In carcinoma cells may have previously undergone measurements (23). Whether these cells will remany tumors, epithelial carcinoma cells appear a partial or complete EMT within primary tu- veal important etiologic mechanisms remains to advance only partway down the road toward mors, having been induced to do so via hetero- unclear; for example, the detection of large numthe mesenchymal state. This yields cancer cells typic signals originating in the tumor-associated bers of CTCs may simply reflect a high level of that concomitantly express epithelial and mesen- stroma; components of the EMT program may aggressiveness of a primary tumor, rather than chymal markers and thus persist in a pheno- then have enabled their physical dissemination. revealing the particular cells that serve as the typic state that is not encountered in normal tissues. Intrinsic Induced Intrinsic versus induced cancer stem cells. The discussions above imply that (i) the stem-cell state is an integral part of the development of metastases, (ii) in many types of EMT carcinomas, entrance into this state is facilitated by passage through an EMT, and (iii) EMTs can be induced in carcinoma cells by contexCells poised to Recruitment of tual signals received, for example, undergo EMT reactive stroma Transition to from the tumor-associated reactive CSC-like state stroma. Taken together, these noCSC with tions imply that CSCs can be formed metastatic potential de novo through the actions of these signals. At the same time, these inResponsive Differentiated Transitioning duced CSCs cannot represent the differentiated cancer cell cancer cell Cancer stem cell Stromal cell Inflammatory cell cancer cell only source of cells in the CSC pool within a tumor: Intrinsic CSCs are likely to exist within tumors from Fig. 2. Acquisition of the metastatic phenotype. Tumors are heterogeneous populations of cells. CSC subtheir very inception, long before re- populations are particularly well poised to complete the metastatic cascade. Two alternative means of generating active stroma and EMTs become im- CSCs are depicted here. Intrinsic CSCs are thought to exist in primary tumors from the very early stages of tumorigenesis and may be the oncogenic derivatives of normal-tissue stem or progenitor cells. Induced CSCs may portant (Fig. 2). We speculate that the presence arise as a consequence of the EMT. In this case, carcinoma cells initially recruit a variety of stromal cells, such as of intrinsic and induced subtypes fibroblasts, myofibroblasts, granulocytes, macrophages, mesenchymal stem cells, and lymphocytes. Together these of CSCs within a tumor may partly cells create a reactive microenvironment that releases factors (e.g., Wnt, transforming growth factor–b, fibroblast growth factor) that cause the neighboring cancer cells to undergo the EMT and acquire CSC-like characteristics. explain the heterogeneity evident in clinical tumor pathology, where highly aggressive tumors (such as the claudin- However, it is plausible that after extravasat- key intermediaries between primary tumors and low and basal-type breast cancers) exhibit a ing, these cells will not encounter an activated metastases. Our understanding of CTCs and the roles normal mammary stem cell gene profile and thus stroma and, in the absence of associated strocontain high numbers of intrinsic CSCs, whereas mal signaling, may well lapse back to a fully that they play in metastatic dissemination is still luminal-type breast cancers correlate with a ma- epithelial state that lacks CSC function. Still, clouded by some major unresolved biological ture mammary luminal cell phenotype and thus such cells cannot afford to jettison CSC function issues and by technical issues arising from decontain low numbers of intrinsic CSCs (20, 21). completely if they are to serve as founders of a tection sensitivities of these rare cells. Circulating As such, in some subtypes of cancer, intrinsic metastatic tumor; this suggests that complex carcinoma cells have diameters (20 to 30 mm) preneoplastic SCs are likely to be present al- mechanisms operate to maintain the mesenchymal/ that are far too large to allow them to pass ready during the early stages of tumorigenesis. CSC state, even in those CSCs whose recent his- through the bores of capillaries (~8-mm diameter), Such SCs, which already should possess some tory suggests that they are induced rather than such as those present in the capillary beds of the lungs (4). By all rights, within minutes of being of the EMT-associated phenotypes, may play intrinsic CSCs. Circulating tumor cells. The blood of many released by primary tumors into the venous cirprominent roles in disseminating carcinoma cells long before frankly malignant tumors have patients with advanced primary carcinomas con- culation, CTCs should be trapped in these captains circulating cancer cells, at least a subset of illaries during their first pass through the heart. developed. Such thinking confines the induced CSCs to which may be in transit from the primary tumor Yet some persist for far longer periods of time later stages of tumorigenesis when the extensive to sites of future metastasis. These circulating tu- [with half-lives of 1 to 2.4 hours (24 )], which reactive stroma is first apparent. Accordingly, mor cells (CTCs) offer the prospect of under- suggests the possibility that only exceptionally www.sciencemag.org

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small or physically plastic CTCs can elude the sieving action of the pulmonary microvasculature and thereby accumulate to substantial steadystate concentrations in the blood. Moreover, if CTCs occasionally travel in multicell clumps of far larger diameter, such clumps must lodge almost immediately in microvessels and thus have extremely short lives in the circulation; such clumps would be strongly underrepresented by current methods that tally CTC numbers in patients. In addition, the tissue factor protein displayed on the surface of individual cancer cells attracts clouds of aggregating platelets (25, 26), which also increase the effective diameters of the cancer cells. This suggests that the CTCs found in the circulation may represent special subpopulations of cancer cells that, for one reason or another, do not trigger platelet aggregation. These platelet cloaks may also complicate the detection of CTCs by occluding the cellsurface marker antigens that are used to identify and separate CTCs from the million-fold greater numbers of nonepithelial cells in the circulation. Epithelial-specific cell-surface markers are in widespread use to detect CTCs (27–29). This represents a potential problem, because it is likely that carcinoma cells that have passed through a partial or complete EMT are no longer detectable by epithelial-specific antigens. Enrichment and detection of CTCs via depletion of hematopoietic cells (using antibodies specific for CD45) may represent one way of circumventing this issue in the future (23). Despite these complications, the ability to isolate CTCs from the circulation may become a powerful tool to study the biology of migrating CSCs, especially if those cells differ from CSCs that reside in the primary tumor site. Moreover, they offer the prospect of creating a highly useful (and relatively noninvasive) diagnostic parameter: By monitoring longitudinally the concentrations of CTCs in a cancer patient, oncologists may be able to determine, from day to day, how effectively an applied therapy has been in reducing the burdens of primary tumors that are presumably the sources of these CTCs. Homing. Once lodged in the capillary bed of a foreign tissue, CTCs may soon extravasate and invade the foreign parenchyma, or they may proliferate intraluminally and eventually rupture the wall of the microvessels in which they are lodged (30–32). The formation of metastases in certain favored target organs may be influenced by structural differences in the capillaries of various tissues. For example, the sinusoid capillaries in the bone marrow are formed from single layers of endothelial cells and are devoid of supporting mural cells; this design is thought to facilitate the normal trafficking of hematopoietic cells in and out of the bone marrow (33). This route may also present a path of least resistance to

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carcinoma cells, and thus may help to explain why the bone marrow is a favored site of metastasis by cancer cells that originate in diverse primary tumors (e.g., breast, prostate, lung, and gastric cancers) (34). In certain tumor types, the layout of the circulation may be the strongest determinant of metastatic tropism. Most frequently cited is the behavior of colorectal carcinomas (CRCs), which have a strong preference for generating liver metastases. In fact, the disseminating CRC cells may be intrinsically poorly adapted for survival in the liver microenvironment. However, because of the portal circulation, which drains from the mesentery directly into the liver, myriad carcinoma cells may be dumped over extended periods of time into the liver microvasculature; on rare occasion, an otherwise low-probability event may then generate a liver metastasis. In these various cases, homing to a particular organ can be considered to be a passive process that is determined by circulation patterns and the physical properties of the vasculature rather than by particular biological properties of the disseminating cancer cell. Organ-specific homing may also constitute an active process, where tissue and cancer cell– specific features determine metastatic dissemination. The expression by metastasizing cancer cells of specific proteins (for example, integrins) seems to play a key role in this process (35–37). We envisage that homing of metastasizing cancer cells may be a combination of both mechanical trapping of cancer cells in the microvasculature of distant organs and cancer cell–mediated adhesion to specific luminally displayed components of the vasculature (Fig. 3). Colonization-Adaptation of the Disseminated Cell to the Microenvironment at the Metastatic Site Although EMT programs may prove critical to the physical dissemination of carcinoma cells, the multiple powers of these programs would not seem capable of addressing the problem of colonization. Instead, colonization seems to represent a far more complex set of phenomena and a relatively small number of unifying, generalizable principles. This complexity can be judged, perhaps simplistically, by listing common tumors and their known metastatic tropisms. For example, as noted above, prostate carcinomas preferentially metastasize to bone, whereas CRCs preferentially metastasize to the liver (38). Nonetheless, these and other primary tumors can also form metastases at additional, alternative tissue sites. In each case, the tissue microenvironment of a primary tumor is likely to differ markedly from that of the secondary site of dissemination, necessitating substantial adaptive moves by recently arrived cancer cells. The details of these adaptive programs would seem to be dictated by the microenvironment of the starting point (the primary tumor) and the micro-

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environment of the landing site (the tissue parenchyma in which a metastasis is founded). This logic suggests that the number of distinct adaptive programs can be gauged by a simple calculation: the product of multiplying the number of metastasizing primary tumor types by the number of distinct sites of dissemination. However, clearly some adaptive moves, such as the activation of certain ensembles of genes, may simultaneously confer an ability to colonize multiple distinct tissues, reducing the diversity of adaptive programs. Moreover, we know that certain lung cancers metastasize quickly to multiple sites, whereas others, such as breast and prostate carcinomas, often take years to develop metastatic colonies and then only in a relatively limited number of sites (38). This suggests that the differentiation programs of certain normal cells, such as those in the lungs, position their neoplastic descendants to adapt readily to foreigntissue microenvironments, whereas other cancer cell types must laboriously cobble together far more complex shifts in gene-expression programs. Moreover, it is unclear how often colonization depends on epigenetic changes versus genetic mutations in cell genomes. Nonetheless, solid progress is being made toward understanding the biochemical adaptations that carcinoma cells must make to thrive in distant tissues. Gene sets have been defined in breast tumor xenografts that can predict homing and colonization of breast cancer cells specifically to the lung, bone, or brain (39–41); conversely, genomic profiling of metastases has been successfully used to predict the sites of primary tumor origin (42). These gene-expression patterns suggest that carcinoma cells within primary breast tumors acquire patterns that enable their subsequent colonization preferentially to specific target organs. These findings are supported by recent studies demonstrating that genetically distinct subpopulations of cells present in primary tumors are responsible for forming metastases (43, 44). Precisely how these gene-expression programs are acquired by the cells within primary tumors is not yet clear. They may be dictated by the differentiation programs of normal cells of origin, by somatic genetic and epigenetic changes that have been selected during multistep tumor progression, or simply stochastically. Interestingly, the discovery of reseeding of primary tumors by their derived metastases (45) raises a fourth possibility: Metastatic cells may contaminate the gene-expression patterns of the corresponding parental primary tumor by introducing gene-expression programs that were selected during earlier metastatic colonization. In general, colonization is an extremely inefficient process, and most cancer cells that successfully translocate from the primary tumor to a secondary site undergo apoptosis within 24 hours of extravasation (46–48). Experimental and clinical data support the notion that the survivors per-

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SPECIALSECTION secondary metastases derived from this shower will soon eclipse the single initiating metastasis that spawned them. Certain microenvironments are almost guaranteed to provide hospitable sites for such disseminating, colonization-competent cancer cells: sites of wound-healing and stroma of alreadyestablished tumors. Strangely enough, one insight has come from dentists who have observed tumors growing out of extracted tooth sockets within weeks of oral surgery (54); the tumors arising in these sites of active wound healing represented the first clinical manifestations of previously undetected, widespread metastatic

site for settling and colonization by disseminated CTCs, including those deriving from the contralaterally implanted tumors and those generated by the tumor itself. These diverse observations underscore the notion that, in contrast to normal tissue stroma, the stromata of aggressive tumors and sites of wound healing can serve as readily colonizable microenvironments that make few adaptive demands on the cells that have disseminated to them. Conclusions One of the most revealing findings of the past decade in cancer research is that cells within any

Homing

Colonization

Mechanical trapping A

Micrometastasis

Macrometastasis

E

F

D

Site-specific adhesion or B chemoattraction

Quiescence

Pre-metastatic niche

C Differentiated cancer cell

Cancer stem cell

Stromal cell

Inflammatory cell

Extracellular matrix

Fig. 3. Adaptation of metastatic cells to a foreign environment. Homing and colonization of a cancer cell to a distant organ are complex processes with many questions still unanswered. CTCs transiting from the primary tumor to a metastatic site can arrive at their destination via a variety of mechanisms: (A) CTCs may become lodged in the capillary beds of specific organs due to size. (B) CTCs may display specific adhesion molecules that enable them to adhere to microvessels in specific organs, or they may respond to a chemoattractive gradient arising from a particular tissue. (C) CTCs may preferentially home to organs where a premetastatic niche has prepared a microenvironment conducive to their survival. (D) Once cancer cells have exited the blood stream (extravasated) they may first experience a period of quiescence (dormancy) while they adapt to their newfound microenvironment. (E) Dormant cells may progress to micrometastatic deposits (perhaps in response to the recruitment of an appropriate stroma or an enhanced ability to respond to proliferative signals present in the host microenvironment) where their size is kept in check because of a balance in proliferation, apoptosis, and phagocytosis by the host-tissue immune system. (F) To develop into a macrometastasis, cancer cells must recruit an adequate blood supply (necessary for growth beyond 1 to 2 mm). The signals or mechanisms responsible for the transition from dormancy to micrometastasis to macrometastasis remain largely unknown.

disease in these patients. Indeed, the similarity between wound-healing environments and the hospitable stroma of tumors was encapsulated in an observation made years ago that tumors are like “wounds that will not heal” (55). This theme has been extended by the recent observations cited above (45) that the cells from contralaterally implanted aggressive tumors in mice can metastasize to one another and at apparently high efficiency. The suggestion here is that the reactive stroma that arose in each of these tumors (and contributed to their locally aggressive phenotypes) also generated a hospitable

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sisting in metastatic sites can exist in at least three alternative states: (i) as solitary viable cancer cells in a quiescent, nonproliferative state (dormancy); (ii) as micrometastases, which remain as small lesions (probably due to a balance between proliferation and apoptosis); or (iii) as actively growing macrometastatic lesions (Fig. 3). Our current understanding of the mechanisms governing the entrance into these three states and the transitions between them is limited because of the experimental challenges of studying dormancy, the extended times required for these processes to reach completion, the paucity of appropriate models, and the technical challenges surrounding the analysis of single cells in vastly larger living tissues (49). Successful colonization is presumed to include the ability to acquire mitogenic stimulation from growth factors and cytokines that are naturally present in the alien microenvironment— to self-renew and generate a large flock of descendants—to recruit the necessary supporting stroma, including an appropriate blood supply (4, 50). Micrometastases composed of actively proliferating cells represent attractive venues for cancer cells to develop complex colonization programs. Thus, we imagine that active cell division is essential for the generation of genetic and epigenetic alterations; once the resulting variants arise in these micrometastases, their novel phenotypes can be tested for an ability to confer selective advantage in the presence of highly demanding, otherwise-inhospitable microenvironments. It is also possible that the tissue microenvironment at the secondary site may change over time as a result of aging, disease, or wounding and that these environmental changes function as triggers that induce the release of disseminated cells from the dormant state. Several models of mouse metastasis now suggest that factors derived from primary tumors can educate distant sites in preparation for, and before the arrival of, metastasizing cancer cells (51–53). In these studies, factors secreted by the primary tumors (e.g., VEGF-A, PlGF, PSAP) are thought to mobilize bone marrow– derived cells that are subsequently attracted to premetastatic sites. The cells of this “premetastatic niche” then release factors (e.g., SDF-1, S1000A8, S100A9) that can attract disseminating tumor cells (Fig. 3). This interesting concept is still in the early stages of investigation. Consequences of successful colonization. The outcome of successful colonization is a rapidly expanding macrometastasis that can now serve as the focus for disseminating a shower of secondary metastases. Importantly, many of the cancer cells that are dispatched from this recently successful metastasis may be invested with a functional colonization program that may empower them to colonize either a limited subset of sites throughout the body or, alternatively, multiple distinct tissue types. The throngs of

given carcinoma display a great deal of heterogeneity. This intratumoral heterogeneity is due, in no small part, to subpopulations of cells that are phenotypically distinct but genetically identical; for instance, cancer cells that are less and more differentiated can share a common set of genetic alterations. These distinct phenotypic states, involving CSCs and non-CSCs, could hold important implications for our understanding of the biology of tumor progression and clinical therapy. For example, the biological attributes of the tumor as a whole may be strongly influenced by its subpopulation of CSCs. These cells may

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drive the self-renewal of the tumor cell population and, in the context of the present discussion, be responsible for the tumor’s invasiveness and metastatic dissemination. Moreover, the observations that (i) EMT programs can drive carcinoma cells into states that approximate the CSC state and (ii) EMTs can be activated by contextual signals experienced by carcinoma cells lead to the notion that great plasticity is likely to exist between the non-CSCs and CSCs within a tumor. Likewise, these various observations could hold important implications for strategies aimed at reducing metastasis and eradicating minimal residual disease, including dormant tumor cells, micrometastases, and macrometastatic deposits. Conventional therapeutics, which efficiently target actively proliferating cells within the primary tumor, have little impact on quiescent or slowly proliferating cancer cells and, thus, on those cells that reside in many micrometastatic colonies (56, 57). The difficulty of treating this residual disease may be compounded by the physiology of certain target organs. For example, the blood-brain barrier may shield metastases within the brain from drugs delivered through the circulatory system (58). CSCs can erect additional barriers to successful treatment. Rapidly accumulating evidence indicates that CSCs exhibit a heightened resistance to drug-induced death (59). This resistance may stem from two sources: (i) SCs often retreat reversibly from the active growth-and-division cycle into states of quiescence, and (ii) even more important may be the intrinsic drug resistance exhibited by the mesenchymal cancer cells that are the products of the EMT and exhibit traits associated with CSCs (60). Thus, various observations of drug-resistant carcinoma cell subpopulations confirm that the surviving cells often exhibit a more mesenchymal phenotype (61). Without eradicating carcinoma cells that have entered into the mesenchymal/CSC state, the oncologist is confronted with neoplastic subpopulations that are capable of regrowing primary tumors and, in addition, dispatching metastatic travelers to distant organ sites. The most elusive aspect of the invasionmetastasis cascade involves the fates of carcinoma cells after they have disseminated and extravasated into the parenchyma of distant organs. We presume that the self-renewal ability of recently disseminated cells is an essential prerequisite to their successful colonization of such distant tissues, but at present the evidence for this is only indirect. Interestingly, in the absence of a reactive stroma and EMT-inducing signals in such distant tissues, recently disseminated cells that may have arrived in a quasi-mesenchymal/CSC state may lapse back to a fully epithelial state and thereby forfeit the “stem-ness” that would seem to be essential for their successful founding of metastases.

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At present, we do not know when and how carcinoma cells acquire the abilities to colonize distant organ sites. Clearly, certain cell-biological programs that have been established and operated in primary tumors may prove advantageous when disseminated carcinoma cells initially confront the microenvironment of distant tissues. Accordingly, we do not know whether the adaptations required for colonization are largely carried by these traveling cells to their sites of metastasis or whether they are instead cobbled together later in sites of dissemination as carcinoma cells struggle to survive and proliferate in foreign, potentially hostile tissue microenvironments. These questions have proven difficult to address because the process of colonization in the great majority of tumors is extraordinarily inefficient. It is unclear whether fully dormant cancer cells are invariably doomed to eventual elimination or whether they may reawaken after months and years and suddenly spawn exuberant tumors. We suspect that only those micrometastases containing proliferating cells are capable of exploring multiple alternative phenotypic states until they stumble on one that enables them to flourish; at present, this notion is deduced from first principles rather than being demonstrated experimentally. The multiplicity of adaptive programs is an issue of great interest: they may be shared by many tumors and at many sites of dissemination. Alternatively, as argued here, these programs represent ad hoc solutions that are dictated by the origins of disseminated cancer cells and the nature of their newfound homes; if so, we may confront myriad distinct adaptive programs that are difficult to rationalize in terms of a common set of underlying biochemical mechanisms. The therapeutic implications of these different scenarios remain to be determined. Still, on a positive note, it is clear that the pace of discovery is rapid and that paths of future exploration are in sight. We now understand much more about the metastatic process than we did even a few short years ago. References and Notes 1. I. J. Fidler, Nat. Rev. Cancer 3, 453 (2003). 2. J. A. Joyce, J. W. Pollard, Nat. Rev. Cancer 9, 239 (2009). 3. D. Hanahan, R. A. Weinberg, Cell 100, 57 (2000). 4. A. F. Chambers, A. C. Groom, I. C. MacDonald, Nat. Rev. Cancer 2, 563 (2002). 5. J. P. Thiery, J. P. Sleeman, Nat. Rev. Mol. Cell Biol. 7, 131 (2006). 6. L. Foulds, Cancer Res. 14, 327 (1954). 7. D. Bonnet, J. E. Dick, Nat. Med. 3, 730 (1997). 8. M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, M. F. Clarke, Proc. Natl. Acad. Sci. U.S.A. 100, 3983 (2003). 9. L. E. Ailles, I. L. Weissman, Curr. Opin. Biotechnol. 18, 460 (2007). 10. E. Charafe-Jauffret et al., Cancer Res. 69, 1302 (2009). 11. R. Pang et al., Cell Stem Cell 6, 603 (2010). 12. P. Marcato et al., Stem Cells 29, 32 (2011). 13. E. Quintana et al., Nature 456, 593 (2008).

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14. K. Ishizawa et al., Cell Stem Cell 7, 279 (2010). 15. J. P. Thiery, H. Acloque, R. Y. Huang, M. A. Nieto, Cell 139, 871 (2009). 16. S. A. Mani et al., Cell 133, 704 (2008). 17. A. P. Morel et al., PLoS ONE 3, e2888 (2008). 18. A. Gal et al., Oncogene 27, 1218 (2008). 19. J. Yang, R. A. Weinberg, Dev. Cell 14, 818 (2008). 20. E. Lim et al.; kConFab, Nat. Med. 15, 907 (2009). 21. A. Prat et al., Breast Cancer Res. 12, R68 (2010). 22. S. Braun et al., N. Engl. J. Med. 353, 793 (2005). 23. K. Pantel, C. Alix-Panabières, S. Riethdorf, Nat. Rev. Clin. Oncol. 6, 339 (2009). 24. S. Meng et al., Clin. Cancer Res. 10, 8152 (2004). 25. E. Camerer et al., Blood 104, 397 (2004). 26. B. Nieswandt, M. Hafner, B. Echtenacher, D. N. Männel, Cancer Res. 59, 1295 (1999). 27. M. Lacroix, Endocr. Relat. Cancer 13, 1033 (2006). 28. K. Pantel, R. H. Brakenhoff, B. Brandt, Nat. Rev. Cancer 8, 329 (2008). 29. M. Mego, S. A. Mani, M. Cristofanilli, Nat. Rev. Clin. Oncol. 7, 693 (2010). 30. S. Ito et al., Int. J. Cancer 93, 212 (2001). 31. C. W. Wong et al., Am. J. Pathol. 161, 749 (2002). 32. E. Sahai, Nat. Rev. Cancer 7, 737 (2007). 33. H. G. Kopp, S. T. Avecilla, A. T. Hooper, S. Rafii, Physiology (Bethesda) 20, 349 (2005). 34. C. Alix-Panabières, S. Riethdorf, K. Pantel, Clin. Cancer Res. 14, 5013 (2008). 35. M. Abdel-Ghany, H. C. Cheng, R. C. Elble, B. U. Pauli, J. Biol. Chem. 276, 25438 (2001). 36. H. Wang et al., J. Cell Biol. 164, 935 (2004). 37. D. M. Brown, E. Ruoslahti, Cancer Cell 5, 365 (2004). 38. K. R. Hess et al., Cancer 106, 1624 (2006). 39. Y. Kang et al., Cancer Cell 3, 537 (2003). 40. A. J. Minn et al., Nature 436, 518 (2005). 41. P. D. Bos et al., Nature 459, 1005 (2009). 42. G. Bloom et al., Am. J. Pathol. 164, 9 (2004). 43. P. J. Campbell et al., Nature 467, 1109 (2010). 44. S. Yachida et al., Nature 467, 1114 (2010). 45. M. Y. Kim et al., Cell 139, 1315 (2009). 46. I. J. Fidler, J. Natl. Cancer Inst. 45, 773 (1970). 47. J. W. Kim et al., Cancer Lett. 213, 203 (2004). 48. K. J. Luzzi et al., Am. J. Pathol. 153, 865 (1998). 49. P. E. Goss, A. F. Chambers, Nat. Rev. Cancer 10, 871 (2010). 50. J. A. Aguirre-Ghiso, Nat. Rev. Cancer 7, 834 (2007). 51. R. N. Kaplan et al., Nature 438, 820 (2005). 52. S. Hiratsuka, A. Watanabe, H. Aburatani, Y. Maru, Nat. Cell Biol. 8, 1369 (2006). 53. S. Y. Kang et al., Proc. Natl. Acad. Sci. U.S.A. 106, 12115 (2009). 54. A. Hirshberg, P. Leibovich, I. Horowitz, A. Buchner, J. Oral Maxillofac. Surg. 51, 1334 (1993). 55. H. F. Dvorak, N. Engl. J. Med. 315, 1650 (1986). 56. G. N. Naumov et al., Breast Cancer Res. Treat. 82, 199 (2003). 57. S. Braun et al., J. Clin. Oncol. 18, 80 (2000). 58. R. J. Weil, D. C. Palmieri, J. L. Bronder, A. M. Stark, P. S. Steeg, Am. J. Pathol. 167, 913 (2005). 59. X. Li et al., J. Natl. Cancer Inst. 100, 672 (2008). 60. E. Buck et al., Mol. Cancer Ther. 6, 532 (2007). 61. C. J. Creighton et al., Proc. Natl. Acad. Sci. U.S.A. 106, 13820 (2009). 62. C.L.C. is supported by the National Health and Medical Research Council of Australia and the Advanced Medical Research Foundation. R.A.W. is supported by the National Cancer Institute, MIT Ludwig Center for Molecular Oncology, and Breast Cancer Research Fund. R.A.W. is a founder and shareholder of Verastem, Inc., a biopharmaceutical company focused on discovering and developing drugs that target cancer stem cells. 10.1126/science.1203543

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showed that persistent activation of the innate, pro-inflammatory arm of immunity could facilitate cellular transformation and promote cancer outgrowth and argued that this effect of immunity precluded its capacity to fulfill a protective function (9, 10). By the 1990s, improved mouse models of immunodeficiency on pure genetic backgrounds became commonplace, permitting a few groups to reassess the role of immunity in cancer control. Interest in cancer immunosurveillance was rekindled by the discovery of the importance of interferon-g (IFN-g) in promoting immunologically induced rejection of transplanted tumor cells (11) and by the demonstration that mice lacking either IFN-g responsiveness (gene-targeted mice lacking either the IFN-g receptor or the STAT1 transcription factor required for IFN receptor signaling) or adaptive immunity [RAG2−/− mice lacking T cells, B cells, and natural killer T (NKT) cells] were more susceptible to carcinogeninduced and spontaneous primary tumor formation (Fig. 1) (12, 13). Other laboratories soon began to report similar results, and collectively these findings documented that the immune system can function as an extrinsic tumor suppressor [(11–17), reviewed in (18)]. We now recognize that the immune system plays at least three distinct roles in preventing cancer: (i) It protects the host against viral infection and hence suppresses virus-induced tumors; (ii) it prevents the establishment of an

REVIEW

Cancer Immunoediting: Integrating Immunity’s Roles in Cancer Suppression and Promotion Robert D. Schreiber,1* Lloyd J. Old,2 Mark J. Smyth3,4 Understanding how the immune system affects cancer development and progression has been one of the most challenging questions in immunology. Research over the past two decades has helped explain why the answer to this question has evaded us for so long. We now appreciate that the immune system plays a dual role in cancer: It can not only suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promote tumor progression either by selecting for tumor cells that are more fit to survive in an immunocompetent host or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth. Here, we discuss a unifying conceptual framework called “cancer immunoediting,” which integrates the immune system’s dual host-protective and tumor-promoting roles. he idea that the immune system can control cancer has been the subject of debate for over a century. In the early 1900s, Paul Ehrlich was perhaps the first to reason that cancer would be quite common in long-lived organisms if not for the protective effects of immunity (1). However, so little was known about the composition and function of the immune system at the time that it was simply not possible to assess the validity of this prediction. It would take nearly 50 years before the idea of immune control of cancer resurfaced, stimulated in large part by an enhanced understanding of the immune system combined with the demonstration of the existence of tumor antigens (2). These advances provided the foundation upon which Burnet and Thomas built their cancer immunosurveillance hypothesis, a concept that formally envisaged that adaptive immunity was responsible for preventing cancer development in immunocompetent hosts (3, 4). However, subsequent studies by Stutman provided little support for this hypothesis. Of particular note were experiments showing that the cancer susceptibility of immunocompetent mice (to both spontaneous and carcinogen-induced tumors) was similar to that of nude mice that had major but not total immunodeficiency (5, 6). On the basis of these findings, the cancer immunosurveillance hypothesis was largely abandoned, and soon additional arguments began to surface

as to why cancer immunosurveillance could not possibly occur. Some investigators argued that tumor cells did not possess the appropriate “danger signals” needed to alert the immune system to the presence of a foreign cell (7), whereas others suggested that the immune system would ignore or be tolerant to a developing tumor because tumor cells were too similar to the normal cells from which they were derived (8). Still others

1 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. 2New York Branch of The Ludwig Institute for Cancer Research at Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. 3Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, 3002 Victoria, Australia. 4Department of Pathology, University of Melbourne, Parkville, 3010 Victoria, Australia.

Fig. 1. The immune status of mice is a critical determinant of their susceptibility to tumors induced by chemical carcinogens. Over the past two decades, numerous studies have established that immunodeficient mice are more tumor prone than are immunocompetent mice after treatment with carcinogens such as MCA. The immunodeficient mice tested in such experiments include gene-targeted mice on pure genetic backgrounds with deficits of innate or adaptive immunity as well as wild-type mice rendered immunodeficient by chronic administration of monoclonal antibodies that, for example, deplete CD4+ and CD8+ T cells or interferon-g. Immunodeficiency has also been found to increase the susceptibility of untreated mice to spontaneously arising tumors and to increase the incidence of tumor formation in mouse genetic models of cancer. Schematic is based on experiments described in (13).

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

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The Cancer Immunoediting Hypothesis The discovery in 2001 that the immune system controls not only tumor quantity but also tumor quality (immunogenicity) (13, 24) prompted a major revision of the cancer immunosurveillance hypothesis. This study revealed that tumors formed in mice that lacked an intact immune system were, as a group, more immunogenic (and hence were classified as “unedited”) than similar tumors derived from immunocompetent mice (and hence were termed “edited”) (Fig. 2). The notion that the immune system not only protects the host

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Fig. 2. Tumors in immunocompetent mice are qualitatively different from tumors in immunodeficient mice. This observation, which led to the formulation of the cancer immunoediting hypothesis, is based on comparative analyses of carcinogen-induced tumors harvested from immunocompetent and immunodeficient mice. In these experiments, tumor cell lines were established from tumors arising in each group of mice, and these cells were then injected into immunodeficient recipient mice or immunocompetent wild-type (WT) recipient mice. Tumor cells from carcinogen-treated WT mice formed progressively growing tumors in both immunodeficient mice (not shown) and naïve syngeneic immunocompetent mice (blue) 100% of the time. In contrast, although tumor cells from carcinogen-treated immunodeficient mice grew progressively when transplanted into immunodeficient mice (not shown), only half of the tumor cell lines were capable of forming progressively growing tumors in naïve syngeneic immunocompetent recipients (purple), whereas the other half of the cell lines were rejected by the recipients (red). Thus, tumors from immunodeficient mice are termed “unedited” and further designated as “progressor” or “regressor” to denote their growth phenotypes after injection into naïve WT recipients. Carcinogen-induced tumors from immunocompetent mice are termed “edited” because they are less immunogenic and show only a progressor growth phenotype. Schematic is based on experiments described in (13). against tumor formation but also shapes tumor immunogenicity is the basis of the cancer immunoediting hypothesis, which stresses the dual host-protective and tumor-promoting actions of immunity on developing tumors. We postulate that the cancer immunoediting process, in its most complex embodiment, proceeds sequentially through three distinct phases that we have termed “elimination,” “equilibrium,” and “escape” (Fig. 3) (18, 24–29). However, in some cases tumor cells may directly enter into either the equilibrium or escape phases without passing through an earlier phase. In addition, external factors may influence the directionality of the flow. The latter consideration may help explain the influences of environmental stress, immune system deterioration accompanying aging, and even immunotherapeutic intervention on tumor cell outgrowth in human cancer patients.

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Elimination. The elimination phase is best described as an updated version of cancer immunosurveillance, in which the innate and adaptive immune systems work together to detect the presence of a developing tumor and destroy it before it becomes clinically apparent. The mechanisms by which the immune system is alerted to the presence of a developing tumor are not fully understood. Among the possibilities are the classical “danger signals” such as Type I IFNs as originally described by Matzinger (7), which we now know are induced early during tumor development. These cytokines activate dendritic cells and promote induction of adaptive anti-tumor immune responses. However, roles for different damage-associated molecular pattern molecules (DAMPs) need also to be considered because they are released either directly from dying tumor cells [such as high mobility group box 1 (HMGB1)]

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Tumor Antigens and Cancer Immunosurveillance A fundamental tenet of tumor immunology in general and of cancer immunosurveillance in particular is that cancer cells express antigens that differentiate them from their nontransformed counterparts. The existence of tumor antigens was first demonstrated by the finding that mice immunized with chemically induced tumors were protected against subsequent rechallenge with the same tumor [reviewed in (2)]. These types of tumor antigens became known as “transplantation rejection antigens,” and similar antigens have since been demonstrated in a wide variety of experimentally induced tumors [such as those induced by different carcinogens, viruses, or ultraviolet (UV) irradiation] and even in spontaneous tumors. Subsequent molecular studies revealed that these antigens were often products of mutated cellular genes, aberrantly expressed normal genes, or genes encoding viral proteins. In the case of human cancer, identification of tumor antigens required the development of novel in vitro detection and cloning methods that used as probes antibodies and cytolytic T lymphocytes (CD8+ T cells) derived from cancer patients that were specific for the autologous tumor (19–22). The human tumor antigens discovered in these and other ways include differentiation antigens (such as melanocyte differentiation antigens), mutational antigens (such as p53), overexpressed cellular antigens (such as HER-2), viral antigens (such as human papillomavirus proteins), and cancer/testis (CT) antigens that are expressed in germ cells of testis and ovary but silent in normal somatic cells (such as MAGE and NYESO-1) (23). Thus, the identification of this large array of immunogenic mouse and human tumor antigens puts to rest the long-held view that tumor antigens are overexpressed normal proteins and therefore were subject to immunological tolerance.

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inflammatory environment that facilitates tumorigenesis by eliminating pathogens and by prompt resolution of inflammation; and (iii) it eliminates tumor cells in certain tissues because nascent transformed cells often co-express ligands for activating receptors on innate immune cells and tumor antigens (see below) that are recognized by immune receptors on lymphocytes of the adaptive immune system. This third role is most pertinent to our discussion.

expressed on the surface of tumor cells. Such ligands bind to activating receptors on innate immune cells, leading to release of pro-inflammatory and immunomodulatory cytokines, which in turn establish a microenvironment that facilitates the

or from damaged tissues (such as hyaluronan fragments) as solid tumors begin to grow invasively (30). A third potential mechanism may involve stress ligands such as RAE-1 and H60 (mouse) or MICA/B (human) that are frequently

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Cancer Immunoediting Fig. 3. The cancer immunoediting concept. Cancer immunoediting is an extrinsic tumor suppressor mechanism that engages only after cellular transformation has occurred and intrinsic tumor suppressor mechanisms have failed. In its most complex form, cancer immunoediting consists of three sequential phases: elimination, equilibrium, and escape. In the elimination phase, innate and adaptive immunity work together to destroy developing tumors long before they become clinically apparent. Many of the immune molecules and cells that participate in the elimination phase have been identified, but more work is needed to determine their exact sequence of action. If this phase goes to completion, then the host remains free of cancer, and elimination thus represents the full extent of the process. If, however, a rare cancer cell variant is not destroyed in the elimination phase, it may then enter the equilibrium phase, in which its outgrowth is prevented by immunologic mechanisms. T cells, IL-12, and IFN-g are required to maintain tumor cells in a state of functional dormancy, whereas NK cells and molecules that participate in the recognition or effector function of cells of innate immunity are not required; this indicates that equilibrium is a function of adaptive immunity only. Editing of tumor immunogenicity occurs in the equilibrium phase. Equilibrium may also represent an end stage of the cancer immunoediting process and may restrain outgrowth of occult cancers for the lifetime of the host. However, as a consequence of constant immune selection pressure placed on genetically unstable tumor cells held in equilibrium, tumor cell variants may emerge that (i) are no longer recognized by adaptive immunity (antigen loss variants or tumors cells that develop defects in antigen processing or presentation), (ii) become insensitive to immune effector mechanisms, or (iii) induce an immunosuppressive state within the tumor microenvironment. These tumor cells may then enter the escape phase, in which their outgrowth is no longer blocked by immunity. These tumor cells emerge to cause clinically apparent disease. [Figure adapted from (18)] www.sciencemag.org

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development of a tumor-specific adaptive immune response (31). Although in some experimental systems, activation of innate immunity can protect against tumor development, in most systems effective cancer immunosurveillance responses require the additional expression of tumor antigens capable of propagating the expansion of effector CD4+ and CD8+ T cells. Thus, coordinated and balanced activation of both innate and adaptive immunity is needed to protect the host against a developing tumor. If tumor cell destruction goes to completion, the elimination phase represents an endpoint of the cancer immunoediting process. The elimination phase has not yet been directly observed in vivo, but its existence has been inferred from the earlier onset or greater penetrance of neoplasia in mice lacking certain immune cell subsets, recognition molecules, effector pathways, or cytokines and by studies comparing tumor initiation, growth, and metastases in wildtype versus immunodeficient mice [reviewed in (18)]. These studies have revealed that the immune components required for effective elimination of any given tumor are dependent on specific characteristics of the tumor, such as how it originated (spontaneous versus carcinogen-induced), its anatomic location, and its rate of growth. Equilibrium. Rare tumor cell variants may survive the elimination phase and enter the equilibrium phase, in which the adaptive immune system prevents tumor cell outgrowth and also sculpts the immunogenicity of the tumor cells. We envisage equilibrium to be the longest phase of the cancer immunoediting process—perhaps extending throughout the life of the host. As such, it may represent a second stable endpoint of cancer immunoediting. In equilibrium, the immune system maintains residual tumor cells in a functional state of dormancy, a term used to describe latent tumor cells that may reside in patients for decades before eventually resuming growth as either recurrent primary tumors or distant metastases (32). Equilibrium thus represents a type of tumor dormancy in which outgrowth of occult tumors is specifically controlled by immunity. An early suggestion that the immune system could maintain tumor cells in a dormant/equilibrium state came from tumor transplantation experiments in which mice were primed with a transplantable tumor and then rechallenged with the same tumor in order to induce tumor latency (33). However, stronger evidence for the existence of an immunologically mediated equilibrium phase came from primary tumorigenesis experiments showing that immunocompetent mice treated with low-dose carcinogen [3′-methylcholanthrene (MCA)] harbored occult cancer cells for an extended time period even when the mice did not develop any apparent tumors (34). When the immune system of these mice was ablated [by administering monoclonal antibodies (mAbs) that

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deplete T cells and IFN-g], tumors rapidly appeared at the original MCA injection site in half of the mice. Tumor cells isolated from these lesions were highly immunogenic and thus resembled unedited sarcoma cells derived from MCA-treated immunodeficient RAG2−/− mice. Further analyses revealed that adaptive immunity—specifically, interleukin-12 (IL-12), IFN-g, CD4+, and CD8+ T cells—but not innate immunity was responsible for maintaining the occult tumor cells in equilibrium. This observation mechanistically distinguishes equilibrium from elimination because the latter displays an obligate requirement for both innate and adaptive immunity. Additional studies with different mouse tumor models have confirmed the capacity of the immune system to control the outgrowth of occult primary carcinomas and metastases for extended periods of time (35, 36). In the low-dose MCA system, equilibrium appears to be the result of both the growth inhibitory and cytocidal actions of immunity on the residual tumor cells (34). Conceivably, the same immune functions also provide the selective pressure that promote outgrowth of tumor cells that have acquired the most immunoevasive mutations. Escape. In the escape phase, tumor cells that have acquired the ability to circumvent immune recognition and/or destruction emerge as progressively growing, visible tumors. Progression from equilibrium to the escape phase can occur because the tumor cell population changes in response to the immune system’s editing functions and/or because the host immune system changes in response to increased cancer-induced immunosuppression or immune system deterioration. Tumor cell escape can occur through many different mechanisms [reviewed in (18, 24–26, 28, 37, 38)]. At the tumor cell level, alterations leading to reduced immune recognition (such as a loss of antigens) or increased resistance to the cytotoxic effects of immunity (for example, through induction of anti-apoptotic mechanisms involving persistent activation of pro-oncogenic transcription factors such as STAT3 or expression of anti-apoptotic effector molecules such as BCL-2) promote tumor outgrowth. Loss of tumor antigen expression is one of the best-studied escape mechanisms, and it can occur in at least three ways: (i) through emergence of tumor cells that lack expression of strong rejection antigens, (ii) through loss of major histocompatibility complex (MHC) class I proteins that present these antigens to tumor-specific T cells, or (iii) through loss of antigen processing function within the tumor cell that is needed to produce the antigenic peptide epitope and load it onto the MHC class I molecule. All of these alterations are probably driven by a combination of genetic instability inherent in all tumor cells and the process of immunoselection (24, 38). The end result is the generation via a Darwinian selection process of poorly immunogenic tumor cell variants that become “invisible”

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to the immune system and thus acquire the capacity to grow progressively. Alternatively, escape may result from the establishment of an immunosuppressive state within the tumor microenvironment (39). Tumor cells can promote the development of such a state by producing immunosuppressive cytokines such as vascular endothelial growth factor (VEGF), transforming growth factor–b (TGF-b), galectin, or indoleamine 2,3-dioxygenase (IDO) and/or by recruiting regulatory immune cells that function as the effectors of immunosuppression [reviewed in (18)]. Regulatory T cells (Treg cells) and myeloid-derived suppressor cells (MDSCs) are two major types of immunosuppressive leukocyte populations that play key roles in inhibiting host-protective antitumor responses. Treg cells are CD4+ T cells that constitutively express CD25 and the transcription factor Foxp3. When stimulated, they inhibit the function of tumorspecific T lymphocytes by producing the immunosuppressive cytokines IL-10 and TGF-b; by expressing the negative co-stimulatory molecules CTLA-4, PD-1, and PD-L1; and by consuming IL-2, a cytokine that is critical for maintaining CTL function. MDSCs are a heterogeneous group of myeloid progenitor cells and immature myeloid cells that inhibit lymphocyte function by inducing Treg cells; producing TGF-b; depleting or sequestering the amino acids arginine, tryptophan, or cysteine required for T cell function; or nitrating T cell receptors or chemokine receptors on tumor-specific T cells. Cancer Immunoediting Versus Inflammation Inflammation is a complex physiological process that normally functions to maintain tissue homeostasis in response to tissue stressors such as infection or tissue damage (40). Acute inflammation (innate immunity) frequently precedes the development of protective adaptive immune responses to pathogens and cancer. Chronic inflammation, on the other hand, has been shown to contribute to tumorigenesis at all stages. It contributes to cancer initiation by generating genotoxic stress, to cancer promotion by inducing cellular proliferation, and to cancer progression by enhancing angiogenesis and tissue invasion (41). On the basis of these observations, it has been proposed that inflammation and tumor immunity are mutually exclusive processes (9, 10). In our view, a more likely interpretation is that tumor-promoting inflammation and protective tumor immunity are dynamically interconnected processes that vie for dominance as tumor cells develop and transit through cancer immunoediting (42). This scenario is supported by data from several different experimental systems. First, although tumor induction in MCA-treated mice requires the participation of pro-inflammatory cytokines/signaling (such as IL-1b, IL-23, or MyD88) the tumors, once formed, became susceptible to control by other components of im-

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munity (such as IFN-g, IFN-a/b, IL-12, or T cells) (43). Thus, tumor-promoting inflammation and cancer immunosurveillance/immunoediting can coexist within the same tumor model. Second, immune components with pro-oncogenic activity can also promote induction of tumor immunity, depending on when they are recruited into the cancer development process. For example, whereas MyD88 and IL-1b clearly promote carcinogen-induced tumorigenesis in mouse models (44–47), the same proteins have the opposite effect at later stages of tumorigenesis— that is, they promote development of protective immune responses against established tumors by facilitating recognition of tumor cells undergoing “immunogenic death” (48–50). This paradoxical role of inflammatory cytokines and the immune response in cancer is also illustrated by the observation that tumor necrosis factor–a (TNF-a) has both tumor-promoting and antitumor activities in mouse and Drosophila tumor models (51) and by more recent work showing that in a mouse melanoma model, IFN-g is required both for UVB-induced tumor formation and for immune rejection of these tumors (52). Lastly, inflammation can play an important role during tumor escape, when inflammatory cells are recruited to the site of a progressively growing tumor, undergo activation by cancer-derived products (such as VEGF), and suppress protective tumor immunity (41). Cancer Immunoediting in Humans Although studies of tumor development in mice served as the main driver for the formulation of the cancer immunoediting hypothesis, evidence has since been obtained indicating that immunoediting also occurs in humans and can alter the course of tumor development in cancer patients. We discuss three key types of evidence supporting this conclusion; more comprehensive summaries can be found in (18, 24). Intratumoral immune responses predict patient prognosis. The strongest evidence of cancer immunoediting in humans comes from reports that correlate the quantity, quality, and spatial distribution of tumor-infiltrating lymphocytes (TILs) with patient survival. Tumor infiltration by IFN-g producing Th1 CD4+ T cells and CD8+ T cells, and the presence of cytokines such as IFN-g and TNF-a that promote tumor control, has been associated with an improved prognosis for patients with many different cancers. A study of melanoma patients provided an early indication that TILs are associated with a favorable patient prognosis (53, 54). A subsequent landmark study by Naito et al. demonstrated that the presence and location of one particular type of TIL, CD8+ T cells, in colon cancers had a particularly important influence on clinical outcome; specifically, accumulation of CD8+ T cells within the tumor predicted improved patient survival, whereas accumulation of the same cells at the tumor margin had no effect

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on survival (55). Subsequent studies in ovarian cancer, melanoma, and colon cancer confirmed this observation and further showed that the ratio and distribution patterns of intratumoral CD8+ T cells and Treg cells were critical determinants of prognosis (56–59). Recent exciting studies of human colon and lung cancers have not only confirmed these observations but have provided quantitative insights into the key variables involved (58, 59). Remarkably, the type and density of lymphocytes infiltrating these cancers was found to be a more powerful prognostic indicator than previous pathological criteria for tumor staging and was even more predictive than correlating disease progression with oncogene expression. Spontaneous immune responses in cancer patients. A major advance to the field of tumor immunology came from the demonstration that cancer patients can develop high levels of antibody and T cell responses to antigens expressed in their tumors [reviewed in (60)]. These immune responses are generally observed in patients with progressively growing tumors, indicating that immune recognition of cancer does not always result in immune protection. However, there is presently no way to know whether such immune responses influence the rate or pattern of tumor growth in these patients and whether these responses represent the footprint of incomplete or ongoing elimination or equilibrium phases of cancer immunoediting. An example of the latter comes from the analysis of individuals with paraneoplastic neurologic disorders (PNDs). PNDs arise as a consequence of antibody and T cell responses against certain autologous tumors that ectopically express proteins normally expressed only in cells of the nervous system (61, 62). This antitumor response develops into an autoimmune response as it attacks normal neurons that express the tumor-associated antigens. The neurologic dysfunctions observed in PND usually become evident before the tumor is discovered. Immunodeficiency is associated with a higher risk of cancer. Immunodeficiency has been linked to increased cancer risk in patients with AIDS and in transplant recipients maintained on immunosuppressants [reviewed in (18, 24)]. Although the cancers arising in these patients are typically those with a viral etiology such as lymphomas (Epstein-Barr virus), Kaposi’s sarcoma (herpesviruses), and cervical cancer (human papilloma viruses), there is at least some evidence that these patients are at greater risk for malignancies of the colon, lung, pancreas, kidney, head and neck, and endocrine system as well as nonmelanoma skin cancers. Melanoma incidence rates are also 2 to 10 times higher than average in renal transplant patients. Interestingly, increased incidences of other cancers—including breast, prostate, ovarian, brain, and testes—have not been observed in immunosuppressed transplant patients. Insights into the role of the immune system in human cancer have also come from anecdotal

reports of cancer being transferred from an organ donor to the immunosuppressed recipient (63, 64). In one study, two individuals received kidney transplants from the same cadaver donor, and both recipients later succumbed to malignant melanoma that was shown by tissue typing to be of donor origin. Medical records revealed that 16 years before her death, the donor had been diagnosed with malignant melanoma and successfully treated. One interpretation of these findings is that the donor’s kidneys contained dormant melanoma cells held in equilibrium by the donor’s immune system. The transfer of the kidney to naïve and immunosuppressed recipients may have removed the immune pressure holding the tumor cells in equilibrium and thus allowed the occult melanoma cells to grow out into clinically apparent cancer. Together, these clinical observations are consistent with the hypothesis that de novo malignancies arise only in certain permissive microenvironments created by immunosuppressive regimens that suspend or severely compromise the elimination and/or equilibrium phases of cancer immunoediting. Cancer Immunoediting in Immunotherapy With our newfound knowledge of the immune system’s capacity to not only recognize and destroy cancer but also to shape cancer immunogenicity, more informed attempts to control cancer via immunological means can now be pursued. It is now well accepted that progressively growing, clinically apparent tumors in cancer patients have developed successful strategies to circumvent the natural, extrinsic tumor-suppressor mechanisms of immunity. Thus to be effective, immunotherapies will have to increase the quality or quantity of immune effector cells, reveal additional protective tumor antigens, and/or eliminate cancerinduced immunosuppressive mechanisms. Multiple forms of immunotherapy are being explored to achieve these objectives. These include (i) vaccine approaches to elicit strong specific immune responses to tumor antigens such as MAGE-3 and NY-ESO-1; (ii) approaches involving adoptive transfer of in vitro expanded, naturally arising, or genetically engineered tumor-specific lymphocytes; (iii) therapeutic administration of monoclonal antibodies such as Rituximab (directed against CD20 on leukemia and lymphoma cells) and Herceptin (directed against HER2 on breast cancer cells) to target and eliminate tumor cells; and (iv) approaches that inhibit or destroy the molecular or cellular mediators of cancerinduced immunosuppression such as CTLA-4, PD-1, or Treg cells. Quantitative analyses performed on patients undergoing various forms of cancer immunotherapy have revealed that the cancer immunoediting process reoccurs either in part or in its entirety during therapy. Specifically, whereas some treated patients display responses that recapitulate the elimination phase of cancer immunoediting (for

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example, they develop increased numbers of tumor-specific T cells with intact effector function, or they show destruction of some or all tumor cells) others show evidence for establishment of a therapeutically induced equilibrium phase, and still others display evidence for development of additional escape mechanisms, such as outgrowth of antigen-loss variants. Recently, the occurrence of all three phases of cancer immunoediting has been documented in a melanoma patient with preexisting NY-ESO-1 immunity undergoing CTLA-4 blockade monotherapy (65). When observed over a 28-month period after initiation of immunotherapy, melanoma lesions could be identified that disappeared (elimination), were held in a protracted state of growth dormancy (equilibrium), or continued to grow (escape). Thus, cancer immunoediting can occur not only when the unmanipulated immune system encounters a developing tumor but also when an established tumor is subjected to immunotherapy. Future Directions We envision that future work on cancer immunoediting will address five major questions: (i) What immune effector processes mediate cancer elimination, equilibrium, and escape? T cells play a critical role in mediating both natural and therapeutically induced cancer immunoediting responses. However, it remains unclear whether they represent the ultimate effectors of these processes. Although activated T cells and other lymphocytes can certainly kill tumor cells, they also elaborate a variety of cytokines such as IFN-g and TNF-a that can exert profound cytostatic and cytocidal effects on tumor cells, activate tumor cytotoxicity in other cell types (such as macrophages) present in the tumor microenvironment, and block tumor angiogenesis. Identifying the molecular mechanisms and targets responsible for cancer elimination, equilibrium, and escape will determine whether the three phases of cancer immunoediting are manifest by similar or distinct effector processes. (ii) How do the antigens of nascent tumors differ from the antigens of established, clinically apparent tumors? Almost all of our knowledge of tumor antigens is based on analyses of advanced cancers in imunocompetent hosts. It is important to identify the antigens expressed in early developing tumors because these are the initial targets of the elimination phase of cancer immunoediting. In addition, it would be interesting to define the antigens of tumors from immunosuppressed individuals because these antigens may not have undergone extensive editing and thus may be similar to the antigens of nascent tumors. Are the major antigens of unedited tumors more likely to be associated with driver or passenger mutations? An answer to this question may provide insights into the capacity of immunity to eliminate developing tumor cells, hold them in an equilibrium state, or facilitate their outgrowth. Can we use

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information from high-throughput screening of cancer genomes and proteomes or other cuttingedge techniques to rapidly identify the mutations and epigenetic changes in unedited and edited cancer cells that result in formation of functionally relevant tumor antigens? (iii) What is the link between the types of antigens expressed in a tumor and the mechanism of cellular transformation? With the exception of viral-mediated oncogenesis, little is known about whether and how the mechanisms leading to cell transformation affect the quality or quantity of tumor antigens. The possibility should be considered that experimental tumors, in which transformation is driven by strong oncogenes, may harbor fewer passenger mutations than do spontaneous tumors (66). Because passenger mutations can produce tumor antigens, oncogene-driven cancer models may therefore not always be optimal models for exploring the immunology of naturally developing tumors. However, a recent study revealed a previously unknown capacity of the immune system to sustain tumor regression upon oncogene inactivation (67). These considerations emphasize the need for further work on defining the relationships between cellular transformation mechanisms and tumor immunogenicity. In the future, careful consideration should be given to the use of cancer models that most closely recapitulate both the biology and immunology of human cancers. (iv) Is a durable state of equilibrium a desirable and attainable endpoint for cancer immunotherapy? We currently know very little about the effector mechanisms that operate in the equilibrium phase. To date, mouse studies reveal that T cells and IFN-g contribute to equilibrium, but the recognition pathways and immune network and mechanisms remain unclear. If these can be identified, it might be possible to develop cancer therapies aimed at recapitulating immunological tumor dormancy. It will also be important to understand what effect conventional interventions, such as surgery, radiotherapy, and chemotherapy have on the equilibrium phase. (v) How can we most effectively inhibit cancerinduced immunosuppressive mechanisms at the tumor site so as to boost the host-protective antitumor effects of preexisting or therapeutically induced immunity without concomitantly inducing life-threatening autoimmunity? Arguably, this may be the most pressing question in all of tumor immunology. Unlike other therapies that target cancer cells, therapies aimed at inhibiting immunosuppression target the immune system itself. An exciting approach being evaluated in clinical trials involves the use of monoclonal antibodies to blockade immunosuppressive molecules such as CTLA-4 or PD-1 expressed by T cells. In a related approach, the effectiveness of monoclonal antibodies that block the PD-1 ligand, PD-L1, which can be expressed on both tumor cells and normal host cells, is also being explored. These types of therapies have been designated “checkpoint block-

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ade” (68). In the case of CTLA-4 blockade, a recent phase III clinical trial reported that therapy with CTLA-4–blocking antibodies imparted a significant survival benefit in approximately onethird of patients with metastatic melanoma, making this drug a promising treatment for cancer (69). The success of the current CTLA-4 blockade clinical trials has stimulated interest in blocking other potential effectors of immunosuppression, including the soluble (such as IDO and TGF-b) and cellular (such as Treg cells and MDSCs) mediators of the process. Clearly, there is much to be learned about the benefits and risks of inhibiting the different immunosuppressive mechanisms that may be concurrently operating in the cancer patient. Conclusion The cancer immunoediting concept attempts to integrate the diverse effects of the immune system on tumor development and outgrowth. With elucidation of the molecular and cellular mechanisms that underlie the elimination, equilibrium, and escape phases of this process, it should be possible to develop new cancer immunotherapies that are safer and more efficacious in a substantial percentage of cancer patients. Given the well-established effects of immunity on cancer development and outgrowth, escape from immune control can now be viewed as one of the “Hallmarks of Cancer” (70). References and Notes 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25.

P. Ehrlich, Ned. Tijdschr. Geneeskd. 5, 273 (1909). L. J. Old, E. A. Boyse, Annu. Rev. Med. 15, 167 (1964). M. Burnet, BMJ 1, 841 (1957). L. Thomas, Cellular and Humoral Aspects of the Hypersensitive States, H. Lawrence, Ed. (Hoeber-Harper, New York, 1959). O. Stutman, Science 183, 534 (1974). O. Stutman, Adv. Cancer Res. 22, 261 (1975). P. Matzinger, Annu. Rev. Immunol. 12, 991 (1994). D. Pardoll, Annu. Rev. Immunol. 21, 807 (2003). F. Balkwill, A. Mantovani, Lancet 357, 539 (2001). M. Karin, Y. Cao, F. R. Greten, Z. W. Li, Nat. Rev. Cancer 2, 301 (2002). A. S. Dighe, E. Richards, L. J. Old, R. D. Schreiber, Immunity 1, 447 (1994). D. H. Kaplan et al., Proc. Natl. Acad. Sci. U.S.A. 95, 7556 (1998). V. Shankaran et al., Nature 410, 1107 (2001). M. J. Smyth et al., J. Exp. Med. 192, 755 (2000). M. J. Smyth et al., J. Exp. Med. 191, 661 (2000). S. E. Street, J. A. Trapani, D. MacGregor, M. J. Smyth, J. Exp. Med. 196, 129 (2002). M. Girardi et al., Science 294, 605 (2001). M. D. Vesely, M. H. Kershaw, R. D. Schreiber, M. J. Smyth, Annu. Rev. Immunol. 10.1146/annurev-immunol031210-101324 (2011). L. J. Old, Cancer Res. 41, 361 (1981). A. Knuth, B. Danowski, H. F. Oettgen, L. J. Old, Proc. Natl. Acad. Sci. U.S.A. 81, 3511 (1984). P. van der Bruggen et al., Science 254, 1643 (1991). U. Sahin et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11810 (1995). M. A. Cheever et al., Clin. Cancer Res. 15, 5323 (2009). G. P. Dunn, A. T. Bruce, H. Ikeda, L. J. Old, R. D. Schreiber, Nat. Immunol. 3, 991 (2002). G. P. Dunn, L. J. Old, R. D. Schreiber, Immunity 21, 137 (2004).

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26. G. P. Dunn, L. J. Old, R. D. Schreiber, Annu. Rev. Immunol. 22, 329 (2004). 27. G. P. Dunn, C. M. Koebel, R. D. Schreiber, Nat. Rev. Immunol. 6, 836 (2006). 28. M. J. Smyth, G. P. Dunn, R. D. Schreiber, Adv. Immunol. 90, 1 (2006). 29. J. B. Swann, M. J. Smyth, J. Clin. Invest. 117, 1137 (2007). 30. G. P. Sims, D. C. Rowe, S. T. Rietdijk, R. Herbst, A. J. Coyle, Annu. Rev. Immunol. 28, 367 (2010). 31. N. Guerra et al., Immunity 28, 571 (2008). 32. J. A. Aguirre-Ghiso, Nat. Rev. Cancer 7, 834 (2007). 33. J. D. Farrar et al., J. Immunol. 162, 2842 (1999). 34. C. M. Koebel et al., Nature 450, 903 (2007). 35. S. Loeser et al., J. Exp. Med. 204, 879 (2007). 36. J. Eyles et al., J. Clin. Invest. 120, 2030 (2010). 37. L. Zitvogel, A. Tesniere, G. Kroemer, Nat. Rev. Immunol. 6, 715 (2006). 38. H. T. Khong, N. P. Restifo, Nat. Immunol. 3, 999 (2002). 39. S. Radoja, T. D. Rao, D. Hillman, A. B. Frey, J. Immunol. 164, 2619 (2000). 40. R. Medzhitov, Nature 454, 428 (2008). 41. S. I. Grivennikov, F. R. Greten, M. Karin, Cell 140, 883 (2010). 42. J. D. Bui, R. D. Schreiber, Curr. Opin. Immunol. 19, 203 (2007). 43. M. W. Teng et al., Proc. Natl. Acad. Sci. U.S.A. 107, 8328 (2010). 44. W. E. Naugler et al., Science 317, 121 (2007). 45. S. Rakoff-Nahoum, R. Medzhitov, Science 317, 124 (2007). 46. J. B. Swann et al., Proc. Natl. Acad. Sci. U.S.A. 105, 652 (2008). 47. Y. Krelin et al., Cancer Res. 67, 1062 (2007). 48. L. Apetoh et al., Nat. Med. 13, 1050 (2007). 49. M. Obeid et al., Nat. Med. 13, 54 (2007). 50. F. Ghiringhelli et al., Nat. Med. 15, 1170 (2009). 51. J. B. Cordero et al., Dev. Cell 18, 999 (2010). 52. M. R. Zaidi et al., Nature 469, 548 (2011). 53. W. H. Clark Jr. et al., J. Natl. Cancer Inst. 81, 1893 (1989). 54. C. G. Clemente et al., Cancer 77, 1303 (1996). 55. Y. Naito et al., Cancer Res. 58, 3491 (1998). 56. E. Sato et al., Proc. Natl. Acad. Sci. U.S.A. 102, 18538 (2005). 57. I. S. van Houdt et al., Int. J. Cancer 123, 609 (2008). 58. F. Pagès et al., N. Engl. J. Med. 353, 2654 (2005). 59. J. Galon et al., Science 313, 1960 (2006). 60. M. Dougan, G. Dranoff, Annu. Rev. Immunol. 27, 83 (2009). 61. M. L. Albert, R. B. Darnell, Nat. Rev. Cancer 4, 36 (2004). 62. M. L. Albert et al., Nat. Med. 4, 1321 (1998). 63. H. Myron Kauffman et al., Transplantation 74, 358 (2002). 64. R. M. MacKie, R. Reid, B. Junor, N. Engl. J. Med. 348, 567 (2003). 65. J. Yuan et al., Cancer Immun. 10, 1 (2010). 66. M. DuPage et al., Cancer Cell 19, 72 (2011). 67. K. Rakhra et al., Cancer Cell 18, 485 (2010). 68. A. J. Korman, K. S. Peggs, J. P. Allison, Adv. Immunol. 90, 297 (2006). 69. F. S. Hodi et al., N. Engl. J. Med. 363, 711 (2010). 70. D. Hanahan, R. A. Weinberg, Cell 144, 646 (2011). 71. We thank M. D. Vesely and M. H. Kershaw for invaluable advice and contributions to this review and E. R. Unanue and P. M. Allen for constructive suggestions. R.D.S. is supported by grants from the National Cancer Institute, the Cancer Research Institute, and the Ludwig Institute for Cancer Research. L.J.O. is supported by The Ludwig Institute for Cancer Research and grants from the Cancer Research Institute. M.J.S. is supported by a National Health and Medical Research Council of Australia (NH&MRC) Australia Fellowship and Program Grant and a grant from the Association for International Cancer Research. We apologize to all the investigators whose research could not be appropriately cited because of the journal’s space limitations. R.D.S. is a co-founder and member of the Board of Directors of and is a paid scientific advisor for Igenica, a biopharmaceutical company dedicated to the discovery and development of antibody-based therapeutics for the treatment of cancer. 10.1126/science.1203486

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Cancer Crusade at 40

Mutations in the RNA Granule Component TDRD7 Cause Cataract and Glaucoma Salil A. Lachke,1* Fowzan S. Alkuraya,1,2,3,4* Stephen C. Kneeland,5* Takbum Ohn,6† Anton Aboukhalil,1,7 Gareth R. Howell,5 Irfan Saadi,1‡ Resy Cavallesco,1 Yingzi Yue,1 Anne C-H. Tsai,8 K. Saidas Nair,5 Mihai I. Cosma,5,9 Richard S. Smith,5 Emily Hodges,10 Suad M. AlFadhli,11 Amal Al-Hajeri,11 Hanan E. Shamseldin,2 AbdulMutalib Behbehani,12 Gregory J. Hannon,10 Martha L. Bulyk,1,13,14 Arlene V. Drack,15 Paul J. Anderson,6 Simon W. M. John,5,16§ Richard L. Maas1§ The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis. n eukaryotic cells, cytoplasmic RNA granules (RGs) function in determining whether mRNAs undergo degradation, stabilization, or intracellular localization (1–3). Lower eukaryotes such as yeast harbor RGs that are classified as either processing bodies (PBs) or stress gran-

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1 Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA. 2Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, 11211, KSA. 3Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh 11533, KSA. 4Department of Pediatrics, King Khalid University Hospital and College of Medicine, King Saud University, Riyadh 11461, KSA. 5Howard Hughes Medical Institute and The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA. 6Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA. 7Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 8Department of Pediatrics, University of Colorado-Denver, Aurora, CO 80045, USA. 9Department of Medicine, Maine Medical Center, Portland, Maine, ME 04102, USA. 10Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA. 11Medical Laboratory Medicine, Faculty of Allied Health Sciences, Kuwait University, Kuwait City 13060, KW. 12 Department of Surgery, Faculty of Medicine, Kuwait University, Alsafat 13110, KW. 13Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA. 14Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology (HST), Harvard Medical School, Boston, MA 02115, USA. 15Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA 52242, USA. 16Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02115 USA.

*These authors contributed equally to this work. †Present address: Division of Natural Sciences, Chosun University, Gwangju 501-759, South Korea. ‡Present address: Center for Regenerative and Developmental Biology, The Forsyth Institute, Cambridge, MA 02142, USA. §To whom correspondence should be addressed. E-mail: [email protected] (R.L.M.); simon.john@jax. org (S.W.M.J.)

ules (SGs) (3, 4), whereas metazoan cells harbor additional classes of RGs (5–7). Somatic cells contain both PBs and transport ribonucleoprotein (RNP) particles and accumulate SGs in response to environmental stress (8, 9). PBs contain components of mRNA decay processes like Xrn1mediated 5′ to 3′ degradation, nonsense-mediated decay (NMD), and microRNA-mediated silencing, and serve as sites where mRNAs can be either stored or degraded (10). In neuronal cells and fibroblasts, RNPs function in the transport and localized translation of mRNAs involved in synapse formation or motility (8, 11). SGs store bulk mRNA during conditions of stress and can interact with PBs to exchange mRNAs that are then directed to translational reinitiation or degradation (9, 12). Lastly, germ cells contain germ cell– specific granules (GCG) that have been implicated in germ cell specification (2, 7). Proteins with roles in development have been associated with RGs (13), but the importance of this association and that of somatic cell RGs in metazoan organogenesis remains unknown. For example, the precise spatial and temporal expression of key transcription factors is essential for normal transcriptional regulation during development. However, whether a similar level of developmental control exists over the expression of factors involved in posttranscriptional mRNA regulation, and whether this control is required for organogenesis, is unclear. We report the identification of Tudor domain–containing 7 protein, or TDRD7, as an RG component with a highly enriched and conserved pattern of developmental expression in the vertebrate ocular lens. In lens, we identify TDRD7 as a component of a unique class of RNPs and show that these TDRD7-RNA granules (TDRD7-RGs) differentially associate

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with lens PBs and RNPs that contain a known RG component, STAU1 (STAU1-RNPs). Furthermore, human TDRD7 mutations result in cataract formation via the misregulation of specific, developmentally critical lens transcripts, and Tdrd7 null mutant mice develop cataract as well as glaucoma, the latter defined by elevated intraocular pressure (IOP) and optic nerve damage. The study of patients with balanced chromosomal rearrangements represents an important entry point to understanding disease mechanisms, and numerous examples exist of disease genes that have been identified by virtue of being disrupted by rearrangement breakpoints (14). As part of the Developmental Genome Anatomy Project (DGAP) (www.bwhpathology.org/dgap), we ascertained a male patient, designated DGAP186, with juvenile cataract and hypospadias who has a de novo balanced paracentric inversion of chromosome 9, 46,XY,inv(9)(q22.33q34.11) (Fig. 1, A and B). We determined that the 9q34.11 breakpoint disrupts the gene NR5A1 (fig. S1). NR5A1 encodes a steroid nuclear receptor protein implicated in hypospadias in mouse and human mutants without cataract (15–17). NR5A1 disruption is therefore likely to explain the reproductive tract phenotype and unlikely to account for the cataract phenotype in DGAP186. Analysis of the 9q22.33 breakpoint revealed that TDRD7 is disrupted (Fig. 1, B and C, and fig. S1), and TDRD7 haploinsufficiency as a direct result of the allelic disruption was observed in DGAP186 lymphoblastoid cells at both the RNA and protein levels, (Fig. 1D and fig. S1). To independently confirm the involvement of TDRD7 in pediatric cataract, we identified a family, F3R, with autosomal recessive congenital cataract. Homozygosity mapping (18) identified a single block of shared homozygosity between the four affected siblings that spanned the TDRD7 locus (Fig. 1E and fig. S2). Furthermore, bidirectional sequencing of TDRD7 uncovered a novel in-frame 3–base pair deletion that removes a highly conserved amino acid, V618 (Fig. 1F and fig. S2). This V618del variant, which was not detected in 320 ethnically matched controls (640 chromosomes), is predicted to disrupt the structure of TDRD7 and is therefore likely to represent a loss-of-function mutation (fig. S2). To assay the endogenous expression of TDRD7, we turned to mouse and chick embryos. In situ hybridization revealed strong and highly specific expression of Tdrd7 transcripts in the developing mouse lens (Fig. 2A and fig. S3). At embryonic day E12.5, Tdrd7 is expressed in differentiating fiber cells in the posterior lens, whereas the anterior epithelium of the lens (AEL) lacks detectable expression. Expression of chick TDRD7 is also high in the developing lens (Fig. 2B). To confirm a direct causal link between TDRD7 haploinsufficiency and cataract formation, we used a replication-competent avian sarcoma (RCAS) viral vector (19) to deliver short hairpins that

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specifically targeted chick TDRD7 to achieve RNA interference–mediated gene knockdown. TDRD7-knockdown short hairpin–mediated RNA interference (shRNA) retroviruses were injected in E2 [Hamburger and Hamilton stage 11 (HH st. 11)] optic vesicles, and the embryos were analyzed for the presence of cataract at E16 (HH st. 42). Injections of green fluorescent protein–expressing control RCAS virus led to highly efficient uptake by lens cells (fig. S3). At E16, a cataract phenotype was observed at significant frequency (17/115, 15%) in chick embryos injected with TDRD7shRNA RCAS virus but not in those that received control virus (1/48, 2%; P < 0.05) (Fig. 2C and fig. S3). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis indicated that cataract formation was observed in lenses in which TDRD7 transcripts were reduced to approximately 40% or less of control levels (fig. S3). To gain further insight into TDRD7 function, we analyzed mutant mice with an N-ethyl-Nnitrosourea (ENU)–induced recessive mutation in Tdrd7. These mice, identified during a screen for glaucoma phenotypes, develop cataracts and high IOP. A nonsense mutation c.2187C>T (Q723X) produces a Tdrd7 null allele, as determined by the absence of TDRD7 protein in homozygous mutants (fig. S4). Within 4 weeks of birth, all Tdrd7 homozygous mutants developed a posterior cataract that became severe with age (Fig. 2, D and E). At later stages, the lens fiber cell compartment developed vacuoles with lens capsule rupture and extrusion of fiber cell mass into the vitreous (Fig. 2, F and G). This feature of the mouse lens phenotype precisely recapitulates the unusual posterior lenticonus (a conical projection of the lens surface) and posterior capsule defects observed in the DGAP186 proband (15). In addition, in some Tdrd7 mutant mice, the mass of fiber cells passed through the pupil into the anterior chamber of the eye (fig. S4). By 4 months of age, iris flattening was detected and anterior chamber depth increased (fig. S5). By 6 months of age, the IOP was elevated in some Tdrd7 mutants, and the incidence of elevated IOP increased with age (Fig. 2H and fig. S5). The ocular drainage structures Schlemm’s canal and the trabecular meshwork normally influence IOP and are located in the angle of the anterior chamber—the angle formed between the iris and the cornea, where the aqueous humor flows out of the anterior chamber. If the egress of aqueous humor is impeded, the accumulated fluid leads to increased IOP, which in turn contributes to retinal ganglion cell (RGC) death and optic nerve atrophy, all of which are hallmarks of glaucoma. In Tdrd7 mutants, the angles are largely normal in morphology with an open-angle configuration, defined by absence of morphologic obstruction (Fig. 2, I and J); the open-angle configuration also predominates in human glaucoma. In Tdrd7 mutant mice, severe optic nerve atrophy characterized by RGC axon loss and excavative remodeling of the optic nerve were observed (Fig. 2, K to N, and fig. S5). Notably, in family F3R, two of the four

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affected individuals developed glaucoma with open angles and increased IOP after cataract extraction, pointing to the Tdrd7 mutant mouse as a potential model for certain aspects of human glaucoma. TDRD7 contains five conserved Tudor class domains and three OST-HTH (Oskar-TDRD7Helix-Turn-Helix)/LOTUS domains (Fig. 1B), which bind methylated arginine residues and RNA, respectively (20). TDRD1, TDRD6, and TDRD7 have been associated with a GCG known as the chromatoid body (CB) that is found in mammalian male germ cells (21, 22). To gain insight into TDRD7’s cellular function, we generated a mouse TDRD7 antibody and analyzed protein expression during mouse embryogenesis. TDRD7 expression between E11.0 and E12.5 becomes markedly enriched in lens fiber cells, where it is expressed in numerous cytoplasmic granules of 0.3 to 0.8 mm diameter (Fig. 3, A and B, and fig. S6). A similar pattern was observed with a second independent TDRD7 antibody (fig. S6). TDRD7 granules were also found in differentiating secondary fiber cells in postnatal day 1 (P1) lens (fig. S6). To deter-

mine whether these TDRD7 positive granules contained RNA, we stained mouse E12.5 lens sections with SYTO RNASelect or Pyronin Y, two RNA-specific stains (Fig. 3C and fig. S7). Costaining with TDRD7 antibody revealed that TDRD7 granules colocalize with RNA in lens fiber cell cytoplasm, and thus constitute bona fide RGs, denoted TDRD7-RGs (Fig. 3, C to E). To establish whether TDRD7 is associated with a specific class of RGs in mouse embryonic lens, we examined the expression of protein markers for different classes of somatic cell RGs (23). Immunostaining of mouse embryonic lens sections with antibodies against the PB markers DCP1A and Ge-1 (23) demonstrated the presence of numerous cytoplasmic PBs in lens fiber cells (Fig. 3F and fig. S8). These structures were also found to contain DDX6/RCK and were dissociated by cycloheximide (CHX) (fig. S9), indicating that they are functional PBs. Costaining these sections with TDRD7 antibody demonstrated that TDRD7-RGs interact with PBs (Fig. 3F and fig. S8). In addition to colocalization, PBs and TDRD7RGs were occasionally juxtaposed to each other,

Fig. 1. TDRD7 mutations in human pediatric cataract. (A) Cataract in DGAP186 (left eye, white arrowhead). (B) Ideogram of normal and inverted chromosome 9 [inv(9)]. Inversion breakpoints are shown by red lines, with a schematic below of TDRD7. Dotted black line marks breakpoint that disrupts TDRD7 within the 2.6-kb region shown and in TDRD7 protein. The black bar indicates TDRD7 genomic probe in Southern analysis. TaqI and StuI refer to the fragments resulting from restriction enzyme digest. The red bar indicates fosmid clone G248P8912G10 used as a TDRD7-specific probe. Exons are indicated by purple boxes. (C) Chromosomal spread of DGAP186 lymphoblastoid cells analyzed by fluorescence in situ hybridization shows split TDRD7-specific red probe, whereas green anchor probe remains intact in inv(9). White arrowheads indicate inverted genomic region that hybridizes to TDRD7-specific probe; white arrow represents noninverted region (see fig. S1). (D) TDRD7 haploinsufficiency is demonstrated by Western blot of DGAP186 lymphoblastoid cells. (E) Pedigree of consanguineous family F3R with congenital cataract (solid symbols, affected status; half-solid symbols, carrier status). (F) Sequence chromatogram shows c.1852_1854del (p.617delVal) mutation (boxed) in family F3R (see fig. S2).

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in an arrangement similar to the docking-type interactions observed for DCP1A and STAU1RNPs in neurons (fig. S8) (24). Quantification of both colocalizing and docking configurations between TDRD7 granules and PBs indicated a modest but significant degree of overall interaction [12 T 4%, mean T SEM, n = 250 PBs; P < 1.5 × 10−5, compared to a model of random distribution throughout the lens (15)]. STAU1 and STAU2, mammalian homologs of the Drosophila RNA-binding protein Staufen, are components of transport RNPs (25–27). Staining with STAU1 antibody revealed the presence of numerous STAU1-positive RNPs in lens fiber cells (Fig. 3G). These colocalized to a high degree (29 T 5%, mean T SEM, n = 258 granules)

with TDRD7 (Fig. 3G and fig. S10). Expression of TDRD7 and STAU1 proteins could be detected as early as E10.5 at the lens vesicle stage (fig. S10). When tested at E11.5, the extent of TDRD7-STAU1 colocalization was highest along the anterior edge of the elongating fiber cell compartment (Fig. 3H), which apposes the AEL at later developmental stages. Similar results were obtained with the second TDRD7 antibody (fig. S10). We also tested for other known components of RGs, namely TIA-1, TIAL1 (TIAR), STAU2, and HuR (ELAVL1) (23), and found that these were not components of mouse lens RGs at E12.5 (fig. S11). Previous studies have indicated that RGs dynamically interact with each other, and they may

Fig. 2. Tdrd7 deficiency causes cataract and glaucoma. (A) In situ hybridization of E12.5 mouse embryo (coronal section) demonstrates Tdrd7 RNA expression in differentiating lens fiber cells (FC) (arrowhead), and its absence in the AEL (arrow). (B) In situ of stage 21 chick embryonic eye (coronal section) shows TDRD7 expression in lens FCs (arrowhead) and absence in AEL. NR, neuroretina; RPE, retinal pigment epithelium. (C) Cataract (white arrowhead) in chick lenses infected with TDRD7-shRNA RCAS virus. (D) Absence of cataract in 1-month-old Tdrd7 heterozygous mouse (control) and (E) cataract in age-matched Tdrd7 null mouse lens. (F) Histology of control lens at 3 months shows no ocular abnormality and (G) a severe cataract in Tdrd7 null mouse lens at same age. Note vacuoles in FC lens compartment (arrow) and rupture of lens capsule that extrudes fiber cell mass (arrowhead) into the vitreous. RD, mild to severe retinal dysplasia, focally present in some mutants. (H) Tdrd7 null (red diamonds), but not control (blue triangles), mice exhibit age-dependent increase in IOP (see fig. S5). (I and J) Histology of iridocorneal angles showing normal morphology and open configuration (*) in both control (I) and mutant (J) eyes. Mutant angles are largely normal and open, but some mutants had mild, focal abnormalities of Schlemm’s canal (SC) and trabecular meshwork (TM). Iris strands that attach to the cornea are common in mice (arrow) but are not continuous around the angle and do not block drainage. Angles of all histologically assessed mice with high IOP at 6 to 12 months were open (n = 3). Co, cornea; Ir, iris. (K to N) At ages >20 months, the optic nerve head and nerve fiber layer (RGC axons, arrows) in control mice (K) appear normal, whereas Tdrd7 null mice (L) have considerable nerve damage with essentially no nerve fiber layer (arrows) and optic nerve head excavation (*). V, vessel. (M and N) Retinal periphery of control mouse (M) has normal cell density (arrows) in the ganglion cell layer (GCL), whereas mutant (N) with glaucomatous excavation of the nerve head has obvious cell loss. INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: [(F) and (G)] 500 mm; [(I) and (J)] 50 mm; [(K) and (L)] 100 mm; [(M) and (N)] 50 mm. www.sciencemag.org

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share components or participate in mRNA exchange (12). STAU1 is especially relevant, as it is a component of all four RG classes and functions in diverse aspects of RNA metabolism, including mRNA transport and mRNA decay (24, 28, 29). We therefore hypothesized that TDRD7 granules, either alone or through their interaction with STAU1RNPs and PBs, might regulate the number of RGs in the cell as well as the expression levels of specific lens transcripts (fig. S12). We further postulated that the misregulation of specific mRNAs due to reduced TDRD7 dosage might cause cataract formation. To test these hypotheses, we first performed TDRD7 knockdowns in a U2OS cell line–based assay previously developed to identify genes critical for RG formation (13). These experiments revealed that Tdrd7 knockdown led to significant reductions in the numbers of SGs and PBs (fig. S13). To determine whether this effect was conserved in lens cell lines with higher numbers of SGs and PBs, we examined the human lens cell line SRA01/04 and quantified the results. In these cells, TDRD7-knockdown produced a dramatic reduction in SGs, indicating that TDRD7 is critical for an appropriate response of these cells to stressful stimuli (Fig. 4A and fig. S14). In contrast, TDRD7 knockdown produced only minimal effects on PB numbers. Statistically significant reductions in SG numbers were also observed in Tdrd7-knockdown experiments in a mouse lens– derived epithelial cell line, 21EM15 (henceforth Tdrd7-KD 21EM15) (30), where the reduction in Tdrd7 expression of 60 T 4% (mean T SEM, n = 3) approximates that in DGAP186 lymphoblastoid cells due to haploinsufficiency (figs. S13 and S14). Next, to identify Tdrd7 deficiency–induced changes in gene expression, we performed microarray analyses in biological triplicate on Tdrd7KD 21EM15 cells and compared the in vitro results to those obtained for Tdrd7 null lenses. In initial microarray experiments, we observed that 21EM15 cells expressed 56 of the top 100 genes that are expressed during embryonic lens development, including Tdrd7. Thus, 21EM15 cells at least partly recapitulate the molecular events during endogenous lens development. In Tdrd7-KD 21EM15 cells, compared to 21EM15 cells infected with control vector, ~6% of expressed genes were differentially regulated at a threshold of a 1.3-fold or greater (table S1). Of particular interest, key genes that encode SG and PB components (G3bp, Hspb1, and Ddx6), mouse homologs of known human cataract genes (Crygs and Epha2), and genes involved in fiber cell differentiation (Prox1) were all significantly downregulated (Fig. 5A and fig. S15). To extend these results in vivo, we further analyzed the Tdrd7 null mouse lens. As expected from Western blot analyses (fig. S4), immunofluorescence confirmed a complete absence of TDRD7 in the lens (Fig. 4B). We then undertook a microarray expression analysis of isolated lenses from Tdrd7 null mutants and littermate controls, focusing on postnatal days P4, three weeks before overt cataract appearance, and P30, when

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cataracts are fully penetrant. To accurately identify Tdrd7-dependent genes that function in lens development, we compared the P4 and P30 Tdrd7 mutant mouse lens microarray data with that obtained from the Tdrd7-KD 21EM15 mouse lens cell line. Comparison of differentially regulated genes (DRGs) from the P4 and P30 Tdrd7 null lens microarray data sets (each versus control), and the DRGs in Tdrd7-KD 21EM15 cells (versus control) identified several biologically relevant genes that were concordantly regulated in all three data sets (Fig. 5, A and B). These DRGs were classified into five distinct functional categories, denoted Classes I to V: Class I, genes involved in SG assembly or function; Class II, genes involved in P-body function and/or encoding helicases; Class III, genes linked to cataract or other ocular phenotypes; Class IV, crystallin genes (apart from Class III); and Class V, genes normally down-regulated during lens fiber cell differentiation (Fig. 5A). Whereas Tdrd7 null lenses and Tdrd7-KD 21EM15 lens cells exhibit overlapping expression, each also exhibits unique categories of gene expression (e.g., Classes IVand V, respectively), and thus constitute complementary systems for revealing the full spectrum of DRGs attributable to Tdrd7 loss-of-function. Of 12 DRGs whose expression was significantly altered in Tdrd7-KD 21EM15 lens cells and that were relevant to lens development or RG function, six (Hspb1, Ddx6, Ddx26, Epha2, Prox1, and Crygs) were also concordantly downregulated in Tdrd7 null lens data sets. Two other genes that were down-regulated in both P4 and P30 Tdrd7 null lenses, Sparc and Crybb3, are associated with cataracts and are thus also of interest (31, 32) (Fig. 5B). Thus, from comparative analyses of the microarray data, we identified eight genes that were significantly down-regulated in the Tdrd7 null lens, Tdrd7-KD 21EM15 lens cells, or both, and that have biologically plausible links to lens development or RG function. To determine whether the altered expression of these eight genes resulted from the direct or indirect action of TDRD7, we used qRT-PCR to confirm the microarray expression changes in P30 Tdrd7 null lens RNA. This analysis confirmed the down-regulation of Crybb3, Hspb1, Sparc, and Epha2 and of several other genes (fig. S15). We then performed RNA immunoprecipitation (RIP) experiments with TDRD7 antibody, followed by RNA isolation and RT-PCR. Because 21EM15 cells substantially recapitulate the expression profile of the developing lens, and there is considerable concordance in DRGs between the corresponding Tdrd7 loss-of-function state, we employed 21EM15 lens cells for these experiments. Crybb3 and Hspb1 transcripts were markedly enriched in the TDRD7 immunoprecipitations, whereas Epha2 transcripts were only modestly enriched and Sparc transcripts were not enriched (Fig. 5C). Thus, based on their strong Tdrd7-expression dependence and evidence for direct binding from the RIP experiments, we conclude that Crybb3 and Hspb1 mRNAs are

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likely to be direct targets of TDRD7 regulatory function in the lens. An attractive model emerges from these results. During lens development, AEL cells migrate into the lens “equatorial zone,” where they differentiate into fiber cells. During fiber cell differentiation, because of the nuclear degradation process that helps promote lens clarity, most AEL-expressed genes cease transcription. By binding and stabilizing specific transcripts, TDRD7 and other RG components may facilitate the translation of crystallin mRNAs necessary to achieve the high protein levels and tight packing that provides for ocular transparency at high refractive index. TDRD7 also functions by maintaining mRNA expression levels of the heat shock gene Hspb1, which encodes a stress response chaperone protein that is a stress granule component that functions in mRNA decay (33, 34). Downregulation of Hspb1 mRNA levels is one of the earliest and quantitatively most striking gene expression changes we detect in Tdrd7 null lenses.

HSPB1 down-regulation at the protein level was detected in P4 and P22 lenses (Fig. 4B and fig. S15). Interestingly, HSPB1 also interacts with several lens crystallin proteins (35) and stabilizes aB-crystallin (36). Moreover, destabilizing mutations in aB-crystallin are a known cause of congenital cataract (OMIM ID 123590). Thus, Crybb3 and Hspb1 are excellent candidates to contribute to cataract formation in Tdrd7 loss-of-function mutants. TDRD7 may also regulate certain transcripts indirectly. For example, TDRD7 deficiency results in reduction of Sparc transcripts, and Sparc null mice develop late-onset cataracts that resemble Tdrd7 null mutant cataracts (31). These results place Tdrd7 function upstream of Sparc mRNA expression and, because Tdrd7 is involved in the regulation of multiple cataract genes (fig. S15), can explain the earlier cataract onset in Tdrd7 mutants compared with Sparc mutants. In addition, Sparc and Epha2 mRNAs have been described as direct targets of STAU1-RNPs, and

Fig. 3. TDRD7 RNA granules interact with STAU1-RNPs in lens fiber cells. (A) Immunofluorescence (IF) of mouse E12.5 lens (coronal section) with TDRD7 antibody demonstrates highly specific punctate expression in lens FCs and absence in AEL. (B) Higher magnification of E12.5 lens FCs stained with TDRD7 antibody shows numerous cytoplasmic granules (arrowheads). (C) Costaining with SYTO RNASelect reveals a similar pattern of cytoplasmic RNA granules (arrowheads). (D) 4´,6´-diamidino-2-phenylindole (DAPI) staining in same section. (E) Merged image [(B) to (D)] reveals colocalization (yellow) of TDRD7 with cytoplasmic RNA granules (see fig. S7). RNA staining of TDRD7 granules is somewhat variable and may reflect dynamic interactions of these granules. Scale bars: (A) 15 mm; (E) 2.5 mm. (F) IF of mouse E12.5 lens shows numerous cytoplasmic granules stained with antibodies against DCP1A (red) and TDRD7 (green). Colocalized granules appear yellow. (Inset) Higher magnification demonstrates colocalization of DCP1A P-bodies and TDRD7 RNA granules. (G) Staining of E12.5 lens with STAU1 antibody reveals numerous granules (red) in FC cytoplasm. These granules colocalize (yellow) to a high degree with TDRD7 granules (green). (Inset) Higher magnification indicates intimate colocalization (yellow, white arrowheads) of TDRD7 and STAU1 granules. (H) At E11.5, TDRD7 and STAU1 expression show a high degree of colocalization (yellow) in the anterior portion of the FC compartment. DAPI-stained nuclei are blue. (Inset) Higher magnification demonstrates high degree of colocalization (yellow) of TDRD7-RGs and STAU1-RNPs. Scale bars: (F) 15 mm; (F inset) 4 mm; (G) 15 mm; (G inset) 1 mm; (H) 10 mm; (H inset) 4 mm.

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RESEARCH ARTICLE Epha2 mutations cause cataracts in both mouse and human (26, 37). Because STAU1-RNPs and TDRD7-RGs interact in the lens, Sparc and Epha2 down-regulation in Tdrd7 mutant lenses may reflect the disruption of this interaction between distinct RNPs and provide an additional mechanism of action for TDRD7 in the lens. A model encompassing both direct and indirect mechanisms of TDRD7 action in the developing lens is depicted in Fig. 5D.

In sum, TDRD7 is an RNA granule component that is highly enriched in the developing lens; TDRD7 perturbation in chick, mouse, and human causes cataract. In the lens, TDRD7-RGs play an essential role in the regulation of specific genes that are critical for lens development, including those responsive to stress, such as Hspb1. Tdrd7 deficiency disturbs this regulatory mechanism and leads to cataract. The RNA granule function of TDRD7 may also be deployed in oth-

er tissues and cell types at various developmental stages. For example, TDRD7 is a component of a testis-specific RNA granule, the chromatoid body (CB) (21), and we observe that Tdrd7 null mice exhibit male sterility due to an arrest in spermatogenesis at the round spermatid stage, likely due to a CB defect (fig. S16). Lastly, Tdrd7 null mice develop elevated IOP and other features of glaucoma as they age. IOP elevation and glaucoma can result from oxidative and other stresses that

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Fig. 4. Tdrd7 is critical for RNA granule formation and HSPB1 expression. (A) SRA01/04 cell line shows increased numbers of P-bodies (detected by DDX6/ RCK antibody) and stress granules (detected by elF3b antibody) in response to oxidative stress (sodium arsenite, 0.1 mM). The numbers of stress granules are markedly reduced, and P-bodies are modestly reduced in TDRD7 knockdown SRA01/04 cells (for quantification, see fig. S14). Scale bar: 5 mm. (B) (Upper panels) IF with TDRD7 antibody demonstrates robust TDRD7-RGs in Tdrd7 heterozygous mouse lens and absence in Tdrd7 null mouse mutant lens. Scale bar: 3 mm. (Lower panels) IF with HSPB1 antibody demonstrates reduction of HSPB1 protein in P4 Tdrd7 null lens (see fig. S15). Scale bar: 20 mm. EZ, equatorial zone; OFZ, organelle-free zone.

Fig. 5. TDRD7 regulates mRNAs critical to lens development and RG function. (A) Microarray analyses of Tdrd7KD 21EM15 mouse lens cells reveals down-regulation of RNA granule genes and human cataract genes and upregulation of other genes normally down-regulated during fiber cell differentiation. Genes are assigned to specific classes, denoted I to V. Jag1 is expressed in fiber cells but is highly restricted. (B) Microarray analyses of mouse Tdrd7 null mutant lens at P4 and P30 reveals down-regulation of RNA granule genes and human cataract genes. Fold changes (FC) are expressed as Log2(FC). Error bars in (A) and (B) represent propagated SEM. (C) RIP using TDRD7 antibody, followed by RT-PCR, reveals Crybb3, Hspb1 enriched in TDRD7 complexes. (D) Model for TDRD7 function in lens development. In lens fiber cells, TDRD7-RGs regulate certain key lens transcripts (e.g., Crybb3 and Hspb1) directly via binding, and others indirectly via interactions with STAU1-NPs, which also bind specific transcripts (e.g., Sparc and Epha2) directly (26). These interactions may allow TDRD7, alone or in conjunction with STAU1, to protect specific mRNAs from the RNA decay mechanisms that normally exist in lens fiber cells, thus selectively stabilizing these mRNAs for translating the high levels of refractive proteins needed for lens transparency.

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References and Notes 1. M. J. Moore, Science 309, 1514 (2005). 2. P. Anderson, N. Kedersha, Nat. Rev. Mol. Cell Biol. 10, 430 (2009). 3. R. Parker, U. Sheth, Mol. Cell 25, 635 (2007). 4. U. Sheth, R. Parker, Science 300, 805 (2003). 5. S. Vasudevan, J. A. Steitz, Cell 128, 1105 (2007). 6. A. Eulalio, I. Behm-Ansmant, E. Izaurralde, Nat. Rev. Mol. Cell Biol. 8, 9 (2007). 7. N. Kotaja, P. Sassone-Corsi, Nat. Rev. Mol. Cell Biol. 8, 85 (2007). 8. M. A. Kiebler, G. J. Bassell, Neuron 51, 685 (2006). 9. J. R. Buchan, R. Parker, Mol. Cell 36, 932 (2009). 10. M. Brengues, D. Teixeira, R. Parker, Science 310, 486 (2005). 11. Y. Oleynikov, R. H. Singer, Curr. Biol. 13, 199 (2003). 12. N. Kedersha et al., J. Cell Biol. 169, 871 (2005). 13. T. Ohn, N. Kedersha, T. Hickman, S. Tisdale, P. Anderson, Nat. Cell Biol. 10, 1224 (2008).

14. A. W. Higgins et al., Am. J. Hum. Genet. 82, 712 (2008). 15. Materials and methods are available as supporting material on Science Online. 16. X. Luo, Y. Ikeda, K. L. Parker, Cell 77, 481 (1994). 17. J. C. Achermann, M. Ito, M. Ito, P. C. Hindmarsh, J. L. Jameson, Nat. Genet. 22, 125 (1999). 18. F. S. Alkuraya, Genet. Med. 12, 236 (2010). 19. S. Harpavat, C. L. Cepko, BMC Dev. Biol. 6, 2 (2006). 20. V. Anantharam, D. Zhang, L. Aravind, Biol. Direct. 5, 13 (2010). 21. M. Hosokawa et al., Dev. Biol. 301, 38 (2007). 22. N. Kotaja et al., Proc. Natl. Acad. Sci. U.S.A. 103, 2647 (2006). 23. N. Kedersha, P. Anderson, Methods Enzymol. 431, 61 (2007). 24. M. Zeitelhofer et al., J. Neurosci. 28, 7555 (2008). 25. F. Roegiers, Y. N. Jan, Trends Cell Biol. 10, 220 (2000). 26. L. Furic, M. Maher-Laporte, L. DesGroseillers, RNA 14, 324 (2008). 27. J. P. Vessey et al., Proc. Natl. Acad. Sci. U.S.A. 105, 16374 (2008). 28. Y. K. Kim, L. Furic, L. Desgroseillers, L. E. Maquat, Cell 120, 195 (2005). 29. S. A. Barbee et al., Neuron 52, 997 (2006). 30. M. S. Haque, J. K. Arora, G. Dikdan, T. W. Lysz, P. S. Zelenka, Mol. Vis. 5, 8 (1999). 31. D. T. Gilmour et al., EMBO J. 17, 1860 (1998). 32. S. A. Riazuddin et al., Invest. Ophthalmol. Vis. Sci. 46, 2100 (2005). 33. N. L. Kedersha, M. Gupta, W. Li, I. Miller, P. Anderson, J. Cell Biol. 147, 1431 (1999). 34. K. S. Sinsimer et al., Mol. Cell. Biol. 28, 5223 (2008).

35. L. Fu, J. J. Liang, J. Biol. Chem. 277, 4255 (2002). 36. L. Fu, J. J. Liang, Biochem. Biophys. Res. Commun. 302, 710 (2003). 37. G. Jun et al., PLoS Genet. 5, e1000584 (2009). 38. S. C. Saccà, A. Izzotti, Prog. Brain Res. 173, 385 (2008). 39. This work was supported by R01 EY10123, R01 HD060050, P01 GM061354, KACST 08-MED497-20, R01 EY11721, The Barbara and Joseph Cohen Foundation, The Peace by Pieces Fund, and the Dubai Harvard Foundation for Medical Research. S.M.A. is supported by a grant from Kuwait University (YM01/09). A.V.D. was supported by a Marjorie Carr Adams Career Development Award from the Foundation Fighting Blindness. A.A. is supported by an American Heart Association Predoctoral Fellowship. S.W.M.J. and G.J.H. are Investigators of the Howard Hughes Medical Institute. We thank C. Cepko for advice on the chick experiments, L. Reinholdt for assistance in assessing male sterility, A. Bell for technical assistance, J. Reddan and V. Reddy for their gifts of lens cell lines, and the patients and their families for participating in this research. All microarray data are deposited in the Gene Expression Omnibus database (www.ncbi.nih.gov/geo), and the accession number for the SuperSeries for all data files is GSE25812.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1571/DC1 Materials and Methods Figs. S1 to S16 Table S1 References 2 August 2010; accepted 24 January 2011 10.1126/science.1195970

REPORTS Baryons at the Edge of the X-ray–Brightest Galaxy Cluster Aurora Simionescu,1* Steven W. Allen,1 Adam Mantz,2 Norbert Werner,1 Yoh Takei,3 R. Glenn Morris,1 Andrew C. Fabian,4 Jeremy S. Sanders,4 Paul E. J. Nulsen,5 Matthew R. George,6 Gregory B. Taylor7,8 Studies of the diffuse x-ray–emitting gas in galaxy clusters have provided powerful constraints on cosmological parameters and insights into plasma astrophysics. However, measurements of the faint cluster outskirts have become possible only recently. Using data from the Suzaku x-ray telescope, we determined an accurate, spatially resolved census of the gas, metals, and dark matter out to the edge of the Perseus Cluster. Contrary to previous results, our measurements of the cluster baryon fraction are consistent with the expected universal value at half of the virial radius. The apparent baryon fraction exceeds the cosmic mean at larger radii, suggesting a clumpy distribution of the gas, which is important for understanding the ongoing growth of clusters from the surrounding cosmic web. alaxy clusters provide critical constraints on cosmological parameters that are independent from those determined using type Ia supernovae, galaxy surveys, and the primordial cosmic microwave background radiation (CMB) (1–3). In particular, knowledge of their baryon content is a key ingredient in the use of clusters as cosmological probes (4, 5). Most baryons reside in the hot, diffuse, x-ray– emitting intracluster medium (ICM). Until recently, x-ray observations have generally targeted

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only the inner parts of clusters, where the emission is brightest, leaving a large fraction of their volumes practically unexplored. Estimates of the gas mass and total mass at large radii have relied on simple model extrapolations of the thermodynamic properties measured at smaller radii. X-ray spectroscopy of the outer regions of galaxy clusters was made possible only recently, with the use of the Suzaku satellite. Because of its much lower instrumental background than that of other x-ray observatories, Suzaku can

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measure the characteristics of the faint emission from cluster outskirts more reliably. Even so, few such observations have been published, and the thermodynamic profiles at large radii are not well resolved (6–11). The Perseus Cluster of galaxies is the brightest extragalactic extended x-ray source. It is a relaxed system, both closer (at a redshift of 0.0183) and substantially brighter than any of the other clusters for which Suzaku has previously been used to study the ICM properties at large radii. Its large angular size mitigates the impact of residual systematic uncertainties in modeling the effects of Suzaku's complex pointspread function (PSF), making the Perseus Cluster an ideal target in which to study cluster outskirts. A large mosaic of Suzaku observations of the Perseus Cluster, with a total exposure time 1 Kavli Institute for Particle Astrophysics and Cosmology, Stanford University, 452 Lomita Mall, Stanford, CA 94305, USA. 2 NASA Goddard Space Flight Center, Greenbelt, MD 20771, USA. 3Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency (JAXA), 3-1-1 Yoshinodai, Sagamihara, Kanagawa 229-8510, Japan. 4Institute of Astronomy, Madingley Road, Cambridge CB3 0HA, UK. 5Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138, USA. 6 Department of Astronomy, University of California, Berkeley, CA 94720, USA. 7Department of Physics and Astronomy, University of New Mexico, Alberquerque, NM 87131, USA. 8National Radio Astronomy Observatory, 1003 Lopezville Rd., Socorro, NM 87801, USA.

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

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damage the aqueous humor drainage tissues (38). It is possible that the IOP elevation in Tdrd7 null mice and in the two human patients who developed glaucoma results, at least in part, from abnormalities in the protective stress response or in other TDRD7-related functions in the drainage tissues. Thus, the data demonstrate that human organogenesis defects can result from perturbation of a distinct, tissue-specific RG component that posttranscriptionally regulates the levels of developmentally critical mRNAs.

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of 260 ks, was obtained in August/September 2009. The pointings extend along two arms from near the cluster center toward the east (E) and northwest (NW), out to a radius of 2°, which corresponds to 2.8 Mpc for a Hubble constant H0 = 70 km/s/Mpc. Here we focus on the data obtained with the three available x-ray imaging spectrometer (XIS) cameras [see supporting online material (SOM) text and Fig. 1]. We extracted spectra from annuli centered on the cluster center. After accounting for background emission, we modeled each spectral region as a single-temperature thermal plasma in collisional ionization equilibrium, with the temperature, metallicity, and spectrum normalization as free parameters. The best-fit radial profiles of temperature and metallicity are presented in Fig. 2. Individual elements are assumed to be present with solar relative abundances (12). For comparison, we also show results previously obtained from an ultradeep Chandra observation of the cluster center (13). The Suzaku and Chandra data sets show excellent agreement where they intersect, and together they measure the temperature and metallicity structure of the intracluster gas with high precision and spatial resolution out to the virial radius (defined here as r200, the radius within which the mean enclosed mass density of the cluster is 200 times the critical density of the universe at the cluster redshift). In the narrow interval spanning 0.95 to 1.05 r200, the temperature is approximately a third of the peak temperature. Along the E arm, between 0.1 and 0.7 Mpc, the temperature is systematically lower than toward the NW, and the x-ray emission is brighter. This thermodynamic feature is known as a “cold front” and typically arises after a merger between the main cluster and a smaller subcluster (14). Our results show that the cluster outskirts are substantially metal-enriched, to a level amounting to approximately one-third of the solar metallicity. Previously, the only measurement of the metallicity close to the virial radius was obtained from a large region spanning 0.5 to 1 r200

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Fig. 1. X-ray surface brightness image of the NW (top) and E (bottom) arm mosaics observed with Suzaku, corrected for vignetting and instrumental background. The dashed white line marks the virial radius; the red circles mark excluded point sources and instrumental artefacts. The images have been rotated so that the cluster center is toward the left.

Fig. 2. Projected temperature (kT) and metallicity (Z) profiles of the Perseus Cluster. Results from Suzaku observations of the NW arm are shown in red and of the E arm in blue. Earlier Chandra measurements of the cluster center (13) are shown in black.

near the compressed outskirts of two interacting clusters (15). This previous result is in agreement with our measurements when converted to the solar abundance units (12) adopted here. From the Suzaku data, we have also determined the electron density, entropy, and pressure profiles, corrected for projection effects under the assumption of spherical symmetry (Fig. 3). Outside the cold front at 0.7 Mpc, there is a good match between the E and NW profiles, with the electron density decreasing steadily with radius, approximately following a power law model ne º r−a with slope a = 1.68 T 0.04. This is consistent with previous results from ROSAT (Roentgen satellite) data extending out to ~1.4 Mpc (16). Standard large-scale structure formation models show that matter is shock-heated as it falls into clusters under the pull of gravity. Simple theoretical models of this process predict that the entropy K should behave as a power law

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with radius K º rb, with b ~ 1.1 to 1.2 (17, 18). Except for the E cold front region, the entropy profile in Perseus roughly follows this expected trend until 2/3 r200. Beyond this radius and until 0.95 r200, both the E and NW arms show a flattening from the power law shape, confirming hints from previous Suzaku results (7). The pressure profile is the most regular of the thermodynamic quantities plotted, and at most radii shows good agreement between the E and NW. At large radii, the pressure profile appears significantly shallower than would be expected by extrapolating the average profile of a sample of clusters studied previously with the XMMNewton satellite within ~ 0.5 r200 (19). Invoking hydrostatic equilibrium of the ICM, the gas pressure can be used to estimate the underlying gravitational potential and total (dark matter plus luminous matter) mass profile of the cluster. Numerical simulations show that the lat-

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ter typically follows a functional form described by Navarro, Frenk, White (20), also known as the NFW profile. We used the data from the NW arm of the Perseus Cluster, which appears dynamically relaxed, to determine the best-fit total mass profile, assuming an NFW form (SOM text).

The best-fit mass model parameters are typical of those predicted from numerical simulations; the NFW model provides a good description of the Suzaku data. Measuring the total mass profile allowed us to calculate the virial radius of the cluster, r200 =

Fig. 3. Deprojected electron density (ne), entropy (K), and pressure (P) profiles toward the NW (red data points) and E (blue data points). The red line shows the NW profiles corrected for clumping. The expected entropy profile from simulations of gravitational collapse (17, 18) is a power law with index b ~ 1.1, overplotted as a black dotted line in the entropy panel. The average profile of a sample of clusters previously studied with the XMM-Newton satellite within ~0.5 r200 (19) is shown with a solid black curve in the pressure panel; its extrapolation to r200 is shown with a dotted black line.

Fig. 4. The integrated, enclosed gas mass fraction profile for the NW arm. The cosmic baryon fraction from WMAP7 (22) is indicated by the horizontal dashed black line; accounting for 12% of the baryons being in stars (23, 24) gives the expected fraction of baryons in the hot gas phase, shown as a solid black line. The values previously measured for a sample of relaxed clusters at smaller radii with Chandra (5) are shown with blue dots. Predictions from numerical simulations (25) are shown in green. The bottom panel shows by how much the electron density should be overestimated in each annulus because of clumping, in order for the cumulative fgas not to exceed the correspondingly colored curves in the plot above.

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1.79 T 0.04 Mpc, the corresponding enclosed total mass M200 = 6.65+0.43−0.46 × 1014 solar masses, and the cumulative gas mass and gas mass-to-total mass fraction, fgas, as a function of radius (Fig. 4). At relatively small radii of 0.2 to 0.3 r200, the measured fgas value is in good agreement with direct measurements from the Chandra X-ray Observatory (5) and measurements of the Sunyaev-Zel'dovich (SZ) effect (21) for two large samples of galaxy clusters. At about half of r200, the integrated gas mass fraction reaches the cosmic mean value computed from the CMB (22), considering that on average 12% of the baryons are in stars (23, 24) and the rest are in the hot x-ray–emitting gas phase. Outside 2/3 of the virial radius, where the entropy also deviates from the expected power law behavior, we find that the apparent fgas exceeds the cosmic mean baryon fraction measured from the CMB (22). The most plausible explanation for this apparent excess of baryons at large radius is gas clumping. In the x-rays, the directly measurable quantity from the intensity of the bremsstrahlung emission is the average of the square of the electron density, < ne2 >, rather than < ne > . If the density is not uniform (that is, the gas is clumpy), which is expected to occur as matter falls into the cluster, the average electron density estimated from the bremsstrahlung intensity will overestimate the true average, affecting the gas density, gas mass fraction, entropy, and pressure profiles. Outside the central region, and inside the radius where clumping becomes important, the measured fgas profile shows good agreement with recent numerical simulations (25), where a semianalytic model was used to calculate the energy transferred to the intracluster gas by supernovae and active galactic nuclei during the galaxy formation process. This model did not include a realistic implementation of gas cooling and does not capture the complex processes in the central cool core of the cluster; the model is therefore not plotted in this region. Extrapolating this model into the outskirts where clumping is important, we used its predictions together with the measured fgas to determine by how much the electron density must be overestimated to produce the difference between the data and the model. This factor (plotted in green in the bottom panel of Fig. 4) reaches a value of up to 4 in the last annulus centered around the virial radius. The dense clumps are likely to be infalling and may be confined by ram pressure. Correcting the electron density using this factor, and accordingly the entropy and pressure profiles, we obtained the red lines shown in Fig. 3. The clumping-corrected entropy profile along the NW arm is consistent with the expected powerlaw profile. Moreover, the clumping-corrected pressure is also consistent with that expected by extrapolating the average profile of a sample of clusters previously studied with XMM-Newton (19). The corrected electron density decreases

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REPORTS

more steeply with increasing radius, with the bestfit power law index becoming a = 2.5. Correcting for clumping therefore seems to offer a simple solution to all the potential puzzles posed, at first glance, by the observed thermodynamic profiles of the Perseus Cluster at large radii. No additional physics is required by the data. Our study shows no evidence for the puzzling deficit of baryons at r ≥ 0.5 r200 inferred from some previous studies of other systems, using lower-quality data and/or extrapolated models (26–30). This suggests that within r < 0.5 r200 the physics of the x-ray–emitting gas is relatively simple, and x-ray measurements can be used robustly for cosmological work. At larger radii, however, the cluster gas is significantly clumped. Numerical simulations predict gas clumping in the cluster outskirts (31). However, the amount of clumping depends on a large number of physical processes in the ICM which are currently uncertain; for example, viscosity, conduction, star formation feedback, and magnetic fields. Although our results were obtained for just one galaxy cluster, it is expected that the observed physical processes are common. Our results therefore provide an anchor for numerical models of ICM physics and for simulations of the formation and ongoing growth of galaxy clusters. An independent measurement of gas clumping can be obtained from the combination of x-ray and SZ observations, which have different dependences on the electron density.

References and Notes 1. S. D. M. White, J. F. Navarro, A. E. Evrard, C. S. Frenk, Nature 366, 429 (1993). 2. A. Vikhlinin et al., Astrophys. J. 692, 1060 (2009). 3. A. Mantz, S. W. Allen, D. Rapetti, H. Ebeling, Mon. Not. R. Astron. Soc. 406, 1759 (2010). 4. S. W. Allen, R. W. Schmidt, H. Ebeling, A. C. Fabian, L. van Speybroeck, Mon. Not. R. Astron. Soc. 353, 457 (2004). 5. S. W. Allen et al., Mon. Not. R. Astron. Soc. 383, 879 (2008). 6. T. H. Reiprich et al., Astron. Astrophys. 501, 899 (2009). 7. M. R. George, A. C. Fabian, J. S. Sanders, A. J. Young, H. R. Russell, Mon. Not. R. Astron. Soc. 395, 657 (2009). 8. M. W. Bautz et al., Pub. Astron. Soc. Jpn. 61, 1117 (2009). 9. A. Hoshino et al., Pub. Astron. Soc. Jpn. 62, 371 (2010). 10. M. Kawaharada et al., Astrophys. J. 714, 423 (2010). 11. K. Sato et al., Pub. Astron. Soc. Jpn. 62, 1423 (2010). 12. U. Feldman, Phys. Scr. 46, 202 (1992). 13. J. S. Sanders, A. C. Fabian, Mon. Not. R. Astron. Soc. 381, 1381 (2007). 14. M. Markevitch, A. Vikhlinin, Phys. Rep. 443, 1 (2007). 15. Y. Fujita et al., Pub. Astron. Soc. Jpn. 60, 343 (2008). 16. S. Ettori, A. C. Fabian, D. A. White, Mon. Not. R. Astron. Soc. 300, 837 (1998). 17. P. Tozzi, C. Norman, Astrophys. J. 546, 63 (2001). 18. G. M. Voit, S. T. Kay, G. L. Bryan, Mon. Not. R. Astron. Soc. 364, 909 (2005). 19. M. Arnaud et al., Astron. Astrophys. 517, A92 (2010). 20. J. F. Navarro, C. S. Frenk, S. D. M. White, ApJ 490, 493 (1997). 21. S. J. LaRoque et al., Astrophys. J. 652, 917 (2006). 22. E. Komatsu et al., Astrophys. J. Suppl. Ser. 192, 18 (2011). 23. Y.-T. Lin, J. J. Mohr, Astrophys. J. 617, 879 (2004). 24. A. H. Gonzalez, D. Zaritsky, A. I. Zabludoff, Astrophys. J. 666, 147 (2007).

From a Single-Band Metal to a High-Temperature Superconductor via Two Thermal Phase Transitions Rui-Hua He,1,2,3* M. Hashimoto,1,2,3* H. Karapetyan,1,2 J. D. Koralek,3,4 J. P. Hinton,3,4 J. P. Testaud,1,2,3 V. Nathan,1,2 Y. Yoshida,5 Hong Yao,1,3,4 K. Tanaka,1,2,3,6 W. Meevasana,1,2,7 R. G. Moore,1,2 D. H. Lu,1,2 S.-K. Mo,3 M. Ishikado,8 H. Eisaki,5 Z. Hussain,3 T. P. Devereaux,1,2† S. A. Kivelson,1† J. Orenstein,3,4† A. Kapitulnik,1,2† Z.-X. Shen1,2† The nature of the pseudogap phase of cuprate high-temperature superconductors is a major unsolved problem in condensed matter physics. We studied the commencement of the pseudogap state at temperature T* using three different techniques (angle-resolved photoemission spectroscopy, polar Kerr effect, and time-resolved reflectivity) on the same optimally doped Bi2201 crystals. We observed the coincident, abrupt onset at T* of a particle-hole asymmetric antinodal gap in the electronic spectrum, a Kerr rotation in the reflected light polarization, and a change in the ultrafast relaxational dynamics, consistent with a phase transition. Upon further cooling, spectroscopic signatures of superconductivity begin to grow close to the superconducting transition temperature (Tc), entangled in an energy-momentum–dependent manner with the preexisting pseudogap features, ushering in a ground state with coexisting orders. s complex oxides, cuprate superconductors belong to a class of materials that exhibit many broken-symmetry states; unraveling the relationship between superconductivity in the cuprates and other possible brokensymmetry states has been a major challenge of condensed matter physics. A possibly related issue concerns the nature of the pseudogap in the

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cuprates and its relationship with superconductivity. Angle-resolved photoemission spectroscopy (ARPES) studies have shown that the pseudogap develops below a temperature T* near the Brillouin zone boundary while preserving a gapless Fermi arc near the zone diagonal (1). A key issue is the extent to which the pseudogap is a consequence of superconducting fluctuations (2–5), which

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25. O. E. Young, P. A. Thomas, C. J. Short, F. Pearce, http:// arxiv.org/abs/1007.0887 (2010). 26. S. Ettori, Mon. Not. R. Astron. Soc. 344, L13 (2003). 27. I. G. McCarthy, R. G. Bower, M. L. Balogh, Mon. Not. R. Astron. Soc. 377, 1457 (2007). 28. N. Afshordi, Y.-T. Lin, D. Nagai, A. J. R. Sanderson, Mon. Not. R. Astron. Soc. 378, 293 (2007). 29. A. Vikhlinin et al., Astrophys. J. 640, 691 (2006). 30. S. Andreon, Mon. Not. R. Astron. Soc. 407, 263 (2010). 31. M. Roncarelli et al., Mon. Not. R. Astron. Soc. 373, 1339 (2006). 32. We thank P. Thomas and O. Young for kindly providing the simulation results shown in Fig. 4. Support for this work was provided by NASA through Einstein Postdoctoral Fellowship grants number PF9-00070 and PF8-90056 awarded by the Chandra X-ray Center, which is operated by the Smithsonian Astrophysical Observatory for NASA under contract NAS8-03060. We acknowledge NASA grants NNX09AV64G and NNX10AR48G, issued through the Suzaku Guest Observer program, and grant NNX08AZ88G. The authors thank the Suzaku operation team and Guest Observer Facility, supported by JAXA and NASA. This work was supported in part by the U.S. Department of Energy under contract number DE-AC02-76SF00515. We also acknowledge the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI no. 22111513) and Chandra award GO0-11138B.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1576/DC1 SOM Text References 11 November 2010; accepted 8 February 2011 10.1126/science.1200331

should exhibit a rough particle-hole symmetry, or another form of (incipient) order (6–12), which typically should induce particle-hole asymmetric spectral changes. Candidate orders include various forms of density wave, nematic, or unconventional magnetic orders that break different combinations of lattice translational (6–8, 13–19), rotational (6, 9, 15, 17, 20–22), and time-reversal (7, 9, 23–26) symmetries. We have focused on crystals of nearly optimally doped (OP) Pb0.55Bi1.5Sr1.6La0.4CuO6+d (Pb-Bi2201, Tc = 38 K, T* = 132 T8 K) (27), and combined the ARPES measurement of the evolution of the band structure over a wide range of

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1 Geballe Laboratory for Advanced Materials, Departments of Physics and Applied Physics, Stanford University, Stanford, CA 94305, USA. 2Stanford Institute for Materials and Energy Sciences, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA. 3Advanced Light Source and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. 4Department of Physics, University of California, Berkeley, CA 94720, USA. 5Nanoelectronics Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8568, Japan. 6Department of Physics, Osaka University, Toyonaka, Osaka 560-0043, Japan. 7School of Physics, Suranaree University of Technology and Synchrotron Light Research Institute, Nakhon Ratchasima, 30000 Thailand. 8 Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan.

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

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Fig. 1. Fermi surface maps measured below Tc at 10 K (left) and above T* at 172 K (right) in the same momentum-space region (flipped for display). Dashed white lines labeled C1 to C7 depict the cuts along which the EDCs shown in C7 Fig. 2, A to N, were measured. Magenta C1 C3 C5 squares labeled P1 to P16 along M-G ky P1 P16 indicate momenta where EDCs in Fig. 2, V and W, were measured. Red and M M Γ blue squares on the left indicate mokX menta of the Fermi-level crossing kF C2 C4 C6 (kF1 and kF2 in Fig. 2, A to G) at 172 K and back-bending kG (black arrows High in Fig. 2, O to S) at 10 K of the dispersion of the EDC maximum along 172 K 10 K cuts C1 to C7. Red and blue circles (>T*) (
temperature, momentum, and energy, with highprecision measurements of the polar Kerr effect (PKE) and time-resolved reflectivity (TRR). Bi2201 was chosen to avoid the complications resulting from bilayer splitting and strong antinodal bosonic mode coupling inherent to Bi2Sr2CaCu2O8+d (Bi2212) (1). Whereas ARPES is a surface probe, PKE enables us to monitor a bulk, thermodynamic (via the fluctuation-dissipation theorem) property that has proven (28) to be a sensitive probe of the onset of a broken-symmetry state, and TRR gives complementary information on the bulk, near-equilibrium dynamics of the system. We will first analyze our ARPES data collected in different temperature regions. Above T*, Pb-Bi2201 has a simple one-band band structure (right side of Fig. 1). For each cut in momentum space perpendicular to G-M [(0,0)-(p,0)] (C1 to C7 in Fig. 1), the only distinct feature in the corresponding Fermi-function–divided (27) energy distribution curves (EDCs) is a maximum (red circles in Fig. 2, A to G). As a function of the y component of the wave vector (ky), the maxima have an approximately parabolic dispersion for

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ky (π) Fig. 2. (A to G) and (H to N) Selected EDCs at 172 K and at 10 K, respectively, for cuts C1 to C7, nearly perpendicular to G-M (Fig. 1). Each EDC corresponds to a white point in the cuts in Fig. 1. EDCs in magenta and orange are located close to kF. (O to U) Dispersions of the EDC features in (A) to (N) for cuts C1 to C7. For each dispersion curve, every other symbol corresponds to an EDC in (A) to (N).

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Error bars are estimated based on the sharpness of features, to be T3 meV minimum and T8 meV maximum [examples shown in (O)] based on different EDC analyses (27). (V and W) EDCs at momenta P1 to P16 along M-G (Fig. 1) at 172 K and 10 K, respectively. Circles denote the EDC shoulder feature (solid green) and the EDC maximum feature at 10 K (blue) and at 172 K (red). SCIENCE

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each cut (red circles in Fig. 2, O to U); the band bottom lies on the G-M axis, and the dispersion crosses the Fermi level (EF) at two momenta, kF (kF1 and kF2). The binding energy of the band

bottom monotonically decreases from near G to M (Fig. 2, O to U). We take the Fermi-level crossings of this single band to define the Fermi surface. Despite the simplicity of the electronic

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Fig. 3. Temperature 1.0 ARPES PKE dependence of Kerr rotation (qK) measured by PKE, in comparison with T(K)= that of the binding en40 60 ergy position of the EDC 80 0 100 130 maximum at kF given by 150 1 172 ARPES [reproduced from -40 fig. S1F and (29)]. ARPES results are normalized to 0.5 kF1 kF2 -80 the 80 K values (free from -0.25 0.0 0.25 the interference of flucky (π) tuating superconductivity). 4 Hashimoto The dashed black curve TRR et. al. is a guide to the eye for the PKE data, showing a 2 mean-field–like critical behavior close to T* [see 0 0.0 additional discussion in 50 100 150 (27)]. (Left inset) Tem50 100 150 perature dependence of Tc T* the transient reflectivity T (K) change measured by TRR (right axis). The dashed black curve (left axis) is reproduced from the main panel. Error bars (if not visible) are smaller than the symbol size. (Right inset) Dispersion of the EDC maximum at various temperatures above Tc, summarizing the results of Figs. 2A and 4A and fig. S1, A to E. All data were taken on samples from the same growth and annealing batch, except those reproduced from (29) on differently annealed samples.

structure above T*, the width and energydependent broadening of the EDC maximum features, along with the familiar strange metal behavior seen in transport, imply that the system is not well described as a Fermi liquid. We now turn to the temperature region below Tc. Here, the entire Fermi surface is gapped except at the nodal points (kF lying on the zone diagonal). In the nodal region, consistent with previous reports (4, 5, 11, 12), a d-wave–like gap along the Fermi surface is observed that we quantify as the energy position of the EDC maximum (blue circles) at kF (Fig. 2, L to N). This maximum is still the only identifiable feature in the EDC. By comparing the EDCs in Fig. 2, E to G, with those in Fig. 2, L to N, we see that the peaks of EDCs near kF are much sharper below Tc than above T*; however (perhaps surprisingly), the peaks well away from kF appear broader but with larger experimental uncertainties (also see Fig. 2, V and W). Away from the nodal region, the dispersion along each cut rises to a minimum binding energy and then bends back (Fig. 2, H to K). These back-bendings (black arrows in Fig. 2, O to S) occur at momenta kG (kG1 and kG2), which are increasingly separated from the Fermi surface (compare blue and red squares on the left side of Fig. 1) toward the antinodes (kF lying on the zone boundary). Note that, for a superconducting gap, as a consequence of the particle-hole symmetry, one would expect kG ≅ kF (fig. S6), as is the case

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Fig. 4. (A and B) M kG2 kF2 22 K 40 K Selected EDCs at 40 K A B E F G and 22 K along cut C1 60 K (Fig. 1). See Fig. 2, A 40 K and H, for data at 172 K and 10 K, and fig. S1, A 22 K to E, for other interme10 K kF1 diate temperatures. (C) 40 K Antinodal EDCs at 10 K 22 K after dividing by the 40 10 K K counterparts, covering M /60 K /60 K /60 K the momentum range indicated by the gray bar -0.1 0.0 -0.1 0.0 -0.1 0.0 in (H), in comparison with Bi2201 Bi2212 those in (D) taken in a 0 D H C M kF2 kG2 similar range at 30 K on an OP Bi2212 sample. kF2 -20 Nondispersive peaks are seen in both cases de-40 spite different sharpness kG2 and energy positions. (E 40 K to G) EDCs at different -60 fixed momenta [specified T (K)= in (A) and (H)] and tem10 -80 peratures around Tc. The 22 10 K/40 K 30 K counterintuitive increase -0.1 0.0 -0.1 0.0 -0.25 0.0 0.25 -0.1 0.0 -0.1 0.0 of the antinodal gap, deky (π) E-EF (eV) fined by the energy position of the EDC maximum in (F) and (G), with temperature rising above Tc, cannot be understood with a specify momenta M, kF2 at 172 K, and kG2 at 10 K. Apparent asymmetry of the single energy scale assumed. (Insets) Corresponding EDCs divided by the 60 K dispersions across M is due to a finite deviation of the cut from the high-symmetry counterpart, showing the peaks losing definition above Tc (fig. S2E). (H) Summary direction and a subtle balance of spectral weight between different features in the for the dispersions of related EDC features across and below Tc. Vertical arrows EDC. All EDC features and error bars are similarly determined as in Fig. 2.

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in the nodal region. The substantial kG−kF misalignment, previously found in a single antinodal cut perpendicular to G-M, has been interpreted as a signature of non–particle-hole symmetric order (29). Our observation here complements that finding by providing an entire momentum-space picture. The width and shape of the EDC near kF change fairly abruptly as a function of position along the Fermi surface, in contrast with the smooth evolution seen above T* (compare magenta curves in Fig. 2, H to N and A to G). Strikingly, the antinodal EDC maxima below Tc are broader than those above T* (Fig. 2, Vand W, and fig. S7I). Such broadening of the antinodal spectra with decreasing temperature, as reported previously (29), is likely intrinsic, as the expected sharpening is observed simultaneously in the nodal spectra at low energy. Around the M point, both the EDC line shape and the dispersion of the EDC maxima are more complex than their counterparts above T*. The dispersion of the EDC maxima has two separate branches, one at relatively low energy, which shows back-bendings at kG, and the other at higher energy around the band bottom on the G-M axis. As one moves from cuts C1 to C7, the apparent discontinuity between the two branches near the antinode vanishes and the two branches merge (Fig. 2, O to U). Both kG (blue squares in Fig. 1) and the band-bottom energy (blue circles in Fig. 2W) evolve smoothly, even as the dispersion at intermediate energies changes dramatically. Along with the changes in the dispersion of the EDC maxima below Tc, a well-defined shoulder feature (green dots in Fig. 2, H to K and W) (27) emerges at low energy in the EDCs. This feature exhibits little dispersion either along G-M (Fig. 2W) or perpendicular to it (Fig. 2, H to J). Unlike the EDC maxima, its dispersion does not continue toward the zone center (Fig. 2W) but instead loses its definition away from the vicinity of the M point (magenta-shaded region in Fig. 1). The changes in the spectral function on cooling do not occur smoothly; instead, abrupt changes in the thermal evolution occur in the neighborhoods of T* and Tc. A detailed temperature-dependent study of an antinodal cut (C1 in Fig. 1) shows that the dispersion of the EDC maxima (blue circles in fig. S1, A to E) exhibit a transformation that begins at a temperature equal (within experimental uncertainty) to the reported values of T* in the literature (27). Specifically, the energies of the EDC maxima at the band bottom and at kF are temperature independent above T* but begin shifting to higher binding energies below T* (fig. S1F). The energy position at kF is a measure of the pseudogap and can be defined as a spectral order parameter that becomes nonzero below T*. Moreover, at temperatures below T* but well above Tc, one can already see the kG−kF misalignment developing (right inset of Fig. 3). These observations are consistent with our previous report on a similar cut in an OP Pb-Bi2201

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sample prepared under a different post-annealing condition (29). It would be natural (as discussed below) to associate the abrupt change in nature of the ARPES spectra with a transition to a broken-symmetry state below T*. To test this idea, we performed PKE measurements on the same crystals with finely spaced temperature steps (27). The results (Fig. 3) are clearly suggestive of a slightly rounded phase transition at T* below which a finite Kerr rotation emerges. A similar transition in the Kerr rotation was previously observed in YBa2Cu3O6+x (YBCO) and was suggested to be linked to a broken-symmetry state that is not necessarily magnetic in nature (25). The smallness of the Kerr rotation in YBCO suggested that it might not reflect the primary order. The present data again show a small Kerr rotation; however, the strong correspondence between the ARPES and PKE data in Fig. 3 allows us to conjecture a phase transition at T* and also to corroborate the interpretation of previously published data on YBCO in terms of a similar transition. We also studied TRR on the same Pb-Bi2201 crystals (27). At temperatures between Tc and T*, we observed a negative signal with a timeresolution–limited turn-on followed by decay on the order of 100 femtoseconds (fig. S3). Additionally, we observed a positive signal that emerges below Tc and decays on a picosecond time scale. These observations are consistent with previous studies of various cuprate families that revealed different dynamics above and below Tc (30), and associated the former with the pseudogap state (31–36). The striking aspects of the TRR results reported here are the clarity of the onset at T* and the direct correspondence with ARPES and PKE. As shown in the left inset of Fig. 3, the magnitude of the negative TRR signal tracks the ARPES and PKE data quite well. This added correspondence further supports the existence of a phase transition at T* to the pseudogap state with near-equilibrium dynamics distinct from superconductivity. Upon cooling below Tc, it is observed in ARPES that the shoulder on the low-energy side of the EDC maximum appears to develop somewhat above Tc but well below T*, and it grows truly distinct only below Tc (compare Fig. 4, A and B, with fig. S1, A to E). To obtain a clearer view of the structure of this feature, we show, in the insets of Fig. 4, E to G, the spectra at low temperatures divided by the one at 60 K—a procedure that converts the shoulder into a small peak. In Fig. 4C, we adopt the same procedure, but dividing by the 40 K spectra instead, and in fig. S2 we have subtracted off an approximate background from the EDCs. All three methods of analysis produce qualitatively similar results, with a small peak at a position that does not disperse appreciably along the exemplary antinodal cut C1. In all cases, the energy and width of the peak do not change considerably with increasing temperature, whereas the peak intensity is strongly temperature dependent; the peak be-

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comes undetectable at a temperature that, although greater than Tc, is nowhere near T*. This behavior of the processed data is highly reminiscent of the behavior of the superconducting coherence peak in Bi2212 (37), although that peak is visible in the raw data (Fig. 4D). The strong and analogous temperature dependences of the ARPES, PKE, and TRR data seen between Tc and T* are most naturally understood if T* is associated with a phase transition to a nonsuperconducting broken-symmetry state (27). Although superconducting fluctuations are observable above Tc (Fig. 4, E to G, and fig. S2E), they appear to peter out at too low temperatures (being negligible already at 60 K) to play a central role in this higher-temperature transition. To complete this picture, it is necessary to identify the nature of the broken-symmetry state and to relate it to the apparently similar electronic changes that occur below T* in other cuprates (27). Below Tc, the nodal arc is gapped with a dwave–like structure suggestive of a dominantly superconducting origin (38). In contrast, in the antinodal region, rather than one order being dominant, or the two gaps of both orders adding in quadrature, the spectral function develops a complex structure with two energy scales below EF of mixed origin, a larger one being primarily associated with the pseudogap order and a smaller one with the superconducting order. To see what can be learned about the pseudogap order from ARPES, we have used a simple mean-field model (27) to compute the expected changes to the band structure induced by various forms of density wave (6–8, 13–19) (fig. S7) or nematic order (6, 15, 17, 20–22) (fig. S8) coexisting with d-wave superconductivity. We note that some (but certainly not all) key aspects of our experimental observations can be qualitatively reproduced, regardless of which of these orders is assumed (27). An essential feature of both the experiment and the fits is the comparable sizes of the superconducting gap and the pseudogap. This implies that the two orders may have a more intimate connection than just competing orders, such as seen in 2H-NbSe2, where the charge density wave gap is at least three times the superconducting gap (39). References and Notes 1. A. Damascelli, Z. Hussain, Z.-X. Shen, Rev. Mod. Phys. 75, 473 (2003). 2. V. J. Emery, S. A. Kivelson, Nature 374, 434 (1995). 3. Y. Wang, L. Li, N. P. Ong, Phys. Rev. B 73, 024510 (2006). 4. K. Nakayama et al., Phys. Rev. Lett. 102, 227006 (2009). 5. J. Meng et al., Phys. Rev. B 79, 024514 (2009). 6. S. A. Kivelson, E. Fradkin, V. J. Emery, Nature 393, 550 (1998). 7. S. Chakravarty, R. B. Laughlin, D. K. Morr, C. Nayak, Phys. Rev. B 63, 094503 (2001). 8. M. Grilli, G. Seibold, A. Di Ciolo, J. Lorenzana, Phys. Rev. B 79, 125111 (2009). 9. C. M. Varma, Phys. Rev. B 55, 14554 (1997). 10. K. Tanaka et al., Science 314, 1910 (2006). 11. T. Kondo et al., Nat. Phys. 7, 21 (2011). 12. J.-H. Ma et al., Phys. Rev. Lett. 101, 207002 (2008). 13. J. M. Tranquada, B. J. Sternlieb, J. D. Axe, Y. Nakamura, S. Uchida, Nature 375, 561 (1995).

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REPORTS 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40.

G. L. Eesley et al., Phys. Rev. Lett. 65, 3445 (1990). P. Gay et al., J. Low Temp. Phys. 117, 1025 (1999). J. Demsar et al., Phys. Rev. Lett. 82, 4918 (1999). R. A. Kaindl et al., Science 287, 470 (2000). N. Gedik et al., Phys. Rev. B 70, 014504 (2004). E. E. M. Chia et al., Phys. Rev. Lett. 99, 147008 (2007). Y. H. Liu et al., Phys. Rev. Lett. 101, 137003 (2008). A. V. Fedorov et al., Phys. Rev. Lett. 82, 2179 (1999). That this is a bulk superconducting effect is corroborated by the magnetic field-dependent suppression of the Knight shift (a measure of the density of states at EF) seen below Tc of Bi2201 in nuclear magnetic resonance (40). The Knight shift was found to drop sharply at T *, exhibiting a similar temperature dependence as those shown in Fig. 3. S. V. Borisenko et al., Phys. Rev. Lett. 102, 166402 (2009). S. Kawasaki, C. Lin, P. L. Kuhns, A. P. Reyes, G. Q. Zheng, Phys. Rev. Lett. 105, 137002 (2010).

The Bonding Electron Density in Aluminum Philip N. H. Nakashima,1,2,3* Andrew E. Smith,1,4 Joanne Etheridge,2,3 Barrington C. Muddle1,2 Aluminum is considered to approach an “ideal” metal or free electron gas. The valence electrons move freely, as if unaffected by the presence of the metal ions. Therefore, the electron redistribution due to chemical bonding is subtle and has proven extremely difficult to determine. Experimental measurements and ab initio calculations have yielded substantially different results. We applied quantitative convergent-beam electron diffraction to aluminum to provide an experimental determination of the bonding electron distribution. Calculation of the electron distribution based on density functional theory is shown to be in close agreement. Our results yield an accurate quantitative correlation between the anisotropic elastic properties of aluminum and the bonding electron and electrostatic potential distributions. he electronic structure associated with chemical bonding affects all properties of materials except radioactivity. In metals, the widely taught concept of an ideal metal or free electron gas is popularly expressed as a regular array of metal ions surrounded by a sea of delocalized valence electrons (1). Although a gross simplification, this model is often sufficient to give a qualitative account of many characteristic properties of metals such as their high electrical and thermal conductivities and lustrous or shiny appearance (1–3). However, it fails to explain the often strong variation in particular properties of different metals that are sensitive to electronic structure (2–4)—for example, the anisotropy of elastic constants. One of the best examples of a free electron gas is aluminum (2, 5). Aluminum accounts for more than 40% of world production in nonferrous metals (6). The large global effort invested in refining aluminum and its alloys to extend property profiles involves the need for a fundamental understanding of metallic structure in solid solutions and structural evolution at the nanoscale in multiphase alloys. Such an effort must begin with accurate knowledge of the electronic structure, chemical bond-

T

ing, and atomic-scale mechanics in pure aluminum (2–4, 7–12), and then ultimately extend to its alloys (2, 8, 9, 13). A difficulty arising from aluminum’s close approximation to a free electron gas is that the perturbation of the total electron distribution by chemical bonds is subtle and difficult to determine. As a consequence, extensive experimental and theoretical studies of the electron density in aluminum have been carried out since the 1920s (7, 8, 14–17) but have failed to reach a consensus on the electronic structure of the chemical bonds. All possible modes are represented in the lit-

41. We thank I. Vishik, W.-S. Lee, L. Taillefer, and M. Greven for helpful discussions, Y. Li and G. Yu for experimental assistance on SQUID, and J.-H. Chu on resistivity measurements. R.-H.H. thanks the SGF for financial support. This work at the Stanford Institute for Materials and Energy Sciences, the Stanford Synchrotron Radiation Lightsource, and the Advanced Light Source is supported by the Department of Energy, Office of Basic Energy Sciences under contracts DE-AC02-76SF00515 and DE-AC02-05CH11231.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1579/DC1 Materials and Methods SOM Text Figs. S1 to S8 References 28 September 2010; accepted 15 February 2011 10.1126/science.1198415

erature, including bridge bonding between nearest neighbors (17), octahedrally centered bonding between second nearest neighbors (7, 17), tetrahedrally centered bonding between nearest neighbors (8, 17), and mixtures of these modes (17). Using quantitative convergent-beam electron diffraction (QCBED), we have made absolute measurements of electronic structure at ambient and liquid helium–cooled temperatures that are of sufficient accuracy, precision, and resolution to establish the bonding electron distribution in aluminum unequivocally. Electron diffraction is intrinsically sensitive to electron distribution by virtue of the strong interaction of the electron with the electrostatic potential of the specimen. Using Wien2K (18), we performed an ab initio density functional theory (DFT) calculation of the electron distribution [using the generalized gradient approximation (GGA) in the full potential linearly augmented plane wave formalism (FPLAPW), incorporating local orbital and local screening potentials (lo + ls)] and found that it agrees closely with the experimental determination by QCBED. The bonding electron and potential distributions determined here have a strong quantitative correlation with the anisotropy of Young’s modulus for aluminum.

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14. J. E. Hoffman et al., Science 295, 466 (2002). 15. C. Howald, H. Eisaki, N. Kaneko, A. Kapitulnik, Proc. Natl. Acad. Sci. U.S.A. 100, 9705 (2003). 16. M. Vershinin et al., Science 303, 1995 (2004). 17. Y. Kohsaka et al., Science 315, 1380 (2007). 18. C. V. Parker et al., Nature 468, 677 (2010). 19. W. D. Wise et al., Nat. Phys. 4, 696 (2008). 20. V. Hinkov et al., Science 319, 597 (2008). 21. R. Daou et al., Nature 463, 519 (2010). 22. M. J. Lawler et al., Nature 466, 347 (2010). 23. A. Kaminski et al., Nature 416, 610 (2002). 24. B. Fauqué et al., Phys. Rev. Lett. 96, 197001 (2006). 25. J. Xia et al., Phys. Rev. Lett. 100, 127002 (2008). 26. Y. Li et al., Nature 455, 372 (2008). 27. Materials and methods are available as supporting material on Science Online. 28. J. Xia, Y. Maeno, P. T. Beyersdorf, M. M. Fejer, A. Kapitulnik, Phys. Rev. Lett. 97, 167002 (2006). 29. M. Hashimoto et al., Nat. Phys. 6, 414 (2010).

Fig. 1. Sets of Fg published since 1929 (14–17) are plotted as points on the graph of Droct at the octahedral site versus Drtet at the tetrahedral site (o and t, respectively, in the inset). The DFT calculations of (7) would lie in the cross-hatched region of Droct > Drtet. The full potential linear muffin tin orbital (FPLMTO) calculations of (8) would lie in the hexagon-hatched region of Drtet > Droct. The present QCBED and Wien2K DFT (GGA/FPLAPW +lo +ls) (18) results are shown as circled crosses. See fig. S1 (17) for references for all points. All Dr values were computed using the Eden Crystallography package (29).

1 ARC Centre of Excellence for Design in Light Metals, Monash University, Victoria 3800, Australia. 2Department of Materials Engineering, Monash University, Victoria 3800, Australia. 3Monash Centre for Electron Microscopy, Monash University, Victoria 3800, Australia. 4School of Physics, Monash University, Victoria 3800, Australia.

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

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Dr ¼ rtotal − rIAM

ð1Þ

where rtotal is the total electron density and rIAM is the density associated with a superposition of free, unbonded atoms, otherwise known as the independent atom model (IAM) (17, 19). Given that the total electron density in a crystal can be expressed as a Fourier sum, rðrÞ ¼ ∑ Fg expð−2pig:rÞ

tal (QCBED) and theoretical (DFT) results suggest that bonding electron density is almost exclusively located in the tetrahedral hole. Experiments relying on x-ray diffraction and the kinematic (single scattering) approximation to interpret the data are fundamentally limited by problems of extinction and scale (20). These can introduce large systematic errors into the determination of the lowest-order bonding-sensitive structure factors (14, 16, 20) and contribute to the large spread of experimental points in Fig. 1 (14–17)

(fig. S1). Previous electron diffraction measurements using the critical voltage method (15–17) are absolute and therefore free of the problems of extinction and scale; however, the method conventionally depends on the validity of the threebeam approximation (21, 22) and it can only access a very limited number of structure factors (F111 and F002 in aluminum to date), insufficient for a unique and self-consistent determination of Dr. In QCBED, electrons are focused into very small volumes, allowing regions of perfect crystal

ð2Þ

g

Fig. 2. An incident cone of electrons is scattered by the crystal potential to form a CBED pattern. The as-captured intensities, I, can be differentiated with respect to scattering angle according to the approach of (24) and then matched with a calculated differential CBED pattern. The parameters refined in fitting are the structure factors, Vg, and phenomenological absorption coefficients, Vg´, of the most strongly scattering crystal planes; crystal thickness, H; and the coordinates of the pattern in reciprocal space. The results for Vg and H are reported for this example near [110] and 200.8 keV electrons. The crystal is oriented to satisfy the 2 –2 0 Bragg condition so as to increase the sensitivity to V022. The error map is in units of standard uncertainty per pixel in the experimental data.

where Fg are the Fourier coefficients of the electron density (structure factors), g are reciprocal lattice vectors, and r is a real space vector within the unit cell, Eq. 1 becomes DrðrÞ ¼ ∑ðFg − FgIAM Þexpð−2pig:rÞ g

ð3Þ

Experimentalists and theorists alike set out to determine the structure factors, Fg, as accurately as possible because the differences from IAM structure factors, FgIAM, are typically very small. In aluminum, the main problem has been to determine the three lowest-order structure factors, F111, F002, and F022, with sufficient accuracy, as these contain all of the bonding information and are the only ones to deviate significantly from IAM calculated values (15, 16). When considering all published sets of Fg including orders up to at least F022 (14–17), it became apparent that the strongest variations in Dr occur at the centers of the tetrahedral and octahedral interstices. Thus, an appropriate way to summarize the literature is to plot each source as a point on a graph of Droct at the center of the octahedral interstices versus Drtet at the center of the tetrahedral interstices. This is done in Fig. 1 (see fig. S1 for individual references). All determinations of Dr throughout the present work were executed on the basis of DF111, DF002, and DF022, where DFg = Fg − FgIAM, because both previous work (15, 16) and our own work (17) suggest that DFg ≡ 0 for F113 and higher orders. All structure factors were converted to T = 0 K (17) and all charge densities presented here are for T = 0 K. The points corresponding to our QCBED and DFT determinations are circled in Fig. 1. Recent ab initio studies by Ogata et al. (7) and Kioussis et al. (8) provide no structure factors. However, the former explicitly describes the high excess total electron density (i.e., positive Dr) in the octahedral hole (also apparent from their 3D plot), whereas the latter explicitly states that excess (i.e., bonding) charge density is preferentially located in the tetrahedral hole. We therefore partition the graph in Fig. 1 to reflect Droct > Drtet for (7) and Drtet > Droct for (8). Although the spread of points in the graph is large, most determinations show Drtet > Droct. Our experimen-

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The deformation electron density, Dr, is defined as

to be probed selectively (17). The incidence of the electrons over a range of angles (Fig. 2) produces a set of diffracted discs containing intensity oscillations that are highly sensitive to the structure factors of the diffracting crystal planes, the crystal thickness, and the proximity of the rele-

vant reciprocal lattice points to the Ewald sphere. CBED patterns are calculated using a full dynamic (multiple) electron scattering theory (17, 23), making extinction and scale issues irrelevant. Quantitative matching of calculated CBED patterns to experimental ones proceeds by refining the

Table 1. A comparison of the four lowest-order structure factors of aluminum determined in the present work (QCBED) at ambient and low temperatures, as well as by Wien2K [DFT with GGA/FPLAPW +lo +ls (18)], the literature (mean of all x-ray diffraction measurements, mean of all ab initio calculations, mean of all critical voltage measurements), and the IAM (19). F111 and F113 were not measured at low temperature, and critical voltage measurements have never extended beyond F002. The Debye-Waller factors, Bambient = 0.81641 Å2 (17, 31) and Blow temp. = 0.52945 Å2 (17), were used for the QCBED measurements at ambient and low temperatures, respectively (17). Uncertainties are given in parentheses as one standard deviation of the mean and apply to the last significant digit. Fg (e–/atom) 1 0 0 1

1 0 2 1

1 2 2 3

Mean of critical Wien2K Mean of Mean of QCBED QCBED voltage DFT (ambient) (low temp.) x-ray expts. ab initio (15–17) (15–17) (this work) (14–17) (this work) (this work) 8.87(1) 8.37(2) 7.32(8) 6.64(6)

8.37(1) 7.31(3)

8.8(2) 8.4(2) 7.3(1) 6.61(9)

8.86(7) 8.40(9) 7.31(7) 6.65(5)

8.87 8.38 7.30 6.64

8.87(2) 8.38(2)

IAM (19) 8.95 8.50 7.31 6.65

Fig. 3. (A and B) The Dr = 0.0275 e– Å−3 isosurface (corresponding to 50% of Drmax for the QCBED determination) is drawn in the lower unit cell for the present QCBED and DFT (Wien2K) determinations, respectively. The top cell is {110} plane-sectioned to show the contour plots of Dr in these planes, which pass through both the tetrahedral and octahedral interstices as defined in the inset of Fig. 1. Contours are at 0.02 e– Å−3 intervals, with the first dark contour showing Dr = 0 e– Å−3. Dark solid contours show Dr > 0 and light dashed lines Dr < 0. The color scale indicates the value of Dr for all surfaces and sections. All Dr values were computed using Eden Crystallography (29). Plots were generated using VESTA (30). Fig. 4. (A) The 20% Drmax (QCBED) isosurface is plotted about a single atom together with principal lattice vectors originating from the atomic site. (B) The DV = 0 (QCBED) isosurface is plotted and the volume enclosed is colored according to the deformation potential due to chemical bonding, DV. The atoms are localized at the maxima of DV. (C and D) Plots of DV = 0 as a surface and colored according to Dr intersected by the surface for our QCBED and DFT work, respectively. (E) pﬃﬃﬃﬃﬃﬃﬃﬃ The surface represents 1/ Euvw (where Euvw is Young’s modulus in direction [uvw]), calculated from the anisotropic elastic constants of aluminum (28). The surface coloring represents the magnitude of Euvw. All Dr and DV values were computed using Eden Crystallography (29). Plots were generated using VESTA (30). www.sciencemag.org

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parameters to which they are most sensitive (i.e., the structure factors of the most strongly scattering crystal planes and the crystal thickness) (17). Figure 2 summarizes our QCBED experiments. We apply the angular difference method (24) to the as-captured intensities, I, to remove most of the deleterious contributions of inelastic scattering from the resulting difference pattern, dI/dq, including thermal diffuse scattering (TDS), which cannot be removed by energy-filtering electron optics. CBED patterns were usually acquired with the crystal oriented to satisfy the Bragg condition of a reflection, thereby increasing sensitivity to the corresponding structure factor. Pattern matching was carried out with the RefineCB program of Zuo (17, 23, 25), which we modified for 2D near-zone-axis differential QCBED. The variable parameters were the crystal potential structure factors, Vg, of the most strongly scattering crystal planes (i.e., V220, V111, and V002 in Fig. 2), their associated absorption potentials, Vg´, the crystal thickness, H, and the coordinates defining the position of the CBED pattern in reciprocal space. This amounts typically to about 20 parameters, which are outnumbered by the ~104 data points being pattern-matched, resulting in a highly constrained solution. Structure factors measured by QCBED in terms of crystal potential, Vg, were converted to electron density structure factors, Fg, according to the Mott formula (17). In Fig. 2, the output calculated pattern has been matched to the experimental pattern well enough that no difference is detectable without the aid of the error map. This is the difference between the input experimental pattern and the refined calculated pattern expressed in units of uncertainty in each pixel of the experimental pattern. Although some systematic difference remains, its magnitude is small enough to ensure that almost all of the information has been extracted. The levels of uncertainty in the refined parameters listed at the bottom of Fig. 2, which derive from a single pattern, only reflect the uncertainty in the global minimum of the optimization criterion, c2 (17), in parameter space. The true precision can only be assessed by repeating structure factor measurements from many CBED patterns collected across a range of experimental conditions. We have collected 156 CBED patterns from different zone axes, scattering geometries, and crystal thicknesses (~150 to ~2000 Å), with different electron energies and at different temperatures, from 99.9999+% pure aluminum foils electropolished to electron transparency. The QCBED processing of all of these data resulted in the measurement of 14 independent structure factors, each one measured a large number of times, from a total of more than 1 million data points, effectively removing the possibility of a non-unique solution (17). The four lowest-order QCBED measured structure factors (converted to Fg and T = 0 K) are compared to previously published results and the present DFT calculation in Table 1 [see table S1 and (17) for all 14 measured structure factors]. The uncertainty of the

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that study (4) and related work (8), rtotal was determined from ab initio theoretical calculations. Given that rtotal is dominated by rIAM (Eq. 1), which contains no bonding information, a complementary alternative to the topological analysis of Eberhart (4) might be to consider Dr only. The reliability of such an approach depends heavily on the accuracy with which rtotal (and thus Dr) can be determined. Figure 4A presents the 0.2Drmax isosurface surrounding each atom in aluminum as determined by QCBED. This level was chosen to show how the bonding density differs in the three principal directions drawn (i.e., [100], [110], and [111]). The [100] vector does not intersect the drawn isosurface, whereas [110] intersects it at the necking point between edge-sharing tetrahedra and [111] passes directly through the main pocket of bonding density and the center of the tetrahedral hole. Thus, among these three directions, bonding is strongest along [111] and weakest along [100]. If Young’s modulus, Euvw, is proportional to Dr in any particular direction [uvw], then Fig. 4A suggests that E100 < E110 < E111, which is indeed the case (28). A more sophisticated method for relating chemical bonding to elastic anisotropy might be developed by considering both Dr and the deformation potential due to bonding, DV, where, analogously to Eq. 1, DV ¼ Vtotal − VIAM

ð4Þ

where Vtotal is the total crystal potential and VIAM is that of a superposition of free (unbonded) atoms. In Fig. 4B, the DV = 0 (QCBED) isosurface is plotted and bounds the positions of the atoms. Figure 4C examines this isosurface plotted around a single atom, and the surface is colored according to Dr that it passes through. The same is done for the present DFT calculation in Fig. 4D. The plots for the present QCBED and DFT determinations bear a strong resemblance to one another. A heuristically determined physical relationship between the DV = 0 isosurface, Dr, and Young’s modulus, Euvw, is detailed in (17). It suggests that Euvw is proportional to Dr in any particular direction and proportional to 1/r2DV=0 (where rDV=0 is the distance from the atomic site the DV = 0 isosurface). To represent this, ﬃﬃﬃﬃﬃﬃﬃﬃﬃ pto 1/ Euvw is plotted as a surface in Fig. 4E. This surface is colored by the magnitude of Euvw determined from the anisotropic elastic constants in (28). Figure 4E is a close match, in terms of form and relative magnitude, to the QCBED- and DFTgenerated plots in Fig. 4, C and D, respectively. A more quantitative analysis of the correlation between Young’s modulus and chemical bonding, as determined by our QCBED measurements and DFT calculation, in the principal directions shown in Fig. 4A is given in fig. S3 and (17). Our work shows both experimentally and theoretically that interatomic bonding is tetrahedrally centered in aluminum, with almost no bonding electron density in the octahedral interstice.

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Bonding is shown to have a direct correlation with mechanical properties—that is, the anisotropic elastic constants—in the present case of aluminum. References and Notes 1. M. Clugston, R. Flemming, Advanced Chemistry (Oxford Univ. Press, Oxford, 2008), p. 83. 2. A. Cottrell, Introduction to the Modern Theory of Metals (Institute of Metals, London, 1988). 3. E. H. Medlin, R. E. Dingle, D. W. Field, Nature 224, 581 (1969). 4. M. E. Eberhart, Acta Mater. 44, 2495 (1996). 5. J. Sprösser-Prou, A. vom Felde, J. Fink, Phys. Rev. B 40, 5799 (1989). 6. World Metal Statistics Yearbook (World Bureau of Metal Statistics, Hertfordshire, UK, 2006), pp.12–68. 7. S. Ogata, J. Li, S. Yip, Science 298, 807 (2002). 8. N. Kioussis, M. Herbranson, E. Collins, M. E. Eberhart, Phys. Rev. Lett. 88, 125501 (2002). 9. R. S. Leigh, Philos. Mag. 42, 139 (1951). 10. J. Hafner, Z. Phys. B 22, 351 (1975). 11. C. Woodward, D. R. Trinkle, L. G. Hector Jr., D. L. Olmsted, Phys. Rev. Lett. 100, 045507 (2008). 12. J. M. MacLaren, S. Crampin, D. D. Vvedensky, M. E. Eberhart, Phys. Rev. Lett. 63, 2586 (1989). 13. M. Morinaga, S. Nasu, H. Adachi, J. Saito, N. Yukawa, J. Phys. Condens. Matter 3, 6817 (1991). 14. N. N. Sirota, Acta Crystallogr. A 25, 223 (1969). 15. W. Hoppe, R. Mason, Eds., Advances in Structure Research by Diffraction Methods (Pergamon, Oxford, 1975), pp. 221–225. 16. A. G. Fox, M. A. Tabbernor, R. M. Fisher, J. Phys. Chem. Solids 51, 1323 (1990). 17. See supporting material on Science Online. 18. P. Blaha, K. Schwarz, P. Sorantin, S. B. Trickey, Comput. Phys. Commun. 59, 399 (1990). 19. P. A. Doyle, P. S. Turner, Acta Crystallogr. A 24, 390 (1968). 20. P. Coppens, X-ray Charge Densities and Chemical Bonding (Oxford Univ. Press, New York, 1997). 21. A. F. Moodie, J. R. Sellar, D. Imeson, C. J. Humphreys, J. Electron Microsc. 26 (suppl.), 191 (1977). 22. A. F. Moodie, C. J. Humphreys, D. Imeson, J. R. Sellar, in Electron Diffraction 1927–1977, P. J. Dobson, J. B. Pendry, C. J. Humphreys, Eds. (Institute of Physics, Bristol, UK, 1978), chap. 3. 23. J. M. Zuo, Rep. Prog. Phys. 67, 2053 (2004). 24. P. N. H. Nakashima, B. C. Muddle, Phys. Rev. B 81, 115135 (2010). 25. J. M. Zuo, M. Kim, M. O’Keeffe, J. C. H. Spence, Nature 401, 49 (1999). 26. N. B. Brockhouse, A. T. Stewart, Phys. Rev. 100, 756 (1955). 27. G. Honjo, S. Kodera, N. Kitamura, J. Phys. Soc. Jpn. 19, 351 (1964). 28. J. F. Nye, Physical Properties of Crystals (Oxford Univ. Press, Oxford, 1985). 29. J. R. Somoza, A. Szöke, H. Szöke, Acta Crystallogr. A 57, 678 (2001). 30. K. Momma, F. Izumi, J. Appl. Crystallogr. 41, 653 (2008). 31. D. L. McDonald, Acta Crystallogr. 23, 185 (1967). 32. We thank M. Weyland for assisting in the collection of the liquid helium–temperature CBED data; A. F. Moodie and A. W. S. Johnson for their expert readership and valuable suggestions; J. M. Zuo for sharing his RefineCB code, which we have modified to execute our QCBED analysis; J. Shih for electropolishing some of the specimens; the Victorian Partnership for Advanced Computing (VPAC); and the Australian Research Council (ARC) for infrastructure grant LEO454166. P.A. thanks V. A. Streltsov, A. W. S. Johnson, and the late E. N. Maslen for their guidance.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1583/DC1 Materials and Methods Figs. S1 to S3 Table S1 References 1 October 2010; accepted 1 February 2011 10.1126/science.1198543

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QCBED measurements of F111 and F002 at 300 K is an order of magnitude lower than published x-ray diffraction measurements and about 5 times as precise as the mean values of published ab initio determinations (Table 1 and table S1). The high uncertainty in F022 measured by QCBED at ambient temperatures and x-ray diffraction is probably due to aluminum’s lowfrequency, transverse acoustic phonon mode with wave vectors in the {0hh} planes associated with the rigid body movement of atomic chains along 〈011〉 (26, 27). To test this hypothesis, we collected 16 CBED patterns near 〈001〉 with the use of a liquid helium specimen stage. The diffuse background and its structure in these patterns were significantly reduced relative to equivalent ambient temperature patterns (17), giving visual evidence of reduced scattering from phonons. The low-temperature QCBED measurements of F022 are much more precise than at ambient temperatures, with the uncertainty now comparable to the uncertainties in the lower-order structure factors measured by QCBED (Table 1 and table S1). This suggests that the spread in F022 measured at ambient temperatures is phonon-mediated. The increased precision of F022 shows that there is almost no bonding information in this structure factor and that deviations from FgIAM only occur for F111 and F002. This implies that critical voltage measurements to date (Table 1 and table S1) are sufficient to determine the bonding electron density distribution in aluminum; however, this conclusion could not have been reached earlier because of the large variation in previous F022 measurements (14–16). Using F111, F002, and F022 from QCBED at ambient temperatures and our Wien2K (18) DFT calculation (GGA/FPLAPW +lo +ls), we obtained the maps of bonding electron density (17) shown in Fig. 3. The Dr = 0.0275 e– Å−3 isosurfaces (corresponding to 0.5Drmax determined by QCBED) show that electron accumulation occurs in the tetrahedral interstices in both cases. The {110} plane-sectioned contour plots show that there is near-zero bonding density in the octahedral holes according to the QCBED measurements, whereas there is slight antibonding density associated with the octahedral hole in the DFT determination. A stronger accumulation in the tetrahedral holes is indicated by QCBED than by the Wien2K DFT calculation. Although there are some quantitative differences in Dr between the QCBED and DFT determinations, the form of the bonding electron distribution is almost the same. Of all previous determinations represented by points in Fig. 1 (and fig. S1), our DFT-calculated Dr is the closest to the present QCBED benchmark Dr measurement. An important problem in materials science is the effect of interatomic bonding on the mechanical properties of materials (2–4, 7–9, 11, 12). The seminal work of Eberhart (4) has demonstrated a strong correlation between the topology of the total electron density, rtotal, and the anisotropy of elastic constants in metals (4, 28). In

Revealing Extraordinary Intrinsic Tensile Plasticity in Gradient Nano-Grained Copper T. H. Fang,* W. L. Li,* N. R. Tao,* K. Lu† Nano-grained (NG) metals are believed to be strong but intrinsically brittle: Free-standing NG metals usually exhibit a tensile uniform elongation of a few percent. When a NG copper film is confined by a coarse-grained (CG) copper substrate with a gradient grain-size transition, tensile plasticity can be achieved in the NG film where strain localization is suppressed. The gradient NG film exhibits a 10 times higher yield strength and a tensile plasticity comparable to that of the CG substrate and can sustain a tensile true strain exceeding 100% without cracking. A mechanically driven grain boundary migration process with a substantial concomitant grain growth dominates plastic deformation of the gradient NG structure. The extraordinary intrinsic plasticity of gradient NG structures offers their potential for use as advanced coatings of bulk materials. xtensive investigations over the past few decades indicated that with a substantial reduction of grain sizes into the nanometer regime, the strength of polycrystalline metals is greatly increased at the expense of their ductility (1, 2). Free-standing nano-grained (NG) metals usually exhibit a very high strength and a very limited tensile ductility (with a uniform elongation of few percent) and almost no work-hardening before catastrophic failure (3). The brittleness is believed to be an intrinsic “Achilles’ heel” of NG metals because the conventional deformation mechanisms cease to operate: Dislocation slip is substantially suppressed by the extremely small grains (which accounts for the extreme strengthening in NG metals) and grain boundary (GB) sliding or diffusional creep is not active enough to accommodate plastic straining at ambient temperature (3). Experimental observations hint that the observed brittleness in NG metals might be extrinsic rather than intrinsic. For instance, dimples have been observed in fracture surfaces of various NG metals, signifying substantial plastic deformation before failure (4, 5). Large plastic strains can be obtained in other deformation modes such as compression and rolling (6, 7). Indeed, limited tensile ductility of NG metals is often attributed to the absence of work-hardening of nano-sized grains, so that strain localization and early necking occur immediately after yielding. Thus, the intrinsic tensile plasticity may have not been revealed due to superimposition of the strain localization and early necking. Intrinsic tensile plasticity of NG samples might be detected provided the strain localization is effectively suppressed. Previous studies (8, 9) showed that confinement by a ductile substrate is effective in suppressing strain localization in NG metal films under tension. Tensile elongation of NG Cu films adherent on a polymer substrate can be enhanced up to

E

Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]

10% before failure through debonding of the film and substrates. A higher ductility is expected if the strain localization in the NG film could be better suppressed. The elastic mismatch and the interface bonding between the film and the matrix are two key parameters controlling the confinement. Consequently, an ideal architecture might be a NG element metal film adherent on a coarsegrained (CG) substrate of the same metal with a graded grain-size transition between them. This gradient architecture without a shape interface between the NG film and the CG substrate, which is elastically homogeneous but plastically gradient, may offer unusual mechanical responses (10) and provide a unique opportunity for reveal-

ing the intrinsic tensile plasticity of NG metals without strain localization. For synthesizing such an architecture, surface nanocrystallization of CG metals by means of surface plastic deformation techniques (11, 12) is a feasible option. Here, we have used a surface mechanical grinding treatment (SMGT) (13) for preparing a NG Cu film with a spatial gradient in grain size on a bulk CG Cu substrate and have achieved a large tensile plasticity in the NG structure and revealed a different governing deformation mechanism. CG Cu dog-bone–shaped tensile bar specimens with a gauge diameter of 6 mm and gauge length of 20 mm were processed by means of SMGT at cryogenic temperature to form a NG surface layer in the gauge section (Fig. 1) (14). After treatment, the topmost layer of the specimens consists of nanosized elongated grains with random crystallographic orientations (Fig. 1, D and E), with an average transversal grain size of about 20 nm and an aspect ratio of 2.0 (Fig. 1F). Transmission electron microscopy (TEM) measurements showed an increasing grain size gradually with an increasing depth. The average transversal grain sizes are <100 nm in the top 60-mm-thick layer and increase to about 300 nm in a depth of 60 to 150 mm. Below 150-mm depth are typical deformation structures in coarse grains, characterized by dislocation tangles or dislocation cells with sizes ranging from submicrometers to micrometers. The thickness of the deformed CG layer is about 500 to 700 mm. In the top 150mm-thick layer, a gradient nano-grained (GNG) structure with grain sizes varying from 20 to 300 nm is formed on the CG substrate.

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Fig. 1. (A) Schematic of the tensile bar sample of which the gauge section was processed by means of SMGT. (B and C) Schematic of the cross-sectional microstructure of the gauge consisting of a GNG layer (dark blue) and a deformed CG layer (blue) on a CG core (light blue). (D) A typical cross-sectional SEM image of a SMGT Cu sample. (E) A cross-sectional bright-field TEM image of microstructures 3 mm below the treated surface. The arrow indicates the processing direction, and the inset shows the electron diffraction pattern. (F) A transversal grain size distribution from TEM measurements in the top 5-mm-deep layer. (G) Variation of average transversal grain (subgrain or cell) sizes along depth from the surface. Error bars represent the standard deviation of grain-size measurements.

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The top 50-mm-thick surface layer was removed from the as-prepared SMGT Cu sample and cut into a dog-bone tensile specimen. Quasi-static tensile tests of the free-standing GNG foil showed a yield strength of ~660 MPa and a uniform elongation of <2% (Fig. 2A). The measured yield strength, about 10 times that of the CG Cu (63 T 3 MPa), is consistent quantitatively with that calculated from the Hall-Petch relation based on the measured grain sizes. Such a strong-and-brittle tensile behavior of the GNG foil is analogous to that reported in the literature (15). Tensile tests of the SMGT bar samples with a GNG/CG architecture (14) showed a yield strength (0.2% offset) of 129 T 17 MPa (Fig. 2A), twice that of the CG sample. This yield strength increment is reasonably attributed to the strong GNG surface layer and the deformed CG layer. Summing up the estimated strengths of the GNG layer, the deformed CG layer, and the CG core following the rule-of-mixture resulted in a yield strength of about 135 MPa, in good agreement with the measured value.

In contrast to the brittle failure of the free-standing GNG foil, quasi-static tension of the GNG/CG bar samples showed that the GNG surface layer deforms coherently with the CG core in the uniform elongation stage without any surface cracking or delaminating, and the surface roughness is slightly changed (Fig. 2B). After necking, the coherent deformation of the GNG layer and CG core continues, analogous to that of the tensile sample with a monolithic CG structure. No surface cracking or delaminating was detected even in the neck region where the true strain exceeds 100%. The deformed GNG/CG sample surface is much smoother than the deformed CG, both during the uniform deformation and after necking (Fig. 2B). From more than two dozen tensile tests, we observe a uniform elongation of 31 T 2% in the GNG/CG sample, which is similar to that of the CG tensile sample with a gauge diameter of 4.5 mm (32 T 2%). Because the diameter of the deformation-free CG core in the GNG/CG tensile bar is about 4.5 mm, it is believed that the tensile

Fig. 2. (A) Quasi-static tensile engineering stress-strain curves for the CG Cu bar sample with a gauge diameter of 4.5 mm, the GNG/CG bar sample, and a free-standing GNG foil sample (the top 50-mm-thick layer was removed from the GNG/CG sample, gauge dimensions: 4 mm by 2 mm by 0.05 mm), respectively. Strain rate is 6 × 10−4 s−1. Inset shows the tensile GNG/CG bar samples before and after tension (with a nominal strain of 30%). (B) Measured surface height variation profiles in gauge sections of the GNG/CG and the CG bar samples before (both with the same surface roughness) and after tension (with a strain of 30%).

plasticity of the GNG/CG sample is limited by the CG substrate whereas the GNG layer has no detrimental influence on plasticity. Hence, tensile plasticity of the GNG layer is, at least, comparable to that of the CG substrate. Apparently, strain localization in the GNG layer under tension is completely suppressed by the CG substrate with a gradient architecture, of which the tensile behaviors differ fundamentally from that of the freestanding NG samples. The confined GNG layer exhibits a 10-times higher yield strength and a tensile ductility comparable to that of the CG substrate. For a direct comparison of the tensile plasticity between the GNG and the CG structures, the top 750-mm-thick layer was removed from the SMGT Cu sample and cut into a thin foil tensile specimen, of which one side is of NG structure and the other is CG. Both sides were chemically polished to a roughness in the nanometer regime. Upon tensile loading, plastic deformation occurred uniformly throughout the foil after yielding at about 280 MPa. Necking is seen in three dimensions in the middle of the tensile specimen at a nominal strain of 20% (Fig. 3, A to C). Distinct surface morphologies have developed on the two surfaces. At a nominal strain of 5%, elongated hollows appeared roughly vertical to the tension direction in the NG surface, submicrometers to micrometers wide and several micrometers long (Fig. 3E). Their depths range from a few to several tens of nanometers. At larger strains, more hollows were formed with larger width. In the neck region (Fig. 3F), hollows are linked, forming a uniform surface morphology with a roughness below 100 nm, without any cracking. In the CG surface, increasing dislocation slip is observed in coarse grains with an increasing strain (Fig. 3H). In the neck region, much larger surface roughness (micrometer-scale) is induced by intensive slip. In addition, small cracks of several micrometers in length were identified at GBs (Fig. 3I), indicating that plastic strain between neighboring grains could not be accommodated by slip and strain localization onsets. Further straining results in

Fig. 3. (A to I) SEM images of a tensile GNG/CG foil sample (the top 750mm-thick layer was removed from the as-prepared GNG/CG sample, gauge dimensions: 3 mm by 1 mm by 0.75 mm) with a nominal strain of 20% [(B) side view; (A and C) planviews of the NG side and the CG side]. Strain rate is 6 × 10−4 s−1. SEM images of both sides of the foil sample with different nominal strains are shown (D), (E), and (F) correspond to the NG side with strains of 0, 5, and 20%; (G), (H), and (I) correspond to the CG side with strains of 0, 5, and 20%, respectively). Inset in (E) is a magnified image of a hollow. Circles in (I) indicate cracks formed at GBs in the CG side. Double-ended arrows indicated tensile directions.

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Fig. 4. (A) A cross-sectional view of one half of the tensile bar GNG/CG sample after failure. (B) A SEM image at position B indicated in (A) with tensile true strains of 24%. (C) and (D) are electron backscattering diffraction (EBSD) images at position C and D in (A), respectively. For (B) to (D), images are from a depth of 5 to 23 mm below the sample surface. (E) A bright-field TEM image of the top layer (2.5 mm below the surface) with a true strain of 33%. The arrow indicates the loading direction. The outlined area indicates a dislocation-free grain. (F) Variation of average transversal grain sizes (determined from SEM and EBSD images) with tensile true strain in the top layer (depth of 0 to 20 mm) and in the subsurface layer (depth of 20 to 40 mm). Error bars represent the standard deviation of grain-size measurements. more cracks that propagate toward the GNG side across the specimen. Plastic deformation is more uniform and better accommodated in the GNG layer than that in the CG, implying that the GNG structure may possess a higher tensile deformability than the CG structure in which cracks form preferentially under the same loading condition. To reveal the deformation mechanism, we examined microstructures in the GNG layer that underwent different strains in the tensile bar samples after failure (Fig. 4A). The true strain (eT) at different positions can be estimated from the gauge diameter (D) by eT = ln(D02/D2) (where D0 is the original gauge diameter). At position B (eT = 24%), microstructures in the GNG layer seem coarser than the as-processed state, but details cannot be clearly imaged under scanning electron microscopy (SEM). At eT = 54% (Fig. 4C), grain growth is apparent in the GNG layer and roughly equiaxed submicrometer-sized grains with random orientations are developed. At eT = 127% (Fig. 4D), grains become even coarser and elongated (roughly along the loading direction, aspect ratio of ~2.0) with a f011g〈112〉 rolling texture. Cross-sectional TEM observations revealed grain coarsening at a strain of 10%, and a large number of submicrometer-sized grains appeared throughout the GNG layer. At eT = 33%, TEM images from different orientations showed that most grains become submicrometer-sized, at which dislocation density is rather low (as in Fig. 4E, some grains are basically dislocation-free as outlined). The area-weighted cumulative grain size distribution in the top GNG layer (fig. S2) and the slightly changed aspect ratio of grains (1.9 to 2.0) indicated a pronounced inhomogeneous grain growth process, i.e., some grains grow preferentially at the expense of others. The same grain growth mechanism was identified in the entire GNG layer at different depths with increasing

strains. The observed grain growth in the GNG layer corresponds to a drop in microhardness from about 1.6 T 0.11 GPa in the top layer before tension to 1.2 T 0.12 GPa at a true strain of 30%. The microstructure observations preclude conventional deformation mechanisms in plastic deformation of the GNG layer, such as dislocation slip and diffusion-controlled processes (e.g., Coble creep). The grain growth, which dominates the plastic deformation of the present sample, can be reasonably interpreted as a mechanically driven GB migration process, similar to previous observations in NG metals (16–23). Mechanically induced grain growth at room temperature has been reported in NG samples under indentation (16, 17), compression (18, 19), and tensile loading (20–23), and has also been seen in molecular dynamic simulations (24). The estimated GB migration velocity in terms of the grain growth data in the GNG layer is of the same order of magnitude as that reported in a NG Al tensile sample (22). But the observed GB displacements, as large as micrometers, are much larger than the reported results (up to submicrometers). Although such a large GB displacement at room temperature is difficult to explain with existing models (25, 26), it could be understood as the result of an energy release due to substantial defect annihilation in the nanostructures, which can accommodate the large plastic strains. With an increasing true strain (or true stress, which scales with true strain), the average grain size increases substantially and tends to saturation when eT > 80% (Fig. 4F). It implies that the dominant plastic deformation mechanism shifts from the mechanically driven GB migration to conventional dislocation slip when grain sizes are large enough, as verified by the observed rolling texture (Fig. 4D). In the top surface layer, grain growth rates are lower, with a smaller saturated

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grain size (~400 nm) than that in the subsurface layer, which might be attributed to the varied grain morphology and GB structures along depth. A large fraction of GBs in the top GNG layer were derived from twin boundaries (TBs) induced by high strain rates, whereas in the subsurface layer with much lower strain rates, most GBs are conventional high-angle boundaries derived from dislocation structures (27). Hence, GBs in the subsurface layer possess a higher excess energy than those TB-like boundaries in the top layer, as verified by diffusivity measurements (28). Our study shows that NG metals are not only strong but also intrinsically ductile as long as strain localization is effectively suppressed. The extraordinary plasticity of the NG structures originates from a deformation mechanism with concomitant mechanically driven growth of nano-sized grains. The intrinsic mechanical properties of NG materials and the GNG/CG architecture provide an approach for enhancing strength-ductility synergy of materials and offer the potential for using gradient NG layers as advanced coatings of bulk materials.

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References and Notes 1. H. Gleiter, Prog. Mater. Sci. 33, 223 (1989). 2. J. Chen, L. Lu, K. Lu, Scr. Mater. 54, 1913 (2006). 3. M. A. Meyers, A. Mishra, D. J. Benson, Prog. Mater. Sci. 51, 427 (2006). 4. K. S. Kumar, S. Suresh, M. F. Chisholm, J. A. Horton, P. Wang, Acta Mater. 51, 387 (2003). 5. A. Hasnaoui, H. Van Swygenhoven, P. M. Derlet, Science 300, 1550 (2003). 6. Y. M. Wang, E. Ma, M. W. Chen, Appl. Phys. Lett. 80, 2395 (2002). 7. L. Lu, M. L. Sui, K. Lu, Science 287, 1463 (2000). 8. Y. Xiang, T. Li, Z. Suo, J. Vlassak, Appl. Phys. Lett. 87, 161910 (2005). 9. N. Lu, X. Wang, Z. Suo, J. Vlassak, J. Mater. Res. 24, 379 (2009). 10. S. Suresh, Science 292, 2447 (2001). 11. K. Lu, J. Lu, J. Mater. Sci. Technol. 15, 193 (1999).

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REPORTS 21. T. J. Rupert, D. S. Gianola, Y. Gan, K. J. Hemker, Science 326, 1686 (2009). 22. M. Legros, D. S. Gianola, K. J. Hemker, Acta Mater. 56, 3380 (2008). 23. G. J. Fan, L. F. Fu, H. Choo, P. K. Liaw, N. D. Browning, Acta Mater. 54, 4781 (2006). 24. J. Schiotz, Mater. Sci. Eng. A 375-377, 975 (2004). 25. J. W. Cahn, Y. Mishin, A. Suzuki, Acta Mater. 54, 4953 (2006). 26. J. C. M. Li, Phys. Rev. Lett. 96, 215506 (2006). 27. K. Wang, N. R. Tao, G. Liu, J. Lu, K. Lu, Acta Mater. 54, 5281 (2006). 28. Z. B. Wang, K. Lu, G. Wilde, S. V. Divinski, Acta Mater. 58, 2376 (2010).

Self-Recognition Among Different Polyprotic Macroions During Assembly Processes in Dilute Solution Tianbo Liu,1* Melissa L. K. Langston,1 Dong Li,1 Joseph M. Pigga,1 Céline Pichon,1 Ana Maria Todea,2 Achim Müller2* We report a self-recognition phenomenon based on an assembly process in a homogeneous dilute aqueous solution of two nano-scaled, spherical polyprotic metal oxide–based macroions (neutral species in crystals), also called Keplerates of the type [(linker)30(pentagon)12]≡[{M(H2O)}30{(Mo)Mo5}12] where M is FeIII or CrIII. Upon deprotonation of the neutral species, the resulting macroions assemble into hollow “blackberry”-type structures through very slow homogeneous dimer-oligomerization processes. Although the geometrical surface structures of the two macroions are practically identical, mixtures of these form homogeneous superstructures, rather than mixed species. The phase separation is based on the difference in macroionic charge densities present during the slow homogeneous dimer or oligomer formation. The surface water ligands’ residence times of CrIII and FeIII differ markedly and lead to very different interfacial water mobilities between the Keplerates. olecules in solution can self-assemble into larger structures through weak interactions (1, 2). For example, lipid molecules of the same type can assemble in water into larger micelles (2). By contrast, shape and polarity complementarity of different molecules can direct self-assembly through molecular recognition (1, 2). One way to explore interaction-based processes in the context of self-recognition (3) is to examine solutions with mixed species that do not exhibit complementary properties to determine whether self-assembly leads to phase separation or creates structures containing different species. To this end, we studied solutions containing two different hydrophilic, porous, metal oxide–based molecular clusters with identical surface structures that bear multiple metal cations (Lewis acid sites) with influential coordinated water ligands. These 2.5-nm-size clusters of the Keplerate type (Fig. 1A) are intermediate in size between simple ions and colloids (4–11), get deprotonated in aqueous solution, and have a

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1 Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA. 2Fakultät für Chemie, Universität Bielefeld, D-33501 Bielefeld, Germany.

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

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distinctive solution behavior: They (but also other giant inorganic ions) tend to spontaneously and reversibly assemble into stable, uniform, singlelayered, shell-like “blackberry” structures instead of existing as discrete ions in dilute solution (12–18). The formation of these structures is mediated by counterion attractions and hydrogen bonding, as opposed to van der Waals forces, hydrophobic interactions, or chemical reactions (15, 18), which in the present case leads to a phase separation, i.e., two superstructures with no interference or crossover (3). We examined aqueous solutions containing the two spherical polyprotic clusters of the type [(linker)30(pentagon)12]≡[{M(H2O)}30{(Mo)Mo5}12], where M is FeIII or CrIII (4, 5) (below abbreviated as {Mo72Fe30} and {Mo72Cr30}; structural details are shown in Fig. 1, and the chemical formulae in Materials and Methods). The clusters have, according to the properties of the 30 characteristic and active M(H2O) groups coordinated to the non-Mo atoms, extremely different mobilities of their surface hydration layers as well as degrees of deprotonation (13–15). Specifically, the water ligands (weak Brønsted acids) release protons, i.e., about seven for {Mo72Fe30} and about five for {Mo72Cr30} in aqueous solutions, which leads to a comparably smaller surface charge of the

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29. We thank X. Si for assistance in sample preparation and J. Tan for assistance in EBSD experiments. We are grateful for financial support of the Ministry of Science and Technology of China (grant 2005CB623604, 2010DFB54010), the National Natural Science Foundation (grants 50890171, 50971122), and the Danish–Chinese Center for Nanometals (grant 50911130230).

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200177/DC1 Materials and Methods Figs. S1 and S2 8 November 2010; accepted 7 February 2011 Published online 17 February 2011; 10.1126/science.1200177

latter (15). Correspondingly, the degree of deprotonation of the clusters changes with the pH. One result is that upon, for example, addition of acid, the size of the blackberry increases monotonically with decreasing pH, i.e., with decreasing charge density on the macroions (13–15). At pH ~3.5 to 4.5, for instance, the superstructures formed in aqueous solution of {Mo72Cr30} have an average hydrodynamic radius (Rh) of ~60 to 80 nm [measured by dynamic light scattering (DLS) and transmission electron microscopy], which is larger than that of the corresponding {Mo72Fe30} blackberries (~20 to 30 nm) (13–15). At low pH (~2.5), both types of clusters stay as discrete (protonated) neutral molecules in aqueous solution without showing the assembly phenomenon. We studied the mixed aqueous solutions of the {Mo72Cr30}- and {Mo72Fe30}-type Keplerates to determine whether they form homogeneous or heterogeneous blackberry-type structures (Fig. 1C). Aqueous solutions containing both types of macroanion (1:1 mass ratio, up to 0.5 mg/ml each) were prepared and then maintained at 30° or 40°C; the assembly processes start slowly and last for several weeks. The resulting two separated modes in the CONTIN analysis (19) of the DLS study indicated the presence of two differently sized large species (Fig. 1C and red curve in Fig. 2A). The two peaks correspond, within the error limit of the CONTIN analysis, to those of the individual solutions containing either {Mo72Cr30} or {Mo72Fe30} at the same pH value (Fig. 2A). We obtained further evidence for selfrecognition in the assembly process by separating the two formed large “final products” in the mixed solutions with a series of filter membranes of different pore sizes, starting with the largest ones [for details, see Materials and Methods (20)]. A CONTIN analysis of fraction A (20) showed a peak at an Rh of ~60 to 80 nm (Fig. 2B), consistent with the peak of the pure {Mo72Cr30} solution. These larger assemblies have, by element analysis, a Cr:Fe mass ratio of ~1:0.02, demonstrating that they are formed almost entirely from {Mo72Cr30}. The CONTIN analysis of fraction B (20) showed a peak at an Rh of ~25 nm (Fig. 2B), i.e., almost identical to that of the pure {Mo72Fe30} solution. However, because of the incomplete membrane separation, these smaller assemblies have, by element analysis, a relative

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12. K. Lu, J. Lu, Mater. Sci. Eng. A 375-377, 38 (2004). 13. W. L. Li, N. R. Tao, K. Lu, Scr. Mater. 59, 546 (2008). 14. Materials and methods are available as supporting material on Science Online. 15. Y. M. Wang et al., Scr. Mater. 48, 1581 (2003). 16. K. Zhang, J. R. Weertman, J. A. Eastman, Appl. Phys. Lett. 85, 5197 (2004). 17. M. Jin, A. M. Minor, E. A. Stach, J. W. Morris Jr., Acta Mater. 52, 5381 (2004). 18. D. Pan, S. Kuwano, T. Fujita, M. W. Chen, Nano Lett. 7, 2108 (2007). 19. S. Brandstetter, K. Zhang, A. Escuadro, J. Weertman, H. Van Swygenhoven, Scr. Mater. 58, 61 (2008). 20. D. S. Gianola et al., Acta Mater. 54, 2253 (2006).

Fig. 1. (A) Structure of the spherical, 2.5-nm-diameter {Mo72M30}-type clusters (4, 5) (M is Fe3+ or Cr3+; ball-andstick representation) built from 12 pentagonal {(Mo)Mo5}type units spanning an icosahedron (in blue) linked by the 30 “active Lewis acid sites” M3+ (large green spheres with the coordinated “active” H2O ligands) spanning the unique icosidodecahedron with equal edges (in green). The two solids, one inside the other (like Russian dolls), are typical for a Keplerate (M, green; Mo, blue; O, red; H (approximately positioned), gray). (B) The unique identical surface structures of the two Keplerates [space-filling model; colors as in (A)] contain only terminal O atoms coordinated to Mo atoms and water ligands coordinated to the M3+ cations like an “oxide water ball.” (C) (Middle) In mixed dilute aqueous solutions, the clusters (polyhedral representation) {Mo72Fe30} (top) and {Mo72Cr30} (bottom) self-assemble into different (i.e., individual) blackberry structures of the Cr30 (yellow) and Fe30 type (blue)—with interfacial water between the macroions (right)—and do not form mixed species (such as the hypothetical structure shown on the left).

Fig. 2. (A) CONTIN analysis of the DLS studies at 90° scattering angle measured for aqueous solutions containing {Mo72Cr30} or {Mo72Fe30} (0.1 mg/ml each), as well as for a solution containing both species (0.1 mg/ml of each) (pH = 4.2). In the mixed solution, the peak due to the larger {Mo72Cr30} assemblies is dominant because larger structures scatter more light. (B) CONTIN analysis of the DLS experiment carried out on the two components after ultrafiltration of the aqueous solution originally containing both {Mo72Cr30} and {Mo72Fe30} (0.1 mg/ml of each) by a series of membranes with different pore sizes; fraction A (red): material retained after filtering by 100,000 dalton-pore size membrane; fraction B (blue): material retained after filtering by 30,000 dalton-pore size membrane.

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mass ratio of Cr:Fe ≈ 3:10. This is due to the overlap between the two “size distributions” (Fig. 2B). (The species with sizes within the “overlap” region stay mostly in fraction B.) A lag phase at the beginning of the assembly process (sometimes days at room temperature) can formally be correlated with a high–activation energy barrier for the dimer and oligomer formations (Fig. 3) (21, 22). The presence of the (less stable) dimers during the lag phase has been confirmed for the {Mo72Fe30} scenario by analytical ultracentrifugation (21) and recently with small-angle x-ray scattering techniques (23). (The stability of the dimers with protons as “counterions” in between is limited because the protons can easily be released.) These dimers eventually form oligomers, although only those of the appropriate type (i.e., with corresponding shape and correct metal content) get integrated into the incomplete superstructure containing the same type of macroions. This phenomenon can be compared with nucleation processes that occur in crystal growth: Larger seeds grow at the expense of the smaller ones, which have a higher free energy and therefore dissolve—a kind of “survival of the fittest.” Correspondingly, oligomers that are not of the appropriate type “dissociate.” Important for the separation could also be that the homogeneous-type dimers and oligomers have slightly lower free energy than the mixed ones. (The assembly process also has some similarity to virus capsid formation (24); see also the legend of fig. S1.) Two other factors related to properties of the M3+(H2O) groups of the macroions with different M were primarily considered to contribute to the self-recognition process. One is the difference in charge density of the two Keplerates based on different degrees of deprotonation of the Cr(H2O) and Fe(H2O) groups. Consequently, the {Mo72Fe30}-type clusters carry more negative charges (see above), resulting in weaker attraction between them during the self-assembly, which leads to smaller blackberry sizes with larger curvatures. The other point is that the 30 identical CrIII(H2O) and FeIII(H2O) groups differ greatly in the lability (i.e., residence time) of their water ligands. {In CrIII complexes with innocent ligands— like [Cr(H2O)6]3+ and {Mo72Cr30}—the ligands’ residence times are eight orders of magnitude larger than those of the corresponding FeIII complexes; the related exchange reaction rate constants are 2.4 × 10−6 and 1.6 × 102 s−1 for [Cr(H2O)6]3+ and [Fe(H2O)6]3+, respectively (25)}. As a result, the interfacial water mobilities and heterogeneities of the macroions in the two superstructures differ markedly (Fig. 4), and the hydrogen bonding network for the {Mo72Cr30}-type blackberries is more dense and stable. [Dynamical and static heterogeneities and their relations seem also to play a role in supercooled water (26).] Experimental evidence supports the different mobilities of the interfacial water networks, because the transport of Ca2+ through the {Mo72Cr30} blackberry membranes (see Supporting Online Material

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and metal content are present, formation of the two different blackberries is accelerated (21, 22). The two Keplerate materials investigated have a very broad range of interest for materials science (7, 11), e.g., as molecular models for Kagomé lattices (28), as well as for quasi-crystals (29), and for geochemical processes regarding the modeling of H2O exchange at iron minerals’ surfaces (30). References and Notes

Fig. 3. Formal demonstration of the blackberry formation (see text).

Fig. 4. Schematic plot demonstrating the different interfacial water mobilities (dynamical heterogeneities) of the {Mo72Cr30} (yellow) and {Mo72Fe30} (blue) blackberry-type assemblies. (Middle) The thick circle layer (blue) around the {Mo72Cr30} clusters (top) illustrates stable, less mobile interfacial water compared to the {Mo72Fe30} scenario (bottom). The average intermacroion distance related to the interfacial water is ~0.9 T 0.4 nm (13).

with fig. S1) is slower than that through the {Mo72Fe30}-type system by a factor of 3 to 4 (27). Furthermore, the importance of hydrogen bonding for the superstructure formation is confirmed by the larger {Mo72Fe30}-type structures formed in H2O compared with those in D2O (25 nm versus 14 nm, figs. S2 and S3a) and the much slower process in D2O (fig. S3b). To rule out the possibility that the selfrecognition is primarily due to kinetic effects (i.e., that one type of Keplerates assembles faster), we analyzed the kinetics of the assembly processes by static light scattering. Our results showed that the two assembly processes occur at almost the same speed (fig. S3), especially in the initial stages (fig. S4). The kinetic curves obtained can approximately be explained by a two-step nucleation-growth process: (i) the macroanions slowly associate into thermodynamically unfavorable intermediate oligomers and dimers; (ii) when enough oligomers of appropriate shape

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1. J.-M. Lehn, Supramolecular Chemistry: Concepts and Perspectives (VCH, Weinheim, Germany, 1995). 2. R. Jelinek Ed., Cellular and Biomolecular Recognition: Synthetic and Non-Biological Molecules (Wiley-VCH Weinheim, Germany, 2009). 3. We refer to self-recognition here (in context with the present assembly process leading to phase separation) according to the definition: “Self-assembly may occur with self-recognition, mixtures of components yielding defined superstructures without interference or crossover” [(1), p. 142]. 4. A. Müller et al., Angew. Chem. Int. Ed. 38, 3238 (1999). 5. A. M. Todea et al., Angew. Chem. Int. Ed. 46, 6106 (2007). 6. D.-L. Long, L. Cronin, Chemistry 12, 3698 (2006). 7. A. Proust, R. Thouvenot, P. Gouzerh, Chem. Commun. (Camb.) 2008, 1837 (2008). 8. A. Müller, S. Roy, in The Chemistry of Nanomaterials: Synthesis, Properties and Applications, C. N. R. Rao, A. Müller, A. K. Cheetham, Eds. (Wiley-VCH, Weinheim, Germany, 2004), pp. 452–475. 9. N. Hall, Chem. Commun. (Camb.) 2003 (no. 7), 803 (2003) (Focus Article). 10. D.-L. Long, E. Burkholder, L. Cronin, Chem. Soc. Rev. 36, 105 (2007). 11. A. Müller, S. Roy, J. Mater. Chem. 15, 4673 (2005). 12. T. Liu, J. Am. Chem. Soc. 124, 10942 (2002). 13. T. Liu, J. Am. Chem. Soc. 126, 406 (2004). 14. T. Liu, Langmuir 26, 9202 (2010).

15. M. L. Kistler, T. Liu, P. Gouzerh, A. M. Todea, A. Müller, Dalton Trans. 26, 5094 (2009). 16. A. Müller et al., Chem. Commun. (Camb.) 19, 1928 (2001). 17. T. Liu, E. Diemann, H. Li, A. W. M. Dress, A. Müller, Nature 426, 59 (2003). 18. M. L. Kistler, A. Bhatt, G. Liu, D. Casa, T. Liu, J. Am. Chem. Soc. 129, 6453 (2007). 19. S. W. Provencher, Biophys. J. 16, 27 (1976). 20. Materials and methods are available as supporting material on Science Online. 21. J. Zhang, D. Li, G. Liu, K. J. Glover, T. Liu, J. Am. Chem. Soc. 131, 15152 (2009). 22. G. Liu, T. Liu, Langmuir 21, 2713 (2005). 23. E. Fratini, A. Faraone, A. M. Todea, P. Baglioni, Inorg. Chim. Acta 363, 4234 (2010). 24. A. Zlotnick, J. M. Johnson, P. W. Wingfield, S. J. Stahl, D. Endres, Biochemistry 38, 14644 (1999). 25. F.-C. Xu, H. R. Krouse, T. W. Swaddle, Inorg. Chem. 24, 267 (1985). 26. J. Holzmann, A. Appelhagen, R. Ludwig, Z. Phys. Chem. 223, 1001 (2009). 27. P. P. Mishra, J. Pigga, T. Liu, J. Am. Chem. Soc. 130, 1548 (2008). 28. I. Rousochatzakis, A. M. Läuchli, F. Mila, Phys. Rev. B 77, 094420 (2008). 29. A. Müller, Nat. Chem. 1, 13 (2009). 30. E. Balogh, A. M. Todea, A. Müller, W. H. Casey, Inorg. Chem. 46, 7087 (2007). 31. T.L. acknowledges support of this work by the National Science Foundation, Lehigh University, and the Alfred P. Sloan Foundation. A.M. acknowledges the continuous support of the Deutsche Forschungsgemeinschaft.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1590/DC1 Materials and Methods Figs. S1 to S5 References 1 December 2010; accepted 11 February 2011 10.1126/science.1201121

Widespread Persistent Thickening of the East Antarctic Ice Sheet by Freezing from the Base Robin E. Bell,1 Fausto Ferraccioli,2 Timothy T. Creyts,1 David Braaten,3 Hugh Corr,2 Indrani Das,1 Detlef Damaske,4 Nicholas Frearson,1 Thomas Jordan,2 Kathryn Rose,2 Michael Studinger,5 Michael Wolovick1 An International Polar Year aerogeophysical investigation of the high interior of East Antarctica reveals widespread freeze-on that drives substantial mass redistribution at the bottom of the ice sheet. Although the surface accumulation of snow remains the primary mechanism for ice sheet growth, beneath Dome A, 24% of the base by area is frozen-on ice. In some places, up to half of the ice thickness has been added from below. These ice packages result from the conductive cooling of water ponded near the Gamburtsev Subglacial Mountain ridges and the supercooling of water forced up steep valley walls. Persistent freeze-on thickens the ice column, alters basal ice rheology and fabric, and upwarps the overlying ice sheet, including the oldest atmospheric climate archive, and drives flow behavior not captured in present models.

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ice sheets. Radar images of ice sheets are characterized by isochronous internal layers associated with changes in the dielectric properties of

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the ice. In the center of ice sheets, these internal layers continue almost to the ice sheet bed (1). Away from the domes and ice divides, these layers disappear in the bottom 10 to 30% of the ice sheet. This homogeneous, echo-free basal layer can be hundreds of meters thick (2) and is considered to be the result of elevated basal temperatures, deformed ice, stagnant ice, or increased layer roughness (3). The absence of reflectors in the base of the ice sheet makes decoding basal processes difficult. In a few locations, near-bed reflectors have been resolved in the echo-free zone, specifically surrounding subglacial mountain peaks east of Dome Fuji (4) and over Lake Vostok (5). Over Lake Vostok, these reflectors are associated with frozen-on lake water sampled in the 5G 1 Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA. 2British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, UK. 3Center for the Remote Sensing of Ice Sheets, Kansas University, Lawrence, KS, USA. 4Bundesanstalt für Geowissenschaften und Rohstoffe, Hannover, Germany. 5Goddard Earth Science and Technology Center, University of Maryland Baltimore County, MD, and NASA Goddard Space Flight Center, MD, USA.

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

Borehole (6) and are attributed to changing fabric in the basal ice (6). During the International Polar Year 2007– 2009, the seven-nation Antarctica’s Gamburtsev Province (AGAP) expedition sought to comprehensively image the ice sheet bed, deep in the interior of Antarctica (7). This study used airborne and ground-based geophysical methods to understand the fundamental structure of Dome A, the top of the East Antarctic ice sheet and the underlying Gamburtsev Mountains (Fig. 1). The Gamburtsev Mountains were a major nucleation point for growth of the Antarctic ice sheet during the Cenozoic (8). Presently, ice at Dome A drains into all the major ice shelves of Antarctica. Processes occurring in the Dome A region have the potential to affect the majority of East Antarctica. Two Twin Otter aircraft were equipped with ice-penetrating radars, laser ranging systems, gravity meters, and magnetometers. The main survey grid included north-south lines spaced 5 km apart, with crossing lines every 33 km. 150-MHz ice-penetrating radars with bandwidths of 15 to 20 MHz produced highresolution images of the internal structure of the East Antarctic ice sheet.

Beneath Dome A, the internal layers extend close to the base of the ice sheet, whereas away from the dome, the bottom ~1000 m of the ice sheet becomes echo-free. Using improved radar technologies, we have imaged distinct near-bed reflectors within the generally echo-free zone. There appear to be two populations of near-bed reflectors, one found adjacent to the high ridges of the Gamburtsev Subglacial Mountains and one found along the steep valley walls. The first population of basal reflectors originates along the high ridges at the valley heads (Fig. 1B). Valley heads are the upstream terminus of the Gamburtsev Mountain valley network. Bright horizontal bed reflectors indicate that subglacial water collects at these valley heads. The basal reflectors separate from the bright, waterfilled ice sheet bed, similar to the divergence of the freeze-on reflector over Lake Vostok. The coherence of these reflectors over adjacent lines confirms that these near-bed reflectors are not artifacts of off-nadir reflections. The origin and lateral extent of these reflectors are best illustrated with radar data along ice flow (Fig. 2, A and B). The valley head basal reflectors can be traced for 50 to 100 km along flow lines (Fig. 1, A to C).

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Fig. 1. Distribution of frozen-on ice in the Dome A region. (A) Surface elevation with location of frozenon ice (orange, valley head; yellow, valley wall). Contours of 50 m show surface elevation from ICESat (Ice, Cloud, and land Elevation Satellite). A black triangle marks Dome A. (B) Valley head, strong reflector– bounded, freeze-on ice packages (orange) overlain on subglacial topography. (C) Valley wall freeze-on ice packages (yellow) overlain on subglacial topography with regions of supercooling (red dots). (Inset, top right) Location of survey (red bar), along with major Antarctic Ice Divides.

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trated diagrammatically in Fig. 3B. The valley head basal ice packages are consistently sourced from ponded subglacial water along the mountain ridges. Along these ridges, where the overlying ice sheet is thin, cold, and has steep thermal gradients, conductive cooling is the dominant process. The conductive cooling over ponded water may cause fractionation of the water chemistry during freeze-on and loss of the sediment load as the water ponds. Fractionation and sediment loss over ponded water are the probable source of the laminations in the valley head packages. Conduc-

tive cooling is probably the primary mechanism responsible for the freeze-on ice at the valley heads, whereas supercooling is likely to be a more rapid process when subglacial water actively moves up steep slopes. As compared to ice frozen-on from a conductively cooling subglacial water body, supercooled ice will have experienced less chemical fractionation or segregation of the suspended sediments. A supercooled package of ice is therefore likely to be a more homogenous basal package. Most of the valley wall units are associated with locations where the supercooling threshold

Fig. 2. Radar images of type examples of valley head (A and B) and valley wall freeze-on ice (C). (C) is in the valley beneath the Chinese Kunlun Station at Dome A. The location of these profiles is shown in Fig. 1. The base of the ice sheet (bed) is marked in red. The top of the frozen-on ice is indicated with yellow arrows.

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The near-bed reflector amplitude is stronger than the return from ice sheet internal layers but weaker than the return from the ice sheet bed. The valley head reflectors are often laminated. Downflow, these reflectors become fragmented and disappear into the background noise of the echo-free zone. We have identified 12 distinct packages of ice bounded by these bright basal reflectors (Fig. 1B). In the Dome A region, ~7% of the base of the ice sheet is characterized by packages of valley head ice. These valley head packages originate where the ice sheet is 2400 to 3000 m thick. The ice package widths range from 2 to 25 km, and the average thickness is ~490 m. The maximum package thickness is 1100 m, where the basal ice constitutes 50% of the ice sheet. The estimated frozen-on ice volume of the basal ice packages ranges from 45 to 1064 km3. The second population of basal reflectors is found primarily along the steep valley walls of the Gamburtsev Mountains. These reflectors define 200- to 500-m-thick packages that drape the downflow side of steep valley walls (Fig. 2C). The valley wall packages have a relatively weak upper reflector underlain by a uniform distribution of point reflectors with no distinct laminations. On the south side of Dome A, ~16% of the base of the ice sheet is characterized by these reflectors. The average thickness of the 24 packages of ice (Fig. 1B) defined by these diffuse reflectors is 350 m. The packages of ice can be traced for 15 to 30 km along the flow direction and have volumes of up to 160 km3. We interpret both populations of basal reflectors and the underlying packages of ice as the result of basal freeze-on. The volume of ice frozen on to the base of the ice sheet at the Gamburtsev Mountains’ valley heads is 8600 km3. The total volume of the valley wall freeze-on ice is ~3900 km3. We can use the 1.7 m/year surface velocity measured at the AGAP field camp at the southern margin of the study area and the along-flow length of these packages to estimate a minimum age for the process. Using this simple approach, the valley head freeze-on process has been persistent for a minimum of 30,000 to 60,000 years, whereas the valley wall freeze-on has been persistent for a minimum of 10,000 to 20,000 years. The process of basal freeze-on has continued through the glacial-interglacial transition. The processes are likely to have been persistent for substantially longer, but older freeze-on ice may not be easily detected by radar. In the southern Dome A region, 27,000 km2 of the 125,500 km2 area surveyed consists of freeze-on ice. A minimum of 24% of the ice sheet base is affected by the freeze-on process. The two populations of basal ice are distinct in the strength of the upper reflector, the structure of the underlying package of ice, and their geographic distribution. There are two possible freeze-on mechanisms (9): conductive cooling or glaciohydraulic supercooling (10). We suggest that the two populations of basal reflectors reflect the two freeze-on mechanisms. These processes are illus-

Fig. 3. Freeze-on processes. (A) Radar image. (B) Schematic of freezeon processes. The location of this profile is shown in Fig. 1.

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has been exceeded (Fig. 1C). We suggest that the valley wall packages are the result of supercooling of subglacial water moving over rough basal topography. The freeze-on at the valley heads is primarily the result of conductive cooling over static bodies of water, whereas the valley wall ice is primarily the result of the hydrologic potential forcing water up steep valley slopes. Because these processes are not mutually exclusive, individual packages of frozen-on ice are likely to have been produced by a combination of the two mechanisms. Alternative mechanisms for producing the contrasting radar signatures found within the frozen-on ice include spatial and temporal changes in basal water availability or chemistry. The addition of hundreds of meters of ice to the base of an ice sheet deforms the overlying ice upward. This upwarping modifies the ice sheet stratigraphy and may affect the surface accumulation by changing the surface slope. The thickest package of frozen-on ice (1110 m) is situated at

the downflow end of a 20-km-long valley floored by a bright horizontal reflector. The internal layers are deformed upward over 410 m at the valley head, conforming to the shape of the accreted ice body and not the underlying topography (Fig. 4). The accretion sites in the Dome A region are typically coincident with 5- to 35-m mounds in the ice surface, indicating linkage between the basal processes and the ice surface morphology. The thick packages of freeze-on ice surrounding Dome A illustrate that basal freeze-on modifies the fundamental structure of ice sheets, thickening the ice column from the base. The freeze-on rates in the Dome A region may be locally greater than the surface accumulation rates. The upwarping of internal layers over accretion sites implies active interaction between basal accretion and the entire ice sheet. The accretioninduced upwarping of basal ice will move old ice to a higher elevation in the ice sheet, increasing the potential of preserving very old ice. Alterna-

Fig. 4. AGAP radar and lidar data over the freezeon ice package (Fig. 1). (A) is the airborne scanning laser profile over the freeze-on ice. The ice surface along this profile is 5 m above the regional slope, whereas on the two upstream lines, a 10to 15-m mound in the ice surface is coincident with the sites of freeze-on. The radar profile (B) illustrates the upward deflection of the internal layers over the accretion site. The accretion plume is 1100 m thick along this profile, and the internal layers are deflected upward 400 m. (C) illustrates the upward deflection of the internal layers. The location of the profile is shown in Fig. 1.

tively, the widespread melt required to support the freeze-on process may have destroyed the ice containing the ancient paleoclimate records. Without the inclusion of basal processes, simple models of ice sheet temperatures cannot accurately predict the location of the oldest ice (11). In East Antarctica, basal freeze-on has continued in the same locations through the last glacial-interglacial transition and has probably been a persistent process since East Antarctica became encased in a large ice sheet 32 million years ago. The simple geometry of the subglacial topography and the stable ice flow in the Dome A region have enabled us to image this process for the first time. Although the surface accumulation, surface slope, and bed morphology vary distinctly on the northern and southern sides of Dome A, throughout the area almost a quarter of the ice sheet base consists of ice freeze-on from the bottom. Widespread freeze-on can change the rheology and modify the flow of the Antarctic and Greenland ice sheets (12, 13). Inclusion of these basal processes is essential to produce robust predictions of future ice sheet change.

References and Notes 1. G. Q. Robin, C. W. M. Swithinbank, B. M. E. Smith, Radio Echo Exploration of the Antarctic Ice Sheet (International Association of Scientific Hydrology Publication 86, 1970), pp. 97–115. 2. G. De Q. Robin, D. J. Drewry, D. T. Meldrum, Philos. Trans. R. Soc. London Ser. B 279, 185 (1977). 3. R. Drews et al., Cryosphere 3, 195 (2009). 4. S. Fujita et al., J. Geophys. Res. 104, 13013 (1999). 5. R. E. Bell et al., Nature 416, 307 (2002). 6. J. A. MacGregor, K. Matsuoka, M. Studinger, Earth Planet. Sci. Lett. 282, 222 (2009). 7. R. E. S. Bell et al., paper presented at the 2009 Full Meeting of the American Geophysical Union, San Francisco, CA, 14 to 18 December 2009. 8. R. M. DeConto, D. Pollard, Nature 421, 245 (2003). 9. T. T. Creyts et al., paper presented at the 2010 Fall Meeting of the American Geophysical Union, San Francisco, CA, 13 to 17 December 2010. 10. R. B. Alley, D. E. Lawson, E. B. Evenson, J. C. Srasser, G. J. Larson, J. Glaciol. 44, 563 (1998). 11. F. Pattyn, Earth Planet. Sci. Lett. 295, 451 (2010). 12. F. Heliere, L. Chung-Chi, H. Corr, D. Vaughan, IEEE Trans. Geosci. Remote Sensing 45, 2573 (2007). 13. D. E. Sugden et al., Nature 328, 238 (1987). 14. We acknowledge the seven nations involved in the AGAP International Polar Year effort for their logistical, financial, and intellectual support. Specifically, the U.S. Antarctic Program of NSF provided support for the logistics, the development of the instrumentation, and the analysis of the data. The Natural Environment Research Council/British Antarctic Survey provided extensive support for deep-field logistics, data collection, and analysis. The Federal Institute for Geosciences and Resources, Germany, and the Polar Research Institute of China provided invaluable support to the program. C. Finn, Gwenn Flowers, M. Fahnestock, and Slawek Tulaczyk provided early reviews of the work. H. Abdi provided technical support in developing the figures.

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8 November 2010; accepted 18 February 2011 Published online 3 March 2011; 10.1126/science.1200109

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Shanti Pappu,1* Yanni Gunnell,2 Kumar Akhilesh,1 Régis Braucher,3 Maurice Taieb,3 François Demory,3 Nicolas Thouveny3 South Asia is rich in Lower Paleolithic Acheulian sites. These have been attributed to the Middle Pleistocene on the basis of a small number of dates, with a few older but disputed age estimates. Here, we report new ages from the excavated site of Attirampakkam, where paleomagnetic measurements and direct 26Al/10Be burial dating of stone artifacts now position the earliest Acheulian levels as no younger than 1.07 million years ago (Ma), with a pooled average age of 1.51 T 0.07 Ma. These results reveal that, during the Early Pleistocene, India was already occupied by hominins fully conversant with an Acheulian technology including handaxes and cleavers among other artifacts. This implies that a spread of bifacial technologies across Asia occurred earlier than previously accepted. he Acheulian is a phase of the Lower Paleolithic typified by assemblages of large cutting tools primarily composed of bifaces. So far, evidence from Africa suggests that it emerged around 1.6 million years ago (Ma). Determining when hominin populations routinely crafting these Acheulian stone tools inhabited

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India is critical for understanding the dispersal of this distinctive technology across Eurasia. Limited evidence has suggested that Acheulian hominins appeared in India substantially later than in Africa or southwest Asia (1–5). Here, we present age estimates obtained from excavations at Attirampakkam (13°13′50″N,

79°53′20″E, 38.35 meters above sea level), an open-air Paleolithic site situated near a meandering tributary stream of the river Kortallaiyar, northwest of Chennai, in southeast India (Fig. 1) (6–9). Attirampakkam was discovered in 1863 by Robert Bruce Foote and is one among a cluster of sites constituting the southernmost extension of the South Asian Acheulian (10). Extensive excavations since 1999 have exposed a sequence of stratified deposits reaching a maximum thickness of ~9 m (fig. S1). In all of the trenches, Acheulian assemblages were encountered continuously within deposits (layers 6 to 8, Fig. 1) derived from eroding Cretaceous shale and sandstone outcrops in the catchment. These floodplain sediments aggraded during occupation, leading to 1 Sharma Centre for Heritage Education, 28, I Main Road, C.I.T. Colony, Mylapore, Chennai 600004, Tamil Nadu, India. 2 Department of Geography, Université Lumière-Lyon 2, CNRSUMR 5600, 5 Avenue. P. Mendès-France, 69676 Bron cedex, France. 3Centre Européen de Recherche et d’Enseignement en Géosciences de l’Environnement (CEREGE), Europôle de l’Arbois, BP 80, 13545 Aix-en-Provence Cedex 04, France.

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

Fig. 1. Location of the Paleolithic site of Attirampakkam (ATM), Tamil Nadu, India. (A) Regional topographic setting, showing the extent of the Kortallaiyar river catchment and major cities. The Allikulli (A) and Satyavedu (S) Hills consist of massive deposits of quartzite cobble beds (i.e., source materials of crucial importance to hominins). Relief in the Precambrian Nagari Hills is formed by resistant quartzite ridges, which themselves supplied the Allikulli and Satyavedu conglomerate beds during the Cretaceous. Map projection: Transverse Mercator. (B) View of the west wall of trench T8 (sampled for paleomagnetic measurements) showing numbered layers 5 to 8, as mentioned in the text. (C) View of step trench GT-01 and trench T8 in the process of excavation. (D) Close-up view of layer 7 in trench T8, showing an in situ biface (bar scale gradations in units of 1 cm). The arrow indicates the magnetic north.

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Early Pleistocene Presence of Acheulian Hominins in South India

repeated burial of artifacts discarded at the site. Alternating sand and silty clay beds lacking paleosols suggest periodic cycles of sediment deposition without lengthy interruptions (6). The suspended silt particles settled out under conditions of low-velocity laminar overbank streamflow, burying the stone tools without displacing them. A disconformable upward sequence of coarse lateritic gravels, clay-rich silts, and finer

lateritic gravels (Fig. 1) overlies layer 6 and contains later Acheulian–to–late Middle Paleolithic assemblages. Such a complete stratified sequence emphasizes the long-term attractiveness of this site (6–9). We obtained 3528 Acheulian artifacts from trench T8, excavated specifically to investigate the deeper layers. The tools were crafted primarily on fine- to coarse-grained quartzite, a source

Fig. 2. Acheulian artifacts in trench T8. Close-ups of artifacts in layer 6 (A) and in layer 7 (B). Artifacts include cleavers (C and D), large flake tool with a cleaverlike working edge (E), handaxes (F and G), trihedral (H), large flake (I), and Kombewa flake (J). Bar scale gradations as in Fig. 1. Table 1. Cosmogenic nuclide concentrations and Acheulian artifact burial ages.

Samples

T8 6074 T8 8824 T7A 6877 T3 B1-14 T3 B1-197 T3 B1-337

Denudation Minimum Denudation Maximum 10 26 before and Be† Al† Depth* burial age‡ before burial§ burial age‡ after burial (cm) (106 at·g–1) (106 at·g–1) –1 (m·My ) (Ma) (Ma) (m·My–1) 755 950 487 532 642 855

0.508 T 0.03 1.131 0.543 T 0.017 1.29 0.436 T 0.02 1.032 0.514 T 0.041 1.493 0.76 T 0.062 2.082 0.429 T 0.034 1.236

T T T T T T

0.254 1.89 0.123 1.75 0.103 1.81 0.072 1.38 0.098 1.39 0.051 1.43

T T T T T T

0.44 0.18 0.2 0.13 0.13 0.13

1.44 1.45 1.87 1.99 1.17 2.44

T T T T T T

0.34 0.15 0.21 0.19 0.11 0.23

2.16 1.93 2.22 1.57 1.52 1.59

T T T T T T

0.5 1.39 0.19 1.43 0.24 1.78 0.15 2.03 0.14 1.17 0.14 2.47

T T T T T T

0.32 0.15 0.2 0.2 0.11 0.23

*All depths obtained from individual trench datums were standardized by reference to a common stratigraphic datum coinciding with the highest point at the site. Density of materials is 2.2 g·cm–3. †Measurement uncertainties are restricted here to analytical uncertainties within 1 SD. ‡Minimum and maximum ages are calculated following (11). The minimum burial age calculations are based on the stratigraphic and geomorphic evidence that the samples were deeply buried in the past but were recently brought nearer the surface by erosion of the topsoil. Maximum burial ages account for postburial production of 10Be and 26Al by muons. In the latter case, denudation is considered constant before and after burial. Burial-age uncertainties (T1s) include systematic errors in half-lives. Spallation productions are 2.88 and 19.03 at·g–1·year–1 for 10Be and 26Al, respectively. Likewise, slow and fast muon contributions are §Calculated background 0.07 (10Be) and 0.46 (26Al) at·g–1·year–1 and 0.03 (10Be) and 0.20 (26Al) at·g–1·year–1, respectively. denudation rates are maximum rates. The low values obtained are consistent with the low-elevation, low-relief topographic setting close to oceanic base level. Further, the fact that 10Be concentrations among artifacts are statistically similar (see SOM text) suggests that the clasts share similar preburial exposure histories, implying that hominins exploited surface scatters of raw material clasts.

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material widely available as cobble and boulder deposits in the near hinterland. Artifacts include retouched and trimmed large cutting tools (>10 cm) including handaxes, cleavers, trihedrals, unifaces, and other retouched/trimmed large flakes, as well as smaller flake tools with only a few artifacts on cobbles (Fig. 2) (9). Large flakes were minimally retouched, generally retaining a small proportion of cortex. Among the bifaces, handaxes predominate and are mainly on end- or obliquely struck large flakes displaying variability in flaking techniques and shapes, with elongate and ovate shapes predominating. Cleavers (parallel-sided, divergent, and convergent) remain scarce, are on flakes, and range from minimally shaped “cleaverflakes” to reduced cleavers. Cores for detachment of large flakes are absent, implying that Acheulian hominins were transporting to Attirampakkam (i) large flakes and (ii) partly to fully shaped tools from surficial quartzite cobble beds used as quarrying sites noted elsewhere in the region. Further shaping and reduction were carried out at Attirampakkam, as indicated by waste flakes that include biface thinning flakes [see supporting online material (SOM) text]. Because they are quartzite, the artifacts were amenable to cosmic-ray exposure dating. This technique is based on the accumulation in quartz exposed at Earth’s surface of rare nuclides produced by neutrons and muons through nuclear reactions induced by high-energy cosmic radiation. Sediment depositional histories can be revealed by using pairs of radioactive cosmogenic nuclides and exploiting their respective half-lives (11). Here, we use 10Be [T1/2 = 1.387 T 0.012 million years (My)] and 26Al (T1/2 = 0.717 T 0.017 My) to date the burial of six quartzite artifacts from layers 6 and 8 (fig. S1). During exposure at the surface, 26Al/10Be ratios vary between ~3.5 and ~7.1, depending on exposure time and local denudation. Given that, before artifact production and burial at the site, hominins initially collected source materials with similar preburial surface exposure histories from the surrounding landscape, the measurement of 26Al/10Be concentration ratios within artifacts will determine their burial age. This approach can be applied to artifacts from Pleistocene archeological sites that were rapidly buried to depths exceeding 5 to 10 m (12) or to older samples in cave sites where production is instantaneously halted by complete shielding from cosmic radiation (13). At Attirampakkam, however, comparatively shallow burial (Table 1) may have failed to interrupt production entirely. The measured nuclide concentrations, therefore, are the sum of the inherited nuclides at the time of deposition, corrected for radioactive decay, and of the concentration produced at a constant depth since burial (11). Depending on the model used for depth-dependent nuclide production by muons, results provide age brackets ranging from a minimum burial-age estimate with a weighted sample mean of 1.51 T 0.07 Ma (see SOM text) to a maximum burial-age estimate with a weighted mean of 1.68 T 0.07 Ma

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(Fig. 3 and Table 1). Given that physical knowledge of the muonic contribution to in situ nuclide production is currently debated (table S1)— and also that Acheulian sites in Africa, southwest Asia, and Pakistan are around or younger than 1.6 Ma (1, 12, 14–17)—we consider the more conservative minimum burial ages (Fig. 3) to be more likely. These minimum ages are corroborated by results from a continuous paleomagnetic profile involving 49 samples collected down the 9-m stratigraphic section of trench T8. Stereographic projections (fig. S2) of the natural remanent magnetization and its stepwise alternating field and thermal demagnetization reveal two components: (i) a relatively stable upward vertical component (negative inclinations), suggesting that the primary magnetization was acquired in a reverse polarity field, but also (ii) highly unstable horizontal components starting from the northern quadrant, thus compatible with a normal polarity declination, then turning during treatments to the western quadrant. This results in a very coherent and systematic distribution of magnetizations along great circles ending in the southern quadrant. This north-south shift observed for most of the measured samples is interpreted to result from an overprint of the reverse polarity primary magnetization, itself of depositional origin, by a secondary magnetization that was acquired in a normal polarity field. This overprint is likely to be a chemical remanent magnetization, its chemical origin being supported by sample resistance to both alternating field and thermal treatments as

well as by the deeply weathered state of the sediment. Such an interpretation suggests that the sediment sequence was deposited before the normal Brunhes chron; that is, before 0.78 Ma (18). Given the cosmogenic burial ages and the nature of the Acheulian assemblage (fig. S3 and S4 and table S2), we correlate the reverse polarity with the Matuyama chron and place it between the base of the Jaramillo (1.07 Ma) and top of the Olduvai (1.77 Ma) normal subchrons, neither of which are detected (Fig. 3). Considered together, the cosmogenic and paleomagnetic results indicate that Acheulian hominins were present in south India before 1.07 Ma. These ages are contemporary with some other Lower Pleistocene Acheulian sites in Africa and southwest Asia. The earliest known dates for the Acheulian (~1.6 to 1.4 Ma) are from East Africa (14, 15). Early Acheulian sites in South Africa have also yielded an age of ~1.6 Ma (12), suggesting rapid and widespread dispersal of this technology across Africa. Closer to India, the age of the Acheulian at ´Ubeidiya (Israel) is estimated at ~1.4 Ma (16), and the sequence at Gesher Benot Ya’aqov was formed between 0.7 and 0.8 Ma (17). In the Bose basin, China, Acheulianlike bifaces date back to ~0.8 Ma (19). In South Asia, there is at present little unequivocal evidence for a pre-Acheulian Early Pleistocene occupation, barring ages of ~2 Ma attributed to artifacts from Riwat (20) and of 2.2 to 0.9 Ma from the Pabbi Hills, Pakistan (21). Estimated ages for the Acheulian near Potwar, Pakistan, are 0.4 to 0.7 Ma (22). Sparse radiometric ages from

Fig. 3. Age constraints on artifact burial stratigraphy at Attirampakkam. Samples are from three separate trenches (T3, T8, and T7A) with a common reference datum representing the top of the sedimentary sequence, which is preserved near the site but was partly eroded at the site because of land-use practices and other factors (Fig. 1; see SOM). Error bars are T2 SD; that is, there is only a 5% chance that the true age falls outside that range. Note that the Cobb Mt. subchron would have been undetectable at the sampling resolution applied.

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sites in India have situated the Acheulian within the Middle Pleistocene, with a few dates suggesting an early Middle to Early Pleistocene age. However, these ages often exceed the limits of confidence of the methods used (2). They include an electron spin resonance (ESR) mean age of 1.27 T 0.17 Ma, assuming linear U uptake, on two herbivore teeth from Isampur (23); an ESR age of ~0.8 Ma (lacking uncertainty envelopes) on calcrete from the Amarpura formation, Rajasthan (24), which has been correlated with the Acheulian site of Singi Talav (4); dates ranging from ~1.4 to 0.67 Ma for the tephra at Bori (Kukdi river) (25); and paleomagnetic measurements with evidence of reversals at the sites of Bori, Morgaon, Gandhigram, Andora, and Nevasa (26). However, the reliability of these ages has, in each case, been questioned on various grounds (5, 27, 28). Likewise, the age and stratigraphic position of artifacts and faunal remains from the Early Pleistocene Dhansi formation along the river Narmada are yet to be firmly established (29). Based on data from controlled excavations and two independent dating methods, our ages from Attirampakkam show that the Acheulian in India is older than previously thought. Evidence from other sites in South Asia should be reconsidered and redated. References and Notes 1. R. Dennell, The Palaeolithic Settlement of Asia (Cambridge Univ. Press, Cambridge, 2009). 2. S. Mishra, Curr. Anthropol. 33, 325 (1992). 3. M. D. Petraglia, B. Allchin, in The Evolution and History of Human Populations in South Asia, M. D. Petraglia, B. Allchin, Eds. (Springer, New York, 2007), pp. 1–20. 4. C. Gaillard, S. Mishra, M. Singh, S. Deo, R. Abbas, Quat. Int. 223–224, 234 (2010). 5. P. R. Chauhan, Quat. Int. 223–224, 248 (2010). 6. Y. Gunnell, C. Rajshekhar, S. Pappu, M. Taieb, A. Kumar, Curr. Sci. 91, 114 (2006). 7. S. Pappu, Y. Gunnell, M. Taieb, J.-P. Brugal, Y. Touchard, Curr. Anthropol. 44, 591 (2003). 8. S. Pappu, Y. Gunnell, M. Taieb, A. Kumar, Man and Environment 29, 1 (2004); www.sharmaheritage.com. 9. S. Pappu, A. Kumar, in Axe age. Acheulian Tool-Making from Quarry to Discard, N. Goren-Inbar, G. Sharon, Eds. (Equinox, London, 2006), pp. 155–180. 10. S. Pappu, A Re-Examination of the Palaeolithic Archaeological Record of Northern Tamil Nadu, South India [British Archaological Reports (BAR) International Series 1003, John and Erica Hedges, Oxford, 2001]. 11. D. E. Granger, P. F. Muzikar, Earth Planet. Sci. Lett. 188, 269 (2001). 12. R. J. Gibbon, D. E. Granger, K. Kuman, T. C. Partridge, J. Hum. Evol. 56, 152 (2009). 13. G. Shen, X. Gao, B. Gao, D. E. Granger, Nature 458, 198 (2009). 14. B. Asfaw et al., Nature 360, 732 (1992). 15. I. de la Torre, R. Mora, J. Martínez-Moreno, J. Anthropol. Archaeol. 27, 244 (2008). 16. O. Bar-Yosef, N. Goren-Inbar, The Lithic Assemblages of ‘Ubeidya: A Lower Palaeolithic Site in the Jordan Valley (Hebrew Univ. of Jerusalem, Jerusalem, 1993). 17. N. Goren-Inbar et al., Science 289, 944 (2000). 18. S. C. Cande, D. V. Kent, J. Geophys. Res. 100, 6093 (1995). 19. H. Yamei et al., Science 287, 1622 (2000). 20. R. W. Dennell, H. M. Rendell, E. Hailwood, Curr. Anthropol. 29, 495 (1988). 21. R. W. Dennell, Early Hominin Landscapes in Northern Pakistan: Investigations in the Pabbi Hills (BAR International Series 1265, Archaeopress, Oxford, 2004). 22. H. Rendell, R. W. Dennell, Curr. Anthropol. 26, 393 (1985). 23. K. Paddayya et al., Curr. Sci. 83, 641 (2002).

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REPORTS Remote Sensing Centre, Bengaluru, (Geosphere Biosphere Program). We thank Uday Raj, S. Ravindranath, C. B. S. Dutt, P. G. Diwakar, and R. Ramesh. We also thank the Archaeological Survey of India, Delhi and Chennai Circle, and the Department of Archaeology, State Government of Tamil Nadu, for licenses to excavate and support; the Institut Universitaire de France for field and analytical costs; M. Arnold and G. Aumaître for assistance with 10Be and 26Al measurements; and the ASTER (Accélérateur pour les Sciences de la Terre, Environnement, Risques) national facility (CEREGE, Aix-en-Provence), which is supported by the Institut National des Sciences de l‘Univers/CNRS, the French

The Buttermilk Creek Complex and the Origins of Clovis at the Debra L. Friedkin Site, Texas Michael R. Waters,1* Steven L. Forman,2 Thomas A. Jennings,3 Lee C. Nordt,4 Steven G. Driese,4 Joshua M. Feinberg,5 Joshua L. Keene,3 Jessi Halligan,3 Anna Lindquist,5 James Pierson,2 Charles T. Hallmark,6 Michael B. Collins,7 James E. Wiederhold3 Compelling archaeological evidence of an occupation older than Clovis (~12.8 to 13.1 thousand years ago) in North America is present at only a few sites, and the stone tool assemblages from these sites are small and varied. The Debra L. Friedkin site, Texas, contains an assemblage of 15,528 artifacts that define the Buttermilk Creek Complex, which stratigraphically underlies a Clovis assemblage and dates between ~13.2 and 15.5 thousand years ago. The Buttermilk Creek Complex confirms the emerging view that people occupied the Americas before Clovis and provides a large artifact assemblage to explore Clovis origins. early 80 years ago, Clovis was identified as the oldest archaeological horizon in North America [~12.8 to 13.1 thousand years ago (ka)]. Decades of subsequent research

N

have advanced our understanding of Clovis chronology, adaptations, and technological organization (1–3). Whereas genetic studies indicate that the first Americans hailed from northeast

Ministry of Research, the Institut de Recherche pour le Développement, and the Commissariat à l‘Énergie Atomique. All three reviewers contributed valuable comments.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1596/DC1 SOM Text Figs. S1 to S4 Tables S1 and S2 References 8 November 2010; accepted 2 February 2011 10.1126/science.1200183

Asia (1), no fluted Clovis points or other diagnostic characteristics of Clovis have been identified there (4). Additionally, fluted points in Alaska are rare, are technologically different, and postdate Clovis (5, 6). These lines of evidence suggest that, although the ultimate ancestors of Clovis originated from northeast Asia (1), important technological developments, including the invention 1 Center for the Study of the First Americans, Departments of Anthropology and Geography, Texas A&M University, 4352 TAMU, College Station, TX 77843–4352, USA. 2Luminescence Dating Research Laboratory, Department of Earth and Environmental Sciences, 845 West Taylor Street (m/c 186),University of Illinois, Chicago, IL 60607–7059, USA. 3Center for the Study of the First Americans, Department of Anthropology, Texas A&M University, 4352 TAMU, College Station, TX 77843– 4352, USA. 4Department of Geology, Baylor University, One Bear Place no. 97354, Waco, TX 76798–7354, USA. 5Department of Geology and Geophysics, Institute for Rock Magnetism, University of Minnesota, Minneapolis, MN 55455–0219, USA. 6 Department of Soil and Crop Science, 2474 TAMU, Texas A&M University, College Station, TX 77843–2474, USA. 7Department of Anthropology, Texas State University, 232 Evans Liberal Arts, San Marcos, TX 78666, USA.

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

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24. A. J. Kailath et al., Radiat. Meas. 32, 371 (2000). 25. S. Mishra, T. R. Venkatesan, S. N. Rajaguru, B. L. K. Somayajulu, Curr. Anthropol. 36, 847 (1995). 26. S. J. Sangode, S. Mishra, S. Naik, S. Deo, Gondwana Geol. Mag. Spec. Vol. 10 (2007), p. 111. 27. S. K. Acharyya, Curr. Sci. 84, 127 (2003). 28. J. A. Westgate et al., Quat. Res. 50, 107 (1998). 29. R. Patnaik et al., J. Hum. Evol. 56, 114 (2009). 30. Excavations at Attirampakkam were funded by the Homi Bhabha Fellowships Council, the Leakey Foundation, the Earthwatch Institute, and the Sharma Centre for Heritage Education. Ongoing studies are being funded by the Indian Space Research Organisation, Regional

Fig. 1. Geomorphic surfaces and excavation areas and trenches (black rectangles and squares) at the Friedkin site. www.sciencemag.org

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REPORTS Buttermilk Creek with some sediment derived from the adjacent slope. Stratification that may have existed within the floodplain deposit has been obscured by pedogenesis (A-Bss horizons). To date the floodplain clays, we obtained 49 optically stimulated luminescence (OSL) ages from two columns (Fig. 2, A and B, and table S2) (7). OSL ages were determined on the quartzdominated signal of the 4- to 11-mm fraction extracted from these deposits by using multiplealiquot (8) and single-aliquot protocols (9). Ages are concordant by these different analytical

Buttermilk Creek (Fig. 1) in a small valley incised into chert-bearing Edwards Limestone about 250 m downstream of the Gault Clovis site (1, 2). We excavated two areas at the Friedkin site: block A on terrace 2 (T-2) and block B [supporting online material (SOM) text] on terrace 1 (T-1). The evidence for pre-Clovis lies in block A. At the base of block A, limestone bedrock is overlain by colluvium (Fig. 2 and fig. S3, 2Bk horizon). Overlying the colluvium is 1.4 m of clay that was deposited incrementally in a floodplain environment by overbank flooding from

of the Clovis fluted point, took place south of the North American continental ice sheets before 13.1 ka from an ancestral pre-Clovis tool assemblage. Over the past few decades, some credible evidence for pre-Clovis occupation of the Americas has emerged, but this evidence is not robust (1). Here, we describe a large assemblage of preClovis artifacts from the Debra L. Friedkin site (41BL1239) in central Texas and address both the pre-Clovis colonization of North America and Clovis origins. The site is located along

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of the alluvial Vertisol (A-Bss) overlying colluvium (2Bk). (D) Photomicrographs illustrating in situ chert microdebitage (CH) larger than the adjacent voids (V) and vertical crack infill (CI) in the Buttermilk Creek Complex layer of the Bss5 horizon. SCIENCE

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hematite and/or goethite at depths >40 cm. This trend of increasing oxidation with depth is indicative of undisturbed, nonsaturated, modern soils. Measurements of natural remnant magnetization are all of normal polarity, and magnetic inclinations are consistent with the latitude of the field site. Thus, on the basis of multiple lines of evidence—pedologic, magnetic, chronologic ordering of dates and diagnostic artifacts, artifact size distribution, and distribution of artifacts with and without calcium carbonate accumulations— we conclude that the artifacts from block A lie in undisturbed contexts and have not worked downward or displaced upward by soil-forming processes. The Buttermilk Creek Complex assemblage consists of 15,528 lithic artifacts—tools (n = 56), macrodebitage (n = 2268), and microdebitage (n = 13,204). The 56 tools include 12 bifaces, 1 discoidal core, 23 edge-modified flake tools, 5 blade fragments, 14 bladelets, and 1 piece of polished hematite (Fig. 4) (SOM text). All flaked stone tools and debitage are Edwards chert. Buttermilk Creek Complex biface technology (Fig. 4) includes three trajectories—preform, chopper/adze, and discoidal core production. Ten of the 12 bifaces are late-stage fragments, and one with a lanceolate-like shape may be a point preform fragment. Another biface is large and thick and may be a chopper or adze. The discoidal flake core has removals from both faces and from all directions. A core rejuvenation flake from a similarly sized discoidal core was also recovered. The assemblage also includes blade technology. Five blade and 14 bladelet fragments were recovered; all have two or three prior blade removal scars. Microscopic analyses indicate that some of the blades and bladelets were used (figs. S12 and S13). No blade or bladelet cores were found, but two possible bladelet core fragments were recovered. Angostura

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pedologic studies show this is not true and that Vertisols are minimally mixed (13). At the Friedkin site, the vertic features of the soil are weakly expressed. Horizonation is preserved within the soil with the amount of clay, organic carbon, and calcium carbonate, as well as color, soil structure, and pH varying systematically with depth (Fig. 2C). Crack infills constitute a minor portion of the soil volume with diameters rapidly decreasing from 2 cm near the surface to <1 cm at a depth of 1 m. Soil material between the crack infills is intact with few rodent burrows and limited root penetration (<2 mm diameter). Only a few cracks reach the Clovis and Buttermilk Creek Complex levels, and crack apertures are <1 mm diameter, smaller than the microdebitage (sizes 6 to 7, table S15) from these levels (Fig. 2D). There is no sorting of artifacts by size from the surface through the Buttermilk Creek Complex layers (fig. S11). Refitted artifacts occur in the Paleoindian and Buttermilk Creek Complex levels (table S16). The number of artifacts with pedogenic calcium carbonate adhering to them (sizes 1 to 5, table S15) increases with depth. No calcium carbonate accumulated on artifacts in the Late Prehistoric and Archaic levels, about 40 to 60% of the artifacts from the Paleoindian levels are coated with CaCO3, and about 90% of the artifacts in the Buttermilk Creek Complex levels have CaCO3 coatings. The percentage of calcium carbonate coatings on artifacts parallels pedogenic CaCO3 occurrence and concentration within the soil horizons. Measurements of mass-normalized magnetic susceptibility show a smooth decrease through the floodplain clays with depth, typical of uninterrupted soil development (14, 15) (fig. S7). Further, the magnetic mineralogy gradually changes from an assemblage dominated by magnetite in the upper 40 cm to an assemblage dominated by

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Fig. 3. OSL ages (1s) from columns 1 and 2 in block A with the positions of diagnostic artifacts and archaeological complexes indicated.

Elevation (m above datum)

approaches (7). The multiple-aliquot analyses, which evaluate equivalent doses for fast, medium, and slow luminescence components (10), yielded a uniform response indicating full solar resetting. Single-aliquot analyses show a normal unimodal distribution also indicative of full solar resetting. All ages are in chronological order, and the two columns of dates correlate in time and depth (Fig. 3). Late Prehistoric, Late Archaic, Early Archaic, Paleoindian (Golondrina and Dalton), Folsom, and Clovis horizons occur in the floodplain clays (figs. S8 and S9). Time-diagnostic artifacts recovered from these horizons are in correct stratigraphic order and correlate with corresponding OSL ages (Fig. 3). The Folsom horizon is identified within a 2.5-cm-thick layer with three Folsom points. The Clovis horizon is defined within a 2.5-cm-thick layer in which Clovis diagnostics were found, including three bifaces with overshot flake removal scars, three channel flakes, and five blade segments. Below the Clovis horizon is a 20-cm-thick layer containing artifacts that represent repeated visits to the site and together define the Buttermilk Creek Complex. Eighteen OSL ages, ranging from ~14 to 17.5 ka, were obtained from this layer, and all but three overlap at one standard deviation (Fig. 2, A and B, and table S2). The most conservative estimate of the age of the Buttermilk Creek Complex is ~13.2 to 15.5 ka, on the basis of the minimum age represented by each of the 18 OSL ages. The high clay content and pedogenic characteristics—including slickensides, surface cracking, and evidence of subsurface microlow and microhigh topography—indicate that the floodplain deposit containing the Buttermilk Creek Complex artifacts is a Vertisol (11) (SOM text). Although Vertisols were once thought to be well mixed by argilliturbation processes (12), more recent

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Twenty-three edge-modified tools were made on flake fragments and include 17 straight-toconvex edged tools, 4 notches, 1 graver, and 1 bifacially worked tool. These tools are generally small, with most (n = 21) falling into debitage size classes 2 through 4 (table S15). The assemblage also includes bend and radially broken artifacts. One biface has radial breaks on three of the four sides, appears to have been intentionally broken, and has evidence of use wear on the radial edges (fig. S13). The graver and one of the retouched flakes were resharpened along a fractured edge, demonstrating that bend and radially broken flakes also served as unifacial tool blanks. Of the macrodebitage with platforms (n = 843), 47% are normal and 51% are biface thinning

flakes, including 1 distal overshot fragment, 3 partial overshots, and 10 end-thinning flakes (SOM text). In addition, we found a polished piece of hematite that is multifaceted with three primary worked surfaces. In general, the Buttermilk Creek Complex tools and cores are small in size and lightweight, a tool kit designed for high residential mobility. Although no organic artifacts were preserved, the Buttermilk Creek Complex stone tools have wear that is indicative of use on both soft and hard materials (SOM text), suggesting that organic materials were also part of this assemblage. The Debra L. Friedkin site provides empirical evidence that people were in North America by 15.5 ka, as suggested by genetic models (1, 16).

The Buttermilk Creek Complex tool kit—bifaces made through core reduction including end thinning, a lanceolate-like preform, a discoidal core, blades, bladelets, radially broken tools, a variety of edge-modified tools (notches, gravers, and scrapers), and ground hematite—also provides an ancestral assemblage from which the biface- and blade-dominated Clovis tool kit (1) could have evolved (SOM text). Artifacts in a similar dated geological context have been reported at the Gault site, Texas (17). Bifaces, flake tools, and debitage dating to 14.2 and 14.8 ka occur at the Schaefer and Hebior sites, Wisconsin, and are associated with the remains of two mammoths (18). At Meadowcroft Rockshelter, Pennsylvania, an assemblage of bladelets, flakes, and one bifacial projectile point date between 13.4 and 15.2 ka (19). At Paisley Caves, Oregon, artifacts and human coprolites occur at 14.1 ka (20). A small number of flakes appear in stratigraphic contexts at Page-Ladson, Florida, that date to 14.4 ka (21). These data are evidence that by 15.5 ka, human populations occupied the continental United States and that they had biface, blade, and bladelet assemblages. The sites of Cactus Hill, Virginia, and Miles Point, Maryland, hint that these technologies may have been present a few millennia earlier (22, 23). This early occupation of North America provides ample time for people to settle into the environments of North America, colonize South America by at least ~14.1 to 14.6 ka (Monte Verde, Chile) (24, 25), develop the Clovis tool kit, and create a base population through which Clovis technology could spread. References and Notes

Fig. 4. Buttermilk Creek Complex artifacts: (a) lanceolate point preform, (b) chopper/adze, (c) discoidal flake core, (d) radially broken flake with notch, (e) graver, (f) flake tool with retouch on a radially broken edge, (g and h) flake tools with marginal edge retouch, (i) polished hematite, (j) bifacially retouched flake, (k) radially/bend broken flake, (l) radially broken biface, (m and n) blade midsections, (o to s) bladelets.

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1. T. Goebel, M. R. Waters, D. H. O’Rourke, Science 319, 1497 (2008). 2. M. R. Waters, T. W. Stafford Jr., Science 315, 1122 (2007). 3. B. A. Bradley, M. B. Collins, A. Hemmings, Clovis Technology (International Monographs in Prehistory no. 17, Ann Arbor, MI, 2010). 4. B. Bradley, D. Stanford, World Archaeol. 36, 459 (2004). 5. C. Young, S. Gilbert-Young, Curr. Res. Pleistocene 24, 154 (2007). 6. B. Hedman, The Raven Bluff Site: Preliminary Findings from a Late Pleistocene Site in the Alaskan Arctic (Bureau of Land Management Technical Report, Fairbanks, AK, 2010). 7. Materials and methods are available as supporting material on Science Online. 8. M. Jain, L. Bøtter-Jensen, A. K. Singhvi, Radiat. Meas. 37, 67 (2003). 9. A. S. Murray, A. G. Wintle, Radiat. Meas. 37, 377 (2003). 10. J. S. Singarayer, R. M. Bailey, Radiat. Meas. 37, 451 (2003). 11. Soil Survey Staff, Soil Taxonomy: A Basic System of Soil Classification for Making and Interpreting Soil Surveys, USDA-NRCS Agriculture Handbook 436 (U.S. Government Printing Office, ed. 2, 1999). 12. W. R. Wood, D. L. Johnson, in Advances in Archaeological Method and Theory, M. B. Schiffer, Ed. (Academic Press, New York, 1978), vol. 1, pp. 315–381. 13. L. Wilding, D. Tessier, in Vertisols: Their Distribution, Properties, Classification and Management, L. Wilding, R. Puentes, Eds. (Texas A&M University Printing Center, College Station, 1988), pp. 55–81. 14. B. A. Maher, H. M. Yu, H. M. Roberts, A. G. Wintle, Quat. Sci. Rev. 22, 445 (2003). 15. N. J. Vidic, M. J. Singer, K. L. Verosub, Palaeogeogr. Palaeoclimatol. Palaeoecol. 211, 271 (2004).

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REPORTS 23. D. L. Lowery, M. A. O’Neal, J. S. Wah, D. P. Wagner, D. J. Stanford, Quat. Sci. Rev. 29, 1472 (2010). 24. T. D. Dillehay, Ed., The Archaeological Context and Interpretation, vol. 2 of Monte Verde: A Late Pleistocene Settlement in Chile (Smithsonian Institution Press, Washington, DC, 1997). 25. J. M. Erlandson, T. J. Braje, M. H. Graham, Island Coastal Archaeol. 3, 277 (2008). 26. We thank the North Star Archaeological Research Program established by J. Cramer and R. Cramer for funding the fieldwork from 2006–2009 and chronological and chemical analyses. Donations from M. Mullins, R. Wilson, R. Shlemon, B. Rotstan, R. Engle, E. Guerri, G. Moore, T. Cloninger, S. Kohntopp, M. Payn, J. Pytkowicz, and the Center for the Study of the First Americans membership and funding from the Chair in First Americans Studies supported the laboratory analyses. L. Pfeiffer and W. Wheless III helped to

Dental Occlusion in a 260-MillionYear-Old Therapsid with Saber Canines from the Permian of Brazil Juan Carlos Cisneros,1,2 Fernando Abdala,3 Bruce S. Rubidge,3 Paula Camboim Dentzien-Dias,4 Ana de Oliveira Bueno2 Anomodonts, a group of herbivorous therapsid “mammal-like reptiles,” were the most abundant tetrapods of the Permian. We present a basal anomodont from South America, a new taxon that has transversally expanded palatal teeth and long saber canines. The function of the saber teeth is unknown, but probable uses include deterring attack from predators and intraspecific display or combat. The complex palatal teeth were used to process high-fiber food and represent early evidence of dental occlusion in a therapsid. This discovery provides new insight into the evolution of heterogeneous dentition in therapsids and broadens our understanding of ecological interactions at the end of the Paleozoic. ritical modifications in terrestrial communities occurred during the Permian period as an increasing variety of herbivorous tetrapods and their predators evolved. This process resulted in the establishment of a modern trophic pyramid by the end of the period (1). A key faunal element in this ecological transformation was the appearance of therapsid “mammallike reptiles” during the Middle Permian (2–4) and the development of heterodont dentition and diverse feeding adaptations within this group, which facilitated their exploitation of different ecological niches (1, 2). By far the most successful Permo-Triassic therapsid lineage in terms of individual abundance was the herbivorous Anomodontia [~130 species (5)], which includes the paraphyletic basal anomodonts and the more derived monophyletic dicynodonts (5). Basal anomodonts comprise only 11 species and are known from MiddleLate Permian rocks of China, Russia, and South

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1 Universidade Federal do Piauí, Centro de Ciências da Natureza, 64049-550, Ininga, Teresina, Piauí, Brazil. 2Universidade Federal do Rio Grande do Sul, Departamento de Paleontologia e Estratigrafia, Avenida Bento Gonçalves 9500, Porto Alegre, Rio Grande do Sul, Brazil. 3Bernard Price Institute for Palaeontological Research, University of the Witwatersrand, Private Bag 3, WITS 2050, Johannesburg, South Africa. 4Universidade Federal do Piauí, Picos, Piauí, Brazil.

Africa (6). Dicynodonts, the most speciose clade within Anomodontia, are known from every continent (6) and have a distinctive keratinous beak that replaced marginal dentition and, together with modification of the occlusal musculature, facilitated propalinal jaw masticatory movements (7 ). This adaptation was probably the key to their successful exploitation of herbivory. Here we report an early basal anomodont from the Permian of South America, Tiarajudens eccentricus gen. et spec. nov. (8) (Fig. 1), a taxon with dentition that is markedly different from all other anomodont and therapsid dental patterns. The skull of Tiarajudens (~225 mm long) is large for a basal anomodont, with a preorbital length only slightly shorter than the postorbital length (~ 45% of cranial length). Most anomodonts have shorter snouts, but these proportions are comparable to those of the South African Anomocephalus (9) and the Chinese Biseridens (6). Tiarajudens has five prominent leaf-shaped upper incisiform teeth (Fig. 1F) with coarse serrations on their crowns. The last tooth in the maxillary row, below the anterior margin of the orbit, is a large canine measuring ~120 mm in length despite post-fossilization damage to the tip. This laterally compressed fang is reniform in basal cross section (Fig. 1E) and features enamel.

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make the project a reality. D. and D. Friedkin allowed excavations on their property and generously supported our field logistics. M. Thompson, S. Woldhagen, R. Lansford, R. McCulloch, and M. Yalch provided help during the excavation. T. Goebel, K. Graf, S. Fiedel, and T. Stafford, and four anonymous individuals reviewed drafts of this paper.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1599/DC1 Materials and Methods SOM Text Figs. S1 to S14 Tables S1 to S16 References 17 December 2010; accepted 7 February 2011 10.1126/science.1201855

Thirteen large, transversally expanded palatal teeth (Fig. 1G) are present on the pterygoid and ectopterygoid, medial to the canine. The crown blades are oblique, arranged in echelon, and the palatal tooth row is straight, oriented 20° in relation to the lateral margin of the jugal. Unworn teeth are tall and chisel-like (Fig. 2A) and old, worn teeth display wide occlusal surfaces (Fig. 2, B and C), subdivided into two uneven wear platforms; a labial wear facet separated by a step from a larger, ventrally convex lingual facet. Palatal teeth have long roots, partially exposed in lateral view (Fig. 1H), and distinct alveoli, indicating thecodont implantation. The presence of a shortened snout and elevated zygoma (Fig. 1) identify Tiarajudens as an anomodont (6 ), but it is the only one yet described to possess large canines. The flattened cross section of these teeth contrasts with the much shorter and thicker tusks of dicynodonts, which are circular in basal cross section and lack enamel (10–12). The leaf-shaped and serrated upper incisiform teeth resemble those of the planteating Russian basal anomodonts Ulemica and Suminia (13, 14), and are also characteristic of other herbivorous groups such as pareiasaurs, ornithischian and prosauropod dinosaurs, and extant iguanid lizards (1, 13). Tiarajudens is also unusual in having large, transversally expanded palatal teeth. Palatal teeth are absent in all basal anomodonts except Biseridens, in which they are small, rounded, and bulbous (6 ). The presence of teeth in the ectopterygoid is a feature unknown in any other therapsid but is characteristic of pelycosaur-grade synapsids such as Edaphosaurus (15). In occlusal aspect, the palatal teeth of Tiarajudens resemble the maxillary molariforms of the Early Permian Diadectes (16), except that they have the codont implantation, evinced by long roots and distinct alveoli. The dentary is not preserved, but wear facets on the palatal teeth suggest that they occluded against mandibular teeth, resembling a mechanism present in Edaphosaurus (15), where a cluster of homodont palatal teeth are opposed to a matching set of lower teeth on the coronoid, anterior coronoid, and prearticular bones (15, 16). The presence of replacement teeth (Figs. 1H and 2A) and their positioning and

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16. E. Tamm et al., PLoS ONE 2, e829 (2007). 17. M. B. Collins, B. A. Bradley, Curr. Res. Pleistocene 25, 70 (2008). 18. D. J. Joyce, Quat. Int. 142–143, 44 (2006). 19. J. M. Adovasio, D. R. Pedler, in Entering America: Northeast Asia and Beringia Before the Last Glacial Maximum, D. M. Madsen, Ed. (Univ. of Utah Press, Salt Lake City, 2004), pp. 139–158. 20. M. T. P. Gilbert et al., Science 320, 786 (2008); 10.1126/ science.1154116. 21. S. D. Webb, Ed., First Floridians and Last Mastodons: The Page-Ladson Site in the Aucilla River (Springer, Dordrecht, Netherlands, 2005). 22. J. M. McAvoy, L. D. McAvoy, Eds., Archaeological Investigations of Site 44SX202, Cactus Hill, Sussex County, Virginia (Research Report Series No. 8, Virginia Department of Historic Resources, Richmond, 1997).

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Fig. 2. (A) Replacement palatal tooth in posterior view (last tooth in the row, apex to the top). (B and C) Reconstruction of fourth palatal tooth, (B), posterior, slightly medial view, (C), occlusal view. (D) and (E) Comparative figures showing a skull reconstruction of T. eccentricus (D) and the skull of the extant saber-toothed water deer Hydropotes sp. (E). Scale bar, 10 mm for (A) to (C). (D) and (E) are not to scale.

Fig. 1. Cranium and dentition of T. eccentricus from the Permian of Brazil (UFRGS PV393P, holotype). (A) Photograph and (B) drawing of the skull in lateral view. (C) Photograph and (D) drawing of the skull in medial view (parasagittal section). (E) Saber canine in basal cross section; anterior is to the left. (F) Incisiforms in lingual view (iii to v indicate tooth positions). (G) Palatal molariforms in occlusal view (stereophotograph). (H) Lateral view of palatal teeth, showing a posterior-to-anterior replacement sequence; replacement teeth are indicated by arrows. Scale bars, 50 mm for (A) to (D) and 10 mm for (E) to (H). Abbreviations: an, angular; ar, articular; c, canine; ect, ectopterygoid; j, jugal; la, lacrimal; lf, left frontal; ln, left nasal; lpm, left premaxilar; mx, maxilla; po, postorbital; pof, postfrontal; prf, prefrontal; pt, pterygoid; q, quadrate; qj, quadratojugal; rf, right frontal; rn, right nasal; rpm, right premaxilar; sa, surangular; sc, scleral ossicles; sm, septomaxilla; sq, squamosal; t, teeth. stages of eruption are consistent with a posteriorto-anterior alternating pattern of substitution. This evidence indicates that Tiarajudens was a sophisticated heterodont herbivore capable of processing high-fiber food through a set of palatal molariform teeth.

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Large saber canines are unexpected in a herbivore. All Permian tetrapods with saber teeth [biarmosuchians, anteosaurid dinocephalians, and gorgonopsians (2, 17, 14)] are mid- to top-level predators. The canines of Tiarajudens are as long as those of the largest gorgonopsian Inostrancevia

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from Russia (14), but relative to skull size the canines of Tiarajudens are larger (1:0.26 for Inostrancevia and 1:0.57 in Tiarajudens). However the saber teeth of Tiarajudens lack the serrations characteristic of the canines of Permian carnivorous therapsids (2, 14). Despite their great length, the canines were not fragile. These could have served to manage food items before processing, to deter attacks from predators (18), or for intraspecific display and combat (18, 19), as seen in extant antlerless water deer (Hydropotes sp.; Fig. 2E) and musk deer (Moschus sp.) from Asia (20, 21). The latter could have been an alternative to the head-butting combat employed by coexisting Permian dinocephalians (22) and extant ruminants. The earliest saber-toothed herbivores hitherto known were Late Paleocene (57 million years ago) uintatheres and the pantodont Titanoides (23). The presence of saber teeth in

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Tiarajudens extends this specialization in terrestrial herbivores back to the Paleozoic, some 260 million years ago. The majority rule consensus tree of a phylogenetic analysis of early anomodonts (Fig. 3) (24) shows Biseridens to be the most basal anomodont, followed by a new Gondwanan monophyletic clade, Anomocephaloidea (8), comprising Tiarajudens and Anomocephalus (see supporting online material). This is followed by a polytomy including the South African Galechirus, the Russian monophyletic group Venyukovioidea (25) (comprising Otsheria, Suminia and Ulemica), and a monophyletic Chainosauria, which incorporates the South African basal anomodonts Patranomodon and Galeops and dicynodonts. The basal position of the Brazilian taxon implies that its saber teeth predate the tusks of dicynodonts and do not represent homologies. The new clade Anomocephaloidea represents a previously unrecognized Gondwanan radiation of large, relatively robust basal anomodonts and contrasts with the Russian Venyukovioidea, formed by small, gracile taxa, including one arboreal form (26). The extensive morphological experimentation observed in T. eccentricus and other basal anomodonts played a major role in the evolution of dicynodonts, the most successful Permo-Triassic tetrapod lineage. References and Notes 1. H.-D. Sues, R. R. Reisz, in Evolution of Herbivory in Terrestrial Vertebrates, H.-D. Sues, Ed. (Cambridge Univ. Press, Cambridge, 2000), pp. 9–41. 2. B. S. Rubidge, C. A. Sidor, Annu. Rev. Ecol. Syst. 32, 449 (2001). 3. J. Liu, B. Rubidge, J. Li, Acta Palaeontol. Pol. 54, 393 (2009). 4. T. S. Kemp, J. Evol. Biol. 19, 1231 (2006). 5. J. Fröbisch, Earth Sci. Rev. 95, 119 (2009).

6. J. Liu, B. Rubidge, J. Li, Proc. Biol. Sci. 277, 285 (2010). 7. K. D. Angielczyk, Paleobiology 30, 268 (2004). 8. Therapsida Broom, 1905; Anomodontia Owen, 1859; Anomocephaloidea taxon nov. Etymology: From the basal anomodont genus name Anomocephalus. Definition: All taxa more related to Anomocephalus africanus Modesto, Rubidge & Welman 1999 than to Otsheria (Venyukovia) netzvetajevi Tchudinov 1960; Tiarajudens eccentricus gen. et sp. nov. Etymology: A combination of Tiarajú, the type locality, and dens (Latin) tooth; eccentricus (Latin), “out of the center,” used here in its modern sense, meaning something that departs from a conventional or established norm or pattern. Holotype: UFRGS PV393P, reposited in the Universidade Federal do Rio Grande do Sul, Porto Alegre. Partial cranium (Fig. 1) and unprepared postcranium. Horizon and locality: Found in an exposure of the Rio do Rasto Formation at Tiarajú District, São Gabriel Municipality, Rio Grande do Sul State, Brazil, Middle Permian. This unit is considered to correlate with the Tapinocephalus Assemblage Zone of the South African Karoo because of the co-occurrence of dinocephalians and pareiasaurs (28). Diagnosis: Large basal anomodont distinguished by the presence of large canines, reniform in basal cross section, featuring enamel, comprising more than 120% of the maximum snout (antorbital) height and more than 60% of total skull length; a row of 13 transversally expanded palatal teeth (average ratio of labiolingualmesiodistal length = 3:1), located in the ectopterygoid and pterygoid, showing uneven wear facets and long roots. 9. S. Modesto, B. Rubidge, J. Welman, Proc. Biol. Sci. 266, 331 (1999). 10. D. F. G. Poole, Q. J. Microsc. Sci. 97, 303 (1956). 11. G. M. King, Philos. Trans. R. Soc. London Ser. B 291, 243 (1981). 12. C. Sullivan, R. R. Reisz, R. M. H. Smith, Proc. Biol. Sci. 270, 173 (2003). 13. N. Rybczynski, R. R. Reisz, Nature 411, 684 (2001). 14. M. F. Ivakhnenko, Paleontol. J. 42, 859 (2008). 15. S. P. Modesto, Palaeontology 38, 213 (1995). 16. R. R. Reisz, J. Exp. Zool. (Mol. Dev. Evol.) 306B, 261 (2006). 17. M. F. Ivakhnenko, Paleontol. J. 39, S393 (2005). 18. T. Caro, Antipredator Defenses in Birds and Mammals (Univ. of Chicago Press, Chicago, 2005).

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19. C. Darwin, The Descent of Man and Selection in Relation to Sex (William Clowes, London, 1871). 20. V. Geist, Deer of the World: Their Evolution, Behaviour, and Ecology (Stackpole Books, Mechanicsburg, PA, 1998). 21. D. J. Emlen, Annu. Rev. Ecol. Evol. Syst. 39, 387 (2008). 22. H. R. Barghusen, Paleobiology 1, 295 (1975). 23. K. D. Rose, The Beginning of the Age of Mammals (Johns Hopkins Univ. Press, Baltimore, MD, 2006). 24. Materials and methods are available as supporting material on Science Online. 25. S. P. Modesto, N. Rybczynski, in The Age of Dinosaurs in Russia and Mongolia, M. J. Benton, M. A. Shishkin, D. M. Unwin, E. N. Kurochkin, Eds. (Cambridge Univ. Press, Cambridge, 2000), chap. 2. 26. J. Fröbisch, R. R. Reisz, Proc. Biol. Sci. 276, 3611 (2009). 27. G. J. Ogg, G. Ogg, F. M. Gradstein, The Concise Geologic Time Scale (Cambridge Univ. Press, Cambridge, 2008). 28. J. C. Cisneros, F. Abdala, M. C. Malabarba, Rev. Bras. Paleontol. 8, 13 (2005). 29. We thank A. E. Figueiredo, M. A. França, T. P. Melo, and W. Velasques-Alves for field support. V. Abdala, K. Angielczyk, L. Backwell, P. Backwell, M. Jennions, T. Kemp, and R. Reisz, are acknowledged for discussion and valuable comments. J.C.C., P.C.D.D., and A.O.B. were funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); F.A. and B.S.R. were funded by the Department of Science and Technology, National Research Foundation, and Palaeontological Scientific Trust. Fieldwork was funded by a CNPq grant (478914/2006-77) awarded to C. L. Schultz.

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Fig. 3. Stratigraphically calibrated phylogeny of Permian basal anomodonts (5) and other therapsids. Circles represent taxa known from a single locality. Values represented above the branches are decay values; values below are from symmetric resampling. The temporal range of Dimetrodon extends to the earliest Permian (Asselian). Abbreviations: Ciste., Cistecephalus; Chang., Changhsingian; Eodicyno., Eodicynodon; K, Karoo Assemblage Zone; Kungu., Kungurian; Prist., Pristerognathus; PTB, PermoTriassic Boundary; Road., Roadian; SGCS, Standard Global Chronostratigraphic Scale (27); Tropi., Tropidostoma; Wuchiaping., Wuchiapingian. Ages are in million years ago.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1603/DC1 Materials and Methods SOM Text Figs. S1 to S8 References 10 November 2010; accepted 2 February 2011 10.1126/science.1200305

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Social and Ecological Synergy: Local Rulemaking, Forest Livelihoods, and Biodiversity Conservation Lauren Persha,1* Arun Agrawal,1 Ashwini Chhatre2 Causal pathways to achieve social and ecological benefits from forests are unclear, because there are few systematic multicountry empirical analyses that identify important factors and their complex relationships with social and ecological outcomes. This study examines biodiversity conservation and forest-based livelihood outcomes using a data set on 84 sites from six countries in East Africa and South Asia. We find both positive and negative relationships, leading to joint wins, losses, and trade-offs depending on specific contextual factors; participation in forest governance institutions by local forest users is strongly associated with jointly positive outcomes for forests in our study. uman-dominated forested landscapes in tropical developing countries provide many different and important ecosystem services and also sustain the livelihoods of large numbers of poor peoples (1). Recognition of the diverse socioeconomic and ecological contributions of forests has prompted many governments to pursue policies for improved livelihoods and conservation outcomes. Notable are forest policy decentralization reforms that transfer ownership and management responsibilities to local forest user organizations (2, 3). Such policies have been introduced in more than two-thirds of the developing world (4), including 35 of 51 countries in sub-Saharan Africa (5), and apply to an estimated one-third of forests in developing countries globally (6). Formalized local participation in forest governance via decentralization is often viewed as a key mechanism to provide incentives to local communities to use forests sustainably through en-

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1 School of Natural Resources and Environment, University of Michigan, 440 Church Street, Ann Arbor, MI 48109, USA. 2Department of Geography, University of Illinois at UrbanaChampaign, 607 South Mathews Avenue, Urbana, IL 61801, USA.

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

hanced local knowledge, stronger accountability, and perceived legitimacy of forest rules (2, 3). Its effectiveness is also debated because of fears that decentralization could lead to resource capture by local elites and remain ineffectual without extensive devolution of rights to local participants and functional linkages between local decentralized institutions and well-crafted macro-level governance institutions (7). More broadly, policy pathways toward joint improvements in sustainable livelihoods and biodiversity conservation continue to be unclear. Despite the inherent complexity of social-ecological contexts for forest systems, current policy responses, particularly in terms of explicit management for trade-offs or synergies across multiple social and ecological goals, are seldom based on careful analysis or evidence of factors that lead to improvements across desired sets of social and ecological outcomes together (8, 9). In scholarly research, relationships between human livelihoods and biodiversity conservation are often conceptualized as diametrically opposite (10, 11), but more studies that undertake explicit quantitative analysis of this relationship are needed (12). There is evidence of trade-offs, but case studies also reveal the potential for synergies (13). However, previous work tends to focus anal-

ysis on these social and ecological outcomes in isolation from each other, rather than in tandem as a single outcome constructed across both social and ecological dimensions. Here we analyze patterns of outcome relationships between forestbased household livelihoods and biodiversity conservation in 84 study sites, and the potential explanations associated with the joint production of these two forest benefits. Our analysis draws on a global data set of social, ecological, and governance data on a wide range of representative forests in human-dominated tropical landscapes that has been compiled by the International Forestry Resources and Institutions (IFRI) research program (14, 15). The 84 cases are drawn from six countries in East Africa (6 in Kenya, 7 in Tanzania, and 17 in Uganda) and South Asia (2 in Bhutan, 27 in India, and 25 in Nepal). We use the nonparametric Chao-1 estimator of tree species richness as an indicator of forest biodiversity. We use the percent of households that depend substantially on the forest for subsistence livelihoods as an indicator of livelihood contributions of the same forest (16). We classify the outcome relationships between tree species richness and forest-based subsistence livelihoods into categories on the basis of above- or below-average levels for each of our two indicator variables, relative to other forests in the same forest type in the data set (16). Our approach focuses on three joint-outcome categories where (i) species richness and livelihoods contributions are both above average (sustainable forest systems); (ii) species richness and livelihoods are both below average (unsustainable forest systems); and (iii) either species richness is above average relative to other forests and livelihoods are below average, or species richness is below average but livelihoods are above average (tradeoff forest systems). We find that all possible combinations of relationships between forest-based subsistence livelihoods and tree species richness are present in the data (Fig. 1A). The existence of multiple patterns of relationships underscores the relevance of analyses that seek to identify factors responsible for facilitating or impeding trade-offs and

Fig. 1. (A) Tree species richness and subsistence livelihoods z scores, standardized within each forest type. Black circles indicate East African cases (n = 30), and gray circles indicate South Asian cases (n = 54). (B) Distribution of sustainable, unsustainable, and trade-off outcomes, by region. H-H, above-average standardized values for tree species richness and subsistence livelihoods; L-L, below-average standardized values for tree species richness and subsistence livelihoods; SL, subsistence livelihoods; TSR, tree species richness.

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synergies across social and ecological outcomes. Most of our 84 cases (60%) are characterized by trade-off relationships, although jointly positive outcomes across biodiversity and livelihoods are also well represented (27% of cases). Jointly negative outcomes are less common (13% of cases) (Fig. 1B). The distribution of outcomes across all possible categories suggests that there is no universally applicable positive or negative association between livelihoods and biodiversity to be found in the studied forests. Outcomes are similarly distributed across the two regions covered in our study (likelihood-ratio Χ2 = 2.77, df = 2, P = 0.251), and region was not a significant predictor of outcomes. We also examined how a set of hypothesized social and ecological mediating factors affects the observed outcomes, using ordered logistic regression analysis to identify important predictors of

the likelihood of obtaining jointly negative, tradeoff, or positive outcomes. Our model included three independent variables that are a focus of much theoretical and empirical work related to biodiversity conservation and sustainable livelihoods outcomes: forest size, formal participation (as conferred through policy) of local forest users in forest rulemaking (hereafter, “rulemaking participation”), and dependence on the forest for extractive commercial livelihoods (in our data, primarily charcoaling, small-scale timber harvesting, fuelwood, and collection of nontimber forest products for cash income). Forest patch size is a key factor relating to potential species richness, as well as to prospects for sustainable forest management due to monitoring and enforcement challenges related to scale (17); rulemaking participation is highlighted as important for obtaining local knowledge necessary to improve forest resources,

Table 1. Marginal effects of ordered logit regression (rulemaking participation held at its median value, all other variables at their means). Dependent variable: tree species richness and subsistence livelihoods joint outcome, with three categories: low-low, trade-off, or high-high. N = 84. Wald test X2(3) = 9.33, P = 0.0252, Independent variable

Marg. effect

Forest size Rulemaking participation Commercial livelihoods

−0.036 −0.119 −0.215

Forest size Rulemaking participation Commercial livelihoods

0.001 −0.158 0.006

Forest size Rulemaking participation Commercial livelihoods

0.035 0.277 0.209

Std. error

promoting legitimacy over forest rules, and engendering management accountability (18); a high level of commercial extractive forest use may be viewed as reducing the likelihood of sustainable forest outcomes across subsistence livelihoods and species richness, due to higher access inequities for poor households and negative impacts on biodiversity objectives (19–22). Our results indicate that forest systems are more likely to have sustainable outcomes (aboveaverage tree species richness and subsistence livelihoods) when local forest users participate in forest rulemaking (z = 2.21, P = 0.027), whereas unsustainable forest system outcomes are more likely when users do not participate in rulemaking (z = −2.62, P = 0.009; Tables 1 and 2). The size of the forest and the extent to which the forest provided commercial livelihoods to households are also important factors associated with

pseudo R2 = 0.0903. Independent variables: “forest size,” logarithm of forest size (ha); “rulemaking participation,” local forest user participation in forest rulemaking (0 = No, 1 = Yes); “commercial livelihoods” (percent of local forest users who depend substantially on the forest for cash income). z

Outcome category: low-low (unsustainable forest systems) 0.019 −1.820 0.069 0.045 −2.620 0.009 0.122 −1.750 0.080 Outcome category: trade-offs 0.014 0.070 0.943 0.101 −1.570 0.117 0.087 0.070 0.944 Outcome category: high-high (sustainable forest systems) 0.018 1.980 0.048 0.125 2.210 0.027 0.092 2.270 0.023

Fig. 2. Predicted probabilities of (A) unsustainable, (B) trade-off, and (C) sustainable forest system outcomes when local forest users participate in forest rulemaking (solid black line) and when they do not participate in forest rulemaking (dashed black line), as forest size increases. The gray shaded area www.sciencemag.org

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[95% CI ]

P>z

X

−0.074 −0.208 −0.456

0.003 −0.030 0.026

5.821 0.000 0.207

−0.027 −0.355 −0.164

0.029 0.039 0.177

5.821 0.000 0.207

0.003 0.031 0.028

0.068 0.522 0.390

5.821 0.000 0.207

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between probability curves in each panel shows the decrease in the predicted probabilities of unsustainable forest systems and trade-offs, and the increase in the predicted probability of sustainable forest systems, when local forest users participate in forest rulemaking. VOL 331

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Outcome category

Unsustainable outcome (low-low) Trade-off outcomes Sustainable outcome (high-high)

Predicted probability of outcome (%): Forest rulemaking participation by local forest users No

Yes

0.17 0.68 0.16

0.05 0.52 0.44

obtaining either sustainable or unsustainable forest system outcomes, with a higher likelihood of sustainable outcomes as forest size and commercial livelihoods dependence increase [Table 1; note that the amount of benefits from commercial livelihoods, though important at the individual level, constitutes a small proportion of livelihoods for many households and is relatively low overall for our data (16)]. To better understand how interactions between forest size and rulemaking participation together affect the likelihood of obtaining jointly positive outcomes, we examined how the predicted probability of each of the three outcomes varies across the range of forest sizes in our data set, in the presence and absence of rulemaking participation by local forest users (Fig. 2). We find that rulemaking participation is associated with a lower probability of less desirable outcomes (unsustainable forest systems and those characterized by trade-offs) and a higher probability of sustainable forest system outcomes, across smaller and larger forests. But our results suggest that participation in rulemaking may be especially important in promoting positive outcomes in small forest fragments, where greater challenges to achieving jointly positive outcomes across biodiversity and livelihoods already exist (Fig. 2). We conclude that working toward formal participation of local forest users in rulemaking processes for use and management of forests from which they draw their livelihoods (irrespective of whether such activities are sanctioned under prevailing rules) is an important way to increase the probability of obtaining more positive outcomes across social and ecological dimensions. Further work is needed to understand the causal mechanisms that underlie such outcomes. One proposed mechanism is that rulemaking participation provides an opportunity for local forest users to contribute more specific and locally relevant information on forest resources and dynamics of use for a given forest, which in turn leads to the construction of rules that are viewed as legitimate and better suited for local forest conditions. Rulemaking participation may also help shift incentive structures for forest users to undertake decisions aimed toward a more balanced prioritization between activities that maintain good forest conditions and benefit flows over longer time horizons (hence biodiversity conservation indirectly) and shorter-term livelihoods benefits.

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Change in probability as local forest users gain rulemaking participation (95% CI) −0.12 (−0.21, −0.03) −0.16 (−0.32, 0.00) 0.28 (0.07, 0.48)

In constructing our model, we also tested for the significance of additional variables, particularly market distance and population density, which have been found in prior work to be associated with biodiversity and livelihoods when treated as independent outcomes. However, we do not find a statistically significant association between these other factors and our joint outcome categories (16). Species richness does not encompass all facets of biodiversity, and species composition is also particularly important for assessing biodiversity conservation (9). Our use of tree species richness as an indicator of biodiversity is supported by a strong positive correlation of this variable with an abundance-based similarity index of tree species composition calculated for each of our cases in comparison with a minimally disturbed reference forest within the same forest type (16). There are some important regional differences in the broader set of biophysical, socioeconomic, and institutional factors associated with the East African versus South Asian cases (table S2). Forests are larger on average in East Africa, and a greater proportion of households rely on the forests for commercial income. We also find differences in the strength of association of some of these explanatory and broader contextual factors between the two regions, even as overall patterns of outcomes in the relationship between tree species diversity and subsistence livelihoods are similar. We suggest that this may point to the likelihood of multiple pathways for achieving these outcomes, differentiated, for instance, across varied regional contexts and key factors that also likely operate at broader scales. Recognition that forested ecosystems simultaneously generate multiple services is embodied in forestry policy decentralization reforms that have doubled the area of forest land under community ownership or management in the past 15 years (6). These efforts rest on an assumption that synergies across multiple forest outcomes can be achieved, yet existing scholarship provides only limited guidance on avenues by which policies might better promote these synergies. Our data and analysis from a large number of cases from different countries, forest types, and social contexts suggest that trade-offs favoring forest conservation objectives or immediate human livelihoods, as well as jointly positive or negative results, are each possible across the many different contexts

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that comprise the human-dominated forested landscapes of East Africa and South Asia. Although achieving desirable outcomes across potentially competing social and ecological objectives is a complex process, our analysis suggests that jointly positive outcomes are far more likely when forest users participate in rulemaking aspects of forest governance. Our findings are particularly relevant for small forest patches in human-dominated landscapes (especially forests under 200 ha), which often present a particular challenge for achieving jointly positive results. References and Notes 1. W. D. Sunderlin et al., World Dev. 33, 1383 (2005). 2. A. M. Larson, F. Soto, Annu. Rev. Environ. Resour. 33, 213 (2008). 3. A. Agrawal, A. Chhatre, R. Hardin, Science 320, 1460 (2008). 4. A. Agrawal, E. Ostrom, Polit. Soc. 29, 485 (2001). 5. E. Barrow, K.-R. Jones, I. Nhantumbo, R. Oyono, M. Savadogo, “Cutomary practices and forest tenure reforms in Africa: Status, issues and lessons” (Rights and Resources Initiative, Washington, DC, 2009). 6. W. D. Sunderlin, J. Hatcher, M. Liddle, “From Exclusion to Ownership? Challenges and opportunities in advancing forest tenure reform” (Rights and Resources Initiative, Washington, DC, 2008). 7. K. P. Andersson, C. C. Gibson, F. Lehoucq, World Dev. 34, 576 (2006). 8. E. Ostrom, Proc. Natl. Acad. Sci. U.S.A. 104, 15181 (2007). 9. T. A. Gardner et al., Ecol. Lett. 12, 561 (2009). 10. W. M. Adams et al., Science 306, 1146 (2004). 11. S. Wunder, World Dev. 29, 1817 (2001). 12. R. L. Chazdon et al., Biotropica 41, 142 (2009). 13. L. Naughton-Treves, M. B. Holland, K. Brandon, Annu. Rev. Environ. Resour. 30, 219 (2005). 14. E. Wollenberg, L. Merino, A. Agrawal, E. Ostrom, Int. Forest. Rev. 9, 670 (2007). 15. www.snre.umich.edu/~ifri 16. Materials and methods are available as supporting material on Science Online. 17. J. L. Hill, P. J. Curran, J. Biogeogr. 30, 1391 (2003). 18. J. C. Ribot, A. Agrawal, A. M. Larson, World Dev. 34, 1864 (2006). 19. B. M. Belcher, Int. Forestry Rev. 7, 82 (2005). 20. B. Belcher, M. Ruiz-Perez, R. Achdiawan, World Dev. 33, 1435 (2005). 21. T. A. Gardner, J. Barlow, N. S. Sodhi, C. A. Peres, Biol. Conserv. 143, 2293 (2010). 22. F. E. Putz, G. M. Blate, K. H. Redford, R. Fimbel, J. Robinson, Conserv. Biol. 15, 7 (2001). 23. We thank E. Ostrom, T. Hayes, two anonymous reviewers, and members of the Development, Sustainable Livelihoods and Conservation (DESULICO) group for comments on an earlier draft of this paper, and R. Kornak and J. England for assistance with data cleaning. We gratefully acknowledge the many researchers affiliated with IFRI as Collaborating Research Centers for their data contributions and ongoing involvement with the IFRI network. This work was funded by the Ford Foundation, the MacArthur Foundation, National Science Foundation grants BCS-0703073 and CNH-0709545, and a University of Michigan OVPR/Rackham grant for an Annual Institute on Joint Outcomes related to Sustainability.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1606/DC1 Materials and Methods Figs. S1 to S3 Tables S1 and S2 References 10.1126/science.1199343

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Table 2. Predicted probabilities of obtaining unsustainable, trade-off, or sustainable forest system outcomes as local forest users gain participation in forest rulemaking.

Impacts of Salmon on Riparian Plant Diversity Morgan D. Hocking* and John D. Reynolds The study of natural gradients in nutrient subsidies between ecosystems allows for predictions of how changes in one system can affect biodiversity in another. We performed a large-scale empirical test of the role of Pacific salmon (Oncorhynchus spp.) in structuring riparian plant communities. A comparison of 50 watersheds in the remote Great Bear Rainforest of British Columbia’s central coast in Canada shows that salmon influence nutrient loading to plants, shifting plant communities toward nutrient-rich species, which in turn decreases plant diversity. These effects are mediated by interactions between salmon density and the physical characteristics of watersheds. Predicting how salmon affect terrestrial ecosystems is central to conservation plans that aim to better integrate ecosystem values into resource management. eclines in fish biomass through overexploitation, habitat degradation, and climate change have important consequences for the structure and functioning of marine ecosystems (1). These changes to the marine environment can extend into coastal transition zones such as estuaries, mangroves, and streams, ultimately affecting the biodiversity and resilience of these communities (2, 3). However, marine species may play an even wider role in coastal ecology through nutrient subsidies to adjacent terrestrial ecosystems (4, 5). Tests are often restricted to small-scale observations or experiments and can overlook how physical and biological processes at local and regional scales may mediate the effects of subsidies on ecosystem structure and functioning (6, 7). Here, we tested the hypothesis that marine subsidies can affect terrestrial plant biodiversity across a large-scale gradient in anadromous fish biomass. Throughout the North Pacific region, the largest cross-ecosystem movement of animals is the annual migration of wild salmon (Oncorhynchus spp.) from the ocean into freshwater streams and lakes, where they spawn and die. Adult salmon deliver marine nutrients to aquatic and terrestrial ecosystems and support the populations of many animal species (8–10). Declines in Pacific salmon populations throughout the 20th century have resulted in less salmon for fisheries and potential shifts in terrestrial ecosystem processes. Strong salmon populations provide benefits for bears (Ursus spp.) and other predators, and there are indications that salmon nutrients can affect riparian production (8–10). Nonetheless, uncertainty remains about the role of salmon in driving ecosystem-level structure and functioning. For example, salmon nutrient subsidies to freshwater systems are often counteracted by the physical disturbance of stream substrates associated with the digging of nests (“redds”), which can result in local nutrient export (11, 12). Also,

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Earth2Ocean Research Group, Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada. *To whom correspondence should be addressed. E-mail: [email protected]

the productivity, size, and types of terrestrial habitats adjacent to salmon streams can mediate the effects of salmon subsidies, which vary markedly across their geographic range (10, 13, 14). We used a large-scale survey from 50 watersheds on the central coast of British Columbia, Canada (15) (fig. S1 and table S1), to provide an empirical test of the relative influence of biotic and abiotic processes (i.e., salmon subsidy and physical habitat) in structuring plant nutrient uptake, community structure, and diversity. This region is relatively unaffected by industrial development and is part of the largest old-growth temperate rainforest in the world. Populations

of chum (O. keta), pink (O. gorbuscha), and coho (O. kisutch) salmon, and smaller numbers of sockeye (O. nerka) and Chinook (O. tshawytscha) salmon, spawn in hundreds of streams on remote islands and the adjacent mainland (16). From fall 2006 to fall 2009, we partnered with members of the Heiltsuk Nation to supplement existing federal programs that monitor salmon populations to derive an index of salmon spawning density (kilograms of salmon biomass per meter of spawning length) for each stream. We repeated all analyses with a 10-year mean (1997 to 2006) in a subset of streams (n = 25) where long-term salmon population data were available, and found very similar results (15) (tables S2 and S3). Intact habitats and low hunting pressure result in strong local populations of black bears (U. americanus), grizzly bears (U. arctos horribilis), wolves (Canis lupus), and other predators, which are important salmon consumers in this region (17, 18). They feed preferentially on energy-rich salmon parts and can transfer more than 50% of the salmon to the forest. Salmon carcasses can also provide pulsed increases in volatilized and in-stream nutrient concentrations, which can enter terrestrial systems via depositional and water-flow pathways. Although plant uptake of salmon nutrients has been demonstrated by testing for elevated heavy nitrogen (d15N) (19–22), the key question is whether and how these subsidies alter riparian plant biodiversity.

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Fig. 1. Relationships between stream-level salmon spawning density (kg m−1) and average foliar d15N (A to D), foliar %N (E to H), and plant abundance (cover/total shrub and herb cover) (I to L) in four dominant understory plant species—false azalea (Menziesia ferruginea), blueberry (Vaccinium alaskaense and V. ovalifolium), false lily-of-the-valley (Maianthemum dilatatum), and salmonberry (Rubus spectabilis)— sampled from 50 watersheds in coastal British Columbia. False azalea and blueberry indicate nutrient-poor soils, whereas false lily and, in particular, salmonberry are more competitive in nutrient-rich sites and are considered nitrophiles (23). Significant relationships are fitted using nonlinear LOESS smoothing.

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REPORTS Table 1. The top-ranked model in each analysis. N, sample size; k, number of parameters; Wi, individual AIC model weights; R2, coefficient of fitted versus observed values for each analysis. Parameter estimates and top model fits are shown in tables S10 to S13 and fig. S4. N

k

Wi

R2

1. Foliar d15N

Salmon density [log ( x + 1)], canopy community, soil moisture index Foliar %N, plant species, foliar %N × plant species Salmon density × plant species Salmon density × foliar %N Salmon density [log (x + 1)], canopy community, watershed size, stem density, site slope Plant species, salmon density × plant species Salmon density × canopy community Salmon density × site slope Salmon density, site slope, distance from stream Canopy community Salmon density × canopy community Salmon density, canopy community, watershed size, stem density Site slope, canopy cover, course woody debris cover, distance from stream Salmon density × site slope

906

21

0.99

0.66

2. Foliar %N

906

20

0.92

0.50

3. Plant communities

1192

13

0.60

0.81

4. Plant diversity

1192

15

0.24

0.51

From June to mid-July 2007, we measured the percent cover of all forest plant species, including shrubs, herbs, ferns, mosses, liverworts, and understory trees (n = 104 species), within 1-m2 plots (n = 1200) placed 5, 15, 25, and 35 m from the stream edge (6 transects per watershed × 50 watersheds). We also collected foliar samples for nutrient and stable isotope analyses from four dominant plant species (Fig. 1). Understory plant community structure was quantified into axes of variation via principal components analysis (PCA) (table S4). The first axis of understory plant community variation at the watershed scale (PC1 = 31.1%) was strongly correlated to an established nitrogen indicator classification system in coastal British Columbia (23) (fig. S2). We used hierarchical linear mixed-effects (LME) modeling (24, 25) and model selection via the Akaike information criterion (AICc) to predict variation within and across watersheds in four dependent variables: (i) leaf d15N signatures, (ii) total leaf nitrogen (%N), (iii) understory plant community structure (PC1), and (iv) understory plant diversity (Margalef’s DMg). First, on the basis of a priori hypotheses of up to 12 separate habitat variables, we derived a best-fit habitatonly model for each of the four dependent variables (15) (tables S5 to S8), informed by the long history of forest habitat analyses in British Columbia (23, 26, 27). For example, we assessed canopy community structure across spatial scales from individual plots to watersheds, including the prevalence of the nitrogen-fixing red alder (Alnus rubra), which can have a strong influence on local nutrient dynamics (14). The first axis of canopy community variation (Canopy PC1 = 36.1%) at the watershed scale described a shift from low-productivity forests dominated by western redcedar (Thuja plicata) to more productive forests dominated by red alder and Sitka spruce (Picea sitchensis). This axis is predicted by a broad climatic gradient and by watershed size (fig. S3), but only weakly by salmon spawning density (15). In the second step of the analysis, for each dependent variable we compared the best-fit

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Parameters in top-ranked model

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Fig. 2. Relationships between stream-level salmon spawning density (kg m−1) and understory plant community structure (PC1) (A and B) and plant diversity (DMg) (C and D) in 50 watersheds in coastal British Columbia. In (A) and (C), relationships are fitted using nonlinear LOESS smoothing; in (B) and (D), they are fitted by distance from the stream (5 to 35 m) split by evenly weighted categories (low, medium, high) of streamlevel salmon spawning density (T SEM).

habitat-only models (hypothesis 1) with salmononly models [log(salmon density in kg m−1); hypothesis 2], salmon density + best-fit habitat models (hypothesis 3), and models containing salmon density, best-fit habitat, and a priori predictions of salmon density × habitat interactions (hypothesis 4) (15) (tables S7 and S9). This enabled a test of the hypothesis that salmon and habitat would interact to structure plant biodiversity. All plant species showed increases in d15N across the watershed-level gradient in salmon spawning density (Fig. 1, A to D). The top foliar d15N model included strong effects of salmon (LME: t42 = 6.4, P < 0.0001), habitat, and salmon × habitat interactions, including interactions with total leaf nitrogen (Table 1 and table S10). This shows that increases in leaf d15N with salmon density are caused by both the d15N-enriched source of nitrogen from salmon (~12‰) and high-

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er total nitrogen availability (e.g., through increased nitrification) (22, 28). Streams with high salmon densities also had plants with high total leaf nitrogen (LME: t42 = 3.0, P = 0.005), driven by strong relationships in two nutrient-rich indicator species, false lily-of-the-valley and salmonberry (Fig. 1, E to H, Table 1, and table S11). We observed positive relationships between leaf d15N and %N, which can be caused by salmon subsidies and gradients in soil nutrients, site productivity, and plant dependence on mycorrhizal fungi for nitrogen uptake (29–31). Changes in the slope of leaf d15N by %N across plant species and the gradient in salmon density imply that although many coastal watersheds may be nitrogen-limited, nitrogen can be saturating when salmon resource subsidies are high. Salmonberry was the only sampled species that increased in abundance across the gradient in

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Table 2. The weight of evidence supporting each hypothesis in all four analyses. Shown are the AIC weights (Wi) when the best model from within each hypothesis is competed against the other three hypotheses. In parentheses are the total numbers of models considered within each hypothesis. Analysis 1. 2. 3. 4.

Foliar d N Foliar %N Plant communities Plant diversity 15

1. Habitat

2. Salmon density

0.0% (7) 0.03% (5) 0.01% (9) 1.4% (6)

0.0% (4) 0.05% (4) 0.0% (4) 0.2% (4)

salmon density (Fig. 1, I to L). Salmonberry also had the highest average foliar d15N and %N, the strongest relationships of d15N and %N with salmon density, and the weakest positive slope of d15N with foliar %N (table S10). Salmonberry is a strong nitrophile and has arbuscular mycorrhizal associations that facilitate plant access to nutrients, especially phosphorus (23, 31). In contrast, false azalea and blueberry decreased in abundance across the gradient in salmon density (Fig. 1, I and J). These species have ericoid mycorrhizal associations that facilitate organic nitrogen uptake in nutrient-limited conditions, and are likely restricted to nutrient-poor microsites such as decaying woody debris in highly productive watersheds. Patterns of abundance and nutrient uptake in individual species translate to shifts in overall plant biodiversity (Fig. 2). There was a shift in dominance from plant species typical of nutrient-poor habitats in watersheds with low salmon density toward plants typical of nutrient-rich habitats in watersheds with high salmon density (LME: t46 = 5.0, P < 0.0001). This shift resulted in a decrease

3. Habitat and salmon density 0.4% 1.3% 16.7% 41.2%

(5) (4) (3) (4)

99.6% 98.6% 83.2% 57.2%

in plant diversity (LME: t46 = –3.4, P = 0.001). The top models predicting understory community structure and diversity had strong effects of salmon density, habitat, and salmon density × habitat interactions (Table 1 and tables S12 and S13). Patterns of plant diversity show that plant communities in salmon-rich watersheds become dominated by a few species (15), in particular the nutrientrich shrubs salmonberry and stink currant (Ribies bracteosum). Shifts in plant communities were observed with distance from the stream edge (to 35 m), although these effects were similar across the gradient in salmon density (Fig. 2, B and D). The explicit incorporation of habitat variables that describe shifts in terrestrial ecosystem structure across multiple spatial scales enables predictions of how habitats may mediate the effects of salmon in terrestrial environments (32). Variation in physical habitats and background nitrogen levels within and across watersheds strongly influenced plant nitrogen uptake and community composition (Fig. 3 and fig. S5). In each analysis, the overwhelming weight of evidence supported either hypothesis 3 (salmon density + habitat) or

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hypothesis 4 (salmon density + habitat + salmon density × habitat interactions) (Table 2). There was very low support (< 1%) for the hypotheses that only habitat or salmon density predict foliar d15N, %N, plant community structure, or diversity. The dominant habitat predictors almost always included the canopy community composition and site slope. Background %N was a key driver of foliar d15N. Interactions between these variables and salmon density show that salmon have lower effects in large watersheds dominated by red alder canopies, where high terrestrial productivity and nitrogen fixation likely overwhelm salmon inputs, and in sites with steep slopes, probably through decreased predator access to salmon and reduced retention of carcasses and nutrients (Fig. 3). There has been a push toward management objectives that incorporate wider ecosystem values related to salmon (33–36). For example, as management strategies begin to incorporate the ecological importance of salmon when setting salmon population targets (34), it has become increasingly important to understand the links between salmon caught in fisheries and changes to

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Fig. 3. Productivity and habitat mediate the effect of salmon on plant biodiversity. (A) Plants growing in less productive sites with lower leaf %N show greater increases in foliar d15N with salmon density than plants growing in more productive sites with higher %N. (B and C) Watersheds dominated by red alder (Alnus rubra) have saturated leaf %N and nutrientrich plant communities relative to watersheds with canopies dominated by western redcedar (Thuja plicata), which show large shifts in nutrient use and community structure across stream salmon spawning density. (D) The streamside slope mediates the decrease in plant diversity across the gradient in salmon density. Predictions are based on top model parameter estimates (tables S10 to S13).

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ecosystem structure. Our study shows that declines in salmon will have the largest ecological impacts on smaller and less productive streams. In contrast, large and shallow-sloped watersheds dominated by nitrogen-fixing red alders are predicted to be more resilient to salmon declines. These considerations enable predictions of impacts of anthropogenic stressors across ecosystem boundaries, which can then be incorporated into ecosystem-based management. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

B. Worm et al., Science 314, 787 (2006). J. B. C. Jackson et al., Science 293, 629 (2001). H. K. Lotze et al., Science 312, 1806 (2006). G. A. Polis, S. D. Hurd, Am. Nat. 147, 396 (1996). D. A. Croll, J. L. Maron, J. A. Estes, E. M. Danner, G. V. Byrd, Science 307, 1959 (2005). J. L. Maron et al., Ecol. Monogr. 76, 3 (2006). L. B. Marczak, R. M. Thompson, J. S. Richardson, Ecology 88, 140 (2007). S. M. Gende, R. T. Edwards, M. F. Willson, M. S. Wipfli, Bioscience 52, 917 (2002). R. J. Naiman, R. E. Bilby, D. E. Schindler, J. M. Helfield, Ecosystems 5, 399 (2002). D. J. Janetski, D. T. Chaloner, S. D. Tiegs, G. A. Lamberti, Oecologia 159, 583 (2009). J. W. Moore, D. E. Schindler, Can. J. Fish. Aquat. Sci. 61, 1582 (2004). J. W. Moore et al., Ecology 88, 1278 (2007). S. D. Tiegs et al., Ecol. Appl. 18, 4 (2008).

14. J. M. Helfield, R. J. Naiman, Oecologia 133, 573 (2002). 15. See supporting material on Science Online. 16. M. H. H. Price, C. T. Darimont, N. F. Temple, S. M. MacDuffee, Can. J. Fish. Aquat. Sci. 65, 2712 (2008). 17. T. E. Reimchen, Can. J. Zool. 78, 448 (2000). 18. C. T. Darimont, P. C. Paquet, T. E. Reimchen, BMC Ecol. 8, 14 (2008). 19. R. E. Bilby, B. R. Fransen, P. A. Bisson, Can. J. Fish. Aquat. Sci. 53, 164 (1996). 20. K. K. Bartz, R. J. Naiman, Ecosystems 8, 529 (2005). 21. C. E. Wilkinson, M. D. Hocking, T. E. Reimchen, Oikos 108, 85 (2005). 22. M. D. Hocking, T. E. Reimchen, Oikos 118, 1307 (2009). 23. K. Klinka, V. J. Krajina, A. Ceska, A. M. Scagel, Indicator Plants of Coastal British Columbia (Univ. of British Columbia Press, Vancouver, 1989). 24. J. M. Diez, H. R. Pulliam, Ecology 88, 3144 (2007). 25. A. F. Zuur, E. N. Ieno, N. J. Walker, A. A. Saveliev, G. M. Smith, Mixed Effects Models and Extensions in Ecology with R (Springer Science+Business Media, New York, 2009). 26. P. A. Alaback, Ecology 63, 1932 (1982). 27. J. Pojar, K. Klinka, D. V. Meidinger, For. Ecol. Manage. 22, 119 (1987). 28. G. Pinay, T. O'Keefe, R. Edwards, R. J. Naiman, Ecosystems 6, 336 (2003). 29. E. A. Hobbie, J. E. Hobbie, Ecosystems 11, 815 (2008). 30. J. M. Craine et al., New Phytol. 183, 980 (2009). 31. J. M. Kranabetter, W. H. MacKenzie, Ecosystems 13, 108 (2010). 32. J. J. Verspoor, D. C. Braun, J. D. Reynolds, Ecosystems 13, 1020 (2010).

CD40 Agonists Alter Tumor Stroma and Show Efficacy Against Pancreatic Carcinoma in Mice and Humans Gregory L. Beatty,1,2,6 Elena G. Chiorean,3 Matthew P. Fishman,1 Babak Saboury,5 Ursina R. Teitelbaum,2,6 Weijing Sun,2,6 Richard D. Huhn,4 Wenru Song,4 Dongguang Li,4 Leslie L. Sharp,4 Drew A. Torigian,2,5 Peter J. O’Dwyer,2,6 Robert H. Vonderheide1,2,6* Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly in pancreatic ductal adenocarcinoma (PDA). Because CD40 activation can reverse immune suppression and drive antitumor T cell responses, we tested the combination of an agonist CD40 antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable PDA and observed tumor regressions in some patients. We reproduced this treatment effect in a genetically engineered mouse model of PDA and found unexpectedly that tumor regression required macrophages but not T cells or gemcitabine. CD40-activated macrophages rapidly infiltrated tumors, became tumoricidal, and facilitated the depletion of tumor stroma. Thus, cancer immune surveillance does not necessarily depend on therapy-induced T cells; rather, our findings demonstrate a CD40-dependent mechanism for targeting tumor stroma in the treatment of cancer. ancreatic ductal adenocarcinoma (PDA) remains an almost universally lethal disease; chemotherapy offers minimal benefit over best supportive care for patients who are not surgical candidates (1). We have previously demonstrated that leukocytes actively infiltrate the stromal compartment of PDA, even at the earliest stages of tumor development, and orchestrate an immune reaction that is immunosuppressive (2). In this study, we investigated the reversibility of this immune suppression, hy-

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pothesizing initially that doing so would unleash tumor-specific cellular immunity to eliminate PDA. Activation of the tumor necrosis factor (TNF) receptor superfamily member CD40 has been shown to be a key regulatory step in the development of T cell–dependent antitumor immunity, which relies on CD40-mediated “licensing” of antigen-presenting cells (APCs) for tumorspecific T cell priming and activation (3–8). On the basis of this premise, we investigated in both humans and mice whether systemic CD40

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33. R. E. Bilby, B. R. Fransen, J. K. Walter, C. J. Cederholm, W. J. Scarlett, Fisheries 26, 6 (2001). 34. DFO (Fisheries and Oceans Canada, Vancouver, 2005). 35. C. T. Darimont et al., Conserv. Lett. 3, 379 (2010). 36. D. E. Schindler et al., Nature 465, 609 (2010). 37. Supported by the Tom Buell Endowment Fund at Simon Fraser University (SFU), a Natural Science and Engineering Research Council of Canada (NSERC) postdoctoral fellowship (M.D.H.), NSERC Discovery and Accelerator grants (J.D.R.), the B.C. Leading Edge Endowment Fund, the Pacific Salmon Foundation, the B.C. Pacific Salmon Forum, and Mountain Equipment Co-op. We thank C. Aries for plant surveys; D. Braun, A. Cooper, E. Darling, N. Dulvy, R. Field, J. Harding, J. Linton, M. Stubbs, W. Palen, J. Verspoor, and the Earth2Ocean Research Group at SFU for discussions and analytical support; A. Albright, J. Barlow, J. Beaudin, J. Gordon-Walker, I. Jansma, E. Nelson, M. Spoljaric, J. Wilson, and the Raincoast Conservation Foundation for field support; R. Carpenter, M. Reid, and others at the Heiltsuk Integrated Resource Management Department; L. Jorgenson from Qqs Projects Society in Bella Bella; and the Heiltsuk Nation for research partnerships in their traditional territory.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1609/DC1 Materials and Methods SOM Text Figs. S1 to S5 Tables S1 to S13 References 30 November 2010; accepted 25 January 2011 10.1126/science.1201079

activation with an agonist CD40 monoclonal antibody (mAb) can circumvent tumor-induced immune suppression and invoke productive T cell–dependent antitumor immunity in PDA. We first evaluated the clinical impact of CD40 activation by performing a clinical trial of the fully human agonist CD40 mAb CP-870,893 (9) in combination with gemcitabine chemotherapy (2′-deoxy-2′,2′-difluorocytidine) for patients with chemotherapy-naïve, surgically incurable PDA (10). We tested CP-870,893 with gemcitabine because chemotherapy delivered before an agonist CD40 mAb can facilitate enhanced tumor antigen presentation by APCs (11–14). Twenty-one patients (90% with metastatic disease) received gemcitabine weekly on days 1, 8, and 15 with CP-870,893 administered on day 3 of each 28day cycle (fig. S1). Treatment was well tolerated overall (fig. S2), and the most common side effect was mild-to-moderate cytokine release syndrome characterized by chills, fevers, rigors, and other symptoms on the day of CP-870,893 1 Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104, USA. 2Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 3Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. 4Pfizer Corporation, New London, CT 06320, USA. 5Department of Radiology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. 6Division of Hematology-Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

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

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infusion. Symptoms were transient (<24 hours) and managed in the outpatient clinic with supportive care. The impact of therapy on the primary and metastatic lesions was evaluated using two standard modalities: (i) [18F]2-fluoro-2-deoxyD-glucose (FDG) avidity on positron emission tomography–computed tomography (PET-CT) as a measure of an on-target biological effect of therapy and (ii) CT imaging for Response Evaluation Criteria in Solid Tumors (RECIST) assessment. Metabolic responses were observed by FDG–PET-CT in both the primary and metastatic lesions in 88% of patients evaluated after two cycles of therapy (fig. S3). By RECIST, 4 out of 21 patients developed a partial response (PR), 11 patients had stable disease (SD), and 4 patients had progressive disease (PD) (Fig. 1A). Two patients were not evaluable on study for tumor response. One of these patients (patient 10031010) was taken off the study protocol after one dose of CP-870,893 because of a grade 4 cerebrovascular accident but recovered neurologically and restarted gemcitabine alone and achieved a PR. The second patient (patient 10031001) had clinical deterioration from disease progression, and posttherapy CT imaging was not obtained. The median progression-free survival (PFS) for the 21 patients was 5.6 months (95% confidence interval, 4.0 months to not estimable), and the median overall survival (OS) was 7.4 months (95% confidence interval, 5.5 to 12.8 months). Median PFS and OS are based

on interim data as of 25 August 2010, with 6 of 21 patients alive. Gemcitabine alone achieves a historical tumor response rate of 5.4%, with median PFS of 2.3 months and median OS of 5.7 months (1). Therefore, treatment with CP870,893 and gemcitabine showed therapeutic efficacy in patients with metastatic PDA. One patient with a PR showed a 46% reduction in the primary pancreatic lesion, complete resolution of a 7.6-cm hepatic metastasis, and 47% reduction in the one remaining hepatic metastasis (Fig. 1B). Upon biopsy after four cycles of therapy, this remaining liver lesion showed no viable tumor. Instead, we observed necrosis and an infiltrate dominated by macrophages with an absence of lymphocytes (Fig. 1C). A second patient with a PR underwent surgical resection of the primary tumor after achieving a complete resolution of all hepatic metastases and a 64% reduction in the primary pancreatic lesion. Histological analysis of the resected primary lesion revealed a cellular infiltrate devoid of lymphocytes (Fig. 1C). Such tumor regression without lymphocyte infiltration was unexpected on the basis of results from implantable tumor models, where CD40-induced antitumor activity is dependent on T cells (6–8). Therefore, to understand the mechanism of CD40mediated tumor regression, we turned to a spontaneous mouse model of PDA. The KPC model of PDA is a genetically engineered mouse model that incorporates the conditional expression of both mutant KrasG12D

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and p53R172H alleles in pancreatic cells (15). Because immunocompetent KPC mice spontaneously develop PDA tumors that display high fidelity to the histopathologic and molecular features of human PDA (15), the KPC model is thought to be relevant for understanding treatment mechanisms in patients (16, 17). We therefore modeled our clinical study in KPC mice by evaluating the therapeutic efficacy of gemcitabine weekly with the agonist CD40 mAb FGK45 administered 48 hours after the first infusion of gemcitabine. Tumor response was monitored by three-dimensional ultrasonography (fig. S4) (10). We found that the combination of FGK45 with gemcitabine induced tumor regression in 30% of mice (Fig. 2A), similar to the objective response rate in our patients. Treatment with FGK45 alone resulted in the same rate of tumor regression, whereas gemcitabine alone did not (Fig. 2A). Activation of the CD40 pathway is a critical event in the development of tumor-specific T cell immunity (18). To test whether CD40 immunotherapy is dependent on T cell activation, we examined the function of T cells isolated from the spleen and pancreas (including tumor and peripancreatic lymph nodes) of KPC mice. Seven days after treatment with FGK45, both CD4+ and CD8+ T cells of KPC mice acquired an increased capacity to secrete cytokines, including interferon-g (IFN-g) and interleukin IL17A (fig. S5A); however, FGK45 induced the same rate of tumor regression in KPC mice in the absence of CD4+ or CD8+ cells or both CD4+

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Fig. 1. Agonist CD40 mAb in combination with gemcitabine 10031016 10061003 induces clinical responses in patients with surgically incurable PDA. C Liver Metastasis Primary Tumor (A) Best overall percentage of change from baseline in tumor target lesion measurement shown as a waterfall plot. *Patient 10061001 was defined as PD because of the appearance of a new nontarget lesion. **Patient 10031001 did not obtain posttherapy scans because of clinical deterioration from disease progression after one dose of CP-870,893. ***Patient 10031010 came off the study after one dose of CP-870,893 but restarted gemcitabine alone and achieved a PR. (B) CT imaging obtained at baseline and end of cycle 3. The primary pancreatic tumor and two metastatic liver lesions are marked by arrows, with the longest dimension annotated. (C) Histopathology of a biopsied metastatic lesion [from patient 10031016 (left)] and a surgically resected primary pancreatic lesion [from patient 10061003 (right)]. Both patients achieved a PR. Patient 10031016 underwent tumor biopsy after completing four cycles of therapy; patient 10061003 underwent surgical resection of the primary tumor after 12 cycles of therapy. Arrows (left) indicate a macrophage-dominated inflammatory infiltrate within extensive tumor necrosis. Arrows (right) identify polymorphonuclear infiltrating cells without lymphocytes; arrowheads mark tumor cells. Scale bars, 50 mm. www.sciencemag.org

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REPORTS ilar overall, with the exception that tumors were smaller in responder animals (Fig. 2, B to E). CD3+ T cells were not observed to infiltrate tumors in KPC mice at baseline or at any time after treatment with FGK45 (Fig. 2, F to H). Instead, CD3+ T cells remained localized to

Fig. 2. Antitumor activity of A agonist CD40 mAb in KPC mice is T cell–independent. (A) KPC mice were treated with gemcitabine or phosphatebuffered saline (PBS) on day 0 and day 7, with control IgG2a or FGK45 administered on day 2. Cohorts of KPC mice receiving treatment with FGK45 were also depleted of CD4+ or CD8+ cells or both CD4+ and CD8+ cells with the use of GK1.5 and 2.43 antibodies, respectively, on days –1, 0, 1, 3, 7, and 10. Percent change in tumor volume from day –1 (baseline) to day +14 is shown for each mouse as a waterfall plot (in comparison with PBS + IgG2a, gemcitabine + FGK45: P < 0.05; gemcitabine + IgG2a: P = 1.00; PBS + FGK45: P < 0.05; FGK45 + GK1.5: P < 0.05; FGK + 2.43: P < 0.05; FGK45 + GK1.5 + 2.43: P < 0.05; Fisher’s exact test). Hematoxylin and eosin (H&E) histology [(B) to (E)] and CD3 immunohistochemistry [(F) to (I)] are shown for tumors from KPC mice treated with IgG2a (B and F) or FGK45 (C to E and G to I). Responders (D), (E), (H), and (I) were defined as FGK45-treated mice that demonstrated tumor regression by ultrasound analysis. FGK45-treated mice with tumor progression by ultrasound were defined as nonresponders (C) and (G). Scale bars, 50 mm. Fig. 3. Agonist CD40 A mAb targets systemic macrophages before their infiltration of tumor. Immunohistochemistry was used to quantify leukocyte infiltration (A) into peripancreatic lymph nodes (A, B, and F) and KPC tumor (A, C, D, E, and G) by staining with a biotinylated goat antibody directed against rat IgG to detect FGK45 or RB68C5 bound to cells at 10 min (A) to (D) or 18 hours (A) and (E) to (G) after treatment. Macrophages were depleted with CELs before FGK45 treatment (A) and (G). LN, peripancreatic lymph node; T, tumor; scale bars, 50 mm. Error bars in (A) represent SD. (H) A heat map displaying the flow cytometric analysis of tumorassociated macrophages from individual KPC mice (columns) for expression of cell surface molecules (rows) 3 days after treatment with control IgG2a compared with FGK45. (Top) The percentage of tumor-associated macrophages with surface molecule expression. (Bottom) Mean fluorescence intensity as the

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peripancreatic lymph nodes situated adjacent to developing tumors (Fig. 2, E and I). Moreover, when the whole pancreas was resected en bloc with peripancreatic lymph nodes, CD3+ T cells did not change in frequency before and after therapy (fig. S5, B and C). Thus, activation of

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number of standard deviations from the mean, which was determined from treatment with control IgG2a. P values are based on Student’s t test; nd, not determined. SCIENCE

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and CD8+ cells (Fig. 2A). This was unexpected given the established link between CD40 activation and the development of productive antitumor T cell immunity (6–8). Histopathological appearance of tumors from responder compared with nonresponder animals at day 14 was sim-

the CD40 pathway does not trigger T cell infiltration into tumors and is insufficient to induce productive antitumor T cell immunity in the KPC model. CD40 is expressed on a wide range of leukocytes including monocytes, tissue macrophages, B cells, and dendritic cells and can also be found on some tumors (19). By immunohistochemistry, we found that PDA cells were CD40-negative, whereas cells in the tumor stroma expressed CD40, particularly F4/80+ tumorassociated macrophages (fig. S6). We therefore investigated the impact of CD40 immunotherapy on F4/80+ macrophages in KPC mice. Within 18 hours of administration, FGK45 induced a transient depletion of macrophages from the peripheral blood, with subsequent accumulation in the spleen (fig. S7, A and B). To test whether CD40 mAb initially engages CD40 on peripheral blood macrophages, which then migrate into tissues, we compared the biodistribution of FGK45 with that of RB6-8C5, a mAb used as a control and specific for Gr-1 expressed on granulocytes and immature myeloid cells in peripheral blood (fig. S7, C and D). Within 2 to

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10 min after systemic administration of RB68C5, Gr-1+ cells in the peripheral blood were found to be coated with antibody (fig. S8A). In contrast, FGK45 was not found on the cell surface of F4/80+ macrophages in peripheral blood (fig. S8A) nor was FGK45 internalized after binding to CD40 (fig. S8B). Instead, at 10 min, FGK45 was observed bound to leukocytes residing within the spleen, lymph nodes, and pancreas of KPC mice (fig. S9A). Within the tumor microenvironment, FGK45 was found initially to localize to peripancreatic lymph nodes (Fig. 3, A and B) and not the tumor (Fig. 3, A and C), in contrast to RB6-8C5, which bound leukocytes in the tumor periphery as early as 10 min after treatment (Fig. 3D). At 18 hours, FGK45 was observed bound to F4/80+ macrophages infiltrating the tumor but not lymph nodes (Fig. 3, A, E, and F; and fig. S9, B and C). The binding of FGK45 to leukocytes within the tumor microenvironment was found to occur in clusters of cells rather than diffusely. We observed no change in the frequency of macrophages in the pancreas after FGK45 treatment (fig. S10). To determine whether FGK45 binds to macro-

phages before their infiltration of tumors, we treated KPC mice with clodronate-encapsulated liposomes (CELs) to deplete systemic macrophages (fig. S11A). Because liposomes are not capable of traversing vascular barriers produced by capillary walls, CELs do not diffuse into tissues (20) and, therefore, do not deplete macrophages within tumors (fig. S11, B to E). When KPC mice were treated with CELs, however, FGK45 labeling was no longer detectable in the tumors (Fig. 3, A and G), despite the continued presence of CD40+ macrophages within the tumor. These findings support the hypothesis that FGK45 binds to macrophages before their migration into tumors. Depending on their phenotype, macrophages can either promote or inhibit tumor progression (21, 22). In the absence of any treatment, we found that tumor-associated macrophages were activated and capable of secreting IL-10, TNF-a, and IL-6 but had reduced expression of MHC class II molecules compared with splenic macrophages from tumor-bearing KPC mice and normal littermates (fig. S12). After treatment with FGK45, macrophages in KPC tumors and spleens

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Fig. 4. CD40 activated C tumor-infiltrating macrophages mediate tumor regression. (A) KPC mice were treated with control IgG2a or FGK45 or FGK45 plus depletion of systemic macrophages by means of CELs. Shown is a waterfall plot displaying percent change in tumor volume from baseline to day +14 (in comparison with IgG2a, FGK45: P < 0.05; FGK45 + CELs: P = 1.00; Fisher’s exact test). (B) F4/80+ tumor-associated macrophages were isolated from KPC mice that had been treated with FGK45 (blue squares) or control IgG2a (green circles) and incubated with KPCderived tumor cell lines. Tumor cell death in vitro was measured by 7-aminoactinomycin D (7-AAD) labeling and flow cytometric analysis at 24 hours. Shown is a representative assay from two independent experiments each performed in triplicate. Means T SD are depicted; *P < 0.05, Student’s t test. (C) Cleaved caspase 3 expression on KPC tumors was determined by immunohistochemistry 18 hours after treatment with control IgG2a (top panel) or FGK45 (bottom panel). (D) to (L) www.sciencemag.org

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KPC tumors were analyzed 18 hours after treatment with control IgG2a (D, F, and H), FGK45 (E, G, and I), or FGK45 + CEL (J, K, and L). Shown are hematoxylin-and-eosin histology (D, E, J); Masson’s trichrome stain to reveal extracellular matrix in blue [(F), (G), and (K)], and immunohistochemistry for collagen I [(H), (I), and (L)]. Scale bars, 50 mm. VOL 331

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in tumor-bearing KPC mice up-regulated MHC class II and the costimulatory molecule CD86 with expression levels peaking at day 3 and returning to baseline after 7 days (Fig. 3H, and figs. S13 and S14). These changes coincided with a cytokine surge on day 1 after FGK45 treatment, with elevated serum levels of IL-12, TNF-a, and IFN-g, but not IL-10, in KPC animals (fig. S15). To determine whether macrophages were necessary for CD40-mediated tumor regression, we depleted systemic macrophages from KPC mice with CELs. Treatment with CELs abolished the capacity of FGK45 to induce tumor regression (Fig. 4A). Macrophages isolated from the pancreas of tumor-bearing KPC animals treated in vivo with FGK45 lysed tumor cells in vitro (Fig. 4B). This finding correlated with in vivo observations of cleaved caspase 3 expression in focal areas of the tumor at 18 hours after treatment with FGK45 (Fig. 4C). At this time after treatment, regions of the tumor stroma and associated fibrosis appeared to be undergoing involution (Fig. 4, D to G). These regions displayed a decrease in collagen I content, consistent with degradation of the tumor matrix (Fig. 4, H and I). In KPC mice depleted of systemic macrophages using CELs, FGK45 treatment failed to induce stromal degradation (Fig. 4, J to L). These findings identify a novel mechanism whereby the CD40 pathway can be harnessed therapeutically to restore tumor immune surveillance by targeting tumor-infiltrating macrophages involved in cancer inflammation. PDA is a common, devastating, and highly lethal tumor for which new therapies are critically needed. Our findings identify a previously

unappreciated role for the CD40 pathway in regulating the immune reaction and fibrosis associated with PDA by reeducation of tumorassociated macrophages. Mechanistically, CD40 agonists altered tumor stroma and, in both mice and humans, showed efficacy against PDA. Although tumor-suppressing macrophages have been previously described (22), their role has been largely linked to the orchestration of T cell antitumor immunity. In this study, CD40 activation was, by itself, insufficient for invoking productive antitumor T cell immunity, and we hypothesize that full engagement of T cell immunity in PDA after CD40 activation will require modulation of additional tumor and host factors or the incorporation of novel vaccines (23). Our results emphasize that tumor immunosurveillance can at times be governed strictly by innate immunity under the regulation of the CD40 pathway and support the continued development of emerging therapeutic strategies that target inflammatory cells and stroma within the tumor microenvironment. References and Notes 1. H. A. Burris 3rd et al., J. Clin. Oncol. 15, 2403 (1997). 2. C. E. Clark et al., Cancer Res. 67, 9518 (2007). 3. S. P. Schoenberger, R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief, Nature 393, 480 (1998). 4. S. R. Bennett et al., Nature 393, 478 (1998). 5. J. P. Ridge, F. Di Rosa, P. Matzinger, Nature 393, 474 (1998). 6. R. R. French, H. T. Chan, A. L. Tutt, M. J. Glennie, Nat. Med. 5, 548 (1999). 7. L. Diehl et al., Nat. Med. 5, 774 (1999). 8. E. M. Sotomayor et al., Nat. Med. 5, 780 (1999). 9. R. H. Vonderheide et al., J. Clin. Oncol. 25, 876 (2007).

Cortical Constriction During Abscission Involves Helices of ESCRT-III–Dependent Filaments Julien Guizetti,1,2 Lothar Schermelleh,3 Jana Mäntler,4 Sandra Maar,1 Ina Poser,4 Heinrich Leonhardt,3 Thomas Müller-Reichert,5,2* Daniel W. Gerlich1,2* After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)–III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III–mediated fission of large membrane structures.

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bscission represents the very final step of cell division in animal cells whereby the two daughter cells are physically

severed from one another. The mechanism of abscission is poorly understood (1–3), but it may involve mechanical tearing (4 ) followed by

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10. Materials and methods are available as supporting material on Science Online. 11. A. K. Nowak, B. W. Robinson, R. A. Lake, Cancer Res. 63, 4490 (2003). 12. R. A. Lake, B. W. Robinson, Nat. Rev. Cancer 5, 397 (2005). 13. D. I. Gabrilovich, Lancet Oncol. 8, 2 (2007). 14. L. Zitvogel et al., J. Clin. Invest. 118, 1991 (2008). 15. S. R. Hingorani et al., Cancer Cell 7, 469 (2005). 16. K. P. Olive et al., Science 324, 1457 (2009). 17. T. Van Dyke, Nat. Med. 16, 976 (2010). 18. R. H. Vonderheide, Clin. Cancer Res. 13, 1083 (2007). 19. C. van Kooten, J. Banchereau, J. Leukoc. Biol. 67, 2 (2000). 20. N. Van Rooijen, A. Sanders, J. Immunol. Methods 174, 83 (1994). 21. L. M. Coussens, Z. Werb, Nature 420, 860 (2002). 22. A. Mantovani, A. Sica, Curr. Opin. Immunol. 22, 231 (2010). 23. E. M. Jaffee et al., J. Clin. Oncol. 19, 145 (2001). 24. We thank C. Abrams, E. Furth, C. June, B. Keith, G. Koretzky, C. Simon, and B. Stanger for helpful discussions. This research was supported by the Abramson Family Cancer Research Institute of the University of Pennsylvania School of Medicine (R.H.V.) and by NIH grants P30 CA016520 (P.J.O. and R.H.V.) and K12 CA076931 (G.L.B.). The clinical study and part of the preclinical studies were supported by funding from Pfizer Corp (to R.H.V. and P.J.D). W. Song, D. Li, and L. L. Sharp are employees of Pfizer Corp. P. J. O’Dwyer discloses receiving honoraria from Pfizer; D. A. Torigian owns stock in Pfizer. CP-870,893 is owned and patented by Pfizer Corp., which manages its distribution. Although now at a new address, R. D. Huhn was affiliated with Pfizer Corp. during the conduct and analysis of this study. Some of the clinical data were presented at the 2010 American Society of Clinical Oncology Annual Meeting.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1612/DC1 Materials and Methods Figs. S1 to S15 References 29 September 2010; accepted 14 February 2011 10.1126/science.1198443

plasma membrane wound healing (5). An alternative model proposes that Golgi- (6) or recycling endosome (7 )–derived vesicles establish membrane separation from within the intercellular bridge. To clarify which events lead to abscission, we imaged live HeLa cells stably expressing enhanced green fluorescent protein (EGFP)–atubulin (Fig. 1, A and B) (8). At the intercellular bridge, microtubule bundles gradually narrowed to a diameter of 0.97 T 0.13 mm (mean T SD; n = 17 cells) and then disassembled on one side 1 Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology Zurich (ETHZ), Schafmattstrasse 18, CH-8093 Zurich, Switzerland. 2Marine Biological Laboratory (MBL), Woods Hole, MA 02543, USA. 3Department of Biology, Center for Integrated Protein Science Munich, Ludwig Maximilians University Munich, Grosshaderner Strasse 2, D-82152 Planegg-Martinsried, Germany. 4Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany. 5Medical Theoretical Center, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, D-01307 Dresden, Germany.

*To whom correspondence should be addressed. E-mail: [email protected] (D.W.G.); mueller-reichert@ tu-dresden.de (T.M.-R.)

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REPORTS tubule disassembly (n = 9), the cortex of the intercellular bridge had ingressed to a narrow stalk, which contained a tightly compressed bundle of microtubules (constriction zone in Fig. 1, F and G, and fig. S1). The constriction site cortex was deformed by regularly spaced electron-dense ripples (Fig. 1G), a previously observed structural feature of intercellular bridges (10). Ripples were absent in earlier stages (n = 5 cells fixed <40 min after furrow ingression; fig. S2). The constriction zone localized 0.95 T 0.41 mm (n = 19) from the center of the midbody. Postabscission midbody remnants contained cytoplasmic regions of matching size (fig. S3), indicating that the

constriction zone is the site of abscission. Thus, assembly of a specialized cortical structure in late-stage intercellular bridges mediates abscission by cortical constriction adjacent to the midbody. We did not detect vesicles at constriction zones (Fig. 1, F and G, and fig. S1). Live-cell imaging of the secretory vesicle marker EGFP-Rab8 (11) showed that Golgi-derived vesicles gradually disappeared from the intercellular bridge long before abscission (Fig. 2, A and B). Disruption of the Golgi apparatus using brefeldin A (12) prevented EGFP-Rab8 targeting to the intercellular bridge (fig. S4) but did not perturb abscission (Fig. 2C). Thus, abscission proceeds by

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adjacent to the midbody. The first microtubule bundle disassembly occurred 49 T 10 min after complete furrow ingression, followed by disassembly of the second bundle ~20 min later. In an abscission timing assay based on cytoplasmic exchange of photoactivatable GFP (PAGFP) between the two sister cells (9), the complete disassembly of the first microtubule bundle coincided with abscission in most cells (45 of 51 within a 5-min sampling time; Fig. 1, C to E). To observe abscission, we imaged live cells until partial disassembly of microtubules at the intercellular bridge occurred, and then we chemically fixed cells for serial thin-section electron microscopy. At all sites of partial micro-

Fig. 1. Abscission proceeds by cortical constriction. (A) Confocal live imaging of HeLa cells expressing EGFP–a-tubulin (first microtubule bundle disassembly at t = 0 min; movie S1). (B) Microtubule bundle diameter in 17 cells. (C) Assay for abscission timing. Repetitive PAGFP photoactivation during imaging (dashed circle). (D) Measured mean fluorescence as labeled in (C) (abscission at t = 0 min). r.u. indicates www.sciencemag.org

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relative units. (E) First microtubule (MT) bundle disassembly coincides with abscission, assayed as in (A) to (D). (F) Correlative live-cell imaging (insets) and transmission electron microscopy. Arrowhead indicates microtubule disassembly. (G) Enlarged serial sections of (F). Arrowheads indicate membrane ripples. Scale bars, 5 mm [(A) and (C)] and 200 nm [(F) and (G)]. VOL 331

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a cortical ingression mechanism independent of Golgi-derived secretion. Cortical constriction requires removal of underlying microtubules. As reported (13, 14), RNA interference (RNAi) depletion of the microtubule-severing enzyme spastin delayed abscission (Fig. 2D and fig. S5, A to C). However, spastin-depleted cells still contained constriction zones with electron-dense ripples (Fig. 2E; n = 7). To test indirect consequences of perturbed microtubule homeostasis in spastin-depleted cells, we added the microtubule-depolymerizing compound T138067 (15) after furrow ingression. This led to efficient suppression of the spastin RNAiinduced abscission delay (Fig. 2D). Thus, microtubules are not required for abscission once the intercellular bridge has formed, and their spastinmediated disassembly is a rate-limiting step of abscission. Super-resolution imaging with three-dimensional structured illumination microscopy (16 ) revealed

that F-actin, a candidate factor for cortical constriction, accumulated adjacent to early-stage midbodies (Fig. 2F and fig. S6A). At later stages, however, F-actin localized only to distal ends of the intercellular bridge, and it was undetectable at constriction sites in 12 out of 14 cells (Fig. 2G and fig. S6B). Depolymerization of F-actin by latrunculin B added after furrow ingression had no effect on abscission (Fig. 2C). Thus, F-actin is unlikely to contribute to cortical constriction during abscission. Endosomal sorting complex required for transport III (ESCRT-III) is an attractive candidate cortical constriction factor, with known membranedeforming activity in reconstituted vesicle budding (17–19). ESCRT-III is required for abscission, and it localizes within intercellular bridges (20, 21). We observed the EGFP-tagged CHMP4B core ESCRT-III subunit, stably expressed from an endogenous promotor, to be at very low levels within early-stage intercellular bridges. Then its

levels increased dramatically until disassembly of the first midbody-associated microtubule bundle (Fig. 3, A and B, and fig. S7, A and B). CHMP4B localized to two narrow cortical rings adjacent to the midbody before microtubule disassembly (Fig. 3C; 13 of 15). Upon microtubule disassembly, CHMP4B and other ESCRT-III core subunits extended toward the constriction site (six of six; Fig. 3D and figs. S7 and S8). Thus, ESCRT-III accumulates in late-stage intercellular bridges as a component of cortical constriction zones. To test ESCRT-III function in cortical constriction, we used RNAi to deplete the CHMP2A core subunit (fig. S5, D and E), and we performed correlative live-cell and electron microscopy. Early midbodies (<40 min after furrow ingression) of CHMP2A-depleted cells had normal morphology (fig. S5F). However, late-stage intercellular bridges (>40 min after furrow ingression) were always devoid of rippled constric-

Fig. 2. Abscission does not require Golgi-derived secretion or F-actin but depends on spastin-mediated microtubule disassembly. (A) Confocal live imaging of HeLa cell expressing Rab8-EGFP and mRFP–atubulin (mRFP is monomeric red fluorescent protein red fluorescent protein). t = 0 min indicates disassembly of the first microtubule bundle (arrowhead). (B) Intercellular bridge Rab8-EGFP fluorescence quantification. Line indicates mean; bars indicate SEM of n = 14 cells. (C) Golgi disruption by 10 mg/ml brefeldin A (added 3 hours before imaging; fig. S4) or 5 mM latrunculin B (added after furrow ingression) does not perturb abscission. Cumulative abscission histogram assayed as in Fig. 1, C and D [complete furrow ingression at t = 0 min; n = 40 for dimethyl sulfoxide (DMSO); n = 41 for brefeldin A; n = 23 for latrunculin B]. (D) Spastin-RNAi (fig. S5, A to C) or control (siScr) cells treated by 2 mM T138067 or DMSO after furrow ingression and probed as in (C). (E) Correlative time-lapse and electron microscopy as in Fig. 1F for a spastin RNAi cell fixed 66 min after furrow ingression. (F and G) Three-dimensional structured illumination microscopy of early-stage (F) or late-stage (G) intercellular bridges stained with fluorescent phalloidin and antibody against a-tubulin (anti–a-tubulin). Scale bars, 1 mm [(A), (F), and (G)] and 200 nm (E).

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tion zones (Fig. 3E; n = 16). Thus, ESCRT-III is required for cortical constriction. Microtubule depolymerization by T138067 when added after furrow ingression did not restore abscission in CHMP2A-depleted cells (Fig. 3F). Thus, microtubule disassembly is not sufficient to split the intercellular bridge, suggesting a direct function of ESCRT-III in abscission, in addition to its known requirement for spastin targeting (14). ESCRT-III–mediated cortical constriction needs coordination with microtubule disassembly. Consistent with this, spastin was 8.7 T 0.9 times more abundant on the side of the midbody that disassembled microtubule bundles (Fig. 3, G and H; n = 16). This supports a model of spatially and temporally coordinated ESCRT-III–driven cortical constriction and spastin-mediated microtubule disassembly. ESCRT-III targeting to the intercellular bridge depends on centrosome protein 55 (CEP55) and

the ESCRT-related protein ALIX (21–23). We found that both EGFP-tagged ALIX (Fig. 3I, 19 of 22) and CEP55 (fig. S8, G and H; 25 of 25) were confined to the midbody, whereas ESCRTIII was excluded from internal midbody regions (Fig. 3, C and D). CEP55 accumulated at the midbody earlier than CHMP4B and ALIX (Fig. 3B and fig. S8, I and J). This indicates a mechanism of sequential targeting to the midbody periphery underlying the assembly of ESCRT-III constriction zones. We next performed electron tomography of high-pressure frozen HeLa cells at the abscission stage. The constriction site contained cortical filaments oriented perpendicular to the underlying microtubules (Fig. 4, A to F). These filaments were 17.3 T 2.5 nm (mean T SD; n = 60 measurements at five constriction zones) wide and regularly spaced at 35.3 T 4.1 nm distances. The cortical filaments formed single or intertwined helices spanning the intercellular bridge (Fig. 4F

and fig. S9; n = 4 constriction zones). Thus, a helical filament system may generate contractile force to narrow the membrane tube of the intercellular bridge. Because of the known polymerization and membrane-deforming activity of ESCRT-III (17, 24–27) and because of our observed colocalization of ESCRT-III with constriction zones, we speculate that the 17-nm-diameter filaments may be composed of polymerized ESCRT-III core components. Supporting this idea, the 17-nm-diameter filaments did not occur in intercellular bridges of high-pressure frozen CHMP2A RNAi cells (n = 7 late-stage intercellular bridges; fig. S10). In contrast, spastin RNAi did not perturb 17-nm filaments (fig. S11). Our study reveals a cortical constriction mechanism of abscission involving ESCRT-III–dependent membrane deformation and spastin-mediated microtubule disassembly (fig. S12). Vesicles, microtubules, and F-actin are not directly required

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Fig. 3. ESCRT-III is required for cortical constriction. (A) Confocal time-lapse imaging of HeLa cell expressing CHMP4BEGFP and mRFP–a-tubulin. Arrowhead indicates disassembly of first midbodyassociated microtubule bundle (t = 0 min; movie S2). (B) CHMP4B-EGFP fluorescence quantification at the intercellular bridge. Line and error bars indicate mean and SEM for n = 16 cells. (C) Structured illumination microscopy of mid-stage intercellular bridge. HeLa cell stably expressing CHMP4B-EGFP was stained with anti–a-tubulin. Maximum intensity projections; dashed lines indicate cross sections. Dark zone indicates midbody position (movie S3). (D) As in (C) for late stage (arrowhead marks constriction zone; movie S4). (E) Electron micrograph of a CHMP2A-RNAi cell fixed 166 min after furrow ingression. (F) T138067 (2 mM) or DMSO was added to CHMP2A-RNAi or control (siScr) cells after furrow ingression and probed as in Fig. 2C. (G) Wide-field microscopy of spastin and a-tubulin immunofluorescence in CHMP4B-EGFP–expressing cell. Arrowhead indicates maximal constriction. (H) Structured illumination microscopy of the cell shown in (G). Maximum intensity projections; dashed line indicates cross section. (I) Structured illumination microscopy of ALIX-EGFP and anti–a-tubulin immunofluorescence of a late-stage intercellular bridge. Scale bars, 1 mm [(A), (C), (D), and (G) to (I)] and 200 nm (E). www.sciencemag.org

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for abscission in HeLa cells, yet our data do not exclude the possibility that they function in preparatory steps. Our data are consistent with a model in which ESCRT-III mediates membrane tube constriction as filament helices spanning the ~1-mm-wide intercellular bridge. Possible forcegenerating mechanisms could emerge during assembly of filaments from inward-directed filament curvature, from lateral skew of sliding helices, or from gradual removal of monomers from filaments. References and Notes 1. F. A. Barr, U. Gruneberg, Cell 131, 847 (2007). 2. P. Steigemann, D. W. Gerlich, Trends Cell Biol. 19, 606 (2009). 3. U. S. Eggert, T. J. Mitchison, C. M. Field, Annu. Rev. Biochem. 75, 543 (2006). 4. K. Burton, D. L. Taylor, Nature 385, 450 (1997). 5. H. Darenfed, C. A. Mandato, Biochem. Cell Biol. 83, 711 (2005). 6. A. Gromley et al., Cell 123, 75 (2005). 7. G. Montagnac, A. Echard, P. Chavrier, Curr. Opin. Cell Biol. 20, 454 (2008). 8. Materials and methods are available as supporting material on Science Online. 9. P. Steigemann et al., Cell 136, 473 (2009). 10. J. M. Mullins, J. J. Biesele, J. Cell Biol. 73, 672 (1977).

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11. A. L. Ang, H. Fölsch, U. M. Koivisto, M. Pypaert, I. Mellman, J. Cell Biol. 163, 339 (2003). 12. R. D. Klausner, J. G. Donaldson, J. Lippincott-Schwartz, J. Cell Biol. 116, 1071 (1992). 13. J. W. Connell, C. Lindon, J. P. Luzio, E. Reid, Traffic 10, 42 (2009). 14. D. Yang et al., Nat. Struct. Mol. Biol. 15, 1278 (2008). 15. B. Shan et al., Proc. Natl. Acad. Sci. U.S.A. 96, 5686 (1999). 16. L. Schermelleh et al., Science 320, 1332 (2008). 17. T. Wollert, C. Wunder, J. Lippincott-Schwartz, J. H. Hurley, Nature 458, 172 (2009). 18. S. Saksena, J. Wahlman, D. Teis, A. E. Johnson, S. D. Emr, Cell 136, 97 (2009). 19. J. H. Hurley, P. I. Hanson, Nat. Rev. Mol. Cell Biol. 11, 556 (2010). 20. E. Morita et al., EMBO J. 26, 4215 (2007). 21. J. G. Carlton, J. Martin-Serrano, Science 316, 1908 (2007); 10.1126/science.1143422. 22. H. H. Lee, N. Elia, R. Ghirlando, J. Lippincott-Schwartz, J. H. Hurley, Science 322, 576 (2008). 23. J. G. Carlton, M. Agromayor, J. Martin-Serrano, Proc. Natl. Acad. Sci. U.S.A. 105, 10541 (2008). 24. P. I. Hanson, R. Roth, Y. Lin, J. E. Heuser, J. Cell Biol. 180, 389 (2008). 25. S. Ghazi-Tabatabai et al., Structure 16, 1345 (2008). 26. S. Lata et al., Science 321, 1354 (2008); 10.1126/ science.116107. 27. R. Pires et al., Structure 17, 843 (2009). 28. We thank A. E. Smith, M. Petronczki, P. Steigemann, M. Glotzer, and Y. Barral for comments on the

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manuscript; S. Prugnaller, A. Boden, G. Fabig, E. Seemann, A. Maiser, J. Neumann, L. Kerr, G. Csucs, and the ETHZ Light Microscopy and RNAi Screening Centres for technical support; U. Rothbauer, J. Gertsch, I. Mellman, B. Pfeiffer, and K. H. Altmann for providing reagents; and A. A. Hyman for support and reagents. This work was supported by Swiss National Science Foundation research grant 3100A0-114120, an ETH-TH grant, a European Young Investigators award of the European Science Foundation to D.W.G., MBL Research Fellowships by the Evelyn and Melvin Spiegel Fund to D.W.G. and T.M.R., a Human Frontier Science Program grant (RGP 0034/2010) to T.M.R., and grants from the Deutsche Forschungsgemeinschaft (MU 1423/2-1 and MU 1423/3-1 to T.M.R. and SFB TR5 to H.L. and L.S.) and from the BioImaging Network and the Nanosystems Initiative Munich to L.S. and H.L. Work in the Hyman and Gerlich laboratories is supported by European Commission grant agreement 241548 (MitoSys).

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1201847/DC1 Materials and Methods SOM Text Figs. S1 to S12 Movies S1 to S6 17 December 2010; accepted 31 January 2011 Published online 10 February 2011; 10.1126/science.1201847

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Fig. 4. Electron tomography of high-pressure frozen cells reveals cortical 17-nm-diameter filaments at constriction zone. (A) Tomographic z-sections of intercellular bridge at mid-abscission stage (movie S5). (B) As in (A) for late stage. (C to F) Three-dimensional reconstructions of abscission structures. Red, microtubules; green, 17-nmdiameter filaments; gray, midbody; yellow, plasma membrane; white balls, open microtubule ends; blue balls, closed microtubule ends. (C) Early stage before microtubule disassembly. (D) Mid-stage with compressed but largely intact microtubule bundle in cell shown in (A). (E) Late stage with almost-complete microtubule disassembly in cell shown in (B). (F) Model of another late-stage abscission site reveals intertwined helices of three cortical 17-nm-wide filaments labeled by different tones of green (movie S6). Scale bars, 200 nm.

FGF19 as a Postprandial, Insulin-Independent Activator of Hepatic Protein and Glycogen Synthesis Serkan Kir,1 Sara A. Beddow,2 Varman T. Samuel,2 Paul Miller,3* Stephen F. Previs,3* Kelly Suino-Powell,4 H. Eric Xu,4 Gerald I. Shulman,2,5 Steven A. Kliewer,1,6† David J. Mangelsdorf 1,7† Fibroblast growth factor (FGF) 19 is an enterokine synthesized and released when bile acids are taken up into the ileum. We show that FGF19 stimulates hepatic protein and glycogen synthesis but does not induce lipogenesis. The effects of FGF19 are independent of the activity of either insulin or the protein kinase Akt and, instead, are mediated through a mitogen-activated protein kinase signaling pathway that activates components of the protein translation machinery and stimulates glycogen synthase activity. Mice lacking FGF15 (the mouse FGF19 ortholog) fail to properly maintain blood concentrations of glucose and normal postprandial amounts of liver glycogen. FGF19 treatment restored the loss of glycogen in diabetic animals lacking insulin. Thus, FGF19 activates a physiologically important, insulin-independent endocrine pathway that regulates hepatic protein and glycogen metabolism. ibroblast growth factor 19 (FGF19, also called FGF15 in rodents) is a member of a subfamily of fibroblast growth factors that govern nutrient metabolism (1). FGF19 is expressed in the distal small intestine, where its synthesis is regulated by the nuclear bile acid receptor, FXR, after the postprandial uptake of bile acids (2, 3). Thus, in response to feeding, the concentration of circulating FGF19 increases (4). FGF19 binds to a receptor complex composed of the FGF receptor 4 (FGFR4) and a coreceptor called b-Klotho, which are both highly expressed in liver. Binding of FGF19 to the FGFR4–b-Klotho complex results in activation of the small guanosine triphosphatase

F

Ras and extracellular signal–regulated protein kinase (ERK) signaling pathway (5, 6). FGF19 plays an important role in hepatic bile acid homeostasis by inhibiting expression of CYP7A1, the first and rate-limiting enzyme in the major bile acid synthesis pathway (2, 3). FGF19 also promotes relaxation and refilling of the gallbladder after a meal (7). In addition to its roles in bile acid metabolism, FGF19 lowers serum glucose and triglycerides in diabetic mice through an unclear mechanism (8). To elucidate whether FGF19 may have other effects on metabolism, we investigated FGF19induced signaling in liver in normoglycemic wild-

Fig. 1. Stimulation by FGF19 of signaling pathways that regulate protein synthesis in liver. (A to C) Mice fasted overnight were injected intravenously (i.v.) with vehicle or 1 mg of FGF19 protein per kg of body weight. This concentration is based on the optimal systemic dose to observe the physiologic effects of FGF19 on bile acid metabolism. The animals were killed 1 hour after the injections. Proteins from liver homogenates were separated by SDS– polyacrylamide gel electrophoresis (SDS-PAGE) and identified by Western blotting with the indicated antibodies. Results represent triplicate experiments. (D and E) Overnight serumstarved HepG2 cells were starved for amino acids in Hank’s buffered salt solution medium for 1 hour. Vehicle, wortmannin (200 nM), rapamycin (20 nM), U0126 (10 mM), or BI-D1870 (10 mM) was added for a further 1-hour treatment. The cells were treated with vehicle or 250 ng/ml FGF19 and harvested after 30 min. www.sciencemag.org

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type animals (9). Because recombinant mouse FGF15 is unstable and has variable bioactivity, we used human FGF19 for these studies. FGF19 increased the phosphorylation of liver ERK1 and ERK2 of mice fasted overnight (Fig. 1A and fig. S1). In contrast, insulin, but not FGF19, induced phosphorylation of the protein kinase Akt (fig. S1), which demonstrated that FGF19 and insulin likely work through independent kinase signaling pathways. However, both FGF19 and insulin stimulated the phosphorylation of the eukaryotic initiation factors eIF4B on Ser422 and eIF4E on Ser209 in liver (Fig. 1B and fig. S1). These proteins are components of the eIF4F complex that mediates binding of mRNA to the ribosome, and their phosphorylation promotes the initiation of translation (10). Treatment of animals with insulin or FGF19 produced similar increases in phosphorylation of Ser235 and Ser236 of ribosomal protein S6 1 Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA. 2Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA. 3 Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, 44106, USA. 4Laboratory of Structural Sciences, Van Andel Research Institute, 333 Bostwick Avenue Northeast, Grand Rapids, MI 49503, USA. 5Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA. 6Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 7Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

*Present address: Exploratory Biomarkers, Atherosclerosis, Merck, 126 East Lincoln Avenue, Rahway, NJ 07065, USA. †To whom correspondence should be addressed. E-mail: [email protected] (D.J.M.); steven.kliewer@ utsouthwestern.edu (S.A.K.)

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Proteins were identified by Western blotting with the indicated antibodies. BID1870 treatment blocks the negative-feedback effect of p90RSK on ERK, which results in increased basal phosporylation of ERK and p90RSK. Numbers below blots represent fold-change relative to the vehicle group. Asterisk (*) marks nonspecific band (C). VOL 331

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(rpS6) (Fig. 1B and fig. S1). Phosphorylation of rpS6 improves the efficiency of global protein synthesis by inducing cap-dependent translation (11). We investigated the kinases that might mediate phosphorylation of eIF4 or rpS6 in response to FGF19. Ser209 of eIF4E is a target for the protein kinase Mnk1, which can be activated by phosphorylation at Thr197 and Thr202 by ERK (12). FGF19 induced phosphorylation of Mnk1, which implicated FGF19 as the upstream stimulus of a Ras-ERK-Mnk1 signaling cascade that activates eIF4E (Fig. 1C). rpS6 and eIF4B are well-known targets of p70 ribosomal S6 kinase (p70S6K), which is activated by insulin (fig. S1). However, FGF19 treatment did not induce the phosphorylation of p70S6K or Akt, which is known to activate mammalian target of rapamycin (mTOR) to stimulate p70S6K (Fig. 1, A and C, and fig. S1). Instead, FGF19 induced the phosphorylation of p90 ribosomal S6 kinase (p90RSK),

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which also is known to phosphorylate rpS6 and eIF4B (13, 14). Because p90RSK is a downstream target of ERK, our results indicate that FGF19 utilizes a Ras-ERK-p90RSK pathway to induce phosphorylation of rpS6 and eIF4B. In human hepatocarcinoma HepG2 and rat hepatoma HII4E cells that express FGFR4 and b-Klotho, FGF19 induced the phosphorylation of fibroblast growth factor substrate 2a (FRS2a), ERK, and p90RSK in a dose-dependent manner (fig. S2A). Likewise, FGF19 treatment increased the phosphorylation of rpS6 and eIF4B in HepG2 cells (Fig. 1D). However, this effect was not inhibited by wortmannin, a potent phosphoinositide 3-kinase (PI3K) inhibitor, or rapamycin, an mTOR inhibitor, which suggested that FGF19 does not act through the AktmTOR-S6K pathway (Fig. 1D and fig. S2, B and C). Indeed, FGF19 treatment failed to induce the phosphorylation of Akt or p70S6K (Fig. 1E and

fig. S2B). In contrast, the ERK pathway inhibitor U0126 and p90RSK inhibitor BI-D1870 (15) completely inhibited FGF19-induced phosphorylation of ERK and p90RSK (Fig. 1E), and both inhibitors blocked basal and FGF19-dependent phosphorylation of rpS6 and eIF4B (Fig. 1D). The above findings link FGF19 to stimulation of protein synthesis in liver. We analyzed protein synthesis in mouse liver using 2H2O labeling (16, 17). When injected into animals, 2H2O equilibrates with body water within 90 min, and 2H incorporates into amino acids. To determine the normal rate of protein synthesis after fasting and refeeding, mice were fasted overnight and then refed or continually fasted for another 6 hours. Refeeding caused a 25% increase in the rate of liver protein synthesis (Fig. 2A) (18). In comparison, injection of FGF19 significantly increased total protein synthesis by 18% (Fig. 2B). The de novo synthesis rate of albumin, the major protein product of liver,

Fig. 2. Increased rates of global protein synthesis and albumin synthesis in mouse liver treated with FGF19. (A) Mice fasted overnight received 0.5 ml 2 H2O intraperitoneally (i.p.); 90 min later, the animals were refed or kept fasted for 6 hours and killed (n = 10). Protein samples were hydrolyzed, and 2 H labeling of alanine was determined by mass spectrometry. (B and C) Mice fed ad libitum received 0.5 ml 2H2O; 90 min later (at 6 p.m.), vehicle or 1 mg/kg FGF19 was injected subcutaneously. The next morning (8 a.m.), animals were injected again with the same dose and, 6 hours later, were

killed (n = 10). Protein samples were hydrolyzed, and 2H labeling of alanine was determined by mass spectrometry. For albumin synthesis, 2H incorporation into plasma albumin was measured in the same way. (D) Over a 3-day period, mice (n = 6) received vehicle or 1 mg/kg FGF19 subcutaneously 3 times at 6 p.m. and once on the day they were killed at 8 a.m. Then, 6 hours after the last injection, the livers were harvested. Plasma albumin levels were determined with a Vitros 250 instrument. Values are means T SEM. Statistics by two-tailed t test. *P < 0.05, **P < 0.005.

Fig. 3. FGF19 inhibits GSK3 signaling to increase liver glycogen in mice. (A) Mice fasted overnight were treated i.v. with vehicle or 1 mg/kg FGF19 and killed 10 min later. Proteins from liver homogenates were separated by SDS-PAGE and identified by Western blotting with the indicated antibodies. Results represent triplicate experiments. (B) The ability of glycogen synthase in the homogenates of the same livers to incorporate radiolabeled uridine 5′-diphosphate–glucose into glycogen in the absence and presence of glucose-6-phosphate was measured, and the ratio was shown as glycogen synthase activity (n = 3). (C) Mice fed ad libitum were injected subcutaneously with vehicle or 1 mg/kg FGF19 at 6 p.m. and the next morning at 8 a.m. Then, 6 hours after the last injection, the animals were killed, and liver weight and glycogen content were determined (n = 6). (D) Liver glycogen content was determined in wildtype and Fgf15 – / – mice fed ad libitum (n = 5). (E) Oral glucose tolerance test in

wild-type and Fgf15 – / – mice (n = 6). Values are means T SEM. Asterisks (*) refer to differences between wild-type and Fgf15 – / – groups; number signs (#) refer to differences between Fgf15 – /– and Fgf15 – / – plus FGF19 groups. Statistics by twotailed t test. *P < 0.05, **P < 0.005, #P < 0.05, ##P < 0.005.

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was increased 40% by FGF19 (Fig. 2C). Moreover, continuous treatment with FGF19 significantly increased plasma albumin levels by 10% (Fig. 2D). Thus, FGF19 is a positive regulator of hepatic protein synthesis. The effects of FGF19 on protein synthesis prompted us to investigate glycogen synthesis, another target of insulin action. Glycogen synthesis in liver is negatively regulated by glycogen synthase kinase (GSK) 3a and GSK3b, which phosphorylate and inhibit the enzyme glycogen synthase (GS). Phosphorylation also inactivates GSK3 kinases, which prevents inhibition of GS and thus increases glycogen synthesis (19). In animals fasted overnight, FGF19 induced phosphorylation of both GSK3a (Ser21) and GSK3b (Ser9), which correlated with decreased phosphorylation of Ser641 and Ser645 on GS (Fig. 3A) and increased GS activity (Fig. 3B). Concomitantly, there was a 30% increase in liver glycogen content that led to a small but significant increase in liver weight in FGF19-treated mice compared with that of control animals (Fig. 3C). FGF19 treatment had no effect on liver cholesterol or triglycerides (fig. S3, A and B), nor did it change plasma insulin or glucagon concentrations, which strengthens the idea that it acts directly on liver (fig. S3, C and D). We also analyzed hepatic glycogen concentration in mice lacking Fgf15 (the mouse ortholog of FGF19). Fed Fgf15 –/– mice had >50% less hepatic glycogen than did wild-type animals (Fig. 3D), which demonstrated the physiologic requirement for FGF15 in maintaining normal glycogen metabolism. Moreover, Fgf15 –/– mice showed impaired glucose uptake from the circulation. FGF19 ad-

ministration completely rescued this phenotype (Fig. 3E). Akt and p90RSK phosphorylate the same residues of GSK3a and b (19–22). To test whether p90RSK might mediate phosphorylation of GSK3 kinases for FGF19, we treated HepG2 cells with FGF19 and either the PI3K or p90RSK inhibitor. FGF19-induced phosphorylation of GSK3 kinases in HepG2 cells was compromised when cells were treated with BI-D1870, but not when treated with wortmannin (Fig. 4A). These data further support the idea that FGF19 acts through an insulin-independent Ras-ERK-p90RSK pathway to regulate glycogen synthesis. Streptozotocin (STZ)–treated mice are severely diabetic and have almost no detectable insulin in the blood (Fig. 4B). STZ treatment reduced liver glycogen content to 50% of that of control animals. FGF19 treatment restored hepatic glycogen amounts and had a significant effect on glucose disposal (Fig. 4C and fig. S4). Insulinindependent effects of FGF19 on the rate of net hepatic glycogen synthesis were also investigated in rats fasted overnight. A hyperglycemic clamp was used in combination with somatostatin to inhibit endogenous insulin and glucagon secretion. Under matched conditions of plasma glucose, insulin, and glucagon concentrations (fig. S5), FGF19 increased net hepatic glycogen synthesis by 70% compared with that in control rats (Fig. 4D). Taken together, these studies suggest that FGF19 acts in parallel to and independent from insulin to govern postprandial metabolism in liver. Like insulin, pharmacologic administration of FGF19 can induce protein and glycogen syn-

thesis, whereas loss of the physiologic hormone in Fgf15 –/– mice results in glucose intolerance and reduced hepatic glycogen. However, whereas insulin reaches its maximum serum concentration within 1 hour of a meal in humans, peak FGF19 levels are achieved ~3 hours after a meal (4) just before glycogen accumulation peaks in the liver (23, 24). Thus, we propose insulin and FGF19 work in a coordinated temporal fashion to facilitate the proper postprandial storage of nutrients. Of the anabolic enterokines (e.g., the incretins, GLP-1 and GIP), FGF19 is unusual in that it mimics insulin action rather than stimulating its release. The different signaling pathways used by FGF19 and insulin permit overlapping but distinct biological effects for the two hormones (Fig. 4E). For example, unlike insulin, FGF19 did not increase hepatic triglycerides (fig. S3B) or induce sterol regulatory element–binding protein, isoform 1c (SREBP-1c)–dependent lipogenic gene expression (fig. S6), which requires the PI3K-Akt-mTOR signaling pathway (25, 26). Indeed, FGF19 appears to be unique in its ability to differentially govern glycogen synthesis and lipogenesis. FGF15 (or FGF19) is required to maintain normal glycogen levels in fed mice, and it uses the alternative Ras-ERK-p90RSK pathway; taken together, these findings may explain the puzzling observation that liver-specific loss of insulin signaling in IRS1-IRS2 null mice does not fully block glycogen storage in response to feeding (27, 28). These results may also help explain the glucose- and insulin-lowering actions of FGF19 in diabetic rodents (8). Thus, pharmacologically targeting the FGF19 pathway might be

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Fig. 4. FGF19-induced glycogen synthesis is independent of insulin. (A) Overnight serum-starved HepG2 cells were pretreated with vehicle, wortmannin (200 nM), or BI-D1870 (10 mM) for 1 hour. The cells were lysed 30 min after vehicle or FGF19 (250 ng/ml) treatment. Proteins were identified by Western blotting with the indicated antibodies. Numbers below blots represent fold-change relative to the vehicle group. (B and C) Mice were treated i.p. with STZ (175 mg/kg). Eight days later, diabetic animals were chosen and treated with vehicle or 1 mg/kg FGF19 i.p. at 6 p.m. for seven consecutive days, and killed 6 hours after the last injection at 8 a.m.(n = 5 to 9). Liver glycogen content and plasma insulin levels were determined. *P < 0.05 is between control and STZvehicle groups; #P < 0.05 is between STZ-vehicle and STZ-FGF19 groups. (D) Three-hour hyperglycemic clamp study was performed on rats fasted overnight (n = 5 to 7). Animals were continuously infused with insulin and somatostatin to maintain low levels of insulin and glucagon and variably infused with glucose to maintain hyperglycemia. Net glycogen synthesis was determined by assessing the glycogen content in the clamped animals subtracted by the glycogen content of unclamped animals that were killed after the same duration of fasting. (E) Insulin and FGF19 act through different signaling pathways to coordinate overlapping but distinct postprandial responses in liver. (B, C, and D) Values are means T SEM. Statistics by two-tailed t test. *P < 0.05, ***P < 0.0005. www.sciencemag.org

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References and Notes 1. A. Beenken, M. Mohammadi, Nat. Rev. Drug Discov. 8, 235 (2009). 2. J. A. Holt et al., Genes Dev. 17, 1581 (2003). 3. T. Inagaki et al., Cell Metab. 2, 217 (2005). 4. T. Lundåsen, C. Gälman, B. Angelin, M. Rudling, J. Intern. Med. 260, 530 (2006). 5. H. Kurosu et al., J. Biol. Chem. 282, 26687 (2007). 6. B. C. Lin, M. Wang, C. Blackmore, L. R. Desnoyers, J. Biol. Chem. 282, 27277 (2007). 7. M. Choi et al., Nat. Med. 12, 1253 (2006). 8. L. Fu et al., Endocrinology 145, 2594 (2004). 9. Materials and methods are available as supporting material on Science Online. 10. A. C. Gingras, B. Raught, N. Sonenberg, Annu. Rev. Biochem. 68, 913 (1999). 11. S. Fumagalli, G. Thomas, in Translational Control of Gene Expression, N. Sonenberg et al., Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000), pp. 695–717.

12. T. Ueda, R. Watanabe-Fukunaga, H. Fukuyama, S. Nagata, R. Fukunaga, Mol. Cell. Biol. 24, 6539 (2004). 13. P. P. Roux et al., J. Biol. Chem. 282, 14056 (2007). 14. D. Shahbazian et al., EMBO J. 25, 2781 (2006). 15. G. P. Sapkota et al., Biochem. J. 401, 29 (2007). 16. D. A. Dufner et al., Am. J. Physiol. Endocrinol. Metab. 288, E1277 (2005). 17. N. Rachdaoui et al., Mol. Cell. Proteomics 8, 2653 (2009). 18. S. R. Anderson, D. A. Gilge, A. L. Steiber, S. F. Previs, Metabolism 57, 347 (2008). 19. P. Cohen, S. Frame, Nat. Rev. Mol. Cell Biol. 2, 769 (2001). 20. C. Sutherland, I. A. Leighton, P. Cohen, Biochem. J. 296, 15 (1993). 21. V. Stambolic, J. R. Woodgett, Biochem. J. 303, 701 (1994). 22. Q. Ding et al., Mol. Cell 19, 159 (2005). 23. M. Krssak et al., Diabetes 53, 3048 (2004). 24. R. Taylor et al., J. Clin. Invest. 97, 126 (1996). 25. S. Li, M. S. Brown, J. L. Goldstein, Proc. Natl. Acad. Sci. U.S.A. 107, 3441 (2010). 26. T. Porstmann et al., Cell Metab. 8, 224 (2008).

Clr4/Suv39 and RNA Quality Control Factors Cooperate to Trigger RNAi and Suppress Antisense RNA Ke Zhang,1 Tamas Fischer,1* Rebecca L. Porter,1† Jothy Dhakshnamoorthy,1 Martin Zofall,1 Ming Zhou,2 Timothy Veenstra,2 Shiv I. S. Grewal1‡ Pervasive transcription of eukaryotic genomes generates a plethora of noncoding RNAs. In fission yeast, the heterochromatin factor Clr4/Suv39 methyltransferase facilitates RNA interference (RNAi)–mediated processing of centromeric transcripts into small interfering RNAs (siRNAs). Clr4 also mediates degradation of antisense RNAs at euchromatic loci, but the underlying mechanism has remained elusive. We show that Clr4 and the RNAi effector RITS (RNA-induced transcriptional silencing) interact with Mlo3, a protein related to mRNA quality control and export factors. Loss of Clr4 impairs RITS interaction with Mlo3, which is required for centromeric siRNA production and antisense suppression. Mlo3 also interacts with the RNA surveillance factor TRAMP, which suppresses antisense RNAs targeted by Clr4 and RNAi. These findings link Clr4 to RNA quality control machinery and suggest a pathway for processing potentially deleterious RNAs through the coordinated actions of RNAi and other RNA processing activities. he widespread transcription of eukaryotic genomes necessitates elaborate quality control and surveillance mechanisms, which monitor RNA biogenesis to detect and destroy aberrant RNA (1–4). In Schizosaccharomyces pombe, methylation of histone H3 lysine 9 (H3K9me) by Clr4 provides binding sites for chromodomain proteins, including Chp1 subunit of an Argonaute (Ago1)–containing RNA-induced transcriptional silencing (RITS) complex required for the processing centromeric transcripts to small interfering RNAs (siRNAs) (5). Loss of Clr4 causes severe defects in centromeric siRNA production (6). However, siRNA can be detected in cells where

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1 Laboratory of Biochemistry and Molecular Biology, National Cancer Institute (NCI), National Institutes of Health, Bethesda, MD 20892, USA. 2Laboratory of Proteomics and Analytical Analysis, NCI, Frederick, MD 21702, USA.

*Present address: Heidelberg University, Biochemistry Center (BZH), 69120 Heidelberg, Germany. †Present address: Department of Pathology, University of Rochester, NY 14642, USA. ‡To whom correspondence should be addressed. E-mail: [email protected]

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H3K9 is mutated to unmethylatable (such as H3K9R) residues (7, 8), suggesting an additional role for Clr4. The Clr4 complex (ClrC) interacts with RITS (6, 8, 9), and in addition to their role at centromeres, these factors suppress antisense transcripts at euchromatic loci (10, 11). A yeast two-hybrid screen using full-length Clr4 as the bait identified Mlo3 (12) as an interacting protein (table S1). Mlo3 is related to Saccharomyces cerevisiae Yra1 and mammalian Aly/REF (13) and is required for nuclear export of RNA (13). Immunoprecipitation analysis detected Mlo3 interacting with Clr4 (Fig. 1A) and another ClrC subunit, Rik1 (fig. S1). Moreover, recombinant Mlo3 bound Clr4, and this interaction was mediated by the amino-terminal (amino acids 1 to 55) and carboxy-terminal (amino acids 134 to 199) regions of Mlo3 (fig. S2), known to bind mRNA export machinery (13). Thus, Clr4 associates with Mlo3 in vitro and in vivo. Given the role of Clr4 in heterochromatin assembly, we investigated whether Mlo3 affects heterochromatic silencing (5). Cells lacking Mlo3 maintain H3K9me and its interacting Swi6/HP1

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27. N. Kubota et al., Cell Metab. 8, 49 (2008). 28. X. Dong et al., J. Clin. Invest. 116, 101 (2006). 29. We thank T. Inagaki, X. Ding, and A. Bookout for their help with animal experiments and M. Cobb and P. Scherer (University of Texas Southwestern Medical Center) for discussion and comments. This research was supported by the Howard Hughes Medical Institute (G.I.S., D.J.M.), the NIH (DK67158 and DK62434 to S.A.K. and D.J.M., DK40936 and U24 DK076169 to G.I.S.), the Robert A. Welch Foundation (I-1275 to D.J.M. and I-558 to S.A.K.), and the Yale and Case Western Reserve University Mouse Metabolic Phenotyping Centers. A Materials Transfer Agreement is required for sharing materials from the Van Andel Institute.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1621/DC1 Materials and Methods Figs. S1 to S6 References 28 September 2010; accepted 20 January 2011 10.1126/science.1198363

protein at levels comparable to wild-type (WT) cells at major heterochromatic loci (fig. S3). However, mlo3∆ resulted in a considerable increase in the levels of centromeric repeat transcripts, although to a lesser extent than in clr4∆ (Fig. 1B). The accumulation of repeat transcripts in mlo3∆ was not linked to enhanced RNA polymerase II (Pol II) transcription. We therefore explored the possibility that Mlo3 mediates processing of repeat RNAs. Because Clr4 interacts with RNA processing complex RITS (6, 8, 9), we investigated whether Mlo3 also interact with RITS. We found that Mlo3 coimmunoprecipitated with Chp1, a subunit of RITS (Fig. 1C). This interaction was not sensitive to DNase I and RNase A treatment but was severely compromised upon loss of Clr4 (Fig. 1C), suggesting that Clr4 connects Mlo3 to RNA interference (RNAi). Indeed, mlo3∆ caused severe reduction in the levels of centromeric siRNAs (Fig. 1D). Thus, in addition to creating H3K9me binding sites for RITS, Clr4 physically and functionally links RITS to Mlo3 to mediate processing of centromeric transcripts. Because histone lysine methyltransferases can methylate nonhistone proteins (14–18), we investigated whether Clr4 methylates Mlo3. Recombinant Clr4 could methylate the carboxyterminal region of Mlo3, but not the aminoterminal or middle region (Fig. 2A). Within the carboxy-terminal region, lysines 165 and 167 are in a sequence context that resembles H3K9. We mutated these and lysines 179 and 180 to alanine. A methylation assay using recombinant Mlo3 carrying single- or double-mutant combinations showed that Clr4 methylates K167 of Mlo3 in vitro (Fig. 2B). Furthermore, Western blot using an antibody generated against methylated Mlo3 peptide recognized Mlo3 purified from WT cells, and the signal was diminished in clr4∆ mutant (fig. S4). To explore the importance of these results, we generated Schizosaccharomyces pombe strain in which Lys165 and Lys167 residues located in close proximity were mutated simultaneously to alanine to generate the mlo3-A mutant. Mutant Mlo3 protein was expressed at WT levels (Fig.

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Fig. 1. Clr4- and RITS-interacting protein Mlo3 is required for the production of centromeric siRNA. (A) Immunoprecipitation (IP) of Mlo3-hemagglutinin (HA) using antibody to HA was followed by Western blotting with antibody to Flag to detect Clr4-Flag. Less than 10% of total Mlo3 is associated with ClrC. (B) Strand-specific RT-PCR of RNA isolated from WT, mlo3D, and clr4D cells. (C) Clr4 facilitates Mlo3 interaction with Chp1. Mlo3-Flag immunoprecipitated fractions from indicated cells were subjected to Western blot analyses with antibody to Chp1. The asterisk indicates a nonspecific band. (D) siRNAs isolated from indicated strains were analyzed by Northern blot with probes specific for dg/dh centromeric repeats (upper) or for tRNA used as a loading control (bottom). bp, base pairs. 134

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Fig. 2. Clr4 methylates Mlo3 to facilitate centromeric siRNA production. (A and B) Methyltransferase assay, performed using recombinant Clr4 with glutathione S-transferase (GST)–Mlo3 as the substrate and S-adenosyl-[methyl-3H]-methionine as the methyl donor. Histone H3 fused to GST (GST-H3) was included as a positive control. Clr4 targets histone H3 more efficiently than Mlo3 in vitro. Fluorography indicates proteins that were methylated by Clr4. (A) Clr4 methylates the carboxy-terminal region of Mlo3. (B) Clr4 methylates Mlo3 at Lys167 in vitro. The GST fused carboxy-terminal region of Mlo3 carrying different mutations were used as the substrates. (C) Mlo3-A expresses at WT levels. WT and mutant Mlo3 were detected by Western analysis using antibody to Flag. Western blot with antibody to tubulin TAT1 was used as a control. (D) siRNAs in indicated strains were examined by Northern blot using a probe corresponding to dg/dh centromeric repeats (top). tRNA was used as a loading control (bottom). www.sciencemag.org

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2C). Interestingly, mlo3-A caused a decrease in levels of centromeric siRNA as compared to WT (Fig. 2D). Further reduction in siRNAs was observed in mlo3-A H3K9R double mutant (Fig. 2D), although a residual signal seemed to be present when compared to clr4∆. Thus, in addition to Clr4 bridging the interaction between RITS and Mlo3, the methylation of Mlo3 might be important for siRNA production. In light of the results described above, we wondered whether Mlo3 also mediates Clr4dependent suppression of antisense RNAs at euchromatic loci. As a prelude to addressing this question, we investigated Mlo3 localization across genome by chromatin immunoprecipitation coupled to microarrays (ChIP-chip). Mlo3 showed a broad distribution at euchromatic loci and a relative depletion at heterochromatic regions (figs. S5 and S6). clr4∆, which causes enhanced Pol II occupancy at centromeric repeats (19, 20), resulted in increased Mlo3 binding at repeat loci (fig. S5), supporting the transcription-coupled loading of mRNA processing and export factors (21–24). At euchromatic loci, Mlo3 preferentially localized at the gene body, and its localization peaked near the 3′end of open reading frames (fig. S6). Expression profiling of mlo3∆ cells on both DNA strands showed dramatic accumulation of antisense RNAs at euchromatic loci (~23.5% of genes) (fig. S7), in particular at convergent genes (fig. S8). Strandspecific reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analyses confirmed elevated levels of antisense RNAs corresponding to read-through transcripts in mlo3∆ cells (Fig. 3, A and B). Therefore, Mlo3 is required for the suppression of antisense RNAs at Pol II-transcribed genes. We also tested the effects of the mlo3-A mutant on antisense RNA levels, in particular at loci targeted by Clr4 and RNAi (10). Because clr4∆ and ago1∆ enhance antisense RNA levels when combined with a variant histone h2a.z∆ (10), we examined the mlo3-A mutant transcriptome with or without H2A.Z. Like clr4∆ and ago1∆, mlo3-A also showed weak up-regulation of antisense RNAs (4.7% genes) (fig. S9). However, the mlo3-A h2a.z∆ double mutant showed a synergistic increase in antisense RNAs (18.6% of genes) (Fig. 3C and figs. S8 and S9). mlo3-A caused antisense RNA up-regulation at the same genomic loci that were affected by clr4∆ or ago1∆ (Fig. 3, C and D, and fig. S8). Moreover, both clr4D and mlo3-A showed similar slight accumulation of poly (A)+ RNA signal in the nucleus (fig. S10), suggesting a further functional connection between Clr4 and Mlo3. Together with the results described above, these results suggest that Clr4 cooperates with Mlo3 and Ago1 to suppress antisense RNA. Because antisense RNA levels in mlo3∆ were consistently higher than in clr4∆ and ago1∆ mutants, we wondered whether Mlo3 has an additional Clr4-independent role in antisense suppression. To address this, we performed immunoaffinity purification of functional, Flag-tagged Mlo3 (Mlo3Flag)(fig. S11A). Mol3 copurified with a complex related to the Trf4p/Air2p/Mtr4p polyadenylation

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(TRAMP) (Fig. 4A and table S2), an exosome cofactor promoting degradation of aberrant RNA (2, 4, 25, 26). All TRAMP subunits, including Cid14, Mtr4 and Air1 (25), could be identified in the Mlo3-Flag purified fraction (Fig. 4A). When a Flag-tagged Cid14 subunit of TRAMP (Cid14-Flag) was purified (fig. S11B), a large number of peptides derived from Mlo3 together with the components of TRAMP were identified (Fig. 4A and table S3). The interaction between Cid14 and Mlo3 was insensitive to DNase I and RNase A treatment (Fig. 4B) and did not require Clr4 (fig. S12). Thus, Mlo3 physically associates with TRAMP, a complex involved in the surveillance and degradation of aberrant RNA by the exosome. In this regard, the antisense profile of mlo3∆ closely

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resembles that of rrp6∆ (Fig. 3, C and E, and fig. S8). To explore the relationship between the factors described above, we compared the distribution of antisense RNA in various mutants. These analyses revealed a strong correlation between antisense up-regulation in clr4∆ ha2.z∆, ago1∆ h2a.z∆, and mlo3-A h2a.z∆, consistent with Mlo3, Clr4, and RNAi cooperating to suppress antisense RNAs (Fig. 3D and fig. S13). In addition, Cid14 targets RNAs affected by Mlo3, Clr4, and Ago1 (fig. S13). Using correlation coefficients (r values) obtained from pairwise comparisons of antisense profiles of different mutants, we clustered mutants in a hierarchical manner according to the similarity of their antisense profiles (Fig. 4C) (10). As shown

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previously (10), components of the Clr6 histone deacetylase and Set2 form a distinct cluster, thereby confirming the functional connection between these factors in suppressing antisense RNAs produced from cryptic promoters (Fig. 4C). Double mutants carrying null alleles of either ClrC subunits (e.g., rik1∆ and clr4∆), RNAi machinery (ago1∆), TRAMP (cid14∆), or mlo3-A mutant in combination with deletion of h2a.z form a tight cluster closely associated with rrp6∆ and mlo3∆. In addition, the mlo3-A single mutant associated closely with clr4∆ and rik1∆ mutants (Fig. 4C). This analysis provides further support for these factors cooperating to suppress antisense RNA. These analyses showing that Clr4 interacts with Mlo3 to promote the processing of centro-

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Fig. 3. Mlo3 is required for suppression of antisense RNAs targeted by Clr4 and by the exosome. (A) Strand-specific RTPCR of RNA isolated from WT and mlo3D. (B) Northern blot analyses of RNA at SPBC16h5.04cyp7. Probes complementary to sense (probe a) or antisense (probe b) strands of SPBC16h5.04 gene are shown by red arrows indicating the probe position and 5′ to 3′ direction. (C) Heat map showing up-regulation of antisense RNA in indicated strains at a representative region of chromosome 2. Transcriptome profiling was performed using tiling microarray. Forward-strand transcripts are shown on top, and reverse-strand transcripts are shown at the bottom. Relative expression values (mutant/WT) were converted into color gradient. (D and E) Density plot comparing changes in antisense RNA levels in clr4Dh2a.zD and mlo3-Ah2a.zD (D), or rrp6D and mlo3D (E). Median antisense ratios were calculated for 842 genes. Pearson’s correlation coefficient (r) and the P value of the linear regression are indicated.

REPORTS A LC-MS/MS of total mixtures protein ( % coverage) Cid14-Flag (82.3%)

Mlo3-Flag (80.9%) Cid14 (16.67%) Mtr4 (4.9%) Air1 (9.3%)

Anti-Flag IP

Input

Cid14-Flag RNaseA/H DNase

_ + + + _ _ + + _ _ _ +

_ _ _

Anti-Flag

Mtr4 (69.74%) Air1 (50.5%) Mlo3 (76.9%)

-0.6

-0.4

-0.2

0.0

0.2

+ _ _

Anti-Mlo3

0.4

0.6

0.8

1.0

D Centromeric/ antisense RNA

mlo3-A cid14 cid14 rik1 clr4 cid14 h2a.z mlo3-A ago1 rrp6 h2a.z mlo3 mlo3 swi6 clr4 rik1 mlo3-A h2a.z swi6 h2a.z ago1 set2 clr6-1 cph1 alp13 WT vs WT h2a.z set1 set1

Mlo3 Mlo3/TRAMP ? Mlo3/Clr4/RITS

RdRP Dicer Exosome

siRNA

meric and antisense RNAs suggest a mechanism for the immediate detection and processing of these noncoding RNAs. Mlo3 may determine the fate of the RNAs by either preparing them for nuclear export or facilitating their destruction (2, 27). Mlo3-associated RNA processing activities, RNAi and TRAMP, might collaborate to degrade RNA (Fig. 4D and fig. S14). Indeed, mlo3∆ (this study) or cid14∆ (25) impair centromeric siRNA production by the RNAi machinery. Although defects in siRNA production could result from sequestering of RNAi proteins by the RNAs accumulating in mlo3∆ and cid14∆ cells, this is unlikely because rrp6∆, which mimics accumulation of RNAs observed in mlo3∆ and cid14∆ (Fig. 3, C and E, and Fig. 4C), does not reduce siRNA production (Fig. 1D). We postulate that Mlo3 and/or Mlo3/TRAMP serve as gatekeepers that channel RNAs into the exosome and/or RNAi pathways. Whereas RNAs targeted by Mlo3/TRAMP could be degraded directly by the exosome, Clr4-mediated interaction between Mlo3 and RITS channels these RNAs into the RNAi pathway (Fig. 4D and fig. S14). Methylation of Mlo3 might influence the recognition of aberrant RNA by factors such as RITS, which also binds H3K9me (6, 8, 9). Clr4 and Mlo3 may function as

a hub that integrates heterochromatin formation with RNA processing through the cooperative action of RNAi, TRAMP, and exosome. Our results expand current views about the functions of the heterochromatin machinery, which not only modifies chromatin but also acts in RNA quality control and surveillance. This is important because the uncontrolled accumulation of noncoding RNA can adversely affect genome stability and modify the epigenetic profiles of genomes (5, 28, 29). References and Notes 1. M. J. Moore, N. J. Proudfoot, Cell 136, 688 (2009). 2. J. Houseley, J. LaCava, D. Tollervey, Nat. Rev. Mol. Cell Biol. 7, 529 (2006). 3. V. N. Kim, G. Dreyfuss, Mol. Cells 12, 1 (2001). 4. F. Wyers et al., Cell 121, 725 (2005). 5. S. I. Grewal, S. Jia, Nat. Rev. Genet. 8, 35 (2007). 6. K. Zhang, K. Mosch, W. Fischle, S. I. Grewal, Nat. Struct. Mol. Biol. 15, 381 (2008). 7. I. Djupedal et al., EMBO J. 28, 3832 (2009). 8. E. L. Gerace, M. Halic, D. Moazed, Mol. Cell 39, 360 (2010). 9. E. H. Bayne et al., Cell 140, 666 (2010). 10. M. Zofall et al., Nature 461, 419 (2009). 11. M. Gullerova, N. J. Proudfoot, Cell 132, 983 (2008). 12. J. P. Javerzat, G. Cranston, R. C. Allshire, Nucleic Acids Res. 24, 4676 (1996). 13. A. G. Thakurta, G. Gopal, J. H. Yoon, L. Kozak, R. Dhar, EMBO J. 24, 2512 (2005). 14. S. Chuikov et al., Nature 432, 353 (2004). 15. S. C. Sampath et al., Mol. Cell 27, 596 (2007).

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

K. Zhang et al., Cell 122, 723 (2005). P. Rathert et al., Nat. Chem. Biol. 4, 344 (2008). J. Huang, S. L. Berger, Curr. Opin. Genet. Dev. 18, 152 (2008). E. S. Chen et al., Nature 451, 734 (2008). T. Fischer et al., Proc. Natl. Acad. Sci. U.S.A. 106, 8998 (2009). D. Libri et al., Mol. Cell. Biol. 22, 8254 (2002). D. Zenklusen, P. Vinciguerra, J. C. Wyss, F. Stutz, Mol. Cell. Biol. 22, 8241 (2002). K. Strässer et al., Nature 417, 304 (2002). H. Hieronymus, P. A. Silver, Nat. Genet. 33, 155 (2003). M. Bühler, W. Haas, S. P. Gygi, D. Moazed, Cell 129, 707 (2007). J. LaCava et al., Cell 121, 713 (2005). T. H. Jensen, M. Rosbash, Nat. Struct. Biol. 10, 10 (2003). X. Li, J. L. Manley, Genes Dev. 20, 1838 (2006). P. Huertas, A. Aguilera, Mol. Cell 12, 711 (2003). We thank R. Dhar and R. Allshire for antibody to Mlo3 and strains, E. Chen for helpful contributions, and F. Reyes-Turcu, N. Komissarova, and M. Lichten for comments on manuscripts and discussions. Microarray data are available at the National Center for Biotechnology Information’s Gene Expression Omnibus repository under accession numbers GSE26999 and GSE17271. This work is supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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C

B

set1 h2a.z set1 WT vs WT alp13 cph1 clr6-1 set2 ago1 h2a.z h2a.z swi6 mlo3-A rik1 clr4 swi6 mlo3 h2a.z mlo3 rrp6 ago1 mlo3-A cid14 h2a.z clr4 rik1 cid14 mlo3-Acid14

Fig. 4. Mlo3 associates with the TRAMP complex, which is involved in antisense RNA suppression. (A) Mass spectrometry analysis of Mlo3-Flag or Cid14Flag purified fractions. A detailed list of the proteins identified is provided in table S2 and S3. (B) TRAMP association with Mlo3 is independent of nucleic acids. IP of Cid14-Flag using antibody to Flag was followed by Western blotting with the indicated antibodies. (C) Hierarchical clustering of mutants on the basis of similarities of their antisense profiles. Pairwise comparisons of antisense profiles were performed by calculating the median antisense ratio (mutant/WT) for 842 genes. Pearson’s correlation coefficients were converted into color codes. Except for mlo3-A and clr6-1 mutants, deletion alleles of genes indicated were used. (D) Model showing processing of centromeric and antisense RNA by the coordinated action of Mlo3, TRAMP, Clr4, RNAi, and the exosome (see also fig. S14). Because low levels of siRNAs are detected in mlo3D cells, it is likely that additional mechanisms also target RNAi to centromeric RNAs.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6024/1624/DC1 Materials and Methods Figs. S1 to S14 Tables S1 to S3 References 5 October 2010; accepted 7 February 2011 10.1126/science.1198712

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LIFE SCIENCE TECHNOLOGIES

AAAS/Science Business Office Feature

NEW PRODUCTS: SYNTHETIC GENOMICS

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This comprehensive oligo manufacturing service provides oligos that are deprotected, desalted, quantified using ultraviolet spectrophotometry, and checked for quality using mass spectrometry as standard, offering the highest quality and yield accuracy currently available. Oligos are available in a range of amounts, from 25 nmol up to 10 µmol with the additional option of large-scale production up to 10 g. In addition to providing standard length oligos, Integrated DNA Technologies’ chemistry expertise allows the production of high-quality, long Ultramer DNA oligos up to 200 base pairs in length, making them ideal for gene construction, cloning, and DNA-directed RNA interference (ddRNAi). Oligos can be delivered in individual tubes, as well as 96- or 384-well plates, either lyophilized or resuspended in liquid, such as water or Tris-EDTA (TE) buffer. Integrated DNA Technologies www.idtdna.com

Genome-Scale DNA Assembly Kit

The GENEART High-Order Genetic Assembly System is a genecloning platform that enables researchers to efficiently assemble up to 10 synthetic or preexisting DNA fragments in yeast (in vivo) to create a seamless sequence up to 110 kilobase pairs (kbp) in length. The system capitalizes on yeast cells’ natural ability to take up and assemble DNA fragments to produce a customized molecule into a vector of choice before it can be transferred into E. coli for downstream applications. The process using GENEART HighOrder Genetic Assembly System involves less than 30 minutes of prep work and three days of incubation to obtain the assembled molecule. The seamless construct is enabled by DNA fragments with end-homology. In cases where end-homology does not exist, synthetic oligonucleotides, or DNA linkers, can be designed so that the yeast cells can ‘stitch’ the fragments together into a seamless sequence. Life Technologies For info: 760-603-7200

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The predesigned CompoZr Knockout Zinc Finger Nuclease (ZFN) technology can knockout any gene in the human genome. Currently, few laboratories create gene knockouts in human cell lines because of the inefficiencies of the natural cellular homologous recombination system and the investment of time and labor required to screen the resulting cells. In contrast, CompoZr ZFNs have been shown to efficiently generate human cell lines with precise and stable gene knockouts in a matter of weeks. CompoZr ZFNs are designed to produce a knockout in each gene by inducing a double-strand break at a defined site within the gene’s first three exons. This double-strand break stimulates the cell’s natural DNA repair pathways, resulting in a permanent, sitespecific deletion or mutation that disrupts the gene’s function. Robust and on-target gene knockout using CompoZr Knockout ZFNs is guaranteed by functional validation prior to shipment and unique enhancements to the DNA-recognition and cleavage ZFN subunits. Sigma Life Science For info: 800-325-3010

Magnet-Based Gene Transfection

The magnefect-LT offers gene transfection with improved efficiencies and cell viabilities, even with difficult cells types including neuronal and primary cells. The system uses a unique technology applying proprietary magnet configurations and oscillating magnet arrays operating at fixed frequency that promotes magnetic nano particle/DNA uptake into cells to improve nonviral gene transfection, while maintaining high levels of cell viability. The system is simple to operate, has low running costs (as low as $0.10 per transfection), and provides improved transfection efficiency and effectiveness over the best currently available cationic lipid agents at short transfection times (<30 minutes). It provides a low cost option for users who are budget constrained but still require the benefits that oscillating magnet-assisted transfection can offer such as the high transfection efficiencies and cell viabilities, short transfection times, and low running costs. nanoTherics Limited For info: +44-(0)-1782-554047

Gene Knockout technology

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GC-Rich Target Amplification

When DNA targets with high GC content are amplified, polymerase chain reaction product formation is often compromised by inadequate strand separation and the propensity for complex secondary structure formation. The use of standard 7-deazadGTP is a notable method for overcoming this problem. TriLink has developed CleanAmp 7-deaza-dGTP, an elegant fusion of the secondary structure-reducing nucleotide analog 7-deaza-dGTP and TriLink’s CleanAmp dNTP Hot Start technology. CleanAmp 7-deaza-dGTP is available individually for amplification of routine GC-rich targets or as the CleanAmp 7-deaza-dGTP Mix, which is recommended for more challenging targets with higher GC content. TriLink BioTechnologies, Inc. For info: 800-863-6801

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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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For info: 800-328-2661

online @sciencecareers.org

POSITIONS OPEN

POSITIONS OPEN

PROFESSOR AND ASSISTANT PROFESSOR Positions in Systems Biology at the Columbia University Medical Center The Columbia Initiative in Systems Biology seeks to recruit new faculty that have experimental and/or computational research programs. Although applications from outstanding candidates in all areas of Systems Biology will be considered, areas of focus include next generation sequencing, genome wide proteomics, systems pharmacology, protein sequence and structure analysis and the Systems Biology of cancer, neurodegenerative, metabolic, and cardiovascular diseases. Recruitments will be made jointly with the Departments of Biochemistry and Molecular Biophysics, Genetics and Development, Microbiology, Pathology, Pharmacology and Physiology, as appropriate. It is anticipated that successful candidates will eventually join a new Department of Systems Biology that is currently in the process of formation. Applications should include curriculum vitae, reprints of no more than three publications, a three to four page description of current research and research goals, and three or more letters of reference. Applications should be addressed to: Ms. Desi Tahiraj, Columbia Initiative in Systems Biology, 1130 St. Nicholas Ave 8th Floor Room 801, New York, NY 10032. Applications can also be sent electronically at e-mail: desi@c2b2. columbia.edu. Columbia University is an Equal Opportunity/ Affirmative Action Employer and encourages applications from women and underrepresented minorities.

POSTDOCTORAL POSITIONS Department of Physiology University of Pennsylvania Postdoctoral position in neurophysiology available to study regulation of BK channels and neuronal excitability by lipid messengers. Patch-clamp electrophysiology experience as demonstrated by publication is required. Knowledge of cell biology, molecular biology, and optical methods is desirable. Send curriculum vitae, reference names, and description of previous research to: Dr. Toshinori Hoshi, Department of Physiology, D100 Richards, 3700 Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104. E-mail: [email protected].

NORTH DAKOTA STATE UNIVERSITY Center for Biopharmaceutical Research and Production Two positions: SENIOR SCIENTIST in Immunology—Experience in development of vaccines for use in humans. Website: http://jobs.ndsu.edu/postings/ 368. SENIOR SCIENTIST in Molecular and Cell Biology—Expertise in RTPCR and molecular shuffling, cloning and vector development. Website: http://jobs. ndsu.edu/postings/373. Screening begins April 15, 2011. North Dakota State University is an Equal Opportunity/ Affirmative Action Employer.

PHYSICIST Lawrence Berkeley National Laboratory has a unique opportunity for a Physicist to participate in a nuclear emission tomography program applied to cardiac diagnostic and environmental remediation research. Must have experience in medical image acquisition, reconstruction, and modeling. Ph.D. or equivalent training is highly preferred. Application website: http://jobs. lbl.gov/details.asp?jid025600. Berkeley Lab is an Affirmative Action/Equal Opportunity Employer.

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FACULTY POSITION in Cancer Biology and Cancer Prevention The Susan Lehman Cullman Laboratory for Cancer Research in the Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey is seeking an outstanding investigator for a tenured or tenure-track position. Applicants should hold a Ph.D. degree or equivalent and have a strong track record for research in the areas of mechanisms of cancer causation and prevention, as evidenced by independent, currently funded research (RO1 or equivalent), high-impact publications, and demonstrated leadership activities in the field. A strong commitment to teaching undergraduate and graduate students and postgraduate trainees will be expected. The successful candidate will also be a member of The Cancer Institute of New Jersey, the Center for Cancer Prevention Research, the Environmental and Occupational Health Sciences Institute, and join a large life sciences community with opportunities to participate in a variety of research and teaching programs. Please send or e-mail curriculum vitae, brief research plan, current and past grant support, and the names and addresses of three references to: Dr. Allan H. Conney, Chair of the Search Committee, Department of Chemical Biology, Ernest Mario School of Pharmacy, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020, e-mail: [email protected]. An Affirmative Action/Equal Opportunity Employer.

PROFESSOR AND ALEXANDER CHAIR in Equine Reproduction College of Veterinary Medicine and Biomedical Sciences Colorado State University The Departments of Biomedical Sciences and Clinical Sciences seek an Associate or Full Professor to fill the John Alexander Endowed Chair in Large Animal Reproduction to conduct and supervise basic and applied research related to equine reproduction, which is a primary research emphasis within the Animal Reproduction and Biotechnology Laboratory (ARBL) and the Equine Reproduction Laboratory (ERL). A Ph.D. is required and D.V.M., M.D., or equivalent is desirable. The successful candidate will lead a substantial externally funded research program in equine reproduction and develop strong collaborations with other faculty. Contributions to teaching veterinary and graduate students and participation in outreach efforts also are expected. Information about the ERL and ARBL, and more details about the position can be found at website: http://www.cvmbs.colostate.edu/bms/arbl/. A letter of application, curriculum vitae, statements of research, teaching and clinical interests, and a list of three references who may be contacted when appropriate should be sent to: Dr. Thomas Hansen, ARBL Director Department of Biomedical Sciences Colorado State University Fort Collins, CO 80523-1683 E-mail: [email protected] Review of applicants will begin June 1, 2011. Colorado State University is an Equal Opportunity/Affirmative Action Employer. Colorado State University conducts background checks on all final candidates.

SENIOR POSTDOCTORAL ASSOCIATE Boston University Required expertise in transgenic rats, ultrasound molecular imaging, arterial stiffness analysis, and sonoporation, FACS, MoFlo, confocal microscopy. M.D. or Ph.D., $40,000/year. Send curriculum vitae to Nancy Clinton, [email protected]. Equal Opportunity Employer/ Affirmative Action.

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Senior Faculty positions available in oncologic sciences and interdisciplinary clinical oncology at the USA Mitchell Cancer Institute in Mobile, Alabama Abraham A. Mitchell Distinguished Cancer Research Investigator Abraham A. Mitchell Distinguished Clinical Cancer Investigator The Opportunity: The successful candidate will bring an established, well-funded, basic/translational and/or clinical research program and will receive a research expansion funding award in the aggregate total of up to $1,500,000 over a 3-5 year time period, with the goal of further growing the individual’s and the institution’s research grant funding base and enhancing translational research links both internally and externally to the USA Mitchell Cancer Institute. Funds from the award can be used for partial salary support for the awardee and/or member(s) of his/her research team, materials and supplies, other operational expenses and equipment. State-of-the-art Facilities: The USA Mitchell Cancer Institute offers a robust, interactive scientiﬁc environment with fully-equipped laboratories and access to core facilities including ﬂow cytometry, mass spectrometry, tissue biobank, BL3 laboratory, genomics, laser dissection, atomic absorption and advanced imaging. An AALAC-approved vivarium with advanced imaging capabilities is located nearby. More information can be found at www.USAMCI.com. Community Environment: Experience the best of the Gulf Coast in Mobile, Alabama! With attractions including Bellingrath Gardens, the Gulf Coast Exploreum Science Center & IMAX Theater and the USS ALABAMA Battleship, exciting outdoor adventures, fresh delicious seafood, Delta excursions, fascinating museums, twenty-one world-class golf courses, white sandy beaches, a thriving arts community, and beautiful historic homes, it will be a pleasure to call the Mobile Bay area, the home of Mardi Gras, your home. Requirements: The successful candidate will have a Ph.D., M.D. or equivalent degree, and an established, well-funded and recognized program of basic/ translational and/or clinical cancer research, and other qualiﬁcations appropriate for tenured appointment as Associate or Full Professor. Application Process: Applicants should send letter of interest and CV to Michael R. Boyd, M.D., Ph.D., 1660 Springhill Avenue, Mobile, Alabama 36604, or e-mail to [email protected].

USA is an Afﬁrmative Action and Equal Opportunity Employer.

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The CEPH and the Centre National de Génotypage in association with the European Sequencing and Genotyping Infrastructure are pleased to announce The 4th Paris Workshop on Genomic Epidemiology Dates: May 30, 31 & June 1, 2011 The Paris Workshops on Genomic Epidemiology are held every two years to introduce researchers to new methodologies that underpin large-scale genomic studies of diseases and other applications in the life sciences, particularly in the context of on-going research funded by the EU. The last two years have witnessed the emergence of powerful new sequencing methodologies with vast consequences for systems approaches in biology. The inclusion of these into epidemiological scale studies allows the rapid identification of biological markers underlying many diseases. This workshop will discuss progress in these and other technologies for biomolecular analysis, and their applications in research and clinical settings. Solutions will be presented for the accumulation, handling and interpretation of huge data sets, including the identification of rare and common genetic variants associated with disease, functional evaluation of genetic variation, understanding of gene networks and epigenomic phenomena in health and disease, pharmacogenomics, gene-gene and gene-environment interactions. Examples of the application of these technologies for epidemiological scale studies in different disease areas will be presented.

focus on cancer research

The 4th Paris Workshop inaugurates a major new EU initiative, The European Sequencing and Genotyping Infrastructure (ESGI). The ESGI groups major European genome centres into a single infrastructure designed to increase European access to the most recent genomic technologies. ESGI platforms and access modalities will be presented at the meeting. Confirmed speakers: Gonçalo Abecasis (U. Michigan, USA), David Balding (U. College London, UK), Robert Feil (IGMM, FR), David Bentley (Illumina, USA) Alvis Brazma (EBI, UK), Anne-Cambon Thomsen (UMR Inserm, FR), Bill Cookson (Imperial College London, UK), Ivo Gut (CNAG, ES), Margret Hoehe (MPI-MG, DE), Richard Houlston (ICR, UK), Norbert Hübner (MDC, DE), Maneesh Jain (Life Technologies, FR), Achillefs Kapanidis (U. Oxford, UK), Peter Laird (U. Southern California, USA), William LaRochelle (Roche, USA), Liang Liming (Harvard, USA), Kerstin Lindblad-Toh (Broad, USA), Yukihide Momozawa (U. Liège, BE), Mats Nilsson (U. Uppsala, SE), Shaun Purcell (MGH, USA), Mark Ratain (U. Chicago, USA), Kathryn Roeder (Carnegie Mellon, USA), Sascha Sauer (MPI-MG, DE), Daniel Schaid (Mayo Clinic, USA), Harold Swerdlow (Sanger, UK), Ann-Christine Syvänen (U. Uppsala, SE), Jenny Taylor (U. Oxford, UK), Mathias Uhlen (KTH, SE), Hubert Vidal (Inserm/Inra, FR), Hugh Watkins (U. Oxford, UK), Dan Weeks (U. Pittsburgh, USA), John Whittaker (GSK, UK), Kurt Zatloukal (Med. U. Graz, AT). Organisers: Ivo Gut, Mark Lathrop, Sascha Sauer and Dan Weeks Place: Maison de la Chimie, 28 rue Saint-Dominique, 75007 Paris, France (Strictly limited to 200 participants) Sponsors: CEPH, CNG and European Commission FP7 projects: ESGI (Infrastructure), READNA, CAGEKID (large-scale collaborative projects) Further information and registration: http://www.cng.fr/workshop2011

SENIOR POSITION IN HUMAN AND MOLECULAR GENETICS (HMG), VCU INSTITUTE OF MOLECULAR MEDICINE (VIMM) AND VCU MASSEY CANCER CENTER (MCC) VIRGINIA COMMONWEALTH UNIVERSITY (VCU) SCHOOL OF MEDICINE, HMG, VIMM & MCC Under the leadership of Dr. Paul B. Fisher the Department of Human and Molecular Genetics (HMG) and the VCU Institute of Molecular Medicine (VIMM) in collaboration with the VCU Massey Cancer Center (MCC) in Richmond, Virginia seeks to recruit a seasoned investigator who focuses on cancer development and progression and whose research bridges the gap between laboratory discovery and clinical trials. Research that employs current genomic discoveries in medicine with an emphasis on translating these ﬁndings into improved approaches for diagnosis and treatment of neoplastic diseases are areas of high priority of this institutional initiative. The ideal candidate will be an experienced investigator who has demonstrated consistent research excellence, with a sustained track record of research funding, high-level publications and administrative experience. We are particularly interested in candidates that have managed multifaceted, interactive research programs using hypothesis-based, innovative approaches to address important health-related areas. Candidates with a sustained record of NIH funding will be given the highest priority. Applicants must have demonstrated experience working in and fostering a diverse faculty, staff, and student environment or commitment to do so as a faculty member at VCU. The appropriate candidate will be recruited at the level of Associate/Full Professor with qualiﬁcations commensurate with tenure. This individual will play a major role in VCU SOM and will hold a senior administrative position in HMG and serve as the Associate Director of the VIMM. Additionally, the appropriate recruit may also serve as a Co-Program leader of one of the programs in the MCC. HMG, VIMM and MCC provide an interactive and collaborative research and educational environment that will facilitate the training of the next generation of research scientists, clinicians and academicians, and will provide a direct conduit for the translation of genetic information from bench-to-bedside. Outstanding state-of-the-art core research facilities with a generous start-up and support package are available for the qualiﬁed applicant. Richmond, VA provides an ideal rural living and cultural environment with affordable housing, outstanding school systems and ready access to other metropolitan areas (including Washington, DC, Baltimore, Philadelphia and New York). Moreover, the City of Richmond and surrounding areas offer a diverse and rich cultural heritage that engenders a high quality of living for its residents. Interested candidates should provide by e-mail (preferably as a single PDF ﬁle): a letter of interest, a curriculum vitae, a description of administrative philosophy, and an outline of research interests and future research directions, with contact information for three to four references to: Dr. Paul B. Fisher ([email protected]) Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, 1101 East Marshall Street, Sanger Hall Building, Room 11-015, Richmond, VA 23298-0033 Review of Applications will begin April 1, 2011 and will continue till the position is ﬁlled. VCU is an EEO/AA Employer. Female, Minorities and persons with disabilities are encouraged to apply.

FOCUS ON CAREERS

AAAS/Science Business Office Feature

CANCER RESEARCH

Translating Cancer Research In The New Millennium

From Left: Karl Saxe, Cheryl Jones, Diana Caracino

Cancer research now reaches far beyond questions of uncontrolled cell division into a more broadly focused ‘total picture’ perspective of a tumor’s proteomic, genomic, and metabolomic landscape. With the dawn of personalized medicine and new technologies allowing next generation sequencing of individual patients’ tumors, a burst of information has been brought forth that needs wrangling in order to advance cancer diagnosis and treatment. This shift has opened new areas of fundamental exploration, and presented scientists with new career opportunities and challenges as they try to navigate the information overload. By Kendall Powell

ven in tough economic times, cancer research has more government backing and private philanthropy donations than many other ﬁelds. The National Cancer Institute’s 2010 budget at $3.1 billion is almost double that of the National Institute for General Medical Sciences. “If I were to bet on a stable or growth area against the ﬁnancial background at the moment, I would be backing biomedical research in cancer,” says Hamish Ryder, director of drug discovery at Cancer Research Technology in London, United Kingdom. Still, the academic job market has become ultracompetitive as universities struggle to ﬁnd the funds for starting up new laboratories, notes Karl Saxe, cell biologist and scientiﬁc program director of cancer cell biology and metastasis for the American Cancer Society in Atlanta, Georgia. Saxe, who also oversees the peer review of postdoctoral fellowship applications, sees a noteworthy trend—that more lab heads are demanding postdocs arrive with their funding already in hand. With this current hiring slowdown, researchers need to be creative about looking for posts. But, the good news is that almost any cellular and molecular training can prepare scientists for a career in cancer research. And researchers who pair a strong scientiﬁc background—especially in areas such as cell signaling, metabolic pathways, epigenetics, or small RNAs—with the ability to make sense of large data sets gain a leg up on a career in cancer research. This combination of skills easily translates into success in areas beyond the academic realm, too. For those making job transitions, “I deﬁnitely recommend thinking about all the different options, including government and the pharma world, which has long shed its big, bad evil image,” says Saxe. “And people with really good doctoral degrees [in biomedical ﬁelds] should be thinking about how to turn themselves into informaticists as well.” Information specialists with sophisticated scientiﬁc training will be essential in the push for personalized medicine, Saxe notes. Large, patient-generated genomic data sets are already the rule at some major cancer centers, and increasingly proteomic and metabolomic data are being added. In addition, clinical testing of new, mutationspeciﬁc therapies demand more sophisticated patient-selection

“We’ve needed to reunify these two sides—[signaling and metabolism]—of cancer biology.”

CREDIT: (MIDDLE) © ISTOCKPHOTO.COM/ERAXION; (BOTTOM) PREETI TANDON

E

David Plas

strategies. In such settings, both drug developers and regulators “will need to be able to talk to each other intelligently,” explains Saxe. He predicts that “people who are really good at combining ‘omics and mathematical modeling into their own thinking [about biological processes]” will have the best chance at success in the individualized medicine age.

GETTING BACK TO METABOLISM BASICS The new ‘omics buzzword is metabolomics. But, David Plas, a cancer researcher at University of Cincinnati in Ohio, says metabolomics is a tool in the larger, burgeoning ﬁeld of cancer metabolism research. Plas studies how dysregulation of AKT signaling impacts cell metabolism and how this leads to apoptosis resistance in cancer cells. He and others hope to capitalize on a renewed interest in the role of metabolism in cancer. continued »

UPCOMING FEATURES Translational Research: Careers/Training Programs—May 13 Focus On Japan—June 10 Biotech and Pharma: Moving Up The Industry Ladder—June 17

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AAAS/Science Business Office Feature

CANCER RESEARCH

Jim Maher

Craig Thompson

In the 1920s, the Nobel Laureate Otto Warburg proposed that aberrant cellular metabolism might be the cause of cancer, after observing that cancer cells consume more glucose than normal cells through the anaerobic glycolysis process even when oxygen is plentiful. But his ideas were overshadowed by the molecular biology revolution of the 1970s that led to the traditional view that cancer results from an imbalance between oncogene drivers and tumor suppressor genes. Plas says that the last decade of research has made cancer cell biologists realize that signal transduction pathways control metabolism in more sophisticated ways than a simple response to energy supply and demand. And, Plas explains, similar to the way current therapies attack signaling pathways that control cell cycle and metastasis, targeting the regulation of metabolism signaling pathways has the potential to serve as an avenue for cancer treatments. “We’ve needed to reunify these two sides—[signaling and metabolism]—of cancer biology,” he says. While Plas works on the metabolism-apoptosis connection, other researchers, from both academia and industry, are probing how cancer cell metabolism affects cell growth and metastasis. One major question: Why does oncogenesis favor glycolysis over the Krebs cycle and oxidative phosphorylation? The answer, perhaps, lies in the fact that glycolysis produces many more of the precursor building blocks that rapidly dividing cells need to synthesize daughter cells. “How do cells acquire enough nutrients to maintain homeostasis and live another day?” asks Craig Thompson, president of Memorial Sloan-Kettering Cancer Center (MSKCC) in New York. “Even normal cells have to go into net growth, where the problem isn’t ATP, but rather the [availability of the necessary] building blocks for making nucleotides, lipids, and proteins all over again.” This question of biosynthesis is pulling another basic cell biology ﬁeld, autophagy, into the cancer spotlight. Autophagy—literally ‘self eating’—is a process by which cells digest their own internal organelles to recycle their building block components. Once thought only critical for single-cell organisms like yeast, now, Thompson notes, mammalian cells are thought to turn to this desperate measure to get through energetically stressful times, like metastasis. Plas says scientists need to not only brush up on their biochemistry equations, reactions, and kinetics, but also be able to handle large data sets and, ideally, have mathematical modeling skills. “I’d

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hire that person in an instant,” says Plas. “I’ll give them the biological problem, they bring the math [skills], and then we’d do some interesting things.” Ryder notes that his own organization and other academic institutes have recently recruited metabolism experts for their research efforts. “We were looking for [researchers with] an excellent background in biochemistry—but one broader than just a focus on glycolysis and the Krebs cycle. A knowledge of the biosynthesis of lipids, nucleotides, and proteins, and how catabolism and anabolism are linked is also needed,” says Ryder. For example, to build upon the observation that some cancer cells seem addicted to particular nutrients, such as speciﬁc amino acids, Ryder depends upon his team’s knowledge of the biochemical processes that occur as nutrients enter the cell, including the key intermediates and enzymes in pathways, to identify proteins that could serve as potential drug targets. Ryder also ﬁnds experience with cell culture, gene knockdown technologies, apoptosis assays, and metabolite proﬁling methods—such as mass spectrometry, quantifying metabolites from cell lysates, and the ability to identify key nodes in signaling pathways regulating metabolism—essential for researchers working on his drug discovery projects at the subsidiary of the Cancer Research UK charity. He notes that the cancer metabolism ﬁeld is likely to have a long shelf life—with some 1,500–2,000 genes playing a role in metabolism—and has a strong potential for cross-purposed discoveries in the pharmaceutical industry between cancer and metabolic disease. “Cancer is a horrible example of what can go wrong in multicellular life,” says Thompson. “Almost everything you could do to understand how cells effectively live together will impinge on cancer treatment and diagnosis. The challenge is going to be using a systems biology approach to deconstruct the complexities of multicellular life.”

TAKING ‘OMICS TO THE NEXT LEVEL Such a systems approach will require scientists who can weave together large and disparate data sets from the various realms on the ‘omics map—genomes, proteomes, transcriptomes, and metabolomes. Researchers grappling with all that data will be Gustavo Salem’s main clients. As vice-president of the Biological Systems Division of Agilent Technologies in Santa continued »

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CREDIT: (FROM LEFT) PHOTO BY MATTHEW SEPTIMUS - COURTESY OF MEMORIAL SLOAN-KETTERING CANCER CENTER; PHOTO: LIZ MAHER

“Almost everything you could do to understand how cells effectively live together will impinge on cancer treatment and diagnosis. The challenge is going to be using a systems biology approach to deconvolute the goals of multicellular life.” —Craig Thompson

Sanford National Breast Cancer Institute

STOCKHOLM, 23-27 SEPTEMBER 2011

www.ecco-org.eu

The 2011 European Multidisciplinary Cancer Congress: the multidisciplinary mass gathering and celebration of European basic, translational and clinical studies From September 23 – 27, the European oncology community will convene in Sweden at the premier European cancer meeting: the 2011 European Multidisciplinary Cancer Congress - the only multidisciplinary and multiprofessional educational opportunity in oncology to take place in Europe. A ‘personalised’ commitment In the era of personalised medicine, one major goal behind the multidisciplinary structure and multi-professional format of

the Congress is to accelerate and advance tailored cancer therapy and care. Key to these efforts will be a ‘personalised’ commitment from you in selecting our Congress as the deﬁnitive platform for data disclosure and exposure and submit an abstract. To view the Advance Programme & Call for Abstracts, submit your data before Regular Abstract Submission closes 18 April and register online today visit: www.ecco-org.eu

In partnership:

Debiopharm Life Sciences Award 2011 The President of the Swiss Federal Institute of Technology in Lausanne (EPFL), the Dean of the EPFL School of Life Sciences, and the Executive Board of Debiopharm Group are pleased to invite applications for the Debiopharm Group Life Sciences Award. The Sponsor, Debiopharm Group™, is a Swiss biopharmaceutical company that focuses on the development of prescription drugs that target unmet medical needs. In 2011, awards will be given to investigators in the field of:

CANCER RESEARCH The main award will honor a European investigator below the age of 45 who has made outstanding contributions in the area of basic or translational oncology. It consists of a total of 50‘000 CHF, of which one fifth is a personal award, while the rest will contribute to support the awardee’s research at his/her home institution. The award will be handed over on the occasion of the Annual EPFL Life Science Symposium "Hallmarks & Horizons of Cancer" to be held 7-10 September 2011 (for more information please visit http://isrec2011.epfl.ch). Candidates are invited to submit their applications no later than May 1st, 2011. A Jury of internationally renowned scientific experts will evaluate the applications. The dossier should be comprised of: • a one-page endorsement letter by the home institution describing the importance of the nominee’s contribution to this field • her/his curriculum vitae, • her/his list of publications, • a one-page outline of her/his plans for future research. All applications are to be sent, exclusively in electronic format and in the form of one single “pdf” file, to: [email protected]

Sanford Health, a non-proﬁt, integrated health care system, invites applications for the position of Director of the Sanford National Breast Cancer Institute. Thanks to a recent $100 million gift from Mr. Denny Sanford, Sanford is seeking an outstanding physician-scientist to lead research and clinical activities for a world-renowned destination for breast cancer care and research. Qualiﬁed individuals will have demonstrated experience and a national reputation for work in clinical care and translational research and program development. Sanford’s research mission includes the utilization of clinical care delivery capacities toward the growth of clinical research and commercialization.

Sanford Health’s 20,500 employees serve more than two million people in six states. The system includes 30 hospitals with a total of 1,600 beds and employs more than 900 physicians in 70-plus specialty areas. Sanford Research/USD includes more than 175 full-time research staff and has grown to include more than $36 million in annual research expenditures. The system expects signiﬁcant growth in this area. With main tertiary, research and administrative centers in Sioux Falls, South Dakota and Fargo, North Dakota, Sanford Health is the largest rural health care system in the United States. Sanford seeks an accomplished clinician scientist and innovative research leader with a track record of building and leading clinical care and research teams. The successful candidate will have had a career as a physician and scientist with progressive administrative responsibilities in building research programs and management functions. It is important that the candidate understand how to integrate research activities within the overall patient care mission of the health system. Finally, the candidate will have a desire to forge strong research collaborations and business relationships, a signiﬁcant track record of extramural funding, publications and the ability to mentor faculty. The role will receive signiﬁcant institutional support, including new, modern laboratory space and stateof-the-art facilities. A comprehensive compensation package will be tailored to the individual’s qualiﬁcations. Sanford Health is an Equal Opportunity/ Affirmative Action Employer. Applicants should have a MD, PhD or MD/PhD degrees. Candidates should submit a detailed curriculum vita, description of research experience and future plans, and the names of three individuals that would provide recommendation. Application materials should be sent to: David A. Pearce, PhD Sanford Health 2301 E. 60th Street North Sioux Falls, SD 57104 For further information, contact (605) 312-6004 or [email protected].

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Integrating basic & translational science, surgery, radiotherapy, medical oncology & care

Director

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The European Multidisciplinary Cancer Congress

FOCUS ON CAREERS

AAAS/Science Business Office Feature

CANCER RESEARCH

Clara, California, Salem is ahead of the curve in integrating the ‘omics sciences. Traditionally, he says, cancer experts have either been doing discrete genomics or proteomics experiments and then collaborating with groups using different approaches, or they have tried a systems biology approach to ask what is changing across the entire biological system for a speciﬁc type of cancer. “Under either scenario, it becomes a bioinformatics nightmare to bring all that information together,” says Salem. Gathering data from multiple vendors’ instruments and then visualizing or analyzing all the data sets simultaneously is not always possible. But, Agilent is hoping to step into that void by providing tools for visualization and analysis across genomics, next generation sequencing, proteomics, and metabolomics. For example, the Mass Proﬁler Professional (for mass spec data) and the Gene Spring GX (for genomic data) are written on the same software platform so that protein hits and gene lists can be compared directly. “This integrated biology approach is really gaining a lot of attention and a lot of steam,” says Salem. “There will be a need for people who can really speak about bioinformatics and computational biology, and who know how to use mass spectrometers and microarrays.” And the information ﬂood has not even begun to crest, as major worldwide projects such as the International Cancer Genome Consortium (based at the Ontario Institute for Cancer Research in Toronto), the Cancer Genome Atlas (based at the National Cancer Institute in Bethesda, Maryland), and the Cancer Genome Project (based at the Wellcome Trust Sanger Institute in Hinxton, United Kingdom) get under way. The genome consortium, for example, aims to catalog the genetic mutations, changes in gene expression, and epigenetic changes across 500 cancer samples from each of 50 different cancer sites. From those reams of data, says Jim Maher, a cancer researcher and associate dean of the Mayo Graduate School in Rochester, Minnesota, the ultimate challenge will be pulling out which changes are actually causative. “Even with comparative genomics and arrays, there will be a lot of information that is not indicative of what’s really going on,” he explains. “To sort that out, we’re going to need really clever biologists.”

PERSONALIZED MEDICINE USING INFORMATICS One sort of clever biologist, the bioinformatics specialist, will be indispensable in cancer research centers, be they academic-, hospital-, or industry-based. Although Simon Vincent, head of personal awards funding at Cancer Research UK in London, says he is reluctant to name any one area as “hot.” He explains that he “would not

FEATURED PARTICIPANTS Agilent Technologies www.agilent.com

Mayo Graduate School www.mayo.edu/mgs

American Cancer Society www.cancer.org

Memorial Sloan-Kettering Cancer Center www.mskcc.org

Cancer Research Technology www.cancertechnology. co.uk Cancer Research UK www.cancerresearchuk.org

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University of Cincinnati www.uc.edu

“There will be a need for people who can really speak about bioinformatics and computational biology, and who know how to use mass spectrometers and microarrays.” —Gustavo Salem have predicted 10 years ago that today it would be metabolism and the Warburg effect.” Though he does point to one area with the promise of staying power: stratiﬁed medicine. “Taking all of the information about the biology of [speciﬁc] tumors and using that to decide the best treatment course for patients and getting them into the right trial—that will have longer term impact,” says Vincent. Cancer Research UK, Europe’s largest private funder of diseasespeciﬁc research, has funneled a signiﬁcant portion, about US$26 million (£16 million), of their approximate US$530 million (£333 million) budget toward patient-stratiﬁcation research. Vincent’s organization will act as a coordination center to bring together its funding mechanisms and ﬁve core research institutes and use the beneﬁts of the United Kingdom’s centralized healthcare system and records. “One of the challenges for scientists highly skilled in data manipulation and informatics is to pull all [these data sets] together, be they genetic, patient, or hospital data sets, to inform research,” says Vincent. In addition, he says, the long history of centralized records makes the United Kingdom a leader in both classical epidemiology and genetic epidemiology of cancer. He notes an early example of the power of bioinformatics in cancer research was the identiﬁcation of the BRAF gene as a driver in melanomas through the Cancer Genome Project. Salem agrees that informaticists will be the gatekeepers of personalized medicine. He points to the WIN Consortium, or Worldwide Innovative Networking in personalized cancer medicine, a group of 22 cancer centers worldwide that will be implementing a common system for gathering microarray data from all their patients, which should enable them to track patients over time and divide data into subpopulations. One of the founding centers, the Institut de cancérologie Gustave-Roussy in Villejuif, France has been collecting patient tumor samples, whisked from the operating room to the hospital’s laboratory in pneumatic tubes, for the last eight years. “More and more centers are becoming interested in getting [multiple] ‘omics data sets from patients,” says Salem. And this trend, which started with cancer research, is rapidly moving towards other ﬁelds, such as neurology, cardiology, and metabolism. “There will be a signiﬁcant increase in the number of people needed to understand the scientiﬁc output from clinical labs,” he explains. Given the complex nature of the changing landscape in cancer research, the researchers who are likely to be in high demand and get the jobs are “the most curious people, who have changed their projects frequently and are not locked into any particular ﬁeld,” says Maher. “And, those who have deliberately cross-trained.” Kendall Powell is a freelance science writer based in Lafayette, Colorado. DOI: 10.1126/science.opms.r1100102

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Please submit a letter describing qualiﬁcations, with a CV and three letters of reference by [30 days from publication date] to: Dr. Joseph Schlessinger, Director, Cancer Biology Institute, Yale Cancer Center, c/o Vickie Johnson, 333 Cedar St., PO Box 208028, New Haven, CT 06520-8028. Yale University is an Equal Opportunity/Afﬁrmative Action Employer.

FULL PROFESSOR DIRECTOR OF THE LUDWIG CENTER FOR CANCER RESEARCH OF THE UNIVERSITY OF LAUSANNE This center (LICR@UNIL, www.unil.ch/licr), representing a new formal collaboration between the Ludwig Institute for Cancer Research and UNIL, will be based at the University’s hospital and biomedical campuses (CHUV and Epalinges). The focus will be on basic, translational, and clinical cancer research, involving current Ludwig-supported faculty with expertise in tumor immuno-biology and immunotherapy as well as future to-be-appointed faculty focused on other aspects of tumor biology, cancer genetics and experimental therapeutics. The Director is anticipated to be an accomplished mid-career cancer scientist with the ability to lead LICR@UNIL as well as attract and mentor new faculty. The Director will also contribute to new developments including the establishment of a new Department of Oncology at CHUV/UNIL, and of an integrated regional cancer center in conjunction with the Swiss Institute of Experimental Cancer Research (ISREC) and its parent institution, the Swiss Federal Institute of Technology Lausanne (EPFL). The operations of the Ludwig Center for Cancer Research will be funded by annual core support from the Ludwig Institute and from UNIL, complemented by support from philanthropic sources, and via Swiss and EU grant mechanisms. Generous support will be available to partially support the Director’s research activities. The successful candidate will have a personal research program focused on mechanisms of cancer with a track record of success and an articulated vision for the future, not only for her/his personal research group, but also for the Ludwig Center and its involvement (along with the parent Ludwig Institute) in developing world-class cancer research in Lausanne. Proficiency in French is not a prerequisite, although a willingness to become so is anticipated. Further information may be obtained from Prof. Patrick Francioli ([email protected]), chair of the search committee. Confidentiality is guaranteed. Applicants should send their curriculum vitae, a list of publications in which the five most significant ones are identified, a summary of the past, present and future research program, and at least three names of reference by May 15th, 2011 for the attention of Prof. Patrick Francioli, Dean of the Faculty of Biology and Medicine, rue du Bugnon 21, CH-1011 Lausanne, Switzerland. The University of Lausanne wishes to promote the access of women to academic careers and encourages applications from women.

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The Yale Comprehensive Cancer Center, its brand new Cancer Biology Institute, and the Yale School of Medicine invite applications from basic science investigators for junior or senior appointments with interests in cancer biology, in the areas of cancer genetics and genomics, signal transduction, structural biology, proteomics, and mass spectrometry, as well as drug discovery. The Cancer Biology Institute is one of 5 newly formed multi-disciplinary research institutes at Yale’s West Campus, a 136 acre parcel located 7 miles from Yale’s New Haven campus that includes 20 buildings and 1.6 million square feet of research, ofﬁce, and warehousing space. Additional West Campus assets include the Institute of Chemical Biology, Institute of Microbial Diversity, Institute of Systems Biology, Institute of Biodesign, as well as core facilities in high throughput cell biology, small molecule discovery, and genomic analysis. The position will have a role in deﬁning and implementing the vision for cancer biology research at Yale going forward. This includes developing translational research opportunities with the recently opened Smilow Cancer Hospital at Yale-New Haven. Appointment at Yale Medical School is available in a number of departments and will be commensurate with a demonstrated record of scholarly achievement. Of particular importance is experience in one or more of the following; a record of original research in cancer biology, independent extramurally funded laboratory investigation for senior applicants, engagement in translational research activities, and/or the development of strong cooperative teams across disciplines. Women and minority candidates are urged to apply.

THE FACULTY OF BIOLOGY AND MEDICINE OF THE UNIVERSITY OF LAUSANNE, SWITZERLAND INVITES APPLICATIONS FOR A POSITION OF

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Yale Cancer Center Cancer Biology Institute

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Van Deuren Endowed Chair for Breast Cancer Research The Medical College of Wisconsin (MCW) invites nominations and applications for the Van Deuren Endowed Chair for Breast Cancer Research and Leader of the developing Breast Cancer Research Program of the MCW Cancer Center. The position is part of the College’s expansion of basic and clinical cancer research, under the leadership of Cancer Center Director Ming You, MD, PhD, which is complemented by a new, state-of-the-art clinical cancer center. The position may be ﬁlled at the rank of Associate or Full Professor (PhD, MD or MD/PhD). We seek an individual with scientiﬁc expertise in basic science, clinical or translational research in breast cancer and with a strong record of NIH funding and publication in high proﬁle cancer journals. The successful candidate should possess dynamic leadership qualities, experience in program development and be a highly motivated interdisciplinary collaborator.

focus on cancer research

The Medical College of Wisconsin (www.mcw.edu) is the largest private research institution in Wisconsin, conducting over $130 million annually in funded research. It is among the fastest growing medical schools in the United States in terms of NIH funding. The Scientist magazine ranked the Medical College of Wisconsin in the top 5 academic institutions for postdoctoral training and in the top 50 best academic centers at which to be a scientist. In addition to a strong core of biomedical science departments, the College is home to nine federally designated Centers for biomedical research. Excellent shared facilities are available including state-of-the-art instrumentation in structural biology, magnetic resonance imaging in human and animal research, molecular genetics and molecular imaging. A development package and research space commensurate with the candidate’s interests will be provided. Salary and other considerations will be competitive and consistent with the College’s commitment to recruiting the best-qualiﬁed individual. Applicants should provide curriculum vitae, statement of research interests, and the names and contact information of three references. Please submit application materials electronically ([email protected]) to: Andrea Brown, MBA, Van Deuren Chair Search, Medical College of Wisconsin, Cancer Center - TBRC, 8701 Watertown Plank Road, Milwaukee, WI 53226.

Assistant, Associate and Full Professor Positions in Cancer Cell Biology The Cancer Center at the Medical College of Wisconsin (MCW) invites applications for tenure-track positions at the Assistant, Associate or Full Professor level. The successful applicant will be expected to develop a program aligned with major ongoing research efforts in Cancer Cell Biology at the MCW Cancer Center. We encourage applications from investigators with expertise in the following areas relevant to cancer: • Cell Signaling • Redox Biology • Mitochondria and Bioenergetics • Inﬂammatory Mediators • Genomic Instability The candidate is expected to establish a vigorous and extramurally funded research program, and participate in collaborative and interdisciplinary projects. Teaching at the graduate level is also expected. Applicants must have a doctoral degree in a relevant area, a minimum of two years of postdoctoral experience, and a strong record of research accomplishments. The MCW Cancer Center is an integrated partnership of more than 200 cancer research scientists and physicians at the Medical College of Wisconsin, Froedtert Hospital, Children’s Hospital of Wisconsin, Clement Zablocki VA Medical Center, and the Blood Center of Wisconsin. The Cancer Center occupies 30,000 sq. ft. of newly completed space dedicated for cancer-related basic science research on the MCW campus, and an additional 50,000 sq. ft. of space adjacent to Froedtert Hospital in the MCW medical complex. Candidates should send by e-mail ([email protected]) a complete curriculum vitae, bibliography, statement of research interests, and names of at least three references to: Andrea Brown, MBA Cancer Cell Biology Search Medical College of Wisconsin Cancer Center - TBRC 8701 Watertown Plank Road Milwaukee, WI 53226

Associate Director of Basic Sciences The Medical College of Wisconsin is actively recruiting for an Associate Director for Basic Sciences in its newly developing Cancer Center. The Associate Director should have a PhD and/or MD degree and a distinguished record of achievement in a basic science discipline of cancer research. This position will be responsible for assisting the basic science program leaders in program development, in identifying areas of potential research collaboration, and in facilitating translational research from the basic science laboratories to the clinic. The Associate Director will also assist in the development of and oversee all basic science core facilities in the Center. The successful candidate will be a member of the Executive Committee of the Cancer Center and report directly to the Center Director. The academic appointment will be in a mutually agreed upon department at the Medical College of Wisconsin where teaching, research and service requirements of a departmental appointment are expected. Rank is open and commensurate with experience. This leader could also potentially qualify for an endowed chair. The successful candidate will bring signiﬁcant extramural funding portfolio and focus on research funding. In addition to an active research program, this position will include limited administrative activities within the Cancer Center and service to the institution. Salary is competitive and will depend on rank and experience. Qualified individuals are encouraged to send, via e-mail ([email protected]), a letter of interest, CV, and contact information for three references to: Andrea Brown, MBA AD of Basic Science Search Medical College of Wisconsin Cancer Center - TBRC 8701 Watertown Plank Road Milwaukee, WI 53226

Searle Professorship in Medicinal Chemistry As part of a campus-wide expansion program in the area of Cancer Drug Discovery, Cancer Experimental Therapeutics, and Chemical Biology, the Department of Medicinal Chemistry at the University of Michigan http: //www.umich.edu/~pharmacy/MedChem/ solicits applications for the John G. Searle Professor of Medicinal Chemistry. This endowed Chair is intended to support a scholar with a proven record of accomplishment and outstanding contributions in research in anticancer drug discovery and development, experimental therapeutics, and the chemistry-biology interface. The Department of Medicinal Chemistry has long been recognized as one of the leading programs in the country in the application of modern Chemical Biology approaches aimed at the discovery and development of drug molecules. The successful candidate will be expected to provide leadership to build on this tradition. This program involves faculty and students from not only the Department of Medicinal Chemistry in the College of Pharmacy but also departments in the School of Medicine, the College of Literature, Science, and the Arts, and the Life Sciences Institute. The successful candidate will closely work with faculty at the University of Michigan Comprehensive Cancer Center with emphasis in anticancer drug discovery and experimental therapeutics of cancer. Potential areas of research may include, but are not limited to, synthetic organic chemistry, bioorganic chemistry, mechanistic enzymology, and structural biochemistry, all of which should include a strong synthetic component focused on exciting medicinal targets and rationales with emphasis on anticancer drug discovery. The successful candidate will be expected to have and maintain a vigorous, externally funded research program and to participate in teaching programs both at the graduate and undergraduate level. Applications, including a curriculum vitae and a list of at least ﬁve references must be electronically submitted to [email protected]. Applications will be reviewed commencing May 1, 2011 and will continue until the position is ﬁlled. The University of Michigan is a non-discriminatory, Afﬁrmative Action Employer and is responsive to the needs of dual career couples.

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DEAN The Graduate School of Biomedical Sciences invites nominations and applications for the position of Dean. Our objective is to recruit an outstanding scientist, educator and experienced leader to support the continued growth of the Graduate School as a leading center of graduate education and biomedical research in the 21st century. The successful candidate will be able to articulate a vision (and the steps needed to translate that vision into action) to enhance our Master’s and Doctoral programs in basic and translational research across the full spectrum of the health sciences, building on the successful history of collaboration with institutions and education programs throughout the Texas Medical Center.

Research Why you u should advertise in this issue of Science: Content: Attract researchers interested in bridging the gap between basic research and effective disease treatments. Reach: Science has over 700,000 print readers each week. 66% of our readership hold doctorate degrees, 60% work in academia, 34% work in the Biotech/Pharma industry. Results: Use Science’s winning formula of unparalleled circulation combined with relevant content.

Tobookyourad,e-mail: US, Canada, Latin America: [email protected] Europe and RoW: [email protected] Japan: [email protected] China, Taiwan: [email protected]

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Originally part of the academic arm of MD Anderson Cancer Center in the 1940s, the Graduate School of Biomedical Sciences (GSBS) was formally established by the Texas Legislature in 1963, and has become the premier academic bridge between the University of Texas components located in the world-renowned Texas Medical Center in Houston, Texas. Its faculty of more than 600 is drawn from The University of Texas MD Anderson Cancer Center and from schools that make up The University of Texas Health Science Center at Houston (including the Medical School, School of Public Health, School of Dentistry, and School of Biomedical Informatics). Total combined research expenditures of the GSBS faculty for the most recently completed academic year was more than $350 million. The current student enrollment is 585. The most recent Assessment of Research-Doctorate Programs by the National Research Council rated several GSBS programs in the top ten percent nationally. The Dean reports to the Presidents of MD Anderson Cancer Center (MD Anderson) and the University of Texas Health Science Center at Houston (UTHealth) and confers with ofﬁcials of those institutions, as well as UT System ofﬁcers and the Texas Higher Education Coordinating Board, on academic and ﬁnancial matters relating to current or future affairs of the administration of the Graduate School. As the chief academic and administrative ofﬁcer for GSBS, the Dean serves as the major advocate and spokesperson for the Graduate School, its faculty and students. S/he has responsibility for the overall leadership of GSBS and for overseeing graduate academic programs in consultation with the Graduate Faculty, its Executive Committee and standing committees, as well as the executive leadership of both MD Anderson and UTHealth. The Dean is responsible for administering the rules and policies of the Graduate School as well as the overall coordination and effectiveness of GSBS programs; providing outstanding leadership that embraces emerging directions in biomedical graduate education; exhibiting intellectual leadership; and building, shaping and leading initiatives in support of the strategic visions developed by MD Anderson and UTHealth. S/he must also be capable of maximizing the opportunities presented by the coexistence of two major research enterprises within a single Graduate School and work to facilitate increasing institutional collaboration. The Dean approves new and renewing appointments to the GSBS faculty and participates as a member of the GSBS Executive Committee. The Dean oversees a staff of 22, including an Associate Dean, Assistant Dean for Admissions, Associate Dean for Academic Affairs, and an Assistant Dean for Student Affairs & Outreach, all of whom report directly to the Dean. S/he will administer the affairs of the Graduate School through subordinates and through direct participation in the functions and activities of GSBS. The Dean will be an active member of the Graduate Faculty and an innovative scientist with outstanding leadership, management, and interpersonal skills; have a record of excellence in teaching and securing extramural funding; and demonstrate a signiﬁcant record of department, college and university service. S/he must have prior experience in a senior leadership position in an academic environment and qualify for an appointment at the level of Professor; the Dean will also be appointed to the John P. McGovern Distinguished Professorship of Biomedical Sciences. Applicants should forward (1) a letter of interest, which explains in detail the candidate’s administrative philosophy and vision; (2) a curriculum vitae; and (3) the names and contact information of ﬁve references to: Search Committee, Dean of the Graduate School of Biomedical Sciences, c/o Rosanne L. Evans, Search Coordinator, Ofﬁce of the Provost and Executive Vice President, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030; E-mail: [email protected]. Review of applicants will begin on February 25, 2011 and will continue until the position is ﬁlled. The University of Texas MD Anderson Cancer Center and the University of Texas Health Science Center at Houston are Equal Opportunity/Afﬁrmative Action Employers.

focus on cancer research

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The University of Texas MD Anderson Cancer Center and The University of Texas Health Science Center at Houston Graduate School of Biomedical Sciences

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THE BREAKTHROUGH TOBY ROBINS BREAST CANCER RESEARCH CENTRE AT THE INSTITUTE OF CANCER RESEARCH, CHELSEA, LONDON The Breakthrough Breast Cancer Research Centre is the first centre in the UK entirely devoted to breast cancer research. The Centre’s goal is to advance research into the causes, diagnosis and treatment of breast cancer. We are located in new laboratory space with excellent core facilities and funding.

Director of the Breakthrough Toby Robins Breast Cancer Research Centre The Director will provide leadership and direction of the Breakthrough Toby Robins Breast Cancer Research Centre towards Breakthrough’s vision of ‘a future free from the fear of breast cancer’. In addition the Director will pursue an independent research programme in Breast Cancer Research which complements the Centre’s existing strengths. He/she will build a thriving research team to deliver the research objectives. The successful candidate will have ambitious aspirations and a bold vision for improving the diagnosis, treatment and prevention of breast cancer, be an outstanding scientist with major achievements in biomedical research and a strong commitment to breast cancer research. He/she must be a strategic and visionary thinker, able to identify and implement powerful and coherent research programmes with clinical potential and be able to lead, inspire and recruit high calibre staff. In addition the successful candidate will have excellent interpersonal skills to manage a wide network of relationships. Informal enquiries should be addressed to Professor Chris Marshall, Director of Research [email protected] [Tel:+44-(0)207-153-5197]. Further information about Breakthrough Breast Cancer can be obtained directly from Dr Norman Freshney, Director of Research Management, Breakthrough Breast Cancer, Weston House, 246 High Holborn, London WC1V 7EX [Tel:+44-(0)207-025-2450]. Formal applications should be sent to Professor Chris Marshall The Institute of Cancer Research, 237 Fulham Rd, London SW3 6JB. Applications should include: · Full CV · Lists of major publications, achievements, major research grants, distinctions. · Brief account of research interests. · Statement of research goals. · Names and addresses of three referees, with an indication of any sensitivities about the timing of their involvement. For further details of how to apply, as well as a job description and personal specification, please visit our website at www.icr.ac.uk/jobs. Alternatively you may call our 24 hour recruitment line on +44-(0)207-153-5475. Closing date: 28th April 2011

FOCUS ON CANCER RESEARCH

POSITIONS OPEN

Postdoctoral Fellow Position in Structural Biology & Cancer Drug Discovery The University of Texas MD Anderson Cancer Center and University of Oxford The Center for Targeted Therapy at The University of Texas MD Anderson Cancer Center, in collaboration with the Structural Genomics Consortium at the University of Oxford, seeks a postdoctoral fellow with experience in protein biochemistry and computational biology for a two-year appointment. Year one of the fellowship investigates the crystal structure of novel, cancer-relevant target proteins in the laboratory of Dr. Stefan Knapp at the Structural Genomics Consortium in Oxford, England. Year two develops novel inhibitors of the same target proteins with Dr. John Ladbury, in collaboration with structural modeling and synthetic chemistry groups, at MD Anderson in Houston, Texas. A supplemental living allowance is offered while at Oxford. Candidates must have a Ph.D., M.D./Ph.D. or equivalent with up to two years post-graduate work. Experience in protein biochemistry and computational biology is required. Interested applicants should forward a curriculum vitae, research statement and three letters of references by email to [email protected] with “Structural Biology Postdoc” in the subject line. MD Anderson Cancer Center is an equal opportunity employer and does not discriminate on the basis of race, color, national origin, gender, sexual orientation, age, religion, disability or veteran status except where such distinction is required by law. All positions at The University of Texas MD Anderson Cancer Center are security sensitive and subject to examination of criminal history record information. Smoke-free and drug-free environment.

Senior Faculty Position Department of Microbiology and Physiological Systems Worcester, MA The Department of Microbiology and Physiological Systems invites applications for a senior faculty position. The candidate should have a track record of internationally recognized research that addresses important questions broadly relevant to infectious disease and encompasses multiple levels of biological organization. Technologies and approaches of particular interest include, but are not limited to, advanced imaging from single molecules to the whole animal, mechanistic mathematical modeling closely informed by experiment, and integrative functional analysis of genome-scale information. The Department of Microbiology and Physiological Systems has strong expertise in bacterial and viral pathogenesis, immunology, and fundamental cellular physiology. The University of Massachusetts Medical School (http://www.umassmed.edu/) is a vibrant, rapidly growing and highly interactive scientific community. Superb resources, a highly competitive start-up package, and generous newly renovated space will be provided to the successful candidate. The Department of Microbiology and Physiological Systems has close ties to clinical departments and a joint clinical appointment is possible for individuals with clinical training. Applicants should submit a cover letter, curriculum vitae, statement of research interests and contact information for three references to http://www.academicjobsonline.org. Inquiries, but not application materials, may be directed to [email protected]. As an equal opportunity and affirmative action employer, UMMS recognizes the power of a diverse community and encourages applications from individuals with varied experiences, perspectives and backgrounds.

NISC Director

NIH Intramural Sequencing Center, National Human Genome Research Institute The National Human Genome Research Institute (NHGRI), a major research component of the National Institutes of Health (NIH) and the Department of Health and Human Services (DHHS), seeks to identify an outstanding Director to lead the N I H Intramural Sequencing Center (NISC), located in Rockville, Maryland. The NISC Director leads a multi-disciplinary genomics facility that emphasizes the generation and analysis of DNA sequence. NISC brings together diverse and unique scientific expertise to perform state-of-the-art genome sequencing and sequence analysis for basic and translational research projects. The NISC Director has the responsibility for an annual budget exceeding $7 million and a staff of ~40. In addition to providing scientific and administrative leadership of this premier research enterprise, the Director is expected to be an internationally recognized, highly collaborative, and accomplished genomics researcher. Applicants must possess a doctoral-level scientific degree. The applicant must have extensive experience in genomics research, computational biology, and large-scale DNA sequencing; this should include a productive track record of high profile publications. S/he must have proven experience in directing and managing a scientific research program, with well-honed administrative and interpersonal skills to meet the demands of both research and program direction. Salary is competitive and will be commensurate with candidate’s experience. A full Federal benefit package is available, including retirement, health and life insurance, long-term care insurance, annual and sick leave, and the thrift savings plan (401K equivalent). Appropriate support for this program will be provided and exceptional candidates may be eligible for tenure. Interested applicants should submit a cover letter that includes a brief description of research and administrative experience, a current curriculum vitae and bibliography, names and contact information of three references, and a brief written vision for leading NISC. Questions about the position and applications themselves should be sent to Ms. Ellen Rolfes via email at [email protected]. Applications must be submitted by May 15, 2011. DHHS and NIH are Equal Opportunity Employers and encourage applications from women and minorities.

NATIONAL HUMAN GENOME RESEARCH INSTITUTE

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES | NATIONAL INSTITUTES OF HEALTH | genome.gov

National Institute of Mental Health Associate Director for Clinical Research Ofﬁce of the Director The National Institute of Mental Health, a major research component of the National Institutes of Health (NIH) and the Department of Health and Human Services (DHHS), is seeking exceptional candidates for the position of Associate Director for Clinical Research (ADCR), Ofﬁce of the Director. The ADCR serves as the administrative and scientiﬁc leader on clinical research issues across the Institute, especially pertaining to the areas of human subject research protections, conﬂict of interest. As part of this purview, the ADCR ensures NIMH human subject research complies with Federal and NIH policies. Working with the NIMH Data Safety Monitoring Board and other NIMH ofﬁces, the ADCR also ensures that conﬂicts of interest are managed appropriately, clinical recruitment milestones are met, and adverse events related to NIMH-sponsored multi-site clinical trials are monitored/acted upon appropriately. The ADCR provides expert advice and guides Institute initiatives for clinical trials, including the scope, design, and standards for data sharing as well as the development of public-private partnerships in support of clinical research. The ADCR Director reports to the Director, NIMH. Working closely with the Director and other senior leadership at NIMH, the ADCR assists in the scientiﬁc and administrative management of an organization with a budget of $1.4 billion and a staff of approximately 1,300. (http://www.nimh.nih.gov) Applicants must have a M.D., and be board certiﬁed in a medical specialty relevant to mental disorders, with research experience in one or more of the Institute’s research areas. In addition, extensive experience in designing, conducting, and publishing clinical research, including treatment trials, is highly desirable to ensure appropriate evaluation of clinical research proposals. Applicants should be known and respected within their profession as distinguished individuals of outstanding capability. Salary is commensurate with experience and accomplishments. Experience with NIH administrative policies, procedures, and operations is highly desirable but not essential. Interested candidates should send a letter of interest, including a brief description of research and administrative experience, a curriculum vitae and bibliography, and the names of at least three references to: Chair, NIMH ADCR Search Committee at [email protected] or at 6001 Executive Blvd, Room 8235, MSC 9669 Bethesda, MD 20892-9669 (for express or courier delivery use Rockville, MD 20852). Review of applications will begin on April 18, 2011, but applications will continue to be accepted and considered until the position is ﬁlled. For questions contact Dr. Thomas Insel, Director, NIMH at [email protected]. The NIH encourages the application and nomination of qualiﬁed women, minorities, and individuals with disabilities. HHS and NIH are Equal Opportunity Employers.

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Faculty Position in Civil and Environmental Engineering at Ecole Polytechnique Fédérale de Lausanne (EPFL) EPFL’s School ENAC (Architecture, Civil and Environmental Engineering) seeks a TenureTrack Assistant Professor in Air Quality Engineering and Atmospheric Chemistry.

mitted to excellence in undergraduate and graduate teaching. Substantial start-up resources will be available. We offer internationally competitive salaries and benefits.

Topics of interest include: Gas-phase atmospheric chemistry and gas-particle interactions; chemical characterization of indoor and outdoor air pollutants; aerosol generation and chemical and physical characterization; instrument development and sampling techniques; health risk assessment; oxidative processes in the atmosphere; aerosol-mediated chemical processes; trace gas removal in the atmosphere; tropospheric ozone production; atmospheric chemistry of climate change gases; iceatmosphere interactions; transport of semivolatile pollutants. Candidates with interests in heterogeneous atmospheric chemistry are especially encouraged to apply.

Applications should include a résumé with a list of publications, a concise statement of research and teaching interests, and the names and addresses (including e-mail) of at least four referees. Applications should be submitted electronically to http://enac.epfl.ch/page2114.html by 1st May 2011 when formal screening of applications will begin.

Successful candidates are expected to initiate independent research programs and be com-

Informal enquiries may be made to: Professor Andrea Rinaldo [email protected] Additional information about EPFL is available at http://www.epfl.ch, http://enac.epfl.ch EPFL is an equal opportunity employer. Women candidates are particularly encouraged to apply.

Junior Faculty Position Department of Microbiology and Physiological Systems Worcester, MA The Department of Microbiology and Physiological Systems invites applications for a junior-level tenure-track faculty position. Candidates should have an outstanding record of achievement in research that addresses important questions broadly relevant to infectious disease and encompasses multiple levels of biological organization. Technologies and approaches of particular interest include, but are not limited to, advanced imaging from single molecules to the whole animal, mechanistic mathematical modeling closely informed by experiment, and integrative functional analysis of genome-scale information. The Department of Microbiology and Physiological Systems is a newly formed department with strong expertise in bacterial and viral pathogenesis, immunology, and fundamental cellular physiology. The University of Massachusetts Medical School (http://www.umassmed.edu/) is a vibrant, rapidly growing and highly interactive scientific community with broad expertise. Superb resources, a highly competitive start-up package, and generous and newly renovated space will be provided to the successful candidate. The Department of Microbiology and Physiological Systems has close ties to clinical departments and a joint clinical appointment is possible for individuals with clinical training. Applicants should submit a cover letter, curriculum vitae, statement of research interests and contact information for three references to http://www.academicjobsonline.org. Inquiries, but not application materials, may be directed to [email protected]. As an equal opportunity and affirmative action employer, UMMS recognizes the power of a diverse community and encourages applications from individuals with varied experiences, perspectives and backgrounds.

Director Gastrointestinal Immunology Program The Department of Medicine at the Georgetown University Medical Center is seeking a dynamic founding director of the Gastrointestinal Immunology Program. The ideal candidate will have an MD or MD/PhD degree, and an established track record of NIH – supported research of gastrointestinal immunology. The candidate will establish and lead a translational effort in immunology to be integrated with clinical and basic science investigators within the Georgetown scientiﬁc community, and the Georgetown - Howard Universities Center for Clinical and Translational Science. A clinical focus and the ability to collaborate with other investigators will be advantageous. Appointment will be at the Professor or Associate Professor level. Please send curriculum vitae to Tolise Miles at [email protected], and contact Ms. Miles for questions via e-mail or by phone at (202) 444-7309. Georgetown University is an Afﬁrmative Action/Equal Opportunity Employer.

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.

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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Aker/Zvonkovic Photography

Our World-Class Research Institute Is Looking for Scientific Leaders

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MEETINGS Computational Biosciences Initiative University of California Los Angeles Open Faculty Appointments UCLA announces a Chancellor’s Initiative in Computational Biosciences with an emphasis in bioinformatics/genomics/computational biology. Building on outstanding faculty in these areas across the College of Letters and Sciences, David Geffen School of Medicine (DGSOM), Henry Samueli School of Engineering and Applied Mathematics (HSSEAS), and School of Public Health (SPH), UCLA will establish a Computational Biosciences Institute that will include: faculty with departmental appointments across campus who are afﬁliated with the recently established Interdepartmental PhD Program in Bioinformatics; a new bioinformatics center that provides the analysis of high throughput data for groups across campus; and computational infrastructure. Over the next few years, we anticipate up to 12 new appointments from across campus associated with the Computational Biosciences Institute. This year we are conducting a search for a junior or senior level appointment in the area of bioinformatics and computational biology. We seek nominations and applications from individuals who have expertise in areas related to bioinformatics, genomics, and computational biology and would bring intellectual leadership and synergy to this new UCLA Institute. The candidate’s research program should in part develop novel computational, quantitative, or bioinformatics methodology to address fundamental issues in biology or biomedical science. Letters of nomination and questions about the position should be sent to Dr. Matteo Pellegrini at [email protected]. Materials should be submitted online as a single pdf through www.mcdb.ucla.edu/compbiosci. Please include a cover letter with a statement of research and vision, names of referees, and CV. Please use job number 0865-1112-01 in all correspondence Applications will be reviewed upon receipt until position is ﬁlled. Candidates who wish to receive full consideration should submit all materials by March 25, 2011. UCLA is California’s largest university, with an enrollment of nearly 38,000 undergraduate and graduate students. The UCLA College of Letters and Science and the university’s 11 professional schools feature renowned faculty and 323 degree programs and majors. The Biosciences at UCLA include more than 300 faculty members, many top ten ranked departments, and is consistently in the top ten in NIH funding. As a campus with a diverse student body, we encourage applications from women, minorities, and individuals with a history of mentoring under-represented minorities in the sciences. UCLA is an Afﬁrmative Action/Equal Opportunity Employer with a strong institutional commitment to the achievement of faculty and staff diversity.

Chair Department of Molecular Microbiology and Immunology The Warren Alpert Medical School Brown University The Warren Alpert Medical School and the Division of Biology and Medicine of Brown University invite applications for the position of Professor and Chair of the Department of Molecular Microbiology and Immunology. This position is to lead an interdisciplinary group of faculty and build a research program in host-pathogen interactions and pathogenesis of disease. The Department contributes to the teaching of undergraduate, graduate, and medical students. Brown University is expanding opportunities for research and educational activities that bridge basic scientists and clinical translational researchers at its afﬁliated hospitals. The next Chair will provide leadership in expanding the research activities of the department and in recruiting and mentoring new faculty. Excellent core research facilities, interdisciplinary graduate programs with external funding, and additional research space are available to foster expansion of the Department. The new Chair should have a record of scholarship and externally-funded research that is recognized internationally, and demonstrated leadership and administrative skills. Applicants should submit their application electronically in a single pdf to: [email protected]. Postal communications should be sent to: Chair, MMI Chair Search Committee, Brown University, Division of Biology and Medicine, Box G-A1, Providence, Rhode Island 02912. Applicants should include a letter describing their vision as the new Chair and future career plans, a curriculum vitae, and ﬁve names of potential external referees with contact information. Review of applications will begin immediately and will continue until the position is ﬁlled. A complete description of the Department and faculty can be found at the Department’s website: http://bms.brown.edu/mmi/. Brown University is an EEO/AA Employer and invites applications from women, minorities, and protected persons.

Islet Society Meeting 17-18 July, 2011 Nordic Sea Hotel, Stockholm

For details, please see

www.isletsociety.org

GENE CENTER MUNICH

The Gene Center of the Ludwig-Maximilians-Universität München (LMU) invites applications for the position of an

Independent Group Leader in Advanced Biological Mass Spectrometry Candidates must have an outstanding record of internationally recognized research accomplishments in advanced biological mass spectrometry, ideally including technologies for the structural characterization of transient multicomponent complexes. The research group is expected to contribute to the establishment of molecular systems biology at the LMU, and to participate in extramural funding networks such as SFBs and programs of the excellence initiative. Candidates are expected to conduct independent research that complements existing research at the Gene Center, to obtain additional extramural funding, and are welcome to participate in teaching (in English or German). Primary selection criteria are research excellence and the potential for scienti ic interactions. The Gene Center offers a stimulating and interdisciplinary environment, and is committed to expand the research focus towards molecular systems biology. Information can be found at www.genzentrum.lmu.de. The position is initially for ive years but may be extended by 2-3 years if funding permits. The LMU seeks to increase the number of women researchers and especially invites quali ied women to apply. The LMU offers a Dual Career Service. Handicapped candidates with equal quali ications will be given preference. Applicants should submit a single pdf- ile with their motivation letter, CV, list of publications, and research proposal before May 30, 2011 to [email protected]. Informal enquiries may be sent to Patrick Cramer ([email protected]).

Boehringer Ingelheim ranks among the world’s 15 leading pharmaceutical corporations. Our vision drives us forward. It helps us to foster value through innovation in our company and to look to the future with constantly renewed commitment and ambition.

Value through Innovation Our family’s science, your family’s health.

BIOTHERAPEUTICS OPENINGS:

For 125 years Boehringer Ingelheim has been committed to the research

RD02111: Scientist II, Immunogen Design/Expression RD02211: Senior Scientist, Mouse Immunization/Adjuvants RD02311: Senior Principal Scientist, Lead Generation (B-cell Biology) RD02411: Scientist III, B-cell Biology/Hybridoma RD02511: Principal Scientist, Binding Assay Development RD02611: Scientist III, Binding Assay Development

and development of innovative medicines that help improve the lives of patients and their families. Research & Development has been the foundation of Boehringer Ingelheim's success and continues to be the major driver of innovative, new medicines for the treatment of diseases with an unmet therapeutic need. We have more than 6,900 highly qualified people working in research & development out of approximately 41,500 Boehringer Ingelheim employees worldwide. Our drug discovery focuses on six major therapeutic areas: respiratory diseases, cardiometabolic diseases, oncology, neurological diseases, immunology & infectious diseases. Biotherapeutics research is a rapidly growing area at Boehringer Ingelheim. The Biotherapeutics department in Ridgefield, CT works with resources in all major therapeutic areas to bring novel biotherapeutic agents to the clinical pipeline. We are looking for enthusiastic and talented individuals to join a highly effective team. Learn more about us at us.boehringer-ingelheim.com

RD02711: Senior Scientist, Bi-specific and Fc Engineering RD02811: Scientist III, Bi-specific and Fc Engineering RD02911: Scientist II, Lead Expression RD03011: Principal Scientist, Downstream Process Assessment (Protein Purification) RD04510: Senior Research Fellow, Lead Generation RD04710: Scientist ll, Lead Generation RD11610: Principal Scientist, Bi-specific Antibody Engineering

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Call for Large-Scale Sequencing Projects The U.S. Department of Energy JGI (DOE JGI) is now accepting letters of intent for complex large-scale genome sequence-based projects to advance the frontiers of DOE-mission science relevant to bioenergy and the environment. The specific emphasis of this call is targeted toward: Plant and Plant-Microbe interactions: Plant phenotypes are likely to be strongly influenced by their associated microbes. Studies are encouraged that explore the interaction of plants with their rhizosphere communities and other microbes or fungi that affect bioenergy-relevant plant phenotypes. Microbial emission and capture of greenhouse gases: Bacteria, archaea, fungi, and algae are important consumers and producers of greenhouse gases in the environment. Studies are sought that will provide insight into global carbon, nitrogen, and methane cycles, and/or suggest novel strategies for carbon capture, nitrogen processing, or methane reduction from environmental sources. Letters of intent will only be accepted electronically and should be submitted at by April 26, 2011. Applicants will be advised whether to prepare a full proposal within two weeks, and full proposals will be due on June 15, 2011. For more information about the Community Sequence Program, the types of proposals being accepted, and to submit a letter of intent, go to: http://go.usa.gov/4Lt

CIRCB

CHANTAL BIYA INTERNATIONAL REFERENCE CENTRE FOR HIV/AIDS RESEARCH ON PREVENTION AND MANAGEMENT Yaounde, Cameroon

Job Opportunity - Position of Scientific Director The “Chantal Biya” International Reference Centre for Research on HIV/AIDS Prevention and Management (CIRCB) is located in Yaounde, the capital city of Cameroon, Central Africa. The CIRCB is affiliated to the Ministry of Public Health of Cameroon. CIRCB is seeking a candidate for full-time position of Scientific Director. The candidate should be holder of a doctoral degree (Ph.D., MD or MD/Ph.D.) in Virology, Immunology, Cell and Molecular Biology, with skills and experience in project management and management of human resources. Candidate should have a documented history of research success with a minimum of 10 years post-doctoral experience, a good track record of publications and a successful academic career. The candidate will be expected to coordinate and supervise all activities of the Scientific Division. Salary: a competitive salary and benefits package will be commensurate with qualifications and experience. This position is available for three to five years on a renewable contract. African scientists working abroad are encouraged to apply. Interested candidates should send their curriculum vitae, a two-page statement of research interests covering past accomplishments, current and planned research goals, complete bibliography, names and addresses of three referees to: Dr. Pierre Joseph FOUDA, MD Administrator [email protected] Prof. Vittorio Colizzi, MD, PhD. Scientific Director [email protected] B.P. 3077, Yaounde, Cameroon. Telephone/Fax: (+237) 2231-5456

Website: www.circb-cameroun.org

Faculty Positions in Pharmacology and Toxicology University of Kansas Medical Center (KUMC) The Department of Pharmacology, Toxicology, and Therapeutics, under the direction of Curtis Klaassen, Professor and Chair (http:// www.kumc.edu/pharmacology/), is continuing its expansion by inviting applications for two Assistant Professor, tenure-track faculty positions to augment the strength of our seventeen recent hires. Preference will be given to candidates who have done research in areas, such as nuclear receptors, hepatotoxicity, xenobiotic disposition (ADME), the metabolic syndrome, nutrition, pharmacogenomics, and epigenetics that complement existing strengths in the department and the medical center. This expansion is supported by a Centers of Biomedical Research Excellence (COBRE) grant entitled “Nuclear Receptors in Liver Function and Dysfunction,” a training grant in Environmental Sciences, and a new Liver Center. The COBRE provides an extensive program of mentoring junior faculty by experienced established senior faculty. A competitive startup package and appropriate space will be offered in a new 200,000 sq. ft. research building. Standard support facilities are present, including biotechnology, second-generation sequencing, transgenics, proteomics, and a state-of-theart brain imaging center. The department also has excellent LC-MS/MS and histopathology facilities. Applications will be reviewed ﬁrst on May 9th, and until the positions are ﬁlled. Applicants must be proﬁcient in the use of the English language. Anticipated appointment date is between July 1 and December 31, 2011. Applicants should provide a C.V., statement of research interests, and names of three references. To review the position description and apply on-line, go to http://jobs.kumc.edu and search for position J0083463. The University of Kansas Medical Center is proud to be an Equal Opportunity/Afﬁrmative Action Employer.

Neuropharmacology Tenure-Track Position Department of Pharmacology College of Medicine University of Saskatchewan Saskatoon, SK S7N 5E5 CANADA Applications are invited for a tenure-track Assistant Professor position to begin September 1, 2011. Preference will be given to candidates with an MD degree and/or a PhD degree in Pharmacology, expertise in Neuropharmacology with postdoctoral training. The position is open to candidates trained in all areas of Neuroscience, but those with expertise in synaptic plasticity, electrophysiology, structure-function of brain circuits, or neuronal stem cells and neuro-regeneration are especially encouraged to apply. The successful candidate will be given state-of-the-art research facilities, will develop an externally funded research program, and will contribute to the Department’s teaching responsibilities. Prior to May 31, 2011, applicants should submit, by e-mail, a Curriculum Vitae, a brief statement of research interests, reprints of recent publications, and the names and addresses of three referees to: Chair, Search Committee, c/o Cindy S. Wruck, [email protected]. All qualiﬁed candidates are encouraged to apply. However, citizens and permanent residents of Canada will be given priority. The University of Saskatchewan is committed to employment equity. Members of designated groups (women, aboriginal people, people with disabilities, and visible minorities) are encouraged to self-identify on their applications.

Make an impact on the Development of Molecularly Guided Cancer Therapy - come work at the National Cancer Institute! Chief, Diagnostic Biomarkers and Technology Branch The Cancer Diagnosis Program (CDP), Division of Cancer Treatment and Diagnosis (DCTD), NCI is seeking an experienced scientist for the position of Chief, Diagnostic Biomarkers and Technology Branch, Health Science Administrator GS-601-15. A PhD or equivalent in a recognized discipline of the health sciences or allied sciences (e.g., chemistry, biochemistry, immunology, genetics, biology, microbiology, molecular biology) is required, as is considerable experience and in-depth knowledge of new technologies and their direct application to the development of diagnostics. Skill in managing multiple projects simultaneously, providing technical direction to ad hoc team members, and clearly articulating issues to professional staff is highly desirable. The CDP fosters the development and validation of molecular diagnostics that inform the treatment of malignant diseases. The Branch Chief reports to the Associate Director, CDP, and supervises program directors in the Branch. The Chief provides leadership in conceptualizing, planning, implementing, managing, and evaluating research programs designed to lead to creation of molecular diagnostics that will improve the treatment and survival of cancer patients. Responsibilities include independent management of complex, multidisciplinary contracts, grants, and cooperative agreements for several programs of national and international scope and impact involving the development of new technologies for cancer diagnosis research. Interaction with other DCTD programs, other government agencies, public health institutions, academia, and private industry as well as travel will be required. Base salary for this position ranges from $123,758 to $155,500 per annum. Beneﬁts include health and life insurance options, retirement, paid holidays and vacation leave. This is an exploratory ad to gauge interest and the candidate pool. If you provide us with your email address we will inform you when a Vacancy Announcement (VA) to ﬁll the position is issued. Please note that copies of school transcripts are required when applying to the VA. Please submit your C.V., a statement of interest and contact information to Administrative Officer, Kat Bern, at [email protected]. For more information about the position, please contact Barbara A. Conley, M.D. at [email protected] or (301) 496-8639. CDP website: http://www.cancerdiagnosis.nci.nih.gov. DHHS, NIH and NCI are Equal Opportunity Employers.

Health  Sustainability  Policy  Technology

LIVING THE

PROMISE

INNOVATIVE THINKING

BREAKTHROUGH RESEARCH REAL-WORLD SOLUTIONS

HEALTH SOLUTIONS UCR neuroscientists are developing powerful new diagnostic tools and targeted treatments for autism, fragile X syndrome and other disorders.

Explore more solutions:

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FOCUS ON CANCER RESEARCH

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CHAIR Department of Physiology The University of Texas Health Science Center at San Antonio (UTHSCSA) invites applications and nominations for the position of Chair of the Department of Physiology. We seek candidates with an outstanding record of scientiﬁc achievement, grant support, and mentoring, and must be qualiﬁed to attain the rank of professor with tenure. Dynamic leadership, communication, interpersonal skills, and keen vision are required. The Department currently consists of twenty-one full-time faculty with clusters of strength in neuroscience, ion channels, aging, and model systems (http://www.physiology.uthscsa.edu). The search, however, is not limited to these research foci, and candidates with scientiﬁc interests in any area relevant to physiology (e.g. cardiovascular, muscle, renal) are encouraged to apply. The Department of Physiology is one of seven Basic Science Departments that comprise the Graduate School of Biomedical Sciences (GSBS), and the GSBS is one of ﬁve component schools of the UTHSCSA. The recent hiring of two new Deans (Medical School and Graduate School), a Health Science Center administration that will focus resources to grow our research mission, and the construction of a new, cutting-edge 200,000 ft2 research building (South Texas Research Facility – STRF) makes this a wonderful opportunity for a visionary leader. UTHSCSA is a Tier One research institution located in the Northwest region of San Antonio and sits as a gateway to the picturesque Texas Hill Country. San Antonio is a vibrant, dynamic, and multicultural city with much to offer including an attractive cost-of-living. Applications should include a Curriculum Vitae, a brief statement of research interests and academic vision, and a list of four references. The deadline for submission of completed applications is September 1, 2011. Send materials electronically to [email protected] or by mail to: Charles P. France, Ph.D., Chair, Search Committee for Physiology Chair, Graduate Dean’s Ofﬁce, MC 7819, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. All faculty appointments are designated as security sensitive positions. The University of Texas Health Science Center at San Antonio is an Equal Employment Opportunity/Afﬁrmative Action Employer.

Faculty Position in Bioengineering at Washington State University The Gene and Linda Voiland School of Chemical Engineering and Bioengineering (ChEBE) at Washington State University invites applications for a tenure-track faculty position at the Assistant/Associate/Full Professor level. The successful applicant will be based at the Pullman campus. Successful candidates must hold a doctorate in bioengineering, chemical engineering, or a closely related discipline. We desire candidates who excel in protein biomolecular engineering, so preference will be given to candidates with a strong record of publication in high impact, peer-refereed, archival journals, a proven ability to attract externally sponsored research funding, a proven record of collaboration, and demonstrated ability to teach undergraduate and graduate courses. We are primarily interested in applicants whose research advances the understanding and application of the role and function of proteins in human health. As such, candidates with interests in cardiac muscle research, tissue engineering, biosensors, stimulus-responsive materials, computational approaches to cellular engineering and protein bioengineering are encouraged to apply. The Voiland School currently has 250 undergraduate students, 41 PhD students, and 14 faculty. This ﬁscal year, faculty in the school have been awarded extramural funds totaling $3.7 million from industry, NSF, DTRA, DOE, NIH, and ONR and have created two new companies from technologies developed as a part of their research. During the next year, we plan to add three additional faculty, including the individual who will be hired into this position. Faculty in the Voiland School are conducting interdisciplinary bioengineering research and have strong interactions with College of Veterinary Medicine and WSU’s newly established School for Global Animal Health. A successful candidate for this position is expected to establish and maintain a funded, innovative and nationally recognized research program, collaborate with others within the School, the University, or other organizations, and develop and teach graduate and undergraduate core and elective courses. Interested candidates should submit a letter of application, curriculum vitae, names and contact information of four references, a statement of research plans and a statement of teaching philosophy online at: www.wsujobs.com/applicants/Central?quickFind=55959. The letter should be addressed to Dr. Wen-Ji Dong, Chair Search Committee, Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164-2710. Screening of application materials will begin immediately and will continue until the position is ﬁlled. Preferred starting date of this new position is August 16, 2011. Washington State University is an Equal Opportunity/Afﬁrmative Action Educator and Employer. Members of ethnic minorities, women, special disabled veterans, veterans of the Vietnam-era, recently separated veterans, and other protected veterans, persons of disability and/or persons age 40 and over are encouraged to apply. WSU is committed to excellence through diversity, has faculty friendly policies including a partner accommodation program, and a NSF ADVANCE Institutional Transformation grant (http://www.excelinse.wsu.edu/). WSU employs only U.S. citizens and lawfully authorized non-U.S. citizens. All new employees must show employment eligibility veriﬁcation as required by the U.S. Citizenship and Immigration Services.

The Gene and Linda Voiland School of Chemical Engineering and Bioengineering

Assistant Professor of Chemical Engineering at Washington State University The Gene and Linda Voiland School of Chemical Engineering and Bioengineering at Washington State University invites applications for a tenure-track position as Assistant Professor of Chemical Engineering. The successful applicant will be based at the Pullman campus. The primary area of research interest is energy, including but not limited to catalysis, reaction engineering, and advanced materials for renewable and sustainable energy. Candidates must hold a Ph.D. in Chemical Engineering or closely related discipline at the time of appointment. We desire candidates who will excel in energy-related research and to teach core and elective courses in chemical engineering so preference will be given to candidates with a strong record of publication in high impact, peer-refereed, archival journals, a proven ability to attract externally sponsored research, a proven record of collaboration, and demonstrated ability to teach undergraduate and graduate courses. The Voiland School currently has 250 undergraduate students, 41 PhD students, and 14 faculty. Our year-to-date award totals at $3.7 million for externally funded research. This research is funded by industry, NSF, DTRA, DOE, NIH, and ONR. To date this year, the faculty have created two new companies from technologies developed as a part of their research. During the next year, we plan to add three additional faculty, including the individual who will be hired into this position. The successful candidate may hold a partial appointment in the Agricultural Research Center, WSU’s agricultural experiment station. Washington State University’s Pullman campus is close to the US DOE’s Paciﬁc Northwest National Laboratory (PNNL), and faculty members at the Pullman campus have excellent opportunities for research collaborations with staff at PNNL including the Institute for Interfacial Catalysis (IIC), as well as access to the state-of-the-art analytical equipment located at the PNNL user facility, the Environmental Molecular Sciences Laboratory (EMSL). Responsibilities for this position include: conducting funded innovative research in renewable and sustainable energy leading to internationally regarded publications, developing and leading a nationally recognized research group focusing in that area, collaborating with others within the School, the University, PNNL, and/or other organizations, and successfully developing and teaching graduate and undergraduate core and elective courses. Submit a letter of application, curriculum vitae, names and contact information of four references, a statement of research plans and a statement of teaching philosophy online at www.wsujobs.com/applicants/Central?quickFind=55985. The application letter should be addressed to Dr. Yong Wang, Voiland Distinguished Professor and Chair Search Committee, Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164 2710. Screening of application materials will begin immediately and will continue until the position is ﬁlled. Preferred starting date of this new position is August 16, 2011. WSU is committed to excellence through diversity, has faculty friendly policies including a partner accommodation program, and a NSF ADVANCE Institutional Transformation grant to increase the advancement of women faculty in science, engineering and math (see http://www.excelinse.wsu.edu/.) WSU is an EEO/AA/ADA Educator and Employer.

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The Gene and Linda Voiland School of Chemical Engineering and Bioengineering

Get a Career Plan that Works. An exceptional career requires insightful planning and management. That’s where Science Careers comes in. From job search to career enhancement, Science Careers has the tools and resources to help you achieve your goals. Get yourself on the right track today and get a real career plan that works. Visit ScienceCareers.org.

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76th Cold Spring Harbor Symposium on Quantitative Biology

Metabolism & Disease June 1 – 6, 2011 Organizers Terri Grodzicker, David Stewart & Bruce Stillman Cold Spring Harbor Laboratory Poster abstracts due March 31, 2011

Speakers Angelika Amon, Massachusetts Institute of Technology Johan Auwerx, Ecole Polytechnique Federale de Lausanne, Switzerland Joseph Bass, Northwestern University Medical School Shelley Berger, University of Pennsylvania David Botstein, Princeton University Michael Brown, UT Southwestern Medical School Joan Brugge, Harvard Medical School Chi Dang, Johns Hopkins University School of Medicine Ronald Evans, HHMI/Salk Institute for Biological Studies Jeffrey Friedman, The Rockefeller University Joseph Goldstein, UT Southwestern Medical Center Eyal Gottlieb, Beatson Institute for Cancer Research, UK Kun-Liang Guan, University of California, San Diego Leonard Guarente, Massachusetts Institute of Technology Grahame Hardie, University of Dundee, UK Takashi Kadowaki, Tokyo University Medical School, Japan William Kaelin, HHMI/Dana-Farber Cancer Institute Barbara Kahn, Beth Israel Hospital C. Ronald Kahn, Joslin Diabetes Center Michael Karin, University of California, San Diego Gerard Karsenty, Columbia University Shigeaki Kato, University of Tokyo, Japan Daniel Kelly, Sanford-Burnham Med. Res. Inst. at Lake Nona Paolo Sassone-Corsi, University of California, Irvine Ulrich Schibler, University of Geneva, Switzerland

Narry Kim, Seoul National University, Korea Mitchell Lazar, University of Pennsylvania

Gregg Semenza, Johns Hopkins University School of Medicine Reuben Shaw, Salk Institute for Biological Studies

Richard Losick, Harvard University

Gerald Shulman, HHMI/Yale Medical School

Tak Mak, Ontario Cancer Institute, Canada Susanne Mandrup, University of Southern Denmark

Pamela Silver, Harvard Medical School

David Mangelsdorf, HHMI/UT Southwestern Medical Center

David Sinclair, Harvard Medical School Nahum Sonenberg, McGill University, Canada

Steven McKnight, UT Southwestern Medical Center

Bruce Spiegelman, Dana-Farber Cancer Institute

Noboru Mizushima, Tokyo Medical & Dental University, Japan Richard Morimoto, Northwestern University Deborah Muoio, Duke University School of Medicine

Craig Thompson, Memorial Sloan-Kettering Cancer Center Peter Tontonoz, HHMI/University of California, Los Angeles Benjamin Tu, UT Southwestern Medical Center

Anders Naar, Harvard Medical School Christopher Newgard, Duke University Medical Center

Matthew Vander Heiden, Massachusetts Institute of Technology Eric Verdin, J. David Gladstone Institutes

Dianne Newman, California Institute of Technology Stephen O’Rahilly, University of Cambridge, UK

Karen Vousden, Beatson Institute for Cancer Research, UK Amy Wagers, Harvard University

Pere Puigserver, Harvard Medical School Joshua Rabinowitz, Princeton University Danny Reinberg, HHMI/NYU School of Medicine Gary Ruvkun, Massachusetts General Hospital

Douglas Wallace, Children’s Hospital of Philadelphia Xiaodong Wang, Zhongguancun Life Science Park, China Eileen White, Rutgers University/The Cancer Institute of NJ Junying Yuan, Harvard Medical School

David Sabatini, Whitehead Institute

Registration, abstract submission and further information: http://www.cshl.edu/meetings

e-mail: [email protected]

phone: 516.367.8346

Image credit: Guy Perkins and Mark Ellisman, NCMIR, UCSD

Cynthia Kenyon, University of California, San Francisco

fax: 516.367.8845

online @sciencecareers.org

POSITIONS OPEN

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From technology specialists to patent attorneys to policy advisers, learn more about the types of careers that scientists can pursue and the skills needed in order to succeed in nonresearch careers.

TWO TENURE-TRACK FACULTY POSITIONS Marine Microbial Ecology and Coastal Processes Old Dominion University_s Department of Ocean, Earth and Atmospheric Sciences (OEAS) seeks to fill two tenure-track faculty positions. One is in the area of marine microbial ecology. Possible research areas include, but are not limited to: microbial contributions to marine productivity, food webs, and biogeochemical cycling as well as microbial proteomics, genomics, and physiology. Skills are particularly sought in the application of developing technologies, including biochemical or molecular biology approaches. The second position lies in the area of coastal processes, with a focus either on observational physical oceanography or coastal dynamics. Specific areas of interest include: coastal and shelf circulation, surf-zone processes, surface- or bottom-boundary layers, sediment transport, air-sea exchange, Quaternary processes, and influences of climate change on coastal systems such as salt marshes, estuaries, and barrier islands. Specific course offerings by the successful applicants will include undergraduate or graduate classes contributing to the department_s overall curriculum as well as courses commensurate with the candidates_ expertise. The department has a robust graduate program with students pursuing either M.S. or Ph.D. degrees. Both positions will be available as soon as July 25, 2011. Colleagues at the assistant-professor level are preferred, but exceptional candidates at the associate level will be considered. Applicants must hold a Ph.D. degree in the oceanographic or related sciences and postdoctoral experience is desirable. The successful candidates must have excellent communications skills and demonstrate strong potential for outstanding accomplishments in research and teaching. Many opportunities exist for disciplinary and interdisciplinary interactions with more than 25 other faculty in OEAS, its Center for Coastal Physical Oceanography, and other departments within the university. Research and training awards to our faculty in FY2010 were in excess of $6.3M. Additional information about the department and its facilities can be found at website: http://sci.odu.edu/oceanography/. Applications, including a cover letter, curriculum vitae, teaching and research statements (one page each), copies of three relevant peer-reviewed publications, and contact information for three references must be submitted electronically to e-mail: [email protected]. Review of applications will begin April 15, 2011 and continue until the positions are filled. The College of Sciences welcomes the opportunity to work with candidates to identify suitable employment opportunities for spouses. Old Dominion University is an Equal Opportunity/Affirmative Action Institution and requires compliance with the Immigration Reform and Control Act of 1986.

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Announcing the 2011 Recipients

The Canada Gairdner Awards are given in 3 categories; International Awards (seminal discoveries/contributions to medical science), the Global Health Award (scientific advances contributing to health in the developing world) and the Wightman Award (leadership in Canadian medicine). International Award

Adrian Peter Bird PhD, The Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh

International Award

Howard Cedar MD, PhD, Department of Developmental Biology and Cancer Research, Hebrew University, Jerusalem

International Award

Aharon Razin PhD, Department of Developmental Biology and Cancer Research, Hebrew University, Jerusalem

For their pioneering discoveries on DNA methylation and its role in gene expression.

International Award

International Award

Global Health Award

Robert Black MD, MPH, Edgar Berman Professor & Chair in International Health, Johns Hopkins University, Baltimore For improving child survival and particularly for critical clinical and epidemiological studies to reduce childhood diarrheal deaths.

Wightman Award Supported nationally by:

Jules A. Hoffmann PhD, Distinguished Class Research Director, CNRS and Group Leader, Member of the Board of Administrators of CNRS, Strasbourg

Shizuo Akira MD, PhD, Director and Professor, WPI Immunology Frontier Research Center, Osaka

Michael Hayden CM, OBC, MB, ChB, PhD, Canada Research Chair in Human Genetics & Molecular Medicine, University of British Columbia, Vancouver

The awards will be presented in Toronto, Canada on October 27, 2011

www.gairdner.org This announcement is supported by:

For their ground breaking discoveries and definition of the family of Toll-like receptors and the array of microbial compounds that they recognize to provide innate resistance to infection.

For his outstanding national and international leadership in medical genetics, entrepreneurship and humanitarianism.

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Angiostatin and Endostatin: Key Players in a Dual Threat Approach to Cancer Treatment John Swarthout Ph.D., Sigma-Aldrich Corporation Cancer is a complex disease characterized by unregulated proliferation, tissue invasion, and metastasis. One promising area of research toward the continued development of anticancer drugs surrounds natural and synthetic angiogenesis inhibitors. Advancing our understanding of the regulatory mechanisms central to tumor angiogenesis will provide fundamental insights into how to target this process for limiting the growth and spread of a tumor. Numerous antiangiogenic factors have been identified, including the endogenous proteins angiostatin and endostatin.1 Angiogenic inhibitors influence the induction of angiogenesis by growth factors, the activity of angiogenic proteinases, endothelial cell proliferation and migration, or microtubule formation. However, the exact manner by which angiostatin and endostatin impede tumorigenesis needs to be completely elucidated. Recently, angiostatin and endostatin became widely available to the research community in recombinant forms, expressed and purified from Pichia pastoris. The increased availability of these proteins will likely facilitate tumor angiogenesis research. Throughout tumorigenesis, cells acquire a set of functional capabilities including self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, tissue invasion and metastasis, and sustained angiogenesis.2 Angiogenesis is the multistep physiological process of new capillary growth from pre-existing blood vessels,3 and is requisite for the growth and spread of cancer.2 In many cancers, the balance that normally exists between angiogenic inducers and angiogenic inhibitors shifts toward the proangiogenic state resulting in the synthesis of new blood vessels (Figure 1).4 Initially cancers co-opt the existing vasculature. Then the angiogenic switch results in the production of factors that induce angiogenic sprouting of the vasculature. Expressed pro-angiogenic factors bind to receptors including vascular endothelial growth factor (VEGF) receptor 2 and neuropilin-1 on the vascular endothelial cells of nearby blood vessels and promote cell proliferation, migration, and invasion into the tumor.2,4,5 This is important as avascular tumors are limited in size and require development of new blood vessels to deliver the necessary oxygen and nutrients, and for removal of cellular waste through the interstitium. Therefore, reducing or inhibiting angiogenesis removes a vital lifeline that allows cancer cells to grow, invade nearby tissue, metastasize, and form new colonies of cancer cells. Both angiostatin and endostatin require enzymatic cleavage from a parent molecule before they are biologically active.1 Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen. It was originally isolated from tumor bearing mice,6 and has both potent antiangiogenic activity and antiproliferative activity toward endothelial and cancer cells.7 Plasminogen contains five kringle (K) domains of ~80 residues each. Studies using recombinant angiostatin demonstrated tumor inhibitory activity resides in a fragment of K1-3,8 and this region forms a central cavity that may contain a protein recognition site essential for activity.9 Recent evidence supports dual antitumor mechanisms for plasminogen derivatives, one affecting angiogenesis and another targeting

Recombinant angiostatin and endostatin proteins, sold by Sigma-Aldrich under license from CMCC Boston. Reference US Patent Nos. 5,854,205; 6,024,688; 5,861,372

Figure 1. The “angiogenic switch” is illustrated as a balance between proangiogenic factors (represented by red spheres) and angiogenesis inhibitors (represented by gray spheres).

tumor cells directly.10 Kringle 5 (K5), like angiostatin, is a byproduct of the proteolytic cleavage of plasminogen. In a recent study, Ansell et al., demonstrated K5 functions as a competitive antagonist of hepatocyte growth factor (HGF).11 HGF contains kringle motifs and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiostatic role, K5 can direct a potent antitumor response with its ability to recruit tumor-associated neutrophils and Natural Killer T cells.12 Endostatin is 20 kDa carboxyl-terminal fragment of type XVIII collagen that is present in walls and basement membranes of blood vessels and plays an important role in endothelial cell adhesion and cytoskeletal organization.13,14 Endogenous endostatin inhibits migration and induces apoptosis in endothelial cells, inhibits tumor growth, and impairs blood vessel maturation in wound healing.15,16 It is thought to interfere with the proangiogenic action of growth factors such as basic fibroblast growth factor and VEGF, and is known to inhibit at least 65 different tumor types.17 Additionally, a study measuring changes in gene expression in human dermal microvascular cells following treatment with endostatin demonstrated a downregulation of several proangiogenic pathways as well as the upregulation of many antiangiogenic genes.17 There are numerous clinical trials underway employing antiangiogenic drugs for the treatment of cancers.18 However, to enhance the survival benefits, there is mounting interest in developing more effective ways to combine antiangiogenic drugs with established chemotherapies.19 Recent studies indicate a combination of antiangiogenic factors demonstrate an additive or synergistic inhibition of angiogenesis and tumor growth.20,21 For example, the in vivo co-production of human endostatin and tissue inhibitor of metalloproteinase-1 demonstrated a synergistic antitumor growth and reduction of metastasis in murine melanoma.22 Continued research will advance our understanding of the antiangiogenic mechanisms of these proteins as well as lead to improved efficacy in anticancer therapies. For a complete list of references and more information, visit:

wherebiobegins.com/angiogenesis

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SYNTHETIC GENOMICS

SYNTHETIC GENOMICS BUILDING A BETTER BACTERIUM

The May 20, 2010, online edition of Science magazine contained pieces on Brownian motion and gravitational waves, small RNAs and drug delivery—items of interest to narrow slices of the research community. One article, though, generated instant worldwide attention. Entitled “Creation of a bacterial cell controlled by a chemically synthesized genome,” the report detailed the world’s first “synthetic cell,” and it was at once praised and panned. Watchdog groups weighed in, as did U.S. President Barack Obama. Powered by advances in DNA synthesis and genome manipulation, the study was merely a proof-of-principle: Mycoplasma mycoides JCVI-syn1.0 has no practical scientific or commercial value. Yet its cobalt blue colonies represent the living embodiment of an entirely new, and previously unimaginable, branch of biology. Welcome to the age of synthetic genomics. By Jeffrey M. Perkel

“This is the control experiment. We are now at stage one.”

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the technology to develop algae capable of cranking out faster, cheaper, and better biofuels and agricultural products, striking a $300 million deal with ExxonMobil Research and Engineering in 2009 to advance that aim. But they’re not there yet. “To my viewpoint,” says Venter, whose eponymous Institute performed the synthetic cell work, “this is the control experiment. We are now at stage one.”

THE SYNTHETIC CELL Mycoplasma mycoides JCVI-syn1.0 was the product of some 15 years and $40 million worth of effort by Venter, Clyde Hutchison, Hamilton Smith, and about two-dozen others at the JCVI. The team ﬁrst sequenced and then chemically synthesized the genome of the bacterium, Mycobacterium mycoides, and then inserted it into a related organism, M. capricolum. In the parlance of synthetic biology, M. capricolum served as a “chassis”—a microbial shell. Loaded with the genetic operating system of its close cousin, it was then “rebooted” to produce a living synthetic cell. Bioethicist Arthur Caplan, writing in Nature, called the work “one of the most important scientiﬁc achievements in the history of mankind.” Others were more measured; New York Times science writer Nicholas Wade called the research “a matter of scale rather than a scientiﬁc breakthrough.” The U.K.’s Daily Mail, in a bit of nuanced headline writing (and while simultaneously invoking the specter of global pandemic as in the Will Smith movie, I Am Legend), declared: “Scientist accused of playing God after

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“Synthetic genomics,” reads the introduction to Synthetic Genomics: Options for Governance, a report by the J. Craig Venter Institute (JCVI), Massachusetts Institute of Technology, and the Center for Strategic & International Studies, “combines methods for the chemical synthesis of DNA with computational techniques to design it.” That doesn’t sound all that different from the standard molecular biology researchers have been doing for decades, and in some respects, it isn’t; what’s different is the incorporation of design and engineering sensibilities—not to mention the scale of the science. “These methods allow scientists to construct genetic material that would be impossible or impractical to produce using more conventional biotechnological approaches.” (See report, www.jcvi.org/cms/research/projects/ syngen-options/overview/) Researchers have been making point mutations, cloning genes, and designing novel biological circuits for years. They can even transplant biological pathways, using what James Collins, a synthetic biologist and Howard Hughes Medical Institute investigator at Boston University calls “genetic engineering on steroids.” (As the JCVI report notes, “There is no clear and unambiguous threshold between synthetic genomics and more conventional approaches to biotechnology.”) But it can be a long, laborious process; by J. Craig Venter’s estimation, DuPont’s development of microbes that can spin glucose into propanediol, a precursor to the company’s Sorona synthetic polymer, required “10 years and well over $100 million.” And that’s just one pathway; rewriting a biological operating system from the ground up is a different matter entirely. Enter synthetic genomics. Fueled by advances in gene building, metagenomics, and bio-circuitry design, researchers are coaxing microbes to do things never before possible—albeit not yet at the genomic scale. But that could soon change; in the not-too-distant future, says Collins, it may be possible to design a minimally functional genome, fold in novel or desired biochemical circuits, synthesize the DNA, and go. Venter has formed a company to do just that; Synthetic Genomics is using

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SYNTHETIC GENOMICS

creating artiﬁcial life by making such as new aminoacyl-tRNA designer microbe from scratch— synthetases. “That’s not somebut could it wipe out humanity?” thing you could do by mutagenThe answer to that question esis or by any sort of simple is undeniably no; Venter’s team genetic engineering methods,” merely recapitulated the genome he says. of M. mycoides (with the addiFirst, though, researchers will tion of a few “watermarks” and have to bone up on their biolother small genetic tweaks) and ogy. Genome sequencing and transplanted it into the functionmetagenomics efforts have ing membranes and cytoplasm of ﬁlled databases to overﬂowa close relative. If M. mycoides ing with novel genes, but recannot wipe out humanity, neisearchers simply don’t know ther can its lab-bred cousin. what many of them do. Even “Excitement and relief...There e To build the genome, Venter less well understood are the were literally thousands of hurdles and his team turned to Blue regulatory layers controlling Heron (acquired by OriGene those activities. Venter’s study, that had to be overcome.” Technologies in 2010). Unlike says Raik Grünberg, a postmost oligonucleotide synthesis doctoral fellow at the Centre ﬁrms, which specialize in crankfor Genomic Regulationing out polymerase chain reaction primers by the thousands, European Molecular Biology Laboratory (CGR-EMBL) SysBlue Heron (and other gene synthesis companies, including tems Biology Unit in Barcelona who develops synthetic biologiGENEART, Gene Oracle, and DNA 2.0) has mastered the art cal circuits, highlights not only a technological development, but of synthesizing relatively long, entirely accurate sequences, and also researchers’ biological ignorance. “It shows that we can stringing them together to create gene-sized fragments on the now write genomes. But at the same moment everyone is realorder of hundreds to thousands of bases. Venter’s group or- izing … we don’t really know what to write.” dered up 1,078 1-kb “cassettes,” the building blocks of the M. Another problem is that it’s one thing to draw a straightforward mycoides genome. pathway on paper; it’s quite another to make it work in practice. The team had already demonstrated they could assemble Unlike the electrical circuits on which those drawings are based, complete genomes, having successfully built both an intact biology simply isn’t binary, but stochastic. Promoters aren’t 100 functional virus (the 5-kb phiX174) and a bacterial genome (the percent on or off, for instance, and operator sequences are not 583-kb M. genitalium). They also showed they could transplant all the same. “It can take only a matter of few days or weeks to a natural (i.e., nonsynthetic) chromosome from one cell to an- design a synthetic gene circuit to look like the schematic,” says other. The next step, synthesizing a genome and transplanting Collins, “but it can take many months to try to actually construct it, should have been simple. Yet according to Venter, the process it so that it functions as desired.” What inevitably follows is a involved “invention after invention after invention of new ways long period of what Collins calls “post-hoc tweaking.” to do things”—everything from synthesis and recombination “That’s where most of us spend most of our time,” he says. to handling bacterial restriction systems. Even DNA manipulaThe effort can pay big dividends, however, as with the biotion proved problematic. “You can’t pipette whole chromosomes engineering of microbes that can synthesize artemisinic acid, without just the shearing forces from pipetting tearing the DNA,” a precursor to the antimalarial drug artemisinin. Artemisinin is he says; as a result, the team took to moving its DNA around in a terpenoid normally extracted from wormwood, a lengthy and agarose plugs. expensive process. University of California-Berkeley Professor Using the synthetic cassettes from Blue Heron, the team as- Jay Keasling led that effort, which took the better part of a desembled the genome via stepwise homologous recombination cade, to provide a rapid, reliable, and low-cost source of the drug. in yeast, building ﬁrst 10-kb pieces, then 100-kb, and ﬁnally the Microbially derived artemisinin, he says, could eventually cost complete 1,077,947-bp chromosome. Highlighting the impor- just a tenth of the native material. “We might be able to save half tance of accurate DNA synthesis, a single error in the dnaA cod- a million children a year,” Keasling says. ing sequence set the team back three months. Keasling’s team started by transplanting a yeast mevalonIn the end, a single bright blue colony signaled success. Upon ate isoprenoid pathway and a synthetic (codon-optimized) receiving the news from project leader Dan Gibson, Venter says amorphadiene synthase gene into E. coli, creating a strain capahe felt “excitement and relief… There were literally thousands of ble of turning sugar into amorphadiene, a precursor to artemisinhurdles that had to be overcome.” ic acid. The next biosynthetic step is a series of oxidation reactions, all of which require a cytochrome-P450. There the team hit THE PROBLEM WITH BIOLOGY a stumbling block, as that enzyme’s identity was unknown. But Venter calls the resulting organism a “synthetic cell,” and the ap- with luck, and some comparative genomics, the team cloned the plications of the technology used to make it run the gamut from necessary gene and transferred it into yeast, giving a strain that bioengineering to basic biology. Chief Scientiﬁc Ofﬁcer Richard could produce artemisinic acid. The ﬁnal step was to migrate the Roberts of New England Biolabs, which supplies reagents sup- entire pathway back into E. coli. porting synthetic biology, suggests one possible use: designing According to Keasling, this work—supported by $42 million organisms in which one of the 64 triplets is reassigned to some from the Bill and Melinda Gates Foundation—represents the novel, non-natural amino acid. That would require a complete ge- culmination of years of genetic tinkering with promoters and rinomic rewrite, as well as the insertion of additional machinery, bosome binding sites, RNA stabilization elements continued » www.sciencemag.org/products

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SYNTHETIC GENOMICS

and transcription factor operators. One key problem, he says, was that one of the intermediates (hydroxymethylglutaryl-CoA) is actually toxic to E. coli. Once the team identiﬁed that step, they tweaked it by both suppressing the biosynthetic enzyme and activating the utilization enzyme. They also constructed a synthetic protein scaffold—a kind of biological assembly line—to “channel” metabolic intermediates from enzyme to enzyme and prevent them from accumulating, increasing output an additional 75-fold. The whole process, Roberts says, represents “probably … the most complicated genetic engineering feat to date.” Keasling licensed the work to a spin-off company called Amyris Biotechnologies, which in turn licensed it to Sanoﬁ Aventis. “Right now, they are scaling up the process, and we should have artemisinin out late this year or early next year,” he says.

RNA SOLUTIONS Such feats of bioengineering highlight the power of synthetic biology. Yet their nearly universal reliance on protein-mediated regulation underscores one of its shortcomings, as well. “There’s a design gap right now in synthetic biology,” says Christina Smolke, assistant professor of bioengineering at Stanford University. Natural biological systems, she says, “have very complex regulatory strategies in play. And they’re layering different mechanisms—not just transcription, but RNA-based mecha-

FEATURED PARTICIPANTS Amyris Biotechnologies www.amyrisbiotech.com

GENEART www.geneart.com

Bill and Melinda Gates Foundation www.gatesfoundation.org

Howard Hughes Medical Institute www.hhmi.org

Blue Heron www.blueheronbio.com

J. Craig Venter Institute www.jcvi.org

Boston University www.bu.edu

Life Technologies www.lifetechnologies.com

Center for Strategic and International Studies www.csis.org

Massachusetts Institute of Technology www.mit.edu

Centre for Genomic Regulation pasteur.crg.es/portal/page/ portal/Internet

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ExxonMobil Research and Engineering www.exxonmobil.com

University of California, San Francisco www.ucsf.edu

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nisms and posttranslational mechanisms. So everything is very tightly regulated.” RNA-based regulators, for instance, have different kinetics and are more malleable than protein, with relatively simple folding rules and a research-friendly modular architecture. They also exact less of an energetic burden on the cell. “As we start to think about genome design,” Smolke says, “issues [such as] the energetic cost of the entire system and how much energy it requires to run all the programs you want to actually get in there, become signiﬁcant.” Smolke’s lab, which builds microbes capable of synthesizing benzylisoquinoline alkaloids (another class of pharmacologically interesting plant-derived compounds), is developing regulatory RNA modules to try to incorporate some subtlety into its synthetic circuitry. In a report published last November in Science, her team described synthetic mini-genes with built-in RNA modules that would, upon sensing the presence of one or more cellsignaling proteins, induce an alternative splicing event that up- or down-regulates the expression of either a ﬂuorescent reporter or a pro-apoptotic gene. According to Smolke, the regulatory modules comprise three elements—a sensor, an actuator, and an information processor that links the two—all contained within a three-exon, two-intron synthetic construct encoding the output gene. The approach is completely generalizable, she says. Her team used the approach to make cells responsive to signaling through disease pathways, but it could be used, for instance, to keep toxic metabolites in check; all the researcher needs do is exchange one sensing element for another. Even the actuator is modular; Smolke’s lab has used microRNAs, antisense RNAs, and even ribozymes. Such regulators could help researcher exert ﬁner control over synthetic systems. But they also add another layer of complexity for those who would design novel genomes. Enter University of California, San Francisco biologist Christopher Voigt. Voigt has been engineering logic circuits, like NOR and XOR gates, from synthetic DNA and E. coli. Yet circuitry represent just half of the problem of programming cells, Voigt says; the other half is software. Just as computer programmers would rather code in high-level languages like C++ than in the 1s and 0s of the computer, so too is it easier to instruct DNA synthesizers in a high level language than in the language of As, Cs, Gs, and Ts—especially when writing sequences the size of a genome. Voigt is now working with Life Technologies to develop a “genetic compiler” and language for programming synthetic genomes. The compiler would reduce human-readable instructions to a series of fundamental components, which could then be strung together in silico and synthesized in vitro. “The idea is for Life Technologies to have it where you write your genetic code like C++ and [the software] converts it into a DNA sequence that they synthesize for you,” he says. In the short term at least, most such work will continue to be done at the level of individual circuits and pathways. But technology evolves, and with it, science itself. Already, new biological vistas are opening. Says Luis Serrano, head of the CRG-EMBL Systems Biology Unit (and Grünberg’s advisor), “If you can make a genome from scratch, now people can play. And by playing you can learn. And by learning we will be able in the future to engineer [genomes] better, or even to design them from scratch.” Jeffrey M. Perkel is a freelance science writer based in Pocatello, Idaho. DOI: 10.1126/science.opms.p1100053

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NEW PRODUCTS: SYNTHETIC GENOMICS

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Advancing materials. Improving the quality of life.

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Abstract Deadline • June 21, 2011 ENERGY AND THE ENVIRONMENT

NANOMATERIALS

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Functional Metal-Oxide Nanostructures

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Carbon Nanotubes, Graphene, and Related Nanostructures

B

Material Challenges in Current and Future Nuclear Technologies Advanced Materials for Fuel Cells

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In Situ Studies of Solid-Oxide Fuel-Cell Materials

BB

Functional Nanowires and Nanotubes

D

Sustainable Synthesis of Nanomaterials

CC

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Advanced Materials for Solar-Fuel Generation

Functional Semiconductor Nanocrystals and Metal-Hybrid Structures

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Mobile Energy

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Transport Properties in Polymer Nanocomposites II

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Applications of Hierarchical 3D Structures

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Fundamental Processes of Solar Harvesting in Excitonic Solar Cells

Mechanical Nanofabrication, Nanopatterning, and Nanoassembly

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Safety and Toxicity Control of Nanomaterials

J

Photonic and Plasmonic Materials for Enhanced Photovoltaic Performance

BIOMATERIALS

K

Materials for High-Performance Photonics

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Bioelectronics—Materials, Properties, and Applications

II

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JJ

Nanofunctional Materials, Nanostructures, and Nanodevices for Cancer Applications

FUNCTIONAL MATERIALS L

Topological Insulator Materials

M

Oxide Semiconductors— Defects, Growth, and Device Fabrication

N

Diamond Electronics and Biotechnology— Fundamentals to Applications V

O

Compound Semiconductors for Generating, Emitting, and Manipulating Energy

P

Ferroelectric and Multiferroic Materials

Q

Magnetoelectric Composites

R

Compliant Electronics and Photonics

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Solution Processing of Inorganic and Hybrid Materials for Electronics and Photonics

T

Large-Area Processing and Patterning for Active Optical and Electronic Devices III

U

Charge Generation/Transport in Organic Semiconductor Materials

V

Multifunctional Polymer-based Materials

W

Phonons in Nanomaterials— Theory, Experiments, and Applications

Y

Advances in Energetic Materials Research

KK

Biomaterials for Tissue Regeneration

LL

Synthetic and Biological Gels

MM Micro- and Nanoscale Processing of Biomedical Materials NN

Nucleation and Growth of Biological and Biomimetic Materials

OO

Multiscale Mechanics of Hierarchical Materials

MATERIALS EXPLORATION PP

Three-Dimensional Tomography of Materials

QQ

Functional Imaging of Materials— Advances in Multifrequency and Multispectral Scanning Probe Microscopy and Analysis

RR

Dynamics in Conﬁned Systems and Functional Interfaces

SS

Properties and Processes at the Nanoscale— Nanomechanics of Material Behavior

TT

Microelectromechanical Systems— Materials and Devices V

UU

Combinatorial and High-throughput Methods in Materials Science

MEETING CHAIRS Cammy R. Abernathy

Paul V. Braun

Masashi Kawasaki

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University of Florida [email protected]ﬂ.edu

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